Patent application title: Plants Having Enhanced Yield-Related Traits and a Method for Making the Same
Inventors:
Christophe Reuzeau (La Chapelle Gonaguet, FR)
Christophe Reuzeau (La Chapelle Gonaguet, FR)
Valerie Frankard (Waterloo, BE)
Ana Isabel Sanz Molinero (Madrid, ES)
Ana Isabel Sanz Molinero (Madrid, ES)
Assignees:
BASF Plant Science GmbH
IPC8 Class: AC12N1582FI
USPC Class:
800287
Class name: Multicellular living organisms and unmodified parts thereof and related processes method of introducing a polynucleotide molecule into or rearrangement of genetic material within a plant or plant part the polynucleotide contains a tissue, organ, or cell specific promoter
Publication date: 2013-12-05
Patent application number: 20130326733
Abstract:
The present invention relates generally to the field of molecular biology
and concerns a method for improving various plant growth characteristics
by modulating expression in a plant of a nucleic acid encoding a GRP
(Growth-Related Protein), specifically a nucleic acid encoding a HAL3
polypeptide, a nucleic acid encoding a MADS15 polypeptide, a nucleic acid
encoding a PLT transcription factor polypeptide, a nucleic acid encoding
a Squamosa promoter binding protein-like 15 (SPL15) transcription factor
polypeptide or a nucleic acid encoding a basic/helix-loop-helix (bHLH)
transcription factor. The present invention also concerns plants having
modulated expression of a nucleic acid encoding a GRP, which plants have
improved growth characteristics relative to corresponding wild type
plants or other control plants. The invention also provides constructs
useful in the methods of the invention.Claims:
1. A method for enhancing a yield-related trait in a plant relative to a
control plant, comprising: (a) increasing expression of: (i) a nucleic
acid encoding a HAL3 polypeptide; (ii) a nucleic acid encoding a MADS15
protein; (iii) a nucleic acid encoding a PLT transcription factor
polypeptide; (iv) a nucleic acid encoding an SPL15 transcription factor
polypeptide; or (v) a nucleic acid encoding a bHLH transcription factor
comprising the amino acid sequence of SEQ ID NO 213, SEQ ID NO 215, SEQ
ID NO: 217, SEQ ID NO: 219, SEQ ID NO 225, SEQ ID NO: 297, SEQ ID NO: 299
or SEQ ID NO: 301, or an orthologue or paralogue thereof; (b) selecting
for a plant having an enhanced yield-related trait relative to a control
plant.
2. The method of claim 1, wherein said increased expression is effected by: (a) introducing and expressing in the plant a nucleic acid encoding a HAL3 polypeptide; (b) introducing and expressing in the plant a nucleic acid encoding a MADS15 protein, wherein said MADS15 protein comprises any one or more of the motifs of SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50 and SEQ ID NO: 51; (c) introducing and expressing in the plant a nucleic acid encoding a PLT transcription factor polypeptide; (d) introducing and expressing in the plant a nucleic acid encoding a SPL15 transcription factor polypeptide; or (e) introducing and expressing in the plant a nucleic acid encoding a bHLH transcription factor comprising the amino acid sequence of SEQ ID NO 213, SEQ ID NO 215, SEQ ID NO: 217, SEQ ID NO: 219, SEQ ID NO 225, SEQ ID NO: 297, SEQ ID NO: 299 or SEQ ID NO: 301, or an orthologue or paralogue thereof.
3. The method of claim 2, wherein: (a) said nucleic acid encoding a HAL3 protein comprises any one of the nucleic acid SEQ ID NOs given in Table A, or a portion thereof, or a sequence capable of hybridizing with any one of the nucleic acids SEQ ID NOs given in Table A; (b) said nucleic acid encoding a MADS15 protein comprises any one of the nucleic acid SEQ ID NOs given in Table D, or a portion thereof, or a sequence capable of hybridixing with any one of the nucleic acids SEQ ID NOs given in Table D; (c) said nucleic acid encoding a PLT transcription factor polypeptide comprises any one of the nucleic acid SEQ ID NOs given in Table I, or a portion thereof, or a sequence capable of hybridizing with any one of the nucleic acids SEQ ID NOs given in Table I; (d) said nucleic acid encoding a SPL15 transcription factor polypeptide comprises any one of the nucleic acid SEQ ID NOs given in Table N, or a portion thereof, or a sequence capable of hybridizing with any one of the nucleic acids SEQ ID NOs given in Table N; or (e) said nucleic acid encoding a bHLH transcription factor is a portion of, an allelic variant of, a splice variant of, or a sequence capable of hybridizing to a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 212, SEQ ID NO: 214, SEQ ID NO: 216, SEQ ID NO: 218, SEQ ID NO: 220, SEQ ID NO: 222, SEQ ID NO: 224, SEQ ID NO: 226, SEQ ID NO: 228, SEQ ID NO: 296, SEQ ID NO: 298 or SEQ ID NO: 300, wherein said portion, allelic variant, splice variant or hybridizing sequence encodes a bHLH transcription factor.
4. The method of claim 2, wherein: (a) said nucleic acid encoding a HAL3 protein encodes an orthologue or paralogue of any of the polypeptide SEQ ID NOs given in Table A; (b) said nucleic acid encoding a MADS15 protein encodes an orthologue or paralogue of any of the polypeptide SEQ ID NOs given in Table D; (c) said nucleic acid encoding a PLT transcription factor encodes an orthologue or paralogue of any of the polypeptide SEQ ID NOs given in Table D; (d) said nucleic acid encoding a SPL15 transcription factor polypeptide encodes an orthologue or paralogue of any of the polypeptide SEQ ID NOs given in Table N.
5. The method of claim 1, wherein: (a) said nucleic acid encodes a HAL3 protein, and wherein said enhanced yield-related trait comprises increased yield and/or early vigour; (b) said nucleic acid encodes MADS15 protein, and wherein said enhanced yield-related trait comprises root biomass; (c) said nucleic acid encodes a PLT transcription factor, and wherein said enhanced yield-related trait comprises increased seed yield; (d) said nucleic acid encodes a SPL15 transcription factor polypeptide, and wherein said enhanced yield-related trait comprises increased seed yield; (e) said nucleic acid encodes a bHLH transcription factor, and wherein said enhanced yield-related trait comprises early vigour.
6. The method of claim 5, wherein: (a) said nucleic acid encodes a HAL3 protein, and wherein said enhanced yield-related trait is increased seed yield comprising increased seed biomass, increased harvest index, and/or increased number of (filled) seeds; (b) said nucleic acid encodes a PLT transcription factor, and wherein said increased seed yield comprises increased seed biomass, increased number of flowers per plant, increased number of (filled) seeds, increased seed filling rate, increased harvest index, increased Thousand Kernel Weight (TKW), increased seed area, increased seed length, and/or increased seed width; (c) said nucleic acid encodes a SPL15 transcription factor polypeptide, and wherein said increased seed yield comprises increased aboveground biomass, increased number of flowers per plant, increased seed yield, increased number of (filled) seeds, increased thousand kernel weight (TKW), and/or increased harvest index.
7. The method of claim 2, wherein: (a) said nucleic acid encoding a HAL3 protein protein is operably linked to a shoot-specific promoter or a beta expansin promoter; (b) said nucleic acid encoding a MADS15 protein is operably linked to a constitutive promoter or a GOS2 promoter; (c) said nucleic acid encoding a PLT transcription factor is operably linked to a constitutive promoter or a GOS2 promoter; (d) said nucleic acid encoding a SPL15 transcription factor polypeptide is operably linked to a constitutive promoter, a high mobility group B (HMGB) promoter, a GOS2 promoter, a rice HMGB promoter, or a rice GOS2 promoter; or (e) said nucleic acid encoding a bHLH transcription factor is operably linked to a constitutive promoter or a GOS2 promoter.
8. The method of claim 2, wherein: (a) said nucleic acid encoding a HAL3 protein is of plant origin, from a dicotyledonous plant, from the family Brassicaceae, from the genus Arabidopsis, or from Arabidopsis thaliana; (b) said nucleic acid encoding a MADS15 protein is of plant origin, from a monocotyledonous plant, from the family Poaceae, from the genus Oryza, or from Oryza sativa; (c) said nucleic acid encoding a PLT transcription factor is of plant origin, from a dicotyledonous plant, from the Brassicaceae family, from the genus Arabidopsis, or from Arabidopsis thaliana; (d) said nucleic acid encoding a SPL15 transcription factor polypeptide is of plant origin, from a dicotyledonous plant, from the family Brassicaceae, or from Arabidopsis thaliana; or (e) said nucleic acid encoding a bHLH transcription factor is of plant origin, from a monocotyledonous plant, from the family Poaceae, from the genus Oryza, or from Oryza sativa.
9. A plant or part thereof obtained by the method of claim 1, or a seed or progeny of said plant, wherein said plant or part thereof, or said seed or progeny, comprises a recombinant nucleic acid encoding a HAL3 polypeptide, a recombinant nucleic acid encoding a MADS15 protein, a recombinant nucleic acid encoding a PLT transcription factor polypeptide operably linked to a constitutive promoter, a recombinant nucleic acid encoding a SPL15 transcription factor polypeptide, or a recombinant nucleic acid encoding a bHLH transcription factor.
10. A construct comprising: (a) (i) a nucleic acid encoding a HAL3 polypeptide (ii) a nucleic acid encoding a MADS15 protein comprising one or more of the motifs of SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50 and SEQ ID NO: 51; (iii) a nucleic acid encoding a PLT transcription factor polypeptide; (iv) a nucleic acid encoding an SPL15 transcription factor polypeptide; or (v) a nucleic acid encoding a bHLH transcription factor comprising the amino acid sequence of SEQ ID NO 213, SEQ ID NO 215, SEQ ID NO: 217, SEQ ID NO: 219, SEQ ID NO 225, SEQ ID NO: 297, SEQ ID NO: 299 or SEQ ID NO: 301, or an orthologue or paralogue thereof; (b) one or more control sequences capable of driving expression of the nucleic acid of (a) in a plant; and optionally (c) a transcription termination sequence.
11. The construct of claim 10, wherein: (a) said nucleic acid encodes a HAL3 polypeptide, and wherein said one or more control sequences is a shoot-specific promoter or a beta expansin promoter; (b) said nucleic acid encodes a MADS15 protein, and wherein said one or more control sequences is a constitutive promoter or a GOS2 promoter; (c) said nucleic acid encodes a PLT transcription factor polypeptide, and wherein said one or more control sequences is a constitutive promoter or a GOS2 promoter; (d) said nucleic acid encodes a SPL15 transcription factor polypeptide, and wherein said one or more control sequences is a constitutive promoter, a HMGB promoter, or a GOS2 promoter; or (e) said nucleic acid encodes a bHLH transcription factor, and wherein said one or more control sequences is a constitutive promoter or a GOS2 promoter.
12. A plant, plant part or plant cell transformed with the construct of claim 10.
13. A plant, plant part or plant cell comprising the construct of claim 10.
14. A method for the production of a transgenic plant having an enhanced yield-related trait relative to a control plant, comprising: (a) introducing and increasing expression in a plant of: (i) a nucleic acid encoding a HAL3 polypeptide; (ii) a nucleic acid encoding a MADS15 protein comprising one or more of the motifs of SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50 and SEQ ID NO: 51; (iii) a nucleic acid encoding a PLT transcription factor polypeptide; (iv) a nucleic acid encoding an SPL15 transcription factor polypeptide; or (v) a nucleic acid encoding a bHLH transcription factor comprising the amino acid sequence of SEQ ID NO 213, SEQ ID NO 215, SEQ ID NO: 217, SEQ ID NO: 219, SEQ ID NO 225, SEQ ID NO: 297, SEQ ID NO: 299 or SEQ ID NO: 301, or an orthologue or paralogue thereof; (b) cultivating the plant under conditions promoting plant growth and development; and (c) selecting for a plant having an enhanced yield-related trait relative to a control plant.
15. The method of claim 14, further comprising obtaining a seed or progeny of said plant.
16. A transgenic plant having an enhanced yield-related trait relative to a control plant resulting from increased expression of: (i) a nucleic acid encoding a HAL3 polypeptide; (ii) a nucleic acid encoding a MADS15 protein comprising one or more of the motifs of SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50 and SEQ ID NO: 51; (iii) a nucleic acid encoding a PLT transcription factor polypeptide; (iv) a nucleic acid encoding an SPL15 transcription factor polypeptide; or (v) a nucleic acid encoding a bHLH transcription factor comprising the amino acid sequence of SEQ ID NO 213, SEQ ID NO 215, SEQ ID NO: 217, SEQ ID NO: 219, SEQ ID NO 225, SEQ ID NO: 297, SEQ ID NO: 299 or SEQ ID NO: 301, or an orthologue or paralogue thereof, in said plant, or a transgenic plant cell obtained from said transgenic plant.
17. The transgenic plant of claim 16, wherein said plant is a crop plant, a monocot plant or a cereal, or wherein said plant is rice, maize, wheat, barley, millet, rye, triticale, sorghum or oats.
18. Harvestable parts of the transgenic plant of claim 16.
19. Products obtained from the transgenic plant of claim 16 and/or from harvestable parts of said trasngenic plant.
20. A method for increasing abiotic stress resistance in a plant relative to a control plant, comprising introducing into a plant the construct of claim 10, and selecting for a plant having increased abiotic stress resistance relative to a control plant, wherein said nucleic acid encodes a PLT transcription factor polypeptide, and wherein said increased abiotic stress resistance is preferably increased nutrient uptake efficiency.
21. An isolated polypeptide comprising: (a) the amino acid sequence of SEQ ID NO: 297, SEQ ID NO: 299, or SEQ ID NO: 301; or (b) an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the amino acid sequence of SEQ ID NO: 297, SEQ ID NO: 299 or SEQ ID NO: 301, wherein said amino acid sequence further comprises a bHLH domain.
22. An isolated nucleic acid molecule comprising: (a) a nucleic acid sequence encoding the isolated polypeptide of claim 21; (b) the nucleic acid sequence of SEQ ID NO: 296, SEQ ID NO: 298 or SEQ ID NO: 300; or (c) the complement of the nucleic acid sequence of SEQ ID NO: 296, SEQ ID NO: 298 or SEQ ID NO: 300.
Description:
RELATED APPLICATIONS
[0001] The present application is a continuation of U.S. patent application Ser. No. 12/294,983 filed on Sep. 29, 2008, which is a national stage application (under 35 U.S.C. 371) of PCT/EP2007/053081, filed Mar. 30, 2007, which claims benefit of European applications 06075822.4, filed Mar. 31, 2006; 06075823.2, filed Mar. 31, 2006; 06075918.0, filed Apr. 10, 2006; 06075957.8, filed Apr. 27, 2006; 06114140.4, filed May 18, 2006; 06117235.9, filed Jul. 14, 2006 and 07100833.8, filed Jan. 19, 2007 and U.S. Provisional applications 60/790,151, filed Apr. 7, 2006; 60/790,116, filed Apr. 7, 2006; 60/792,225, filed Apr. 14, 2006; 60/798,602, filed May 8, 2006; 60/801,687, filed May 19, 2006 and 60/889,958, filed Feb. 15, 2007. The entire contents of each of these applications are hereby incorporated by reference herein.
SUBMISSION OF SEQUENCE LISTING
[0002] The Sequence Listing associated with this application is filed in electronic format via EFS-Web and hereby incorporated by reference into the specification in its entirety. The name of the text file containing the Sequence Listing is Sequence_Listing--32279--00060. The size of the text file is 523 KB, and the text file was created on Jun. 3, 2013.
[0003] The present invention relates generally to the field of molecular biology and concerns a method for improving various plant growth characteristics by modulating expression in a plant of a nucleic acid encoding a GRP (Growth-Related Protein). The present invention also concerns plants having modulated expression of a nucleic acid encoding a GRP, which plants have improved growth characteristics relative to corresponding wild type plants or other control plants. The invention also provides constructs useful in the methods of the invention.
[0004] The ever-increasing world population and the dwindling supply of arable land available for agriculture fuels research towards increasing the efficiency of agriculture. Conventional means for crop and horticultural improvements utilise selective breeding techniques to identify plants having desirable characteristics. However, such selective breeding techniques have several drawbacks, namely that these techniques are typically labour intensive and result in plants that often contain heterogeneous genetic components that may not always result in the desirable trait being passed on from parent plants. Advances in molecular biology have allowed mankind to modify the germplasm of animals and plants. Genetic engineering of plants entails the isolation and manipulation of genetic material (typically in the form of DNA or RNA) and the subsequent introduction of that genetic material into a plant. Such technology has the capacity to deliver crops or plants having various improved economic, agronomic or horticultural traits.
[0005] A trait of particular economic interest is increased yield. Yield is normally defined as the measurable produce of economic value from a crop. This may be defined in terms of quantity and/or quality. Yield is directly dependent on several factors, for example, the number and size of the organs, plant architecture (for example, the number of branches), seed production, leaf senescence and more. Root development, nutrient uptake, stress tolerance and early vigour may also be important factors in determining yield. Optimizing the abovementioned factors may therefore contribute to increasing crop yield.
[0006] Seed yield is a particularly important trait, since the seeds of many plants are important for human and animal nutrition. Crops such as corn, rice, wheat, canola and soybean account for over half the total human caloric intake, whether through direct consumption of the seeds themselves or through consumption of meat products raised on processed seeds. They are also a source of sugars, oils and many kinds of metabolites used in industrial processes. Seeds contain an embryo (the source of new shoots and roots) and an endosperm (the source of nutrients for embryo growth during germination and during early growth of seedlings). The development of a seed involves many genes, and requires the transfer of metabolites from the roots, leaves and stems into the growing seed. The endosperm, in particular, assimilates the metabolic precursors of carbohydrates, oils and proteins and synthesizes them into storage macromolecules to fill out the grain.
[0007] Another important trait for many crops is early vigour. Improving early vigour is an important objective of modern rice breeding programs in both temperate and tropical rice cultivars. Long roots are important for proper soil anchorage in water-seeded rice. Where rice is sown directly into flooded fields, and where plants must emerge rapidly through water, longer shoots are associated with vigour. Where drill-seeding is practiced, longer mesocotyls and coleoptiles are important for good seedling emergence. The ability to engineer early vigour into plants would be of great importance in agriculture. For example, poor early vigor has been a limitation to the introduction of maize (Zea mays L.) hybrids based on Corn Belt germplasm in the European Atlantic.
[0008] A further important trait is that of improved abiotic stress tolerance. Abiotic stress is a primary cause of crop loss worldwide, reducing average yields for most major crop plants by more than 50% (Wang et al., Planta (2003) 218: 1-14). Abiotic stresses may be caused by drought, salinity, extremes of temperature, chemical toxicity and oxidative stress. The ability to improve plant tolerance to abiotic stress would be of great economic advantage to farmers worldwide and would allow for the cultivation of crops during adverse conditions and in territories where cultivation of crops may not otherwise be possible.
[0009] Crop yield may therefore be increased by optimising one of the above-mentioned factors.
[0010] Depending on the end use, the modification of certain yield traits may be favoured over others. For example for applications such as forage or wood production, or bio-fuel resource, an increase in the vegetative parts of a plant may be desirable, and for applications such as flour, starch or oil production, an increase in seed parameters may be particularly desirable. Even amongst the seed parameters, some may be favoured over others, depending on the application. Various mechanisms may contribute to increasing seed yield, whether that is in the form of increased seed size or increased seed number.
[0011] One approach to increasing yield (seed yield and/or biomass) in plants may be through modification of the inherent growth mechanisms of a plant, such as the cell cycle or various signalling pathways involved in plant growth or in defense mechanisms.
[0012] It has now been found that various growth characteristics may be improved in plants by modulating expression in a plant of a nucleic acid encoding a GRP (Growth-Related Protein) in a plant. The GRP may be one of the following: a MADS-box transcription factor (OsMADS15), a PLT transcription factor, a bHLH transcription factor, an SPL15 transcription factor, and a halotolerance protein (HAL3).
BACKGROUND
Halotolerance Proteins
[0013] Many enzymatic processes in a cell require the involvement of Coenzyme A (CoA or CoASH). Coenzyme A (CoASH) itself is a highly polar molecule, consisting of adenosine 3',5'-diphosphate linked to 4-phosphopantethenic acid (Vitamin B5) and thence to β-mercaptoethylamine. The free SH group may be esterified, depending on the process in which CoA is involved. The pathway of CoA synthesis has been elucidated in bacteria, animals and plants. In plants, the conversion of the CoA precursor 4'-phosphopantothenoyl-cysteine (PPC) into 4'-phosphopantetheine is catalysed by the flavoprotein 4'-phosphopantothenoyl-cysteine decarboxylase (PPCDC or HAL3, Kupke et al. J. Biol. Chem. 276, 19190-19196, 2001). The gene encoding HAL3 proteins may be part of a small gene family: the Arabidopsis genome comprises two isoforms (AtHAL3a and AtHAL3b; Espinoza-Ruiz et al. Plant J. 20, 529-539, 1999), in tobacco 3 HAL3 genes are present (Yonamine et al. J. Exp. Bot. 55, 387-395, 2004), though in rice HAL3 is a single copy gene. It is suggested that AtHAL3 and other HAL3 proteins function as a trimer, with each monomer having a flavin mononucleotide (FMN) bound (Albert et al. Structure 8, 961-969, 2000). The FMN cofactor is postulated to play a role in the redox reaction generating 4'-phosphopantetheine.
[0014] The molecular function of HAL3 proteins is not fully elucidated yet. The HAL3 proteins show homology to the yeast SIS2 protein, which is involved in halotolerance (Ferrando et al., Mol. Cell. Biol. 15, 5470-5481). Plants or plant cells ectopically expressing HAL3 show improved salt, osmotic or lithium stress tolerance (Espinoza-Ruiz et al., 1999; Yonamine et al., 2004). Tobacco HAL3a overexpression in BY2 cells reportedly caused an increase in intracellular proline content, which may contribute to the salt tolerant phenotype (Yonamine et al., 2004). Furthermore, Arabidopsis plants overexpressing AtHAL3a displayed a faster growth rate than the wild type plants (Espinoza-Ruiz et al., 1999). The AtHAL3b protein was shown to interact with the cell cycle protein CDKB1:1 (WO 00/36124), therefore it was postulated that AtHAL3b is useful for conferring salt stress tolerance to plants and for increasing the growth rate of plants under salt stress conditions Surprisingly, it has now been found that preferentially increasing expression of a nucleic acid encoding a HAL3 polypeptide in expanding tissues gives plants having increased yield relative to control plants, which yield increase is at least 10% compared to the control plants.
Transcription Factors
[0015] Transcription factors are usually defined as proteins that bind to a cis-regulatory element (eg. an enhancer, a TATA box) and that, in association with RNA polymerase, are capable of activating and/or repressing transcription. The Arabidopsis genome codes for at least 1533 transcriptional regulators, which account for ˜5.9% of its estimated total number of genes (Riechmann et al., 2000 (Science Vol. 290, 2105-2109)).
MADS15
[0016] The MADS-box genes constitute a large gene family of eukaryotic transcriptional regulators involved in diverse aspects of yeast, plant and animal development. MADS-box genes encode a strongly conserved MADS domain responsible for DNA binding to specific boxes in the regulatory region of their target genes. The gene family can be divided into two main lineages, type I and type II. Type II genes are also named MIKC-type proteins, referring to the four functional domains they possess (FIG. 5, (Jack, Plant Mol. Biol. 46, 515-520, 2001)):
[0017] MADS for DNA binding, about 60 amino acids (highly conserved) located at the N-terminal end of the protein;
[0018] I for intervening domain (less conserved), involved in the selective formation of MADS dimer;
[0019] K for keratin domain (well conserved) responsible for dimerisation;
[0020] C for C-terminal region (variable in sequence and length) involved in transcriptional activation, or in the formation of a multimeric transcription factor complex.
[0021] Over 100 MADS-box genes have been identified in Arabidopsis, and have been phylogenetically classified into 12 clades, each clade having specific deviations from the MADS consensus (Thiessen et al. J. Mol. Evol. 43, 484-516, 1996). OsMADS15 belongs to the SQUA clade (for SQUAMOSA, from Antirrhinum majus). Genes of the SQUA clade are classified as A function organ identity genes with reference to the ABC floral organ identity specification model, proposed by Coen and Meyerowitz in 1991 (Nature 353, 31-7). Besides OsMADS15, rice OsMADS14, OsMADS18, and OsMADS20 are also part of the SQUA clade.
[0022] The SQUA clade in dicotyledonous plants (dicots) is subdivided into two subgroups, the AP1 and the FUL subclades (in which OsMADS15 clusters). These subclades diverge essentially with respect to the specific amino acid motifs located at the C-terminus of their respective proteins (Litt and Irish, Genetics 165, 821-833, 2003). In addition to the presence of a specific AP1 amino acid motif, the dicot AP1 clade related proteins usually comprise a farnesylation motif at their C-terminus (this motif is CAAX, where C is cysteine, A is usually an aliphatic amino acid, and X is methionine, glutamine, serine, cysteine or alanine). In monocotyledonous plants (monocots), the SQUA clade proteins are also subdivided into two main groups, which may be distinguished based on conserved C-terminal motifs located within the last 15 amino acids of the proteins: LPPWMLS (for example OsMADS15, SEQ ID NO: 117) and LPPWMLR (for example OsMADS18). In contrast to dicot sequences of the SQUA clade, monocot sequences of the SQUA clade do not possess a farnesylation motif at their C-terminus.
[0023] OsMADS15 was postulated to function in a complex with other proteins to control organ formation. However, so far no mutants with a visible phenotype have been identified; no experimental data have been presented relating to ectopic expression or down-regulated expression of OsMADS15, except for the data presented in WO 01/14559 (EP1209232, U.S. Pat. No. 6,995,302). In this disclosure, transgenic Fagopyrum esculentum plants expressing OsMADS15 in sense direction showed increased branching, whereas transgenics expressing the antisense construct had decreased growth and suppressed branching. Kalanchoe daigremontiana transformed with the sense construct had leaf development around the roots, which was taken as an indication of increased branching. The authors stated that no changes were observed in terms of flower development and structure of these flowers. The orthologue of OsMADS15 in Arabidopsis is APETALA1 (AP1). Ectopic expression of AP1 results in a decrease of flowering time, and AP1 mutants exhibit delayed flowering and have abnormal flowers.
PLT
[0024] The AP2(apetala2)/ERF (ethylene-responsive responsive element-binding factor) family comprises transcription factors with at least one highly conserved DNA binding domain, the AP2 domain. The AP2 domain was originally described in APETALA2 (AP2), an Arabidopsis protein involved in developmental programs such as meristem identity regulation and floral organ specification (Jofuku et al., (1994) Plant Cell 6, 1211-1225). AP2/ERF proteins are divided into subfamilies based on whether they contain one (ERF subfamily) or two (AP2 subfamily) DNA binding domains (FIG. 12). More than 140 AP2/ERF genes have been identified in the Arabidopsis thaliana genome (Reichmann et al. (2000) Science 290: 2105-2110), amongst which up to 18 belong to the AP2 subfamily (Kim et al. (2006) Mol Bio Evol 23(1): 107-120).
[0025] Two Arabidopsis AP2 subfamily transcription factor polypeptides, encoded by the Plethora 1 (PLT1) and Plethora 2 (PLT2) genes, collectively called PLT genes, have been shown to be required for stem cell specification and maintenance specifically in the root meristem. In RT-PCR analysis, PLT transcripts were mainly detected in roots, indicating that PLT expression is strongly associated with root identity. Ectopic PLT expression in the Arabidopsis embryo induces homeotic transformation of apical domains into root stem cells, roots, or hypocotyls (Aida et al (2004) Cell 119:109-120).
[0026] US patent application 2004/0045049 and international patent application WO03/013227 provide the nucleic acid sequence encoding the Arabidopsis PLT1 transcription factor (referred to in the applications as G1793). Overexpression of G1793 (using the CaMV 35S promoter) in Arabidopsis was reported to produce alterations in cotyledon morphology, a mild reduction in overall plant size and thin inflorescences (possibly with abnormal flowers) compared to wild type controls. G1793 overexpressors produced more seed oil than control plants. Nucleic acid sequences encoding potential Glycine max, Oryza sativa and Zea mays orthologs are provided.
[0027] International patent application WO 03/002751 provides for a Glycine max nucleic acid sequence encoding a PLT polypeptide, with sequence similarity to a corn nucleic acid sequence identified by gene profiling (by DNA microarray).
[0028] International patent application WO 05/075655 provides for Oryza sativa and Zea mays nucleic acid sequences with sequence similarity to the Arabidopsis PLT genes.
bHLH
[0029] The basic/helix-loop-helix (bHLH) proteins are a superfamily of transcription factors that bind as dimers to specific DNA target sites and that have been well characterized in non-plant eukaryotes as important regulatory components in diverse biological processes. The distinguishing characteristic of the bHLH family is a bipartite domain consisting of approximately 60 amino acids. This bipartite domain is comprised of a DNA-binding basic region, which binds to a consensus hexanucleotide E-box and two α-helices separated by a variable loop region. The two α-helices promote dimerization, allowing the formation of homo- and heterodimers between different family members. While the bHLH domain is evolutionarily conserved, there is little sequence similarity between clades beyond the domain.
[0030] Bailey et al., 2003 (The Plant Cell, Vol. 15, 2497-2501) report the total number of detected bHLH genes in Arabidopsis thaliana to be 162, making bHLH genes one of the largest families of transcription factors in Arabidopsis; the rice genome reportedly contains 131 bHLH genes (Buck and Atchley, 2003 (J. Mol. Evol. 56:742-750)). Heim et al., 2003 (Mol. Biol. Evol. 20(5):735-747) identified 12 subfamilies of bHLH genes from Arabidopsis thaliana based on structural similarities.
[0031] The bHLH proteins from plants that have been characterized have been reported to function in anthocyanin biosynthesis, phytochrome signaling, globulin expression, fruit dehiscence, carpel and epidermal development (Buck and Atchley, 2003).
SPL
[0032] The Squamosa Qromoter binding protein-like (SPL) transcription factor polypeptides are structurally diverse proteins that share a highly conserved DNA binding domain (DBD) of about 80 amino acid residues in length (Klein et al. (1996) Mol Gen Genet. 259: 7-16; Cardon et al. (1999) Gene 237: 91-104). The SPL transcription factor DNA consensus sequence binding site in the promoter of target genes is 5'-TNCGTACAA-3' where N represents any base. Within the SPL DBD are ten conserved cysteine (Cys) or histidine (His) residues (see FIG. 23) of which eight are zinc coordinating residues binding two zinc ions necessary for the formation of SPL specific zinc finger tertiary structure (Yamasaki et al. (2004) J Mol Biol 337: 49-63). A second conserved feature within the SPL DBD is a bipartite nuclear localisation signal. Outside of the DBD, a micro RNA (miRNA) target motif (miR156) is found in most of the nucleic acid sequences encoding SPL transcription factor polypeptides (either in the coding region, or the 3' UTR) across the plant kingdom (Rhoades et al. (2002) Cell 110: 513-520). miRNAs control SPL gene expression post-transcriptionally by targeting SPL encoding mRNAs for degradation or by translational repression.
[0033] Riechmann et al. (Science 290: 2105-2109, 2000) report 16 SPL transcription factor polypeptides in Arabidopsis thaliana, with little sequence similarity between themselves (apart from the abovementioned features), the size of the deduced SPL polypeptide ranging from 131 to 927 amino acids. Nevertheless, pairs of SPL transcription factor polypeptides sharing higher sequence homology were detected within the SPL family of this plant (Cardon et al. (1999)).
[0034] The SPL transcription factor polypeptides (only found in plants) characterized to date have been shown to function in plant development, in particular in flower development. Transgenic plants overexpressing an SPL3 transcription factor polypeptide were reported to flower earlier (Cardon et al. (1997) Plant J 12: 367-377).
[0035] In European patent application EP1033405, the nucleic acid and deduced polypeptide sequences of the SPL15 transcription factor polypeptide are presented.
[0036] In international patent application WO03013227, a nucleic acid sequence (and deduced polypeptide sequence; internal reference G2346) is presented that encodes part of the SPL15 transcription factor polypeptide however 38 amino acids from the C-terminal end of the SPL15 transcription factor. Transgenic Arabidopsis thaliana plants constitutively overexpressing the modified SPL15 transcription factor (or G2346) polypeptide have slightly enlarged cotyledons. At later stages of development, the same plants are reported to show no consistent differences from control plants.
SUMMARY OF THE INVENTION
[0037] Surprisingly, it has now been found that preferentially increasing expression of a nucleic acid encoding a HAL3 polypeptide in expanding tissues gives plants having enhanced yield-related traits, in particular increased early vigour and increased seed yield relative to control plants, which seed yield increase is at least 10% compared to the control plants.
[0038] According to another embodiment of present invention, there is provided a method for enhancing yield-related traits, in particular for increasing early vigour of a plant and increasing seed yield of a plant, relative to control plants, comprising preferentially increasing expression of a nucleic acid encoding a HAL3 polypeptide in expanding tissues of a plant.
[0039] Surprisingly, it has now been found that modulating expression of a nucleic acid encoding a MADS15 polypeptide gives plants having enhanced yield-related traits, in particular increased yield relative to control plants.
[0040] According one embodiment, there is provided a method for increasing plant yield relative to control plants, comprising increasing expression of a nucleic acid encoding a MADS15 polypeptide in a plant. The increased yield comprised increased vegetative biomass but not increased seed yield.
[0041] According to another embodiment, the present invention provides methods for increasing yield of a plant relative to control plants, comprising decreasing the level and/or activity of an endogenous MADS15 polypeptide. The increased yield comprised higher seed yield, but did not comprise increased vegetative biomass.
[0042] Surprisingly, it has now been found that increasing expression of a nucleic acid encoding a PLT transcription factor polypeptide gives plants having enhanced yield related traits, in particular increased yield relative to control plants.
[0043] According to a further embodiment, there is provided a method for increasing plant yield relative to control plants, comprising increasing expression in a plant of a nucleic acid encoding a PLT transcription factor polypeptide.
[0044] Surprisingly, it has now been found that modulating expression in a plant of a nucleic acid encoding a particular class of bHLH transcription factor gives plants having enhanced yield-related traits relative to control plants. The particular class of bHLH transcription factor suitable for enhancing yield-related traits in plants is described in detail below.
[0045] According to a further embodiment, the present invention provides a method for enhancing yield-related traits in plants relative to control plants, comprising modulating expression in a plant of a nucleic acid encoding a particular class of bHLH transcription factor.
[0046] Surprisingly, it has now been found that increasing expression in a plant of a nucleic acid encoding an SPL15 transcription factor polypeptide gives plants having enhanced yield related traits, in particular increased yield relative to control plants.
[0047] According to a further embodiment, the invention provides a method for increasing yield in plants relative to control plants, comprising increasing expression in a plant of a nucleic acid encoding an SPL15 transcription factor polypeptide.
DEFINITIONS
Polypeptide(s)/Protein(s)
[0048] The terms "polypeptide" and "protein" are used interchangeably herein and refer to amino acids in a polymeric form of any length.
Polynucleotide(s)/Nucleic Acid(s)/Nucleic Acid Sequence(s)/Nucleotide Sequence(s)
[0049] The terms "polynucleotide(s)", "nucleic acid sequence(s)", "nucleotide sequence(s)", "nucleic acid(s)" are used interchangeably herein and refer to nucleotides, either ribonucleotides or deoxyribonucleotides or a combination of both, in a polymeric form of any length.
Control Plant(s)
[0050] The choice of suitable control plants is a routine part of an experimental setup and may include corresponding wild type plants or corresponding plants without the gene of interest. The control plant is typically of the same plant species or even of the same variety as the plant to be assessed. The control plant may also be a nullizygote of the plant to be assessed. A "control plant" as used herein refers not only to whole plants, but also to plant parts, including seeds and seed parts.
Homoloque(s)
[0051] "Homologues" of a protein encompass peptides, oligopeptides, polypeptides, proteins and enzymes having amino acid substitutions, deletions and/or insertions relative to the unmodified protein in question and having similar biological and functional activity as the unmodified protein from which they are derived.
[0052] A deletion refers to removal of one or more amino acids from a protein.
[0053] An insertion refers to one or more amino acid residues being introduced into a predetermined site in a protein. Insertions may comprise N-terminal and/or C-terminal fusions as well as intra-sequence insertions of single or multiple amino acids. Generally, insertions within the amino acid sequence will be smaller than N- or C-terminal fusions, of the order of about 1 to 10 residues. Examples of N- or C-terminal fusion proteins or peptides include the binding domain or activation domain of a transcriptional activator as used in the yeast two-hybrid system, phage coat proteins, (histidine)-6-tag, glutathione S-transferase-tag, protein A, maltose-binding protein, dihydrofolate reductase, Tag100 epitope, c-myc epitope, FLAG®-epitope, lacZ, CMP (calmodulin-binding peptide), HA epitope, protein C epitope and VSV epitope.
[0054] A substitution refers to replacement of amino acids of the protein with other amino acids having similar properties (such as similar hydrophobicity, hydrophilicity, antigenicity, propensity to form or break α-helical structures or β-sheet structures). Amino acid substitutions are typically of single residues, but may be clustered depending upon functional constraints placed upon the polypeptide; insertions will usually be of the order of about 1 to 10 amino acid residues. The amino acid substitutions are preferably conservative amino acid substitutions. Conservative substitution tables are well known in the art (see for example Creighton (1984) Proteins. W.H. Freeman and Company and Table 1 below).
TABLE-US-00001 TABLE 1 Examples of conserved amino acid substitutions Conservative Conservative Residue Substitutions Residue Substitutions Ala Ser Leu Ile; Val Arg Lys Lys Arg; Gln Asn Gln; His Met Leu; Ile Asp Glu Phe Met; Leu; Tyr Gln Asn Ser Thr; Gly Cys Ser Thr Ser; Val Glu Asp Trp Tyr Gly Pro Tyr Trp; Phe His Asn; Gln Val Ile; Leu Ile Leu, Val
[0055] Amino acid substitutions, deletions and/or insertions may readily be made using peptide synthetic techniques well known in the art, such as solid phase peptide synthesis and the like, or by recombinant DNA manipulation. Methods for the manipulation of DNA sequences to produce substitution, insertion or deletion variants of a protein are well known in the art. For example, techniques for making substitution mutations at predetermined sites in DNA are well known to those skilled in the art and include M13 mutagenesis, T7-Gen in vitro mutagenesis (USB, Cleveland, Ohio), QuickChange Site Directed mutagenesis (Stratagene, San Diego, Calif.), PCR-mediated site-directed mutagenesis or other site-directed mutagenesis protocols.
Derivatives
[0056] "Derivatives" include peptides, oligopeptides, polypeptides which may, compared to the amino acid sequence of the naturally-occurring form of the protein, such as the protein of interest, comprise substitutions of amino acids with non-naturally occurring amino acid residues, or additions of non-naturally occurring amino acid residues. "Derivatives" of a protein also encompass peptides, oligopeptides, polypeptides which comprise naturally occurring altered (glycosylated, acylated, prenylated, phosphorylated, myristoylated, sulphated etc.) or non-naturally altered amino acid residues compared to the amino acid sequence of a naturally-occurring form of the polypeptide. A derivative may also comprise one or more non-amino acid substituents or additions compared to the amino acid sequence from which it is derived, for example a reporter molecule or other ligand, covalently or non-covalently bound to the amino acid sequence, such as a reporter molecule which is bound to facilitate its detection, and non-naturally occurring amino acid residues relative to the amino acid sequence of a naturally-occurring protein.
Ortholoque(s)/Paraloque(s)
[0057] Orthologues and paralogues encompass evolutionary concepts used to describe the ancestral relationships of genes. Paralogues are genes within the same species that have originated through duplication of an ancestral gene and orthologues are genes from different organisms that have originated through speculation.
Domain
[0058] The term "domain" refers to a set of amino acids conserved at specific positions along an alignment of sequences of evolutionarily related proteins. While amino acids at other positions can vary between homologues, amino acids that are highly conserved at specific positions indicate amino acids that are likely essential in the structure, stability or function of a protein. Identified by their high degree of conservation in aligned sequences of a family of protein homologues, they can be used as identifiers to determine if any polypeptide in question belongs to a previously identified polypeptide family.
Motif/Consensus Sequence/Signature
[0059] The term "motif" or "consensus sequence" or "signature" refers to a short conserved region in the sequence of evolutionarily related proteins. Motifs are frequently highly conserved parts of domains, but may also include only part of the domain, or be located outside of conserved domain (if all of the amino acids of the motif fall outside of a defined domain).
Hybridisation
[0060] The term "hybridisation" as defined herein is a process wherein substantially homologous complementary nucleotide sequences anneal to each other. The hybridisation process can occur entirely in solution, i.e. both complementary nucleic acids are in solution. The hybridisation process can also occur with one of the complementary nucleic acids immobilised to a matrix such as magnetic beads, Sepharose beads or any other resin. The hybridisation process can furthermore occur with one of the complementary nucleic acids immobilised to a solid support such as a nitro-cellulose or nylon membrane or immobilised by e.g. photolithography to, for example, a siliceous glass support (the latter known as nucleic acid arrays or microarrays or as nucleic acid chips). In order to allow hybridisation to occur, the nucleic acid molecules are generally thermally or chemically denatured to melt a double strand into two single strands and/or to remove hairpins or other secondary structures from single stranded nucleic acids.
[0061] The term "stringency" refers to the conditions under which a hybridisation takes place. The stringency of hybridisation is influenced by conditions such as temperature, salt concentration, ionic strength and hybridisation buffer composition. Generally, low stringency conditions are selected to be about 30° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. Medium stringency conditions are when the temperature is 20° C. below Tm, and high stringency conditions are when the temperature is 10° C. below Tm. High stringency hybridisation conditions are typically used for isolating hybridising sequences that have high sequence similarity to the target nucleic acid sequence. However, nucleic acids may deviate in sequence and still encode a substantially identical polypeptide, due to the degeneracy of the genetic code. Therefore medium stringency hybridisation conditions may sometimes be needed to identify such nucleic acid molecules.
[0062] The Tm is the temperature under defined ionic strength and pH, at which 50% of the target sequence hybridises to a perfectly matched probe. The Tm is dependent upon the solution conditions and the base composition and length of the probe. For example, longer sequences hybridise specifically at higher temperatures. The maximum rate of hybridisation is obtained from about 16° C. up to 32° C. below Tm. The presence of monovalent cations in the hybridisation solution reduce the electrostatic repulsion between the two nucleic acid strands thereby promoting hybrid formation; this effect is visible for sodium concentrations of up to 0.4M (for higher concentrations, this effect may be ignored). Formamide reduces the melting temperature of DNA-DNA and DNA-RNA duplexes with 0.6 to 0.7° C. for each percent formamide, and addition of 50% formamide allows hybridisation to be performed at 30 to 45° C., though the rate of hybridisation will be lowered. Base pair mismatches reduce the hybridisation rate and the thermal stability of the duplexes. On average and for large probes, the Tm decreases about 1° C. per % base mismatch. The Tm may be calculated using the following equations, depending on the types of hybrids:
1) DNA-DNA hybrids (Meinkoth and Wahl, Anal. Biochem., 138: 267-284, 1984):
Tm=81.5° C.+16.6×log10[Na.sup.+]a+0.41×%[G/Cb]-500.time- s.[Lc]-1-0.61×% formamide
2) DNA-RNA or RNA-RNA hybrids:
Tm=79.8+18.5 (log10[Na.sup.+]a)+0.58 (% G/Cb)+11.8 (% G/Cb)2-820/Lc
3) oligo-DNA or oligo-RNAd hybrids: For <20 nucleotides: Tm=2 (ln) For 20-35 nucleotides: Tm=22+1.46 (ln)
[0063] a or for other monovalent cation, but only accurate in the 0.01-0.4 M range.
[0064] b only accurate for % GC in the 30% to 75% range.
[0065] c L=length of duplex in base pairs.
[0066] d Oligo, oligonucleotide; ln, effective length of primer=2×(no. of G/C)+(no. of A/T).
[0067] Non-specific binding may be controlled using any one of a number of known techniques such as, for example, blocking the membrane with protein containing solutions, additions of heterologous RNA, DNA, and SDS to the hybridisation buffer, and treatment with Rnase. For non-homologous probes, a series of hybridizations may be performed by varying one of (i) progressively lowering the annealing temperature (for example from 68° C. to 42° C.) or (ii) progressively lowering the formamide concentration (for example from 50% to 0%). The skilled artisan is aware of various parameters which may be altered during hybridisation and which will either maintain or change the stringency conditions.
[0068] Besides the hybridisation conditions, specificity of hybridisation typically also depends on the function of post-hybridisation washes. To remove background resulting from non-specific hybridisation, samples are washed with dilute salt solutions. Critical factors of such washes include the ionic strength and temperature of the final wash solution: the lower the salt concentration and the higher the wash temperature, the higher the stringency of the wash. Wash conditions are typically performed at or below hybridisation stringency. A positive hybridisation gives a signal that is at least twice of that of the background. Generally, suitable stringent conditions for nucleic acid hybridisation assays or gene amplification detection procedures are as set forth above. More or less stringent conditions may also be selected. The skilled artisan is aware of various parameters which may be altered during washing and which will either maintain or change the stringency conditions.
[0069] For example, typical high stringency hybridisation conditions for DNA hybrids longer than 50 nucleotides encompass hybridisation at 65° C. in 1×SSC or at 42° C. in 1×SSC and 50% formamide, followed by washing at 65° C. in 0.3×SSC. Examples of medium stringency hybridisation conditions for DNA hybrids longer than 50 nucleotides encompass hybridisation at 50° C. in 4×SSC or at 40° C. in 6×SSC and 50% formamide, followed by washing at 50° C. in 2×SSC. The length of the hybrid is the anticipated length for the hybridising nucleic acid. When nucleic acids of known sequence are hybridised, the hybrid length may be determined by aligning the sequences and identifying the conserved regions described herein. 1×SSC is 0.15M NaCl and 15 mM sodium citrate; the hybridisation solution and wash solutions may additionally include 5×Denhardt's reagent, 0.5-1.0% SDS, 100 μg/ml denatured, fragmented salmon sperm DNA, 0.5% sodium pyrophosphate.
[0070] For the purposes of defining the level of stringency, reference can be made to Sambrook et al. (2001) Molecular Cloning: a laboratory manual, 3rd Edition Cold Spring Harbor Laboratory Press, CSH, New York or to Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989 and yearly updates).
Splice Variant
[0071] The term "splice variant" as used herein encompasses variants of a nucleic acid sequence in which selected introns and/or exons have been excised, replaced, displaced or added, or in which introns have been shortened or lengthened. Such variants will be ones in which the biological activity of the protein is substantially retained; this may be achieved by selectively retaining functional segments of the protein. Such splice variants may be found in nature or may be manmade. Methods for predicting and isolating such splice variants are well known in the art (see for example Foissac and Schiex, BMC Bioinformatics. 2005; 6: 25).
Allelic Variant
[0072] Alleles or allelic variants are alternative forms of a given gene, located at the same chromosomal position. Allelic variants encompass Single Nucleotide Polymorphisms (SNPs), as well as Small Insertion/Deletion Polymorphisms (INDELs). The size of INDELs is usually less than 100 bp. SNPs and INDELs form the largest set of sequence variants in naturally occurring polymorphic strains of most organisms.
Gene Shuffling/Directed Evolution
[0073] Gene shuffling or directed evolution consists of iterations of DNA shuffling followed by appropriate screening and/or selection to generate variants of nucleic acids or portions thereof encoding proteins having a modified biological activity (Castle et al., (2004) Science 304(5674): 1151-4; U.S. Pat. Nos. 5,811,238 and 6,395,547).
Regulatory Element/Control Sequence/Promoter
[0074] The terms "regulatory element", "control sequence" and "promoter" are all used interchangeably herein and are to be taken in a broad context to refer to regulatory nucleic acid sequences capable of effecting expression of the sequences to which they are ligated. The term "promoter" typically refers to a nucleic acid control sequence located upstream from the transcriptional start of a gene and which is involved in recognising and binding of RNA polymerase and other proteins, thereby directing transcription of an operably linked nucleic acid. Encompassed by the aforementioned terms are transcriptional regulatory sequences derived from a classical eukaryotic genomic gene (including the TATA box which is required for accurate transcription initiation, with or without a CCAAT box sequence) and additional regulatory elements (i.e. upstream activating sequences, enhancers and silencers) which alter gene expression in response to developmental and/or external stimuli, or in a tissue-specific manner. Also included within the term is a transcriptional regulatory sequence of a classical prokaryotic gene, in which case it may include a -35 box sequence and/or -10 box transcriptional regulatory sequences. The term "regulatory element" also encompasses a synthetic fusion molecule or derivative that confers, activates or enhances expression of a nucleic acid molecule in a cell, tissue or organ.
[0075] A "plant promoter" comprises regulatory elements, which mediate the expression of a coding sequence segment in plant cells. Accordingly, a plant promoter need not be of plant origin, but may originate from viruses or micro-organisms, for example from viruses which attack plant cells. The "plant promoter" can also originate from a plant cell, e.g. from the plant which is transformed with the nucleic acid sequence to be expressed in the inventive process and described herein. This also applies to other "plant" regulatory signals, such as "plant" terminators. The promoters upstream of the nucleotide sequences useful in the methods of the present invention can be modified by one or more nucleotide substitution(s), insertion(s) and/or deletion(s) without interfering with the functionality or activity of either the promoters, the open reading frame (ORF) or the 3'-regulatory region such as terminators or other 3' regulatory regions which are located away from the ORF. It is furthermore possible that the activity of the promoters is increased by modification of their sequence, or that they are replaced completely by more active promoters, even promoters from heterologous organisms. For expression in plants, the nucleic acid molecule must, as described above, be linked operably to or comprise a suitable promoter which expresses the gene at the right point in time and with the required spatial expression pattern.
[0076] For the identification of functionally equivalent promoters, the promoter strength and/or expression pattern of a candidate promoter may be analysed for example by operably linking the promoter to a reporter gene and assaying the expression level and pattern of the reporter gene in various tissues of the plant. Suitable well-known reporter genes include for example beta-glucuronidase or beta galactosidase. The promoter activity is assayed by measuring the enzymatic activity of the beta-glucuronidase or beta-galactosidase. The promoter strength and/or expression pattern may then be compared to that of a reference promoter (such as the one used in the methods of the present invention). Alternatively, promoter strength may be assayed by quantifying mRNA levels or by comparing mRNA levels of the nucleic acid used in the methods of the present invention, with mRNA levels of housekeeping genes such as 18S rRNA, using methods known in the art, such as Northern blotting with densitometric analysis of autoradiograms, quantitative real-time PCR or RT-PCR (Heid et al., 1996 Genome Methods 6: 986-994). Generally by "weak promoter" is intended a promoter that drives expression of a coding sequence at a low level. By "low level" is intended at levels of about 1/10,000 transcripts to about 1/100,000 transcripts, to about 1/500,0000 transcripts per cell. Conversely, a "strong promoter" drives expression of a coding sequence at high level, or at about 1/10 transcripts to about 1/100 transcripts to about 1/1,000 transcripts per cell.
Operably Linked
[0077] The term "operably linked" as used herein refers to a functional linkage between the promoter sequence and the gene of interest, such that the promoter sequence is able to initiate transcription of the gene of interest.
Constitutive Promoter
[0078] A "constitutive promoter" refers to a promoter that is transcriptionally active during most, but not necessarily all, phases of growth and development and under most environmental conditions, in at least one cell, tissue or organ. Table 2 below gives examples of constitutive promoters.
TABLE-US-00002 TABLE 2 Examples of constitutive promoters Gene Source Reference Actin McElroy et al, Plant Cell, 2: 163-171, 1990 HMGB WO 2004/070039 CAMV 35S Odell et al, Nature, 313: 810-812, 1985 CaMV 19S Nilsson et al., Physiol. Plant. 100: 456-462, 1997 GOS2 de Pater et al, Plant J Nov; 2(6): 837-44, 1992, WO 2004/065596 Ubiquitin Christensen et al, Plant Mol. Biol. 18: 675-689, 1992 Rice cyclophilin Buchholz et al, Plant Mol Biol. 25(5): 837-43, 1994 Maize H3 histone Lepetit et al, Mol. Gen. Genet. 231: 276-285, 1992 Alfalfa H3 Wu et al. Plant Mol. Biol. 11: 641-649, 1988 histone Actin 2 An et al, Plant J. 10(1); 107-121, 1996 34S FMV Sanger et al., Plant. Mol. Biol., 14, 1990: 433-443 Rubisco U.S. Pat. No. 4,962,028 small subunit OCS Leisner (1988) Proc Natl Acad Sci USA 85(5): 2553 SAD1 Jain et al., Crop Science, 39 (6), 1999: 1696 SAD2 Jain et al., Crop Science, 39 (6), 1999: 1696 nos Shaw et al. (1984) Nucleic Acids Res. 12(20): 7831-7846 V-ATPase WO 01/14572 Super promoter WO 95/14098 G-box proteins WO 94/12015
Ubiquitous Promoter
[0079] A ubiquitous promoter is active in substantially all tissues or cells of an organism.
Developmentally-Regulated Promoter
[0080] A developmentally-regulated promoter is active during certain developmental stages or in parts of the plant that undergo developmental changes.
Inducible Promoter
[0081] An inducible promoter has induced or increased transcription initiation in response to a chemical (for a review see Gatz 1997, Annu. Rev. Plant Physiol. Plant Mol. Biol., 48:89-108), environmental or physical stimulus, or may be "stress-inducible", i.e. activated when a plant is exposed to various stress conditions, or a "pathogen-inducible" i.e. activated when a plant is exposed to exposure to various pathogens.
Organ-Specific/Tissue-Specific Promoter
[0082] An organ-specific or tissue-specific promoter is one that is capable of preferentially initiating transcription in certain organs or tissues, such as the leaves, roots, seed tissue etc. For example, a "root-specific promoter" is a promoter that is transcriptionally active predominantly in plant roots, substantially to the exclusion of any other parts of a plant, whilst still allowing for any leaky expression in these other plant parts. Promoters able to initiate transcription in certain cells only are referred to herein as "cell-specific".
[0083] A green tissue-specific promoter as defined herein is a promoter that is transcriptionally active predominantly in green tissue, substantially to the exclusion of any other parts of a plant, whilst still allowing for any leaky expression in these other plant parts.
[0084] Examples of green tissue-specific promoters which may be used to perform the methods of the invention are shown in Table 3 below.
TABLE-US-00003 TABLE 3 Examples of green tissue-specific promoters Gene Expression Reference Maize Orthophosphate dikinase Leaf specific Fukavama et al., 2001 Maize Phosphoenolpyruvate Leaf specific Kausch et al., 2001 carboxylase Rice Phosphoenolpyruvate Leaf specific Liu et al., 2003 carboxylase Rice small subunit Rubisco Leaf specific Nomura et al., 2000 rice beta expansin EXBP9 Shoot specific WO 2004/070039 Pigeonpea small subunit Leaf specific Panguluri et al., 2005 Rubisco Pea RBCS3A Leaf specific
[0085] Another example of a tissue-specific promoter is a meristem-specific promoter, which is transcriptionally active predominantly in meristematic tissue, substantially to the exclusion of any other parts of a plant, whilst still allowing for any leaky expression in these other plant parts
Terminator
[0086] The term "terminator" encompasses a control sequence which is a DNA sequence at the end of a transcriptional unit which signals 3' processing and polyadenylation of a primary transcript and termination of transcription. The terminator can be derived from the natural gene, from a variety of other plant genes, or from T-DNA. The terminator to be added may be derived from, for example, the nopaline synthase or octopine synthase genes, or alternatively from another plant gene, or less preferably from any other eukaryotic gene.
Modulation
[0087] The term "modulation" means in relation to expression or gene expression, a process in which the expression level is changed by said gene expression in comparison to the control plant, preferably the expression level is increased. The original, unmodulated expression may be of any kind of expression of a structural RNA (rRNA, tRNA) or mRNA with subsequent translation. The term "modulating the activity" shall mean any change of the expression of the inventive nucleic acid sequences or encoded proteins, which leads to increased yield and/or increased growth of the plants.
Increased Expression/Overexpression
[0088] The term "increased expression" or "overexpression" as used herein means any form of expression that is additional to the original wild-type expression level.
[0089] Methods for increasing expression of genes or gene products are well documented in the art and include, for example, overexpression driven by appropriate promoters, the use of transcription enhancers or translation enhancers. Isolated nucleic acids which serve as promoter or enhancer elements may be introduced in an appropriate position (typically upstream) of a non-heterologous form of a polynucleotide so as to upregulate expression of a nucleic acid encoding the polypeptide of interest. For example, endogenous promoters may be altered in vivo by mutation, deletion, and/or substitution (see, Kmiec, U.S. Pat. No. 5,565,350; Zarling et al., PCT/US93/03868), or isolated promoters may be introduced into a plant cell in the proper orientation and distance from a gene of the present invention so as to control the expression of the gene.
[0090] If polypeptide expression is desired, it is generally desirable to include a polyadenylation region at the 3'-end of a polynucleotide coding region. The polyadenylation region can be derived from the natural gene, from a variety of other plant genes, or from T-DNA. The 3' end sequence to be added may be derived from, for example, the nopaline synthase or octopine synthase genes, or alternatively from another plant gene, or less preferably from any other eukaryotic gene.
[0091] An intron sequence may also be added to the 5' untranslated region (UTR) or the coding sequence of the partial coding sequence to increase the amount of the mature message that accumulates in the cytosol. Inclusion of a spliceable intron in the transcription unit in both plant and animal expression constructs has been shown to increase gene expression at both the mRNA and protein levels up to 1000-fold (Buchman and Berg (1988) Mol. Cell. biol. 8: 4395-4405; Callis et al. (1987) Genes Dev 1:1183-1200). Such intron enhancement of gene expression is typically greatest when placed near the 5' end of the transcription unit. Use of the maize introns Adh1-S intron 1, 2, and 6, the Bronze-1 intron are known in the art. For general information see: The Maize Handbook, Chapter 116, Freeling and Walbot, Eds., Springer, N.Y. (1994).
Endogenous Gene
[0092] Reference herein to an "endogenous" gene not only refers to the gene in question as found in a plant in its natural form (i.e., without there being any human intervention), but also refers to that same gene (or a substantially homologous nucleic acid/gene) in an isolated form subsequently (re)introduced into a plant (a transgene). For example, a transgenic plant containing such a transgene may encounter a substantial reduction of the transgene expression and/or substantial reduction of expression of the endogenous gene.
Decreased Expression
[0093] Reference herein to "reduction or substantial elimination" is taken to mean a decrease in endogenous gene expression and/or polypeptide levels and/or polypeptide activity relative to control plants. The reduction or substantial elimination is in increasing order of preference at least 10%, 20%, 30%, 40% or 50%, 60%, 70%, 80%, 85%, 90%, or 95%, 96%, 97%, 98%, 99% or more reduced compared to that of control plants.
[0094] For the reduction or substantial elimination of expression an endogenous gene in a plant, a sufficient length of substantially contiguous nucleotides of a nucleic acid sequence is required. In order to perform gene silencing, this may be as little as 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10 or fewer nucleotides, alternatively this may be as much as the entire gene (including the 5' and/or 3' UTR, either in part or in whole). The stretch of substantially contiguous nucleotides may be derived from the nucleic acid encoding the protein of interest (target gene), or from any nucleic acid capable of encoding an orthologue, paralogue or homologue of the protein of interest. Preferably, the stretch of substantially contiguous nucleotides is capable of forming hydrogen bonds with the target gene (either sense or antisense strand), more preferably, the stretch of substantially contiguous nucleotides has, in increasing order of preference, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity to the target gene (either sense or antisense strand). A nucleic acid sequence encoding a (functional) polypeptide is not a requirement for the various methods discussed herein for the reduction or substantial elimination of expression of an endogenous gene.
[0095] This reduction or substantial elimination of expression may be achieved using routine tools and techniques. A preferred method for the reduction or substantial elimination of endogenous gene expression is by introducing and expressing in a plant a genetic construct into which the nucleic acid (in this case a stretch of substantially contiguous nucleotides derived from the gene of interest, or from any nucleic acid capable of encoding an orthologue, paralogue or homologue of any one of the protein of interest) is cloned as an inverted repeat (in part or completely), separated by a spacer (non-coding DNA).
[0096] In such a preferred method, expression of the endogenous gene is reduced or substantially eliminated through RNA-mediated silencing using an inverted repeat of a nucleic acid or a part thereof (in this case a stretch of substantially contiguous nucleotides derived from the gene of interest, or from any nucleic acid capable of encoding an orthologue, paralogue or homologue of the protein of interest), preferably capable of forming a hairpin structure. The inverted repeat is cloned in an expression vector comprising control sequences. A non-coding DNA nucleic acid sequence (a spacer, for example a matrix attachment region fragment (MAR), an intron, a polylinker, etc.) is located between the two inverted nucleic acids forming the inverted repeat. After transcription of the inverted repeat, a chimeric RNA with a self-complementary structure is formed (partial or complete). This double-stranded RNA structure is referred to as the hairpin RNA (hpRNA). The hpRNA is processed by the plant into siRNAs that are incorporated into an RNA-induced silencing complex (RISC). The RISC further cleaves the mRNA transcripts, thereby substantially reducing the number of mRNA transcripts to be translated into polypeptides. For further general details see for example, Grierson et al. (1998) WO 98/53083; Waterhouse et al. (1999) WO 99/53050).
[0097] Performance of the methods of the invention does not rely on introducing and expressing in a plant a genetic construct into which the nucleic acid is cloned as an inverted repeat, but any one or more of several well-known "gene silencing" methods may be used to achieve the same effects.
[0098] One such method for the reduction of endogenous gene expression is RNA-mediated silencing of gene expression (downregulation). Silencing in this case is triggered in a plant by a double stranded RNA sequence (dsRNA) that is substantially similar to the target endogenous gene. This dsRNA is further processed by the plant into about 20 to about 26 nucleotides called short interfering RNAs (siRNAs). The siRNAs are incorporated into an RNA-induced silencing complex (RISC) that cleaves the mRNA transcript of the endogenous target gene, thereby substantially reducing the number of mRNA transcripts to be translated into a polypeptide. Preferably, the double stranded RNA sequence corresponds to a target gene.
[0099] Another example of an RNA silencing method involves the introduction of nucleic acid sequences or parts thereof (in this case a stretch of substantially contiguous nucleotides derived from the gene of interest, or from any nucleic acid capable of encoding an orthologue, paralogue or homologue of the protein of interest) in a sense orientation into a plant. "Sense orientation" refers to a DNA sequence that is homologous to an mRNA transcript thereof. Introduced into a plant would therefore be at least one copy of the nucleic acid sequence. The additional nucleic acid sequence will reduce expression of the endogenous gene, giving rise to a phenomenon known as co-suppression. The reduction of gene expression will be more pronounced if several additional copies of a nucleic acid sequence are introduced into the plant, as there is a positive correlation between high transcript levels and the triggering of co-suppression.
[0100] Another example of an RNA silencing method involves the use of antisense nucleic acid sequences. An "antisense" nucleic acid sequence comprises a nucleotide sequence that is complementary to a "sense" nucleic acid sequence encoding a protein, i.e. complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA transcript sequence. The antisense nucleic acid sequence is preferably complementary to the endogenous gene to be silenced. The complementarity may be located in the "coding region" and/or in the "non-coding region" of a gene. The term "coding region" refers to a region of the nucleotide sequence comprising codons that are translated into amino acid residues. The term "non-coding region" refers to 5' and 3' sequences that flank the coding region that are transcribed but not translated into amino acids (also referred to as 5' and 3' untranslated regions).
[0101] Antisense nucleic acid sequences can be designed according to the rules of Watson and Crick base pairing. The antisense nucleic acid sequence may be complementary to the entire nucleic acid sequence (in this case a stretch of substantially contiguous nucleotides derived from the gene of interest, or from any nucleic acid capable of encoding an orthologue, paralogue or homologue of the protein of interest), but may also be an oligonucleotide that is antisense to only a part of the nucleic acid sequence (including the mRNA 5' and 3' UTR). For example, the antisense oligonucleotide sequence may be complementary to the region surrounding the translation start site of an mRNA transcript encoding a polypeptide. The length of a suitable antisense oligonucleotide sequence is known in the art and may start from about 50, 45, 40, 35, 30, 25, 20, 15 or 10 nucleotides in length or less. An antisense nucleic acid sequence according to the invention may be constructed using chemical synthesis and enzymatic ligation reactions using methods known in the art. For example, an antisense nucleic acid sequence (e.g., an antisense oligonucleotide sequence) may be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acid sequences, e.g., phosphorothioate derivatives and acridine substituted nucleotides may be used. Examples of modified nucleotides that may be used to generate the antisense nucleic acid sequences are well known in the art. Known nucleotide modifications include methylation, cyclization and `caps` and substitution of one or more of the naturally occurring nucleotides with an analogue such as inosine. Other modifications of nucleotides are well known in the art.
[0102] The antisense nucleic acid sequence can be produced biologically using an expression vector into which a nucleic acid sequence has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest). Preferably, production of antisense nucleic acid sequences in plants occurs by means of a stably integrated nucleic acid construct comprising a promoter, an operably linked antisense oligonucleotide, and a terminator.
[0103] The nucleic acid molecules used for silencing in the methods of the invention (whether introduced into a plant or generated in situ) hybridize with or bind to mRNA transcripts and/or genomic DNA encoding a polypeptide to thereby inhibit expression of the protein, e.g., by inhibiting transcription and/or translation. The hybridization can be by conventional nucleotide complementarity to form a stable duplex, or, for example, in the case of an antisense nucleic acid sequence which binds to DNA duplexes, through specific interactions in the major groove of the double helix. Antisense nucleic acid sequences may be introduced into a plant by transformation or direct injection at a specific tissue site. Alternatively, antisense nucleic acid sequences can be modified to target selected cells and then administered systemically. For example, for systemic administration, antisense nucleic acid sequences can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface, e.g., by linking the antisense nucleic acid sequence to peptides or antibodies which bind to cell surface receptors or antigens. The antisense nucleic acid sequences can also be delivered to cells using the vectors described herein.
[0104] According to a further aspect, the antisense nucleic acid sequence is an a-anomeric nucleic acid sequence. An a-anomeric nucleic acid sequence forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual b-units, the strands run parallel to each other (Gaultier et al. (1987) Nucl Ac Res 15: 6625-6641). The antisense nucleic acid sequence may also comprise a 2'-o-methylribonucleotide (Inoue et al. (1987) Nucl Ac Res 15, 6131-6148) or a chimeric RNA-DNA analogue (Inoue et al. (1987) FEBS Lett. 215, 327-330).
[0105] The reduction or substantial elimination of endogenous gene expression may also be performed using ribozymes. Ribozymes are catalytic RNA molecules with ribonuclease activity that are capable of cleaving a single-stranded nucleic acid sequence, such as an mRNA, to which they have a complementary region. Thus, ribozymes (e.g., hammerhead ribozymes (described in Haselhoff and Gerlach (1988) Nature 334, 585-591) can be used to catalytically cleave mRNA transcripts encoding a polypeptide, thereby substantially reducing the number of mRNA transcripts to be translated into a polypeptide. A ribozyme having specificity for a nucleic acid sequence can be designed (see for example: Cech et al. U.S. Pat. No. 4,987,071; and Cech et al. U.S. Pat. No. 5,116,742). Alternatively, mRNA transcripts corresponding to a nucleic acid sequence can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules (Bartel and Szostak (1993) Science 261, 1411-1418). The use of ribozymes for gene silencing in plants is known in the art (e.g., Atkins et al. (1994) WO 94/00012; Lenne et al. (1995) WO 95/03404; Lutziger et al. (2000) WO 00/00619; Prinsen et al. (1997) WO 97/13865 and Scott et al. (1997) WO 97/38116).
[0106] Gene silencing may also be achieved by insertion mutagenesis (for example, T-DNA insertion or transposon insertion) or by strategies as described by, among others, Angell and Baulcombe ((1999) Plant J 20(3): 357-62), (Amplicon VIGS WO 98/36083), or Baulcombe (WO 99/15682).
[0107] Gene silencing may also occur if there is a mutation on an endogenous gene and/or a mutation on an isolated gene/nucleic acid subsequently introduced into a plant. The reduction or substantial elimination may be caused by a non-functional polypeptide. For example, a polypeptide may bind to various interacting proteins; one or more mutation(s) and/or truncation(s) may therefore provide for a polypeptide that is still able to bind interacting proteins (such as receptor proteins) but that cannot exhibit its normal function (such as signalling ligand).
[0108] A further approach to gene silencing is by targeting nucleic acid sequences complementary to the regulatory region of the gene (e.g., the promoter and/or enhancers) to form triple helical structures that prevent transcription of the gene in target cells. See Helene, C., Anticancer Drug Res. 6, 569-84, 1991; Helene et al., Ann. N.Y. Acad. Sci. 660, 27-36 1992; and Maher, L. J. Bioassays 14, 807-15, 1992.
[0109] Other methods, such as the use of antibodies directed to an endogenous polypeptide for inhibiting its function in planta, or interference in the signalling pathway in which a polypeptide is involved, will be well known to the skilled man. In particular, it can be envisaged that manmade molecules may be useful for inhibiting the biological function of a target polypeptide, or for interfering with the signalling pathway in which the target polypeptide is involved.
[0110] Alternatively, a screening program may be set up to identify in a plant population natural variants of a gene, which variants encode polypeptides with reduced activity. Such natural variants may also be used for example, to perform homologous recombination.
[0111] Artificial and/or natural microRNAs (miRNAs) may be used to knock out gene expression and/or mRNA translation. Endogenous miRNAs are single stranded small RNAs of typically 19-24 nucleotides long. They function primarily to regulate gene expression and/or mRNA translation. Most plant microRNAs (miRNAs) have perfect or near-perfect complementarity with their target sequences. However, there are natural targets with up to five mismatches. They are processed from longer non-coding RNAs with characteristic fold-back structures by double-strand specific RNases of the Dicer family. Upon processing, they are incorporated in the RNA-induced silencing complex (RISC) by binding to its main component, an Argonaute protein. MiRNAs serve as the specificity components of RISC, since they base-pair to target nucleic acids, mostly mRNAs, in the cytoplasm. Subsequent regulatory events include target mRNA cleavage and destruction and/or translational inhibition. Effects of miRNA overexpression are thus often reflected in decreased mRNA levels of target genes.
[0112] Artificial microRNAs (amiRNAs), which are typically 21 nucleotides in length, can be genetically engineered specifically to negatively regulate gene expression of single or multiple genes of interest. Determinants of plant microRNA target selection are well known in the art. Empirical parameters for target recognition have been defined and can be used to aid in the design of specific amiRNAs, Schwab R, 2005. Convenient tools for design and generation of amiRNAs and their precursors are also available to the public, Schwab et al., 2006.
[0113] For optimal performance, the gene silencing techniques used for reducing expression in a plant of an endogenous gene requires the use of nucleic acid sequences from monocotyledonous plants for transformation of monocotyledonous plants, and from dicotyledonous plants for transformation of dicotyledonous plants. Preferably, a nucleic acid sequence from any given plant species is introduced into that same species. For example, a nucleic acid sequence from rice is transformed into a rice plant. However, it is not an absolute requirement that the nucleic acid sequence to be introduced originates from the same plant species as the plant in which it will be introduced. It is sufficient that there is substantial homology between the endogenous target gene and the nucleic acid to be introduced.
[0114] Described above are examples of various methods for the reduction or substantial elimination of expression in a plant of an endogenous gene. A person skilled in the art would readily be able to adapt the aforementioned methods for silencing so as to achieve reduction of expression of an endogenous gene in a whole plant or in parts thereof through the use of an appropriate promoter, for example.
Selectable Marker (Gene)/Reporter Gene
[0115] "Selectable marker", "selectable marker gene" or "reporter gene" includes any gene that confers a phenotype on a cell in which it is expressed to facilitate the identification and/or selection of cells that are transfected or transformed with a nucleic acid construct of the invention. These marker genes enable the identification of a successful transfer of the nucleic acid molecules via a series of different principles. Suitable markers may be selected from markers that confer antibiotic or herbicide resistance, that introduce a new metabolic trait or that allow visual selection. Examples of selectable marker genes include genes conferring resistance to antibiotics (such as nptII that phosphorylates neomycin and kanamycin, or hpt, phosphorylating hygromycin, or genes conferring resistance to, for example, bleomycin, streptomycin, tetracyclin, chloramphenicol, ampicillin, gentamycin, geneticin (G418), spectinomycin or blasticidin), to herbicides (for example bar which provides resistance to Basta®; aroA or gox providing resistance against glyphosate, or the genes conferring resistance to, for example, imidazolinone, phosphinothricin or sulfonylurea), or genes that provide a metabolic trait (such as manA that allows plants to use mannose as sole carbon source or xylose isomerase for the utilisation of xylose, or antinutritive markers such as the resistance to 2-deoxyglucose). Expression of visual marker genes results in the formation of colour (for example β-glucuronidase, GUS or β-galactosidase with its coloured substrates, for example X-Gal), luminescence (such as the luciferin/luceferase system) or fluorescence (Green Fluorescent Protein, GFP, and derivatives thereof). This list represents only a small number of possible markers. The skilled worker is familiar with such markers. Different markers are preferred, depending on the organism and the selection method.
[0116] It is known that upon stable or transient integration of nucleic acids into plant cells, only a minority of the cells takes up the foreign DNA and, if desired, integrates it into its genome, depending on the expression vector used and the transfection technique used. To identify and select these integrants, a gene coding for a selectable marker (such as the ones described above) is usually introduced into the host cells together with the gene of interest. These markers can for example be used in mutants in which these genes are not functional by, for example, deletion by conventional methods. Furthermore, nucleic acid molecules encoding a selectable marker can be introduced into a host cell on the same vector that comprises the sequence encoding the polypeptides of the invention or used in the methods of the invention, or else in a separate vector. Cells which have been stably transfected with the introduced nucleic acid can be identified for example by selection (for example, cells which have integrated the selectable marker survive whereas the other cells die).
[0117] Since the marker genes, particularly genes for resistance to antibiotics and herbicides, are no longer required or are undesired in the transgenic host cell once the nucleic acids have been introduced successfully, the process according to the invention for introducing the nucleic acids advantageously employs techniques which enable the removal or excision of these marker genes. One such a method is what is known as co-transformation. The co-transformation method employs two vectors simultaneously for the transformation, one vector bearing the nucleic acid according to the invention and a second bearing the marker gene(s). A large proportion of transformants receives or, in the case of plants, comprises (up to 40% or more of the transformants), both vectors. In case of transformation with Agrobacteria, the transformants usually receive only a part of the vector, i.e. the sequence flanked by the T-DNA, which usually represents the expression cassette. The marker genes can subsequently be removed from the transformed plant by performing crosses. In another method, marker genes integrated into a transposon are used for the transformation together with desired nucleic acid (known as the Ac/Ds technology). The transformants can be crossed with a transposase source or the transformants are transformed with a nucleic acid construct conferring expression of a transposase, transiently or stable. In some cases (approx. 10%), the transposon jumps out of the genome of the host cell once transformation has taken place successfully and is lost. In a further number of cases, the transposon jumps to a different location. In these cases the marker gene must be eliminated by performing crosses. In microbiology, techniques were developed which make possible, or facilitate, the detection of such events. A further advantageous method relies on what is known as recombination systems; whose advantage is that elimination by crossing can be dispensed with. The best-known system of this type is what is known as the Cre/lox system. Cre1 is a recombinase that removes the sequences located between the loxP sequences. If the marker gene is integrated between the loxP sequences, it is removed once transformation has taken place successfully, by expression of the recombinase. Further recombination systems are the HIN/HIX, FLP/FRT and REP/STB system (Tribble et al., J. Biol. Chem., 275, 2000: 22255-22267; Velmurugan et al., J. Cell Biol., 149, 2000: 553-566). A site-specific integration into the plant genome of the nucleic acid sequences according to the invention is possible. Naturally, these methods can also be applied to microorganisms such as yeast, fungi or bacteria.
Transgenic/Transgene/Recombinant
[0118] For the purposes of the invention, "transgenic", "transgene" or "recombinant" means with regard to, for example, a nucleic acid sequence, an expression cassette, gene construct or a vector comprising the nucleic acid sequence or an organism transformed with the nucleic acid sequences, expression cassettes or vectors according to the invention, all those constructions brought about by recombinant methods in which either
[0119] (a) the nucleic acid sequences encoding proteins useful in the methods of the invention, or
[0120] (b) genetic control sequence(s) which is operably linked with the nucleic acid sequence according to the invention, for example a promoter, or
[0121] (c) a) and b) are not located in their natural genetic environment or have been modified by recombinant methods, it being possible for the modification to take the form of, for example, a substitution, addition, deletion, inversion or insertion of one or more nucleotide residues. The natural genetic environment is understood as meaning the natural genomic or chromosomal locus in the original plant or the presence in a genomic library. In the case of a genomic library, the natural genetic environment of the nucleic acid sequence is preferably retained, at least in part. The environment flanks the nucleic acid sequence at least on one side and has a sequence length of at least 50 bp, preferably at least 500 bp, especially preferably at least 1000 bp, most preferably at least 5000 bp. A naturally occurring expression cassette--for example the naturally occurring combination of the natural promoter of the nucleic acid sequences with the corresponding nucleic acid sequence encoding a polypeptide useful in the methods of the present invention, as defined above--becomes a transgenic expression cassette when this expression cassette is modified by non-natural, synthetic ("artificial") methods such as, for example, mutagenic treatment. Suitable methods are described, for example, in U.S. Pat. No. 5,565,350 or WO 00/15815.
[0122] A transgenic plant for the purposes of the invention is thus understood as meaning, as above, that the nucleic acids used in the method of the invention are not at their natural locus in the genome of said plant, it being possible for the nucleic acids to be expressed homologously or heterologously. However, as mentioned, transgenic also means that, while the nucleic acids according to the invention or used in the inventive method are at their natural position in the genome of a plant, the sequence has been modified with regard to the natural sequence, and/or that the regulatory sequences of the natural sequences have been modified. Transgenic is preferably understood as meaning the expression of the nucleic acids according to the invention at an unnatural locus in the genome, i.e. homologous or, preferably, heterologous expression of the nucleic acids takes place. Preferred transgenic plants are mentioned herein.
Transformation
[0123] The term "introduction" or "transformation" as referred to herein encompasses the transfer of an exogenous polynucleotide into a host cell, irrespective of the method used for transfer. Plant tissue capable of subsequent clonal propagation, whether by organogenesis or embryogenesis, may be transformed with a genetic construct of the present invention and a whole plant regenerated there from. The particular tissue chosen will vary depending on the clonal propagation systems available for, and best suited to, the particular species being transformed. Exemplary tissue targets include leaf disks, pollen, embryos, cotyledons, hypocotyls, megagametophytes, callus tissue, existing meristematic tissue (e.g., apical meristem, axillary buds, and root meristems), and induced meristem tissue (e.g., cotyledon meristem and hypocotyl meristem). The polynucleotide may be transiently or stably introduced into a host cell and may be maintained non-integrated, for example, as a plasmid. Alternatively, it may be integrated into the host genome. The resulting transformed plant cell may then be used to regenerate a transformed plant in a manner known to persons skilled in the art.
[0124] The transfer of foreign genes into the genome of a plant is called transformation. Transformation of plant species is now a fairly routine technique. Advantageously, any of several transformation methods may be used to introduce the gene of interest into a suitable ancestor cell. The methods described for the transformation and regeneration of plants from plant tissues or plant cells may be utilized for transient or for stable transformation. Transformation methods include the use of liposomes, electroporation, chemicals that increase free DNA uptake, injection of the DNA directly into the plant, particle gun bombardment, transformation using viruses or pollen and microprojection. Methods may be selected from the calcium/polyethylene glycol method for protoplasts (Krens, F. A. et al., (1982) Nature 296, 72-74; Negrutiu I et al. (1987) Plant Mol Biol 8: 363-373); electroporation of protoplasts (Shillito R. D. et al. (1985) Bio/Technol 3, 1099-1102); microinjection into plant material (Crossway A et al., (1986) Mol. Gen. Genet. 202: 179-185); DNA or RNA-coated particle bombardment (Klein T M et al., (1987) Nature 327: 70) infection with (non-integrative) viruses and the like. Transgenic plants, including transgenic crop plants, are preferably produced via Agrobacterium-mediated transformation. An advantageous transformation method is the transformation in planta. To this end, it is possible, for example, to allow the agrobacteria to act on plant seeds or to inoculate the plant meristem with agrobacteria. It has proved particularly expedient in accordance with the invention to allow a suspension of transformed agrobacteria to act on the intact plant or at least on the flower primordia. The plant is subsequently grown on until the seeds of the treated plant are obtained (Clough and Bent, Plant J. (1998) 16, 735-743). Methods for Agrobacterium-mediated transformation of rice include well known methods for rice transformation, such as those described in any of the following: European patent application EP 1198985 A1, Aldemita and Hodges (Planta 199: 612-617, 1996); Chan et al. (Plant Mol Biol 22 (3): 491-506, 1993), Hiei et al. (Plant J 6 (2): 271-282, 1994), which disclosures are incorporated by reference herein as if fully set forth. In the case of corn transformation, the preferred method is as described in either Ishida et al. (Nat. Biotechnol 14(6): 745-50, 1996) or Frame et al. (Plant Physiol 129(1): 13-22, 2002), which disclosures are incorporated by reference herein as if fully set forth. Said methods are further described by way of example in B. Jenes et al., Techniques for Gene Transfer, in: Transgenic Plants, Vol. 1, Engineering and Utilization, eds. S. D. Kung and R. Wu, Academic Press (1993) 128-143 and in Potrykus Annu. Rev. Plant Physiol. Plant Molec. Biol. 42 (1991) 205-225). The nucleic acids or the construct to be expressed is preferably cloned into a vector, which is suitable for transforming Agrobacterium tumefaciens, for example pBin19 (Bevan et al., Nucl. Acids Res. 12 (1984) 8711). Agrobacteria transformed by such a vector can then be used in known manner for the transformation of plants, such as plants used as a model, like Arabidopsis (Arabidopsis thaliana is within the scope of the present invention not considered as a crop plant), or crop plants such as, by way of example, tobacco plants, for example by immersing bruised leaves or chopped leaves in an agrobacterial solution and then culturing them in suitable media. The transformation of plants by means of Agrobacterium tumefaciens is described, for example, by Hofgen and Willmitzer in Nucl. Acid Res. (1988) 16, 9877 or is known inter alia from F. F. White, Vectors for Gene Transfer in Higher Plants; in Transgenic Plants, Vol. 1, Engineering and Utilization, eds. S. D. Kung and R. Wu, Academic Press, 1993, pp. 15-38.
[0125] In addition to the transformation of somatic cells, which then have to be regenerated into intact plants, it is also possible to transform the cells of plant meristems and in particular those cells which develop into gametes. In this case, the transformed gametes follow the natural plant development, giving rise to transgenic plants. Thus, for example, seeds of Arabidopsis are treated with agrobacteria and seeds are obtained from the developing plants of which a certain proportion is transformed and thus transgenic [Feldman, K A and Marks M D (1987). Mol Gen Genet. 208:274-289; Feldmann K (1992). In: C Koncz, N-H Chua and J Shell, eds, Methods in Arabidopsis Research. Word Scientific, Singapore, pp. 274-289]. Alternative methods are based on the repeated removal of the inflorescences and incubation of the excision site in the center of the rosette with transformed agrobacteria, whereby transformed seeds can likewise be obtained at a later point in time (Chang (1994). Plant J. 5: 551-558; Katavic (1994). Mol Gen Genet, 245: 363-370). However, an especially effective method is the vacuum infiltration method with its modifications such as the "floral dip" method. In the case of vacuum infiltration of Arabidopsis, intact plants under reduced pressure are treated with an agrobacterial suspension [Bechthold, N (1993). C R Acad Sci Paris Life Sci, 316: 1194-1199], while in the case of the "floral dip" method the developing floral tissue is incubated briefly with a surfactant-treated agrobacterial suspension [Clough, S J and Bent, A F (1998). The Plant J. 16, 735-743]. A certain proportion of transgenic seeds are harvested in both cases, and these seeds can be distinguished from non-transgenic seeds by growing under the above-described selective conditions. In addition the stable transformation of plastids is of advantages because plastids are inherited maternally is most crops reducing or eliminating the risk of transgene flow through pollen. The transformation of the chloroplast genome is generally achieved by a process which has been schematically displayed in Klaus et al., 2004 [Nature Biotechnology 22 (2), 225-229]. Briefly the sequences to be transformed are cloned together with a selectable marker gene between flanking sequences homologous to the chloroplast genome. These homologous flanking sequences direct site specific integration into the plastome. Plastidal transformation has been described for many different plant species and an overview is given in Bock (2001) Transgenic plastids in basic research and plant biotechnology. J Mol. Biol. 2001 Sep. 21; 312 (3):425-38 or Maliga, P (2003) Progress towards commercialization of plastid transformation technology. Trends Biotechnol. 21, 20-28. Further biotechnological progress has recently been reported in form of marker free plastid transformants, which can be produced by a transient co-integrated maker gene (Klaus et al., 2004, Nature Biotechnology 22(2), 225-229).
T-DNA Activation Taming
[0126] T-DNA activation tagging (Hayashi et al. Science (1992) 1350-1353), involves insertion of T-DNA, usually containing a promoter (may also be a translation enhancer or an intron), in the genomic region of the gene of interest or 10 kb up- or downstream of the coding region of a gene in a configuration such that the promoter directs expression of the targeted gene.
[0127] Typically, regulation of expression of the targeted gene by its natural promoter is disrupted and the gene falls under the control of the newly introduced promoter. The promoter is typically embedded in a T-DNA. This T-DNA is randomly inserted into the plant genome, for example, through Agrobacterium infection and leads to modified expression of genes near the inserted T-DNA. The resulting transgenic plants show dominant phenotypes due to modified expression of genes close to the introduced promoter.
TILLING
[0128] TILLING (Targeted Induced Local Lesions In Genomes) is a mutagenesis technology useful to generate and/or identify nucleic acids encoding proteins with modified expression and/or activity. TILLING also allows selection of plants carrying such mutant variants. These mutant variants may exhibit modified expression, either in strength or in location or in timing (if the mutations affect the promoter for example). These mutant variants may exhibit higher activity than that exhibited by the gene in its natural form. TILLING combines high-density mutagenesis with high-throughput screening methods. The steps typically followed in TILLING are: (a) EMS mutagenesis (Redei G P and Koncz C (1992) In Methods in Arabidopsis Research, Koncz C, Chua N H, Schell J, eds. Singapore, World Scientific Publishing Co, pp. 16-82; Feldmann et al., (1994) In Meyerowitz E M, Somerville C R, eds, Arabidopsis. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., pp 137-172; Lightner J and Caspar T (1998) In J Martinez-Zapater, J Salinas, eds, Methods on Molecular Biology, Vol. 82. Humana Press, Totowa, N.J., pp 91-104); (b) DNA preparation and pooling of individuals; (c) PCR amplification of a region of interest; (d) denaturation and annealing to allow formation of heteroduplexes; (e) DHPLC, where the presence of a heteroduplex in a pool is detected as an extra peak in the chromatogram; (f) identification of the mutant individual; and (g) sequencing of the mutant PCR product. Methods for TILLING are well known in the art (McCallum et al., (2000) Nat Biotechnol 18: 455-457; reviewed by Stemple (2004) Nat Rev Genet. 5(2): 145-50).
Homologous Recombination
[0129] Homologous recombination allows introduction in a genome of a selected nucleic acid at a defined selected position. Homologous recombination is a standard technology used routinely in biological sciences for lower organisms such as yeast or the moss Physcomitrella. Methods for performing homologous recombination in plants have been described not only for model plants (Offring a et al. (1990) EMBO J. 9(10): 3077-84) but also for crop plants, for example rice (Terada et al. (2002) Nat Biotech 20(10): 1030-4; lida and Terada (2004) Curr Opin Biotech 15(2): 132-8).
Yield
[0130] The term "yield" in general means a measurable produce of economic value, typically related to a specified crop, to an area, and to a period of time. Individual plant parts directly contribute to yield based on their number, size and/or weight, or the actual yield is the yield per acre for a crop and year, which is determined by dividing total production (includes both harvested and appraised production) by planted acres.
Early Vigour
[0131] Early vigour (active healthy well-balanced growth especially during early stages of plant growth) may result from increased plant fitness due to, for example, the plants being better adapted to their environment (i.e. optimizing the use of energy resources and partitioning between shoot and root). Plants having early vigour also show increased seedling survival and a better establishment of the crop, which often results in highly uniform fields (with the crop growing in uniform manner, i.e. with the majority of plants reaching the various stages of development at substantially the same time), and often better and higher yield. Therefore, early vigour may be determined by measuring various factors, such as thousand kernel weight, percentage germination, percentage emergence, seedling growth, seedling height, root length, root and shoot biomass and many more.
Increase/Improve/Enhance
[0132] The terms "increase", "improve" or "enhance" are interchangeable and shall mean in the sense of the application at least a 5%, 6%, 7%, 8%, 9% or 10%, preferably at least 15% or 20%, more preferably 25%, 30%, 35% or 40% more yield and/or growth in comparison to control plants as defined herein.
Seed Yield
[0133] Increased seed yield may manifest itself as one or more of the following: a) an increase in seed biomass (total seed weight) which may be on an individual seed basis and/or per plant and/or per hectare or acre; b) increased number of flowers per plant; c) increased number of (filled) seeds; d) increased seed filling rate (which is expressed as the ratio between the number of filled seeds divided by the total number of seeds); e) increased harvest index, which is expressed as a ratio of the yield of harvestable parts, such as seeds, divided by the total biomass; and f) increased thousand kernel weight (TKW), which is extrapolated from the number of filled seeds counted and their total weight. An increased TKW may result from an increased seed size and/or seed weight, and may also result from an increase in embryo and/or endosperm size.
[0134] An increase in seed yield may also be manifested as an increase in seed size and/or seed volume. Furthermore, an increase in seed yield may also manifest itself as an increase in seed area and/or seed length and/or seed width and/or seed perimeter. Increased yield may also result in modified architecture, or may occur because of modified architecture.
Plant
[0135] The term "plant" as used herein encompasses whole plants, ancestors and progeny of the plants and plant parts, including seeds, shoots, stems, leaves, roots (including tubers), flowers, and tissues and organs, wherein each of the aforementioned comprise the gene/nucleic acid of interest. The term "plant" also encompasses plant cells, suspension cultures, callus tissue, embryos, meristematic regions, gametophytes, sporophytes, pollen and microspores, again wherein each of the aforementioned comprises the gene/nucleic acid of interest.
[0136] Plants that are particularly useful in the methods of the invention include all plants which belong to the superfamily Viridiplantae, in particular monocotyledonous and dicotyledonous plants including fodder or forage legumes, ornamental plants, food crops, trees or shrubs selected from the list comprising Acer spp., Actinidia spp., Abelmoschus spp., Agave sisalana, Agropyron spp., Agrostis stolonifera, Allium spp., Amaranthus spp., Ammophila arenaria, Ananas comosus, Annona spp., Apium graveolens, Arachis spp, Artocarpus spp., Asparagus officinalis, Avena spp. (e.g. Avena sativa, Avena fatua, Avena byzantina, Avena fatua var. sativa, Avena hybrida), Averrhoa carambola, Bambusa sp., Benincasa hispida, Bertholletia excelsea, Beta vulgaris, Brassica spp. (e.g. Brassica napus, Brassica rapa ssp. [canola, oilseed rape, turnip rape]), Cadaba farinosa, Camellia sinensis, Canna indica, Cannabis sativa, Capsicum spp., Carex elata, Carica papaya, Carissa macrocarpa, Carya spp., Carthamus tinctorius, Castanea spp., Ceiba pentandra, Cichorium endivia, Cinnamomum spp., Citrullus lanatus, Citrus spp., Cocos spp., Coffea spp., Colocasia esculenta, Cola spp., Corchorus sp., Coriandrum sativum, Corylus spp., Crataegus spp., Crocus sativus, Cucurbita spp., Cucumis spp., Cynara spp., Daucus carota, Desmodium spp., Dimocarpus longan, Dioscorea spp., Diospyros spp., Echinochloa spp., Elaeis (e.g. Elaeis guineensis, Elaeis oleifera), Eleusine coracana, Erianthus sp., Eriobotrya japonica, Eucalyptus sp., Eugenia uniflora, Fagopyrum spp., Fagus spp., Festuca arundinacea, Ficus carica, Fortunella spp., Fragaria spp., Ginkgo biloba, Glycine spp. (e.g. Glycine max, Soja hispida or Soja max), Gossypium hirsutum, Helianthus spp. (e.g. Helianthus annuus), Hemerocallis fulva, Hibiscus spp., Hordeum spp. (e.g. Hordeum vulgare), Ipomoea batatas, Juglans spp., Lactuca sativa, Lathyrus spp., Lens culinaris, Linum usitatissimum, Litchi chinensis, Lotus spp., Luffa acutangula, Lupinus spp., Luzula sylvatica, Lycopersicon spp. (e.g. Lycopersicon esculentum, Lycopersicon lycopersicum, Lycopersicon pyriforme), Macrotyloma spp., Malus spp., Malpighia emarginata, Mammea americana, Mangifera indica, Manihot spp., Manilkara zapota, Medicago sativa, Melilotus spp., Mentha spp., Miscanthus sinensis, Momordica spp., Morus nigra, Musa spp., Nicotiana spp., Olea spp., Opuntia spp., Ornithopus spp., Oryza spp. (e.g. Oryza sativa, Oryza latifolia), Panicum miliaceum, Panicum virgatum, Passiflora edulis, Pastinaca sativa, Pennisetum sp., Persea spp., Petroselinum crispum, Phalaris arundinacea, Phaseolus spp., Phleum pratense, Phoenix spp., Phragmites australis, Physalis spp., Pinus spp., Pistacia vera, Pisum spp., Poa spp., Populus spp., Prosopis spp., Prunus spp., Psidium spp., Punica granatum, Pyrus communis, Quercus spp., Raphanus sativus, Rheum rhabarbarum, Ribes spp., Ricinus communis, Rubus spp., Saccharum spp., Salix sp., Sambucus spp., Secale cereale, Sesamum spp., Sinapis sp., Solanum spp. (e.g. Solanum tuberosum, Solanum integrifolium or Solanum lycopersicum), Sorghum bicolor, Spinacia spp., Syzygium spp., Tagetes spp., Tamarindus indica, Theobroma cacao, Trifolium spp., Triticosecale rimpaui, Triticum spp. (e.g. Triticum aestivum, Triticum durum, Triticum turgidum, Triticum hybernum, Triticum macha, Triticum sativum or Triticum vulgare), Tropaeolum minus, Tropaeolum majus, Vaccinium spp., Vicia spp., Vigna spp., Viola odorata, Vitis spp., Zea mays, Zizania palustris, Ziziphus spp., amongst others.
DETAILED DESCRIPTION OF THE INVENTION
Detailed Description for HAL3
[0137] According to a first embodiment of the present invention, there is provided a method for increasing plant yield relative to control plants, comprising preferentially increasing expression of a nucleic acid encoding a HAL3 polypeptide in expanding tissues of a plant.
[0138] Any reference hereinafter to a "protein useful in the methods of the invention" is taken to mean a HAL3 polypeptide as defined herein. Any reference hereinafter to a "nucleic acid useful in the methods of the invention" is taken to mean a nucleic acid capable of encoding such an HAL3 polypeptide. The nucleic acid to be introduced into a plant (and therefore useful in performing the methods of the invention) is any nucleic acid encoding the type of protein which will now be described, hereafter also named "HAL3 nucleic acid" or "HAL3 gene".
[0139] A "reference", "reference plant", "control", "control plant", "wild type" or "wild type plant" is in particular a cell, a tissue, an organ, a plant, or a part thereof, which was not produced according to the method of the invention. Accordingly, the terms "wild type", "control" or "reference" are exchangeable and can be a cell or a part of the plant such as an organelle or tissue, or a plant, which was not modified or treated according to the herein described method according to the invention. Accordingly, the cell or a part of the plant such as an organelle or a plant used as wild type, control or reference corresponds to the cell, plant or part thereof as much as possible and is in any other property but in the result of the process of the invention as identical to the subject matter of the invention as possible. Thus, the wild type, control or reference is treated identically or as identical as possible, saying that only conditions or properties might be different which do not influence the quality of the tested property. That means in other words that the wild type denotes (1) a plant, which carries the unaltered or not modulated form of a gene or allele or (2) the starting material/plant from which the plants produced by the process or method of the invention are derived.
[0140] Preferably, any comparison between the wild type plants and the plants produced by the method of the invention is carried out under analogous conditions. The term "analogous conditions" means that all conditions such as, for example, culture or growing conditions, assay conditions (such as buffer composition, temperature, substrates, pathogen strain, concentrations and the like) are kept identical between the experiments to be compared.
[0141] The "reference", "control", or "wild type" is preferably a subject, e.g. an organelle, a cell, a tissue, a plant, which was not modulated, modified or treated according to the herein described process of the invention and is in any other property as similar to the subject matter of the invention as possible. The reference, control or wild type is in its genome, transcriptome, proteome or metabolome as similar as possible to the subject of the present invention. Preferably, the term "reference-" "control-" or "wild type-"-organelle, -cell, -tissue or plant, relates to an organelle, cell, tissue or plant, which is nearly genetically identical to the organelle, cell, tissue or plant, of the present invention or a part thereof preferably 95%, more preferred are 98%, even more preferred are 99,00%, in particular 99,10%, 99,30%, 99,50%, 99,70%, 99,90%, 99,99%, 99, 999% or more. Most preferable the "reference", "control", or "wild type" is preferably a subject, e.g. an organelle, a cell, a tissue, a plant, which is genetically identical to the plant, cell organelle used according to the method of the invention except that nucleic acid molecules or the gene product encoded by them are changed, modulated or modified according to the inventive method.
[0142] In case, a control, reference or wild type differing from the subject of the present invention only by not being subject of the method of the invention can not be provided, a control, reference or wild type can be a plant in which the cause for the modulation of the activity conferring the increase of the metabolites as described under examples.
[0143] The increase referred to the activity of the polypeptide amounts in a cell, a tissue, a organelle, an organ or an organism or a part thereof preferably to at least 5%, preferably to at least 10% or at to least 15%, especially preferably to at least 20%, 25%, 30% or more, very especially preferably are to at least 40%, 50% or 60%, most preferably are to at least 70% or more in comparison to the control, reference or wild type.
[0144] The term "increased yield" is taken to mean an increase in biomass (weight) of one or more parts of a plant, which may include aboveground (harvestable) parts and/or (harvestable) parts below ground. Preferably, the increase in yield is at least 10% over the yield of corresponding wild type plants.
[0145] In particular, such harvestable parts are seeds, and performance of the methods of the invention results in plants having increased seed yield relative to the seed yield of control plants.
[0146] The term "expression" or "gene expression" means the appearance of a phenotypic trait as a consequence of the transcription of a specific gene or specific genes. The term "expression" or "gene expression" in particular means the transcription of a gene or genes into structural RNA (rRNA, tRNA) or mRNA with subsequent translation of the latter into a protein. The process includes transcription of DNA, processing of the resulting mRNA product and its translation into an active protein.
[0147] Performance of the methods of the invention gives plants having enhanced yield-related traits. In particular performance of the methods of the invention gives plants having increased yield, especially increased seed yield relative to control plants. The terms "yield" and "seed yield" are described in more detail in the "definitions" section herein.
[0148] Reference herein to enhanced yield-related traits is taken to mean an increase in biomass (weight) of one or more parts of a plant, which may include aboveground (harvestable) parts and/or (harvestable) parts below ground. In particular, such harvestable parts are seeds, and performance of the methods of the invention results in plants having increased seed yield relative to the seed yield of suitable control plants.
[0149] Taking corn as an example, a yield increase may be manifested as one or more of the following: increase in the number of plants per hectare or acre, an increase in the number of ears per plant, an increase in the number of rows, number of kernels per row, kernel weight, thousand kernel weight, ear length/diameter, increase in the seed filling rate (which is the number of filled seeds divided by the total number of seeds and multiplied by 100), among others. Taking rice as an example, a yield increase may manifest itself as an increase in one or more of the following: number of plants per hectare or acre, number of panicles per plant, number of spikelets per panicle, number of flowers (florets) per panicle (which is expressed as a ratio of the number of filled seeds over the number of primary panicles), increase in the seed filling rate (which is the number of filled seeds divided by the total number of seeds and multiplied by 100), increase in thousand kernel weight, among others.
[0150] According to a preferred feature of the present invention, performance of the methods of the invention gives plants having increased seed yield relative to control plants. Therefore according to the present invention, there is provided a method for increasing seed yield in plants relative to the seed yield of control plants, the method comprising preferentially increasing expression of a nucleic acid encoding a HAL3 polypeptide in shoot tissues, preferably in expanding tissues of a plant shoot.
[0151] Since the transgenic plants according to the present invention have increased yield, it is likely that these plants exhibit an increased growth rate (during at least part of their life cycle), relative to the growth rate of control plants at a corresponding stage in their life cycle.
[0152] The increased growth rate may be specific to one or more parts of a plant (including seeds), or may be throughout substantially the whole plant. Plants having an increased growth rate may have a shorter life cycle. The life cycle of a plant may be taken to mean the time needed to grow from a dry mature seed up to the stage where the plant has produced dry mature seeds, similar to the starting material. This life cycle may be influenced by factors such as early vigour, growth rate, greenness index, flowering time and speed of seed maturation. The increase in growth rate may take place at one or more stages in the life cycle of a plant or during substantially the whole plant life cycle. Increased growth rate during the early stages in the life cycle of a plant may reflect enhanced vigour. The increase in growth rate may alter the harvest cycle of a plant allowing plants to be sown later and/or harvested sooner than would otherwise be possible (a similar effect may be obtained with earlier flowering time). If the growth rate is sufficiently increased, it may allow for the further sowing of seeds of the same plant species (for example sowing and harvesting of rice plants followed by sowing and harvesting of further rice plants all within one conventional growing period). Similarly, if the growth rate is sufficiently increased, it may allow for the further sowing of seeds of different plants species (for example the sowing and harvesting of corn plants followed by, for example, the sowing and optional harvesting of soybean, potato or any other suitable plant). Harvesting additional times from the same rootstock in the case of some crop plants may also be possible. Altering the harvest cycle of a plant may lead to an increase in annual biomass production per acre (due to an increase in the number of times (say in a year) that any particular plant may be grown and harvested). An increase in growth rate may also allow for the cultivation of transgenic plants in a wider geographical area than their wild-type counterparts, since the territorial limitations for growing a crop are often determined by adverse environmental conditions either at the time of planting (early season) or at the time of harvesting (late season). Such adverse conditions may be avoided if the harvest cycle is shortened. The growth rate may be determined by deriving various parameters from growth curves, such parameters may be: T-Mid (the time taken for plants to reach 50% of their maximal size) and T-90 (time taken for plants to reach 90% of their maximal size), amongst others.
[0153] According to a preferred feature of the present invention, performance of the methods of the invention gives plants having an increased growth rate relative to control plants. Therefore, according to the present invention, there is provided a method for increasing the growth rate of plants, which method comprises modulating expression, preferably increasing expression, in a plant of a nucleic acid encoding a HAL3 polypeptide as defined herein.
[0154] An increase in yield and/or growth rate occurs whether the plant is under non-stress conditions or whether the plant is exposed to various stresses compared to control plants. Plants typically respond to exposure to stress by growing more slowly. In conditions of severe stress, the plant may even stop growing altogether. Mild stress on the other hand is defined herein as being any stress to which a plant is exposed which does not result in the plant ceasing to grow altogether without the capacity to resume growth. Mild stress in the sense of the invention leads to a reduction in the growth of the stressed plants of less than 40%, 35% or 30%, preferably less than 25%, 20% or 15%, more preferably less than 14%, 13%, 12%, 11% or 10% or less in comparison to the control plant under non-stress conditions. Due to advances in agricultural practices (irrigation, fertilization, pesticide treatments) severe stresses are not often encountered in cultivated crop plants. As a consequence, the compromised growth induced by mild stress is often an undesirable feature for agriculture. Mild stresses are the everyday biotic and/or abiotic (environmental) stresses to which a plant is exposed. Abiotic stresses may be due to drought or excess water, anaerobic stress, salt stress, chemical toxicity, oxidative stress and hot, cold or freezing temperatures. The abiotic stress may be an osmotic stress caused by a water stress (particularly due to drought), salt stress, oxidative stress or an ionic stress. Biotic stresses are typically those stresses caused by pathogens, such as bacteria, viruses, fungi and insects.
[0155] In particular, the methods of the present invention may be performed under non-stress conditions or under conditions of mild drought to give plants having increased yield relative to control plants. As reported in Wang et al. (Planta (2003) 218: 1-14), abiotic stress leads to a series of morphological, physiological, biochemical and molecular changes that adversely affect plant growth and productivity. Drought, salinity, extreme temperatures and oxidative stress are known to be interconnected and may induce growth and cellular damage through similar mechanisms. Rabbani et al. (Plant Physiol (2003) 133: 1755-1767) describes a particularly high degree of "cross talk" between drought stress and high-salinity stress. For example, drought and/or salinisation are manifested primarily as osmotic stress, resulting in the disruption of homeostasis and ion distribution in the cell. Oxidative stress, which frequently accompanies high or low temperature, salinity or drought stress, may cause denaturing of functional and structural proteins. As a consequence, these diverse environmental stresses often activate similar cell signaling pathways and cellular responses, such as the production of stress proteins, up-regulation of anti-oxidants, accumulation of compatible solutes and growth arrest. The term "non-stress" conditions as used herein are those environmental conditions that allow optimal growth of plants. Persons skilled in the art are aware of normal soil conditions and climatic conditions for a given location.
[0156] Performance of the methods of the invention gives plants grown under non-stress conditions or under mild drought conditions increased yield relative to suitable control plants grown under comparable conditions. Therefore, according to the present invention, there is provided a method for increasing yield in plants grown under non-stress conditions or under mild drought conditions, which method comprises increasing expression in a plant of a nucleic acid encoding a HAL3 polypeptide.
[0157] Performance of the methods of the invention gives plants grown under conditions of nutrient deficiency, particularly under conditions of nitrogen deficiency, increased yield relative to control plants grown under comparable conditions. Therefore, according to the present invention, there is provided a method for increasing yield in plants grown under conditions of nutrient deficiency, which method comprises increasing expression in a plant of a nucleic acid encoding a HAL3 polypeptide. Nutrient deficiency may result from a lack or excess of nutrients such as nitrogen, phosphates and other phosphorous-containing compounds, potassium, calcium, cadmium, magnesium, manganese, iron and boron, amongst others.
[0158] According to a second preferred feature of the invention, performance of the methods of the invention gives plants having increased plant vigour relative to control plants, particularly during the early stages of plant development (typically three, four weeks post germination in the case of rice and maize, but this will vary from species to species) leading to early vigour. Therefore, according to the present invention, there is provided a method for increasing the plant early vigour, which method comprises modulating, preferably increasing expression in a plant of a nucleic acid encoding a HAL3 polypeptide. Preferably the increase in seedling vigour is achieved by expressing the nucleic acid encoding the HAL3 polypeptide under the control of a shoot specific promoter. There is also provided a method for producing plants having early vigour relative to control plants, which method comprises modulating, preferably increasing, expression in a plant of a nucleic acid encoding a HAL3 polypeptide.
[0159] Early vigour may also result from increased plant fitness due to, for example, the plants being better adapted to their environment (i.e. optimizing the use of energy resources and partitioning between shoot and root). Plants having early vigour also show increased seedling survival and a better establishment of the crop, which often results in highly uniform fields (with the crop growing in uniform manner, i.e. with the majority of plants reaching the various stages of development at substantially the same time), and often better and higher yield. Therefore, early vigour may be determined by measuring various factors, such as thousand kernel weight, percentage germination, percentage emergence, seedling growth, seedling height, root length, root and shoot biomass and many more.
[0160] The methods of the invention are advantageously applicable to any plant.
[0161] Plants that are particularly useful in the methods of the invention include all plants which belong to the superfamily Viridiplantae, in particular monocotyledonous and dicotyledonous plants including fodder or forage legumes, ornamental plants, food crops, trees or shrubs. According to a preferred embodiment of the present invention, the plant is a crop plant. Examples of crop plants include soybean, sunflower, canola, alfalfa, rapeseed, cotton, tomato, potato and tobacco. Further preferably, the plant is a monocotyledonous plant. Examples of monocotyledonous plants include sugarcane. More preferably the plant is a cereal. Examples of cereals include rice, maize, wheat, barley, millet, rye, triticale, sorghum and oats.
[0162] Other advantageous plants are selected from the group consisting of Asteraceae such as the genera Helianthus, Tagetes e.g. the species Helianthus annus [sunflower], Tagetes lucida, Tagetes erecta or Tagetes tenuifolia [Marigold], Brassicaceae such as the genera Brassica, Arabadopsis e.g. the species Brassica napus, Brassica rapa ssp. [canola, oilseed rape, turnip rape] or Arabidopsis thaliana. Fabaceae such as the genera Glycine e.g. the species Glycine max, Soja hispida or Soja max [soybean]. Linaceae such as the genera Linum e.g. the species Linum usitatissimum, [flax, linseed]; Poaceae such as the genera Hordeum, Secale, Avena, Sorghum, Oryza, Zea, Triticum e.g. the species Hordeum vulgare [barley]; Secale cereale [rye], Avena sativa, Avena fatua, Avena byzantina, Avena fatua var. sativa, Avena hybrida [oat], Sorghum bicolor [Sorghum, millet], Oryza sativa, Oryza latifolia [rice], Zea mays [corn, maize] Triticum aestivum, Triticum durum, Triticum turgidum, Triticum hybernum, Triticum macha, Triticum sativum or Triticum vulgare [wheat, bread wheat, common wheat]; Solanaceae such as the genera Solanum, Lycopersicon e.g. the species Solanum tuberosum [potato], Lycopersicon esculentum, Lycopersicon lycopersicum, Lycopersicon pyriforme, Solanum integrifolium or Solanum lycopersicum [tomato].
[0163] The term "HAL3 polypeptide" as defined herein refers to a flavoprotein belonging to the superfamily HFCD (Homo-oligomeric Flavin containing Cys Decarboxylases; Kupke J. Biol. Chem. 276, 27597-27604, 2001). These proteins share a flavin-binding motif, conserved active-site residues and are trimeric or dodecameric enzymes. HAL3 proteins preferably comprise from N-terminus to C-terminus (i) a substrate binding helix, (ii) an insertion His motif, (iii) a PXMNXXMW motif and (iv) a substrate recognition clamp (FIG. 1). These four domains are predicted to be involved in substrate binding, the insertion His motif comprises a conserved His residue that is involved in the active site whereas the sequence in the substrate recognition clamp may be somewhat variable in sequence and length (Blaesse et al., EMBO J. 19, 6299-6310, 2000). The structural features (i) to (iv) are also described in Kupke et al. (2001), which disclosure is incorporated herein by reference. Typically, HAL3 polypeptides are capable of binding of FMN cofactors.
[0164] Typically the substrate binding helix is comprised in sequence motif 1 with the following consensus sequence (SEQ ID NO: 6):
TABLE-US-00004 (K/R)PR(V/I) (I/L)LAA(S/T)GSVA(A/S) (I/M/V)KF (G/E/A/S) (N/S/I)L(C/V/A) (H/R/G) (C/S/I) (F/L) (T/S/C) (E/D/Q)WA(E/D)V(R/K)AV(V/A/S).
[0165] The insertion His motif, the PXMNXXMW motif and the substrate recognition clamp are part of sequence motif 2 with the following consensus sequence (SEQ ID NO: 7):
TABLE-US-00005 VLHIELR(R/K/Q)WAD(V/I/A) (L/M) (V/I)IAPLSANTL(G/A) KIAGG(L/M)CDNLLTC(I/V)(I/V)RAWD(Y/F) (T/S/N/D/K)KP (L/F/M/I)F(V/A)APAMNT(L/F)MW(N/S/T)NPFT(E/S/Q/A) (R/K)H(L/F/I) (X1) (X2) (L/I/M) (D/N/S) (E/L/Q) (L/M)G(I/V/L) (T/S/A/I)L(I/V)PP(I/V/T) (K/T/S)K (R/T/K)LACGD(Y/H)G(N/T)GAM(A/S)E
wherein X1 may be any amino acid, preferably X1 is one of L, V, E, H, D, M, I or Q and wherein X2 may be any amino acid, preferably X2 is one of s, L, T, A, or V.
[0166] These motifs form part of a larger conserved region in the protein, as an example, the conserved region of Arabidopsis HAL3a is given as SEQ ID NO: 8. HAL3 polypeptides useful in the methods of the present invention have in increasing order of preference at least 65%, 70%, 75%, 80%, 85%, 90% or 95% sequence identity to the conserved region represented by SEQ ID NO: 8.
[0167] The conserved region in HAL3 proteins, as exemplified in SEQ ID NO: 8 and comprising the above described features (i) to (iv), encompasses a Flavoprotein domain which may be identified using specialised databases e.g. SMART (Schultz et al. (1998) Proc. Natl. Acad. Sci. USA 95, 5857-5864; Letunic et al. (2002) Nucleic Acids Res 30, 242-244), InterPro (Mulder et al., (2003) Nucl. Acids. Res. 31, 315-318), Prosite (Bucher and Bairoch (1994), A generalized profile syntax for biomolecular sequences motifs and its function in automatic sequence interpretation. (In) ISMB-94; Proceedings 2nd International Conference on Intelligent Systems for Molecular Biology. Altman R., Brutlag D., Karp P., Lathrop R., Searls D., Eds., pp 53-61, AAAIPress, Menlo Park; Hulo et al., Nucl. Acids. Res. 32:D134-D137, (2004)) or Pfam (Bateman et al., Nucleic Acids Research 30(1): 276-280 (2002)). This FMN-binding domain (Pfam entry PF02441, InterPro entry IPRO03382) is typically found in flavoprotein enzymes. The terms "domain" and "motif" are defined in the definitions section above.
[0168] The conserved region in SEQ ID NO: 2, as represented by SEQ ID NO: 8, may also in other HAL3 proteins be identified, using methods for the alignment of sequences for comparison as described hereinabove. In some instances, the default parameters may be adjusted to modify the stringency of the search. For example using BLAST, the statistical significance threshold (called "expect" value) for reporting matches against database sequences may be increased to show less stringent matches. This way, short nearly exact matches may be identified, including the conserved motifs 1 and 2 (SEQ ID NO: 6 and 7), or matches with one or more conservative change at any position.
[0169] HAL3 polypeptides (at least in their native form) and their homologues typically catalyse the decarboxylation of 4'-phosphopantothenoylcysteine to 4'-phosphopantetheine, a step in coenzyme A biosynthesis, which reaction may be tested in a biochemical assay; alternatively, HAL3 activity may be assayed by a complementation test with a dfp mutant E. coli strain (Kupke et al 2001; Yonamine et al 2004). The enzyme is also able to decarboxylate pantothenoylcysteine to pantothenoylcysteamine. Furthermore, the protein is involved in conferring salt and osmotic stress tolerance to plants, which feature may be useful in a bioassay for HAL3.
[0170] SEQ ID NO: 2 (encoded by SEQ ID NO: 1) is an example of a HAL3 polypeptide comprising from N-terminus to C-terminus features (i) a substrate binding helix, (ii) an insertion His motif, (iii) a PXMNXXMW motif and (iv) a substrate recognition clamp; and having in increasing order of preference at least 65%, 70%, 75%, 80%, 85%, 90% or 95% sequence identity to the conserved region represented by SEQ ID NO: 8. Further examples of HAL3 polypeptides comprising features (i) to (iv) are given in Table A in the examples section below:
[0171] Homologues of a HAL3 polypeptide may also be used to perform the methods of invention. Homologues (or homologous proteins) may readily be identified using routine techniques well known in the art, such as by sequence alignment. Methods for the alignment of sequences for comparison are well known in the art, such methods include GAP, BESTFIT, BLAST, FASTA and TFASTA. GAP uses the algorithm of Needleman and Wunsch ((1970) J Mol Biol 48: 443-453) to find the alignment of two complete sequences that maximizes the number of matches and minimizes the number of gaps. The BLAST algorithm (Altschul et al. (1990) J Mol Biol 215: 403-10) calculates percent sequence identity and performs a statistical analysis of the similarity between the two sequences. The software for performing BLAST analysis is publicly available through the National Centre for Biotechnology Information. Homologues may readily be identified using, for example, the ClustalW multiple sequence alignment algorithm (version 1.83), with the default pairwise alignment parameters, and a scoring method in percentage. Global percentages of similarity and identity may also be determined using one of the methods available in the MatGAT software package (Campanella et al., BMC Bioinformatics. 2003 Jul. 10; 4:29. MatGAT: an application that generates similarity/identity matrices using protein or DNA sequences.). Minor manual editing may be performed to optimise alignment between conserved motifs, as would be apparent to a person skilled in the art.
[0172] The sequence identity values may be determined over the entire conserved domain (as indicated above) or over the full length nucleic acid or amino acid sequence using the programs mentioned above using the default parameters.
[0173] Homologues also include orthologues and paralogues. Orthologues and paralogues may easily be found by performing a so-called reciprocal blast search. This may be done by a first BLAST involving submitting a query sequence (for example, SEQ ID NO: 1 or SEQ ID NO: 2) for a BLAST search against any sequence database, such as the publicly available NCBI database. BLASTN or TBLASTX (using standard default values) may be used when starting from a nucleotide sequence and BLASTP or TBLASTN (using standard default values) may be used when starting from a protein sequence. The BLAST results may optionally be filtered. The full-length sequences of either the filtered results or non-filtered results are then submitted to a second BLAST search (BLAST back) against sequences from the organism from which the query sequence is derived (where the query sequence is SEQ ID NO: 1 or SEQ ID NO: 2 the second BLAST would therefore be against Arabidopsis sequences). The results of the first and second BLAST searches are then compared. A paralogue is identified if a high-ranking hit from the first BLAST is from the same species as from which the query sequence is derived; an orthologue is identified if a high-ranking hit is not from the same species as from which the query sequence is derived. Preferred orthologues are orthologues of AtHAL3a (SEQ ID NO: 2), AtHAL3b (SEQ ID NO: 10) or of the long form of AtHAL3b (SEQ ID NO: 40). High-ranking hits are those having a low E-value. The lower the E-value, the more significant the score (or in other words the lower the chance that the hit was found by chance). Computation of the E-value is well known in the art. In the case of large families, ClustalW may be used, followed by a neighbour joining tree, to help visualize clustering of related genes and to identify orthologues and paralogues. In addition to E-values, comparisons are also scored by percentage identity. Percentage identity refers to the number of identical nucleotides (or amino acids) between the two compared nucleic acid (or polypeptide) sequences over a particular length. Preferably, HAL3 polypeptide homologues have in increasing order of preference at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more sequence identity or similarity (functional identity) to an unmodified HAL3 polypeptide as represented by SEQ ID NO: 2. Preferably, HAL3 polypeptide homologues are as represented by the sequences referred to in Table A.
[0174] The HAL3 polypeptide useful in the methods of the present invention may be a derivative of SEQ ID NO: 2. "Derivatives" include peptides, oligopeptides, polypeptides which may, compared to the amino acid sequence of the naturally-occurring form of the protein, such as the one presented in SEQ ID NO: 2, comprise substitutions of amino acids with non-naturally occurring amino acid residues, or additions of non-naturally occurring amino acid residues. Derivatives of the proteins as represented by the sequences listed in Table A are further examples which may be suitable for use in the methods of the invention
[0175] Examples of nucleic acids encoding HAL3 polypeptides include but are not limited to those represented by the sequences listed in Table A. Variants of nucleic acids encoding HAL3 polypeptides may be suitable for use in the methods of the invention. Suitable variants include portions of nucleic acids encoding HAL3 polypeptides and/or nucleic acids capable of hybridising with nucleic acids/genes encoding HAL3 polypeptides. Further variants include splice variants and allelic variants of nucleic acids encoding HAL3 polypeptides.
[0176] The term "portion" as defined herein refers to a piece of DNA encoding a polypeptide comprising from N-terminus to C-terminus features (i) a substrate binding helix, (ii) an insertion His motif, (iii) a PXMNXXMW motif and (iv) a substrate recognition clamp; and having in increasing order of preference at least 65%, 70%, 75%, 80%, 85%, 90% or 95% sequence identity to the conserved region represented by SEQ ID NO: 8.
[0177] A portion may be prepared, for example, by making one or more deletions to a nucleic acid encoding a HAL3 polypeptide. The portions may be used in isolated form or they may be fused to other coding (or non coding) sequences in order to, for example, produce a protein that combines several activities. When fused to other coding sequences, the resulting polypeptide produced upon translation may be bigger than that predicted for the HAL3 portion. Preferably, the portion codes for a polypeptide with substantially the same biological activity as the HAL3 polypeptide of SEQ ID NO: 2. The portion is typically at least 50, 100, 150 or 200 nucleotides in length, preferably at least 250, 300, 350 or 400 nucleotides in length, more preferably at least 450, 500, 550, 600 or 650 nucleotides in length.
[0178] Preferably, the portion is a portion of a nucleic acid as represented by the sequences listed in Table A. Most preferably the portion is a portion of a nucleic acid as represented by SEQ ID NO: 1.
[0179] The terms "fragment", "fragment of a sequence" or "part of a sequence" "portion" or "portion thereof" mean a truncated sequence of the original sequence referred to. The truncated sequence (nucleic acid or protein sequence) can vary widely in length; the minimum size being a sequence of sufficient size to provide a sequence with at least a comparable function and/or activity of the original sequence referred to or hybidizing with the nucleic acid molecule of the invention or used in the process of the invention under stringend conditions, while the maximum size is not critical. In some applications, the maximum size usually is not substantially greater than that required to provide the desired activity and/or function(s) of the original sequence. A comparable function means at least 40%, 45% or 50%, preferably at least 60%, 70%, 80% or 90% or more of the original sequence.
[0180] Another variant of a nucleic acid encoding a HAL3 polypeptide, useful in the methods of the present invention, is a nucleic acid capable of hybridising under reduced stringency conditions, preferably under stringent conditions, with a probe derived from the nucleic acid as defined hereinbefore, which hybridising sequence encodes a polypeptide comprising from N-terminus to C-terminus features (i) a substrate binding helix, (ii) an insertion His motif, (iii) a PXMNXXMW motif and (iv) a substrate recognition clamp; and has in increasing order of preference at least 65%, 70%, 75%, 80%, 85%, 90% or 95% sequence identity to the conserved region represented by SEQ ID NO: 8.
[0181] Preferably, the hybridising sequence is one that is capable of hybridising to a nucleic acid as represented by (or to probes derived from) the sequences listed in Table A, or to a portion of any of these sequences (the target sequence). Most preferably the hybridising sequence is capable of hybridising to SEQ ID NO: 1 (or to probes derived therefrom). Probes are generally less than 700 bp or 600 bp in length, preferably less than 500, 400 bp, 300 bp 200 bp or 100 bp in length. Commonly, probe lengths for DNA-DNA hybridisations such as Southern blotting, vary between 100 and 500 bp, whereas the hybridising region in probes for DNA-DNA hybridisations such as in PCR amplification generally are shorter than 50 but longer than 10 nucleotides, preferably they are 15, 20, 25, 30, 35, 40, 45 or 50 bp in length.
[0182] The HAL3 polypeptide may be encoded by a splice variant. The term "splice variant" as used herein encompasses variants of a nucleic acid sequence in which selected introns and/or exons have been excised, replaced, displaced or added, or in which introns have been shortened or lengthened. Such variants will be ones in which the substantial biological activity of the protein is retained, which may be achieved by selectively retaining functional segments of the protein. Such splice variants may be found in nature or may be manmade. Methods for making such splice variants are well known in the art.
[0183] Preferred splice variants are splice variants of the nucleic acid encoding a HAL3 polypeptide comprising from N-terminus to C-terminus (i) a substrate binding helix, (ii) an insertion His motif, (iii) a PXMNXXMW motif and (iv) a substrate recognition clamp; and having in increasing order of preference at least 65%, 70%, 75%, 80%, 85%, 90% or 95% sequence identity to the conserved region represented by SEQ ID NO: 8.
[0184] Further preferred splice variants of nucleic acids encoding HAL3 polypeptides comprising features as defined hereinabove are splice variants of a nucleic acid as represented by any one of the sequences listed in Table A. Most preferred is a splice variant of a nucleic acid sequence as represented by SEQ ID NO: 1.
[0185] The HAL3 polypeptide may also be encoded by an allelic variant of a nucleic acid encoding a polypeptide comprising from N-terminus to C-terminus (i) a substrate binding helix, (ii) an insertion His motif, (iii) a PXMNXXMW motif and (iv) a substrate recognition clamp; and having in increasing order of preference at least 65%, 70%, 75%, 80%, 85%, 90% or 95% sequence identity to the conserved region represented by SEQ ID NO: 8.
[0186] Preferred allelic variants of nucleic acids encoding HAL3 polypeptides comprising features as defined hereinabove are allelic variants of a nucleic acid as represented by any one of the sequences listed in Table A. Most preferred is an allelic variant of a nucleic acid sequence as represented by SEQ ID NO: 1.
[0187] Directed evolution (or gene shuffling) may also be used to generate variants of nucleic acids encoding HAL3 polypeptides. Site-directed mutagenesis may be used to generate variants of nucleic acids encoding HAL3 polypeptides. Several methods are available to achieve site-directed mutagenesis, the most common being PCR based methods (Current Protocols in Molecular Biology. Wiley Eds.).
[0188] Nucleic acids encoding HAL3 polypeptides may be derived from any natural or artificial source. The nucleic acid may be modified from its native form in composition and/or genomic environment through deliberate human manipulation. Preferably the HAL3 nucleic acid is from a plant, further preferably from a dicotyledonous plant, more preferably from the family Brassicaceae, most preferably the nucleic acid is from Arabidopsis thaliana.
[0189] The increased expression of a nucleic acid encoding a HAL3 polypeptide, preferentially in shoots of a plant, may be performed by introducing a genetic modification (preferably in the locus of a HAL3 gene). The locus of a gene as defined herein is taken to mean a genomic region, which includes the gene of interest and 10 KB up- or downstream of the coding region.
[0190] The genetic modification may be introduced, for example, by any one (or more) of the following methods: T-DNA activation, TILLING and homologous recombination (as described in the definitions section) or by introducing and increasing expression a nucleic acid encoding a HAL3 polypeptide preferentially in expanding tissues of a plant. Following introduction of the genetic modification, there follows an optional step of selecting for increased expression of a nucleic acid encoding a HAL3 factor polypeptide preferentially in expanding tissues, which increased expression gives plants having increased yield.
[0191] T-DNA activation tagging results in transgenic plants that show dominant phenotypes due to modified expression of genes close to the introduced promoter. The promoter to be introduced may be any promoter capable of increasing expression of the nucleic acid encoding a HAL3 polypeptide preferentially in expanding tissues of a plant.
[0192] A genetic modification may also be introduced in the locus of a gene encoding a HAL3 polypeptide using the technique of TILLING (Targeted Induced Local Lesions In Genomes). Plants carrying such mutant variants have increased expression of a nucleic acid encoding a HAL3 polypeptide preferentially in expanding plant tissues.
[0193] T-DNA activation and TILLING are examples of technologies that enable the generation of genetic modifications comprising preferentially increasing expression of a nucleic acid encoding a HAL3 polypeptide in expanding tissues of plants.
[0194] The effects of the invention may also be reproduced using homologous recombination. The nucleic acid to be targeted is preferably the region controlling the natural expression of a nucleic acid encoding a HAL3 polypeptide in a plant. A weak promoter specific for expression in shoots is introduced into this region, replacing it partly or substantially all of it.
[0195] A preferred method for introducing a genetic modification (which in this case need not be in the locus of a HAL3 gene) is to introduce and express preferentially in the shoot of a plant a nucleic acid encoding a HAL3 polypeptide, as defined hereinabove. The nucleic acid to be introduced into a plant may be a full-length nucleic acid or may be a portion or a hybridising sequence or another nucleic acid variant as hereinbefore defined.
[0196] The methods of the invention rely on preferentially increased expression of a nucleic acid encoding a HAL3 polypeptide in shoot tissue of a plant, preferably in the cell expansion zone of vegetative shoots.
[0197] The invention also provides genetic constructs and vectors to facilitate introduction and/or preferential expression of the nucleic acid sequences useful in the methods according to the invention, in shoots, preferably in expanding tissues of a plant shoots.
[0198] Therefore, there is provided a gene construct comprising:
[0199] (i) a nucleic acid encoding a HAL3 polypeptide as defined hereinabove;
[0200] (ii) one or more control sequences, of which at least one is a shoot-specific promoter, operably linked to the nucleic acid of (i).
[0201] Constructs useful in the methods according to the present invention may be constructed using recombinant DNA technology well known to persons skilled in the art. The gene constructs may be inserted into vectors, which may be commercially available, suitable for transforming into plants and suitable for expression of the gene of interest in the transformed cells. The invention therefore provides use of a gene construct as defined hereinabove in the methods of the invention.
[0202] Plants are transformed with a vector comprising the sequence of interest (i.e., a nucleic acid encoding a HAL3 polypeptide). The skilled artisan is well aware of the genetic elements that must be present on the vector in order to successfully transform, select and propagate host cells containing the sequence of interest. The sequence of interest is operably linked to one or more control sequences (at least to a promoter).
[0203] Suitable promoters, which are functional in plants, are generally known. They may take the form of constitutive or inducible promoters. Suitable promoters can enable the development- and/or tissue-specific expression in multi-celled eukaryotes; thus, leaf-, root-, flower-, seed-, stomata-, tuber- or fruit-specific promoters may advantageously be used in plants.
[0204] Different plant promoters usable in plants are promoters such as, for example, the USP, the LegB4-, the DC3 promoter or the ubiquitin promoter from parsley.
[0205] For expression in plants, the nucleic acid molecule must, as described above, be linked operably to or comprise a suitable promoter which expresses the gene at the right point in time and in a cell- or tissue-specific manner. Usable promoters are constitutive promoters (Benfey et al., EMBO J. 8 (1989) 2195-2202), such as those which originate from plant viruses, such as 35S CAMV (Franck et al., Cell 21 (1980) 285-294), 19S CaMV (see also U.S. Pat. No. 5,352,605 and WO 84/02913), 34S FMV (Sanger et al., Plant. Mol. Biol., 14, 1990: 433-443), the parsley ubiquitin promoter, or plant promoters such as the Rubisco small subunit promoter described in U.S. Pat. No. 4,962,028 or the plant promoters PRP1 [Ward et al., Plant. Mol. Biol. 22 (1993)], SSU, PGEL1, OCS [Leisner (1988) Proc Natl Acad Sci USA 85(5): 2553-2557], lib4, usp, mas [Comai (1990) Plant Mol Biol 15 (3):373-381], STLS1, ScBV (Schenk (1999) Plant Mol Biol 39(6):1221-1230), B33, SAD1 or SAD2 (flax promoters, Jain et al., Crop Science, 39 (6), 1999: 1696-1701) or nos [Shaw et al. (1984) Nucleic Acids Res. 12(20):7831-7846]. Further examples of constitutive plant promoters are the sugarbeet V-ATPase promoters (WO 01/14572). Examples of synthetic constitutive promoters are the Super promoter (WO 95/14098) and promoters derived from G-boxes (WO 94/12015). If appropriate, chemical inducible promoters may furthermore also be used, compare EP-A 388186, EP-A 335528, WO 97/06268. Stable, constitutive expression of the proteins according to the invention a plant can be advantageous. However, inducible expression of the polypeptide of the invention is advantageous, if a late expression before the harvest is of advantage, as metabolic manipulation may lead to plant growth retardation.
[0206] The expression of plant genes can also be facilitated via a chemical inducible promoter (for a review, see Gatz 1997, Annu. Rev. Plant Physiol. Plant Mol. Biol., 48:89-108). Chemically inducible promoters are particularly suitable when it is desired to express the gene in a time-specific manner. Examples of such promoters are a salicylic acid inducible promoter (WO 95/19443), and abscisic acid-inducible promoter (EP 335 528), a tetracyclin-inducible promoter (Gatz et al. (1992) Plant J. 2, 397-404), a cyclohexanol- or ethanol-inducible promoter (WO 93/21334) or others as described herein.
[0207] Other suitable promoters are those which react to biotic or abiotic stress conditions, for example the pathogen-induced PRP1 gene promoter (Ward et al., Plant. Mol. Biol. 22 (1993) 361-366), the tomato heat-inducible hsp80 promoter (U.S. Pat. No. 5,187,267), the potato chill-inducible alpha-amylase promoter (WO 96/12814) or the wound-inducible pinll promoter (EP-A-0 375 091) or others as described herein.
[0208] Preferred promoters are in particular those which bring gene expression in tissues and organs, in seed cells, such as endosperm cells and cells of the developing embryo. Suitable promoters are the oilseed rape napin gene promoter (U.S. Pat. No. 5,608,152), the Vicia faba USP promoter (Baeumlein et al., Mol Gen Genet, 1991, 225 (3): 459-67), the Arabidopsis oleosin promoter (WO 98/45461), the Phaseolus vulgaris phaseolin promoter (U.S. Pat. No. 5,504,200), the Brassica Bce4 promoter (WO 91/13980), the bean arc5 promoter, the carrot DcG3 promoter, or the Legumin B4 promoter (LeB4; Baeumlein et al., 1992, Plant Journal, 2 (2): 233-9), and promoters which bring about the seed-specific expression in monocotyledonous plants such as maize, barley, wheat, rye, rice and the like. Advantageous seed-specific promoters are the sucrose binding protein promoter (WO 00/26388), the phaseolin promoter and the napin promoter. Suitable promoters which must be considered are the barley Ipt2 or Ipt1 gene promoter (WO 95/15389 and WO 95/23230), and the promoters described in WO 99/16890 (promoters from the barley hordein gene, the rice glutelin gene, the rice oryzin gene, the rice prolamin gene, the wheat gliadin gene, the wheat glutelin gene, the maize zein gene, the oat glutelin gene, the sorghum kasirin gene and the rye secalin gene). Further suitable promoters are Amy32b, Amy 6-6 and Aleurain [U.S. Pat. No. 5,677,474], Bce4 (oilseed rape) [U.S. Pat. No. 5,530,149], glycinin (soya) [EP 571 741], phosphoenolpyruvate carboxylase (soya) [JP 06/62870], ADR12-2 (soya) [WO 98/08962], isocitrate lyase (oilseed rape) [U.S. Pat. No. 5,689,040] or α-amylase (barley) [EP 781 849]. Other promoters which are available for the expression of genes in plants are leaf-specific promoters such as those described in DE-A 19644478 or light-regulated promoters such as, for example, the pea petE promoter.
[0209] Further suitable plant promoters are the cytosolic FBPase promoter or the potato ST-LSI promoter (Stockhaus et al., EMBO J. 8, 1989, 2445), the Glycine max phosphoribosylpyrophosphate amidotransferase promoter (GenBank Accession No. U87999) or the node-specific promoter described in EP-A-0 249 676.
[0210] In a preferred embodiment, the nucleic acid sequence encoding a HAL3 polypeptide is operably linked to a shoot a shoot specific promoter. A shoot-specific promoter refers to any promoter able to drive expression of the gene of interest in vegetative plant shoot tissues. Preferably, the shoot specific promoter is able to preferentially drive expression of the gene of interest in expanding tissues of vegetative shoots, that is in the cell expansion zone of vegetative shoots. Reference herein to "preferentially" driving expression in the shoot is taken to mean driving expression of any sequence operably linked thereto in the cell expansion zone of vegetative shoots substantially to the exclusion of driving expression elsewhere in the plant, apart from any residual expression due to leaky promoter expression. The shoot-specific promoter may be either a natural or a synthetic promoter.
[0211] Preferably, the shoot-specific promoter is a promoter isolated from a beta-expansin gene, such as a rice beta expansin EXBP9 promoter (WO 2004/070039), as represented by SEQ ID NO: 5 or a promoter of similar strength and/or a promoter with a similar expression pattern as the rice beta expansin promoter (i.e. a functionally equivalent promoter).
[0212] It should be clear that the applicability of the present invention is not restricted to the nucleic acid encoding a HAL3 polypeptide represented by SEQ ID NO: 1, nor is the applicability of the invention restricted to expression of a nucleic acid encoding a HAL3 polypeptide when driven by a beta expansin promoter.
[0213] Optionally, one or more terminator sequences (also a control sequence) may be used in the construct introduced into a plant. Additional regulatory elements may include transcriptional as well as translational enhancers. Those skilled in the art will be aware of terminator and enhancer sequences that may be suitable for use in performing the invention. Such sequences would be known or may readily be obtained by a person skilled in the art.
[0214] The genetic constructs of the invention may further include an origin of replication sequence that is required for maintenance and/or replication in a specific cell type. One example is when a genetic construct is required to be maintained in a bacterial cell as an episomal genetic element (e.g. plasmid or cosmid molecule). Preferred origins of replication include, but are not limited to, the f1-ori and colE1.
[0215] Other control sequences (besides promoter, enhancer, silencer, intron sequences, 3'UTR and/or 5'UTR regions) may be protein and/or RNA stabilizing elements.
[0216] For the detection and/or selection of the successful transfer of the nucleic acid sequences as depicted in the sequence protocol and used in the process of the invention, it is advantageous to use marker genes (=reporter genes). These marker genes enable the identification of a successful transfer of the nucleic acid molecules via a series of different principles, for example via visual identification with the aid of fluorescence, luminescence or in the wavelength range of light which is discernible for the human eye, by a resistance to herbicides or antibiotics, via what are known as nutritive markers (auxotrophism markers) or antinutritive markers, via enzyme assays or via phytohormones. Examples of such markers which may be mentioned are GFP (=green fluorescent protein); the luciferin/luceferase system, the β-galactosidase with its colored substrates, for example X-Gal, the herbicide resistances to, for example, imidazolinone, glyphosate, phosphinothricin or sulfonylurea, the antibiotic resistances to, for example, bleomycin, hygromycin, streptomycin, kanamycin, tetracyclin, chloramphenicol, ampicillin, gentamycin, geneticin (G418), spectinomycin or blasticidin, to mention only a few, nutritive markers such as the utilization of mannose or xylose, or antinutritive markers such as the resistance to 2-deoxyglucose. This list is a small number of possible markers. The skilled worker is very familiar with such markers. Different markers are preferred, depending on the organism and the selection method.
[0217] The present invention also encompasses plants obtainable by the methods according to the present invention. The present invention therefore provides plants, plant parts or plant cells thereof obtainable by the method according to the present invention, which plants or parts or cells thereof comprise a nucleic acid transgene encoding a HAL3 polypeptide under the control of a shoot-specific promoter.
[0218] The invention also provides a method for the production of transgenic plants having increased yield relative to control plants, comprising introduction and preferential expression of a nucleic acid encoding a HAL3 polypeptide in the shoot of a plant.
[0219] Host plants for the nucleic acids or the vector used in the method according to the invention, the expression cassette or construct or vector are, in principle, advantageously all plants, which are capable of synthesizing the polypeptides used in the inventive method.
[0220] More specifically, the present invention provides a method for the production of transgenic plants having increased yield which method comprises:
[0221] (i) introducing and preferentially expressing a nucleic acid encoding a HAL3 polypeptide in the shoot of a plant; and
[0222] (ii) cultivating the plant cell under conditions promoting plant growth and development.
[0223] The nucleic acid may be introduced directly into a plant cell or into the plant itself (including introduction into a tissue, organ or any other part of a plant). According to a preferred feature of the present invention, the nucleic acid is preferably introduced into a plant by transformation.
[0224] The transfer of foreign genes into the genome of a plant is called transformation. In doing this the methods described for the transformation and regeneration of plants from plant tissues or plant cells are utilized for transient or stable transformation. To select transformed plants, the plant material obtained in the transformation is, as a rule, subjected to selective conditions so that transformed plants can be distinguished from untransformed plants. For example, the seeds obtained in the above-described manner can be planted and, after an initial growing period, subjected to a suitable selection by spraying. A further possibility consists in growing the seeds, if appropriate after sterilization, on agar plates using a suitable selection agent so that only the transformed seeds can grow into plants. Further advantageous transformation methods, in particular for plants, are known to the skilled worker and are described herein below.
[0225] Generally after transformation, plant cells or cell groupings are selected for the presence of one or more markers which are encoded by plant-expressible genes co-transferred with the gene of interest, following which the transformed material is regenerated into a whole plant.
[0226] As mentioned Agrobacteria transformed with an expression vector according to the invention may also be used in the manner known per se for the transformation of plants such as experimental plants like Arabidopsis or crop plants, such as, for example, cereals, maize, oats, rye, barley, wheat, soya, rice, cotton, sugarbeet, canola, sunflower, flax, hemp, potato, tobacco, tomato, carrot, bell peppers, oilseed rape, tapioca, cassava, arrow root, tagetes, alfalfa, lettuce and the various tree, nut, and grapevine species, in particular oil-containing crop plants such as soya, peanut, castor-oil plant, sunflower, maize, cotton, flax, oilseed rape, coconut, oil palm, safflower (Carthamus tinctorius) or cocoa beans, for example by bathing scarified leaves or leaf segments in an agrobacterial solution and subsequently growing them in suitable media.
[0227] In addition to the transformation of somatic cells, which then has to be regenerated into intact plants, it is also possible to transform the cells of plant meristems and in particular those cells which develop into gametes. In this case, the transformed gametes follow the natural plant development, giving rise to transgenic plants. Thus, for example, seeds of Arabidopsis are treated with agrobacteria and seeds are obtained from the developing plants of which a certain proportion is transformed and thus transgenic [Feldman, K A and Marks M D (1987). Mol Gen Genet. 208:274-289; Feldmann K (1992). In: C Koncz, N-H Chua and J Shell, eds, Methods in Arabidopsis Research. Word Scientific, Singapore, pp. 274-289]. Alternative methods are based on the repeated removal of the influorescences and incubation of the excision site in the center of the rosette with transformed agrobacteria, whereby transformed seeds can likewise be obtained at a later point in time (Chang (1994). Plant J. 5: 551-558; Katavic (1994). Mol Gen Genet, 245: 363-370). However, an especially effective method is the vacuum infiltration method with its modifications such as the "floral dip" method. In the case of vacuum infiltration of Arabidopsis, intact plants under reduced pressure are treated with an agrobacterial suspension [Bechthold, N (1993). C R Acad Sci Paris Life Sci, 316: 1194-1199], while in the case of the "floral dip" method the developing floral tissue is incubated briefly with a surfactant-treated agrobacterial suspension [Clough, S J and Bent, A F (1998). The Plant J. 16, 735-743]. A certain proportion of transgenic seeds are harvested in both cases, and these seeds can be distinguished from nontransgenic seeds by growing under the above-described selective conditions. In addition the stable transformation of plastids is of advantages because plastids are inherited maternally is most crops reducing or eliminating the risk of transgene flow through pollen. The transformation of the chloroplast genome is generally achieved by a process, which has been schematically displayed in Klaus et al., 2004 [Nature Biotechnology 22 (2), 225-229]. Briefly the sequences to be transformed are cloned together with a selectable marker gene between flanking sequences homologous to the chloroplast genome. These homologous flanking sequences direct site specific integration into the plastome. Plastidal transformation has been described for many different plant species and an overview can be taken from Bock (2001) Transgenic plastids in basic research and plant biotechnology. J Mol Biol. 2001 Sep. 21; 312 (3):425-38 or Maliga, P (2003) Progress towards commercialization of plastid transformation technology. Trends Biotechnol. 21, 20-28. Further biotechnological progress has recently been reported in form of marker free plastid transformants, which can be produced by a transient cointegrated maker gene (Klaus et al., 2004, Nature Biotechnology 22(2), 225-229).
[0228] The genetically modified plant cells can be regenerated via all methods with which the skilled worker is familiar. Suitable methods can be found in the abovementioned publications by S. D. Kung and R. Wu, Potrykus or Hofgen and Willmitzer.
[0229] Following DNA transfer and regeneration, putatively transformed plants may be evaluated, for instance using Southern analysis, for the presence of the gene of interest, copy number and/or genomic organisation. Alternatively or additionally, expression levels of the newly introduced DNA may be monitored using Northern and/or Western analysis, or quantitative PCR, all techniques being well known to persons having ordinary skill in the art.
[0230] The generated transformed plants may be propagated by a variety of means, such as by clonal propagation or classical breeding techniques. For example, a first generation (or T1) transformed plant may be selfed to give homozygous second generation (or T2) transformants, and the T2 plants further propagated through classical breeding techniques.
[0231] The generated transformed organisms may take a variety of forms. For example, they may be chimeras of transformed cells and non-transformed cells; clonal transformants (e.g., all cells transformed to contain the expression cassette); grafts of transformed and untransformed tissues (e.g., in plants, a transformed rootstock grafted to an untransformed scion).
[0232] The present invention clearly extends to any plant cell or plant produced by any of the methods described herein, and to all plant parts and propagules thereof. The present invention extends further to encompass the progeny of a primary transformed or transfected cell, tissue, organ or whole plant that has been produced by any of the aforementioned methods, the only requirement being that progeny exhibit the same genotypic and/or phenotypic characteristic(s) as those produced by the parent in the methods according to the invention.
[0233] The invention also includes host cells containing an isolated nucleic acid encoding a HAL3 polypeptide. Preferred host cells according to the invention are plant cells.
[0234] The invention also extends to harvestable parts of a plant such as, but not limited to seeds, leaves, fruits, flowers, stems, rhizomes, tubers and bulbs. The invention furthermore relates to products derived, preferably directly derived, from a harvestable part of such a plant, such as dry pellets or powders, oil, fat and fatty acids, starch or proteins.
[0235] The present invention also encompasses use of nucleic acids encoding HAL3 polypeptides and use of HAL3 polypeptides in increasing plant yield as defined hereinabove in the methods of the invention.
[0236] Nucleic acids encoding HAL3 polypeptides, or HAL3 polypeptides, may find use in breeding programmes in which a DNA marker is identified which may be genetically linked to a HAL3 gene. The nucleic acids/genes, or the HAL3 polypeptides may be used to define a molecular marker. This DNA or protein marker may then be used in breeding programmes to select plants having increased yield as defined hereinabove in the methods of the invention.
[0237] Allelic variants of a HAL3 nucleic acid/gene may also find use in marker-assisted breeding programmes. Such breeding programmes sometimes require introduction of allelic variation by mutagenic treatment of the plants, using for example EMS mutagenesis; alternatively, the programme may start with a collection of allelic variants of so called "natural" origin caused unintentionally. Identification of allelic variants then takes place, for example, by PCR. This is followed by a step for selection of superior allelic variants of the sequence in question and which give increased yield. Selection is typically carried out by monitoring growth performance of plants containing different allelic variants of the sequence in question. Growth performance may be monitored in a greenhouse or in the field. Further optional steps include crossing plants in which the superior allelic variant was identified with another plant. This could be used, for example, to make a combination of interesting phenotypic features.
[0238] A nucleic acid encoding a HAL3 polypeptide may also be used as probes for genetically and physically mapping the genes that they are a part of, and as markers for traits linked to those genes. Such information may be useful in plant breeding in order to develop lines with desired phenotypes. Such use of HAL3 nucleic acids requires only a nucleic acid sequence of at least 15 nucleotides in length. The HAL3 nucleic acids may be used as restriction fragment length polymorphism (RFLP) markers. Southern blots (Sambrook J, Fritsch E F and Maniatis T (1989) Molecular Cloning, A Laboratory Manual) of restriction-digested plant genomic DNA may be probed with the HAL3 nucleic acids. The resulting banding patterns may then be subjected to genetic analyses using computer programs such as MapMaker (Lander et al. (1987) Genomics 1: 174-181) in order to construct a genetic map. In addition, the nucleic acids may be used to probe Southern blots containing restriction endonuclease-treated genomic DNAs of a set of individuals representing parent and progeny of a defined genetic cross. Segregation of the DNA polymorphisms is noted and used to calculate the position of the HAL3 nucleic acid in the genetic map previously obtained using this population (Botstein et al. (1980) Am. J. Hum. Genet. 32:314-331).
[0239] The production and use of plant gene-derived probes for use in genetic mapping is described in Bernatzky and Tanksley (1986) Plant Mol. Biol. Reporter 4: 37-41. Numerous publications describe genetic mapping of specific cDNA clones using the methodology outlined above or variations thereof. For example, F2 intercross populations, backcross populations, randomly mated populations, near isogenic lines, and other sets of individuals may be used for mapping. Such methodologies are well known to those skilled in the art.
[0240] The nucleic acid probes may also be used for physical mapping (i.e., placement of sequences on physical maps; see Hoheisel et al. In: Non-mammalian Genomic Analysis: A Practical Guide, Academic press 1996, pp. 319-346, and references cited therein).
[0241] In another embodiment, the nucleic acid probes may be used in direct fluorescence in situ hybridisation (FISH) mapping (Trask (1991) Trends Genet. 7:149-154). Although current methods of FISH mapping favour use of large clones (several kb to several hundred kb; see Laan et al. (1995) Genome Res. 5:13-20), improvements in sensitivity may allow performance of FISH mapping using shorter probes.
[0242] A variety of nucleic acid amplification-based methods for genetic and physical mapping may be carried out using the nucleic acids. Examples include allele-specific amplification (Kazazian (1989) J. Lab. Clin. Med. 11:95-96), polymorphism of PCR-amplified fragments (CAPS; Sheffield et al. (1993) Genomics 16:325-332), allele-specific ligation (Landegren et al. (1988) Science 241:1077-1080), nucleotide extension reactions (Sokolov (1990) Nucleic Acid Res. 18:3671), Radiation Hybrid Mapping (Walter et al. (1997) Nat. Genet. 7:22-28) and Happy Mapping (Dear and Cook (1989) Nucleic Acid Res. 17:6795-6807). For these methods, the sequence of a nucleic acid is used to design and produce primer pairs for use in the amplification reaction or in primer extension reactions. The design of such primers is well known to those skilled in the art. In methods employing PCR-based genetic mapping, it may be necessary to identify DNA sequence differences between the parents of the mapping cross in the region corresponding to the instant nucleic acid sequence. This, however, is generally not necessary for mapping methods.
[0243] The methods according to the present invention result in plants having increased yield, as described hereinbefore. This increased yield may also be combined with other economically advantageous traits, such as further yield-enhancing traits, tolerance to other abiotic and biotic stresses, traits modifying various architectural features and/or biochemical and/or physiological features.
Detailed Description for MADS15 Up
[0244] Surprisingly, it has now been found that modulating expression in a plant of a nucleic acid encoding a MADS15 polypeptide gives plants having enhanced yield-related traits relative to control plants. The particular class of MADS15 polypeptides suitable for enhancing yield-related traits in plants is described in detail below.
[0245] The present invention provides a method for enhancing yield-related traits in plants relative to control plants, comprising modulating expression in a plant of a nucleic acid encoding a MADS15 polypeptide.
[0246] Any reference hereinafter to a "protein useful in the methods of the invention" is taken to mean a MADS15 polypeptide as defined herein. Any reference hereinafter to a "nucleic acid useful in the methods of the invention" is taken to mean a nucleic acid capable of encoding such a MADS15 polypeptide.
[0247] A preferred method for modulating (preferably, increasing) expression of a nucleic acid encoding a protein useful in the methods of the invention is by introducing and expressing in a plant a nucleic acid encoding a protein useful in the methods of the invention as defined below.
[0248] The nucleic acid to be introduced into a plant (and therefore useful in performing the methods of the invention) is any nucleic acid encoding the type of protein which will now be described, hereafter also named "MADS15 nucleic acid" or "MADS15 gene".
[0249] The term "MADS15 polypeptide" as defined herein refers to a protein that falls in the group of FUL-like MADS box proteins as delineated by Adam et al. (J. Mol. Evol. 62, 15-31). FUL-like MADS box proteins are part of the SQUAMOSA subfamily and have the typical MIKCc architecture: a MADS domain (M) at the N-terminus, followed by an intervening domain (I) involved in dimerisation, a highly conserved keratin domain (K) and a variable domain (C) at the C-terminus, which is responsible for binding of interacting proteins or transcriptional activation (De Bodt et al. Trends in Plant Science 8, 475-483).
[0250] Plant MADS15 polypeptides may also be identified by the presence of certain conserved motifs. The presence of these conserved motifs may be identified using methods for the alignment of sequences for comparison as described hereinabove. In some instances, the default parameters may be adjusted to modify the stringency of the search. For example using BLAST, the statistical significance threshold (called "expect" value) for reporting matches against database sequences may be increased to show less stringent matches. This way, short nearly exact matches may be identified. Upon identification of a MADS15 polypeptide by the presence of these motifs, a person skilled in the art may easily derive the corresponding nucleic acid encoding the polypeptide comprising the relevant motifs, and use a sufficient length of contiguous nucleotides of the same to perform any one or more of the gene silencing methods described above (for the reduction or substantial elimination of an endogenous MADS15 gene expression).
[0251] Typically, the presence of at least one of the motifs 1 to 4 should be sufficient to identify any query sequence as a MADS15, however, the presence of at least motifs 1 and 2 is preferred. The consensus sequence provided is based on the monocot sequences given in the sequence listing. A person skilled in the art would be well aware that the consensus sequence may vary somewhat if further or different sequences (for example from dicotyledonous plants) were used for comparison.
[0252] Motif 1, located in the MADS domain (SEQ ID NO: 48):
TABLE-US-00006 L(L/V)KKA(H/N)EIS(VI)L(C/Y)DAE(V/I/L) (A/G) (L/A/V) (I/V) (I/V)FS(T/P/A/N)KGKLYE(Y/F) (A/S) (T/S)(D/N/E)S(K/C/R/S)M(D/E) (N/I/R/K)IL(E/D)R.
[0253] Preferably, this conserved sequence motif 1 has the sequence:
TABLE-US-00007 LLKKAHEISVLCDAEVA (L/A/V)I(I/V)FS(T/P)KGKLYEYATDS (C/R)M(D/E)(R/K)ILER,
most preferably the conserved sequence motif 1 has the sequence:
TABLE-US-00008 LLKKAHEISVLCDAEVAAIVFSPKGKLYEYATDSRMDKILER
[0254] Motif 2, located in the K domain (SEQ ID NO: 49):
TABLE-US-00009 KLK(A/S) (K/R) (V/I)E(A/T/S) (L/I) (Q/N) (K/R/N) (S/C/R) (Q/H) (R/K)HLMGE
[0255] Preferably, this conserved sequence motif 2 has the sequence:
TABLE-US-00010 KLKAK(V/I)E(A/T) (L/I)QK(S/C) (Q/H) (R/K)HLMGE
[0256] More preferably, this conserved sequence motif 2 has the sequence:
TABLE-US-00011 KLKAKIETIQKCHKHLMGE
[0257] Optionally, the MADS15 polypeptide or its homologue may comprise in the C domain motif 3 and/or motif 4:
TABLE-US-00012 motif 3 (SEQ ID NO: 50): Q(P/Q/V/A)QTS (S/F) (S/F) (S/F) (S/F) (S/C/F) (F/M) motif 4 (SEQ ID NO: 51): (G/A/V/L) (L/P)XWMX(S/H)
wherein the first X residue in motif 4 may be any amino acid, but preferably L, P or H; and wherein the second X residue may be any amino acid, but preferably V or L.
[0258] Alternatively, the C-domain (starting behind the keratin domain, i.e. at R175 in SEQ ID NO: 44) may be characterised by the increased occurrence of glutamine (normally 3.93%, here at least 8.77% with a maximum of 26.73%), in addition, the content in alanine, proline, and/or serine may also be increased (above 7.8%, 4.85% and 6.89% respectively).
[0259] Alternatively formulated, a preferred MADS15 protein useful in the methods of the present invention comprises at least an N-terminal MADS domain corresponding to the SMART domain SM00432 (Pfam PF00319) followed by a Keratin domain corresponding to the K-box region defined in Pfam as PF01486, more preferably, the MADS15 protein has in its MADS domain the conserved signature of SEQ ID NO: 48 and in its K-domain the conserved signature of SEQ ID NO: 49, most preferably, the MADS15 protein has the sequence given in SEQ ID NO: 44.
[0260] Examples of proteins useful in the methods of the invention and nucleic acids encoding the same are given below in table D of Example 8.
[0261] Also useful in the methods of the invention are homologues of any one of the amino acid sequences given in table D.
[0262] Also useful in the methods of the invention are derivatives of any one of the polypeptides given in table D or orthologues or paralogues of any of the aforementioned SEQ ID NOs. A preferred derivative is a derivative of SEQ ID NO: 44. Derivatives of the polypeptides given in table D are further examples which may be suitable for use in the methods of the invention. Derivatives useful in the methods of the present invention preferably have similar biological and functional activity as the unmodified protein from which they are derived.
[0263] The invention is illustrated by transforming plants with the Oryza sativa nucleic acid sequence represented by SEQ ID NO: 43, encoding the polypeptide sequence of SEQ ID NO: 44, however performance of the invention is not restricted to these sequences. The methods of the invention may advantageously be performed using any nucleic acid encoding a protein useful in the methods of the invention as defined herein, including orthologues and paralogues, such as any of the nucleic acid sequences given in table D. The amino acid sequences given in table D may be considered to be orthologues and paralogues of the MADS15 polypeptide represented by SEQ ID NO: 44.
[0264] Orthologues and paralogues may easily be found by performing a so-called reciprocal blast search. Typically, this involves a first BLAST involving BLASTing a query sequence (for example using any of the sequences listed in table D) against any sequence database, such as the publicly available NCBI database. BLASTN or TBLASTX (using standard default values) are generally used when starting from a nucleotide sequence, and BLASTP or TBLASTN (using standard default values) when starting from a protein sequence. The BLAST results may optionally be filtered. The full-length sequences of either the filtered results or non-filtered results are then BLASTed back (second BLAST) against sequences from the organism from which the query sequence is derived (where the query sequence is SEQ ID NO: 43 or SEQ ID NO: 44, the second BLAST would therefore be against Oryza sativa sequences). The results of the first and second BLASTs are then compared. A paralogue is identified if a high-ranking hit from the first blast is from the same species as from which the query sequence is derived, a BLAST back then ideally results in the query sequence as highest hit; an orthologue is identified if a high-ranking hit in the first BLAST is not from the same species as from which the query sequence is derived, and preferably results upon BLAST back in the query sequence being among the highest hits.
[0265] High-ranking hits are those having a low E-value. The lower the E-value, the more significant the score (or in other words the lower the chance that the hit was found by chance). Computation of the E-value is well known in the art. In addition to E-values, comparisons are also scored by percentage identity. Percentage identity refers to the number of identical nucleotides (or amino acids) between the two compared nucleic acid (or polypeptide) sequences over a particular length. In the case of large families, ClustalW may be used, followed by a neighbour joining tree, to help visualize clustering of related genes and to identify orthologues and paralogues.
[0266] Table D gives examples of orthologues and paralogues of the MADS15 protein represented by SEQ ID NO 44. Further orthologues and paralogues may readily be identified using the BLAST procedure described above.
[0267] The proteins of the invention are identifiable by the presence of the conserved MADS and/or keratin domain(s) (shown in FIG. 5). Specialist databases exist for the identification of domains, for example, SMART (Schultz et al. (1998) Proc. Natl. Acad. Sci. USA 95, 5857-5864; Letunic et al. (2002) Nucleic Acids Res 30, 242-244, InterPro (Mulder et al., (2003) Nucl. Acids. Res. 31, 315-318, Prosite (Bucher and Bairoch (1994), A generalized profile syntax for biomolecular sequences motifs and its function in automatic sequence interpretation. (In) ISMB-94; Proceedings 2nd International Conference on Intelligent Systems for Molecular Biology. Altman R., Brutlag D., Karp P., Lathrop R., Searls D., Eds., pp 53-61, AAAIPress, Menlo Park; Hulo et al., Nucl. Acids. Res. 32:D134-D137, (2004), or Pfam (Bateman et al., Nucleic Acids Research 30(1): 276-280 (2002). A set of tools for in silico analysis of protein sequences is available on the ExPASY proteomics server (hosted by the Swiss Institute of Bioinformatics (Gasteiger et al., ExPASy: the proteomics server for in-depth protein knowledge and analysis, Nucleic Acids Res. 31:3784-3788 (2003)).
[0268] Domains may also be identified using routine techniques, such as by sequence alignment. Methods for the alignment of sequences for comparison are well known in the art, such methods include GAP, BESTFIT, BLAST, FASTA and TFASTA. GAP uses the algorithm of Needleman and Wunsch ((1970) J Mol Biol 48: 443-453) to find the global (i.e. spanning the complete sequences) alignment of two sequences that maximizes the number of matches and minimizes the number of gaps. The BLAST algorithm (Altschul et al. (1990) J Mol Biol 215: 403-10) calculates percent sequence identity and performs a statistical analysis of the similarity between the two sequences. The software for performing BLAST analysis is publicly available through the National Centre for Biotechnology Information (NCBI). Homologues may readily be identified using, for example, the ClustalW multiple sequence alignment algorithm (version 1.83), with the default pairwise alignment parameters, and a scoring method in percentage. Global percentages of similarity and identity may also be determined using one of the methods available in the MatGAT software package (Campanella et al., BMC Bioinformatics. 2003 Jul. 10; 4:29. MatGAT: an application that generates similarity/identity matrices using protein or DNA sequences.). Minor manual editing may be performed to optimise alignment between conserved motifs, as would be apparent to a person skilled in the art. Furthermore, instead of using full-length sequences for the identification of homologues, specific domains (such as the MADS or keratin domain, or one of the motifs defined above) may be used as well. The sequence identity values, which are indicated below in Example 10 as a percentage were determined over the entire nucleic acid or amino acid sequence, and/or over selected domains or conserved motif(s), using the programs mentioned above using the default parameters.
[0269] Furthermore, a MADS15 protein may also be identifiable by its ability or inability to bind DNA and to interact with other proteins. DNA-binding activity and protein-protein interactions may readily be determined in vitro or in vivo using techniques well known in the art. Examples of in vitro assays for DNA binding activity include: gel retardation analysis using known MADS-box DNA binding domains (West et al. (1998) Nucl Acid Res 26(23): 5277-87), or yeast one-hybrid assays. An example of an in vitro assay for protein-protein interactions is the yeast two-hybrid analysis (Fields and Song (1989) Nature 340:245-6). Proteins known to interact with OsMADS15 include other MADS proteins (such as those involved in the flowering signalling complex), receptor like kinases such a Clavata1 and Clavata2, Erecta, BRI1 or RSK. Further details are provided in Example 13.
[0270] Nucleic acids encoding proteins useful in the methods of the invention need not be full-length nucleic acids, since performance of the methods of the invention does not rely on the use of full-length nucleic acid sequences. Examples of nucleic acids suitable for use in performing the methods of the invention include the nucleic acid sequences given in table D, but are not limited to those sequences. Nucleic acid variants may also be useful in practising the methods of the invention. Examples of such nucleic acid variants include portions of nucleic acids encoding a protein useful in the methods of the invention, nucleic acids hybridising to nucleic acids encoding a protein useful in the methods of the invention, splice variants of nucleic acids encoding a protein useful in the methods of the invention, allelic variants of nucleic acids encoding a protein useful in the methods of the invention and variants of nucleic acids encoding a protein useful in the methods of the invention that are obtained by gene shuffling. The terms portion, hybridising sequence, splice variant, allelic variant and gene shuffling will now be described.
[0271] According to the present invention, there is provided a method for enhancing yield-related traits in plants, comprising introducing and expressing in a plant a portion of any one of the nucleic acid sequences given in table D, or a portion of a nucleic acid encoding an orthologue, paralogue or homologue of any of the amino acid sequences given in table D.
[0272] Portions useful in the methods of the invention, encode a polypeptide falling within the definition of a nucleic acid encoding a protein useful in the methods of the invention as defined herein and having substantially the same biological activity as the amino acid sequences given in table D. Preferably, the portion is a portion of any one of the nucleic acids given in table D. The portion is typically at least 400 consecutive nucleotides in length, preferably at least 600 consecutive nucleotides in length, more preferably at least 700 consecutive nucleotides in length and most preferably at least 800 consecutive nucleotides in length, the consecutive nucleotides being of any one of the nucleic acid sequences given in table D. Most preferably the portion is a portion of the nucleic acid of SEQ ID NO: 43. Preferably, the portion encodes an amino acid sequence comprising (any one or more of) the MADS and keratin domain as defined herein.
[0273] A portion of a nucleic acid encoding a MADS15 protein as defined herein may be prepared, for example, by making one or more deletions to the nucleic acid. The portions may be used in isolated form or they may be fused to other coding (or non coding) sequences in order to, for example, produce a protein that combines several activities. When fused to other coding sequences, the resultant polypeptide produced upon translation may be bigger than that predicted for the MADS15 protein portion.
[0274] Another nucleic acid variant useful in the methods of the invention is a nucleic acid capable of hybridising, under reduced stringency conditions, preferably under stringent conditions, with a nucleic acid encoding a MADS15 protein as defined herein, or with a portion as defined herein.
[0275] Hybridising sequences useful in the methods of the invention, encode a polypeptide having a MADS and/or keratin domain (see the alignment of FIG. 6) and having substantially the same biological activity as the MADS15 protein represented by any of the amino acid sequences given in table D. The hybridising sequence is typically at least 400 consecutive nucleotides in length, preferably at least 600 consecutive nucleotides in length, more preferably at least 700 consecutive nucleotides in length and most preferably at least 800 consecutive nucleotides in length, the consecutive nucleotides being of any one of the nucleic acid sequences given in table D. Preferably, the hybridising sequence is one that is capable of hybridising to any of the nucleic acids given in table D, or to a portion of any of these sequences, a portion being as defined above. Most preferably, the hybridising sequence is capable of hybridising to a nucleic acid as represented by SEQ ID NO: 43 or to a portion thereof. Preferably, the hybridising sequence encodes an amino acid sequence comprising any one or more of the motifs or domains as defined herein.
[0276] According to the present invention, there is provided a method for enhancing yield-related traits in plants, comprising introducing and expressing in a plant a nucleic acid capable of hybridizing to any one of the nucleic acids given in table D, or comprising introducing and expressing in a plant a nucleic acid capable of hybridising to a nucleic acid encoding an orthologue, paralogue or homologue of any of the nucleic acid sequences given in table D.
[0277] Another nucleic acid variant useful in the methods of the invention is a splice variant encoding a MADS15 protein as defined hereinabove.
[0278] According to the present invention, there is provided a method for enhancing yield-related traits in plants, comprising introducing and expressing in a plant a splice variant of any one of the nucleic acid sequences given in table D, or a splice variant of a nucleic acid encoding an orthologue, paralogue or homologue of any of the amino acid sequences given in table D.
[0279] Preferred splice variants are splice variants of a nucleic acid represented by SEQ ID NO: 43 or a splice variant of a nucleic acid encoding an orthologue or paralogue of SEQ ID NO: 44. Preferably, the amino acid sequence encoded by the splice variant comprises any one or more of the motifs or domains as defined herein.
[0280] Another nucleic acid variant useful in performing the methods of the invention is an allelic variant of a nucleic acid encoding a MADS15 protein as defined hereinabove. The allelic variants useful in the methods of the present invention have substantially the same biological activity as the MADS15 protein of SEQ ID NO: 44.
[0281] According to the present invention, there is provided a method for enhancing yield-related traits in plants, comprising introducing and expressing in a plant an allelic variant of any one of the nucleic acids given in table D, or comprising introducing and expressing in a plant an allelic variant of a nucleic acid encoding an orthologue, paralogue or homologue of any of the amino acid sequences given in table D.
[0282] Preferably, the allelic variant is an allelic variant of SEQ ID NO: 43 or an allelic variant of a nucleic acid encoding an orthologue or paralogue of SEQ ID NO: 44. Preferably, the amino acid sequence encoded by the allelic variant comprises any one or more of the motifs or domains as defined herein.
[0283] A further nucleic acid variant useful in the methods of the invention is a nucleic acid variant obtained by gene shuffling. Gene shuffling or directed evolution may also be used to generate variants of nucleic acids encoding MADS15 proteins as defined above.
[0284] According to the present invention, there is provided a method for enhancing yield-related traits in plants, comprising introducing and expressing in a plant a variant of any one of the nucleic acid sequences given in table D, or comprising introducing and expressing in a plant a variant of a nucleic acid encoding an orthologue, paralogue or homologue of any of the amino acid sequences given in table D, which variant nucleic acid is obtained by gene shuffling.
[0285] Preferably, the variant nucleic acid obtained by gene shuffling encodes an amino acid sequence comprising any one or more of the motifs or domains as defined herein.
[0286] Furthermore, nucleic acid variants may also be obtained by site-directed mutagenesis. Several methods are available to achieve site-directed mutagenesis, the most common being PCR based methods (Current Protocols in Molecular Biology. Wiley Eds.).
[0287] Nucleic acids encoding MADS15 proteins may be derived from any natural or artificial source. The nucleic acid may be modified from its native form in composition and/or genomic environment through deliberate human manipulation. Preferably the MADS15-encoding nucleic acid is from a plant, further preferably from a monocotyledonous plant, more preferably from the Poaceae family, most preferably the nucleic acid is from Oryza sativa.
[0288] Any reference herein to a MADS15 protein is therefore taken to mean a MADS15 protein as defined above. Any nucleic acid encoding such a MADS15 protein is suitable for use in performing the methods of the invention.
[0289] The present invention also encompasses plants or parts thereof (including seeds) obtainable by the methods according to the present invention. The plants or parts thereof comprise a nucleic acid transgene encoding a MADS15 protein as defined above.
[0290] The invention also provides genetic constructs and vectors to facilitate introduction and/or expression of the nucleic acid sequences useful in the methods according to the invention, in a plant. The gene constructs may be inserted into vectors, which may be commercially available, suitable for transforming into plants and suitable for expression of the gene of interest in the transformed cells. The invention also provides use of a gene construct as defined herein in the methods of the invention.
[0291] More specifically, the present invention provides a construct comprising
[0292] (a) nucleic acid encoding MADS15 protein as defined above;
[0293] (b) one or more control sequences capable of driving expression of the nucleic acid sequence of (a); and optionally
[0294] (c) a transcription termination sequence.
[0295] Plants are transformed with a vector comprising the sequence of interest (i.e., a nucleic acid encoding a MADS15 polypeptide as defined herein. The skilled artisan is well aware of the genetic elements that must be present on the vector in order to successfully transform, select and propagate host cells containing the sequence of interest. The sequence of interest is operably linked to one or more control sequences (at least to a promoter).
[0296] Advantageously, any type of promoter may be used to drive expression of the nucleic acid sequence.
[0297] The promoter may be a constitutive promoter; alternatively, the promoter may be an inducible promoter, i.e. having induced or increased transcription initiation in response to a chemical or a stress-inducible promoter, or a pathogen-induced promoter.
[0298] Additionally or alternatively, the promoter may be an organ-specific or tissue-specific promoter, or the promoter may be a ubiquitous promoter, or the promoter may be developmentally regulated. Furthermore, the promoter may be organ-specific or tissue-specific or cell-specific.
[0299] Preferably, the MADS15 nucleic acid or variant thereof is operably linked to a constitutive promoter. A preferred constitutive promoter is one that is also substantially ubiquitously expressed. Further preferably the promoter is derived from a plant, more preferably a monocotyledonous plant. Most preferred is use of a GOS2 promoter (for example from rice, SEQ ID NO: 47 or SEQ ID NO: 108). It should be clear that the applicability of the present invention is not restricted to the MADS15 nucleic acid represented by SEQ ID NO: 43, nor is the applicability of the invention restricted to expression of a MADS15 nucleic acid when driven by a GOS2 promoter. Examples of other constitutive promoters or functionally equivalent promters which may also be used to drive expression of a MADS15 nucleic acid are shown in the definitions section.
[0300] Optionally, one or more terminator sequences may be used in the construct introduced into a plant. Additional regulatory elements may include transcriptional as well as translational enhancers. Those skilled in the art will be aware of terminator and enhancer sequences that may be suitable for use in performing the invention. Such sequences would be known or may readily be obtained by a person skilled in the art.
[0301] An intron sequence may also be added to the 5' untranslated region (UTR) or in the coding sequence to increase the amount of the mature message that accumulates in the cytosol.
[0302] Other control sequences (besides promoter, enhancer, silencer, intron sequences, 3'UTR and/or 5'UTR regions) may be protein and/or RNA stabilizing elements. Such sequences would be known or may readily be obtained by a person skilled in the art.
[0303] The genetic constructs of the invention may further include an origin of replication sequence that is required for maintenance and/or replication in a specific cell type. One example is when a genetic construct is required to be maintained in a bacterial cell as an episomal genetic element (e.g. plasmid or cosmid molecule). Preferred origins of replication include, but are not limited to, the f1-ori and colE1.
[0304] For the detection of the successful transfer of the nucleic acid sequences as used in the methods of the invention and/or selection of transgenic plants comprising these nucleic acids, it is advantageous to use marker genes (or reporter genes). Therefore, the genetic construct may optionally comprise a selectable marker gene.
[0305] The invention also provides a method for the production of transgenic plants having enhanced yield-related traits relative to control plants, comprising introduction and expression in a plant of any nucleic acid encoding a MADS15 protein as defined hereinabove.
[0306] More specifically, the present invention provides a method for the production of transgenic plants having increased yield, which method comprises:
[0307] (i) introducing and expressing in a plant or plant cell a MADS15 nucleic acid or variant thereof; and
[0308] (ii) cultivating the plant cell under conditions promoting plant growth and development.
[0309] The nucleic acid may be introduced directly into a plant cell or into the plant itself (including introduction into a tissue, organ or any other part of a plant). According to a preferred feature of the present invention, the nucleic acid is preferably introduced into a plant by transformation.
[0310] The genetically modified plant cells can be regenerated via all methods with which the skilled worker is familiar. Suitable methods can be found in the abovementioned publications by S. D. Kung and R. Wu, Potrykus or Hofgen and Willmitzer.
[0311] Generally after transformation, plant cells or cell groupings are selected for the presence of one or more markers which are encoded by plant-expressible genes co-transferred with the gene of interest, following which the transformed material is regenerated into a whole plant. To select transformed plants, the plant material obtained in the transformation is, as a rule, subjected to selective conditions so that transformed plants can be distinguished from untransformed plants. For example, the seeds obtained in the above-described manner can be planted and, after an initial growing period, subjected to a suitable selection by spraying. A further possibility consists in growing the seeds, if appropriate after sterilization, on agar plates using a suitable selection agent so that only the transformed seeds can grow into plants. Alternatively, the transformed plants are screened for the presence of a selectable marker such as the ones described above.
[0312] Following DNA transfer and regeneration, putatively transformed plants may also be evaluated, for instance using Southern analysis, for the presence of the gene of interest, copy number and/or genomic organisation. Alternatively or additionally, expression levels of the newly introduced DNA may be monitored using Northern and/or Western analysis, both techniques being well known to persons having ordinary skill in the art.
[0313] The generated transformed plants may be propagated by a variety of means, such as by clonal propagation or classical breeding techniques. For example, a first generation (or T1) transformed plant may be selfed and homozygous second-generation (or T2) transformants selected, and the T2 plants may then further be propagated through classical breeding techniques.
[0314] The generated transformed organisms may take a variety of forms. For example, they may be chimeras of transformed cells and non-transformed cells; clonal transformants (e.g., all cells transformed to contain the expression cassette); grafts of transformed and untransformed tissues (e.g., in plants, a transformed rootstock grafted to an untransformed scion).
[0315] The present invention clearly extends to any plant cell or plant produced by any of the methods described herein, and to all plant parts and propagules thereof. The present invention extends further to encompass the progeny of a primary transformed or transfected cell, tissue, organ or whole plant that has been produced by any of the aforementioned methods, the only requirement being that progeny exhibit the same genotypic and/or phenotypic characteristic(s) as those produced by the parent in the methods according to the invention.
[0316] The invention also includes host cells containing an isolated nucleic acid encoding a MADS15 protein as defined hereinabove. Preferred host cells according to the invention are plant cells.
[0317] Host plants for the nucleic acids or the vector used in the method according to the invention, the expression cassette or construct or vector are, in principle, advantageously all plants, which are capable of synthesizing the polypeptides used in the inventive method.
[0318] The invention also extends to harvestable parts of a plant such as, but not limited to seeds, leaves, fruits, flowers, stems, rhizomes, roots, tubers and bulbs. The invention furthermore relates to products derived, preferably directly derived, from a harvestable part of such a plant, such as dry pellets or powders, oil, fat and fatty acids, starch or proteins.
[0319] According to a preferred feature of the invention, the modulated expression is increased expression.
[0320] As mentioned above, a preferred method for modulating (preferably, increasing) expression of a nucleic acid encoding a MADS15 protein is by introducing and expressing in a plant a nucleic acid encoding a MADS15 protein; however the effects of performing the method, i.e. enhancing yield-related traits may also be achieved using other well known techniques. A description of some of these techniques will now follow.
[0321] One such technique is T-DNA activation tagging. The resulting transgenic plants show dominant phenotypes due to modified expression of genes close to the introduced promoter.
[0322] The effects of the invention may also be reproduced using the technique of TILLING (Targeted Induced Local Lesions In Genomes), or by homologous recombination.
[0323] Reference herein to enhanced yield-related traits is taken to mean an increase in biomass (weight) of one or more parts of a plant, which may include aboveground (harvestable) parts and/or (harvestable) parts below ground.
[0324] Taking corn as an example, a yield increase may be manifested as one or more of the following: increase in the number of plants established per hectare or acre, an increase in the number of ears per plant, an increase in the number of rows, number of kernels per row, kernel weight, thousand kernel weight, ear length/diameter, increase in the seed filling rate (which is the number of filled seeds divided by the total number of seeds and multiplied by 100), among others. Taking rice as an example, a yield increase may manifest itself as an increase in one or more of the following: number of plants per hectare or acre, number of panicles per plant, number of spikelets per panicle, number of flowers (florets) per panicle (which is expressed as a ratio of the number of filled seeds over the number of primary panicles), increase in the seed filling rate (which is the number of filled seeds divided by the total number of seeds and multiplied by 100), increase in thousand kernel weight, among others.
[0325] In a particular embodiment, such harvestable parts are roots, and performance of the methods of the invention results in plants having increased root growth relative to the root growth of suitable control plants. Root development is an essential determinant of plant growth and crop yield since the root is the main channel to extract nutrients from the environment.
[0326] Increased root yield may for example manifest itself as one of more of the following: a) increased amount of root biomass (whereby a discrimination between thick roots and thin roots may be made by defining a certain treshold), b) increased average root diameter, c) increased total root biomass, and d) increased root biomass/shoot biomass ratio. For the purpose of this invention, it should be understood that the term `root growth` encompasses all aspects of growth of the different parts that make up the root system at different stages of its development, both in monocotyledonous and dicotyledonous plants. It is to be understood that enhanced growth of the root can result from enhanced growth of one or more of its parts including the primary root, lateral roots, adventitious roots, etc. all of which fall within the scope of this invention.
[0327] Since the transgenic plants according to the present invention have increased yield, it is likely that these plants exhibit an increased growth rate (during at least part of their life cycle), relative to the growth rate of control plants at a corresponding stage in their life cycle. The increased growth rate may be specific to one or more parts of a plant (including seeds), or may be throughout substantially the whole plant. Plants having an increased growth rate may have a shorter life cycle. The life cycle of a plant may be taken to mean the time needed to grow from a dry mature seed up to the stage where the plant has produced dry mature seeds, similar to the starting material. This life cycle may be influenced by factors such as early vigour, growth rate, greenness index, flowering time and speed of seed maturation. The increase in growth rate may take place at one or more stages in the life cycle of a plant or during substantially the whole plant life cycle. Increased growth rate during the early stages in the life cycle of a plant may reflect enhanced vigour. The increase in growth rate may alter the harvest cycle of a plant allowing plants to be sown later and/or harvested sooner than would otherwise be possible (a similar effect may be obtained with earlier flowering time). If the growth rate is sufficiently increased, it may allow for the further sowing of seeds of the same plant species (for example sowing and harvesting of rice plants followed by sowing and harvesting of further rice plants all within one conventional growing period). Similarly, if the growth rate is sufficiently increased, it may allow for the further sowing of seeds of different plants species (for example the sowing and harvesting of corn plants followed by, for example, the sowing and optional harvesting of soy bean, potato or any other suitable plant). Harvesting additional times from the same rootstock in the case of some crop plants may also be possible. Altering the harvest cycle of a plant may lead to an increase in annual biomass production per acre (due to an increase in the number of times (say in a year) that any particular plant may be grown and harvested). An increase in growth rate may also allow for the cultivation of transgenic plants in a wider geographical area than their wild-type counterparts, since the territorial limitations for growing a crop are often determined by adverse environmental conditions either at the time of planting (early season) or at the time of harvesting (late season). Such adverse conditions may be avoided if the harvest cycle is shortened. The growth rate may be determined by deriving various parameters from growth curves, such parameters may be: T-Mid (the time taken for plants to reach 50% of their maximal size) and T-90 (time taken for plants to reach 90% of their maximal size), amongst others.
[0328] According to a preferred feature of the present invention, performance of the methods of the invention gives plants having an increased growth rate relative to control plants. Therefore, according to the present invention, there is provided a method for increasing the growth rate of plants, which method comprises modulating expression, preferably increasing expression, in a plant of a nucleic acid encoding a MADS15 protein as defined herein.
[0329] An increase in yield and/or growth rate occurs whether the plant is under non-stress conditions or whether the plant is exposed to various stresses compared to control plants. Plants typically respond to exposure to stress by growing more slowly. In conditions of severe stress, the plant may even stop growing altogether. Mild stress on the other hand is defined herein as being any stress to which a plant is exposed which does not result in the plant ceasing to grow altogether without the capacity to resume growth. Mild stress in the sense of the invention leads to a reduction in the growth of the stressed plants of less than 40%, 35% or 30%, preferably less than 25%, 20% or 15%, more preferably less than 14%, 13%, 12%, 11% or 10% or less in comparison to the control plant under non-stress conditions. Due to advances in agricultural practices (irrigation, fertilization, pesticide treatments) severe stresses are not often encountered in cultivated crop plants. As a consequence, the compromised growth induced by mild stress is often an undesirable feature for agriculture. Mild stresses are the everyday biotic and/or abiotic (environmental) stresses to which a plant is exposed. Abiotic stresses may be due to drought or excess water, anaerobic stress, salt stress, chemical toxicity, oxidative stress and hot, cold or freezing temperatures. The abiotic stress may be an osmotic stress caused by a water stress (particularly due to drought), salt stress, oxidative stress or an ionic stress. Biotic stresses are typically those stresses caused by pathogens, such as bacteria, viruses, fungi and insects.
[0330] In particular, the methods of the present invention may be performed under non-stress conditions or under conditions of mild drought to give plants having increased yield relative to control plants. As reported in Wang et al. (Planta (2003) 218: 1-14), abiotic stress leads to a series of morphological, physiological, biochemical and molecular changes that adversely affect plant growth and productivity. Drought, salinity, extreme temperatures and oxidative stress are known to be interconnected and may induce growth and cellular damage through similar mechanisms. Rabbani et al. (Plant Physiol (2003) 133: 1755-1767) describes a particularly high degree of "cross talk" between drought stress and high-salinity stress. For example, drought and/or salinisation are manifested primarily as osmotic stress, resulting in the disruption of homeostasis and ion distribution in the cell. Oxidative stress, which frequently accompanies high or low temperature, salinity or drought stress, may cause denaturing of functional and structural proteins. As a consequence, these diverse environmental stresses often activate similar cell signaling pathways and cellular responses, such as the production of stress proteins, up-regulation of anti-oxidants, accumulation of compatible solutes and growth arrest. The term "non-stress" conditions as used herein are those environmental conditions that allow optimal growth of plants. Persons skilled in the art are aware of normal soil conditions and climatic conditions for a given location.
[0331] Performance of the methods of the invention gives plants grown under non-stress conditions or under mild drought conditions increased yield relative to control plants grown under comparable conditions. Therefore, according to the present invention, there is provided a method for increasing yield in plants grown under non-stress conditions or under mild drought conditions, which method comprises increasing expression in a plant of a nucleic acid encoding a MADS15 polypeptide.
[0332] Performance of the methods of the invention gives plants grown under conditions of nutrient deficiency, particularly under conditions of nitrogen deficiency, increased yield relative to control plants grown under comparable conditions. Therefore, according to the present invention, there is provided a method for increasing yield in plants grown under conditions of nutrient deficiency, which method comprises increasing expression in a plant of a nucleic acid encoding a MADS15 polypeptide. Nutrient deficiency may result from a lack or excess of nutrients such as nitrogen, phosphates and other phosphorous-containing compounds, potassium, calcium, cadmium, magnesium, manganese, iron and boron, amongst others.
[0333] In a preferred embodiment of the invention, the increase in yield and/or growth rate occurs according to the methods of the present invention under non-stress conditions.
[0334] The methods of the invention are advantageously applicable to any plant.
[0335] Plants that are particularly useful in the methods of the invention include all plants which belong to the superfamily Viridiplantae, in particular monocotyledonous and dicotyledonous plants including fodder or forage legumes, ornamental plants, food crops, trees or shrubs. According to a preferred embodiment of the present invention, the plant is a crop plant. Examples of crop plants include soybean, sunflower, canola, alfalfa, rapeseed, cotton, tomato, potato and tobacco. Further preferably, the plant is a monocotyledonous plant. Examples of monocotyledonous plants include sugarcane. More preferably the plant is a cereal. Examples of cereals include rice, maize, wheat, barley, millet, rye, triticale, sorghum and oats.
[0336] The present invention also encompasses use of nucleic acids encoding the MADS15 protein described herein and use of these MADS15 proteins in enhancing yield-related traits in plants.
[0337] Nucleic acids encoding the MADS15 protein described herein, or the MADS15 proteins themselves, may find use in breeding programmes in which a DNA marker is identified which may be genetically linked to a MADS15-encoding gene. The nucleic acids/genes, or the MADS15 proteins themselves may be used to define a molecular marker. This DNA or protein marker may then be used in breeding programmes to select plants having enhanced yield-related traits as defined hereinabove in the methods of the invention.
[0338] Allelic variants of a MADS15 protein-encoding nucleic acid/gene may also find use in marker-assisted breeding programmes. Such breeding programmes sometimes require introduction of allelic variation by mutagenic treatment of the plants, using for example EMS mutagenesis; alternatively, the programme may start with a collection of allelic variants of so called "natural" origin caused unintentionally. Identification of allelic variants then takes place, for example, by PCR. This is followed by a step for selection of superior allelic variants of the sequence in question and which give increased yield. Selection is typically carried out by monitoring growth performance of plants containing different allelic variants of the sequence in question. Growth performance may be monitored in a greenhouse or in the field. Further optional steps include crossing plants in which the superior allelic variant was identified with another plant. This could be used, for example, to make a combination of interesting phenotypic features.
[0339] Nucleic acids encoding MADS15 proteins may also be used as probes for genetically and physically mapping the genes that they are a part of, and as markers for traits linked to those genes. Such information may be useful in plant breeding in order to develop lines with desired phenotypes. Such use of MADS15 protein-encoding nucleic acids requires only a nucleic acid sequence of at least 15 nucleotides in length. The MADS15 protein-encoding nucleic acids may be used as restriction fragment length polymorphism (RFLP) markers. Southern blots (Sambrook J, Fritsch E F and Maniatis T (1989) Molecular Cloning, A Laboratory Manual) of restriction-digested plant genomic DNA may be probed with the MADS15 protein-encoding nucleic acids. The resulting banding patterns may then be subjected to genetic analyses using computer programs such as MapMaker (Lander et al. (1987) Genomics 1: 174-181) in order to construct a genetic map. In addition, the nucleic acids may be used to probe Southern blots containing restriction endonuclease-treated genomic DNAs of a set of individuals representing parent and progeny of a defined genetic cross. Segregation of the DNA polymorphisms is noted and used to calculate the position of the MADS15 protein-encoding nucleic acid in the genetic map previously obtained using this population (Botstein et al. (1980) Am. J. Hum. Genet. 32:314-331).
[0340] The production and use of plant gene-derived probes for use in genetic mapping is described in Bernatzky and Tanksley (1986) Plant Mol. Biol. Reporter 4: 37-41. Numerous publications describe genetic mapping of specific cDNA clones using the methodology outlined above or variations thereof. For example, F2 intercross populations, backcross populations, randomly mated populations, near isogenic lines, and other sets of individuals may be used for mapping. Such methodologies are well known to those skilled in the art.
[0341] The nucleic acid probes may also be used for physical mapping (i.e., placement of sequences on physical maps; see Hoheisel et al. In: Non-mammalian Genomic Analysis: A Practical Guide, Academic press 1996, pp. 319-346, and references cited therein).
[0342] In another embodiment, the nucleic acid probes may be used in direct fluorescence in situ hybridisation (FISH) mapping (Trask (1991) Trends Genet. 7:149-154). Although current methods of FISH mapping favour use of large clones (several kb to several hundred kb; see Laan et al. (1995) Genome Res. 5:13-20), improvements in sensitivity may allow performance of FISH mapping using shorter probes.
[0343] A variety of nucleic acid amplification-based methods for genetic and physical mapping may be carried out using the nucleic acids. Examples include allele-specific amplification (Kazazian (1989) J. Lab. Clin. Med. 11:95-96), polymorphism of PCR-amplified fragments (CAPS; Sheffield et al. (1993) Genomics 16:325-332), allele-specific ligation (Landegren et al. (1988) Science 241:1077-1080), nucleotide extension reactions (Sokolov (1990) Nucleic Acid Res. 18:3671), Radiation Hybrid Mapping (Walter et al. (1997) Nat. Genet. 7:22-28) and Happy Mapping (Dear and Cook (1989) Nucleic Acid Res. 17:6795-6807). For these methods, the sequence of a nucleic acid is used to design and produce primer pairs for use in the amplification reaction or in primer extension reactions. The design of such primers is well known to those skilled in the art. In methods employing PCR-based genetic mapping, it may be necessary to identify DNA sequence differences between the parents of the mapping cross in the region corresponding to the instant nucleic acid sequence. This, however, is generally not necessary for mapping methods.
[0344] The methods according to the present invention result in plants having enhanced yield-related traits, as described hereinbefore. These traits may also be combined with other economically advantageous traits, such as further yield-enhancing traits, tolerance to other abiotic and biotic stresses, traits modifying various architectural features and/or biochemical and/or physiological features.
Detailed Description of MADS15 Down
[0345] It has now surprisingly been found that decreasing the level and/or activity of an endogenous MADS15 polypeptide gives plants having enhanced yield-related traits, in particular increased yield relative, to corresponding wild type plants. The present invention therefore provides methods for enhancing yield related traits, particularly for increasing yield of a plant relative to control plants, comprising decreasing the level and/or activity of an endogenous MADS15 polypeptide. Reference herein to "control plants" is taken to mean corresponding wild type plants in which there is no reduction in activity of the endogenous MADS15 polypeptide.
[0346] Advantageously, performance of the methods according to the present invention results in plants having increased yield, particularly increased seed yield and/or increased biomass, relative to corresponding wild type plants.
[0347] The term "increased yield" as defined herein is taken to mean an increase in biomass (weight) of one or more parts of a plant, which may include aboveground (harvestable) parts and/or (harvestable) parts below ground.
[0348] In particular, such harvestable parts include vegetative biomass and/or seeds, and performance of the methods of the invention results in plants having increased yield (in vegetative biomass and/or seed) relative to the yield of control plants.
[0349] Taking corn as an example, a yield increase may be manifested as one or more of the following: increase in the number of plants per hectare or acre, an increase in the number of ears per plant, an increase in the number of rows, number of kernels per row, kernel weight, thousand kernel weight, ear length/diameter, increase in the seed filling rate (which is the number of filled seeds divided by the total number of seeds and multiplied by 100), among others. Taking rice as an example, a yield increase may manifest itself as an increase in one or more of the following: number of plants per hectare or acre, number of panicles per plant, number of spikelets per panicle, number of flowers (florets) per panicle (which is expressed as a ratio of the number of filled seeds over the number of primary panicles), increase in the seed filling rate (which is the number of filled seeds divided by the total number of seeds and multiplied by 100), increase in thousand kernel weight, among others.
[0350] The increase in seed yield may also be manifested as an increase in seed size and/or seed volume. This may increase the amount, or change the composition of, substances in the seed, such as oils, proteins and carbohydrates.
[0351] According to a preferred feature, performance of the methods of the invention result in plants having increased yield, particularly increased biomass and/or seed yield. Therefore, according to the present invention, there is provided a method for increasing plant yield, which method comprises decreasing the level of activity of a MADS15 polypeptide or a homologue thereof, preferably by downregulating expression of a MADS15 gene or a homologue thereof.
[0352] Since the transgenic plants according to the present invention have increased yield, it is likely that these plants exhibit an increased growth rate (during at least part of their life cycle), relative to the growth rate of corresponding wild type plants at a corresponding stage in their life cycle. The increased growth rate may be specific to one or more parts of a plant (including seeds), or may be throughout substantially the whole plant. Plants having an increased growth rate may have a shorter life cycle. The life cycle of a plant may be taken to mean the time needed to grow from a dry mature seed up to the stage where the plant has produced dry mature seeds, similar to the starting material. This life cycle may be influenced by factors such as early vigour, growth rate, flowering time and speed of seed maturation. An increase in growth rate may take place at one or more stages in the life cycle of a plant or during substantially the whole plant life cycle. Increased growth rate during the early stages in the life cycle of a plant may reflect enhanced vigour. The increase in growth rate may alter the harvest cycle of a plant allowing plants to be sown later and/or harvested sooner than would otherwise be possible (a similar effect may be obtained with earlier flowering time). If the growth rate is sufficiently increased, it may allow for the further sowing of seeds of the same plant species (for example sowing and harvesting of rice plants followed by sowing and harvesting of further rice plants all within one conventional growing period). Similarly, if the growth rate is sufficiently increased, it may allow for the further sowing of seeds of different plants species (for example the sowing and harvesting of corn plants followed by, for example, the sowing and optional harvesting of soy bean, potato or any other suitable plant). Harvesting additional times from the same rootstock in the case of some crop plants may also be possible. Altering the harvest cycle of a plant may lead to an increase in annual biomass production per acre (due to an increase in the number of times (say in a year) that any particular plant may be grown and harvested). An increase in growth rate may also allow for the cultivation of transgenic plants in a wider geographical area than their wild-type counterparts, since the territorial limitations for growing a crop are often determined by adverse environmental conditions either at the time of planting (early season) or at the time of harvesting (late season). Such adverse conditions may be avoided if the harvest cycle is shortened. The growth rate may be determined by deriving various parameters from growth curves, such parameters may be: T-Mid (the time taken for plants to reach 50% of their maximal size) and T-90 (time taken for plants to reach 90% of their maximal size), amongst others.
[0353] Performance of the methods of the invention gives plants having an increased growth rate. Therefore, according to the present invention, there is provided a method for increasing the growth rate of plants relative to control plants, which method comprises preferentially reducing the expression level and/or activity of an endogenous MADS15 gene in a plant.
[0354] An increase in yield and/or growth rate occurs whether the plant is under non-stress conditions or whether the plant is exposed to various stresses compared to control plants. Plants typically respond to exposure to stress by growing more slowly. In conditions of severe stress, the plant may even stop growing altogether. Mild stress on the other hand is defined herein as being any stress to which a plant is exposed which does not result in the plant ceasing to grow altogether without the capacity to resume growth. Mild stress in the sense of the invention leads to a reduction in the growth of the stressed plants of less than 40%, 35% or 30%, preferably less than 25%, 20% or 15%, more preferably less than 14%, 13%, 12%, 11% or 10% or less in comparison to the control plant under non-stress conditions. Due to advances in agricultural practices (irrigation, fertilization, pesticide treatments) severe stresses are not often encountered in cultivated crop plants. As a consequence, the compromised growth induced by mild stress is often an undesirable feature for agriculture. Mild stresses are the everyday biotic and/or abiotic (environmental) stresses to which a plant is exposed. Abiotic stresses may be due to drought or excess water, anaerobic stress, salt stress, chemical toxicity, oxidative stress and hot, cold or freezing temperatures. The abiotic stress may be an osmotic stress caused by a water stress (particularly due to drought), salt stress, oxidative stress or an ionic stress. Biotic stresses are typically those stresses caused by pathogens, such as bacteria, viruses, fungi and insects.
[0355] In particular, the methods of the present invention may be performed under non-stress conditions or under conditions of mild drought to give plants having increased yield relative to control plants. As reported in Wang et al. (Planta (2003) 218: 1-14), abiotic stress leads to a series of morphological, physiological, biochemical and molecular changes that adversely affect plant growth and productivity. Drought, salinity, extreme temperatures and oxidative stress are known to be interconnected and may induce growth and cellular damage through similar mechanisms. Rabbani et al. (Plant Physiol (2003) 133: 1755-1767) describes a particularly high degree of "cross talk" between drought stress and high-salinity stress. For example, drought and/or salinisation are manifested primarily as osmotic stress, resulting in the disruption of homeostasis and ion distribution in the cell. Oxidative stress, which frequently accompanies high or low temperature, salinity or drought stress, may cause denaturing of functional and structural proteins. As a consequence, these diverse environmental stresses often activate similar cell signaling pathways and cellular responses, such as the production of stress proteins, up-regulation of anti-oxidants, accumulation of compatible solutes and growth arrest. The term "non-stress" conditions as used herein are those environmental conditions that allow optimal growth of plants. Persons skilled in the art are aware of normal soil conditions and climatic conditions for a given location.
[0356] Performance of the methods of the invention gives plants grown under non-stress conditions or under mild drought conditions increased yield relative to control plants grown under comparable conditions. Therefore, according to the present invention, there is provided a method for increasing yield in plants grown under non-stress conditions or under mild drought conditions, which method comprises reducing expression of an endogenous MADS15 gene in a plant.
[0357] Performance of the methods of the invention gives plants grown under conditions of nutrient deficiency, particularly under conditions of nitrogen deficiency, increased yield relative to control plants grown under comparable conditions. Therefore, according to the present invention, there is provided a method for increasing yield in plants grown under conditions of nutrient deficiency, which method comprises decreasing expression in a plant of a nucleic acid encoding a MADS15 polypeptide. Nutrient deficiency may result from a lack or excess of nutrients such as nitrogen, phosphates and other phosphorous-containing compounds, potassium, calcium, cadmium, magnesium, manganese, iron and boron, amongst others.
[0358] In a preferred embodiment of the invention, the increase in yield and/or growth rate occurs according to the methods of the present invention under non-stress conditions.
[0359] The methods of the invention are advantageously applicable to any plant. Plants that are particularly useful in the methods of the invention include all plants which belong to the superfamily Viridiplantae, in particular monocotyledonous and dicotyledonous plants including fodder or forage legumes, ornamental plants, food crops, trees or shrubs. According to a preferred embodiment of the present invention, the plant is a crop plant.
[0360] Examples of crop plants include soybean, sunflower, canola, alfalfa, rapeseed, cotton, tomato, potato and tobacco. Further preferably, the plant is a monocotyledonous plant. Examples of monocotyledonous plants include sugarcane. More preferably the plant is a cereal. Examples of cereals include rice, maize, wheat, barley, millet, rye, triticale, sorghum and oats.
[0361] Reference herein to a "decrease" in level of an endogenous MADS15 protein in a plant is taken to mean a reduction in protein concentration or substantial elimination of an endogenous MADS15 protein relative to endogenous MADS15 protein levels found in corresponding wild type plants. This reduction or substantial elimination may result in reduced or substantially abolished MADS15 protein activity in a plant.
[0362] Reference herein to a "decrease" in activity of an endogenous MADS15 protein in a plant is taken to mean a reduction in MADS15 protein activity or substantial elimination of activity of an endogenous MADS15 protein relative to endogenous MADS15 protein activity levels found wild type plants.
[0363] Preferably, the reduction in endogenous MADS15 protein level and/or activity is obtained by downregulating the expression of the endogenous MADS15 gene.
[0364] Reference herein to an "endogenous" MADS15 gene not only refers to MADS15 genes as found in a plant in its natural form (i.e., without there being any human intervention), but also refers to isolated MADS15 genes subsequently introduced into a plant. For example, a transgenic plant containing a MADS15 transgene may encounter a reduction or substantial elimination of the MADS15 transgene expression and/or reduced or substantial elimination of expression of an endogenous MADS15 gene.
[0365] This reduction (or substantial elimination) of endogenous MADS15 gene expression may be achieved using any one or more of several well-known gene silencing methods. "Gene silencing" or "downregulation" of expression, as used herein, refers to a reduction or the substantial elimination of MADS15 gene expression and/or MADS15 polypeptide levels and/or MADS15 polypeptide activity.
[0366] One such method for reduction or substantial elimination of endogenous MADS15 gene expression is RNA-mediated downregulation of gene expression (RNA silencing). Silencing in this case is triggered in a plant by a double stranded RNA molecule (dsRNA) that is substantially homologous to a target MADS15 gene. This dsRNA is further processed by the plant into about 21 to about 26 nucleotides called short interfering RNAs (siRNAs). The siRNAs are incorporated into an RNA-induced silencing complex (RISC) that cleaves the mRNA of a MADS15 target gene, thereby reducing or substantially eliminating the number of MADS15 mRNAs to be translated into a MADS15 protein.
[0367] One example of an RNA silencing method involves the introduction of coding sequences or parts thereof in a sense orientation into a plant. The additional gene, or part thereof, will silence an endogenous MADS15 gene, giving rise to a phenomenon known as co-suppression. The reduction of MADS15 gene expression will be more pronounced if several additional copies are introduced into the plant, as there is a positive correlation between high transcript levels and the triggering of co-suppression.
[0368] Another example of an RNA silencing method involves the use of antisense MADS15 nucleic acid sequences.
[0369] The antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection). Preferably, production of antisense nucleic acids in plants occurs by means of a stably integrated transgene comprising a promoter operative for constitutive expression in plants, an antisense oligonucleotide, and a terminator.
[0370] A preferred method for reduction or substantial elimination of endogenous MADS15 gene expression via RNA silencing is by using an expression vector into which a MADS15 gene or fragment thereof has been cloned as an inverted repeat (in part or completely), separated by a spacer (non-coding DNA).
[0371] In still another embodiment, the reduction or substantial elimination of endogenous MADS15 exprssion may be obtained by using ribozymes. For example, a derivative of a Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in an MADS15-encoding mRNA.
[0372] Gene silencing may also be achieved by insertion mutagenesis or if there is a mutation on the endogenous MADS15 gene and/or a mutation on an isolated MADS15 gene subsequently introduced into a plant. The reduction or substantial elimination of MADS15 protein activity may be caused by a non-functional MADS15. For example, MADS15 binds to various interacting proteins; one or more mutation(s) and/or truncation(s) within the MADS box of a MADS15 may therefore provide for a MADS15 protein that is still able to bind interacting proteins but that cannot exhibit its normal function as transcription factor.
[0373] A further approach to gene silencing is by targeting nucleotide sequences complementary to the regulatory region of the MADS15 gene (e.g., the MADS15 promoter and/or enhancers) to form triple helical structures that prevent transcription of the MADS15 gene in target cells.
[0374] Still another approach to gene silencing is described by Hiratsu et al. (Plant J. 34, 733-739, 2003). This method does not depend on sequence homology to the targeted gene but involves the use of a repression sequence domain in transcriptional gene fusions, and has been used to modify traits of agronomic interest (Fujita et al., Plant Cell 17, 3470-3488, 2005 and Mitsuda at al., Plant Cell 17, 2993-3006, 2005). Typically, a nucleotide chimeric fusion is made between a gene encoding a protein capable of positively influencing the expression of the targeted gene (such as a transcription activator), and a nucleotide fragment encoding a repression domain. Upon expression of the chimeric gene fusion, the expression of the targeted gene is repressed, usually in a dominant negative fashion. Repression domains are well known in the art, for example the EAR motif present in some AP2 and Zinc finger transcription factor. Methods based on repression domains are well suited to overcome gene redundancy for the targeted gene in the plant species of choice.
[0375] Described above are examples of various methods for gene silencing (for the reduction or substantial elimination of endogenous MADS15 gene expression. The methods of the invention rely on the reduction of expression of an endogenous MADS15 gene in a plant. A person skilled in the art would readily be able to adapt the aforementioned methods for silencing so as to achieve gene silencing in a whole plant or in parts thereof through the use of an appropriate promoter, for example.
[0376] It should be noted that the essence of the present invention resides in the advantageous and surprising results found upon reduction or substantial elimination of endogenous MADS15 gene expression in a plant, and is not limited to any particular method for such reduction or substantial elimination of endogenous MADS15 protein activity. The activity of a MADS15 polypeptide may also be decreased or eliminated by introducing a genetic modification (preferably in the locus of a MADS15 gene). The locus of a gene as defined herein is taken to mean a genomic region, which includes the gene of interest and 10 kb up- or down stream of the coding region.
[0377] The genetic modification may be introduced, for example, by any one (or more) of the following methods: T-DNA inactivation, TILLING, site-directed mutagenesis, directed evolution, homologous recombination. Following introduction of the genetic modification, there follows a step of selecting for decreased activity of a MADS15 polypeptide, which decrease in activity gives plants having increased yield.
[0378] T-DNA inactivation tagging involves insertion of a T-DNA, in the genomic region of the gene of interest or 10 kb up- or downstream of the coding region of a gene in a configuration such that the T-DNA inhibits expression of the targeted gene. Typically, regulation of expression of the targeted gene by its natural promoter is disrupted. The T-DNA is randomly inserted into the plant genome, for example, through Agrobacterium infection and leads to down-regulated expression of genes near the inserted T-DNA. The resulting transgenic plants show phenotypes due to inhibited expression of genes close to the introduced T-DNA.
[0379] A genetic modification may also be introduced in the locus of a MADS15 gene using the technique of TILLING (Targeted Induced Local Lesions In Genomes).
[0380] Site-directed mutagenesis and random mutagenesis may be used to generate variants of MADS15 nucleic acids. Several methods are available to achieve site-directed mutagenesis, the most common being PCR based methods (current protocols in molecular biology. Wiley Eds.).
[0381] Directed evolution may also be used to generate variants of MADS15 nucleic acids. This consists of iterations of DNA shuffling followed by appropriate screening and/or selection to generate variants of MADS15 nucleic acids or variants thereof encoding MADS15 polypeptides having a modified (here decreased or abolished) biological activity (Castle et al., (2004) Science 304(5674): 1151-4; U.S. Pat. Nos. 5,811,238 and 6,395,547).
[0382] T-DNA activation, TILLING, site-directed mutagenesis and directed evolution are examples of technologies that enable the generation of novel alleles and MADS15 variants.
[0383] The effects of the invention may also be reproduced using homologous recombination. The nucleic acid to be targeted may be an allele encoding an inactive protein or a protein with decreased activity, used to replace the endogenous gene or may be introduced in addition to the endogenous gene, and needs to be targeted to the locus of the MADS15 gene.
[0384] Other methods, such as the use of antibodies directed to the endogenous MADS15 for inhibiting its function in planta, or interference in the signalling pathway in which MADS15 is involved, will be well known to the skilled man. Alternatively, a screening program may be set up to identify natural variants of a MADS15 gene, which variants have reduced MADS15 activity, or no MADS15 activity at all. Such natural variants may also be used in the methods of the present invention.
[0385] For optimal performance, the gene silencing techniques used for the reduction or substantial elimination of endogenous MADS15 gene expression requires the use of MADS15 nucleic acid sequences from monocotyledonous plants for transformation into monocotyledonous plants. Preferably, a MADS15 nucleic acid from any given plant species is introduced into that same species. For example, a MADS15 nucleic acid from rice (be it a full length MADS15 sequence or a fragment) is transformed into a rice plant. The MADS15 nucleic acid need not be introduced into the same plant variety.
[0386] Reference herein to a "MADS15 gene" or a "MADS15 nucleic acid" is taken to mean a polymeric form of a deoxyribonucleotide or a ribonucleotide polymer of any length, either double- or single-stranded, or analogues thereof, that have the essential characteristic of a natural ribonucleotide in that they can hybridise to nucleic acids in a manner similar to naturally occurring polynucleotides. A "MADS15 gene" or a "MADS15 nucleic acid" refers to a sufficient length of substantially contiguous nucleotides of a MADS15-encoding gene to perform gene silencing; this may be as little as 20 or fewer nucleotides. A gene encoding a (functional) protein is not a requirement for the various methods discussed above for the reduction or substantial elimination of expression of an endogenous MADS15 gene.
[0387] The methods of the invention may be performed using a sufficient length of substantially contiguous nucleotides of a MADS15 gene/nucleic acid, which may consist of 20 or fewer nucleotides, which may be from any part of the MADS15 gene/nucleic acid, such as the 5' end of the coding region that is well conserved amongst the MADS15 gene family, or encoding one of the conserved motifs described below.
[0388] MADS15 genes are well known in the art and useful in the methods of the invention are substantially contiguous nucleotides of the plant MADS15 genes/nucleic acid described in Moon et al. (Plant Physiol. 120, 1193-1204, 1999).
[0389] Other MADS15 gene/nucleic acid sequences may also be used in the methods of the invention, and may readily be identified by a person skilled in the art. MADS15 polypeptides may be identified by the presence of one or more of several well-known features (see below). Upon identification of a MADS15 polypeptide, a person skilled in the art could easily derive, using routine techniques, the corresponding encoding nucleic acid sequence and use a sufficient length of contiguous nucleotides of the same to perform any one or more of the gene silencing methods described above.
[0390] The term "MADS15 polypeptide or homologue thereof" as defined herein refers to a protein that falls in the group of FUL-like MADS box proteins as delineated by Adam et al. (J. Mol. Evol. 62, 15-31). FUL-like MADS box proteins are part of the SQUAMOSA subfamily and have the typical MIKCc architecture: a MADS domain (M) at the N-terminus, followed by an intervening domain (I) involved in dimerisation, a highly conserved keratin domain (K) and a variable domain (C) at the C-terminus, which is responsible for binding of interacting proteins or transcriptional activation (De Bodt et al. Trends in Plant Science 8, 475-483).
[0391] Plant MADS15 polypeptides may also be identified by the presence of certain conserved motifs. The presence of these conserved motifs may be identified using methods for the alignment of sequences for comparison as described hereinabove. In some instances, the default parameters may be adjusted to modify the stringency of the search. For example using BLAST, the statistical significance threshold (called "expect" value) for reporting matches against database sequences may be increased to show less stringent matches. This way, short nearly exact matches may be identified. Upon identification of a MADS15 polypeptide by the presence of these motifs, a person skilled in the art may easily derive the corresponding nucleic acid encoding the polypeptide comprising the relevant motifs, and use a sufficient length of contiguous nucleotides of the same to perform any one or more of the gene silencing methods described above (for the reduction or substantial elimination of an endogenous MADS15 gene expression).
[0392] Typically, the presence of at least one of the motifs 1 to 4 should be sufficient to identify any query sequence as a MADS15, however the presence of at least motifs 1 and 2 is preferred. The consensus sequence provided is based on the monocot sequences given in the sequence listing. A person skilled in the art would be well aware that the consensus sequence may vary somewhat if further or different sequences (for example from dicot sequences) were used for comparison.
[0393] Motif 1, located in the MADS domain (SEQ ID NO: 114):
TABLE-US-00013 L(L/V)KKA(H/N)EIS(VI)L(C/Y)DAE(V/I/L) (A/G) (L/A/V) (I/V) (I/V)FS(T/P/A/N)KGKLYE(Y/F) (A/S) (T/S) (D/N/E)S(K/C/R/S)M(D/E) (N/I/R/K)IL(E/D)R.
[0394] Preferably, this conserved sequence motif 1 has the sequence:
TABLE-US-00014 LLKKAHEISVLCDAEVA(L/A/V)I(I/V)FS(T/P)KGKLYEYATDS (C/R)M(D/E) (R/K)ILER,
most preferably the conserved sequence motif 1 has the sequence:
TABLE-US-00015 LLKKAHEISVLCDAEVAAIVFSPKGKLYEYATDSRMDKILER
[0395] Motif 2, located in the K domain (SEQ ID NO: 115):
TABLE-US-00016 KLK(A/S) (K/R) (V/I)E(A/T/S) (L/I) (Q/N) (K/R/N) (S/C/R) (Q/H) (R/K)HLMGE
[0396] Preferably, this conserved sequence motif 2 has the sequence:
TABLE-US-00017 KLKAK(V/I)E(A/T) (L/I)QK(S/C) (Q/H) (R/K)HLMGE
[0397] More preferably, this conserved sequence motif 2 has the sequence:
TABLE-US-00018 KLKAKIETIQKCHKHLMGE
[0398] Optionally, the MADS15 polypeptide or its homologue may comprise in the C domain motif 3 and/or motif 4:
TABLE-US-00019 motif 3 (SEQ ID NO: 116): Q(P/Q/V/A)QTS(S/F) (S/F) (S/F) (S/F) (S/C/F) (F/M) motif 4 (SEQ ID NO: 117): (G/A/V/L) (L/P)XWMX(S/H)
wherein the first X residue in motif 4 may be any amino acid, but preferably L, P or H; and wherein the second X residue may be any amino acid, but preferably V or L.
[0399] Alternatively, the C-domain (starting behind the keratin domain, i.e. at R175 in SEQ ID NO: 110) may be characterised by the increased occurrence of glutamine (normally 3.93%, here at least 8.77% with a maximum of 26.73%), in addition, the content in alanine, proline, and/or serine may also be increased (above 7.8%, 4.85% and 6.89% respectively).
[0400] Alternatively formulated, a preferred MADS15 protein useful in the methods of the present invention comprises at least an N-terminal MADS domain corresponding to the SMART domain SM00432 (Pfam PF00319) followed by a Keratin domain corresponding to the K-box region defined in Pfam as PF01486, more preferably, the MADS15 protein has in its MADS domain the conserved signature of SEQ ID NO: 114 and in its K-domain the conserved signature of SEQ ID NO: 115, most preferably, the MADS15 protein has the sequence given in SEQ ID NO: 110.
[0401] Homologues, as defined above, may readily be identified using routine techniques well known in the art, such as by sequence alignment; homologues of OsMADS15 may have been named differently in various plant species, therefore the gene/protein names should not be used for identifying orthologues or paralogues. Methods for the alignment of sequences for comparison are well known in the art, such methods include GAP, BESTFIT, BLAST, FASTA and TFASTA. GAP uses the algorithm of Needleman and Wunsch ((1970) J Mol Biol 48: 443-453) to find the alignment of two complete sequences that maximizes the number of matches and minimizes the number of gaps. The BLAST algorithm (Altschul et al. (1990) J Mol Biol 215: 403-10) calculates percent sequence identity and performs a statistical analysis of the similarity between the two sequences. The software for performing BLAST analysis is publicly available through the National Centre for Biotechnology Information. Homologous sequences may readily be identified using, for example, the ClustalW multiple sequence alignment algorithm (version 1.83), with the default pairwise alignment parameters, and a scoring method in percentage. Minor manual editing may be performed to optimise alignment between conserved motifs (see below), as would be apparent to a person skilled in the art.
[0402] The various structural domains in a MADS15 protein may be identified using specialised databases e.g. SMART (Schultz et al. (1998) Proc. Natl. Acad. Sci. USA 95, 5857-5864; Letunic et al. (2002) Nucleic Acids Res 30, 242-244), InterPro (Mulder et al., (2003) Nucl. Acids. Res. 31, 315-318;), Prosite (Bucher and Bairoch (1994), A generalized profile syntax for biomolecular sequences motifs and its function in automatic sequence interpretation. (In) ISMB-94; Proceedings 2nd International Conference on Intelligent Systems for Molecular Biology. Altman R., Brutlag D., Karp P., Lathrop R., Searls D., Eds., pp 53-61, AAAIPress, Menlo Park; Hulo et al., Nucl. Acids. Res. 32:D134-D137, (2004)) or Pfam (Bateman et al., Nucleic Acids Research 30(1): 276-280 (2002)).
[0403] Furthermore, a MADS15 protein may also be identifiable by its ability to bind DNA and to interact with other proteins. DNA-binding activity and protein-protein interactions may readily be determined in vitro or in vivo using techniques well known in the art. Examples of in vitro assays for DNA binding activity include: gel retardation analysis using known MADS-box DNA binding domains (West et al. (1998) Nucl Acid Res 26(23): 5277-87), or yeast one-hybrid assays. An example of an in vitro assay for protein-protein interactions is the yeast two-hybrid analysis (Fields and Song (1989) Nature 340:245-6). Proteins known to interact with OsMADS15 include other MADS proteins (such as those involved in the flowering signalling complex), receptor like kinases such a Clavata1 and Clavata2, Erecta, BR11 or RSK.
[0404] Therefore upon identification of a MADS15 polypeptide using one or several of the features described above, a person skilled in the art may easily derive the corresponding nucleic acid encoding the polypeptide, and use a sufficient length of substantially contiguous nucleotides of the same to perform any one or more of the gene silencing methods described above (for the reduction or substantial elimination of an endogenous MADS15 gene expression).
[0405] Preferred for use in the methods of the invention is a sufficient length of substantially contiguous nucleotides of SEQ ID NO: 109 (OsMADS15), or the use of a sufficient length of substantially contiguous nucleotides of a nucleic acid sequence encoding an orthologue or paralogue of OsMADS15 (SEQ ID NO: 109). Examples of such orthologues and paralogues of OsMADS15 are provided in Table D below. Close homologues of OsMADS15 are the proteins represented by SEQ ID NO: 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171 and 173.
[0406] Orthologues in, for example, monocot plant species may easily be found by performing a so-called reciprocal blast search. This may be done by a first blast involving blasting a query sequence (for example, SEQ ID NO: 109 or SEQ ID NO: 110) against any sequence database, such as the publicly available NCBI database. BLASTN or TBLASTX (using standard default values) may be used when starting from a nucleotide sequence and BLASTP or TBLASTN (using standard default values) may be used when starting from a protein sequence. The BLAST results may optionally be filtered. The full-length sequences of either the filtered results or non-filtered results are then BLASTed back (second BLAST) against sequences from the organism from which the query sequence is derived (where the query sequence is SEQ ID NO: 109 or SEQ ID NO: 110 the second blast would therefore be against rice sequences). The results of the first and second BLASTs are then compared. A paralogue is identified if a high-ranking hit from the first blast is from the same species as from which the query sequence is derived; an orthologue is identified if a high-ranking hit is not from the same species as from which the query sequence is derived. High-ranking hits are those having a low E-value. The lower the E-value, the more significant the score (or in other words the lower the chance that the hit was found by chance). Computation of the E-value is well known in the art. In the case of large families, ClustalW may be used, followed by a neighbour joining tree, to help visualize clustering of related genes and to identify orthologues and paralogues.
[0407] The source of the substantially contiguous nucleotides of a MADS15 gene/nucleic acid may be any plant source or artificial source. For optimal performance, the gene silencing techniques used for the reduction or substantial elimination of endogenous MADS15 gene expression requires the use of MADS15 sequences from monocotyledonous plants for transformation into monocotyledonous plants. Preferably, MADS15 sequences from the family Poaceae are transformed into plants of the family Poaceae. Further preferably, a MADS15 nucleic acid from rice (be it a full length MADS15 sequence or a fragment) is transformed into a rice plant. The MADS15 nucleic acid need not be introduced into the same plant variety. Most preferably, the MADS15 nucleic acid from rice is a sufficient length of substantially contiguous nucleotides of SEQ ID NO: 109 (OsMADS15) or a sufficient length of substantially contiguous nucleotides of a nucleic acid sequence encoding an orthologue or paralogue of OsMADS15 (SEQ ID NO: 110). As mentioned above, a person skilled in the art would be well aware of what would constitute a sufficient length of substantially contiguous nucleotides to perform any of the gene silencing methods defined hereinabove, this may be as little as 20 or fewer substantially contiguous nucleotides in some cases.
[0408] The invention also provides genetic constructs and vectors to facilitate introduction and/or expression of the nucleotide sequences useful in the methods according to the invention.
[0409] Therefore, there is provided a gene construct comprising one or more control sequences capable of driving expression of a sense and/or antisense MADS15 nucleic acid sequence in a plant so as to silence an endogenous MADS15 gene in the plant; and optionally a transcription termination sequence. Preferably, the control sequence is a constitutive and ubiquitous promoter.
[0410] A preferred construct for gene silencing is one comprising an inverted repeat of a MADS15 gene or fragment thereof, preferably capable of forming a hairpin structure, which inverted repeat is under the control of a constitutive promoter.
[0411] Therefore, the invention provides a construct comprising:
[0412] (a) a MADS15 nucleic acid capable of forming a hairpin structure;
[0413] (b) one or more control sequences capable of driving expression of the nucleic acid sequence of (a); and optionally
[0414] (c) a transcription termination sequence.
[0415] Constructs useful in the methods according to the present invention may be created using recombinant DNA technology well known to persons skilled in the art. The gene constructs may be inserted into vectors, which may be commercially available, suitable for transforming into plants and suitable for expression of the gene of interest in the transformed cells. The invention therefore provides use of a gene construct as defined hereinabove in the methods of the invention.
[0416] The sequence of interest is operably linked to one or more control sequences (at least to a promoter) capable of increasing expression in a plant.
[0417] Advantageously, any type of promoter may be used to drive expression of the nucleic acid sequence. The promoter may be an inducible promoter, i.e. having induced or increased transcription initiation in response to a developmental, chemical, environmental or physical stimulus. Additionally or alternatively, the promoter may be a tissue-preferred or cell-preferred promoter, i.e. one that is capable of preferentially initiating transcription in certain tissues, such as the leaves, roots, seed tissue etc, or even in specific cells. Promoters able to initiate transcription in certain tissues or cells only are referred to herein as "tissue-specific", respectively "cell-specific".
[0418] Preferably, the MADS15 nucleic acid or functional variant thereof is operably linked to a constitutive promoter. Preferably the promoter is a ubiquitous promoter and is expressed predominantly throughout the plant. Preferably, the constitutive promoter capable of preferentially expressing the nucleic acid throughout the plant has a comparable expression profile to a GOS2 promoter. More preferably, the constitutive promoter has the same expression profile as the rice GOS2 promoter, most preferably, the promoter capable of preferentially expressing the nucleic acid throughout the plant is the GOS2 promoter from rice (SEQ ID NO: 113 or SEQ ID NO: 174). It should be clear that the applicability of the present invention is not restricted to the MADS15 nucleic acid represented by SEQ ID NO: 109, nor is the applicability of the invention restricted to expression of a MADS15 nucleic acid when driven by a GOS2 promoter. An alternative constitutive promoter that is useful in the methods of the present invention is the high mobility group protein promoter (PRO0170, SEQ ID NO: 40 in WO2004070039). Examples of other constitutive promoters that may also be used to drive expression of a MADS15 nucleic acid are shown in the definitions section.
[0419] Optionally, one or more terminator sequences may also be used in the construct introduced into a plant. Additional regulatory elements may include transcriptional as well as translational enhancers. Those skilled in the art will be aware of terminator and enhancer sequences that may be suitable for use in performing the invention. Such sequences would be known or may readily be obtained by a person skilled in the art.
[0420] The genetic constructs of the invention may further include an origin of replication sequence that is required for maintenance and/or replication in a specific cell type. One example is when a genetic construct is required to be maintained in a bacterial cell as an episomal genetic element (e.g. plasmid or cosmid molecule). Preferred origins of replication include, but are not limited to, the f1-ori and colE1. The genetic construct may optionally comprise a selectable marker gene.
[0421] The present invention also encompasses plants including plant parts obtainable by the methods according to the present invention having increased yield relative to control plants and which have reduced or substantially eliminated expression of an endogenous MADS15 gene.
[0422] The invention furthermore provides a method for the production of transgenic plants having increased yield relative to control plants, which transgenic plants have reduced or substantially eliminated expression of an endogenous MADS15 gene.
[0423] More specifically, the present invention provides a method for the production of transgenic plants having increased seed yield which method comprises:
[0424] introducing and expressing in a plant, plant part or plant cell a gene construct comprising one or more control sequences capable of preferentially driving expression of an inverted repeat MADS15 nucleic acid sequence in a plant so as to silence an endogenous MADS15 gene in the plant; and
[0425] cultivating the plant, plant part or plant cell under conditions promoting plant growth and development.
[0426] Preferably, the construct introduced into a plant is one comprising an inverted repeat (in part or complete) of a MADS15 gene or fragment thereof, preferably capable of forming a hairpin structure.
[0427] According to a preferred feature of the present invention, the construct is introduced into a plant by transformation.
[0428] Generally after transformation, plant cells or cell groupings are selected for the presence of one or more markers which are encoded by plant-expressible genes co-transferred with the gene of interest, following which the transformed material is regenerated into a whole plant.
[0429] Following DNA transfer and regeneration, putatively transformed plants may be evaluated, for instance using Southern analysis, for the presence of the gene of interest, copy number and/or genomic organisation. Alternatively or additionally, expression levels of the newly introduced DNA may be monitored using Northern and/or Western analysis, or quantitative PCR, all techniques being well known to persons having ordinary skill in the art.
[0430] The generated transformed plants may be propagated by a variety of means, such as by clonal propagation or classical breeding techniques. For example, a first generation (or T1) transformed plant may be selfed to give homozygous second generation (or T2) transformants, and the T2 plants further propagated through classical breeding techniques.
[0431] The generated transformed organisms may take a variety of forms. For example, they may be chimeras of transformed cells and non-transformed cells; clonal transformants (e.g., all cells transformed to contain the expression cassette); grafts of transformed and untransformed tissues (e.g., in plants, a transformed rootstock grafted to an untransformed scion).
[0432] The present invention clearly extends to any plant cell or plant produced by any of the methods described herein, and to all plant parts and propagules thereof. The present invention extends further to encompass the progeny of a primary transformed or transfected cell, tissue, organ or whole plant that has been produced by any of the aforementioned methods, the only requirement being that progeny exhibit the same genotypic and/or phenotypic characteristic(s) as those produced by the parent in the methods according to the invention.
[0433] The invention also extends to harvestable parts of a plant such as seeds and products derived, preferably directly derived, from a harvestable part of such a plant, such as dry pellets or powders, oil, fat and fatty acids, starch or proteins.
[0434] The present invention also encompasses use of MADS15 nucleic acids for the reduction or substantial elimination of endogenous MADS15 gene expression in a plant for increasing plant seed yield as defined hereinabove.
[0435] Nucleic acids encoding the MADS15 protein described herein, or the MADS15 proteins themselves, may find use in breeding programmes in which a DNA marker is identified which may be genetically linked to a MADS15-encoding gene. The nucleic acids/genes, or the MADS15 proteins themselves may be used to define a molecular marker. This DNA or protein marker may then be used in breeding programmes to select plants having enhanced yield-related traits as defined hereinabove in the methods of the invention.
[0436] Allelic variants of a MADS15 protein-encoding nucleic acid/gene may also find use in marker-assisted breeding programmes. Such breeding programmes sometimes require introduction of allelic variation by mutagenic treatment of the plants, using for example EMS mutagenesis; alternatively, the programme may start with a collection of allelic variants of so called "natural" origin caused unintentionally. Identification of allelic variants then takes place, for example, by PCR. This is followed by a step for selection of superior allelic variants of the sequence in question and which give increased yield. Selection is typically carried out by monitoring growth performance of plants containing different allelic variants of the sequence in question. Growth performance may be monitored in a greenhouse or in the field. Further optional steps include crossing plants in which the superior allelic variant was identified with another plant. This could be used, for example, to make a combination of interesting phenotypic features.
[0437] Nucleic acids encoding MADS15 proteins may also be used as probes for genetically and physically mapping the genes that they are a part of, and as markers for traits linked to those genes. Such information may be useful in plant breeding in order to develop lines with desired phenotypes. Such use of MADS15 protein-encoding nucleic acids requires only a nucleic acid sequence of at least 15 nucleotides in length. The MADS15 protein-encoding nucleic acids may be used as restriction fragment length polymorphism (RFLP) markers. Southern blots (Sambrook J, Fritsch E F and Maniatis T (1989) Molecular Cloning, A Laboratory Manual) of restriction-digested plant genomic DNA may be probed with the MADS15 protein-encoding nucleic acids. The resulting banding patterns may then be subjected to genetic analyses using computer programs such as MapMaker (Lander et al. (1987) Genomics 1: 174-181) in order to construct a genetic map. In addition, the nucleic acids may be used to probe Southern blots containing restriction endonuclease-treated genomic DNAs of a set of individuals representing parent and progeny of a defined genetic cross. Segregation of the DNA polymorphisms is noted and used to calculate the position of the MADS15 protein-encoding nucleic acid in the genetic map previously obtained using this population (Botstein et al. (1980) Am. J. Hum. Genet. 32:314-331).
[0438] The production and use of plant gene-derived probes for use in genetic mapping is described in Bernatzky and Tanksley (1986) Plant Mol. Biol. Reporter 4: 37-41. Numerous publications describe genetic mapping of specific cDNA clones using the methodology outlined above or variations thereof. For example, F2 intercross populations, backcross populations, randomly mated populations, near isogenic lines, and other sets of individuals may be used for mapping. Such methodologies are well known to those skilled in the art.
[0439] The nucleic acid probes may also be used for physical mapping (i.e., placement of sequences on physical maps; see Hoheisel et al. In: Non-mammalian Genomic Analysis: A Practical Guide, Academic press 1996, pp. 319-346, and references cited therein).
[0440] In another embodiment, the nucleic acid probes may be used in direct fluorescence in situ hybridisation (FISH) mapping (Trask (1991) Trends Genet. 7:149-154). Although current methods of FISH mapping favour use of large clones (several kb to several hundred kb; see Laan et al. (1995) Genome Res. 5:13-20), improvements in sensitivity may allow performance of FISH mapping using shorter probes.
[0441] A variety of nucleic acid amplification-based methods for genetic and physical mapping may be carried out using the nucleic acids. Examples include allele-specific amplification (Kazazian (1989) J. Lab. Clin. Med. 11:95-96), polymorphism of PCR-amplified fragments (CAPS; Sheffield et al. (1993) Genomics 16:325-332), allele-specific ligation (Landegren et al. (1988) Science 241:1077-1080), nucleotide extension reactions (Sokolov (1990) Nucleic Acid Res. 18:3671), Radiation Hybrid Mapping (Walter et al. (1997) Nat. Genet. 7:22-28) and Happy Mapping (Dear and Cook (1989) Nucleic Acid Res. 17:6795-6807). For these methods, the sequence of a nucleic acid is used to design and produce primer pairs for use in the amplification reaction or in primer extension reactions. The design of such primers is well known to those skilled in the art. In methods employing PCR-based genetic mapping, it may be necessary to identify DNA sequence differences between the parents of the mapping cross in the region corresponding to the instant nucleic acid sequence. This, however, is generally not necessary for mapping methods.
[0442] The methods according to the present invention result in plants having enhanced yield-related traits, as described hereinbefore. These traits may also be combined with other economically advantageous traits, such as further yield-enhancing traits, tolerance to other abiotic and biotic stresses, traits modifying various architectural features and/or biochemical and/or physiological features.
Detailed Description of PLT
[0443] Surprisingly, it has now been found that increasing expression of a nucleic acid encoding a PLT transcription factor polypeptide gives plants having enhanced yield-related traits, in particular increased yield relative to control plants.
[0444] According to the present invention, there is provided a method for increasing plant yield relative to control plants, comprising increasing expression in a plant of a nucleic acid encoding a PLT transcription factor polypeptide.
[0445] The term "increased yield" as defined herein is taken to mean an increase in biomass (weight) of one or more parts of a plant, which may include aboveground (harvestable) parts and/or (harvestable) parts below ground.
[0446] In particular, such harvestable parts are seeds, and performance of the methods of the invention results in plants having increased seed yield relative to the seed yield of control plants.
[0447] Taking corn as an example, a yield increase may be manifested as one or more of the following: increase in the number of plants per hectare or acre, an increase in the number of ears per plant, an increase in the number of rows, number of kernels per row, kernel weight, thousand kernel weight, ear length/diameter, increase in the seed filling rate (which is the number of filled seeds divided by the total number of seeds and multiplied by 100), among others. Taking rice as an example, a yield increase may manifest itself as an increase in one or more of the following: number of plants per hectare or acre, number of panicles per plant, number of spikelets per panicle, number of flowers (florets) per panicle (which is expressed as a ratio of the number of filled seeds over the number of primary panicles), increase in the seed filling rate (which is the number of filled seeds divided by the total number of seeds and multiplied by 100), increase in thousand kernel weight, among others.
[0448] According to a preferred feature of the present invention, performance of the methods of the invention gives plants having increased seed yield relative to control plants. Therefore according to the present invention, there is provided a method for increasing seed yield in plants relative to the seed yield of control plants, the method comprising increasing expression in a plant of a nucleic acid encoding a PLT transcription factor polypeptide.
[0449] Since the transgenic plants according to the present invention have increased yield, it is likely that these plants exhibit an increased growth rate (during at least part of their life cycle), relative to the growth rate of corresponding wild type plants at a corresponding stage in their life cycle. The increased growth rate may be specific to one or more parts of a plant (including seeds), or may be throughout substantially the whole plant. A plant having an increased growth rate may even exhibit early flowering. The increase in growth rate may take place at one or more stages in the life cycle of a plant or during substantially the whole plant life cycle. Increased growth rate during the early stages in the life cycle of a plant may reflect enhanced vigour (increased seedling vigor at emergence). The increase in growth rate may alter the harvest cycle of a plant allowing plants to be sown later and/or harvested sooner than would otherwise be possible. If the growth rate is sufficiently increased, it may allow for the further sowing of seeds of the same plant species (for example sowing and harvesting of rice plants followed by sowing and harvesting of further rice plants all within one conventional growing period). Similarly, if the growth rate is sufficiently increased, it may allow for the further sowing of seeds of different plants species (for example the sowing and harvesting of corn plants followed by, for example, the sowing and optional harvesting of soy bean, potato or any other suitable plant). Harvesting additional times from the same rootstock in the case of some crop plants may also be possible. Altering the harvest cycle of a plant may lead to an increase in annual biomass production per acre (due to an increase in the number of times (say in a year) that any particular plant may be grown and harvested). An increase in growth rate may also allow for the cultivation of transgenic plants in a wider geographical area than their wild-type counterparts, since the territorial limitations for growing a crop are often determined by adverse environmental conditions either at the time of planting (early season) or at the time of harvesting (late season). Such adverse conditions may be avoided if the harvest cycle is shortened. The growth rate may be determined by deriving various parameters from growth curves, such parameters may be: T-Mid (the time taken for plants to reach 50% of their maximal size) and T-90 (time taken for plants to reach 90% of their maximal size), amongst others.
[0450] Performance of the methods of the invention gives plants having an increased growth rate. Therefore, according to the present invention, there is provided a method for increasing the growth rate of plants, which method comprises increasing expression in a plant of a nucleic acid encoding a PLT transcription factor polypeptide.
[0451] An increase in yield and/or growth rate occurs whether the plant is under non-stress conditions or whether the plant is exposed to various stresses compared to control plants. Plants typically respond to exposure to stress by growing more slowly. In conditions of severe stress, the plant may even stop growing altogether. Mild stress on the other hand is defined herein as being any stress to which a plant is exposed which does not result in the plant ceasing to grow altogether without the capacity to resume growth. Mild stress in the sense of the invention leads to a reduction in the growth of the stressed plants of less than 40%, 35% or 30%, preferably less than 25%, 20% or 15%, more preferably less than 14%, 13%, 12%, 11% or 10% or less in comparison to the control plant under non-stress conditions. Due to advances in agricultural practices (irrigation, fertilization, pesticide treatments) severe stresses are not often encountered in cultivated crop plants. As a consequence, the compromised growth induced by mild stress is often an undesirable feature for agriculture. Mild stresses are the everyday biotic and/or abiotic (environmental) stresses to which a plant is exposed. Abiotic stresses may be due to drought or excess water, anaerobic stress, salt stress, chemical toxicity, oxidative stress and hot, cold or freezing temperatures. The abiotic stress may be an osmotic stress caused by a water stress (particularly due to drought), salt stress, oxidative stress or an ionic stress. Biotic stresses are typically those stresses caused by pathogens, such as bacteria, viruses, fungi and insects.
[0452] In particular, the methods of the present invention may be performed under non-stress conditions or under conditions of mild drought to give plants having increased yield relative to control plants. As reported in Wang et al. (Planta (2003) 218: 1-14), abiotic stress leads to a series of morphological, physiological, biochemical and molecular changes that adversely affect plant growth and productivity. Drought, salinity, extreme temperatures and oxidative stress are known to be interconnected and may induce growth and cellular damage through similar mechanisms. Rabbani et al. (Plant Physiol (2003) 133: 1755-1767) describes a particularly high degree of "cross talk" between drought stress and high-salinity stress. For example, drought and/or salinisation are manifested primarily as osmotic stress, resulting in the disruption of homeostasis and ion distribution in the cell. Oxidative stress, which frequently accompanies high or low temperature, salinity or drought stress, may cause denaturing of functional and structural proteins. As a consequence, these diverse environmental stresses often activate similar cell signaling pathways and cellular responses, such as the production of stress proteins, up-regulation of anti-oxidants, accumulation of compatible solutes and growth arrest. The term "non-stress" conditions as used herein are those environmental conditions that allow optimal growth of plants. Persons skilled in the art are aware of normal soil conditions and climatic conditions for a given location.
[0453] Performance of the methods of the invention gives plants grown under non-stress conditions or under mild drought conditions enhanced yield-related traits relative to control plants grown under comparable conditions. Therefore, according to the present invention, there is provided a method for enhancing yield-related traits, in particular for increasing yield, in plants grown under non-stress conditions or under mild drought conditions, which method comprises increasing expression in a plant of a nucleic acid encoding a PLT transcription factor polypeptide.
[0454] The methods of the invention are advantageously applicable to any plant.
[0455] Plants that are particularly useful in the methods of the invention include all plants which belong to the superfamily Viridiplantae, in particular monocotyledonous and dicotyledonous plants including fodder or forage legumes, ornamental plants, food crops, trees or shrubs. According to a preferred embodiment of the present invention, the plant is a crop plant. Examples of crop plants include soybean, sunflower, canola, alfalfa, rapeseed, cotton, tomato, potato and tobacco. Examples of typical crop plants grown for oil production include soybean, sunflower, cotton, canola, peanuts or palm. Examples of typical crop plants grown for starch production include rice, wheat, barley, corn or potato. Further preferably, the plant is a monocotyledonous plant. Examples of monocotyledonous plants include sugarcane. More preferably the plant is a cereal. Examples of cereals include rice, maize, wheat, barley, millet, rye, triticale, sorghum and oats.
[0456] The term "PLT transcription factor polypeptide" as defined herein refers to any polypeptide comprising in increasing order of preference at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the AP2 domain represented by SEQ ID NO: 191.
[0457] Preferably the PLT transcription factor polypeptide of choice is a PLT transcription factor polypeptide that in its natural genetic environment is essentially expressed in the roots (below ground parts) of the plant.
[0458] Further preferably, the PLT transcription factor polypeptide comprises either one motif but preferably both of motif1 as represented by SEQ ID NO: 192 PK(V/L)(A/E)DFLG and motif 2 as represented by SEQ ID NO: 209: (V/L)FX(M/V)WN(D/E), wherein X may be any amino acid; preferably motif 2 has the sequence of SEQ ID NO: 193 (V/L)F(T/S/N)(M/V)WN(D/E).
[0459] Examples of PLT transcription factor polypeptides comprising (i) in increasing order of preference at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the AP2 domain represented by SEQ ID NO: 191 are proteins represented by the sequences given in Table I in the examples section. Preferred examples are represented by SEQ ID NO: 176 and SEQ ID NO: 178.
[0460] The AP2 domain in a PLT transcription factor polypeptide may be identified using specialised databases e.g. SMART (Schultz et al. (1998) Proc. Natl. Acad. Sci. USA 95, 5857-5864; Letunic et al. (2002) Nucleic Acids Res 30, 242-244), InterPro (Mulder et al., (2003) Nucl. Acids. Res. 31, 315-318), Prosite (Bucher and Bairoch (1994), A generalized profile syntax for biomolecular sequences motifs and its function in automatic sequence interpretation. (In) ISMB-94; Proceedings 2nd International Conference on Intelligent Systems for Molecular Biology. Altman R., Brutlag D., Karp P., Lathrop R., Searls D., Eds., pp 53-61, AAAIPress, Menlo Park; Hulo et al., Nucl. Acids. Res. 32:D134-D137, (2004)) or Pfam (Bateman et al., Nucleic Acids Research 30(1): 276-280 (2002)). The AP2 domain of a PLT transcription factor comprises two repeats R1 and R2, each of about 68 amino acids and separated by a linker region (FIG. 13).
[0461] The AP2 domain as represented by SEQ ID NO: 191 may be identified using methods for the alignment of sequences for comparison as described hereinabove. In some instances, the default parameters may be adjusted to modify the stringency of the search. For example using BLAST, the statistical significance threshold (called "expect" value) for reporting matches against database sequences may be increased to show less stringent matches. This way, short nearly exact matches may be identified, including motif1 as represented by SEQ ID NO: 192 and motif 2 as represented by SEQ ID NO: 209, preferably as represented by SEQ ID NO: 193.
[0462] Homologues of a PLT transcription factor polypeptide may also be used to perform the methods of invention. Homologues (or homologous proteins) may readily be identified using routine techniques well known in the art, such as by sequence alignment. Methods for the alignment of sequences for comparison are well known in the art, such methods include GAP, BESTFIT, BLAST, FASTA and TFASTA. GAP uses the algorithm of Needleman and Wunsch ((1970) J Mol Biol 48: 443-453) to find the alignment of two complete sequences that maximizes the number of matches and minimizes the number of gaps. The BLAST algorithm (Altschul et al. (1990) J Mol Biol 215: 403-10) calculates percent sequence identity and performs a statistical analysis of the similarity between the two sequences. The software for performing BLAST analysis is publicly available through the National Centre for Biotechnology Information. Homologues may readily be identified using, for example, the ClustalW multiple sequence alignment algorithm (version 1.83), with the default pairwise alignment parameters, and a scoring method in percentage. Minor manual editing may be performed to optimise alignment between conserved motifs, as would be apparent to a person skilled in the art.
[0463] Homologues also include orthologues and paralogues. Orthologues and paralogues may easily be found by performing a so-called reciprocal blast search. This may be done by a first BLAST involving BLASTing a query sequence (for example, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177 or SEQ ID NO: 178) against any sequence database, such as the publicly available NCBI database. BLASTN or TBLASTX (using standard default values) may be used when starting from a nucleotide sequence and BLASTP or TBLASTN (using standard default values) may be used when starting from a protein sequence. The BLAST results may optionally be filtered. The full-length sequences of either the filtered results or non-filtered results are then BLASTed back (second BLAST) against sequences from the organism from which the query sequence is derived (where the query sequence is SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177 or SEQ ID NO: 178, the second BLAST would therefore be against Arabidopsis thaliana sequences). The results of the first and second BLASTs are then compared. A paralogue is identified if a high-ranking hit from the first BLAST is from the same species as from which the query sequence is derived; an orthologue is identified if a high-ranking hit is not from the same species as from which the query sequence is derived. High-ranking hits are those having a low E-value. The lower the E-value, the more significant the score (or in other words the lower the chance that the hit was found by chance). Computation of the E-value is well known in the art. In the case of large families, ClustalW may be used, followed by a neighbour joining tree, to help visualize clustering of related genes and to identify orthologues and paralogues. In addition to E-values, comparisons are also scored by percentage identity. Percentage identity refers to the number of identical nucleotides (or amino acids) between the two compared nucleic acid (or polypeptide) sequences over a particular length. PLT transcription factor polypeptide homologues have in increasing order of preference at least 25%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more sequence identity or similarity (functional identity) to an unmodified PLT transcription factor polypeptide as represented by SEQ ID NO: 176 or SEQ ID NO: 178. Percentage identity between PLT transcription factor polypeptide homologues outside of the AP2 domain is reputedly low. Preferably, PLT transcription factor polypeptide homologues comprise an AP2 domain with in increasing order of preference at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the AP2 domain represented by SEQ ID NO: 191. Further preferably, PLT transcription factor polypeptide homologues are as represented by SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO: 184, SEQ ID NO: 186, SEQ ID NO: 188, SEQ ID NO: 200 and SEQ ID NO: 202.
[0464] The PLT polypeptide may be a derivative of SEQ ID NO: 176 or SEQ ID NO: 178. "Derivatives" include peptides, oligopeptides, polypeptides which may, compared to the amino acid sequence of the naturally-occurring form of the protein, such as the one presented in SEQ ID NO: 176 or SEQ ID NO: 178, comprise substitutions of amino acids with non-naturally occurring amino acid residues, or additions of non-naturally occurring amino acid residues. Derivatives of SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO: 184, SEQ ID NO: 186, SEQ ID NO: 188, SEQ ID NO: 200 and SEQ ID NO: 202 are further examples which may be suitable for use in the methods of the invention provided that they have the same or similar biological activity.
[0465] Furthermore, PLT transcription factor polypeptides (at least in their native form) typically have DNA-binding activity and an activation domain. A person skilled in the art may easily determine the presence of an activation domain and DNA-binding activity using routine techniques and procedures. Proteins interacting with PLT transcription factor polypeptides (as, for example, in transcriptional complexes) may easily be identified using standard techniques for a person skilled in the art.
[0466] Examples of nucleic acids encoding PLT transcription factor polypeptides include but are not limited to those represented by any one of: SEQ ID NO: 175, SEQ ID NO: 177, SEQ ID NO: 179, SEQ ID NO: 181, SEQ ID NO: 183, SEQ ID NO: 185, SEQ ID NO: 187, SEQ ID NO: 199 and SEQ ID NO: 201. Variants of nucleic acids encoding PLT transcription factor polypeptides may be suitable for use in the methods of the invention provided that they have the same or similar biological activity. Suitable variants include portions of nucleic acids encoding PLT transcription factor polypeptides and/or nucleic acids capable of hybridising with nucleic acids/genes encoding PLT transcription factor polypeptides. Further variants include splice variants and allelic variants of nucleic acids encoding PLT transcription factor polypeptides.
[0467] The term "portion" as defined herein refers to a piece of DNA encoding a polypeptide comprising in increasing order of preference at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the AP2 domain represented by SEQ ID NO: 191. The portion may further comprise either one motif but preferably both of motif1 as represented by SEQ ID NO: 192 and/or motif 2 as represented by SEQ ID NO: 209; preferably, motif 2 has the sequences as represented by SEQ ID NO: 193.
[0468] A portion may be prepared, for example, by making one or more deletions to a nucleic acid encoding a PLT transcription factor polypeptide. The portions may be used in isolated form or they may be fused to other coding (or non coding) sequences in order to, for example, produce a protein that combines several activities. When fused to other coding sequences, the resulting polypeptide produced upon translation may be bigger than that predicted for the PLT transcription factor portion. Preferably, the portion codes for a polypeptide with substantially the same biological activity as the PLT transcription factor polypeptides of SEQ ID NO: 176 and SEQ ID NO: 178.
[0469] Preferably, the portion is a portion of a nucleic acid as represented by any one of the sequences listed in Table I of the Examples. Most preferably the portion is a portion of a nucleic acid as represented by SEQ ID NO: 175 and SEQ ID NO: 177.
[0470] Another variant of a nucleic acid encoding a PLT transcription factor polypeptide, useful in the methods of the present invention, is a nucleic acid capable of hybridising under reduced stringency conditions, preferably under stringent conditions, with a probe derived from the nucleic acid as defined hereinbefore, which hybridising sequence encodes a polypeptide comprising in increasing order of preference at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the AP2 domain represented by SEQ ID NO: 191. The hybridizing sequence may further comprise either one motif but preferably both of motif1 as represented by SEQ ID NO: 192 and/or motif 2 as represented by SEQ ID NO: 209, preferably as represented by SEQ ID NO: 193.
[0471] Preferably, the hybridising sequence is one that is capable of hybridising to a nucleic acid as represented by (or to probes derived from) any one of the sequences listed in Table I of the Examples, or to a portion of any of the aforementioned sequences (the target sequence). Most preferably the hybridising sequence is capable of hybridising to SEQ ID NO: 175 or to SEQ ID NO: 177 (or to probes derived therefrom). Probes are generally less than 1000 bp in length, preferably less than 500 bp in length. Commonly, probe lengths for DNA-DNA hybridisations such as Southern blotting, vary between 100 and 500 bp, whereas the hybridising region in probes for DNA-DNA hybridisations such as in PCR amplification generally are shorter than 50 but longer than 10 nucleotides.
[0472] The PLT transcription factor polypeptide may be encoded by a splice variant. The term "splice variant" as used herein encompasses variants of a nucleic acid sequence in which selected introns and/or exons have been excised, replaced, displaced or added, or in which introns have been shortened or lengthened. Such variants will be ones in which the substantial biological activity of the protein is retained, which may be achieved by selectively retaining functional segments of the protein. Such splice variants may be found in nature or may be manmade. Methods for making such splice variants are well known in the art.
[0473] Preferred splice variants are splice variants of the nucleic acid encoding a PLT transcription factor polypeptide comprising in increasing order of preference at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the AP2 domain represented by SEQ ID NO: 191. Splice variants may further comprise either one motif but preferably both of motif1 as represented by SEQ ID NO: 192 and/or motif 2 as represented by SEQ ID NO: 209, preferably as represented by SEQ ID NO: 193.
[0474] Further preferred splice variants of nucleic acids encoding PLT transcription factor polypeptides comprising features as defined hereinabove are splice variants of a nucleic acid as represented by any one of the sequences listed in Table I of the Examples. Most preferred is a splice variant of a nucleic acid sequence as represented by SEQ ID NO: 175 and SEQ ID NO: 177.
[0475] The PLT transcription factor polypeptide may also be encoded by an allelic variant of a nucleic acid encoding a polypeptide comprising from comprising in increasing order of preference at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the AP2 domain represented by SEQ ID NO: 191. The allelic variant may further comprise either one motif but preferably both of motif1 as represented by SEQ ID NO: 192 and/or motif 2 as represented by SEQ ID NO: 209, preferably as represented by SEQ ID NO: 193.
[0476] Preferred allelic variants of nucleic acids encoding PLT transcription factor polypeptides comprising features as defined hereinabove are splice variants of a nucleic acid as represented by any one of the sequences listed in Table I of the Examples. Most preferred is an allelic variant of a nucleic acid sequence as represented by SEQ ID NO: 175 and SEQ ID NO: 177.
[0477] The increase in expression of a nucleic acid encoding a PLT transcription factor polypeptide leads to raised corresponding mRNA or polypeptide levels, which could equate to raised activity of the PLT transcription factor polypeptide; or the activity may also be raised when there is no change in polypeptide levels, or even when there is a reduction in polypeptide levels. This may occur when the intrinsic properties of the polypeptide are altered, for example, by making mutant versions that are more active that the wild type polypeptide.
[0478] Directed evolution (or gene shuffling) may be used to generate variants of nucleic acids encoding PLT transcription factor polypeptides. This consists of iterations of DNA shuffling followed by appropriate screening and/or selection to generate variants of nucleic acids or portions thereof encoding PLT transcription factor polypeptides or homologues or portions thereof having an modified biological activity (Castle et al., (2004) Science 304(5674): 1151-4; U.S. Pat. Nos. 5,811,238 and 6,395,547).
[0479] Site-directed mutagenesis may be used to generate variants of nucleic acids encoding PLT transcription factor polypeptides. Several methods are available to achieve site-directed mutagenesis, the most common being PCR based methods (current protocols in molecular biology. Wiley Eds.).
[0480] Directed evolution and site-directed mutagenesis are examples of technologies that enable the generation of variants of nucleic acids encoding PLT transcription factor polypeptides with modified activity useful to perform the methods of the invention.
[0481] Nucleic acids encoding PLT transcription factor polypeptides may be derived from any natural or artificial source. The nucleic acid may be modified from its native form in composition and/or genomic environment through deliberate human manipulation. Preferably the PLT transcription factor nucleic acid is from a plant, preferably from a dicotyledonous plant, further preferably from the Brassicaceae family, more preferably from the Arabidopsis genus, most preferably the nucleic acid is from Arabidopsis thaliana.
[0482] The methods of the invention rely on increased expression of a nucleic acid encoding a PLT transcription factor polypeptide in a plant. The nucleic acid may be a full-length nucleic acid or may be a portion or a hybridising sequence or another nucleic acid variant as hereinbefore defined.
[0483] The methods of the invention rely on increased expression of a nucleic acid encoding a PLT transcription factor polypeptide in a plant.
[0484] The invention also provides genetic constructs and vectors to facilitate introduction and/or expression in a plant of the nucleic acid sequences useful in the methods according to the invention.
[0485] Therefore, there is provided a gene construct comprising:
[0486] (i) A nucleic acid encoding a PLT transcription factor polypeptide as defined hereinabove;
[0487] (ii) One or more control sequences, of which at least one is a medium strength constitutive promoter.
[0488] Constructs useful in the methods according to the present invention may be constructed using recombinant DNA technology well known to persons skilled in the art. The gene constructs may be inserted into vectors, which may be commercially available, suitable for transforming into plants and suitable for expression of the gene of interest in the transformed cells. The invention therefore provides use of a gene construct as defined hereinabove in the methods of the invention.
[0489] Plants are transformed with a vector comprising the sequence of interest (i.e., a nucleic acid encoding a PLT transcription factor polypeptide). The skilled artisan is well aware of the genetic elements that must be present on the vector in order to successfully transform, select and propagate host cells containing the sequence of interest. The sequence of interest is operably linked to one or more control sequences (at least to a promoter).
[0490] The nucleic acid encoding a PLT transcription factor polypeptide or variant thereof is operably linked to a constitutive promoter, preferably a medium strength constitutive promoter. A constitutive promoter is transcriptionally active during most, but not necessarily all, phases of its growth and development and is substantially ubiquitously expressed at moderate levels. Promoter strength and/or expression pattern can be analysed as described in the definitions section. The promoter strength and/or expression pattern can for example be compared to that of a well-characterised shoot preferred reference promoter, such as the Cab27 promoter (weak expression, GenBank AP004700) or the putative protochlorophyllid reductase promoter (strong expression, GenBank AL606456). Preferably the promoter used in the methods of the present invention is derived from a plant, further preferably a monocotyledonous plant. Most preferred is use of a GOS2 promoter (from rice) (SEQ ID NO: 194 or alternatively SEQ ID NO: 210). It should be clear that the applicability of the present invention is not restricted to the nucleic acid represented by SEQ ID NO: 175 or SEQ ID NO: 177, nor is the applicability of the invention restricted to expression of a nucleic acid encoding a PLT transcription factor polypeptide when driven by a GOS2 promoter. Examples of other constitutive promoters that may also be used to drive expression of a nucleic acid encoding a PLT transcription factor polypeptide are shown in the definitions section.
[0491] Optionally, one or more terminator sequences (also a control sequence) may be used in the construct introduced into a plant. Additional regulatory elements may include transcriptional as well as translational enhancers. Those skilled in the art will be aware of terminator and enhancer sequences that may be suitable for use in performing the invention. Other control sequences (besides promoter, enhancer, silencer, intron sequences, 3'UTR and/or 5'UTR regions) may be protein and/or RNA stabilizing elements. Such sequences would be known or may readily be obtained by a person skilled in the art.
[0492] The genetic constructs of the invention may further include an origin of replication sequence that is required for maintenance and/or replication in a specific cell type. One example is when a genetic construct is required to be maintained in a bacterial cell as an episomal genetic element (e.g. plasmid or cosmid molecule). Preferred origins of replication include, but are not limited to, the f1-ori and colE1. The genetic construct may optionally comprise a selectable marker gene.
[0493] The present invention also encompasses plants obtainable by the methods according to the present invention. The present invention therefore provides plants, plant parts or plant cells thereof obtainable by the method according to the present invention, which plants or parts or cells thereof comprise a nucleic acid transgene encoding a PLT transcription factor polypeptide under the control of a constitutive promoter, preferably a non-viral constitutive promoter.
[0494] The invention also provides a method for the production of transgenic plants having increased yield relative to control plants, comprising introduction and preferential expression of a nucleic acid encoding a PLT transcription factor polypeptide in a plant.
[0495] More specifically, the present invention provides a method for the production of transgenic plants having increased yield which method comprises:
[0496] (i) introducing and expressing a nucleic acid encoding a PLT transcription factor polypeptide in a plant; and
[0497] (ii) cultivating the plant cell under conditions promoting plant growth and development.
[0498] The nucleic acid may be introduced directly into a plant cell or into the plant itself (including introduction into a tissue, organ or any other part of a plant). According to a preferred feature of the present invention, the nucleic acid is preferably introduced into a plant by transformation.
[0499] Generally after transformation, plant cells or cell groupings are selected for the presence of one or more markers which are encoded by plant-expressible genes co-transferred with the gene of interest, following which the transformed material is regenerated into a whole plant. Following DNA transfer and regeneration, putatively transformed plants may be evaluated, for instance using Southern analysis, for the presence of the gene of interest, copy number and/or genomic organisation. Alternatively or additionally, expression levels of the newly introduced DNA may be monitored using Northern and/or Western analysis, or quantitative PCR, all techniques being well known to persons having ordinary skill in the art.
[0500] The generated transformed plants may be propagated by a variety of means, such as by clonal propagation or classical breeding techniques. For example, a first generation (or T1) transformed plant may be selfed to give homozygous second generation (or T2) transformants, and the T2 plants further propagated through classical breeding techniques.
[0501] The generated transformed organisms may take a variety of forms. For example, they may be chimeras of transformed cells and non-transformed cells; clonal transformants (e.g., all cells transformed to contain the expression cassette); grafts of transformed and untransformed tissues (e.g., in plants, a transformed rootstock grafted to an untransformed scion).
[0502] The present invention clearly extends to any plant cell or plant produced by any of the methods described herein, and to all plant parts and propagules thereof. The present invention extends further to encompass the progeny of a primary transformed or transfected cell, tissue, organ or whole plant that has been produced by any of the aforementioned methods, the only requirement being that progeny exhibit the same genotypic and/or phenotypic characteristic(s) as those produced by the parent in the methods according to the invention.
[0503] The invention also includes host cells containing an isolated nucleic acid encoding a PLT transcription factor polypeptide. Preferred host cells according to the invention are plant cells.
[0504] The invention furthermore extends to harvestable parts of a plant such as, but not limited to seeds, leaves, fruits, flowers, stems, rhizomes, tubers and bulbs. The invention furthermore relates to products derived, preferably directly derived, from a harvestable part of such a plant, such as dry pellets or powders, oil, fat and fatty acids, starch or proteins.
[0505] The present invention also encompasses use of nucleic acids encoding PLT transcription factor polypeptides and use of PLT transcription factor polypeptides in increasing plant yield as defined hereinabove in the methods of the invention.
[0506] The increased expression of a nucleic acid encoding a PLT transcription factor polypeptide may be performed by introducing a genetic modification (preferably in the locus of a PLT transcription factor gene). The locus of a gene as defined herein is taken to mean a genomic region, which includes the gene of interest and 10 KB up- or downstream of the coding region.
[0507] The genetic modification may be introduced by alternative methods as the one described hereinabove, for example, by any one (or more) of the following: T-DNA activation, TILLING and homologous recombination. Following introduction of the genetic modification, there follows an optional step of selecting for increased expression of a nucleic acid encoding a PLT transcription factor polypeptide, which increased expression gives plants having increased yield.
[0508] T-DNA activation tagging results in transgenic plants that show dominant phenotypes due to modified expression of genes close to the introduced promoter. The promoter to be introduced a medium stength constitutive promoter capable of increasing expression of the nucleic acid encoding a PLT transcription factor polypeptide in a plant.
[0509] A genetic modification may also be introduced in the locus of a gene encoding a PLT transcription factor polypeptide using the technique of TILLING (Targeted Induced Local Lesions In Genomes).
[0510] The effects of the invention may also be reproduced using homologous recombination. The nucleic acid to be introduced (which may be a nucleic acid encoding a PLT transcription factor polypeptide or variant thereof as hereinbefore defined) is targeted to the locus of a PLT gene.
[0511] The nucleic acid to be targeted may be an improved allele used to replace the endogenous gene or may be introduced in addition to the endogenous gene.
[0512] T-DNA activation, TILLING and homologous recombination are examples of technologies that enable the generation of genetic modifications comprising increasing expression of a nucleic acid encoding a PLT transcription factor polypeptide in plants.
[0513] Nucleic acids encoding PLT transcription factor polypeptides, or PLT transcription factor polypeptides, may find use in breeding programmes in which a DNA marker is identified which may be genetically linked to a PLT transcription factor gene. The nucleic acids/genes, or the PLT transcription factor polypeptides may be used to define a molecular marker. This DNA or protein marker may then be used in breeding programmes to select plants having increased yield as defined hereinabove in the methods of the invention.
[0514] Allelic variants of a PLT transcription factor nucleic acid/gene may also find use in marker-assisted breeding programmes. Such breeding programmes sometimes require introduction of allelic variation by mutagenic treatment of the plants, using for example EMS mutagenesis; alternatively, the programme may start with a collection of allelic variants of so called "natural" origin caused unintentionally. Identification of allelic variants then takes place, for example, by PCR. This is followed by a step for selection of superior allelic variants of the sequence in question and which give increased yield. Selection is typically carried out by monitoring growth performance of plants containing different allelic variants of the sequence in question. Growth performance may be monitored in a greenhouse or in the field. Further optional steps include crossing plants in which the superior allelic variant was identified with another plant. This could be used, for example, to make a combination of interesting phenotypic features.
[0515] A nucleic acid encoding a PLT transcription factor polypeptide may also be used as probes for genetically and physically mapping the genes that they are a part of, and as markers for traits linked to those genes. Such information may be useful in plant breeding in order to develop lines with desired phenotypes. Such use of PLT transcription factor nucleic acids requires only a nucleic acid sequence of at least 15 nucleotides in length. The PLT transcription factor nucleic acids may be used as restriction fragment length polymorphism (RFLP) markers. Southern blots (Sambrook J, Fritsch E F and Maniatis T (1989) Molecular Cloning, A Laboratory Manual) of restriction-digested plant genomic DNA may be probed with the PLT transcription factor nucleic acids. The resulting banding patterns may then be subjected to genetic analyses using computer programs such as MapMaker (Lander et al. (1987) Genomics 1: 174-181) in order to construct a genetic map. In addition, the nucleic acids may be used to probe Southern blots containing restriction endonuclease-treated genomic DNAs of a set of individuals representing parent and progeny of a defined genetic cross. Segregation of the DNA polymorphisms is noted and used to calculate the position of the PLT transcription factor nucleic acid in the genetic map previously obtained using this population (Botstein et al. (1980) Am. J. Hum. Genet. 32:314-331).
[0516] The production and use of plant gene-derived probes for use in genetic mapping is described in Bernatzky and Tanksley (1986) Plant Mol. Biol. Reporter 4: 37-41. Numerous publications describe genetic mapping of specific cDNA clones using the methodology outlined above or variations thereof. For example, F2 intercross populations, backcross populations, randomly mated populations, near isogenic lines, and other sets of individuals may be used for mapping. Such methodologies are well known to those skilled in the art.
[0517] The nucleic acid probes may also be used for physical mapping (i.e., placement of sequences on physical maps; see Hoheisel et al. In: Non-mammalian Genomic Analysis: A Practical Guide, Academic press 1996, pp. 319-346, and references cited therein).
[0518] In another embodiment, the nucleic acid probes may be used in direct fluorescence in situ hybridisation (FISH) mapping (Trask (1991) Trends Genet. 7:149-154). Although current methods of FISH mapping favor use of large clones (several kb to several hundred kb; see Laan et al. (1995) Genome Res. 5:13-20), improvements in sensitivity may allow performance of FISH mapping using shorter probes.
[0519] A variety of nucleic acid amplification-based methods for genetic and physical mapping may be carried out using the nucleic acids. Examples include allele-specific amplification (Kazazian (1989) J. Lab. Clin. Med. 11:95-96), polymorphism of PCR-amplified fragments (CAPS; Sheffield et al. (1993) Genomics 16:325-332), allele-specific ligation (Landegren et al. (1988) Science 241:1077-1080), nucleotide extension reactions (Sokolov (1990) Nucleic Acid Res. 18:3671), Radiation Hybrid Mapping (Walter et al. (1997) Nat. Genet. 7:22-28) and Happy Mapping (Dear and Cook (1989) Nucleic Acid Res. 17:6795-6807). For these methods, the sequence of a nucleic acid is used to design and produce primer pairs for use in the amplification reaction or in primer extension reactions. The design of such primers is well known to those skilled in the art. In methods employing PCR-based genetic mapping, it may be necessary to identify DNA sequence differences between the parents of the mapping cross in the region corresponding to the instant nucleic acid sequence. This, however, is generally not necessary for mapping methods.
[0520] The methods according to the present invention result in plants having increased yield, as described hereinbefore. This increased yield may also be combined with other economically advantageous traits, such as further yield-enhancing traits, tolerance to other abiotic and biotic stresses, traits modifying various architectural features and/or biochemical and/or physiological features.
Detailed Description of bHLH
[0521] Surprisingly, it has now been found that modulating expression in a plant of a nucleic acid encoding a particular class of bHLH transcription factor gives plants having enhanced yield-related traits relative to control plants. The particular class of bHLH transcription factor suitable for enhancing yield-related traits in plants is described in detail below.
[0522] The present invention provides a method for enhancing yield-related traits in plants relative to control plants, comprising modulating expression in a plant of a nucleic acid encoding a particular class of bHLH transcription factor.
[0523] A preferred method for modulating (preferably, increasing) expression of a nucleic acid encoding a bHLH transcription factor is by introducing and expressing in a plant a nucleic acid encoding a particular class of bHLH transcription factor as further defined below.
[0524] The nucleic acid to be introduced into a plant (and therefore useful in performing the methods of the invention) is any nucleic acid encoding the type of bHLH transcription factor which will now be described. A bHLH transcription factor as defined herein refers to a polypeptide represented by any one of SEQ ID NO 213, SEQ ID NO 215, SEQ ID NO: 217, SEQ ID NO: 219, SEQ ID NO 225, SEQ ID NO: 297, SEQ ID NO: 299 and SEQ ID NO: 301. The invention is illustrated by transforming plants with the Oryza sativa sequence represented by SEQ ID NO: 212, encoding the polypeptide of SEQ ID NO: 213. SEQ ID NO: 215 from Oryza sativa (encoded by SEQ ID NO: 214) and SEQ ID NO: 217 from Oryza sativa (encoded by SEQ ID NO: 216) are paralogues of the polypeptide of SEQ ID NO: 213. SEQ ID NO: 219 from Arabidopsis thaliana (encoded by SEQ ID NO: 218) and SEQ ID NO: 225 from Arabidopsis thaliana (encoded by SEQ ID NO: 224) are orthologues of the polypeptide of SEQ ID NO: 213. SEQ ID NO: 221 from Arabidopsis thaliana (encoded by SEQ ID NO: 220) and SEQ ID NO: 223 from Arabidopsis thaliana (encoded by SEQ ID NO: 222) are variants of SEQ ID NO: 218 and SEQ ID NO: 219.
[0525] Orthologues and paralogues may easily be found by performing a so-called reciprocal blast search. Typically this involves a first BLAST involving BLASTing a query sequence (for example, SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 214, SEQ ID NO: 215, SEQ ID NO: 216, SEQ ID NO: 217, SEQ ID NO: 218, SEQ ID NO: 219, SEQ ID NO: 224 or SEQ ID NO: 225) against any sequence database, such as the publicly available NCBI database. BLASTN or TBLASTX (using standard default values) are generally used when starting from a nucleotide sequence, and BLASTP or TBLASTN (using standard default values) when starting from a protein sequence. The BLAST results may optionally be filtered. The full-length sequences of either the filtered results or non-filtered results are then BLASTed back (second BLAST) against sequences from the organism from which the query sequence is derived (where the query sequence is SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 214, SEQ ID NO: 215, SEQ ID NO: 216 or SEQ ID NO: 217, the second BLAST would therefore be against Oryza sequences; where the query sequence is SEQ ID NO: 218, SEQ ID NO: 219, SEQ ID NO: 224 or SEQ ID NO: 225, the second BLAST would therefore be against Arabidopsis sequences). The results of the first and second BLASTs are then compared. A paralogue is identified if a high-ranking hit from the first blast is from the same species as from which the query sequence is derived, a BLAST back then ideally results in the query sequence as highest hit; an orthologue is identified if a high-ranking hit in the first BLAST is not from the same species as from which the query sequence is derived, and preferably results upon BLAST back in the query sequence being among the highest hits.
[0526] High-ranking hits are those having a low E-value. The lower the E-value, the more significant the score (or in other words the lower the chance that the hit was found by chance). Computation of the E-value is well known in the art. In addition to E-values, comparisons are also scored by percentage identity. Percentage identity refers to the number of identical nucleotides (or amino acids) between the two compared nucleic acid (or polypeptide) sequences over a particular length. Preferably the score is greater than 50, more preferably greater than 100; and preferably the E-value is less than e-5, more preferably less than e-6. An example detailing the identification of orthologues and paralogues is given in Example 31 and Example 30 respectively herein. In the case of large families, ClustalW may be used, followed by a neighbour joining tree, to help visualize clustering of related genes and to identify orthologues and paralogues.
[0527] Examples of orthologues obtained by the BLAST procedure mentioned above are SEQ ID NO: 227 from Medicago truncatula (encoded by SEQ ID NO: 226) and SEQ ID NO: 229 from Hordeum vulgare (encoded by SEQ ID NO: 228). Further orthologues and paralogues may readily be identified using the BLAST procedure described above and by following the procedure given in the Examples section.
[0528] The proteins represented by SEQ ID NO: 297, 299 and 301 were hitherto unknown. Therefore, the invention also provides hitherto unknown bHLH transcription factors and bHLH transcription factor-encoding nucleic acids.
[0529] According to a further embodiment of the present invention, there is therefore provided an isolated nucleic acid molecule comprising:
[0530] (i) a nucleic acid represented by any one of SEQ ID NO: 296, SEQ ID NO: 298 and SEQ ID NO: 300;
[0531] (ii) the complement of a nucleic acid represented by any one of SEQ ID NO: 296, SEQ ID NO: 298 and SEQ ID NO: 300;
[0532] (iii) a nucleic acid encoding a bHLH transcription factor having (a) in increasing order of preference, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the amino acid sequence represented by any one of SEQ ID NO: 297, SEQ ID NO: 299 and SEQ ID NO: 301, and (b) a bHLH domain.
[0533] According to a further embodiment of the present invention, there is also provided an isolated polypeptide comprising:
[0534] (i) an amino acid sequence represented by any one of SEQ ID NO: 297, SEQ ID NO: 299 and SEQ ID NO: 301;
[0535] (ii) an amino acid sequence having (a) in increasing order of preference, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any one of the amino acid sequences represented by SEQ ID NO: 297, SEQ ID NO: 299 and SEQ ID NO: 301, and (b) a bHLH domain;
[0536] (iii) derivatives of any of the amino acid sequences given in (i) or (ii) above.
[0537] The polypeptides represented by any one of SEQ ID NO 213, SEQ ID NO 215, SEQ ID NO: 217, SEQ ID NO: 219, SEQ ID NO 221, SEQ ID NO 223, SEQ ID NO 225, SEQ ID NO: 227, SEQ ID NO: 229, SEQ ID NO: 297, SEQ ID NO: 299, SEQ ID NO: 301, or orthologues or paralogues of any of the aforementioned SEQ ID NOs, all comprise a bHLH domain.
[0538] bHLH domains are well known in the art and may readily be identified by persons skilled in the art. The family is defined by a bHLH signature domain, which consists of 60 or so amino acids with two functionally distinct regions. A basic region, located at the N-terminal end of the domain, is involved in DNA binding and consists of 15 or so amino acids with a high number of basic residues. An HLH region, at the C-terminal end, functions as a dimerization domain and mainly comprises hydrophobic residues that form two amphipathic helices separated by a loop region of variable sequence and length.
[0539] A bHLH domain may be identified using methods for the alignment of sequences for comparison. In some instances, default parameters may be adjusted to modify the stringency of the search. For example using BLAST, the statistical significance threshold (called E-value) for reporting matches against database sequences may be increased to show less stringent matches. In this way, short nearly exact matches may be identified.
[0540] Methods for the alignment of sequences for comparison are well known in the art, such methods include GAP, BESTFIT, BLAST, FASTA and TFASTA. GAP uses the algorithm of Needleman and Wunsch ((1970) J Mol Biol 48: 443-453) to find the global (over the whole the sequence) alignment of two sequences that maximizes the number of matches and minimizes the number of gaps. The BLAST algorithm (Altschul et al. (1990) J Mol Biol 215: 403-10) calculates percent sequence identity and performs a statistical analysis of the similarity between the two sequences. The software for performing BLAST analysis is publicly available through the National Centre for Biotechnology Information (NCBI). Homologues may readily be identified using, for example, the ClustalW multiple sequence alignment algorithm (version 1.83), with the default pairwise alignment parameters, and a scoring method in percentage. Minor manual editing may be performed to optimise alignment between conserved motifs, as would be apparent to a person skilled in the art.
[0541] Specialist databases also exist for the identification of domains. The bHLH domain in a bHLH transcription factor may be identified using, for example, SMART (Schultz et al. (1998) Proc. Natl. Acad. Sci. USA 95, 5857-5864; Letunic et al. (2002) Nucleic Acids Res 30, 242-244), InterPro (Mulder et al., (2003) Nucl. Acids. Res. 31, 315-318), Prosite (Bucher and Bairoch (1994), A generalized profile syntax for biomolecular sequences motifs and its function in automatic sequence interpretation. (In) ISMB-94; Proceedings 2nd International Conference on Intelligent Systems for Molecular Biology. Altman R., Brutlag D., Karp P., Lathrop R., Searls D., Eds., pp 53-61, AAAIPress, Menlo Park; Hulo et al., Nucl. Acids. Res. 32:D134-D137, (2004)) or Pfam (Bateman et al., Nucleic Acids Research 30(1): 276-280 (2002)). The HLH domain structure in the InterPro database is designated IPR001092 and IPR011598; PF00010 in the Pfam database; SM00353 in the SMART database and PS50888 in the PROSITE database. Furthermore, the alignment shown in FIG. 18 highlights the bHLH domain of bHLH transcription factors useful in the methods of the invention.
[0542] The polypeptides represented by any one of SEQ ID NO 213, SEQ ID NO 215, SEQ ID NO: 217, SEQ ID NO: 219, SEQ ID NO 225, SEQ ID NO: 297, SEQ ID NO: 299, SEQ ID NO: 301 or orthologues or paralogues of any of the aforementioned SEQ ID NOs, typically exhibit considerable sequence divergence outside of the conserved bHLH domain.
[0543] FIG. 19a is a matrix showing the overall similarities and identities (in bold) of the bHLH type proteins described above. Even though the identities appear to be relatively low, polypeptides having sequence identity falling within the ranges shown in the matrix may be taken to be orthologues or paralogues of any of SEQ ID NO 213, SEQ ID NO 215, SEQ ID NO: 217, SEQ ID NO: 219 or SEQ ID NO 225; this may be confirmed by the reciprocal blast procedure described above. Typically, nucleic acids encoding bHLH transcription factors useful in the methods of the invention have, in increasing order of preference, at least 13%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more sequence identity to the bHLH transcription factors represented by any one of SEQ ID NO 213, SEQ ID NO 215, SEQ ID NO: 217, SEQ ID NO: 219 or SEQ ID NO 225.
[0544] The matrix shown in FIG. 19b shows similarities and identities (in bold) over the bHLH domain, where of course the values are higher than when considering full length proteins. Typically, nucleic acids encoding bHLH transcription factors useful in the methods of the invention have bHLH domains having, in increasing order of preference, at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more sequence identity to the bHLH domain of any one of the polypeptides represented by any one of SEQ ID NO 213, SEQ ID NO 215, SEQ ID NO: 217, SEQ ID NO: 219 or SEQ ID NO 225.
[0545] A pattern of amino acids, termed a 5-9-13 configuration, may be found at three positions within the basic region of the bHLH domain (see FIG. 4 of Heim et al., 2003 (Mol. Biol. Evol. 20(5):735-747) and FIG. 18 herein where three upwardly pointing arrows show the configuration). The polypeptides represented by any one of SEQ ID NO 213, SEQ ID NO 215, SEQ ID NO: 217, SEQ ID NO: 219, SEQ ID NO 225, SEQ ID NO: 297, SEQ ID NO: 299, SEQ ID NO: 301 or orthologues or paralogues of any of the aforementioned SEQ ID NOs, preferably comprise a K/R ER configuration, more preferably an RER configuration, within the bHLH domain, typically within the basic region of the domain. The presence of this configuration is not a prerequisite to performing the methods of the invention, therefore some variation in the K/R ER configuration is acceptable. Arabidopsis bHLH polypeptides were grouped into twelve subfamilies according to structural similarities (see FIG. 4 of Heim et al., 2003). Members of group IX constitute bHLH polypeptides having an RER configuration. Furthermore, one member of Group VI comprises an RER configuration.
[0546] The polypeptides represented by any one of SEQ ID NO 213, SEQ ID NO 215, SEQ ID NO: 217, SEQ ID NO: 219, SEQ ID NO 225, SEQ ID NO: 297, SEQ ID NO: 299, SEQ ID NO: 301 or orthologues or paralogues of any of the aforementioned SEQ ID NOs, may also comprise (in addition to a bHLH domain and optionally a K/R ER 5-9-13 motif, preferably an RER motif) a domain designated PFB26111 (see Pfam database). The domain is also indicated in FIG. 18.
[0547] Typically, nucleic acids encoding bHLH transcription factors (as defined above) having a bHLH domain and having, in increasing order of preference, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more sequence identity to the PFB26111 domain (see FIG. 18) of any one of the polypeptides represented by any one of SEQ ID NO 213, SEQ ID NO 215, SEQ ID NO: 217, SEQ ID NO: 219, SEQ ID NO 225, SEQ ID NO: 297, SEQ ID NO: 299, or SEQ ID NO: 301 are useful in the methods of the invention.
[0548] Nucleic acids encoding the polypeptides represented by any one of SEQ ID NO 213, SEQ ID NO 215, SEQ ID NO: 217, SEQ ID NO: 219, SEQ ID NO 225, SEQ ID NO: 297, SEQ ID NO: 299, SEQ ID NO: 301, or orthologues or paralogues of any of the aforementioned SEQ ID NOs, need not be full-length nucleic acids, since performance of the methods of the invention does not rely on the use of full length nucleic acid sequences. Examples of nucleic acids suitable for use in performing the methods of the invention include but are not limited to those represented by any one of: SEQ ID NO: 212, SEQ ID NO: 214, SEQ ID NO: 216, SEQ ID NO: 218, SEQ ID NO: 220, SEQ ID NO: 222, SEQ ID NO: 224, SEQ ID NO: 226, SEQ ID NO: 228, SEQ ID NO: 296, SEQ ID NO: 298, and SEQ ID NO: 300. Nucleic acid variants may also be useful in practising the methods of the invention. Examples of such nucleic acid variants include portions of nucleic acids encoding a bHLH transcription factor as defined herein, splice variants of nucleic acids encoding a bHLH transcription factor as defined herein, allelic variants of nucleic acids encoding a bHLH transcription factor as defined herein and variants of nucleic acids encoding a bHLH transcription factor as defined herein that are obtained by gene shuffling. The terms portion, splice variant, allelic variant and gene shuffling will now be described.
[0549] A portion of a nucleic acids encoding a bHLH transcription factor as defined herein may be prepared, for example, by making one or more deletions to the nucleic acid. The portions may be used in isolated form or they may be fused to other coding (or non coding) sequences in order to, for example, produce a protein that combines several activities. When fused to other coding sequences, the resultant polypeptide produced upon translation may be bigger than that predicted for the bHLH transcription factor portion.
[0550] According to the present invention, there is provided a method for enhancing yield-related traits in plants, comprising introducing and expressing in a plant a portion of a nucleic acid encoding a bHLH transcription factor represented by any of SEQ ID NO 213, SEQ ID NO 215, SEQ ID NO: 217, SEQ ID NO: 219, SEQ ID NO 225, SEQ ID NO: 297, SEQ ID NO: 299, SEQ ID NO: 301, or a portion of a nucleic acid encoding orthologues, paralogues or homologues of any of the aforementioned SEQ ID NOs.
[0551] Portions useful in the methods of the invention, encode a polypeptide having a bHLH domain (as described above) and having substantially the same biological activity as the bHLH transcription factor represented by any of SEQ ID NO 213, SEQ ID NO 215, SEQ ID NO: 217, SEQ ID NO: 219, SEQ ID NO 225, SEQ ID NO: 297, SEQ ID NO: 299, SEQ ID NO: 301, or orthologues or paralogues of any of the aforementioned SEQ ID NOs. The portion is typically at least 150 consecutive nucleotides in length, preferably at least 300 consecutive nucleotides in length, more preferably at least 400 consecutive nucleotides in length and most preferably at least 500 consecutive nucleotides in length. Preferably, the portion is a portion of a nucleic acid as represented by any one of SEQ ID NO: 212, SEQ ID NO: 214, SEQ ID NO: 216, SEQ ID NO: 218, SEQ ID NO: 220, SEQ ID NO: 222, SEQ ID NO: 224, SEQ ID NO: 226, SEQ ID NO: 228, SEQ ID NO: 296, SEQ ID NO: 298, and SEQ ID NO: 300. Most preferably the portion is a portion of a nucleic acid as represented by SEQ ID NO: 212.
[0552] Another nucleic acid variant useful in the methods of the invention, is a nucleic acid capable of hybridising under reduced stringency conditions, preferably under stringent conditions, with a nucleic acid encoding a bHLH transcription factor as defined herein, or a with a portion as defined herein.
[0553] Hybridising sequences useful in the methods of the invention, encode a polypeptide having a bHLH domain (as described above) and having substantially the same biological activity as the bHLH transcription factor represented by any of SEQ ID NO 213, SEQ ID NO 215, SEQ ID NO: 217, SEQ ID NO: 219, SEQ ID NO 225, SEQ ID NO: 297, SEQ ID NO: 299, SEQ ID NO: 301 or having substantially the same biological activity as orthologues or paralogues of any of the aforementioned SEQ ID NOs. The hybridising sequence is typically at least 150 consecutive nucleotides in length, preferably at least 300 consecutive nucleotides in length, more preferably at least 400 consecutive nucleotides in length and most preferably at least 500 consecutive nucleotides in length. Preferably, the hybridising sequence is one that is capable of hybridising to any of the nucleic acids represented by SEQ ID NO: 212, SEQ ID NO: 214, SEQ ID NO: 216, SEQ ID NO: 218, SEQ ID NO: 220, SEQ ID NO: 222, SEQ ID NO: 224, SEQ ID NO: 226, SEQ ID NO: 228, SEQ ID NO: 296, SEQ ID NO: 298, and SEQ ID NO: 300 or to a portion of any of the aforementioned sequences, a portion being as defined above. Most preferably the hybridising sequence is capable of hybridising to a nucleic acid as represented by SEQ ID NO: 212, or to portions thereof.
[0554] According to the present invention, there is provided a method for enhancing yield-related traits in plants, comprising introducing and expressing in a plant a nucleic acid capable of hybridizing to a nucleic acid encoding a bHLH transcription factor represented by any of SEQ ID NO 213, SEQ ID NO 215, SEQ ID NO: 217, SEQ ID NO: 219, SEQ ID NO 225, SEQ ID NO: 297, SEQ ID NO: 299, SEQ ID NO: 301, or comprising introducing and expressing in a plant a nucleic acid capable of hybridising to a nucleic acid encoding an orthologue, paralogue or homologue of any of the aforementioned SEQ ID NOs.
[0555] Another nucleic acid variant useful in the methods of the invention is a splice variant encoding a bHLH transcription factor as defined hereinabove.
[0556] According to the present invention, there is provided a method for enhancing yield-related traits in plants, comprising introducing and expressing in a plant a splice variant of a nucleic acid encoding a bHLH transcription factor represented by any of SEQ ID NO 213, SEQ ID NO 215, SEQ ID NO: 217, SEQ ID NO: 219, SEQ ID NO 225, SEQ ID NO: 297, SEQ ID NO: 299, and SEQ ID NO: 301, or a splice variant of a nucleic acid encoding an orthologue, paralogue or homologue of any of the aforementioned SEQ ID NOs.
[0557] Preferred splice variants are splice variants of a nucleic acid encoding bHLH transcription factor represented by any of SEQ ID NO 213, SEQ ID NO 215, SEQ ID NO: 217, SEQ ID NO: 219, SEQ ID NO 225, SEQ ID NO: 297, SEQ ID NO: 299, and SEQ ID NO: 301, or splice variants encoding orthologues or paralogues of any of the aforementioned SEQ ID NOs. Further preferred are splice variants of nucleic acids represented by any one of SEQ ID NO: 212, SEQ ID NO: 214, SEQ ID NO: 216, SEQ ID NO: 218, SEQ ID NO: 220, SEQ ID NO: 222, SEQ ID NO: 224, SEQ ID NO: 226, SEQ ID NO: 228, SEQ ID NO: 296, SEQ ID NO: 298, and SEQ ID NO: 300. Most preferred is a splice variant of a nucleic acid as represented by SEQ ID NO: 212.
[0558] Another nucleic acid variant useful in performing the methods of the invention is an allelic variant of a nucleic acid encoding a bHLH transcription factor as defined hereinabove.
[0559] According to the present invention, there is provided a method for enhancing yield-related traits in plants, comprising introducing and expressing in a plant an allelic variant of a nucleic acid encoding a bHLH transcription factor represented by any of SEQ ID NO 213, SEQ ID NO 215, SEQ ID NO: 217, SEQ ID NO: 219, SEQ ID NO 225, SEQ ID NO: 297, SEQ ID NO: 299, and SEQ ID NO: 301, or comprising introducing and expressing in a plant an allelic variant of a nucleic acid encoding an orthologue, paralogue or homologue of any of the aforementioned SEQ ID NOs.
[0560] The allelic variant may be an allelic variant of a nucleic acid encoding a bHLH transcription factor represented by any of SEQ ID NO 213, SEQ ID NO 215, SEQ ID NO: 217, SEQ ID NO: 219, SEQ ID NO 225, SEQ ID NO: 297, SEQ ID NO: 299, and SEQ ID NO: 301, or an allelic variants of a nucleic acid encoding orthologues or paralogues of any of the aforementioned SEQ ID NOs. Further preferred are allelic variants of nucleic acids represented by any one of SEQ ID NO: 212, SEQ ID NO: 214, SEQ ID NO: 216, SEQ ID NO: 218, SEQ ID NO: 220, SEQ ID NO: 222, SEQ ID NO: 224, SEQ ID NO: 226 and SEQ ID NO: 228, SEQ ID NO: 296, SEQ ID NO: 298, SEQ ID NO: 300. Most preferred is an allelic variant of a nucleic acid as represented by SEQ ID NO: 212.
[0561] A further nucleic acid variant useful in the methods of the invention is a nucleic acid variant obtained by gene shuffling. Gene shuffling or directed evolution may also be used to generate variants of nucleic acids encoding bHLH transcription factors as defined above.
[0562] According to the present invention, there is provided a method for enhancing yield-related traits in plants, comprising introducing and expressing in a plant a variant of a nucleic acid encoding a bHLH transcription factor represented by any of SEQ ID NO 213, SEQ ID NO 215, SEQ ID NO: 217, SEQ ID NO: 219, SEQ ID NO 225, SEQ ID NO: 297, SEQ ID NO: 299, SEQ and ID NO: 301, or comprising introducing and expressing in a variant of a nucleic acid encoding an orthologue, paralogue or homologue of any of the aforementioned SEQ ID NOs, which nucleic acid is obtained by gene shuffling.
[0563] Furthermore, nucleic acid variants may also be obtained by site-directed mutagenesis. Several methods are available to achieve site-directed mutagenesis, the most common being PCR based methods (current protocols in molecular biology. Wiley Eds.).
[0564] Also useful in the methods of the invention are nucleic acids encoding homologues of any one of the amino acids represented by SEQ ID NO 213, SEQ ID NO 215, SEQ ID NO: 217, SEQ ID NO: 219, SEQ ID NO 225, SEQ ID NO: 297, SEQ ID NO: 299, SEQ ID NO: 301 or orthologues or paralogues of any of the aforementioned SEQ ID NOs.
[0565] Also useful in the methods of the invention are nucleic acids encoding derivatives of any one of the amino acids represented by SEQ ID NO 213, SEQ ID NO 215, SEQ ID NO: 217, SEQ ID NO: 219, SEQ ID NO 225, SEQ ID NO: 297, SEQ ID NO: 299, SEQ ID NO: 301 or orthologues or paralogues of any of the aforementioned SEQ ID NOs. Derivatives of SEQ ID NO: 215, SEQ ID NO: 217, SEQ ID NO: 219, SEQ ID NO: 221, SEQ ID NO: 223, SEQ ID NO: 225, SEQ ID NO: 227 and SEQ ID NO: 229, SEQ ID NO: 297, SEQ ID NO: 299, SEQ ID NO: 301 are further examples which may be suitable for use in the methods of the invention
[0566] Furthermore, bHLH transcription factors (at least in their native form) typically have DNA-binding activity and an activation domain. A person skilled in the art may easily determine the presence of an activation domain and DNA-binding activity using routine tools and techniques.
[0567] Nucleic acids encoding bHLH transcription factors may be derived from any natural or artificial source. The nucleic acid may be modified from its native form in composition and/or genomic environment through deliberate human manipulation. Preferably the bHLH transcription factor-encoding nucleic acid is from a plant, further preferably from a monocot, more preferably from the Poaceae family, most preferably the nucleic acid is from Oryza sativa.
[0568] Any reference herein to a bHLH transcription factor is therefore taken to mean a bHLH transcription factor as defined above. Any nucleic acid encoding such a bHLH transcription factor is suitable for use in performing the methods of the invention.
[0569] The present invention also encompasses plants or parts thereof (including plant cells) obtainable by the methods according to the present invention. The plants or parts thereof comprise a nucleic acid transgene encoding a bHLH transcription factor as defined above.
[0570] The invention also provides genetic constructs and vectors to facilitate introduction and/or expression of the nucleic acid sequences useful in the methods according to the invention, in a plant.
[0571] Therefore, there is provided a gene construct comprising:
[0572] (i) Any nucleic acid encoding a bHLH-type transcription factor as defined hereinabove;
[0573] (ii) One or more control sequences operably liked to the nucleic acid of (i).
[0574] Constructs useful in the methods according to the present invention may be constructed using recombinant DNA technology well known to persons skilled in the art. The gene constructs may be inserted into vectors, which may be commercially available, suitable for transforming into plants and suitable for expression of the gene of interest in the transformed cells. The invention therefore provides use of a gene construct as defined hereinabove in the methods of the invention.
[0575] Plants are transformed with a vector comprising the sequence of interest (i.e., a nucleic acid encoding a bHLH-type transcription factor). The skilled artisan is well aware of the genetic elements that must be present on the vector in order to successfully transform, select and propagate host cells containing the sequence of interest. The sequence of interest is operably linked to one or more control sequences (at least to a promoter).
[0576] Advantageously, any type of promoter, whether natural or synthetic, may be used to drive expression of the nucleic acid sequence. The promoter may be an inducible promoter, i.e. having induced or increased transcription initiation in response to a developmental, chemical, environmental or physical stimulus. The promoter may be a tissue-specific promoter, i.e. one that is capable of preferentially initiating transcription in certain tissues, such as the leaves, roots, seed tissue etc.
[0577] According to one preferred feature of the invention, the nucleic acid encoding a bHLH-type transcription factor is operably linked to a constitutive promoter. A constitutive promoter is transcriptionally active during most but not necessarily all phases of its growth and development and is substantially ubiquitously expressed. The constitutive promoter is preferably a GOS2 promoter, more preferably the constitutive promoter is a rice GOS2 promoter, further preferably the constitutive promoter is represented by a nucleic acid sequence substantially similar to SEQ ID NO: 230 or SEQ ID NO: 233, most preferably the constitutive promoter is as represented by SEQ ID NO: 233.
[0578] It should be clear that the applicability of the present invention is not restricted to the bHLH transcription factor-encoding nucleic acid represented by SEQ ID NO: 212, nor is the applicability of the invention restricted to expression of a such a bHLH transcription factor-encoding nucleic acid when driven by a GOS2 promoter. Examples of other constitutive promoters which may also be used perform the methods of the invention are shown in the definitions section.
[0579] Optionally, one or more terminator sequences (also a control sequence) may be used in the construct introduced into a plant. Additional regulatory elements may include transcriptional as well as translational enhancers. Those skilled in the art will be aware of terminator and enhancer sequences that may be suitable for use in performing the invention. Such sequences would be known or may readily be obtained by a person skilled in the art.
[0580] The genetic constructs of the invention may further include an origin of replication sequence that is required for maintenance and/or replication in a specific cell type. One example is when a genetic construct is required to be maintained in a bacterial cell as an episomal genetic element (e.g. plasmid or cosmid molecule). Preferred origins of replication include, but are not limited to, the f1-ori and colE1. Other control sequences (besides promoter, enhancer, silencer, intron sequences, 3'UTR and/or 5'UTR regions) may be protein and/or RNA stabilizing elements. The genetic construct may optionally comprise a selectable marker gene.
[0581] The invention also provides a method for the production of transgenic plants having enhanced yield-related traits relative to control plants, comprising introduction and expression in a plant of any nucleic acid encoding a bHLH-type transcription factor polypeptide as defined hereinabove.
[0582] More specifically, the present invention provides a method for the production of transgenic plants having enhanced yield-related traits, which method comprises:
[0583] (i) introducing and expressing a nucleic acid encoding a bHLH-type transcription factor (as defined herein) in a plant cell; and
[0584] (ii) cultivating the plant cell under conditions promoting plant growth and development.
[0585] The nucleic acid may be introduced directly into a plant cell or into the plant itself (including introduction into a tissue, organ or any other part of a plant). According to a preferred feature of the present invention, the nucleic acid is preferably introduced into a plant by transformation.
[0586] Generally after transformation, plant cells or cell groupings are selected for the presence of one or more markers which are encoded by plant-expressible genes co-transferred with the gene of interest, following which the transformed material is regenerated into a whole plant.
[0587] Following DNA transfer and regeneration, putatively transformed plants may be evaluated, for instance using Southern analysis, for the presence of the gene of interest, copy number and/or genomic organisation. Alternatively or additionally, expression levels of the newly introduced DNA may be monitored using Northern and/or Western analysis, or quantitative PCR, all techniques being well known to persons having ordinary skill in the art.
[0588] The generated transformed plants may be propagated by a variety of means, such as by clonal propagation or classical breeding techniques. For example, a first generation (or T1) transformed plant may be selfed to give homozygous second generation (or T2) transformants, and the T2 plants further propagated through classical breeding techniques.
[0589] The generated transformed organisms may take a variety of forms. For example, they may be chimeras of transformed cells and non-transformed cells; clonal transformants (e.g., all cells transformed to contain the expression cassette); grafts of transformed and untransformed tissues (e.g., in plants, a transformed rootstock grafted to an untransformed scion).
[0590] The present invention clearly extends to any plant cell or plant produced by any of the methods described herein, and to all plant parts and propagules thereof. The present invention extends further to encompass the progeny of a primary transformed or transfected cell, tissue, organ or whole plant that has been produced by any of the aforementioned methods, the only requirement being that progeny exhibit the same genotypic and/or phenotypic characteristic(s) as those produced by the parent in the methods according to the invention.
[0591] The invention also includes host cells containing an isolated nucleic acid encoding a bHLH transcription factor as defined hereinabove. Preferred host cells according to the invention are plant cells.
[0592] The invention also extends to harvestable parts of a plant such as, but not limited to seeds, leaves, fruits, flowers, stems, rhizomes, tubers and bulbs. The invention furthermore relates to products derived, preferably directly derived, from a harvestable part of such a plant, such as dry pellets or powders, oil, fat and fatty acids, starch or proteins.
[0593] According to a preferred feature of the invention, the modulated expression is increased expression.
[0594] As mentioned above, a preferred method for modulating (preferably, increasing) expression of a nucleic acid encoding a bHLH transcription factor is by introducing and expressing in a plant a nucleic acid encoding a bHLH transcription factor; however the effects of performing the method, i.e. enhancing yield-related traits may also be achieved using other well known techniques. A description of some of these techniques will now follow.
[0595] One such technique is T-DNA activation tagging. The resulting transgenic plants show dominant phenotypes due to modified expression of genes close to the introduced promoter. The effects of the invention may also be reproduced using the technique of TILLING (Targeted Induced Local Lesions In Genomes). The effects of the invention may also be reproduced using homologous recombination.
[0596] Reference herein to the term enhanced yield-related traits is taken to mean an increase in biomass (weight) of one or more parts of a plant, which may include aboveground (harvestable) parts and/or (harvestable) parts below ground.
[0597] In particular, such harvestable parts are seeds, and performance of the methods of the invention results in plants having increased seed yield relative to the seed yield of control plants.
[0598] Taking corn as an example, a yield increase may be manifested as one or more of the following: increase in the number of plants established per hectare or acre, an increase in the number of ears per plant, an increase in the number of rows, number of kernels per row, kernel weight, thousand kernel weight, ear length/diameter, increase in the seed filling rate (which is the number of filled seeds divided by the total number of seeds and multiplied by 100), among others. Taking rice as an example, a yield increase may manifest itself as an increase in one or more of the following: number of plants per hectare or acre, number of panicles per plant, number of spikelets per panicle, number of flowers (florets) per panicle (which is expressed as a ratio of the number of filled seeds over the number of primary panicles), increase in the seed filling rate (which is the number of filled seeds divided by the total number of seeds and multiplied by 100), increase in thousand kernel weight, among others.
[0599] Since the transgenic plants according to the present invention have increased yield, it is likely that these plants exhibit an increased growth rate (during at least part of their life cycle), relative to the growth rate of corresponding wild type plants at a corresponding stage in their life cycle. The increased growth rate may be specific to one or more parts of a plant (including seeds), or may be throughout substantially the whole plant. A plant having an increased growth rate may even exhibit early flowering. The increase in growth rate may take place at one or more stages in the life cycle of a plant or during substantially the whole plant life cycle. Increased growth rate during the early stages in the life cycle of a plant may reflect enhanced vigour (increased seedling vigor at emergence). The increase in growth rate may alter the harvest cycle of a plant allowing plants to be sown later and/or harvested sooner than would otherwise be possible. If the growth rate is sufficiently increased, it may allow for the further sowing of seeds of the same plant species (for example sowing and harvesting of rice plants followed by sowing and harvesting of further rice plants all within one conventional growing period). Similarly, if the growth rate is sufficiently increased, it may allow for the further sowing of seeds of different plants species (for example the sowing and harvesting of corn plants followed by, for example, the sowing and optional harvesting of soy bean, potato or any other suitable plant). Harvesting additional times from the same rootstock in the case of some crop plants may also be possible. Altering the harvest cycle of a plant may lead to an increase in annual biomass production per acre (due to an increase in the number of times (say in a year) that any particular plant may be grown and harvested). An increase in growth rate may also allow for the cultivation of transgenic plants in a wider geographical area than their wild-type counterparts, since the territorial limitations for growing a crop are often determined by adverse environmental conditions either at the time of planting (early season) or at the time of harvesting (late season). Such adverse conditions may be avoided if the harvest cycle is shortened. The growth rate may be determined by deriving various parameters from growth curves, such parameters may be: T-Mid (the time taken for plants to reach 50% of their maximal size) and T-90 (time taken for plants to reach 90% of their maximal size), amongst others.
[0600] An increase in yield and/or growth rate occurs whether the plant is under non-stress conditions or whether the plant is exposed to various stresses compared to control plants. Plants typically respond to exposure to stress by growing more slowly. In conditions of severe stress, the plant may even stop growing altogether. Mild stress on the other hand is defined herein as being any stress to which a plant is exposed which does not result in the plant ceasing to grow altogether without the capacity to resume growth. Mild stress in the sense of the invention leads to a reduction in the growth of the stressed plants of less than 40%, 35% or 30%, preferably less than 25%, 20% or 15%, more preferably less than 14%, 13%, 12%, 11% or 10% or less in comparison to the control plant under non-stress conditions. Due to advances in agricultural practices (irrigation, fertilization, pesticide treatments) severe stresses are not often encountered in cultivated crop plants. As a consequence, the compromised growth induced by mild stress is often an undesirable feature for agriculture. Mild stresses are the everyday biotic and/or abiotic (environmental) stresses to which a plant is exposed. Abiotic stresses may be due to drought or excess water, anaerobic stress, salt stress, chemical toxicity, oxidative stress and hot, cold or freezing temperatures. The abiotic stress may be an osmotic stress caused by a water stress (particularly due to drought), salt stress, oxidative stress or an ionic stress. Biotic stresses are typically those stresses caused by pathogens, such as bacteria, viruses, fungi and insects.
[0601] In particular, the methods of the present invention may be performed under non-stress conditions or under conditions of mild drought to give plants having increased yield relative to control plants. As reported in Wang et al. (Planta (2003) 218: 1-14), abiotic stress leads to a series of morphological, physiological, biochemical and molecular changes that adversely affect plant growth and productivity. Drought, salinity, extreme temperatures and oxidative stress are known to be interconnected and may induce growth and cellular damage through similar mechanisms. Rabbani et al. (Plant Physiol (2003) 133: 1755-1767) describes a particularly high degree of "cross talk" between drought stress and high-salinity stress. For example, drought and/or salinisation are manifested primarily as osmotic stress, resulting in the disruption of homeostasis and ion distribution in the cell. Oxidative stress, which frequently accompanies high or low temperature, salinity or drought stress, may cause denaturing of functional and structural proteins. As a consequence, these diverse environmental stresses often activate similar cell signaling pathways and cellular responses, such as the production of stress proteins, up-regulation of anti-oxidants, accumulation of compatible solutes and growth arrest. The term "non-stress" conditions as used herein are those environmental conditions that allow optimal growth of plants. Persons skilled in the art are aware of normal soil conditions and climatic conditions for a given location.
[0602] Performance of the methods of the invention gives plants grown under non-stress conditions or under mild drought conditions increased yield relative to control plants grown under comparable conditions. Therefore, according to the present invention, there is provided a method for increasing yield in plants grown under non-stress conditions or under mild drought conditions, which method comprises increasing expression in a plant of a nucleic acid encoding a bHLH transcription factor.
[0603] Performance of the methods of the invention gives plants grown under conditions of nutrient deficiency, particularly under conditions of nitrogen deficiency, increased yield relative to control plants grown under comparable conditions. Therefore, according to the present invention, there is provided a method for increasing yield in plants grown under conditions of nutrient deficiency, which method comprises increasing expression in a plant of a nucleic acid encoding a MADS15 polypeptide. Nutrient deficiency may result from a lack or excess of nutrients such as nitrogen, phosphates and other phosphorous-containing compounds, potassium, calcium, cadmium, magnesium, manganese, iron and boron, amongst others.
[0604] According to a preferred feature of the present invention, performance of the methods of the invention gives plants having an increased growth rate relative to control plants, particularly during the early stages of plant development (typically three weeks post germination in the case of rice and maize, but this will vary from species to species) leading to early vigour. Therefore, according to the present invention, there is provided a method for increasing the growth rate of plants, which method comprises modulating, preferably increasing, expression in a plant of a nucleic acid encoding a bHLH transcription factor. The present invention therefore also provides a method for obtaining plants having early vigour relative to control plants, which method comprises modulating, preferably increasing, expression in a plant of a nucleic acid encoding a bHLH transcription factor.
[0605] Early vigour may also result from or be manifested as increased plant fitness relative to control plants due to, for example, the plants being better adapted to their environment (i.e. being more able to cope with various abiotic or biotic stress factors). Plants having early vigour also show better establishment of the crop (with the crop growing in uniform manner, i.e. with the majority of plants reaching the various stages of development at substantially the same time), and show better growth and often better yield. Early vigour may be determined by measuring various factors, such as seedling growth rate, thousand kernel weight, percentage germination, percentage emergence, seedling height, root length and shoot biomass and many more.
[0606] The methods of the invention are advantageously applicable to any plant.
[0607] Plants that are particularly useful in the methods of the invention include all plants which belong to the superfamily Viridiplantae, in particular monocotyledonous and dicotyledonous plants including fodder or forage legumes, ornamental plants, food crops, trees or shrubs. According to a preferred embodiment of the present invention, the plant is a crop plant. Examples of crop plants include soybean, sunflower, canola, alfalfa, rapeseed, cotton, tomato, potato and tobacco. Further preferably, the plant is a monocotyledonous plant. Examples of monocotyledonous plants include sugarcane. More preferably the plant is a cereal. Examples of cereals include rice, maize, wheat, barley, millet, rye, triticale, sorghum and oats. Plants in which early vigour is a particularly desirably trait include rice, maize, wheat, sunflower, sorghum.
[0608] The present invention also encompasses use of nucleic acids encoding bHLH transcription factors and use of bHLH transcription factor polypeptides in enhancing yield-related traits.
[0609] Nucleic acids encoding bHLH transcription factor polypeptides, or bHLH transcription factors themselves, may find use in breeding programmes in which a DNA marker is identified which may be genetically linked to a bHLH transcription factor-encoding gene. The nucleic acids/genes, or the bHLH transcription factors themselves may be used to define a molecular marker. This DNA or protein marker may then be used in breeding programmes to select plants having increased yield as defined hereinabove in the methods of the invention.
[0610] Allelic variants of a bHLH transcription factor-encoding acid/gene may also find use in marker-assisted breeding programmes. Such breeding programmes sometimes require introduction of allelic variation by mutagenic treatment of the plants, using for example EMS mutagenesis; alternatively, the programme may start with a collection of allelic variants of so called "natural" origin caused unintentionally. Identification of allelic variants then takes place, for example, by PCR. This is followed by a step for selection of superior allelic variants of the sequence in question and which give increased yield. Selection is typically carried out by monitoring growth performance of plants containing different allelic variants of the sequence in question. Growth performance may be monitored in a greenhouse or in the field. Further optional steps include crossing plants in which the superior allelic variant was identified with another plant. This could be used, for example, to make a combination of interesting phenotypic features.
[0611] A nucleic acid encoding a bHLH transcription factor may also be used as probes for genetically and physically mapping the genes that they are a part of, and as markers for traits linked to those genes. Such information may be useful in plant breeding in order to develop lines with desired phenotypes. Such use of bHLH transcription factor encoding nucleic acids requires only a nucleic acid sequence of at least 15 nucleotides in length. The bHLH transcription factor encoding nucleic acids may be used as restriction fragment length polymorphism (RFLP) markers. Southern blots (Sambrook J, Fritsch E F and Maniatis T (1989) Molecular Cloning, A Laboratory Manual) of restriction-digested plant genomic DNA may be probed with the bHLH transcription factor encoding nucleic acids. The resulting banding patterns may then be subjected to genetic analyses using computer programs such as MapMaker (Lander et al. (1987) Genomics 1: 174-181) in order to construct a genetic map. In addition, the nucleic acids may be used to probe Southern blots containing restriction endonuclease-treated genomic DNAs of a set of individuals representing parent and progeny of a defined genetic cross. Segregation of the DNA polymorphisms is noted and used to calculate the position of the bHLH transcription factor encoding nucleic acid in the genetic map previously obtained using this population (Botstein et al. (1980) Am. J. Hum. Genet. 32:314-331).
[0612] The production and use of plant gene-derived probes for use in genetic mapping is described in Bernatzky and Tanksley (1986) Plant Mol. Biol. Reporter 4: 37-41. Numerous publications describe genetic mapping of specific cDNA clones using the methodology outlined above or variations thereof. For example, F2 intercross populations, backcross populations, randomly mated populations, near isogenic lines, and other sets of individuals may be used for mapping. Such methodologies are well known to those skilled in the art.
[0613] The nucleic acid probes may also be used for physical mapping (i.e., placement of sequences on physical maps; see Hoheisel et al. In: Non-mammalian Genomic Analysis: A Practical Guide, Academic press 1996, pp. 319-346, and references cited therein).
[0614] In another embodiment, the nucleic acid probes may be used in direct fluorescence in situ hybridisation (FISH) mapping (Trask (1991) Trends Genet. 7:149-154). Although current methods of FISH mapping favor use of large clones (several kb to several hundred kb; see Laan et al. (1995) Genome Res. 5:13-20), improvements in sensitivity may allow performance of FISH mapping using shorter probes.
[0615] A variety of nucleic acid amplification-based methods for genetic and physical mapping may be carried out using the nucleic acids. Examples include allele-specific amplification (Kazazian (1989) J. Lab. Clin. Med. 11:95-96), polymorphism of PCR-amplified fragments (CAPS; Sheffield et al. (1993) Genomics 16:325-332), allele-specific ligation (Landegren et al. (1988) Science 241:1077-1080), nucleotide extension reactions (Sokolov (1990) Nucleic Acid Res. 18:3671), Radiation Hybrid Mapping (Walter et al. (1997) Nat. Genet. 7:22-28) and Happy Mapping (Dear and Cook (1989) Nucleic Acid Res. 17:6795-6807). For these methods, the sequence of a nucleic acid is used to design and produce primer pairs for use in the amplification reaction or in primer extension reactions. The design of such primers is well known to those skilled in the art. In methods employing PCR-based genetic mapping, it may be necessary to identify DNA sequence differences between the parents of the mapping cross in the region corresponding to the instant nucleic acid sequence. This, however, is generally not necessary for mapping methods.
[0616] The methods according to the present invention result in plants having enhnaced yield-related traits, as described hereinbefore. These traits may also be combined with other economically advantageous traits, such as further yield-enhancing traits, tolerance to other abiotic and biotic stresses, traits modifying various architectural features and/or biochemical and/or physiological features.
Detailed Description of SPL15
[0617] Surprisingly, it has now been found that increasing expression in a plant of a nucleic acid encoding an SPL15 transcription factor polypeptide gives plants having enhanced yield related traits, particularly increased yield, relative to control plants. Therefore, the invention provides a method for enhancing yield related traits in particular for increasing yield in plants relative to control plants, comprising increasing expression in a plant of a nucleic acid encoding an SPL15 transcription factor polypeptide.
[0618] A preferred method for increasing expression of a nucleic acid encoding a SPL15 transcription factor polypeptide is by introducing and expressing in a plant a nucleic acid encoding a SPL15 transcription factor polypeptide.
[0619] The term "SPL15 transcription factor polypeptide" as defined herein refers to a polypeptide comprising from N-terminal to C-terminal: (i) Motif 1 as represented by SEQ ID NO: 276; and (ii) in increasing order of preference at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98% sequence identity to the SPL DBD represented by SEQ ID NO: 277; and (iii) Motif 2 as represented by SEQ ID NO: 278.
[0620] The most conserved amino acids within Motif 1 are XLXFGXXXYFX, and within Motif 2 DSXXALSLLSX (where X is a specified subset of amino acids differing for each position, as presented in SEQ ID NO: 276 and SEQ ID NO: 277). Within Motif 1 and Motif 2, are allowed one or more conservative change at any position, and/or one, two or three non-conservative change(s) at any position.
[0621] Additionally, the SPL15 transcription factor polypeptide may comprise any one or both of the following: (a) a G/S rich stretch preceding the SPL DBD; and (b) the W(S/T)L tripeptide at the C-terminal end of the polypeptide.
[0622] An example of an SPL15 transcription polypeptide as defined hereinabove comprising from N-terminal to C-terminal: (i) Motif 1 as represented by SEQ ID NO: 276; and (ii) in increasing order of preference at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98% sequence identity to the SPL DBD represented by SEQ ID NO: 277; and (iii) Motif 2 as represented by SEQ ID NO: 278; and additionally comprising: (a) a G/S rich stretch preceding the SPL DBD; and (b) the W(S/T)L tripeptide at the C-terminal end of the polypeptide, is represented as in SEQ ID NO: 235. Further such examples are represented by any one of SEQ ID NO: 237, SEQ ID NO: 239, SEQ ID NO: 241, SEQ ID NO: 243, SEQ ID NO: 245, SEQ ID NO: 247, SEQ ID NO: 249, SEQ ID NO: 251, SEQ ID NO: 253, SEQ ID NO: 255, SEQ ID NO: 257, SEQ ID NO: 259, SEQ ID NO: 261, SEQ ID NO: 263, SEQ ID NO: 283, SEQ ID NO: 285, SEQ ID NO: 287 or orthologues or paralogues of any of the aforementioned SEQ ID NOs. The invention is illustrated by transforming plants with the Arabidopsis thaliana sequence represented by SEQ ID NO: 234, encoding the polypeptide of SEQ ID NO: 235. SEQ ID NO: 237 from Arabidopsis thaliana (encoded by SEQ ID NO: 236) is a paralogue of the polypeptide of SEQ ID NO: 235. SEQ ID NO: 239 (encoded by SEQ ID NO: 238, from Aquilegia formosa×Aquilegia pubescens), SEQ ID NO: 241 (encoded by SEQ ID NO: 240, from Gossypium hirsutum), SEQ ID NO: 243 (encoded by SEQ ID NO: 242, from Ipomoea nil), SEQ ID NO: 245 (encoded by SEQ ID NO: 244, from Lactuca sativa), SEQ ID NO: 247 (encoded by SEQ ID NO: 246, from Malus domestica), SEQ ID NO: 249 (encoded by SEQ ID NO: 248, from Medicago truncatula), SEQ ID NO: 251 (encoded by SEQ ID NO: 250, from Nicotiana bentamiana), SEQ ID NO: 253 (encoded by SEQ ID NO: 252, from Oryza sativa), SEQ ID NO: 255 (encoded by SEQ ID NO: 254, from Oryza sativa), SEQ ID NO: 257 (encoded by SEQ ID NO: 256, from Solanum tuberosum SEQ ID NO: 259 (encoded by SEQ ID NO: 258, from Vitis vinifera), SEQ ID NO: 261 (encoded by SEQ ID NO: 260, from Zea mays), SEQ ID NO: 263 (encoded by SEQ ID NO: 262, Zea mays), SEQ ID NO: 283 (encoded by SEQ ID NO: 282, Brassica rapa), SEQ ID NO: 285 (encoded by SEQ ID NO: 284, Glycine max), and SEQ ID NO: 287 (encoded by SEQ ID NO: 286, Populus tremuloides) are orthologues of the polypeptide of SEQ ID NO: 235.
[0623] Orthologues and paralogues may easily be found by performing a so-called reciprocal blast search. This may be done by a first BLAST involving BLASTing a query sequence (for example, SEQ ID NO: 234 or SEQ ID NO: 235) against any sequence database, such as the publicly available NCBI database. BLASTN or TBLASTX (using standard default values) may be used when starting from a nucleotide sequence and BLASTP or TBLASTN (using standard default values) may be used when starting from a polypeptide sequence. The BLAST results may optionally be filtered. The full-length sequences of either the filtered results or non-filtered results are then BLASTed back (second BLAST) against sequences from the organism from which the query sequence is derived (where the query sequence is SEQ ID NO: 234 or SEQ ID NO: 235, the second BLAST would therefore be against Arabidopsis sequences). The results of the first and second BLASTs are then compared. A paralogue is identified if a high-ranking hit from the first BLAST is from the same species as from which the query sequence is derived, a BLAST back then ideally results in the query sequence as highest hit (besides itself); an orthologue is identified if a high-ranking hit in the first BLAST is not from the same species as from which the query sequence is derived and preferably results upon BLAST back in the query sequence amongst the highest hits. High-ranking hits are those having a low E-value. The lower the E-value, the more significant the score (or in other words the lower the chance that the hit was found by chance). Computation of the E-value is well known in the art. In addition to E-values, comparisons are also scored by percentage identity. Percentage identity refers to the number of identical nucleotides (or amino acids) between the two compared nucleic acid (or polypeptide) sequences over a particular length. An example detailing the identification of orthologues and paralogues is given in Example 41. In the case of large families, ClustalW may be used, followed by a neighbour joining tree, to help visualize clustering of related genes and to identify orthologues and paralogues. In FIG. 22, the SPL15 transcription factor polypeptide paralogues and orthologues cluster together.
[0624] The polypeptides represented by any one of SEQ ID NO: 237, SEQ ID NO: 239, SEQ ID NO: 241, SEQ ID NO: 243, SEQ ID NO: 245, SEQ ID NO: 247, SEQ ID NO: 249, SEQ ID NO: 251, SEQ ID NO: 253, SEQ ID NO: 255, SEQ ID NO: 257, SEQ ID NO: 259, SEQ ID NO: 261, SEQ ID NO: 263, SEQ ID NO: 283, SEQ ID NO: 285, SEQ ID NO: 287, or orthologues or paralogues of any of the aforementioned SEQ ID NOs, all comprise an SPL DBD having, in increasing order of preference, at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98% sequence identity to the SPL15 DBD represented by SEQ ID NO: 277.
[0625] SPL DBDs are well known in the art and may readily be identified by persons skilled in the art. A SPL DBD may be identified using methods for the alignment of sequences for comparison. Methods for the alignment of sequences for comparison include GAP, BESTFIT, BLAST, FASTA and TFASTA. GAP uses the algorithm of Needleman and Wunsch ((1970) J Mol Biol 48: 443-453) to find the alignment of two complete sequences that maximizes the number of matches and minimizes the number of gaps. The BLAST algorithm (Altschul et al. (1990) J Mol Biol 215: 403-10) calculates percent sequence identity and performs a statistical analysis of the similarity between the two sequences. The software for performing BLAST analysis is publicly available through the National Centre for Biotechnology Information. Homologues may readily be identified using, for example, the ClustalW multiple sequence alignment algorithm (version 1.83) available at GenomeNet service at the Kyoto University Bioinformatics Center, with the default pairwise alignment parameters, and a scoring method in percentage. Minor manual editing may be performed to optimise alignment between conserved motifs, as would be apparent to a person skilled in the art. The alignment shown in FIGS. 23 and 24 highlight the SPL DBD in SPL15 transcription factor polypeptides. Preferably, SPL15 transcription factor polypeptides useful in the methods of the invention comprise an SPL DBD having, in increasing order of preference, at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98% sequence identity to the SPL DBD represented by SEQ ID NO: 277.
[0626] In some instances, default parameters may be adjusted to modify the stringency of the search. For example using BLAST, the statistical significance threshold (called "expect" value) for reporting matches against database sequences may be increased to show less stringent matches. In this way, short nearly exact matches may be identified. Motif 1 as represented by SEQ ID NO: 276 and Motif 2 as represented by SEQ ID NO: 278 both comprised in the SPL15 transcription factor polypeptides useful in the methods of the invention may be identified this way (FIG. 24). Within Motif 1 and Motif 2, are allowed one or more conservative change at any position, and/or one, two or three non-conservative change(s) at any position. The W(S/T)L tripeptide at the C-terminal end of the polypeptide may likewise be identified (FIG. 24).
[0627] Special databases also exisit for the identification of domains. The SPL DBD in a SPL15 transcription factor polypeptide may be identified using, for example, SMART (Schultz et al. (1998) Proc. Natl. Acad. Sci. USA 95, 5857-5864; Letunic et al. (2002) Nucleic Acids Res 30, 242-244; hosted by the EMBL at Heidelberg, Germany), InterPro (Mulder et al., (2003) Nucl. Acids. Res. 31, 315-318; hosted by the European Bioinformatics Institute (EBI) in the United Kingdom), Prosite (Bucher and Bairoch (1994), A generalized profile syntax for biomolecular sequences motifs and its function in automatic sequence interpretation. (In) ISMB-94; Proceedings 2nd International Conference on Intelligent Systems for Molecular Biology. Altman R., Brutlag D., Karp P., Lathrop R., Searls D., Eds., pp 53-61, AAAIPress, Menlo Park; Hulo et al., Nucl. Acids. Res. 32: D134-D137, (2004), The ExPASy proteomics server is provided as a service to the scientific community (hosted by the Swiss Institute of Bioinformatics (SIB) in Switzerland) or Pfam (Bateman et al., Nucleic Acids Research 30(1): 276-280 (2002), hosted by the Sanger Institute in the United Kingdom). The SPL DBD in the InterPro database is designated IPR004333, PF03110 in the Pfam database and PS51141 in the PROSITE database.
[0628] Furthermore, the presence of G/S rich stretch preceding the SPL DBD may also readily be identified (FIG. 24). Primary amino acid composition (in %) to determine if a polypeptide domain is rich in specific amino acids may be calculated using software programs from the ExPASy server, in particular the ProtParam tool (Gasteiger E et al. (2003) ExPASy: the proteomics server for in-depth protein knowledge and analysis. Nucleic Acids Res 31:3784-3788). The composition of the protein of interest may then be compared to the average amino acid composition (in %) in the Swiss-Prot Protein Sequence data bank. Within this databank, the average Gly (G) and Ser (E) content are both of 6.9% (adding up to 13.8%). As an example, the G/S rich stretch preceding the SPL DBD of SEQ ID NO: 235 contains 14.5% of G and 25.5% of S (adding up to 40%). As defined herein, a G/S rich stretch has a combined G and S content (in % terms) above that found in the average amino acid composition (in % terms) of the proteins in the Swiss-Prot Protein Sequence. Both G and S belong to the category of very small amino acids.
[0629] The nucleic acid encoding the polypeptides represented by any one of SEQ ID NO: 235, SEQ ID NO: 237, SEQ ID NO: 239, SEQ ID NO: 241, SEQ ID NO: 243, SEQ ID NO: 245, SEQ ID NO: 247, SEQ ID NO: 249, SEQ ID NO: 251, SEQ ID NO: 253, SEQ ID NO: 255, SEQ ID NO: 257, SEQ ID NO: 259, SEQ ID NO: 261, SEQ ID NO: 263, SEQ ID NO: 283, SEQ ID NO: 285, SEQ ID NO: 287, or orthologues or paralogues of any of the aforementioned SEQ ID NOs, need not be full-length nucleic acids, since performance of the methods of the invention does not rely on the use of full length nucleic acid sequences. Furthermore, examples of nucleic acids suitable for use in performing the methods of the invention include but are not limited to those represented by any one of: SEQ ID NO: 234, SEQ ID NO: 236, SEQ ID NO: 238, SEQ ID NO: 240, SEQ ID NO: 242, SEQ ID NO: 244, SEQ ID NO: 246, SEQ ID NO: 248, SEQ ID NO: 250, SEQ ID NO: 252, SEQ ID NO: 254, SEQ ID NO: 256, SEQ ID NO: 258, SEQ ID NO: 260, SEQ ID NO: 262, SEQ ID NO: 282, SEQ ID NO: 284 and SEQ ID NO: 286. Nucleic acid variants may also be useful in practising the methods of the invention. Examples of such variants include portions of nucleic acids, splice variants, allelic variants either naturally occurring or obtained by DNA manipulation.
[0630] A portion may be prepared, for example, by making one or more deletions to a nucleic acid encoding a SPL15 transcription factor polypeptide as defined hereinabove. The portions may be used in isolated form or they may be fused to other coding (or non coding) sequences in order to, for example, produce a protein that combines several activities. When fused to other coding sequences, the resultant polypeptide produced upon translation may be bigger than that predicted for the SPL15 transcription factor portion. Portions useful in the methods of the invention, encode an SPL15 transcription factor polypeptide (as described above) and having substantially the same biological activity as the SPL15 transcription factor polypeptide represented by any of SEQ ID NO: 235, SEQ ID NO: 237, SEQ ID NO: 239, SEQ ID NO: 241, SEQ ID NO: 243, SEQ ID NO: 245, SEQ ID NO: 247, SEQ ID NO: 249, SEQ ID NO: 251, SEQ ID NO: 253, SEQ ID NO: 255, SEQ ID NO: 257, SEQ ID NO: 259, SEQ ID NO: 261, SEQ ID NO: 263, SEQ ID NO: 283, SEQ ID NO: 285, SEQ ID NO: 287, or orthologues or paralogues of any of the aforementioned SEQ ID NOs. Examples of portions may include the nucleotides encoding Motif 1 as represented by SEQ ID NO: 276, in increasing order of preference at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98% sequence identity to the SPL DBD represented by SEQ ID NO: 277, and Motif 2 as represented by SEQ ID NO: 278. Portions may additionally include nucleotides encoding the G/S rich stretch or the W(S/T)L tripeptide (but not necessarily the nucleotides encoding the amino acid sequences between any of these). The portion is typically at least 250 nucleotides in length, preferably at least 500 nucleotides in length, more preferably at least 750 nucleotides in length and most preferably at least 1000 nucleotides in length. Preferably, the portion is a portion of a nucleic acid as represented by any one of SEQ ID NO: 234, SEQ ID NO: 236, SEQ ID NO: 238, SEQ ID NO: 240, SEQ ID NO: 242, SEQ ID NO: 244, SEQ ID NO: 246, SEQ ID NO: 248, SEQ ID NO: 250, SEQ ID NO: 252, SEQ ID NO: 254, SEQ ID NO: 256, SEQ ID NO: 258, SEQ ID NO: 260, SEQ ID NO: 262, SEQ ID NO: 282, SEQ ID NO: 284 and SEQ ID NO: 286. Most preferably the portion is a portion of a nucleic acid as represented by SEQ ID NO: 234.
[0631] Another nucleic acid variant useful in the methods of the invention, is a nucleic acid capable of hybridising under reduced stringency conditions, preferably under stringent conditions, with a nucleic acid encoding a SPL15 transcription factor polypeptide as defined hereinabove, or a with a portion as defined hereinabove.
[0632] Hybridising sequences useful in the methods of the invention, encode a polypeptide comprising from N-terminal to C-terminal: (i) Motif 1 as represented by SEQ ID NO: 276; and (ii) in increasing order of preference at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98% sequence identity to the SPL DBD represented by SEQ ID NO: 277; and (iii) Motif 2 as represented by SEQ ID NO: 278, and having substantially the same biological activity as the SPL15 transcription factor polypeptides represented by SEQ ID NO: 235, SEQ ID NO: 237, SEQ ID NO: 239, SEQ ID NO: 241, SEQ ID NO: 243, SEQ ID NO: 245, SEQ ID NO: 247, SEQ ID NO: 249, SEQ ID NO: 251, SEQ ID NO: 253, SEQ ID NO: 255, SEQ ID NO: 257, SEQ ID NO: 259, SEQ ID NO: 261, SEQ ID NO: 263, SEQ ID NO: 283, SEQ ID NO: 285, SEQ ID NO: 287, or orthologues or paralogues of any of the aforementioned SEQ ID NOs. The hybridising sequence is typically at least 250 nucleotides in length, preferably at least 500 nucleotides in length, more preferably at least 750 nucleotides in length and most preferably at least 1000 nucleotides in length. Preferably, the hybridising sequence is one that is capable of hybridising to any of the nucleic acids represented by SEQ ID NO: 234, SEQ ID NO: 236, SEQ ID NO: 238, SEQ ID NO: 240, SEQ ID NO: 242, SEQ ID NO: 244, SEQ ID NO: 246, SEQ ID NO: 248, SEQ ID NO: 250, SEQ ID NO: 252, SEQ ID NO: 254, SEQ ID NO: 256, SEQ ID NO: 258, SEQ ID NO: 260, SEQ ID NO: 262, SEQ ID NO: 282, SEQ ID NO: 284 and SEQ ID NO: 286, or to a portion of any of the aforementioned sequences, a portion being as defined above. Most preferably the hybridising sequence is capable of hybridising to SEQ ID NO: 234, or to portions thereof.
[0633] Another nucleic acid variant useful in the methods of the invention is a splice variant encoding a SPL15 transcription factor polypeptide as defined hereinabove.
[0634] Preferred splice variants are splice variants of a nucleic acid encoding SPL15 transcription factor polypeptide represented by any of SEQ ID NO: 235, SEQ ID NO: 237, SEQ ID NO: 239, SEQ ID NO: 241, SEQ ID NO: 243, SEQ ID NO: 245, SEQ ID NO: 247, SEQ ID NO: 249, SEQ ID NO: 251, SEQ ID NO: 253, SEQ ID NO: 255, SEQ ID NO: 257, SEQ ID NO: 259, SEQ ID NO: 261, SEQ ID NO: 263, SEQ ID NO: 283, SEQ ID NO: 285, or SEQ ID NO: 287, or splice variants encoding orthologues or paralogues of any of the aforementioned SEQ ID NO: 234, SEQ ID NO: 236, SEQ ID NO: 238, SEQ ID NO: 240, SEQ ID NO: 242, SEQ ID NO: 244, SEQ ID NO: 246, SEQ ID NO: 248, SEQ ID NO: 250, SEQ ID NO: 252, SEQ ID NO: 254, SEQ ID NO: 256, SEQ ID NO: 258, SEQ ID NO: 260, SEQ ID NO: 262, SEQ ID NO: 282, SEQ ID NO: 284 and SEQ ID NO: 286. Most preferred is a splice variant of a nucleic acid as represented by SEQ ID NO: 234.
[0635] Another nucleic acid variant useful in performing the methods of the invention is an allelic variant of a nucleic acid encoding a SPL15 transcription factor polypeptide as defined hereinabove.
[0636] The allelic variant may be an allelic variant of a nucleic acid encoding a SPL15 transcription factor polypeptide represented by any of SEQ ID NO: 235, SEQ ID NO: 237, SEQ ID NO: 239, SEQ ID NO: 241, SEQ ID NO: 243, SEQ ID NO: 245, SEQ ID NO: 247, SEQ ID NO: 249, SEQ ID NO: 251, SEQ ID NO: 253, SEQ ID NO: 255, SEQ ID NO: 257, SEQ ID NO: 259, SEQ ID NO: 261, SEQ ID NO: 263, SEQ ID NO: 283, SEQ ID NO: 285, or SEQ ID NO: 287, or an allelic variant of a nucleic acid encoding orthologues or paralogues of any of the aforementioned SEQ ID NOs. Further preferred are allelic variants of nucleic acids represented by any one of SEQ ID NO: 234, SEQ ID NO: 236, SEQ ID NO: 238, SEQ ID NO: 240, SEQ ID NO: 242, SEQ ID NO: 244, SEQ ID NO: 246, SEQ ID NO: 248, SEQ ID NO: 250, SEQ ID NO: 252, SEQ ID NO: 254, SEQ ID NO: 256, SEQ ID NO: 258, SEQ ID NO: 260, SEQ ID NO: 262, SEQ ID NO: 282, SEQ ID NO: 284 and SEQ ID NO: 286. Most preferred is an allelic variant of a nucleic acid as represented by SEQ ID NO: 234.
[0637] A further nucleic acid variant useful in the methods of the invention is a nucleic acid variant obtained by gene shuffling. Gene shuffling or directed evolution may also be used to generate variants of nucleic acids encoding SPL15 transcription factor polypeptides as defined above.
[0638] Furthermore, nucleic acid variants may also be obtained by site-directed mutagenesis. Several methods are available to achieve site-directed mutagenesis, the most common being PCR based methods (Current Protocols in Molecular Biology. Wiley (Eds)).
[0639] Also useful in the methods of the invention are nucleic acids encoding homologues of any one of the amino acids represented by SEQ ID NO: 235, SEQ ID NO: 237, SEQ ID NO: 239, SEQ ID NO: 241, SEQ ID NO: 243, SEQ ID NO: 245, SEQ ID NO: 247, SEQ ID NO: 249, SEQ ID NO: 251, SEQ ID NO: 253, SEQ ID NO: 255, SEQ ID NO: 257, SEQ ID NO: 259, SEQ ID NO: 261, SEQ ID NO: 263, SEQ ID NO: 283, SEQ ID NO: 285, SEQ ID NO: 287, or orthologues or paralogues of any of the aforementioned SEQ ID NOs.
[0640] Also useful in the methods of the invention are nucleic acids encoding derivatives of any one of the amino acids represented by SEQ ID NO: 235, SEQ ID NO: 237, SEQ ID NO: 239, SEQ ID NO: 241, SEQ ID NO: 243, SEQ ID NO: 245, SEQ ID NO: 247, SEQ ID NO: 249, SEQ ID NO: 251, SEQ ID NO: 253, SEQ ID NO: 255, SEQ ID NO: 257, SEQ ID NO: 259, SEQ ID NO: 261 SEQ ID NO: 263, SEQ ID NO: 283, SEQ ID NO: 285, SEQ ID NO: 287, or orthologues or paralogues of any of the aforementioned SEQ ID NOs. "Derivatives" include peptides, oligopeptides, polypeptides which may, compared to the amino acid sequence of the naturally-occurring form of the protein, such as the one presented in SEQ ID NO: 235, comprise substitutions of amino acids with non-naturally occurring amino acid residues, or additions of non-naturally occurring amino acid residues.
[0641] Furthermore, SPL15 transcription factor polypeptides (at least in their native form) typically have DNA-binding activity and an activation domain. A person skilled in the art may easily determine the presence of an activation domain and DNA-binding activity using routine tools and techniques.
[0642] Nucleic acids encoding SPL15 transcription factor polypeptides may be derived from any natural or artificial source. The nucleic acid may be modified from its native form in composition and/or genomic environment through deliberate human manipulation. Preferably the SPL15 transcription factor polypeptide-encoding nucleic acid is from a plant, further preferably from a dicot, more preferably from the Brassicacea family, most preferably the nucleic acid is from Arabidopsis thaliana.
[0643] Any reference herein to a SPL15 transcription factor polypeptide is therefore taken to mean a SPL15 transcription factor peptide as defined above. Any nucleic acid encoding such a SPL15 transcription factor polypeptide is suitable for use in performing the methods of the invention.
[0644] The invention also provides genetic constructs and vectors to facilitate introduction and/or expression of the nucleic acid sequences useful in the methods according to the invention, in a plant.
[0645] Therefore, there is provided a gene construct comprising:
[0646] (i) A nucleic acid encoding a SPL15 transcription factor polypeptide as defined hereinabove;
[0647] (ii) One or more control sequences operably liked to the nucleic acid of (i).
[0648] Constructs useful in the methods according to the present invention may be constructed using recombinant DNA technology well known to persons skilled in the art. The gene constructs may be inserted into vectors, which may be commercially available, suitable for transforming into plants and suitable for expression of the gene of interest in the transformed cells. The invention therefore provides use of a gene construct as defined hereinabove in the methods of the invention.
[0649] Plants are transformed with a vector comprising the sequence of interest (i.e., a nucleic acid encoding a SPL15 transcription factor polypeptide). The skilled artisan is well aware of the genetic elements that must be present on the vector in order to successfully transform, select and propagate host cells containing the sequence of interest. The sequence of interest is operably linked to one or more control sequences (at least to a promoter).
[0650] Advantageously, any type of promoter, whether natural or synthetic, may be used to drive expression of the nucleic acid sequence. The promoter may be an inducible promoter, i.e. having induced or increased transcription initiation in response to a developmental, chemical, environmental or physical stimulus. The promoter may be a tissue-specific promoter, i.e. one that is capable of preferentially initiating transcription in certain tissues, such as the leaves, roots, seed tissue etc.
[0651] According to the invention, the nucleic acid encoding a SPL15 transcription factor polypeptide is operably linked to a constitutive promoter. The constitutive promoter is preferably a HMGB (high mobility group B) promoter, more preferably the constitutive promoter is a rice HMGB promoter, further preferably the constitutive promoter is represented by a nucleic acid sequence substantially similar to SEQ ID NO: 279, most preferably the constitutive promoter is as represented by SEQ ID NO: 279 or SEQ ID NO: 294.
[0652] It should be clear that the applicability of the present invention is not restricted to the nucleic acid encoding an SPL15 transcription factor polypeptide as represented by SEQ ID NO: 234, nor is the applicability of the invention restricted to expression of a such nucleic acid encoding an SPL15 transcription factor polypeptide when driven by a HMGB promoter. Examples of other constitutive promoters which may also be used perform the methods of the invention are shown in the definitions section.
[0653] Additional regulatory elements for increasing expression of nucleic acids or genes, or gene products, may include transcriptional as well as translational enhancers. Those skilled in the art will be aware of terminator and enhancer sequences that may be suitable for use in performing the invention. Other control sequences (besides promoter, enhancer, silencer, intron sequences, 3'UTR and/or 5'UTR regions) may be protein and/or RNA stabilizing elements. Such sequences would be known or may readily be obtained by a person skilled in the art. Optionally, one or more terminator sequences (also a control sequence) may be used in the construct introduced into a plant.
[0654] An intron sequence may also be added to the 5' untranslated region (UTR) or the coding sequence of the partial coding sequence to increase the amount of the mature message that accumulates in the cytosol.
[0655] The genetic constructs of the invention may further include an origin of replication sequence that is required for maintenance and/or replication in a specific cell type. One example is when a genetic construct is required to be maintained in a bacterial cell as an episomal genetic element (e.g. plasmid or cosmid molecule). Preferred origins of replication include, but are not limited to, the f1-ori and colE1.
[0656] The genetic construct may optionally comprise a selectable marker gene.
[0657] The invention also provides a method for the production of transgenic plants having increased yield relative to control plants, comprising introduction and expression in a plant of a nucleic acid encoding a SPL15 transcription factor polypeptide as defined hereinabove.
[0658] More specifically, the present invention provides a method for the production of transgenic plants having increased yield relative to control plants, which method comprises:
[0659] (i) introducing and expressing a nucleic acid encoding a SPL15 transcription factor polypeptide in a plant cell; and
[0660] (ii) cultivating the plant cell under conditions promoting plant growth and development.
[0661] The nucleic acid may be introduced directly into a plant cell or into the plant itself (including introduction into a tissue, organ or any other part of a plant). According to a preferred feature of the present invention, the nucleic acid is preferably introduced into a plant by transformation.
[0662] Generally after transformation, plant cells or cell groupings are selected for the presence of one or more markers which are encoded by plant-expressible genes co-transferred with the gene of interest, following which the transformed material is regenerated into a whole plant.
[0663] Following DNA transfer and regeneration, putatively transformed plants may be evaluated, for instance using Southern analysis, for the presence of the gene of interest, copy number and/or genomic organisation. Alternatively or additionally, expression levels of the newly introduced DNA may be monitored using Northern and/or Western analysis, or quantitative PCR, all techniques being well known to persons having ordinary skill in the art.
[0664] The generated transformed plants may be propagated by a variety of means, such as by clonal propagation or classical breeding techniques. For example, a first generation (or T1) transformed plant may be selfed to give homozygous second generation (or T2) transformants, and the T2 plants further propagated through classical breeding techniques.
[0665] The generated transformed organisms may take a variety of forms. For example, they may be chimeras of transformed cells and non-transformed cells; clonal transformants (e.g., all cells transformed to contain the expression cassette); grafts of transformed and untransformed tissues (e.g., in plants, a transformed rootstock grafted to an untransformed scion).
[0666] The present invention clearly extends to any plant cell or plant produced by any of the methods described herein, and to all plant parts and propagules thereof. The present invention extends further to encompass the progeny of a primary transformed or transfected cell, tissue, organ or whole plant that has been produced by any of the aforementioned methods, the only requirement being that progeny exhibit the same genotypic and/or phenotypic characteristic(s) as those produced by the parent in the methods according to the invention.
[0667] The invention also includes host cells containing an isolated nucleic acid encoding a SPL15 transcription factor polypeptide as defined hereinabove. Preferred host cells according to the invention are plant cells.
[0668] The invention also extends to harvestable parts of a plant such as, but not limited to seeds, leaves, fruits, flowers, stems, rhizomes, tubers and bulbs. The invention furthermore relates to products derived, preferably directly derived, from a harvestable part of such a plant, such as dry pellets or powders, oil, fat and fatty acids, starch or proteins.
[0669] As mentioned above, a preferred method for increasing expression of a nucleic acid encoding a SPL15 transcription factor polypeptide is by introducing and expressing in a plant a nucleic acid encoding a SPL15 transcription factor polypeptide; however the effects of performing the method, i.e. increasing yield, may also be achieved using other well known techniques. A description of some of these techniques will now follow.
[0670] One such technique is T-DNA activation tagging. The resulting transgenic plants show dominant phenotypes due to modified expression of genes close to the introduced promoter.
[0671] The effects of the invention may also be reproduced using the technique of TILLING (Targeted Induced Local Lesions In Genomes).
[0672] The effects of the invention may also be reproduced using homologous recombination.
[0673] "Increased yield" as defined herein is taken to mean an increase in biomass (weight) of one or more parts of a plant, which may include aboveground (harvestable) parts and/or (harvestable) parts below ground.
[0674] In particular, such harvestable parts include vegetative biomass and/or seeds, and performance of the methods of the invention results in plants having increased yield (in vegetative biomass and/or seed) relative to the yield of control plants.
[0675] Taking corn as an example, a yield increase may be manifested as one or more of the following: increase in the number of plants per hectare or acre, an increase in the number of ears per plant, an increase in the number of rows, number of kernels per row, kernel weight, thousand kernel weight, ear length/diameter, increase in the seed filling rate (which is the number of filled seeds divided by the total number of seeds and multiplied by 100), among others. Taking rice as an example, a yield increase may manifest itself as an increase in one or more of the following: number of plants per hectare or acre, number of panicles per plant, number of spikelets per panicle, number of flowers (florets) per panicle (which is expressed as a ratio of the number of filled seeds over the number of primary panicles), increase in the seed filling rate (which is the number of filled seeds divided by the total number of seeds and multiplied by 100), increase in thousand kernel weight, among others.
[0676] Since the transgenic plants according to the present invention have increased yield, it is likely that these plants exhibit an increased growth rate (during at least part of their life cycle), relative to the growth rate of corresponding wild type plants at a corresponding stage in their life cycle. The increased growth rate may be specific to one or more parts of a plant (including seeds), or may be throughout substantially the whole plant. A plant having an increased growth rate may even exhibit early flowering. The increase in growth rate may alter the harvest cycle of a plant allowing plants to be sown later and/or harvested sooner than would otherwise be possible. If the growth rate is sufficiently increased, it may allow for the further sowing of seeds of the same plant species (for example sowing and harvesting of rice plants followed by sowing and harvesting of further rice plants all within one conventional growing period). Similarly, if the growth rate is sufficiently increased, it may allow for the further sowing of seeds of different plants species (for example the sowing and harvesting of corn plants followed by, for example, the sowing and optional harvesting of soy bean, potato or any other suitable plant). Harvesting additional times from the same rootstock in the case of some crop plants may also be possible. Altering the harvest cycle of a plant may lead to an increase in annual biomass production per acre (due to an increase in the number of times (say in a year) that any particular plant may be grown and harvested). An increase in growth rate may also allow for the cultivation of transgenic plants in a wider geographical area than their wild-type counterparts, since the territorial limitations for growing a crop are often determined by adverse environmental conditions either at the time of planting (early season) or at the time of harvesting (late season). Such adverse conditions may be avoided if the harvest cycle is shortened. The growth rate may be determined by deriving various parameters from growth curves, such parameters may be: T-Mid (the time taken for plants to reach 50% of their maximal size) and T-90 (time taken for plants to reach 90% of their maximal size), amongst others.
[0677] According to a preferred feature of the present invention, performance of the methods of the invention gives plants having an increased growth rate relative to control plants. Therefore, according to the present invention, there is provided a method for increasing the growth rate of plants, which method comprises increasing expression in a plant of a nucleic acid encoding a SPL15 transcription factor polypeptide
[0678] An increase in yield and/or growth rate occurs whether the plant is under non-stress conditions or whether the plant is exposed to various stresses compared to control plants. Plants typically respond to exposure to stress by growing more slowly. In conditions of severe stress, the plant may even stop growing altogether. Mild stress on the other hand is defined herein as being any stress to which a plant is exposed which does not result in the plant ceasing to grow altogether without the capacity to resume growth. Mild stress in the sense of the invention leads to a reduction in the growth of the stressed plants of less than 40%, 35% or 30%, preferably less than 25%, 20% or 15%, more preferably less than 14%, 13%, 12%, 11% or 10% or less in comparison to the control plant under non-stress conditions. Due to advances in agricultural practices (irrigation, fertilization, pesticide treatments) severe stresses are not often encountered in cultivated crop plants. As a consequence, the compromised growth induced by mild stress is often an undesirable feature for agriculture. Mild stresses are the everyday biotic and/or abiotic (environmental) stresses to which a plant is exposed. Abiotic stresses may be due to drought or excess water, anaerobic stress, salt stress, chemical toxicity, oxidative stress and hot, cold or freezing temperatures. The abiotic stress may be an osmotic stress caused by a water stress (particularly due to drought), salt stress, oxidative stress or an ionic stress. Biotic stresses are typically those stresses caused by pathogens, such as bacteria, viruses, fungi and insects.
[0679] In particular, the methods of the present invention may be performed under non-stress conditions or under conditions of mild drought to give plants having increased yield relative to control plants. As reported in Wang et al. (Planta (2003) 218: 1-14), abiotic stress leads to a series of morphological, physiological, biochemical and molecular changes that adversely affect plant growth and productivity. Drought, salinity, extreme temperatures and oxidative stress are known to be interconnected and may induce growth and cellular damage through similar mechanisms. Rabbani et al. (Plant Physiol (2003) 133: 1755-1767) describes a particularly high degree of "cross talk" between drought stress and high-salinity stress. For example, drought and/or salinisation are manifested primarily as osmotic stress, resulting in the disruption of homeostasis and ion distribution in the cell. Oxidative stress, which frequently accompanies high or low temperature, salinity or drought stress, may cause denaturing of functional and structural proteins. As a consequence, these diverse environmental stresses often activate similar cell signaling pathways and cellular responses, such as the production of stress proteins, up-regulation of anti-oxidants, accumulation of compatible solutes and growth arrest. The term "non-stress" conditions as used herein are those environmental conditions that allow optimal growth of plants. Persons skilled in the art are aware of normal soil conditions and climatic conditions for a given location.
[0680] Performance of the methods of the invention gives plants grown under non-stress conditions or under mild drought conditions increased yield relative to control plants grown under comparable conditions. Therefore, according to the present invention, there is provided a method for increasing yield in plants grown under non-stress conditions or under mild drought conditions, which method comprises increasing expression in a plant of a nucleic acid encoding a MADS15 polypeptide.
[0681] Performance of the methods of the invention gives plants grown under conditions of nutrient deficiency, particularly under conditions of nitrogen deficiency, increased yield relative to control plants grown under comparable conditions. Therefore, according to the present invention, there is provided a method for increasing yield in plants grown under conditions of nutrient deficiency, which method comprises increasing expression in a plant of a nucleic acid encoding a MADS15 polypeptide. Nutrient deficiency may result from a lack or excess of nutrients such as nitrogen, phosphates and other phosphorous-containing compounds, potassium, calcium, cadmium, magnesium, manganese, iron and boron, amongst others.
[0682] The methods of the invention are advantageously applicable to any plant.
[0683] Plants that are particularly useful in the methods of the invention include all plants which belong to the superfamily Viridiplantae, in particular monocotyledonous and dicotyledonous plants including fodder or forage legumes, ornamental plants, food crops, trees or shrubs. According to a preferred embodiment of the present invention, the plant is a crop plant. Examples of crop plants include soybean, sunflower, canola, alfalfa, rapeseed, cotton, tomato, potato and tobacco. Further preferably, the plant is a monocotyledonous plant. Examples of monocotyledonous plants include sugarcane. More preferably the plant is a cereal. Examples of cereals include rice, maize, wheat, barley, millet, rye, triticale, sorghum and oats.
[0684] The present invention also encompasses plants obtainable by the methods according to the present invention. The present invention therefore provides plants, parts and cells from such plants obtainable by the methods according to the present invention, which plants or parts or cells comprise a nucleic acid transgene encoding a SPL15 transcription factor polypeptide as defined above.
[0685] The present invention also encompasses use of nucleic acids encoding SPL15 transcription factor polypeptides in increasing yield in a plant compared to yield in a control plant.
[0686] One such use relates to increasing yield of plants, yield being defined as defined herein above. Yield may in particular include one or more of the following: increased aboveground biomass, increased number of flowers per panicle, increased seed yield, increased total number of seeds, increased number of filled seeds, increased thousand kernel weight (TKW) and increased harvest index.
[0687] Nucleic acids encoding SPL15 transcription factor polypeptides may find use in breeding programmes in which a DNA marker is identified which may be genetically linked to a gene encoding SPL15 transcription factor polypeptide. Nucleic acids encoding SPL15 transcription factor polypeptides may be used to define a molecular marker. This marker may then be used in breeding programmes to select plants having increased seed yield. The nucleic acids encoding SPL15 transcription factor polypeptides may be, for example, a nucleic acid as represented by any one of SEQ ID NO: 234, SEQ ID NO: 236, SEQ ID NO: 238, SEQ ID NO: 240, SEQ ID NO: 242, SEQ ID NO: 244, SEQ ID NO: 246, SEQ ID NO: 248, SEQ ID NO: 250, SEQ ID NO: 252, SEQ ID NO: 254, SEQ ID NO: 256, SEQ ID NO: 258, SEQ ID NO: 260, SEQ ID NO: 262, SEQ ID NO: 282, SEQ ID NO: 284 and SEQ ID NO: 286.
[0688] Allelic variants of a nucleic acid encoding an SPL15 transcription factor polypeptide may also find use in marker-assisted breeding programmes. Such breeding programmes sometimes require introduction of allelic variation by mutagenic treatment of the plants, using for example EMS mutagenesis; alternatively, the programme may start with a collection of allelic variants of so called "natural" origin caused unintentionally. Identification of allelic variants then takes place, for example, by PCR. This is followed by a step for selection of superior allelic variants of the sequence in question and which give increased seed yield. Selection is typically carried out by monitoring growth performance of plants containing different allelic variants of the sequence in question, for example, different allelic variants of any one of SEQ ID NO: 234, SEQ ID NO: 236, SEQ ID NO: 238, SEQ ID NO: 240, SEQ ID NO: 242, SEQ ID NO: 244, SEQ ID NO: 246, SEQ ID NO: 248, SEQ ID NO: 250, SEQ ID NO: 252, SEQ ID NO: 254, SEQ ID NO: 256, SEQ ID NO: 258, SEQ ID NO: 260, SEQ ID NO: 262, SEQ ID NO: 282, SEQ ID NO: 284 and SEQ ID NO: 286. Growth performance may be monitored in a greenhouse or in the field. Further optional steps include crossing plants, in which the superior allelic variant was identified, with another plant. This could be used, for example, to make a combination of interesting phenotypic features.
[0689] Nucleic acids encoding SPL15 transcription factor polypeptides may also be used as probes for genetically and physically mapping the genes that they are a part of, and as markers for traits linked to those genes. Such information may be useful in plant breeding in order to develop lines with desired phenotypes. Such use of nucleic acids encoding SPL15 transcription factor polypeptides requires only a nucleic acid sequence of at least 15 nucleotides in length. The nucleic acids encoding SPL15 transcription factor polypeptides may be used as restriction fragment length polymorphism (RFLP) markers. Southern blots (Sambrook J, Fritsch E F and Maniatis T (1989) Molecular Cloning, A Laboratory Manual) of restriction-digested plant genomic DNA may be probed with a nucleic acid encoding SPL15 transcription factor polypeptide. The resulting banding patterns may then be subjected to genetic analyses using computer programs such as MapMaker (Lander et al. (1987) Genomics 1: 174-181) in order to construct a genetic map. In addition, the nucleic acid may be used to probe Southern blots containing restriction endonuclease-treated genomic DNAs of a set of individuals representing parent and progeny of a defined genetic cross. Segregation of the DNA polymorphisms is noted and used to calculate the position of the nucleic acid encoding SPL15 transcription factor polypeptide in the genetic map previously obtained using this population (Botstein et al. (1980) Am. J. Hum. Genet. 32: 314-331).
[0690] The production and use of plant gene-derived probes for use in genetic mapping is described in Bernatzky and Tanksley (GENETICS 112 (4): 887-898, 1986). Numerous publications describe genetic mapping of specific cDNA clones using the methodology outlined above or variations thereof. For example, F2 intercross populations, backcross populations, randomly mated populations, near isogenic lines (NIL), and other sets of individuals may be used for mapping. Such methodologies are well known to those skilled in the art.
[0691] The nucleic acid probes may also be used for physical mapping (i.e., placement of sequences on physical maps; see Hoheisel et al. In: Non-mammalian Genomic Analysis: A Practical Guide, Academic press 1996, pp. 319-346, and references cited therein).
[0692] In another embodiment, the nucleic acid probes may be used in direct fluorescence in situ hybridization (FISH) mapping (Trask (1991) Trends Genet. 7:149-154). Although current methods of FISH mapping favour use of large clones (several kb to several hundred kb; see Laan et al. (1995) Genome Res. 5:13-20), improvements in sensitivity may allow performance of FISH mapping using shorter probes.
[0693] A variety of nucleic acid amplification-based methods for genetic and physical mapping may be carried out using the nucleic acids. Examples include allele-specific amplification (Kazazian (1989) J. Lab. Clin. Med. 11:95-96), polymorphism of PCR-amplified fragments (CAPS; Sheffield et al. (1993) Genomics 16:325-332), allele-specific ligation (Landegren et al. (1988) Science 241:1077-1080), nucleotide extension reactions (Sokolov (1990) Nucleic Acid Res. 18:3671), Radiation Hybrid Mapping (Walter et al. (1997) Nat. Genet. 7:22-28) and Happy Mapping (Dear and Cook (1989) Nucleic Acid Res. 17:6795-6807). For these methods, the sequence of a nucleic acid is used to design and produce primer pairs for use in the amplification reaction or in primer extension reactions. The design of such primers is well known to those skilled in the art. In methods employing PCR-based genetic mapping, it may be necessary to identify DNA sequence differences between the parents of the mapping cross in the region corresponding to the instant nucleic acid sequence. This, however, is generally not necessary for mapping methods.
[0694] The methods according to the present invention result in plants having increased yield, as described hereinbefore. These traits may also be combined with other economically advantageous traits, such as further yield-increasing traits, tolerance to other abiotic and biotic stresses, traits modifying various architectural features and/or biochemical and/or physiological features.
DESCRIPTION OF FIGURES
[0695] The present invention will now be described with reference to the following figures in which:
[0696] FIG. 1 represents the schematic structure of the AtHAL3a polypeptide comprising from N-terminus to C-terminus (i) the substrate binding helix (single underlined), (ii) the insertion His motif (double underlined), (iii) the PXMNXXMW motif (dotted) and (iv) the substrate recognition clamp (wave underlined). The N-terminal and C-terminal ends of the HAL3 proteins are not very conserved.
[0697] FIG. 2 shows a binary vector pEXP::HAL3, for increased expression in Oryza sativa of an Arabidopsis thaliana HAL3 nucleic acid under the control of a beta expansin promoter.
[0698] FIG. 3 shows a multiple alignment of a number of HAL3 sequences from Arabidopsis thaliana AtHAL3b (At_NP--973994), Sorghum bicolor (Sb), Arabidopsis thaliana AtHAL3a (Ath0218), Gossipium hirsutum (Cg), Hordeum vulgare (Hv), Oryza sativa (Os), Vitis vinifera (Vv), Glycine max (Gm), Solanum tuberosum (St), Zea mays (Zm), and Pinus sp (Pg).
[0699] FIG. 4 details examples of sequences useful in performing the methods according to the present invention: SEQ ID NO: 1 and SEQ ID NO: 2 represent the nucleic acid sequence and the protein sequence of AtHAl3a. SEQ ID NO: 3 and SEQ ID NO: 4 are the sequences of the forward and reverse primers used to isolate the AtHAL3 gene. SEQ ID NO: 5 is the sequence of the beta expansin promoter used in the methods of the present invention. SEQ ID NO: 9 to SEQ ID NO: 42 represent examples of full length or partial DNA/protein sequences, useful in the methods of the invention or for isolating corresponding full length sequences. In some cases, sequences were assembled from EST sequences, with lesser quality sequencing. As a consequence, a few nucleic acid substitutions may be expected.
[0700] FIG. 5 (A) shows the domain structure of the MADS15 protein, (B) represents the sequence of SEQ ID NO: 44 with the MADS domain in bold and the keratin domain in italics. Between the MADS domain and the keratin, the intervening domain is located while the C-domain is located C-terminally of the keratin domain.
[0701] FIG. 6 shows a multiple alignment of various MADS15 proteins. The asterisks indicate identical amino acids, the colons represent highly conservative substitutions, the dots represent less conserved substitutions.
[0702] FIG. 7 shows the binary vector for increased expression in Oryza sativa of an Oryza sativa MADS15 protein-encoding nucleic acid under the control of a GOS2 promoter.
[0703] FIG. 8 details examples of sequences useful in performing the methods according to the present invention.
[0704] FIG. 9 is a schematic representation of a full-length OsMADS15 polypeptide. The typical domains (M, MADS; I, intervening domain; K, keratin K-box region; C, variable C-terminal region) are indicated, the lines above and below the diagram show the regions of the protein involved in DNA binding, dimerisation and in multimerisation with interacting proteins.
[0705] FIG. 10 shows the binary vector pGOS::MADS15hp for MADS15 RNA silencing in Oryza sativa, using a hairpin construct under the control of a constitutive promoter (GOS2).
[0706] FIG. 11 details examples of sequences useful in performing the methods according to the present invention, or useful in isolating such sequences. Sequences may result from public EST assemblies, with lesser quality sequencing. As a consequence, a few nucleic acid substitutions may be expected. The start (ATG) and stop codons delimit the nucleic acid sequences when these encode full-length MADS15 polypeptides. However both 5' and 3' UTR may also be used for the performing the methods of the invention.
[0707] FIG. 12 shows the schematic classification of the AP2/ERF transcription factor polypeptides according to the domains present: the AP2 subfamily with two AP2 repeats and the ERF subfamily with only one AP2 repeat. The ERF subfamily is further subdivided into transcription factors comprising a B3 domain in addition to the AP2 repeat (called RAV), or not. The PLT transcription factor polypeptides belong to the AP2 subfamily with two AP2 repeats.
[0708] FIG. 13 represents a schematic presentation of the PLT transcription factor polypeptide structure. The AP2 domain comprises the two AP2 repeats (boxed) separated by a linker region. The approximate positions of the nuclear localisation signal (NLS), of motif 1 PK(V/L)(A/E)DFLG and of motif 2 (V/L)FX(M/V)WN(D/E) are marked as boxes.
[0709] FIG. 14 is an alignment of PLT transcription factor polypeptide sequences (from Table 4), compared to other AP2 domain transcription factor polypeptides (from Table 4 below). The nuclear localisation signal (NLS), motif 1 PK(V/L)(A/E)DFLG and motif 2 (V/L)FX(M/V)WN(D/E) are boxed. Identical residues are blackened, conservative residues are grayed.
TABLE-US-00020 TABLE 4 AP2 domain transcription factor polypeptides aligned against the PLT transcription factor polypeptides used to perform the methods of the invention. Name NCBI accession number Source Arath_ANT NM_119937 Arabidopsis thaliana Arath_BBM NM_121749.1 Arabidopsis thaliana Brana_BBM1 AF317904 Brassica napus Brana_BBM2 AF317905 Brassica napus Medtr_AP2 BBM AY899909 Medicago truncatula Nicta_ANT like AY461432 Nicotiana tabacum Orysa_AP2 XM_473084 Oryza sativa Pinth_ANTL1 AB101585 Pinus thunbergii Zeama_AP2 AY109146.1 Zea mays
[0710] FIG. 15 is a photograph of 40 seeds from a control plant (left) compared to 40 seeds from a transgenic plant with increased expression of a PLT transcription factor polypeptide.
[0711] FIG. 16 shows a binary vector pGOS2::PLT, for increased expression in Oryza sativa of an Oryza sativa PLT transcription factor nucleic acid under the control of a GOS2 promoter.
[0712] FIG. 17 details examples of sequences useful in performing the methods according to the present invention (SEQ ID NO: 175 to SEQ ID NO: 188). Partial sequences (SEQ ID NO: 189 and SEQ ID NO: 190) useful in isolating corresponding full length sequences are also presented.
[0713] FIG. 18 shows an alignment of bHLH transcription factors as defined hereinabove. The sequences were aligned using AlignX program from Vector NTI suite (InforMax, Bethesda, Md.). Multiple alignment was done with a gap opening penalty of 10 and a gap extension of 0.01. Minor manual editing was also carried out where necessary to better position some conserved regions. The bHLH domain is indicated within the solid box and the PFB26111 domain is indicated within the dashed box. Also indicated by the three upwardly pointing arrows is the 5-9-13 configuration (K/R ER). The single downwardly pointing arrow indicates the glutamic acid reside for recognition of the E-BOX.
[0714] FIG. 19 shows a matrix of similarity and identity between bHLH transcription factors from various species. Percentage identity is shown in bold. FIG. 19a is a matrix over full length sequences and FIG. 19b is a matrix over the bHLH domain only. More details are provided in Example 34.
[0715] FIG. 20 shows a binary vector pGOS2::bHLH, for increased expression in Oryza sativa of an Oryza sativa bHLH transcription factor-encoding nucleic acid under the control of a GOS2 promoter.
[0716] FIG. 21 details examples of sequences useful in performing the methods according to the present invention.
[0717] FIG. 22 Neighbour-joining tree output after a multiple sequence alignment of all Arabidopsis thaliana SPL transcription factor polypeptides and of SPL15 transcription factor polypeptide orthologues using CLUSTAL W (1.83) (at GenomeNet service at the Kyoto University Bioinformatics Center), and default values (Blosum 62 as weight matrix, gap open penalty of 10; gap extension penalty of 0.05). Arabidopsis thaliana SPL15 transcription factor polypeptide clusters with the other SPL15 transcription factor polypeptide orthologues and paralogues, as shown by the curly bracket.
[0718] FIG. 23 shows an alignment of the DNA-binding domain (DBD) of SPL15 transcription factor polypeptide orthologues and paralogs. The sequences were aligned using AlignX program from Vector NTI suite (InforMax, Bethesda, Md.). Multiple alignment was done with a gap opening penalty of 10 and a gap extension of 0.01. The conserved Cys and His residues involved in zinc ion binding are boxed. The bipartite nuclear localization signal (NLS) is underlined.
[0719] FIG. 24 is an alignment of SPL15 transcription factor polypeptide orthologues and paralogues. The sequences were aligned using AlignX program from Vector NTI suite (InforMax, Bethesda, Md.). Multiple alignment was done with a gap opening penalty of 10 and a gap extension of 0.01. Minor manual editing was also carried out where necessary to better position some conserved regions. The three main characterized domains, from N-terminal to C-terminal, are boxed and identified as Motif 1, the SPL DNA binding domain and Motif 2. Additionally, the G/S rich stretch preceding the SPL DBD is marked with Xs and the W(S/T)L tripeptide at the C-terminal end of the polypeptide also boxed.
[0720] FIG. 25 shows a binary vector pHMGB::SPL15, for increased expression in Oryza sativa of an Arabidopsis thaliana nucleic acid encoding an SPL15 transcription factor polypeptide under the control of an HMGB promoter.
[0721] FIG. 26 details examples of sequences useful in performing the methods according to the present invention.
EXAMPLES
[0722] The present invention will now be described with reference to the following examples, which are by way of illustration alone. The following examples are not intended to completely define or otherwise limit the scope of the invention.
[0723] DNA manipulation: unless otherwise stated, recombinant DNA techniques are performed according to standard protocols described in (Sambrook (2001) Molecular Cloning: a laboratory manual, 3rd Edition Cold Spring Harbor Laboratory Press, CSH, New York) or in Volumes 1 and 2 of Ausubel et al. (1994), Current Protocols in Molecular Biology, Current Protocols. Standard materials and methods for plant molecular work are described in Plant Molecular Biology Labfax (1993) by R.D.D. Croy, published by BIOS Scientific Publications Ltd (UK) and Blackwell Scientific Publications (UK).
Example Section A
HAL3
Example 1
Identification of Sequences Related to the Nucleic Acid Sequence Used in the Methods of the Invention
[0724] Sequences (full length cDNA, ESTs or genomic) related to the nucleic acid sequence used in the methods of the present invention were identified amongst those maintained in the Entrez Nucleotides database at the National Center for Biotechnology Information (NCBI) using database sequence search tools, such as the Basic Local Alignment Tool (BLAST) (Altschul et al. (1990) J. Mol. Biol. 215:403-410; and Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402). The program is used to find regions of local similarity between sequences by comparing nucleic acid or polypeptide sequences to sequence databases and by calculating the statistical significance of matches. For example, the polypeptide encoded by the nucleic acid of the present invention was used for the TBLASTN algorithm, with default settings and the filter to ignore low complexity sequences set off. The output of the analysis was viewed by pairwise comparison, and ranked according to the probability score (E-value), where the score reflect the probability that a particular alignment occurs by chance (the lower the E-value, the more significant the hit). In addition to E-values, comparisons were also scored by percentage identity. Percentage identity refers to the number of identical nucleotides (or amino acids) between the two compared nucleic acid (or polypeptide) sequences over a particular length. In some instances, the default parameters may be adjusted to modify the stringency of the search. For example the E-value may be increased to show less stringent matches. This way, short nearly exact matches may be identified.
[0725] Table A provides a list of nucleic acid sequences related to the nucleic acid sequence used in the methods of the present invention.
TABLE-US-00021 TABLE A Examples of HAL3 polypeptides: Nucleic acid Protein Plant Source SEQ ID NO: SEQ ID NO: Arabidopsis thaliana 9 10 Oryza sativa 11 12 Triticum aestivum 13 14 Zea mays 15 16 Picea abies 17 18 Brassica napus 19 20 Brassica oleracea 21 22 Nicotiana tabacum 23 24 Solanum tuberosum 25 26 Glycine max 27 28 Vitis vinifera 29 30 Hordeum vulgare 31 32 Gossypium hirsutum 33 34 Sorghum bicolor 35 36 Lycopersicon esculentum 37 38 Arabidopsis thaliana 39 40 Pinus sp. 41 42
[0726] In some instances, related sequences have tentatively been assembled and publicly disclosed by research institutions, such as The Institute for Genomic Research (TIGR). The Eukaryotic Gene Orthologs (EGO) database may be used to identify such related sequences, either by keyword search or by using the BLAST algorithm with the nucleic acid or polypeptide sequence of interest.
Example 2
Cloning of the Nucleic Acid Sequence Used in the Methods of the Invention
[0727] The nucleic acid sequence used in the methods of the invention was amplified by PCR using as template a custom-made Arabidopsis thaliana seedlings cDNA library (in pCMV Sport 6.0; Invitrogen, Paisley, UK). PCR was performed using Hifi Taq DNA polymerase in standard conditions, using 200 ng of template in a 50 μl PCR mix. The primers used were prm00957 (SEQ ID NO: 3; sense, start codon in bold:
TABLE-US-00022 5' aaaaagcaggctcacaatggagaatgggaaaagagac 3')
and prm00958 (SEQ ID NO: 4; reverse, complementary,:
TABLE-US-00023 5' agaaagctgggttggttttaactagttccaccg 3'),
which include the AttB sites for Gateway recombination. The amplified PCR fragment was purified also using standard methods. The first step of the Gateway procedure, the BP reaction, was then performed, during which the PCR fragment recombines in vivo with the pDONR201 plasmid to produce, according to the Gateway terminology, an "entry clone", pHAL3. Plasmid pDONR201 was purchased from Invitrogen, as part of the Gateway® technology.
Example 3
Expression Vector Construction
[0728] The entry clone pHAL3 was subsequently used in an LR reaction with pEXP, a destination vector used for Oryza sativa transformation. This vector contains as functional elements within the T-DNA borders: a plant selectable marker; a screenable marker expression cassette; and a Gateway cassette intended for LR in vivo recombination with the nucleic acid sequence of interest already cloned in the entry clone. A rice beta expansin promoter (SEQ ID NO: 5) for shoot-specific expression was located upstream of this Gateway cassette.
[0729] After the LR recombination step, the resulting expression vector pEXP::HAL3 (FIG. 2) was transformed into Agrobacterium strain LBA4044 and subsequently to Oryza sativa plants.
Example 4
Crop Transformation
[0730] The transformed Agrobacterium containing the expression vectors were used independently to transform Oryza sativa plants. Mature dry seeds of the rice japonica cultivar Nipponbare were dehusked. Sterilization was carried out by incubating for one minute in 70% ethanol, followed by 30 minutes in 0.2% HgCl2, followed by a 6 times 15 minutes wash with sterile distilled water. The sterile seeds were then germinated on a medium containing 2,4-D (callus induction medium). After incubation in the dark for four weeks, embryogenic, scutellum-derived calli were excised and propagated on the same medium. After two weeks, the calli were multiplied or propagated by subculture on the same medium for another 2 weeks. Embryogenic callus pieces were sub-cultured on fresh medium 3 days before co-cultivation (to boost cell division activity).
[0731] Agrobacterium strain LBA4404 containing the expression vector was used for co-cultivation. Agrobacterium was inoculated on AB medium with the appropriate antibiotics and cultured for 3 days at 28° C. The bacteria were then collected and suspended in liquid co-cultivation medium to a density (OD600) of about 1. The suspension was then transferred to a Petri dish and the calli immersed in the suspension for 15 minutes. The callus tissues were then blotted dry on a filter paper and transferred to solidified, co-cultivation medium and incubated for 3 days in the dark at 25° C. Co-cultivated calli were grown on 2,4-D-containing medium for 4 weeks in the dark at 28° C. in the presence of a selection agent. During this period, rapidly growing resistant callus islands developed. After transfer of this material to a regeneration medium and incubation in the light, the embryogenic potential was released and shoots developed in the next four to five weeks. Shoots were excised from the calli and incubated for 2 to 3 weeks on an auxin-containing medium from which they were transferred to soil. Hardened shoots were grown under high humidity and short days in a greenhouse.
[0732] The primary transformants were transferred from a tissue culture chamber to a greenhouse. After a quantitative PCR analysis to verify copy number of the T-DNA insert, only single copy transgenic plants that exhibit tolerance to the selection agent were kept for harvest of T1 seed. Seeds were then harvested three to five months after transplanting. The method yielded single locus transformants at a rate of over 50% (Aldemita and Hodges1996, Chan et al. 1993, Hiei et al. 1994).
Corn Transformation
[0733] Transformation of maize (Zea mays) is performed with a modification of the method described by Ishida et al. (1996) Nature Biotech 14(6): 745-50. Transformation is genotype-dependent in corn and only specific genotypes are amenable to transformation and regeneration. The inbred line A188 (University of Minnesota) or hybrids with A188 as a parent are good sources of donor material for transformation, but other genotypes can be used successfully as well. Ears are harvested from corn plant approximately 11 days after pollination (DAP) when the length of the immature embryo is about 1 to 1.2 mm. Immature embryos are cocultivated with Agrobacterium tumefaciens containing the expression vector, and transgenic plants are recovered through organogenesis. Excised embryos are grown on callus induction medium, then maize regeneration medium, containing the selection agent (for example imidazolinone but various selection markers can be used). The Petri plates are incubated in the light at 25° C. for 2-3 weeks, or until shoots develop. The green shoots are transferred from each embryo to maize rooting medium and incubated at 25° C. for 2-3 weeks, until roots develop. The rooted shoots are transplanted to soil in the greenhouse. T1 seeds are produced from plants that exhibit tolerance to the selection agent and that contain a single copy of the T-DNA insert.
Wheat Transformation
[0734] Transformation of wheat is performed with the method described by Ishida et al. (1996) Nature Biotech 14(6): 745-50. The cultivar Bobwhite (available from CIMMYT, Mexico) is commonly used in transformation. Immature embryos are co-cultivated with Agrobacterium tumefaciens containing the expression vector, and transgenic plants are recovered through organogenesis. After incubation with Agrobacterium, the embryos are grown in vitro on callus induction medium, then regeneration medium, containing the selection agent (for example imidazolinone but various selection markers can be used). The Petri plates are incubated in the light at 25° C. for 2-3 weeks, or until shoots develop. The green shoots are transferred from each embryo to rooting medium and incubated at 25° C. for 2-3 weeks, until roots develop. The rooted shoots are transplanted to soil in the greenhouse. T1 seeds are produced from plants that exhibit tolerance to the selection agent and that contain a single copy of the T-DNA insert.
Soybean Transformation
[0735] Soybean is transformed according to a modification of the method described in the Texas A&M U.S. Pat. No. 5,164,310. Several commercial soybean varieties are amenable to transformation by this method. The cultivar Jack (available from the Illinois Seed foundation) is commonly used for transformation. Soybean seeds are sterilised for in vitro sowing. The hypocotyl, the radicle and one cotyledon are excised from seven-day old young seedlings. The epicotyl and the remaining cotyledon are further grown to develop axillary nodes. These axillary nodes are excised and incubated with Agrobacterium tumefaciens containing the expression vector. After the cocultivation treatment, the explants are washed and transferred to selection media. Regenerated shoots are excised and placed on a shoot elongation medium. Shoots no longer than 1 cm are placed on rooting medium until roots develop. The rooted shoots are transplanted to soil in the greenhouse. T1 seeds are produced from plants that exhibit tolerance to the selection agent and that contain a single copy of the T-DNA insert.
Rapeseed/Canola Transformation
[0736] Cotyledonary petioles and hypocotyls of 5-6 day old young seedling are used as explants for tissue culture and transformed according to Babic et al. (1998, Plant Cell Rep 17: 183-188). The commercial cultivar Westar (Agriculture Canada) is the standard variety used for transformation, but other varieties can also be used. Canola seeds are surface-sterilized for in vitro sowing. The cotyledon petiole explants with the cotyledon attached are excised from the in vitro seedlings, and inoculated with Agrobacterium (containing the expression vector) by dipping the cut end of the petiole explant into the bacterial suspension. The explants are then cultured for 2 days on MSBAP-3 medium containing 3 mg/l BAP, 3% sucrose, 0.7% Phytagar at 23° C., 16 hr light. After two days of co-cultivation with Agrobacterium, the petiole explants are transferred to MSBAP-3 medium containing 3 mg/l BAP, cefotaxime, carbenicillin, or timentin (300 mg/l) for 7 days, and then cultured on MSBAP-3 medium with cefotaxime, carbenicillin, or timentin and selection agent until shoot regeneration. When the shoots are 5-10 mm in length, they are cut and transferred to shoot elongation medium (MSBAP-0.5, containing 0.5 mg/l BAP). Shoots of about 2 cm in length are transferred to the rooting medium (MS0) for root induction. The rooted shoots are transplanted to soil in the greenhouse. T1 seeds are produced from plants that exhibit tolerance to the selection agent and that contain a single copy of the T-DNA insert.
Alfalfa Transformation
[0737] A regenerating clone of alfalfa (Medicago sativa) is transformed using the method of (McKersie et al., 1999 Plant Physiol 119: 839-847). Regeneration and transformation of alfalfa is genotype dependent and therefore a regenerating plant is required. Methods to obtain regenerating plants have been described. For example, these can be selected from the cultivar Rangelander (Agriculture Canada) or any other commercial alfalfa variety as described by Brown D C W and A Atanassov (1985. Plant Cell Tissue Organ Culture 4: 111-112). Alternatively, the RA3 variety (University of Wisconsin) has been selected for use in tissue culture (Walker et al., 1978 Am J Bot 65:654-659). Petiole explants are cocultivated with an overnight culture of Agrobacterium tumefaciens C58C1 pMP90 (McKersie et al., 1999 Plant Physiol 119: 839-847) or LBA4404 containing the expression vector. The explants are cocultivated for 3 d in the dark on SH induction medium containing 288 mg/L Pro, 53 mg/L thioproline, 4.35 g/L K2SO4, and 100 μm acetosyringinone. The explants are washed in half-strength Murashige-Skoog medium (Murashige and Skoog, 1962) and plated on the same SH induction medium without acetosyringinone but with a suitable selection agent and suitable antibiotic to inhibit Agrobacterium growth. After several weeks, somatic embryos are transferred to BOi2Y development medium containing no growth regulators, no antibiotics, and 50 g/L sucrose. Somatic embryos are subsequently germinated on half-strength Murashige-Skoog medium. Rooted seedlings were transplanted into pots and grown in a greenhouse. T1 seeds are produced from plants that exhibit tolerance to the selection agent and that contain a single copy of the T-DNA insert.
Example 5
Evaluation Procedure
5.1 Evaluation Setup
[0738] Approximately 30 independent T0 rice transformants were generated. The primary transformants were transferred from a tissue culture chamber to a greenhouse for growing and harvest of T1 seed. Seven events, of which the T1 progeny segregated 3:1 for presence/absence of the transgene, were retained. For each of these events, approximately 10 T1 seedlings containing the transgene (hetero- and homo-zygotes) and approximately 10 T1 seedlings lacking the transgene (nullizygotes) were selected by monitoring visual marker expression. The transgenic plants and the corresponding nullizygotes were grown side-by-side at random positions. Greenhouse conditions were of shorts days (12 hours light), 28° C. in the light and 22° C. in the dark, and a relative humidity of 70%.
[0739] Four T1 events were further evaluated in the T2 generation following the same evaluation procedure as for the T1 generation but with more individuals per event. From the stage of sowing until the stage of maturity the plants were passed several times through a digital imaging cabinet. At each time point digital images (2048×1536 pixels, 16 million colours) were taken of each plant from at least 6 different angles.
5.2 Statistical Analysis: t Test and F test
[0740] A two factor ANOVA (analysis of variants) was used as a statistical model for the overall evaluation of plant phenotypic characteristics. An F test was carried out on all the parameters measured of all the plants of all the events transformed with the gene of the present invention. The F test was carried out to check for an effect of the gene over all the transformation events and to verify for an overall effect of the gene, also known as a global gene effect. The threshold for significance for a true global gene effect was set at a 5% probability level for the F test. A significant F test value points to a gene effect, meaning that it is not only the mere presence or position of the gene that is causing the differences in phenotype.
[0741] To check for an effect of the genes within an event, i.e., for a line-specific effect, a t-test was performed within each event using data sets from the transgenic plants and the corresponding null plants. "Null plants" or "null segregants" or "nullizygotes" are the plants treated in the same way as the transgenic plant, but from which the transgene has segregated. Null plants can also be described as the homozygous negative transformed plants. The threshold for significance for the t-test is set at 10% probability level. The results for some events can be above or below this threshold. This is based on the hypothesis that a gene might only have an effect in certain positions in the genome, and that the occurrence of this position-dependent effect is not uncommon. This kind of gene effect is also named herein a "line effect of the gene". The p-value is obtained by comparing the t-value to the t-distribution or alternatively, by comparing the F-value to the F-distribution. The p-value then gives the probability that the null hypothesis (i.e., that there is no effect of the transgene) is correct.
Example 6
Evaluation Results
[0742] The mature primary panicles were harvested, counted, bagged, barcode-labeled and then dried for three days in an oven at 37° C. The panicles were then threshed and all the seeds were collected and counted. The filled husks were separated from the empty ones using an air-blowing device. The empty husks were discarded and the remaining fraction was counted again. The filled husks were weighed on an analytical balance. The number of filled seeds was determined by counting the number of filled husks that remained after the separation step. The total seed yield (total seed weight) was measured by weighing all filled husks harvested from a plant. Total seed number per plant was measured by counting the number of husks harvested from a plant. The harvest index (HI) in the present invention is defined as the ratio between the total seed yield and the above ground area (mm2), multiplied by a factor 106. The total number of flowers per panicle as defined in the present invention is the ratio between the total number of seeds and the number of mature primary panicles. The seed fill rate as defined in the present invention is the proportion (expressed as a %) of the number of filled seeds over the total number of seeds (or florets).
[0743] As presented in Table B, the seed yield, number of filled seeds and harvest index are increased in the transgenic plants preferentially expressing a nucleic acid encoding a HAL3 polypeptide in the shoot, compared to control plants.
[0744] Table B shows the average yield increase (total seed weight), increase in number of filled seeds and increase of harvest index in percent, calculated from the transgenic events compared to control plants, in the T1 generation
TABLE-US-00024 TABLE B parameters % increase p-value Total seed weight 14 0.0072 Number of filled seeds 15 0.0052 Harvest Index 10 0.0093
Example 7
Early Vigour Evaluation Results
[0745] Early vigour was determined by counting the total number of pixels from aboveground plant parts discriminated from the background. This value was averaged for the pictures taken on the same time point from different angles and was converted to a physical surface value expressed in square mm by calibration. The results described below are for plants three weeks post-germination.
[0746] Plant early vigour (as determined by aboveground area) was seen in six out of seven events of the T1 generation, with an overall increase in aboveground area for transgenic seedlings of 27% compared to control plants. Four of these T1 events were further evaluated in the T2 generation, and all four of these events gave an increase in aboveground area for transgenic seedlings compared to control plants, with an overall increase in aboveground area for transgenic seedlings of 33% compared to control plants. The results were also shown to be statistically significant with the p-value from the F-test being lower than 0.0001 (T2 generation) indicating that the effect seen is likely due to the transgene rather than the position of the gene or a line effect, see table C.
TABLE-US-00025 TABLE C parameters % increase p-value Early vigour 33 0.0000
Example Section B
MADS15 Upregulated
Example 8
Identification of Sequences Related to SEQ ID NO: 43 and SEQ ID NO: 44
[0747] Sequences (full length cDNA, ESTs or genomic) related to SEQ ID NO: 43 and/or protein sequences related to SEQ ID NO: 44 were identified amongst those maintained in the Entrez Nucleotides database at the National Center for Biotechnology Information (NCBI) using database sequence search tools, such as the Basic Local Alignment Tool (BLAST) (Altschul et al. (1990) J. Mol. Biol. 215:403-410; and Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402). The program is used to find regions of local similarity between sequences by comparing nucleic acid or polypeptide sequences to sequence databases and by calculating the statistical significance of matches. The polypeptide encoded by SEQ ID NO: 43 was used for the TBLASTN algorithm, with default settings and the filter to ignore low complexity sequences set off. The output of the analysis was viewed by pairwise comparison, and ranked according to the probability score (E-value), where the score reflects the probability that a particular alignment occurs by chance (the lower the E-value, the more significant the hit). In addition to E-values, comparisons were also scored by percentage identity. Percentage identity refers to the number of identical nucleotides (or amino acids) between the two compared nucleic acid (or polypeptide) sequences over a particular length. In some instances, the default parameters may be adjusted to modify the stringency of the search.
[0748] In addition to the publicly available nucleic acid sequences available at NCBI, proprietary sequence databases are also searched following the same procedure as described herein above.
[0749] Table D provides a list of nucleic acid and protein sequences related to the nucleic acid sequence as represented by SEQ ID NO: 43 and the protein sequence represented by SEQ ID NO: 44.
TABLE-US-00026 TABLE D Nucleic acid sequences related to the nucleic acid sequence (SEQ ID NO: 43) useful in the methods of the present invention, and the corresponding deduced polypeptides. database SEQ ID NO: accession name source organism nucl/protein number VRN-H1 Hordeum vulgare 52/53 AAW82995 m5 Hordeum vulgare 54/55 CAB97352 TaMADS#11 Triticum aestivum 56/57 BAA33457 TaVRT-1 Triticum aestivum 58/59 AAP33790 AP1 Triticum monococcum 60/61 AAO72630 VRN-A1 Triticum aestivum 62/63 AAW73222 MADS1 Lolium perenne 64/65 AAO45873 MADS1 Lolium temulentum 66/67 AAD10625 m15 Zea mays 68/69 CAD23408 m4 Zea mays 70/71 CAD23417 RMADS211 Oryza sativa 72/73 AAS59822 MADS14 Oryza sativa 74/75 AAF19047 Mads2 Dendrocalamus latiflorus 76/77 AAR32119 Mads1 Dendrocalamus latiflorus 78/79 AAR32118 mads3 Zea mays 80/81 AAG43200 SbMADS2 Sorghum bicolor 82/83 AAB50181 ZAP1 Zea mays 84/85 AAB00081 MADS2 Lolium temulentum 86/87 AAD10626 MADS2 Lolium perenne 88/89 AAO45874 m8 Hordeum vulgare 90/91 CAB97354 FDRMADS3 Oryza sativa 92/93 AAL09473 MADS15 Oryza sativa 94/95 AAL09473 TvFL2 Tradescantia virginiana 96/97 AAP83415 TvFL1 Tradescantia virginiana 98/99 AAP83414 TvFL3 Tradescantia virginiana 100/101 AAP83416 SQUA1 Elaeis guineensis 102/103 AAQ03221 AIFL Allium sp. 104/105 AAP83362 DOMADS2 Dendrobium grex 106/107 AAF13261
Example 9
Alignment of Relevant Polypeptide Sequences
[0750] AlignX from the Vector NTI (Invitrogen) is based on the popular Clustal algorithm of progressive alignment (Thompson et al. (1997) Nucleic Acids Res 25:4876-4882; Chema et al. (2003). Nucleic Acids Res 31:3497-3500). A phylogenetic tree can be constructed using a neighbour-joining clustering algorithm. Default values are for the gap open penalty of 10, for the gap extension penalty of 0.1 and the selected weight matrix is Blosum 62 (if polypeptides are aligned).
[0751] The result of the multiple sequence alignment using polypeptides relevant in identifying the ones useful in performing the methods of the invention is shown in FIG. 6.
Example 10
Calculation of Global Percentage Identity Between Polypeptide Sequences Useful in Performing the Methods of the Invention
[0752] Global percentages of similarity and identity between full length polypeptide sequences useful in performing the methods of the invention were determined using one of the methods available in the art, the MatGAT (Matrix Global Alignment Tool) software (BMC Bioinformatics. 2003 4:29. MatGAT: an application that generates similarity/identity matrices using protein or DNA sequences. Campanella J J, Bitincka L, Smalley J; software hosted by Ledion Bitincka). MatGAT software generates similarity/identity matrices for DNA or protein sequences without needing pre-alignment of the data. The program performs a series of pair-wise alignments using the Myers and Miller global alignment algorithm (with a gap opening penalty of 12, and a gap extension penalty of 2), calculates similarity and identity using for example Blosum 62 (for polypeptides), and then places the results in a distance matrix. Sequence similarity is shown in the bottom half of the dividing line and sequence identity is shown in the top half of the diagonal dividing line.
[0753] Parameters used in the comparison were:
[0754] Scoring matrix: Blosum62
[0755] First Gap: 12
[0756] Extending gap: 2
[0757] Results of the software analysis are shown in Table E for the global similarity and identity over the full length of the polypeptide sequences (excluding the partial polypeptide sequences). Percentage identity is given above the diagonal and percentage similarity is given below the diagonal.
[0758] The percentage identity between the polypeptide sequences useful in performing the methods of the invention can be as low as 40% amino acid identity compared to SEQ ID NO: 44.
TABLE-US-00027 TABLE E MatGAT results for global similarity and identity over the full length of the polypeptide sequences. 1 2 3 4 5 6 7 8 9 10 11 12 13 1. SEQID44 70.8 70.0 70.4 70.4 71.5 71.5 70.1 71.6 71.9 72.3 71.5 73.0 2. SEQID53 77.2 99.2 96.3 96.3 95.5 95.1 87.0 86.2 80.6 80.2 81.0 82.9 3. SEQID55 76.4 99.2 95.5 95.5 94.7 94.3 86.2 85.4 79.8 79.4 80.2 82.1 4. SEQID57 76.0 96.7 95.9 98.8 98.0 97.5 87.0 86.6 81.8 81.4 80.6 82.5 5. SEQID59 76.4 97.1 96.3 99.2 98.0 98.0 87.0 86.6 81.8 81.4 80.6 82.5 6. SEQID61 77.2 96.7 95.9 98.4 98.8 99.6 87.9 87.4 82.6 82.6 81.0 82.9 7. SEQID63 77.2 96.3 95.5 98.0 98.8 99.6 87.4 87.0 82.2 82.2 80.6 82.5 8. SEQID65 76.4 92.2 91.4 92.2 92.7 93.5 93.1 97.6 81.7 80.9 82.3 83.8 9. SEQID67 76.8 92.7 91.8 92.2 92.7 93.5 93.1 98.8 81.3 81.7 82.3 83.8 10. SEQID69 79.4 87.3 86.5 89.0 89.0 89.4 89.0 88.2 88.2 93.5 83.8 85.8 11. SEQID71 79.4 87.3 86.5 89.0 89.0 89.4 89.0 86.9 87.3 97.1 83.8 85.8 12. SEQID73 80.1 88.1 87.4 86.6 87.0 87.4 87.0 88.5 88.1 91.3 90.9 96.4 13. SEQID75 79.8 89.8 89.0 88.6 89.0 89.4 89.0 90.7 90.2 93.5 93.1 96.4 14. SEQID77 79.8 91.8 91.0 91.4 91.8 92.6 92.2 90.6 90.2 91.8 91.4 93.3 95.1 15. SEQID79 80.1 92.2 91.4 91.8 92.2 93.0 92.6 91.0 90.6 92.2 91.8 93.7 95.5 16. SEQID81 86.3 79.3 78.5 77.8 78.1 78.9 78.9 77.0 77.0 81.9 80.7 80.7 80.7 17. SEQID85 85.7 76.9 76.2 76.6 76.9 77.7 77.7 75.5 75.1 80.2 79.1 79.1 79.1 18. SEQID87 89.1 78.2 77.4 79.3 79.7 79.7 80.1 78.2 77.8 82.8 80.8 81.6 80.8 19. SEQID89 89.5 78.2 77.4 78.9 78.9 79.3 79.3 78.5 78.2 83.1 81.2 82.0 81.2 20. SEQID91 85.9 75.4 74.6 75.7 75.7 76.4 76.4 75.4 74.6 79.3 78.6 79.0 79.0 21. SEQID93 91.8 73.0 72.3 73.8 73.8 74.5 74.5 73.0 73.0 76.0 76.8 78.3 76.4 22. SEQID95 99.6 76.8 76.0 76.0 76.0 76.8 76.8 76.0 75.7 78.7 79.0 79.8 79.4 23. SEQID103 75.3 82.8 82.0 81.6 82.0 82.4 82.4 84.4 84.4 82.4 82.4 85.4 84.0 24. SEQID107 70.4 77.3 76.5 76.5 74.5 77.3 76.1 74.9 74.9 76.5 75.7 75.1 77.3 14 15 16 17 18 19 20 21 22 23 24 1. SEQID44 72.7 73.0 82.1 82.2 87.4 87.7 82.5 90.3 99.6 64.7 56.7 2. SEQID53 84.6 85.0 70.7 68.9 71.3 71.3 68.8 65.2 70.4 70.1 61.0 3. SEQID55 83.7 84.1 70.0 68.1 70.5 70.5 68.1 64.4 69.7 69.3 60.2 4. SEQID57 85.8 86.2 70.4 69.6 72.4 72.4 69.6 65.5 70.0 69.7 62.0 5. SEQID59 85.8 86.2 70.7 69.6 72.4 72.4 69.6 65.5 70.0 70.1 61.5 6. SEQID61 87.0 87.4 71.1 70.0 72.4 72.4 70.3 67.2 71.2 70.9 62.5 7. SEQID63 86.6 87.0 71.1 70.0 72.8 72.4 70.3 67.2 71.2 70.9 61.8 8. SEQID65 85.7 86.1 70.5 69.3 71.4 71.8 69.3 66.8 69.8 71.8 60.7 9. SEQID67 85.7 86.1 71.2 70.1 72.1 72.5 68.6 66.0 70.5 71.8 60.7 10. SEQID69 84.9 85.3 71.5 71.4 73.9 74.3 69.9 67.8 71.9 71.8 62.5 11. SEQID71 84.5 84.9 71.5 71.4 73.6 73.9 69.6 69.3 71.9 72.2 61.0 12. SEQID73 89.3 89.7 72.0 71.1 72.4 72.8 69.9 68.0 71.2 71.9 60.2 13. SEQID75 91.5 91.9 73.0 71.8 73.9 74.3 70.7 68.3 72.7 72.0 60.6 14. SEQID77 99.6 71.9 71.1 73.6 73.6 69.9 67.8 72.7 73.3 61.8 15. SEQID79 99.6 72.2 71.4 73.9 73.9 70.3 68.2 72.7 73.7 62.2 16. SEQID81 79.3 79.6 93.4 81.9 82.3 79.0 76.8 81.8 64.3 56.8 17. SEQID85 77.7 78.0 96.3 82.5 82.9 81.3 77.0 81.9 63.6 57.2 18. SEQID87 80.5 80.8 87.4 86.1 99.6 88.4 82.5 87.0 64.6 58.0 19. SEQID89 80.5 80.8 87.8 86.4 99.6 88.8 82.9 87.4 64.6 58.0 20. SEQID91 76.8 77.2 86.2 88.0 90.2 90.6 77.5 82.1 62.7 55.8 21. SEQID93 76.0 76.4 83.3 82.1 85.4 85.8 81.5 89.9 61.0 54.1 22. SEQID95 79.8 79.8 85.9 85.3 88.8 89.1 85.5 91.4 64.3 56.3 23. SEQID103 84.8 85.2 75.2 73.6 75.9 75.9 73.6 72.3 74.9 70.1 24. SEQID107 78.5 78.9 71.5 71.4 72.8 72.8 70.3 67.4 70.0 82.4
Example 11
Identification of Domains Comprised in Polypeptide Sequences Useful in Performing the Methods of the Invention
[0759] The Integrated Resource of Protein Families, Domains and Sites (InterPro) database is an integrated interface for the commonly used signature databases for text- and sequence-based searches. The InterPro database combines these databases, which use different methodologies and varying degrees of biological information about well-characterized proteins to derive protein signatures. Collaborating databases include SWISS-PROT, PROSITE, TrEMBL, PRINTS, Propom and Pfam, Smart and TIGRFAMs. Interpro is hosted at the European Bioinformatics Institute in the United Kingdom.
[0760] The results of the InterPro scan of the polypeptide sequence as represented by SEQ ID NO: 44 are presented in Table F.
TABLE-US-00028 TABLE F InterPro scan results of the polypeptide sequence as represented by SEQ ID NO: 44 Database Accession number Accession name PRINTS PR00404 MADSDOMAIN PFAM PF00319 SRF-TF SMART SM00432 MADS PROFILE PS50066 MADS_BOX_2 SUPERFAMILY SSF55455 SRF-like PFAM PF01486 K-box SUPERFAMILY SSF46589 tRNA-binding arm PANTHER PTHR11945 MADS BOX PROTEIN PANTHER PTHR11945:SF19 MADS BOX PROTEIN
Example 12
Topology Prediction of the Polypeptide Sequences Useful in Performing the Methods of the Invention (Subcellular Localization, Transmembrane . . . )
[0761] TargetP 1.1 predicts the subcellular location of eukaryotic proteins. The location assignment is based on the predicted presence of any of the N-terminal pre-sequences: chloroplast transit peptide (cTP), mitochondrial targeting peptide (mTP) or secretory pathway signal peptide (SP). Scores on which the final prediction is based are not really probabilities, and they do not necessarily add to one. However, the location with the highest score is the most likely according to TargetP, and the relationship between the scores (the reliability class) may be an indication of how certain the prediction is. The reliability class (RC) ranges from 1 to 5, where 1 indicates the strongest prediction. TargetP is maintained at the server of the Technical University of Denmark.
[0762] For the sequences predicted to contain an N-terminal presequence a potential cleavage site can also be predicted.
[0763] A number of parameters were selected, such as organism group (non-plant or plant), cutoff sets (none, predefined set of cutoffs, or user-specified set of cutoffs), and the calculation of prediction of cleavage sites (yes or no).
[0764] The results of TargetP 1.1 analysis of the polypeptide sequence as represented by SEQ ID NO: 44 are presented Table G. The "plant" organism group has been selected, no cutoffs defined, and the predicted length of the transit peptide requested. The subcellular localization of the polypeptide sequence as represented by SEQ ID NO: 44 may be the cytoplasm or nucleus, no transit peptide is predicted.
TABLE-US-00029 TABLE G TargetP 1.1 analysis of the polypeptide sequence as represented by SEQ ID NO: 44 Length (AA) 267 Chloroplastic transit peptide 0.088 Mitochondrial transit peptide 0.492 Secretory pathway signal peptide 0.046 Other subcellular targeting 0.655 Predicted Location Chloroplastic Reliability class 5 Predicted transit peptide length /
[0765] Many other algorithms can be used to perform such analyses, including:
[0766] ChloroP 1.1 hosted on the server of the Technical University of Denmark;
[0767] Protein Prowler Subcellular Localisation Predictor version 1.2 hosted on the server of the Institute for Molecular Bioscience, University of Queensland, Brisbane, Australia;
[0768] PENCE Proteome Analyst PA-GOSUB 2.5 hosted on the server of the University of Alberta, Edmonton, Alberta, Canada;
[0769] TMHMM, hosted on the server of the Technical University of Denmark
Example 13
Assay Related to the Polypeptide Sequences Useful in Performing the Methods of the Invention
[0770] Lim et al. (PMB 44, 513-527, 2000) describe two assays for measuring protein-protein interactions that may be applied to MADS15. In the yeast two-hybrid assay, a truncated form of MADS1 (comprising the complete MADS box, the I- and K-domain but lacking the C-terminal part of the C-domain) was used as bait to test interaction with MADS15. In the in vitro pull-down assay, GST and GST-fused MADS1 proteins were produced and immobilised on glutathione Sepharose 4B. The resin-bound GST/GST-MADS1 was mixed with 35S-labeled MADS15 proteins or fragments thereof. After washing, the interacting proteins were eluted and analysed by SDS-PAGE. A person skilled in the art will appreciate that the MADS15 protein may be used as bait in the two-hybrid screen or may be immobilised on glutathione resin as well. Furthermore, a MADS15 protein, when used according to the methods of the present invention, will result in increased root yield in rice, measured as an increased ratio of root biomass over shoot biomass.
Example 14
Cloning of Nucleic Acid Sequence as Represented by SEQ ID NO: 43
[0771] The Oryza sativa MADS15 gene was amplified by PCR using as template an Oryza sativa seedling cDNA library (Invitrogen, Paisley, UK). After reverse transcription of RNA extracted from seedlings, the cDNAs were cloned into pCMV Sport 6.0. Average insert size of the bank was 1.5 kb and the original number of clones was of the order of 1.59×107 cfu. Original titer was determined to be 9.6×105 cfu/ml after first amplification of 6×1011 cfu/ml. After plasmid extraction, 200 ng of template was used in a 50 μl PCR mix. Primers prm06892 (SEQ ID NO: 45; sense, start codon in bold, AttB1 site in italic: 5'-ggggacaagtttgtacaaaaaagcaggcttaaaca atgggcgggggaaggt-3') and prm06893 (SEQ ID NO: 46; reverse, complementary, AttB2 site in italic: 5'-ggggaccactttgtacaagaaagctgggtttggccgacgacgacgac-3'), which include the AttB sites for Gateway recombination, were used for PCR amplification. PCR was performed using Hifi Taq DNA polymerase in standard conditions. A PCR fragment of around 915 bp was amplified and purified also using standard methods. The first step of the Gateway procedure, the BP reaction, was then performed, during which the PCR fragment recombines in vivo with the pDONR201 plasmid to produce, according to the Gateway terminology, an "entry clone", pMADS15. Plasmid pDONR201 was purchased from Invitrogen, as part of the Gateway® technology.
Example 15
Expression Vector Construction Using the Nucleic Acid Sequence as Represented by SEQ ID NO: 43
[0772] The entry clone pMADS15 was subsequently used in an LR reaction with pGOS2, a destination vector used for Oryza sativa transformation. This vector contains as functional elements within the T-DNA borders: a plant selectable marker; a screenable marker expression cassette; and a Gateway cassette intended for LR in vivo recombination with the nucleic acid sequence of interest already cloned in the entry clone. A rice GOS2 promoter (SEQ ID NO: 108, alternatively, SEQ ID NO: 47 is equally useful) for constitutive expression was located upstream of this Gateway cassette.
[0773] After the LR recombination step, the resulting expression vector pGOS2::MADS15 (FIG. 7) was transformed into Agrobacterium strain LBA4044 according to methods well known in the art.
Example 16
Plant Transformation
Rice Transformation
[0774] The Agrobacterium containing the expression vector was used to transform Oryza sativa plants. Mature dry seeds of the rice japonica cultivar Nipponbare were dehusked. Sterilization was carried out by incubating for one minute in 70% ethanol, followed by 30 minutes in 0.2% HgCl2, followed by a 6 times 15 minutes wash with sterile distilled water. The sterile seeds were then germinated on a medium containing 2,4-D (callus induction medium). After incubation in the dark for four weeks, embryogenic, scutellum-derived calli were excised and propagated on the same medium. After two weeks, the calli were multiplied or propagated by subculture on the same medium for another 2 weeks. Embryogenic callus pieces were sub-cultured on fresh medium 3 days before co-cultivation (to boost cell division activity).
[0775] Agrobacterium strain LBA4404 containing the expression vector was used for co-cultivation. Agrobacterium was inoculated on AB medium with the appropriate antibiotics and cultured for 3 days at 28° C. The bacteria were then collected and suspended in liquid co-cultivation medium to a density (OD600) of about 1. The suspension was then transferred to a Petri dish and the calli immersed in the suspension for 15 minutes. The callus tissues were then blotted dry on a filter paper and transferred to solidified, co-cultivation medium and incubated for 3 days in the dark at 25° C. Co-cultivated calli were grown on 2,4-D-containing medium for 4 weeks in the dark at 28° C. in the presence of a selection agent. During this period, rapidly growing resistant callus islands developed. After transfer of this material to a regeneration medium and incubation in the light, the embryogenic potential was released and shoots developed in the next four to five weeks. Shoots were excised from the calli and incubated for 2 to 3 weeks on an auxin-containing medium from which they were transferred to soil. Hardened shoots were grown under high humidity and short days in a greenhouse.
[0776] The primary transformants were transferred from a tissue culture chamber to a greenhouse. After a quantitative PCR analysis to verify copy number of the T-DNA insert, only single copy transgenic plants that exhibit tolerance to the selection agent were kept for harvest of T1 seed. Seeds were then harvested three to five months after transplanting. The method yielded single locus transformants at a rate of over 50% (Aldemita and Hodges1996, Chan et al. 1993, Hiei et al. 1994).
Corn Transformation
[0777] Transformation of maize (Zea mays) Is performed with a modification of the method described by Ishida et al. (1996) Nature Biotech 14(6): 745-50. Transformation is genotype-dependent in corn and only specific genotypes are amenable to transformation and regeneration. The inbred line A188 (University of Minnesota) or hybrids with A188 as a parent are good sources of donor material for transformation, but other genotypes can be used successfully as well. Ears are harvested from corn plant approximately 11 days after pollination (DAP) when the length of the immature embryo is about 1 to 1.2 mm. Immature embryos are cocultivated with Agrobacterium tumefaciens containing the expression vector, and transgenic plants are recovered through organogenesis. Excised embryos are grown on callus induction medium, then maize regeneration medium, containing the selection agent (for example imidazolinone but various selection markers can be used). The Petri plates are incubated in the light at 25° C. for 2-3 weeks, or until shoots develop. The green shoots are transferred from each embryo to maize rooting medium and incubated at 25° C. for 2-3 weeks, until roots develop. The rooted shoots are transplanted to soil in the greenhouse. T1 seeds are produced from plants that exhibit tolerance to the selection agent and that contain a single copy of the T-DNA insert.
Wheat Transformation
[0778] Transformation of wheat is performed with the method described by Ishida et al. (1996) Nature Biotech 14(6): 745-50. The cultivar Bobwhite (available from CIMMYT, Mexico) is commonly used in transformation. Immature embryos are co-cultivated with Agrobacterium tumefaciens containing the expression vector, and transgenic plants are recovered through organogenesis. After incubation with Agrobacterium, the embryos are grown in vitro on callus induction medium, then regeneration medium, containing the selection agent (for example imidazolinone but various selection markers can be used). The Petri plates are incubated in the light at 25° C. for 2-3 weeks, or until shoots develop. The green shoots are transferred from each embryo to rooting medium and incubated at 25° C. for 2-3 weeks, until roots develop. The rooted shoots are transplanted to soil in the greenhouse. T1 seeds are produced from plants that exhibit tolerance to the selection agent and that contain a single copy of the T-DNA insert.
Soybean Transformation
[0779] Soybean is transformed according to a modification of the method described in the Texas A&M U.S. Pat. No. 5,164,310. Several commercial soybean varieties are amenable to transformation by this method. The cultivar Jack (available from the Illinois Seed foundation) is commonly used for transformation. Soybean seeds are sterilised for in vitro sowing. The hypocotyl, the radicle and one cotyledon are excised from seven-day old young seedlings. The epicotyl and the remaining cotyledon are further grown to develop axillary nodes. These axillary nodes are excised and incubated with Agrobacterium tumefaciens containing the expression vector. After the cocultivation treatment, the explants are washed and transferred to selection media. Regenerated shoots are excised and placed on a shoot elongation medium. Shoots no longer than 1 cm are placed on rooting medium until roots develop. The rooted shoots are transplanted to soil in the greenhouse. T1 seeds are produced from plants that exhibit tolerance to the selection agent and that contain a single copy of the T-DNA insert.
Rapeseed/Canola Transformation
[0780] Cotyledonary petioles and hypocotyls of 5-6 day old young seedling are used as explants for tissue culture and transformed according to Babic et al. (1998, Plant Cell Rep 17: 183-188). The commercial cultivar Westar (Agriculture Canada) is the standard variety used for transformation, but other varieties can also be used. Canola seeds are surface-sterilized for in vitro sowing. The cotyledon petiole explants with the cotyledon attached are excised from the in vitro seedlings, and inoculated with Agrobacterium (containing the expression vector) by dipping the cut end of the petiole explant into the bacterial suspension. The explants are then cultured for 2 days on MSBAP-3 medium containing 3 mg/l BAP, 3% sucrose, 0.7% Phytagar at 23° C., 16 hr light. After two days of co-cultivation with Agrobacterium, the petiole explants are transferred to MSBAP-3 medium containing 3 mg/l BAP, cefotaxime, carbenicillin, or timentin (300 mg/l) for 7 days, and then cultured on MSBAP-3 medium with cefotaxime, carbenicillin, or timentin and selection agent until shoot regeneration. When the shoots are 5-10 mm in length, they are cut and transferred to shoot elongation medium (MSBAP-0.5, containing 0.5 mg/l BAP). Shoots of about 2 cm in length are transferred to the rooting medium (MS0) for root induction. The rooted shoots are transplanted to soil in the greenhouse. T1 seeds are produced from plants that exhibit tolerance to the selection agent and that contain a single copy of the T-DNA insert.
Alfalfa Transformation
[0781] A regenerating clone of alfalfa (Medicago sativa) is transformed using the method of (McKersie et al., 1999 Plant Physiol 119: 839-847). Regeneration and transformation of alfalfa is genotype dependent and therefore a regenerating plant is required. Methods to obtain regenerating plants have been described. For example, these can be selected from the cultivar Rangelander (Agriculture Canada) or any other commercial alfalfa variety as described by Brown D C W and A Atanassov (1985. Plant Cell Tissue Organ Culture 4: 111-112). Alternatively, the RA3 variety (University of Wisconsin) has been selected for use in tissue culture (Walker et al., 1978 Am J Bot 65:654-659). Petiole explants are cocultivated with an overnight culture of Agrobacterium tumefaciens C58C1 pMP90 (McKersie et al., 1999 Plant Physiol 119: 839-847) or LBA4404 containing the expression vector. The explants are cocultivated for 3 d in the dark on SH induction medium containing 288 mg/L Pro, 53 mg/L thioproline, 4.35 g/L K2SO4, and 100 μm acetosyringinone. The explants are washed in half-strength Murashige-Skoog medium (Murashige and Skoog, 1962) and plated on the same SH induction medium without acetosyringinone but with a suitable selection agent and suitable antibiotic to inhibit Agrobacterium growth. After several weeks, somatic embryos are transferred to BOi2Y development medium containing no growth regulators, no antibiotics, and 50 g/L sucrose. Somatic embryos are subsequently germinated on half-strength Murashige-Skoog medium. Rooted seedlings were transplanted into pots and grown in a greenhouse. T1 seeds are produced from plants that exhibit tolerance to the selection agent and that contain a single copy of the T-DNA insert.
Example 17
Phenotypic Evaluation Procedure
17.1 Evaluation Setup
[0782] Approximately 35 independent TO rice transformants were generated. The primary transformants were transferred from a tissue culture chamber to a greenhouse for growing and harvest of T1 seed. Six events, of which the T1 progeny segregated 3:1 for presence/absence of the transgene, were retained. For each of these events, approximately 10 T1 seedlings containing the transgene (hetero- and homo-zygotes) and approximately 10 T1 seedlings lacking the transgene (nullizygotes) were selected by monitoring visual marker expression. The transgenic plants and the corresponding nullizygotes were grown side-by-side at random positions. Greenhouse conditions were of shorts days (12 hours light), 28° C. in the light and 22° C. in the dark, and a relative humidity of 70%.
[0783] Four T1 events were further evaluated in the T2 generation following the same evaluation procedure as for the T1 generation but with more individuals per event. From the stage of sowing until the stage of maturity the plants were passed several times through a digital imaging cabinet. At each time point digital images (2048×1536 pixels, 16 million colours) were taken of each plant from at least 6 different angles.
17.2 Statistical Analysis: F-Test
[0784] A two factor ANOVA (analysis of variants) was used as a statistical model for the overall evaluation of plant phenotypic characteristics. An F-test was carried out on all the parameters measured of all the plants of all the events transformed with the gene of the present invention. The F-test was carried out to check for an effect of the gene over all the transformation events and to verify for an overall effect of the gene, also known as a global gene effect. The threshold for significance for a true global gene effect was set at a 5% probability level for the F-test. A significant F-test value points to a gene effect, meaning that it is not only the mere presence or position of the gene that is causing the differences in phenotype.
[0785] To check for an effect of the genes within an event, i.e., for a line-specific effect, a t-test was performed within each event using data sets from the transgenic plants and the corresponding null plants. "Null plants" or "null segregants" or "nullizygotes" are the plants treated in the same way as the transgenic plant, but from which the transgene has segregated. Null plants can also be described as the homozygous negative transformed plants. The threshold for significance for the t-test is set at 10% probability level. The results for some events can be above or below this threshold. This is based on the hypothesis that a gene might only have an effect in certain positions in the genome, and that the occurrence of this position-dependent effect is not uncommon. This kind of gene effect is also named herein a "line effect of the gene". The p-value is obtained by comparing the t-value to the t-distribution or alternatively, by comparing the F-value to the F-distribution. The p-value then gives the probability that the null hypothesis (i.e., that there is no effect of the transgene) is correct.
17.3 Parameters Measured
[0786] From the stage of sowing until the stage of maturity the plants were passed several times through a digital imaging cabinet. At each time point digital images (2048×1536 pixels, 16 million colours) were taken of each plant from at least 6 different angles.
[0787] The plant aboveground area (or leafy biomass) was determined by counting the total number of pixels on the digital images from aboveground plant parts discriminated from the background. This value was averaged for the pictures taken on the same time point from the different angles and was converted to a physical surface value expressed in square mm by calibration. Experiments show that the aboveground plant area measured this way correlates with the biomass of plant parts above ground. The above ground area is the area measured at the time point at which the plant had reached its maximal leafy biomass. The early vigour is the plant (seedling) aboveground area three weeks post-germination. Increase in root biomass is expressed as an increase in total root biomass (measured as maximum biomass of roots observed during the lifespan of a plant); or as an increase in the root/shoot index (measured as the ratio between root biomass and shoot biomass in the period of active growth of root and shoot).
Example 18
Results of the Phenotypic Evaluation of the Transgenic Plants
[0788] Upon analysis of the plants as described above, the inventors found that plants transformed with the MADS15 gene construct had a higher root yield, expressed as root/shoot index, compared to plants lacking the MADS15 transgene. The increase was 9.4% (p-value 0.0207) in T1 and 25.7% (p-value 0.0002) in T2. The p-values show that the increases were significant.
Example Section C
MADS15 Down-Regulated
[0789] See also Examples 8 to 13 for the identification and characterisation of MADS15 related sequences.
Example 19
Gene Cloning
[0790] The Oryza sativa MADS15 gene was amplified by PCR using as template an Oryza sativa seedling cDNA library (Invitrogen, Paisley, UK). After reverse transcription of RNA extracted from seedlings, the cDNAs were cloned into pCMV Sport 6.0. Average insert size of the bank was 1.5 kb and the original number of clones was of the order of 1.59×107 cfu. Original titer was determined to be 9.6×105 cfu/ml after first amplification of 6×1011 cfu/ml. After plasmid extraction, 200 ng of template was used in a 50 μl PCR mix. Primers prm06892 (SEQ ID NO: 111; sense, start codon in bold, AttB1 site in italic: '-ggggacaagtttgtacaaaaaagcaggcttaaacaatggg cgggggaaggt-3') and prm06893 (SEQ ID NO: 112; reverse, complementary, AttB2 site in italic: 5'-ggggaccactttgtacaagaaagctgggtttggccgacgacgacgac-3'), which include the AttB sites for Gateway recombination, were used for PCR amplification. PCR was performed using Hifi Taq DNA polymerase in standard conditions. A PCR fragment of around 915 bp was amplified and purified also using standard methods. The PCR fragment was subsequently used for the preparation of a hairpin construct, using techniques known in the art. The first step of the Gateway procedure, the BP reaction, was then performed, during which the hairpin construct recombines in vivo with the pDONR201 plasmid to produce, according to the Gateway terminology, an "entry clone", pMADS15hp. Plasmid pDONR201 was purchased from Invitrogen, as part of the Gateway® technology.
Example 20
Vector Construction
[0791] The entry clone pMADS15hp was subsequently used in an LR reaction with p01519, a destination vector for the inverted repeat construct. This vector contains as functional elements within the T-DNA borders: a plant selectable marker; a screenable marker expression cassette; and a Gateway cassette intended for LR in vivo recombination such that the sequence of interest from the entry clone is integrated as an inverted repeat. A rice GOS2 promoter (SEQ ID NO: 174, alternatively, SEQ ID NO: 113 is equally useful) for constitutive expression was located upstream of this Gateway cassette.
[0792] After the LR recombination step, the resulting expression vector with the inverted repeat, FIG. 10) were transformed into Agrobacterium strain LBA4044 and subsequently to Oryza sativa plants.
Example 21
Plant Transformation
Rice Transformation
[0793] The Agrobacterium containing the expression vector was used to transform Oryza sativa plants. Mature dry seeds of the rice japonica cultivar Nipponbare were dehusked. Sterilization was carried out by incubating for one minute in 70% ethanol, followed by 30 minutes in 0.2% HgCl2, followed by a 6 times 15 minutes wash with sterile distilled water. The sterile seeds were then germinated on a medium containing 2,4-D (callus induction medium). After incubation in the dark for four weeks, embryogenic, scutellum-derived calli were excised and propagated on the same medium. After two weeks, the calli were multiplied or propagated by subculture on the same medium for another 2 weeks. Embryogenic callus pieces were sub-cultured on fresh medium 3 days before co-cultivation (to boost cell division activity).
[0794] Agrobacterium strain LBA4404 containing the expression vector was used for co-cultivation. Agrobacterium was inoculated on AB medium with the appropriate antibiotics and cultured for 3 days at 28° C. The bacteria were then collected and suspended in liquid co-cultivation medium to a density (OD600) of about 1. The suspension was then transferred to a Petri dish and the calli immersed in the suspension for 15 minutes. The callus tissues were then blotted dry on a filter paper and transferred to solidified, co-cultivation medium and incubated for 3 days in the dark at 25° C. Co-cultivated calli were grown on 2,4-D-containing medium for 4 weeks in the dark at 28° C. in the presence of a selection agent. During this period, rapidly growing resistant callus islands developed. After transfer of this material to a regeneration medium and incubation in the light, the embryogenic potential was released and shoots developed in the next four to five weeks. Shoots were excised from the calli and incubated for 2 to 3 weeks on an auxin-containing medium from which they were transferred to soil. Hardened shoots were grown under high humidity and short days in a greenhouse.
[0795] The primary transformants were transferred from a tissue culture chamber to a greenhouse. After a quantitative PCR analysis to verify copy number of the T-DNA insert, only single copy transgenic plants that exhibit tolerance to the selection agent were kept for harvest of T1 seed.
[0796] Seeds were then harvested three to five months after transplanting. The method yielded single locus transformants at a rate of over 50% (Aldemita and Hodges1996, Chan et al. 1993, Hiei et al. 1994).
Corn Transformation
[0797] Transformation of maize (Zea mays) is performed with a modification of the method described by Ishida et al. (1996) Nature Biotech 14(6): 745-50. Transformation is genotype-dependent in corn and only specific genotypes are amenable to transformation and regeneration. The inbred line A188 (University of Minnesota) or hybrids with A188 as a parent are good sources of donor material for transformation, but other genotypes can be used successfully as well. Ears are harvested from corn plant approximately 11 days after pollination (DAP) when the length of the immature embryo is about 1 to 1.2 mm. Immature embryos are cocultivated with Agrobacterium tumefaciens containing the expression vector, and transgenic plants are recovered through organogenesis. Excised embryos are grown on callus induction medium, then maize regeneration medium, containing the selection agent (for example imidazolinone but various selection markers can be used). The Petri plates are incubated in the light at 25° C. for 2-3 weeks, or until shoots develop. The green shoots are transferred from each embryo to maize rooting medium and incubated at 25° C. for 2-3 weeks, until roots develop. The rooted shoots are transplanted to soil in the greenhouse. T1 seeds are produced from plants that exhibit tolerance to the selection agent and that contain a single copy of the T-DNA insert.
Wheat Transformation
[0798] Transformation of wheat is performed with the method described by Ishida et al. (1996) Nature Biotech 14(6): 745-50. The cultivar Bobwhite (available from CIMMYT, Mexico) is commonly used in transformation. Immature embryos are co-cultivated with Agrobacterium tumefaciens containing the expression vector, and transgenic plants are recovered through organogenesis. After incubation with Agrobacterium, the embryos are grown in vitro on callus induction medium, then regeneration medium, containing the selection agent (for example imidazolinone but various selection markers can be used). The Petri plates are incubated in the light at 25° C. for 2-3 weeks, or until shoots develop. The green shoots are transferred from each embryo to rooting medium and incubated at 25° C. for 2-3 weeks, until roots develop. The rooted shoots are transplanted to soil in the greenhouse. T1 seeds are produced from plants that exhibit tolerance to the selection agent and that contain a single copy of the T-DNA insert.
Soybean Transformation
[0799] Soybean is transformed according to a modification of the method described in the Texas A&M U.S. Pat. No. 5,164,310. Several commercial soybean varieties are amenable to transformation by this method. The cultivar Jack (available from the Illinois Seed foundation) is commonly used for transformation. Soybean seeds are sterilised for in vitro sowing. The hypocotyl, the radicle and one cotyledon are excised from seven-day old young seedlings. The epicotyl and the remaining cotyledon are further grown to develop axillary nodes. These axillary nodes are excised and incubated with Agrobacterium tumefaciens containing the expression vector. After the cocultivation treatment, the explants are washed and transferred to selection media. Regenerated shoots are excised and placed on a shoot elongation medium. Shoots no longer than 1 cm are placed on rooting medium until roots develop. The rooted shoots are transplanted to soil in the greenhouse. T1 seeds are produced from plants that exhibit tolerance to the selection agent and that contain a single copy of the T-DNA insert.
Rapeseed/Canola Transformation
[0800] Cotyledonary petioles and hypocotyls of 5-6 day old young seedling are used as explants for tissue culture and transformed according to Babic et al. (1998, Plant Cell Rep 17: 183-188). The commercial cultivar Westar (Agriculture Canada) is the standard variety used for transformation, but other varieties can also be used. Canola seeds are surface-sterilized for in vitro sowing. The cotyledon petiole explants with the cotyledon attached are excised from the in vitro seedlings, and inoculated with Agrobacterium (containing the expression vector) by dipping the cut end of the petiole explant into the bacterial suspension. The explants are then cultured for 2 days on MSBAP-3 medium containing 3 mg/l BAP, 3% sucrose, 0.7% Phytagar at 23° C., 16 hr light. After two days of co-cultivation with Agrobacterium, the petiole explants are transferred to MSBAP-3 medium containing 3 mg/l BAP, cefotaxime, carbenicillin, or timentin (300 mg/l) for 7 days, and then cultured on MSBAP-3 medium with cefotaxime, carbenicillin, or timentin and selection agent until shoot regeneration. When the shoots are 5-10 mm in length, they are cut and transferred to shoot elongation medium (MSBAP-0.5, containing 0.5 mg/l BAP). Shoots of about 2 cm in length are transferred to the rooting medium (MS0) for root induction. The rooted shoots are transplanted to soil in the greenhouse. T1 seeds are produced from plants that exhibit tolerance to the selection agent and that contain a single copy of the T-DNA insert.
Alfalfa Transformation
[0801] A regenerating clone of alfalfa (Medicago sativa) is transformed using the method of (McKersie et al., 1999 Plant Physiol 119: 839-847). Regeneration and transformation of alfalfa is genotype dependent and therefore a regenerating plant is required. Methods to obtain regenerating plants have been described. For example, these can be selected from the cultivar Rangelander (Agriculture Canada) or any other commercial alfalfa variety as described by Brown D C W and A Atanassov (1985. Plant Cell Tissue Organ Culture 4: 111-112). Alternatively, the RA3 variety (University of Wisconsin) has been selected for use in tissue culture (Walker et al., 1978 Am J Bot 65:654-659). Petiole explants are cocultivated with an overnight culture of Agrobacterium tumefaciens C58C1 pMP90 (McKersie et al., 1999 Plant Physiol 119: 839-847) or LBA4404 containing the expression vector. The explants are cocultivated for 3 d in the dark on SH induction medium containing 288 mg/L Pro, 53 mg/L thioproline, 4.35 g/L K2SO4, and 100 μm acetosyringinone. The explants are washed in half-strength Murashige-Skoog medium (Murashige and Skoog, 1962) and plated on the same SH induction medium without acetosyringinone but with a suitable selection agent and suitable antibiotic to inhibit Agrobacterium growth. After several weeks, somatic embryos are transferred to BOi2Y development medium containing no growth regulators, no antibiotics, and 50 g/L sucrose. Somatic embryos are subsequently germinated on half-strength Murashige-Skoog medium. Rooted seedlings were transplanted into pots and grown in a greenhouse. T1 seeds are produced from plants that exhibit tolerance to the selection agent and that contain a single copy of the T-DNA insert.
Example 22
Evaluation Methods of Plants Transformed with the MADS15 Inverted Repeat Under Control of the Rice GOS2 Promoter
[0802] Approximately 15 to 20 independent T0 rice transformants were generated. The primary transformants were transferred from a tissue culture chamber to a greenhouse for growing and harvest of T1 seed. Eight events for the inverted repeat construct of which the T1 progeny segregated 3:1 for presence/absence of the transgene, were retained. For each of these events, approximately 10 T1 seedlings containing the transgene (hetero- and homozygotes) and approximately 10 T1 seedlings lacking the transgene (nullizygotes) were selected by monitoring visual marker expression. The selected T1 plants were transferred to a greenhouse. Each plant received a unique barcode label to link unambiguously the phenotyping data to the corresponding plant. The selected T1 plants were grown on soil in 10 cm diameter pots under the following environmental settings: photoperiod=11.5 h, daylight intensity=30,000 lux or more, daytime temperature=28° C. or higher, night time temperature=22° C., relative humidity=60-70%. Transgenic plants and the corresponding nullizygotes were grown side-by-side at random positions. From the stage of sowing until the stage of maturity the plants were passed several times through a digital imaging cabinet. At each time point digital images (2048×1536 pixels, 16 million colours) were taken of each plant from at least 6 different angles.
[0803] The plant aboveground area (or leafy biomass) was determined by counting the total number of pixels on the digital images from aboveground plant parts discriminated from the background. This value was averaged for the pictures taken on the same time point from the different angles and was converted to a physical surface value expressed in square mm by calibration. Experiments show that the aboveground plant area measured this way correlates with the biomass of plant parts above ground. The Areamax is the above ground area at the time point at which the plant had reached its maximal leafy biomass.
[0804] The mature primary panicles were harvested, bagged, barcode-labelled and then dried for three days in the oven at 37° C. The panicles were then threshed and all the seeds collected. The filled husks were separated from the empty ones using an air-blowing device. After separation, both seed lots were then counted using a commercially available counting machine. The empty husks were discarded. The filled husks were weighed on an analytical balance and the cross-sectional area of the seeds was measured using digital imaging. This procedure resulted in the set of the following seed-related parameters:
[0805] The flowers-per-panicle is a parameter estimating the average number of florets per panicle on a plant, derived from the number of total seeds divided by the number of first panicles. The tallest panicle and all the panicles that overlapped with the tallest panicle when aligned vertically, were considered as first panicles and were counted manually. The number of filled seeds was determined by counting the number of filled husks that remained after the separation step. The total seed yield (total seed weight) was measured by weighing all filled husks harvested from a plant. Total seed number per plant was measured by counting the number of husks harvested from a plant and corresponds to the number of florets per plant. These parameters were derived in an automated way from the digital images using image analysis software and were analysed statistically. Individual seed parameters (including width, length, area, weight) were measured using a custom-made device consisting of two main components, a weighing and imaging device, coupled to software for image analysis.
[0806] A two factor ANOVA (analyses of variance) corrected for the unbalanced design was used as statistical model for the overall evaluation of plant phenotypic characteristics. An F-test was carried out on all the parameters measured of all the plants of all the events transformed with that gene. The F-test was carried out to check for an effect of the gene over all the transformation events and to verify for an overall effect of the gene, also named herein "global gene effect". If the value of the F test shows that the data are significant, than it is concluded that there is a "gene" effect, meaning that not only presence or the position of the gene is causing the effect. The threshold for significance for a true global gene effect is set at 5% probability level for the F test.
[0807] To check for an effect of the genes within an event, i.e., for a line-specific effect, a t-test was performed within each event using data sets from the transgenic plants and the corresponding null plants. "Null plants" or "null segregants" or "nullizygotes" are the plants treated in the same way as the transgenic plant, but from which the transgene has segregated. Null plants can also be described as the homozygous negative transformed plants. The threshold for significance for the t-test is set at 10% probability level. The results for some events can be above or below this threshold. This is based on the hypothesis that a gene might only have an effect in certain positions in the genome, and that the occurrence of this position-dependent effect is not uncommon. This kind of gene effect is also named herein a "line effect of the gene". The p-value is obtained by comparing the t-value to the t-distribution or alternatively, by comparing the F-value to the F-distribution. The p-value then gives the probability that the null hypothesis (i.e., that there is no effect of the transgene) is correct.
Example 23
Measurement of Yield-Related Parameters for the Inverted Repeat Construct Transformants
[0808] Upon analysis of the seeds as described above, the inventors found that plants transformed with the hairpin MADS15 gene construct had a higher seed yield, expressed as number of filled seeds, total weight of seeds, total number of seeds, and flowers per panicle, compared to plants lacking the MADS15 transgene, whereas the plants transformed with the sense MADS15 gene construct showed opposite effects. The p-values show that the increases were significant.
[0809] The results obtained for plants in the T1 generation are summarised in Table H, which represent the mean values for all the tested lines:
TABLE-US-00030 TABLE H % difference p-value number of filled seeds +122 0.0000 total weight of seeds +112 0.0000 total number of seeds +27 0.0000 flowers per panicle +25 0.0000
Example Section D
PLT
Example 24
Identification of Sequences Related to the Nucleic Acid Sequence Used in the Methods of the Invention
[0810] Sequences (full length cDNA, ESTs or genomic) related to the nucleic acid sequence used in the methods of the present invention were identified amongst those maintained in the Entrez Nucleotides database at the National Center for Biotechnology Information (NCBI) using database sequence search tools, such as the Basic Local Alignment Tool (BLAST) (Altschul et al. (1990) J. Mol. Biol. 215:403-410; and Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402). The program is used to find regions of local similarity between sequences by comparing nucleic acid or polypeptide sequences to sequence databases and by calculating the statistical significance of matches. For example, the polypeptide encoded by the nucleic acid of the present invention was used for the TBLASTN algorithm, with default settings and the filter to ignore low complexity sequences set off. The output of the analysis was viewed by pairwise comparison, and ranked according to the probability score (E-value), where the score reflect the probability that a particular alignment occurs by chance (the lower the E-value, the more significant the hit). In addition to E-values, comparisons were also scored by percentage identity. Percentage identity refers to the number of identical nucleotides (or amino acids) between the two compared nucleic acid (or polypeptide) sequences over a particular length. In some instances, the default parameters may be adjusted to modify the stringency of the search. For example the E-value may be increased to show less stringent matches. This way, short nearly exact matches may be identified.
[0811] The Table I below provides a list of nucleic acid sequences related to the nucleic acid sequence used in the methods of the present invention.
TABLE-US-00031 TABLE I nucleic acid sequences related to the nucleic acid sequence used in the methods of the present invention Nucleic acid Polypeptide NCBI accession Name SEQ ID NO SEQ ID NO number Source organism Arath_PLT1 175 176 full length NM_112975 Arabidopsis thaliana (At3g20840) Arath_PLT2 177 178 full length NM_103997 Arabidopsis thaliana (At1g51190) Glyma_PLT 179 180 full length BU964973.1 Glycine max CA783156.1 BM309051.1 BM309377.1 Glyma_PLT2 181 182 full length BU926204.1 Glycine max BU547204.1 CA783156.1 BU927164.1 Medtr_PLT 183 184 full length AC144930.20 Medicago truncatula Orysa_PLT 185 186 full length NM_190301 Oryza sativa Zeama_PLT 187 188 full length CS155772.1 Zea mays Lotco_PLT 189 190 partial AP007400 Lotus corniculatus Poptr_PLT I 199 200 full length scaff_III.1595 Populus tremuloides Poptr_PLT II 201 202 full length scaff_I.328 Populus tremuloides Vitvi_PLT 203 204 partial AM469514 Vitis vinifera Brana_PLT 205 206 partial CN730825 Brassica napus Phaco_PLT 207 208 partial CA902624.1| Phaseolus coccineus
[0812] In some instances, related sequences have tentatively been assembled and publicly disclosed by research institutions, such as The Institute for Genomic Research (TIGR). The Eukaryotic Gene Orthologs (EGO) database may be used to identify such related sequences, either by keyword search or by using the BLAST algorithm with the nucleic acid or polypeptide sequence of interest.
Example 25
Cloning of the Nucleic Acid Sequences Used in the Methods of the Invention
[0813] The nucleic acid sequences used in the methods of the invention were amplified by PCR using as template a custom-made Arabidopsis thaliana cDNA library made starting from RNA extracted from different tissues (in pCMV Sport 6.0; Invitrogen, Paisley, UK). PCR was performed using Hifi Taq DNA polymerase in standard conditions, using 200 ng of template in a 50 μl PCR mix. The primers used to amplify SEQ ID NO: 175 (PLT1) were prm08180 (SEQ ID NO: 195; sense, start codon in bold, AttB1 site in italic: 5' GGGGACAAGTTTGTA CAAAAAAGCAGGCTTAAACAATGATCAATCCACACGGTG 3') and prm08181 (SEQ ID NO: 196; reverse, complementary, AttB2 site in italic: 5' GGGGACCACTTTGTACAAGAAAG CTGGGTTCCTTGTTTACTCATTCCACA 3'), which include the AttB sites for Gateway recombination. The primers used to amplify SEQ ID NO: 177 (PLT2) were prm08182 (SEQ ID NO: 197; sense, start codon in bold, AttB1 site in italic: 5'GGGGACAAGTTTGTACAAAAAAGCAGGCTTAAACAATGAATTCTAACA ACTGGCTC 3') and prm08183 (SEQ ID NO: 198; reverse, complementary, AttB2 site in italic: 5' GGGGACCACTTTGTACAAGAAAGCTGGGTTCATCTTTTATTCATTCCACA 3'),
[0814] The amplified PCR fragments were purified also using standard methods. The first step of the Gateway procedure, the BP reaction, was then performed, during which the PCR fragment recombines in vivo with the pDONR201 plasmid to produce, according to the Gateway terminology, an "entry clone", pPLT1 for SEQ ID NO: 175 and pPLT2 for SEQ ID NO: 177. Plasmid pDONR201 was purchased from Invitrogen, as part of the Gateway® technology.
Example 26
Expression Vector Construction
[0815] The entry clones pPLT1 and pPLT2 were subsequently used in an LR reaction with destination vectors used for Oryza sativa transformation. A first vector contains as functional elements within the T-DNA borders: a plant selectable marker; a screenable marker expression cassette; and a Gateway cassette intended for LR in vivo recombination with the nucleic acid sequence of interest already cloned in the entry clone. A rice constitutive promoter, a GOS2 promoter (SEQ ID NO: 210, alternatively SEQ ID NO: 194 is equally useful) was located upstream of this Gateway cassette.
[0816] A second vector contains the same functional elements within the T-DNA borders: a plant selectable marker; a screenable marker expression cassette; and a Gateway cassette intended for LR in vivo recombination with the nucleic acid sequence of interest already cloned in the entry clone. A rice promoter for expression in meristems, an MT promoter (SEQ ID NO: 211) (PRO0126) was located upstream of this Gateway cassette.
[0817] After the LR recombination step, the resulting expression vectors, pGOS2::PLT1, pGOS2; PLT2, pMT::PLT1 and pMT::PLT2 (FIG. 16 shows the construct with the GOS2 promoter) were independently transformed into Agrobacterium strain LBA4044 and subsequently to Oryza sativa plants. Transformed rice plants were allowed to grow and were then examined for the parameters described below.
Example 27
Crop Transformation
[0818] The transformed Agrobacterium icontaining the expression vectors were used independently to transform Oryza sativa plants. Mature dry seeds of the rice japonica cultivar Nipponbare were dehusked. Sterilization was carried out by incubating for one minute in 70% ethanol, followed by 30 minutes in 0.2% HgCl2, followed by a 6 times 15 minutes wash with sterile distilled water. The sterile seeds were then germinated on a medium containing 2,4-D (callus induction medium). After incubation in the dark for four weeks, embryogenic, scutellum-derived calli were excised and propagated on the same medium. After two weeks, the calli were multiplied or propagated by subculture on the same medium for another 2 weeks. Embryogenic callus pieces were sub-cultured on fresh medium 3 days before co-cultivation (to boost cell division activity).
[0819] Agrobacterium strain LBA4404 containing the expression vector was used for co-cultivation. Agrobacterium was inoculated on AB medium with the appropriate antibiotics and cultured for 3 days at 28° C. The bacteria were then collected and suspended in liquid co-cultivation medium to a density (OD600) of about 1. The suspension was then transferred to a Petri dish and the calli immersed in the suspension for 15 minutes. The callus tissues were then blotted dry on a filter paper and transferred to solidified, co-cultivation medium and incubated for 3 days in the dark at 25° C. Co-cultivated calli were grown on 2,4-D-containing medium for 4 weeks in the dark at 28° C. in the presence of a selection agent. During this period, rapidly growing resistant callus islands developed. After transfer of this material to a regeneration medium and incubation in the light, the embryogenic potential was released and shoots developed in the next four to five weeks. Shoots were excised from the calli and incubated for 2 to 3 weeks on an auxin-containing medium from which they were transferred to soil. Hardened shoots were grown under high humidity and short days in a greenhouse.
[0820] Approximately 35 independent T0 rice transformants were generated for one construct. The primary transformants were transferred from a tissue culture chamber to a greenhouse. After a quantitative PCR analysis to verify copy number of the T-DNA insert, only single copy transgenic plants that exhibit tolerance to the selection agent were kept for harvest of T1 seed. Seeds were then harvested three to five months after transplanting. The method yielded single locus transformants at a rate of over 50% (Aldemita and Hodges1996, Chan et al. 1993, Hiei et al. 1994).
Corn Transformation
[0821] Transformation of maize (Zea mays) is performed with a modification of the method described by Ishida et al. (1996) Nature Biotech 14(6): 745-50. Transformation is genotype-dependent in corn and only specific genotypes are amenable to transformation and regeneration. The inbred line A188 (University of Minnesota) or hybrids with A188 as a parent are good sources of donor material for transformation, but other genotypes can be used successfully as well. Ears are harvested from corn plant approximately 11 days after pollination (DAP) when the length of the immature embryo is about 1 to 1.2 mm. Immature embryos are cocultivated with Agrobacterium tumefaciens containing the expression vector, and transgenic plants are recovered through organogenesis. Excised embryos are grown on callus induction medium, then maize regeneration medium, containing the selection agent (for example imidazolinone but various selection markers can be used). The Petri plates are incubated in the light at 25° C. for 2-3 weeks, or until shoots develop. The green shoots are transferred from each embryo to maize rooting medium and incubated at 25° C. for 2-3 weeks, until roots develop. The rooted shoots are transplanted to soil in the greenhouse. T1 seeds are produced from plants that exhibit tolerance to the selection agent and that contain a single copy of the T-DNA insert.
Wheat Transformation
[0822] Transformation of wheat is performed with the method described by Ishida et al. (1996) Nature Biotech 14(6): 745-50. The cultivar Bobwhite (available from CIMMYT, Mexico) is commonly used in transformation. Immature embryos are co-cultivated with Agrobacterium tumefaciens containing the expression vector, and transgenic plants are recovered through organogenesis. After incubation with Agrobacterium, the embryos are grown in vitro on callus induction medium, then regeneration medium, containing the selection agent (for example imidazolinone but various selection markers can be used). The Petri plates are incubated in the light at 25° C. for 2-3 weeks, or until shoots develop. The green shoots are transferred from each embryo to rooting medium and incubated at 25° C. for 2-3 weeks, until roots develop. The rooted shoots are transplanted to soil in the greenhouse. T1 seeds are produced from plants that exhibit tolerance to the selection agent and that contain a single copy of the T-DNA insert.
Soybean Transformation
[0823] Soybean is transformed according to a modification of the method described in the Texas A&M U.S. Pat. No. 5,164,310. Several commercial soybean varieties are amenable to transformation by this method. The cultivar Jack (available from the Illinois Seed foundation) is commonly used for transformation. Soybean seeds are sterilised for in vitro sowing. The hypocotyl, the radicle and one cotyledon are excised from seven-day old young seedlings. The epicotyl and the remaining cotyledon are further grown to develop axillary nodes. These axillary nodes are excised and incubated with Agrobacterium tumefaciens containing the expression vector. After the cocultivation treatment, the explants are washed and transferred to selection media. Regenerated shoots are excised and placed on a shoot elongation medium. Shoots no longer than 1 cm are placed on rooting medium until roots develop. The rooted shoots are transplanted to soil in the greenhouse. T1 seeds are produced from plants that exhibit tolerance to the selection agent and that contain a single copy of the T-DNA insert.
Rapeseed/Canola Transformation
[0824] Cotyledonary petioles and hypocotyls of 5-6 day old young seedling are used as explants for tissue culture and transformed according to Babic et al. (1998, Plant Cell Rep 17: 183-188). The commercial cultivar Westar (Agriculture Canada) is the standard variety used for transformation, but other varieties can also be used. Canola seeds are surface-sterilized for in vitro sowing. The cotyledon petiole explants with the cotyledon attached are excised from the in vitro seedlings, and inoculated with Agrobacterium (containing the expression vector) by dipping the cut end of the petiole explant into the bacterial suspension. The explants are then cultured for 2 days on MSBAP-3 medium containing 3 mg/l BAP, 3% sucrose, 0.7% Phytagar at 23° C., 16 hr light. After two days of co-cultivation with Agrobacterium, the petiole explants are transferred to MSBAP-3 medium containing 3 mg/l BAP, cefotaxime, carbenicillin, or timentin (300 mg/l) for 7 days, and then cultured on MSBAP-3 medium with cefotaxime, carbenicillin, or timentin and selection agent until shoot regeneration. When the shoots are 5-10 mm in length, they are cut and transferred to shoot elongation medium (MSBAP-0.5, containing 0.5 mg/l BAP). Shoots of about 2 cm in length are transferred to the rooting medium (MS0) for root induction. The rooted shoots are transplanted to soil in the greenhouse. T1 seeds are produced from plants that exhibit tolerance to the selection agent and that contain a single copy of the T-DNA insert.
Alfalfa Transformation
[0825] A regenerating clone of alfalfa (Medicago sativa) is transformed using the method of (McKersie et al., 1999 Plant Physiol 119: 839-847). Regeneration and transformation of alfalfa is genotype dependent and therefore a regenerating plant is required. Methods to obtain regenerating plants have been described. For example, these can be selected from the cultivar Rangelander (Agriculture Canada) or any other commercial alfalfa variety as described by Brown D C W and A Atanassov (1985. Plant Cell Tissue Organ Culture 4: 111-112). Alternatively, the RA3 variety (University of Wisconsin) has been selected for use in tissue culture (Walker et al., 1978 Am J Bot 65:654-659). Petiole explants are cocultivated with an overnight culture of Agrobacterium tumefaciens C58C1 pMP90 (McKersie et al., 1999 Plant Physiol 119: 839-847) or LBA4404 containing the expression vector. The explants are cocultivated for 3 d in the dark on SH induction medium containing 288 mg/L Pro, 53 mg/L thioproline, 4.35 g/L K2SO4, and 100 μm acetosyringinone. The explants are washed in half-strength Murashige-Skoog medium (Murashige and Skoog, 1962) and plated on the same SH induction medium without acetosyringinone but with a suitable selection agent and suitable antibiotic to inhibit Agrobacterium growth. After several weeks, somatic embryos are transferred to BOi2Y development medium containing no growth regulators, no antibiotics, and 50 g/L sucrose. Somatic embryos are subsequently germinated on half-strength Murashige-Skoog medium. Rooted seedlings were transplanted into pots and grown in a greenhouse. T1 seeds are produced from plants that exhibit tolerance to the selection agent and that contain a single copy of the T-DNA insert.
Example 28
Phenotypic Evaluation Procedure
28.1 Evaluation Setup
Evaluation in Normal Growth Conditions
[0826] The primary transformants were transferred from a tissue culture chamber to a greenhouse for growing and harvest of T1 seed. Six events, of which the T1 progeny segregated 3:1 for presence/absence of the transgene, were retained. For each of these events, approximately 10 T1 seedlings containing the transgene (hetero- and homo-zygotes) and approximately 10 T1 seedlings lacking the transgene (nullizygotes) were selected by monitoring visual marker expression. The transgenic plants and the corresponding nullizygotes were grown side-by-side at random positions. Greenhouse conditions were of shorts days (12 hours light), 28° C. in the light and 22° C. in the dark, and a relative humidity of 70%.
Evaluation Under Reduced Nitrogen Availability
[0827] The rice plants were grown in potting soil as under normal conditions except for the nutrient solution. The pots were watered from transplantation to maturation with a specific nutrient solution containing reduced N nitrogen (N) content, usually between 7 to 8 times less. The rest of the cultivation (plant maturation, seed harvest) was the same as for plants not grown under abiotic stress.
28.2 Statistical Analysis: F-Test
[0828] A two factor ANOVA (analysis of variants) was used as a statistical model for the overall evaluation of plant phenotypic characteristics. An F-test was carried out on all the parameters measured of all the plants of all the events transformed with the gene of the present invention. The F-test was carried out to check for an effect of the gene over all the transformation events and to verify for an overall effect of the gene, also known as a global gene effect. The threshold for significance for a true global gene effect was set at a 5% probability level for the F-test. A significant F-test value points to a gene effect, meaning that it is not only the mere presence or position of the gene that is causing the differences in phenotype.
[0829] To check for an effect of the genes within an event, i.e., for a line-specific effect, a t-test was performed within each event using data sets from the transgenic plants and the corresponding null plants. "Null plants" or "null segregants" or "nullizygotes" are the plants treated in the same way as the transgenic plant, but from which the transgene has segregated. Null plants can also be described as the homozygous negative transformed plants. The threshold for significance for the t-test is set at 10% probability level. The results for some events can be above or below this threshold. This is based on the hypothesis that a gene might only have an effect in certain positions in the genome, and that the occurrence of this position-dependent effect is not uncommon. This kind of gene effect is also named herein a "line effect of the gene". The p-value is obtained by comparing the t-value to the t-distribution or alternatively, by comparing the F-value to the F-distribution. The p-value then gives the probability that the null hypothesis (i.e., that there is no effect of the transgene) is correct.
28.3 Parameters Measured
[0830] Biomass-Related Parameter Measurement From the stage of sowing until the stage of maturity the plants were passed several times through a digital imaging cabinet. At each time point digital images (2048×1536 pixels, 16 million colours) were taken of each plant from at least 6 different angles.
[0831] The plant aboveground area (or leafy biomass, areamax) was determined by counting the total number of pixels on the digital images from aboveground plant parts discriminated from the background. This value was averaged for the pictures taken on the same time point from the different angles and was converted to a physical surface value expressed in square mm by calibration. Experiments show that the aboveground plant area measured this way correlates with the biomass of plant parts above ground. The above ground area is the area measured at the time point at which the plant had reached its maximal leafy biomass. The early vigour is the plant (seedling) aboveground area three weeks post-germination. Increase in root biomass is expressed as an increase in total root biomass (measured as maximum biomass of roots observed during the lifespan of a plant); or as an increase in the root/shoot index (measured as the ratio between root mass and shoot mass in the period of active growth of root and shoot).
Seed-Related Parameter Measurements
[0832] The mature primary panicles were harvested, counted, bagged, barcode-labelled and then dried for three days in an oven at 37° C. The panicles were then threshed and all the seeds were collected and counted. The filled husks were separated from the empty ones using an air-blowing device. The empty husks were discarded and the remaining fraction was counted again. The filled husks were weighed on an analytical balance. The number of filled seeds was determined by counting the number of filled husks that remained after the separation step. The total seed yield was measured by weighing all filled husks harvested from a plant. Total seed number per plant was measured by counting the number of husks harvested from a plant. Thousand Kernel Weight (TKW) is extrapolated from the number of filled seeds counted and their total weight. The Harvest Index (HI) in the present invention is defined as the ratio between the total seed yield and the above ground area (mm2), multiplied by a factor 106. The total number of flowers per panicle as defined in the present invention is the ratio between the total number of seeds and the number of mature primary panicles. The seed fill rate as defined in the present invention is the proportion (expressed as a %) of the number of filled seeds over the total number of seeds (or florets).
Example 29
Results of the Phenotypic Evaluation of the Transgenic Plants
29.1 Results of the Phenotypic Evaluation of the Transgenics Plants Grown Under Normal Growth Conditions, Expressing Either PLT1 or PLT2 Nucleic Acid Sequence Under the Control of a Constitutive Promoter
[0833] The TKW measurement results of the T1 seeds of PLT1 and PLT2 transgenic rice plants grown under normal growth conditions, are shown in Table J, in absolute values (averaged events) and as a percentage compard to wild type plants. A substantial increase in TKW is observed for the transgenic seeds containing either construct, compared to wild type seeds.
TABLE-US-00032 TABLE J Results of TKW measurements of the T1 seeds of PLT1 and PLT2 transgenic plants grown under normal growth conditions. TKW (g) % increase PLT1 transgenics 0.0290 16% PLT2 transgenics 0.0288 15% WT 0.0250
[0834] Individual seed parameters (including width, length, area, weight) were measured using a custom-made device consisting of two main components, a weighing and imaging device, coupled to software for image analysis.
[0835] The seed area and seed length measurement results of T1 seeds of PLT1 and PLT2 transgenic rice plants are shown in Table K in absolute values (averaged events) and as a percentage compard to wild type plants. A clear increase in both seed area and seed length is observed for the transgenic seeds containing either construct, compared to wild type seeds.
TABLE-US-00033 TABLE K Results of seed area and seed length measurements of the T1 seeds of PLT1 and PLT2 transgenic plants grown under normal growth conditions. Seed area (mm2) % increase Seed length (mm) % increase PLT1 transgenics 27.60 12% 9.4 13% PLT2 transgenics 27.35 11% 9.4 13% WT 24.56 8.3
[0836] Seed width was not significantly affected on the T1 seeds of the PLT and PLT2 transgenic plants (data not shown).
29.2 Results of the Phenotypic Evaluation of the Transgenics Plants Grown Under Reduced Nitrogen Availability Conditions, Expressing Either PLT1 or PLT2 Nucleic Acid Sequence Under the Control of a Constitutive Promoter
[0837] The TKW measurement results of the T1 seeds of PLT1 and PLT2 transgenic rice plants grown under reduced nitrogen availability conditions, are shown in Table L, in absolute values (averaged events) and as a percentage compard to wild type plants. A substantial increase in TKW is observed for the transgenic seeds containing either construct, compared to wild type seeds.
TABLE-US-00034 TABLE L Results of TKW measurements of the T1 seeds of PLT1 and PLT2 transgenic plants grown under reduced nitrogen availability conditions. TKW (g) % increase PLT1 transgenics 0.0295 12% PLT2 transgenics 0.0278 8% WT 0.0256
29.3 Results of the Phenotypic Evaluation of the Transqenics Plants Grown Under Normal Growth Conditions, Expressing PLT2 Nucleic Acid Sequence Under the Control of a Meristem-Specific Promoter
[0838] The TKW measurement results of the T1 seeds PLT2 transgenic rice plants grown under normal growth conditions, expressing PLT2 nucleic acid sequence under the control of a meristem-specific promoter, are shown in Table M, in absolute values (averaged events) and as a percentage compard to wild type plants. A substantial increase in TKW is observed for the transgenic seeds containing the construct, compared to wild type seeds.
TABLE-US-00035 TABLE M Results of TKW measurements of the T1 seeds of PLT2 transgenic plants grown under normal growth conditions, expressing PLT2 nucleic acid sequence under the control of a mersitem-specific promoter. TKW (g) % increase PLT2 transgenics 0.0263 3% WT 0.0254
Example Section E
bHLH
Example 30
Identification of Paralogues of the bHLH of SEQ ID NO: 213 in Rice
[0839] SEQ ID 2 was used to search for paralogues in the rice genome using a BLASTP algorithm used to perform similarity searches of a protein query sequence to a given database of protein sequences. The protein database queried corresponded to the rice proteome of the MIPS institute, the MIPS Oryza sativa Database (MOsDB), comprising 59,712 sequences; 27,051,637 total letters. Results were ranked according to highest similarity as determined from highest score and lowest e-value. Hits identifying bHLH protein sequences paralogous to SEQ ID NO: 2 had a score of at least 50 and an e-value lower than e-05. Pairwise alignments between the query sequence and the de novo identified paralogues are shown below.
TABLE-US-00036 Query: SEQID 2 Smallest Sum High Probability Sequences producing High-scoring Segment Pairs: Score P(N) N 9629.m00093|protein Helix-loop-helix DNA-binding domain, . . . 1132 1.3e-115 1 9629.m00090|protein Helix-loop-helix DNA-binding domain, . . . 305 5.7e-28 1 9630.m01262|protein Helix-loop-helix DNA-binding domain, . . . 189 1.1e-15 1 9629.m07159|protein Helix-loop-helix DNA-binding domain, . . . 112 3.4e-05 1 >9629.m00093|protein Helix-loop-helix DNA-binding domain, putative Length = 225 Score = 1132 (403.5 bits), Expect = 1.3e-115, P = 1.3e-115 Identities = 224/224 (100%), Positives = 224/224 (100%) Query: 1 MKSRKNSTTSTKAAGSCHTSSSGGGGGGGNCYSSSSSKMERKDVEKNRRLHMKGLCLKLS 60 MKSRKNSTTSTKAAGSCHTSSSGGGGGGGNCYSSSSSKMERKDVEKNRRLHMKGLCLKLS Sbjct: 1 MKSRKNSTTSTKAAGSCHTSSSGGGGGGGNCYSSSSSKMERKDVEKNRRLHMKGLCLKLS 60 Query: 61 SLIPAAAPRRHHHHYSTSSSSSPPSSTKEAVTQLDHLEQAAAYIKQLKGRIDELKKRKQQ 120 SLIPAAAPRRHHHHYSTSSSSSPPSSTKEAVTQLDHLEQAAAYIKQLKGRIDELKKRKQQ Sbjct: 61 SLIPAAAPRRHHHHYSTSSSSSPPSSTKEAVTQLDHLEQAAAYIKQLKGRIDELKKRKQQ 120 Query: 121 AAALTTSTSNGGGGGMPVVEVRCQDGTLDVVVVSEAIREERERAVRLHEVIGVLEEEGAE 180 AAALTTSTSNGGGGGMPVVEVRCQDGTLDVVVVSEAIREERERAVRLHEVIGVLEEEGAE Sbjct: 121 AAALTTSTSNGGGGGMPVVEVRCQDGTLDVVVVSEAIREERERAVRLHEVIGVLEEEGAE 180 Query: 181 VVNASFSVVGDKIFYTLHSQALCSRIGLDASRVSHRLRNLLLQY 224 VVNASFSVVGDKIFYTLHSQALCSRIGLDASRVSHRLRNLLLQY Sbjct: 181 VVNASFSVVGDKIFYTLHSQALCSRIGLDASRVSHRLRNLLLQY 224 >9629.m00090|protein Helix-loop-helix DNA-binding domain, putative Length = 363 Score = 305 (112.4 bits), Expect = 5.7e-28, P = 5.7e-28 Identities = 87/230 (37%), Positives = 126/230 (54%) Query: 19 TSSSGGGGGGGNCYSSSSSKMERKDVEKNRRLHMKGLCLKLSSLIPAAAPRRHHHHYSTS 78 TSSSG G +++++ ERK++E+ RR MKGLC+KL+SLIP + H S Sbjct: 18 TSSSGSGASS----TAAAAAAERKEMERRRRQDMKGLCVKLASLIP-----KEHCSMSKM 68 Query: 79 SSSSPPSSTKEAVTQLDHLEQAAAYIKQLKGRIDELKKRKQQ----------------AA 122 ++S TQL L++AAAYIK+LK R+DEL ++ AA Sbjct: 69 QAASR--------TQLGSLDEAAAYIKKLKERVDELHHKRSMMSITSSRCRSGGGGGPAA 120 Query: 123 ALTTSTSNGGGGGMPVVEVRCQDGTLDVVVVSEAIRE------------ERERAVRLHEV 170 A STS GGGG ++ VV V + ++E R V+ H+V Sbjct: 121 AAGQSTSGGGGGEEEEEDMTRTTAAAAVVEVRQHVQEGSLISLDVVLICSAARPVKFHDV 180 Query: 171 IGVLEEEGAEVVNASFSVVGDKIFYTLHSQALCSRIGLDASRVSHRLRNL 220 I VLEEEGA++++A+FS+ +YT++S+A SRIG++ASR+S RLR L Sbjct: 181 ITVLEEEGADIISANFSLAAHNFYYTIYSRAFSSRIGIEASRISERLRAL 230 >9630.m01262|protein Helix-loop-helix DNA-binding domain, putative Length = 211 Score = 189 (71.6 bits), Expect = 1.1e-15, P = 1.1e-15 Identities = 66/214 (30%), Positives = 102/214 (47%) Query: 21 SSGGGGGGGNCYSSSSSKMERKDVEKNRRLHMKGLCLKLSSLIPAAAPRRHH-HHYSTSS 79 S+GGGGGGG K +RK E+ RR M L L SL+ +A P + +S S+ Sbjct: 7 SAGGGGGGG--------KPDRKTTERIRREQMNKLYSHLDSLVRSAPPTVNSIPSHSNSN 58 Query: 80 SSSPPSSTK------EAVTQLDHLEQAAAYIKQLKGRIDELKKRKQQ---AAALTTSTSN 130 S + A T+ D L AA YI+Q + R+D L+++K++ +S+S+ Sbjct: 59 SKYHQRKLRILGGAAAATTRPDRLGVAAEYIRQTQERVDMLREKKRELTGGGGGGSSSSS 118 Query: 131 GGGGGM---PVVEVRCQDGTLDVVVVSEAIREERERAVRLHEVIGVLEEEGAEVVNASFS 187 G G P VEV+ L ++ + A + H + +E+ G +V NA FS Sbjct: 119 GAGAATAAAPEVEVQHLGSGLHAILFTGAPPTD---GASFHRAVRAVEDAGGQVQNAHFS 175 Query: 188 VVGDKIFYTLHSQALCSRIGLDASRVSHRLRNLL 221 V G K YT+H+ G++ RV RL+ + Sbjct: 176 VAGAKAVYTIHAMIGDGYGGIE--RVVQRLKEAI 207
Example 31
Identification of Orthologues of the bHLH of SEQ ID NO: 213 in Arabidopsis thaliana
[0840] SEQ ID 2 was used to search for orthologues in the Arabidopsis genome using a BLASTP algorithm used to perform a similarity search of a protein query sequence to a given database of protein sequences. The protein database queried corresponded to the Arabidopsis proteome of the MIPS institute, the MIPS Arabidopsis thaliana database (MAtDB) comprising 26,735 sequences; 11,317,104 total letters. Results were ranked according to highest similarity as determined from highest score and lowest e-value. Hits identifying bHLH protein sequences orthologous to SEQ ID2 had a score of at least 50 and an e-value lower than e-05. Pairwise alignments between the query sequence and the de novo identified orthologue are shown below.
TABLE-US-00037 Query: SEQID 2 Database: /home/data/blast/orgs_sets/arabi 26,735 sequences; 11,317,104 total letters. Smallest Sum High Probability Sequences producing High-scoring Segment Pairs: Score P(N) N At1g10585 unknown protein 180 4.5e-15 1 At4g20970 hypothetical protein 149 8.7e-12 1 At4g25410 putative protein 118 2.0e-06 1 At5g51780 putative bHLH transcription factor (bHLH036) 105 1.5e-05 1 At5g51790 putative protein 110 1.5e-05 1 >At1g10585 unknown protein Length = 122 Score = 180 (68.4 bits), Expect = 4.5e-15, P = 4.5e-15 Identities = 38/119 (31%), Positives = 72/119 (60%) Query: 106 QLKGRIDELKKRKQQAAALTTSTSNGGGGGMPVVEVRCQDGTLDVVVVSEAIREERERAV 165 QLK ++ LK++K+ G +P + +R +D T+++ ++ + + +R V Sbjct: 3 QLKENVNYLKEKKRTLLQGELGNLYEGSFLLPKLSIRSRDSTIEMNLIMD-LNMKR---V 58 Query: 166 RLHEVIGVLEEEGAEVVNASFSVVGDKIFYTLHSQALCSRIGLDASRVSHRLRNLLLQY 224 LHE++ + EEEGA+V++A+ + D+ YT+ +QA+ SRIG+D SR+ R+R ++ Y Sbjct: 59 MLHELVSIFEEEGAQVMSANLQNLNDRTTYTIIAQAIISRIGIDPSRIEERVRKIIYGY 117 >At4g20970 hypothetical protein Length = 167 Score = 149 (57.5 bits), Expect = 8.7e-12, P = 8.7e-12 Identities = 49/171 (28%), Positives = 86/171 (50%) Query: 33 SSSSSKMERKDVEKNRRLHMKGLCLKLSSLIPAAAPRRHHHHYSTSSSSSPPSSTKEAVT 92 + S ++RK VEKNRR+ MK L +L SL+P HH ST + P EA Sbjct: 8 TGQSRSVDRKTVEKNRRMQMKSLYSELISLLP--------HHSSTEPLTLP-DQLDEAAN 58 Query: 93 QLDHLEQAAAYIKQLKGRI---DELKKRKQQ-AAALTTSTSNGGGGGMPVVEVRCQDGTL 148 + L+ ++ K + L+K ++++++S +P +E++ + G++ Sbjct: 59 YIKKLQVNVEKKRERKRNLVATTTLEKLNSVGSSSVSSSVDVSVPRKLPKIEIQ-ETGSI 117 Query: 149 DVVVVSEAIREERERAVRLHEVIGVLEEE-GAEVVNASFSVVGDKIFYTLH 198 + + ++ E E+I VL EE GAE+ +A +S+V D +F+TLH Sbjct: 118 FHIFLVTSL----EHKFMFCEIIRVLTEELGAEITHAGYSIVDDAVFHTLH 164
Example 32
Identification of a bHLH from Medicago truncatula
[0841] The question to be addressed was whether the query sequence (SEQ ID NO: 227 from Medicago truncatula) was a bHLH polypeptide according to the definition applied herein. SEQ ID 227 was compared to the Arabidopsis proteome database of the MIPS institute.
[0842] Comparison was carried out using the BLASTP 2.0 MP-WashU algorithm, which performs similarity searches of a protein query sequence to a given database of protein sequences. (Reference: Gish, W. (1996-2002)). The parameters used in the comparison were E value 10 Cutoff score (S2): 56
[0843] The protein database queried corresponded to the Arabidopsis proteome of the MIPS institute comprising 26,735 sequences (Database: /home/data/blast/orgs_sets/arabi 26,735 sequences; 11,317,104 total letters). Results were ranked according to highest similarity as determined from highest score and lowest e-value. The first Hit identified (underlined in the alignment) corresponded to SEQ ID NO: 225 (At4g20970) indicating that the sequence is a bHLH polypeptide according to the definition applied herein. Pairwise alignments between the query sequence from Medicago and the first hit corresponding to the de novo identified bHLH are shown below.
[0844] BLASTP 2.0 MP-WashU [9 Sep. 2002] [decunix4.0-ev56-132LPF64 2002-09-09T17:45:09]
TABLE-US-00038 Query = SEQ ID NO: 227 (140 letters) Database: /home/data/blast/orgs_sets/arabi 26,735 sequences; 11,317,104 total letters. Searching . . . 10 . . . 20 . . . 30 . . . 40 . . . 50 . . . 60 . . . 70 . . . 80 . . . 90 . . . 100% done Smallest Sum High Probability Sequences producing High-scoring Segment Pairs: Score P(N) N At4g20970 hypothetical protein 248 2.8e-22 1 At5g51780 putative bHLH transcription factor (bHLH036) 103 6.5e-07 1 At1g10585 unknown protein 100 1.4e-06 1 At4g25400 putative bHLH transcription factor (bHLH118) 99 7.3e-06 1 >At4g20970 hypothetical protein Length = 167 Score = 248 (92.4 bits), Expect = 2.8e-22, P = 2.8e-22 Identities = 52/123 (42%), Positives = 82/123 (66%) Query: 7 EAISVPDQLKEATNYIKKLQINLEKMKEKKNFLLG---IQRPN------VNLNRNQKMGL 57 E +++PDQL EA NYIKKLQ+N+EK +E+K L+ +++ N V+ + + + Sbjct: 45 EPLTLPDQLDEAANYIKKLQVNVEKKRERKRNLVATTTLEKLNSVGSSSVSSSVDVSVPR 104 Query: 58 KSPKIKIQQIGLVLEVVLITGLESQFLFSETFRVLHEE-GVDIVNASYKVNEDSVFHSIH 116 K PKI+IQ+ G + + L+T LE +F+F E RVL EE G +I +A Y + +D+VFH++H Sbjct: 105 KLPKIEIQETGSIFHIFLVTSLEHKFMFCEIIRVLTEELGAEITHAGYSIVDDAVFHTLH 164 Query: 117 CQV 119 C+V Sbjct: 165 CKV 167
Example 33
Identification of Sequences Related to the Nucleic Acid Sequence Used in the Methods of the Invention
[0845] bHLH Sequences, whether nucleotide (full length cDNA, ESTs or genomic) or protein (full length or partial polypeptides), were used to identified other bHLH nucleotide or proteins sequences amongst those maintained in the Entrez Nucleotides database at the National Center for Biotechnology Information (NCBI) using database sequence search tools, such as the Basic Local Alignment Tool (BLAST) (Altschul et al. (1990) J. Mol. Biol. 215:403-410; and Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402). The program was used to find regions of local similarity between sequences by comparing nucleic acid or polypeptide sequences to sequence databases and by calculating the statistical significance of matches. For example, the polypeptide encoded by SEQ ID NO: 213 was used as query to the nr (non-redundant: (Database: All GenBank+EMBL+DDBJ+PDB sequences (but no EST, STS, GSS,environmental samples or phase 0, 1 or 2 HTGS sequences) 3,819,973 sequences; 16,928,533,343 total letters) database sequence using the TBLASTN algorithm, with default settings and the filter to ignore low complexity sequences. The output of the analysis (shown below) was viewed by pairwise comparison, and ranked according to the probability score (E-value), where the score reflect the probability that a particular alignment occurs by chance (the lower the E-value, the more significant the hit). In addition to E-values, comparisons were also scored by percentage identity. Percentage identity refers to the number of identical nucleotides (or amino acids) between the two compared nucleic acid (or polypeptide) sequences over a particular length.
[0846] Hits on the database identifying BHLH proteins produced a score of at least 50 and an e-value equal to or lower than e-05.
TABLE-US-00039 Score E Sequences producing significant alignments: (Bits) Value gi|55765745|ref|NM_183508.2|Oryza sativa (japonica cultivar-gro 440 1e-121 gi|42821746|dbj|AK109432.2|Oryza sativa (japonica cultivar-g... 440 1e-121 gi|55769744|ref|XM_549862.1|Oryza sativa (japonica cultivar-gro 247 1e-105 gi|58530787|dbj|AP008207.1|Oryza sativa (japonica cultivar-g... 263 2e-68 gi|17385651|dbj|AP002845.3|Oryza sativa (japonica cultivar-g... 263 2e-68 gi|32980356|dbj|AK070332.1|Oryza sativa (japonica cultivar-g... 251 9e-65 gi|34894135|ref|NM_183504.1|Oryza sativa (japonica cultivar-gro 137 3e-30 gi|15293050|gb|AY050959.1|Arabidopsis thaliana unknown prote... 80.1 6e-16 gi|79340923|ref|NM_100934.2|Arabidopsis thaliana transcripti... 87.8 2e-15 gi|58531195|dbj|AP008214.1|Oryza sativa (japonica cultivar-g... 51.6 4e-13 gi|42408246|dbj|AP004557.3|Oryza sativa (japonica cultivar-g... 51.6 4e-13 gi|42408168|dbj|AP004376.3|Oryza sativa (japonica cultivar-g... 51.6 4e-13 gi|22328837|ref|NM_118215.2|Arabidopsis thaliana DNA binding... 78.2 1e-12 gi|7268888|emb|AL161554.2|ATCHRIV54 Arabidopsis thaliana DNA chr 77.8 2e-12 gi|5262774|emb|AL080282.1|ATT13K14 Arabidopsis thaliana DNA c... 77.8 2e-12 gi|50906596|ref|XM_464787.1|Oryza sativa (japonica cultivar-gro 77.0 3e-12
Example 34
Determination of Global Similarity and Identity Between bHLH Transcription Factors
[0847] Global percentages similarity and identity between bHLH polypeptides was determined using the MatGAT software (BMC Bioinformatics. 2003 Jul. 10; 4:29. MatGAT: an application that generates similarity/identity matrices using protein or DNA sequences. Campanella J J, Bitincka L, Smalley J.). MatGAT software generates similarity/identity matrices for DNA or protein sequences without needing pre-alignment of the data. The program performs a series of pair-wise alignments, calculates similarity and identity, and then places the results in a distance matrix.
[0848] Parameters used in the comparison were:
[0849] Scoring matrix: Blosum62
[0850] First Gap: 12
[0851] Extending gap: 2
[0852] Results are shown in FIG. 19a.
Example 35
Determination of Global Similarity and Identity Between bHLH Domains in bHLH Polypeptides
[0853] bHLH domains were mapped using the SMART software (Schultz et al. (1998) Proc. Natl. Acad. Sci. USA 95, 5857-5864; Letunic et al. (2006) Nucleic Acids Res 34, D257-D260). Smart software is available through EMBL institute(European Molecular Biology Laboratory (EMBL).
[0854] The sequences of the bHLH domain used are given below.
TABLE-US-00040 Hv_BHLH KESEKERRKRMKALCEKLASLIPREHCCSTTDTMTQLGSLDVGASYIKKL KERVDE OS_NP_908393_BHLH KEMERRRRQDMKGLCVKLASLIPKEHCSMSKMQAASRTQLGSLDEAAAYI KKLKERVDE OS_NP_908397.1_BHLH KDVEKNRRLHMKGLCLKLSSLIPAAAPRRHHHHYSTSSSSSPPSSTKEAV TQLDHLEQAAAYIKQLKGRIDE Os_XP_464787.1_BHLH KTTERIRREQMNKLYSHLDSLVRSAPPTVNSIPSHSNSNSKYHQRKLRIL GGAAAATTRPDRLGVAAEYIRQTQERVDM AT1G10585_bHLH nlrekdrrmrmkhlfsilsshvsptrklpvphlidqatsymiqlkenvny At4g20970_bHLH KTVEKNRRMQMKSLYSELISLLPHHSSTEPLTLPDQLDEAANYIKKLQVN VEK
[0855] Global percentages of similarity and identity between bHLH domains of bHLH polypeptides were determined using the software and parameters described in Example 34.
[0856] Results are shown in FIG. 19b.
Example 36
Cloning of the Nucleic Acid Sequence Used in the Methods of the Invention
[0857] The nucleic acid sequence used in the methods of the invention was amplified by PCR using as template a custom-made Oryza sativa seedlings cDNA library (in pCMV Sport 6.0; Invitrogen, Paisley, UK). PCR was performed using Hifi Taq DNA polymerase in standard conditions, using 200 ng of template in a 50 μl PCR mix. The primers used were prm06808 (SEQ ID NO: 231; sense, start codon in bold, AttB1 site in italic: 5'-ggggacaagtttgtacaaaaaagcaggcttaaacaatgaagagcaggaagaacagc 3') and prm06809 (SEQ ID NO: 232; reverse, complementary, AttB2 site in italic: 5' ggggaccactttgtacaagaaagctgggtgcagagtgaaagagtggtgtg 3'), which include the AttB sites for Gateway recombination. The amplified PCR fragment was purified also using standard methods. The first step of the Gateway procedure, the BP reaction, was then performed, during which the PCR fragment recombines in vivo with the pDONR201 plasmid to produce, according to the Gateway terminology, an "entry clone", pbHLH. Plasmid pDONR201 was purchased from Invitrogen, as part of the Gateway® technology.
Example 37
Expression Vector Construction
[0858] The entry clone p076 was subsequently used in an LR reaction with pGOS2, a destination vector used for Oryza sativa transformation. This vector contains as functional elements within the T-DNA borders: a plant selectable marker; a screenable marker expression cassette; and a Gateway cassette intended for LR in vivo recombination with the nucleic acid sequence of interest already cloned in the entry clone. A rice GOS2 promoter (SEQ ID NO: 233, alternatively, SEQ ID NO: 230 is equally useful) for constitutive expression (internal reference PRO0129) was located upstream of this Gateway cassette.
[0859] After the LR recombination step, the resulting expression vector pGOS2::bHLH (FIG. 20) was transformed into Agrobacterium strain LBA4044 and subsequently to Oryza sativa plants.
Example 38
Plant Transformation
Rice Transformation
[0860] The Agrobacterium containing the expression vector was used to transform Oryza sativa plants. Mature dry seeds of the rice japonica cultivar Nipponbare were dehusked. Sterilization was carried out by incubating for one minute in 70% ethanol, followed by 30 minutes in 0.2% HgCl2, followed by a 6 times 15 minutes wash with sterile distilled water. The sterile seeds were then germinated on a medium containing 2,4-D (callus induction medium). After incubation in the dark for four weeks, embryogenic, scutellum-derived calli were excised and propagated on the same medium. After two weeks, the calli were multiplied or propagated by subculture on the same medium for another 2 weeks. Embryogenic callus pieces were sub-cultured on fresh medium 3 days before co-cultivation (to boost cell division activity).
[0861] Agrobacterium strain LBA4404 containing the expression vector was used for co-cultivation. Agrobacterium was inoculated on AB medium with the appropriate antibiotics and cultured for 3 days at 28° C. The bacteria were then collected and suspended in liquid co-cultivation medium to a density (OD600) of about 1. The suspension was then transferred to a Petri dish and the calli immersed in the suspension for 15 minutes. The callus tissues were then blotted dry on a filter paper and transferred to solidified, co-cultivation medium and incubated for 3 days in the dark at 25° C. Co-cultivated calli were grown on 2,4-D-containing medium for 4 weeks in the dark at 28° C. in the presence of a selection agent. During this period, rapidly growing resistant callus islands developed. After transfer of this material to a regeneration medium and incubation in the light, the embryogenic potential was released and shoots developed in the next four to five weeks. Shoots were excised from the calli and incubated for 2 to 3 weeks on an auxin-containing medium from which they were transferred to soil. Hardened shoots were grown under high humidity and short days in a greenhouse.
[0862] Approximately 30 independent T0 rice transformants were generated for one construct. The primary transformants were transferred from a tissue culture chamber to a greenhouse. After a quantitative PCR analysis to verify copy number of the T-DNA insert, only single copy transgenic plants that exhibit tolerance to the selection agent were kept for harvest of T1 seed. Seeds were then harvested three to five months after transplanting. The method yielded single locus transformants at a rate of over 50% (Aldemita and Hodges1996, Chan et al. 1993, Hiei et al. 1994).
Corn Transformation
[0863] Transformation of maize (Zea mays) is performed with a modification of the method described by Ishida et al. (1996) Nature Biotech 14(6): 745-50. Transformation is genotype-dependent in corn and only specific genotypes are amenable to transformation and regeneration. The inbred line A188 (University of Minnesota) or hybrids with A188 as a parent are good sources of donor material for transformation, but other genotypes can be used successfully as well. Ears are harvested from corn plant approximately 11 days after pollination (DAP) when the length of the immature embryo is about 1 to 1.2 mm. Immature embryos are cocultivated with Agrobacterium tumefaciens containing the expression vector, and transgenic plants are recovered through organogenesis. Excised embryos are grown on callus induction medium, then maize regeneration medium, containing the selection agent (for example imidazolinone but various selection markers can be used). The Petri plates are incubated in the light at 25° C. for 2-3 weeks, or until shoots develop. The green shoots are transferred from each embryo to maize rooting medium and incubated at 25° C. for 2-3 weeks, until roots develop. The rooted shoots are transplanted to soil in the greenhouse. T1 seeds are produced from plants that exhibit tolerance to the selection agent and that contain a single copy of the T-DNA insert.
Wheat Transformation
[0864] Transformation of wheat is performed with the method described by Ishida et al. (1996) Nature Biotech 14(6): 745-50. The cultivar Bobwhite (available from CIMMYT, Mexico) is commonly used in transformation. Immature embryos are co-cultivated with Agrobacterium tumefaciens containing the expression vector, and transgenic plants are recovered through organogenesis. After incubation with Agrobacterium, the embryos are grown in vitro on callus induction medium, then regeneration medium, containing the selection agent (for example imidazolinone but various selection markers can be used). The Petri plates are incubated in the light at 25° C. for 2-3 weeks, or until shoots develop. The green shoots are transferred from each embryo to rooting medium and incubated at 25° C. for 2-3 weeks, until roots develop. The rooted shoots are transplanted to soil in the greenhouse. T1 seeds are produced from plants that exhibit tolerance to the selection agent and that contain a single copy of the T-DNA insert.
Soybean Transformation
[0865] Soybean is transformed according to a modification of the method described in the Texas A&M U.S. Pat. No. 5,164,310. Several commercial soybean varieties are amenable to transformation by this method. The cultivar Jack (available from the Illinois Seed foundation) is commonly used for transformation. Soybean seeds are sterilised for in vitro sowing. The hypocotyl, the radicle and one cotyledon are excised from seven-day old young seedlings. The epicotyl and the remaining cotyledon are further grown to develop axillary nodes. These axillary nodes are excised and incubated with Agrobacterium tumefaciens containing the expression vector. After the cocultivation treatment, the explants are washed and transferred to selection media. Regenerated shoots are excised and placed on a shoot elongation medium. Shoots no longer than 1 cm are placed on rooting medium until roots develop. The rooted shoots are transplanted to soil in the greenhouse. T1 seeds are produced from plants that exhibit tolerance to the selection agent and that contain a single copy of the T-DNA insert.
Rapeseed/Canola Transformation
[0866] Cotyledonary petioles and hypocotyls of 5-6 day old young seedling are used as explants for tissue culture and transformed according to Babic et al. (1998, Plant Cell Rep 17: 183-188). The commercial cultivar Westar (Agriculture Canada) is the standard variety used for transformation, but other varieties can also be used. Canola seeds are surface-sterilized for in vitro sowing. The cotyledon petiole explants with the cotyledon attached are excised from the in vitro seedlings, and inoculated with Agrobacterium (containing the expression vector) by dipping the cut end of the petiole explant into the bacterial suspension. The explants are then cultured for 2 days on MSBAP-3 medium containing 3 mg/l BAP, 3% sucrose, 0.7% Phytagar at 23° C., 16 hr light. After two days of co-cultivation with Agrobacterium, the petiole explants are transferred to MSBAP-3 medium containing 3 mg/l BAP, cefotaxime, carbenicillin, or timentin (300 mg/l) for 7 days, and then cultured on MSBAP-3 medium with cefotaxime, carbenicillin, or timentin and selection agent until shoot regeneration. When the shoots are 5-10 mm in length, they are cut and transferred to shoot elongation medium (MSBAP-0.5, containing 0.5 mg/l BAP). Shoots of about 2 cm in length are transferred to the rooting medium (MS0) for root induction. The rooted shoots are transplanted to soil in the greenhouse. T1 seeds are produced from plants that exhibit tolerance to the selection agent and that contain a single copy of the T-DNA insert.
Alfalfa Transformation
[0867] A regenerating clone of alfalfa (Medicago sativa) is transformed using the method of (McKersie et al., 1999 Plant Physiol 119: 839-847). Regeneration and transformation of alfalfa is genotype dependent and therefore a regenerating plant is required. Methods to obtain regenerating plants have been described. For example, these can be selected from the cultivar Rangelander (Agriculture Canada) or any other commercial alfalfa variety as described by Brown D C W and A Atanassov (1985. Plant Cell Tissue Organ Culture 4: 111-112). Alternatively, the RA3 variety (University of Wisconsin) has been selected for use in tissue culture (Walker et al., 1978 Am J Bot 65:654-659). Petiole explants are cocultivated with an overnight culture of Agrobacterium tumefaciens C58C1 pMP90 (McKersie et al., 1999 Plant Physiol 119: 839-847) or LBA4404 containing the expression vector. The explants are cocultivated for 3 d in the dark on SH induction medium containing 288 mg/L Pro, 53 mg/L thioproline, 4.35 g/L K2SO4, and 100 μm acetosyringinone. The explants are washed in half-strength Murashige-Skoog medium (Murashige and Skoog, 1962) and plated on the same SH induction medium without acetosyringinone but with a suitable selection agent and suitable antibiotic to inhibit Agrobacterium growth. After several weeks, somatic embryos are transferred to BOi2Y development medium containing no growth regulators, no antibiotics, and 50 g/L sucrose. Somatic embryos are subsequently germinated on half-strength Murashige-Skoog medium. Rooted seedlings were transplanted into pots and grown in a greenhouse. T1 seeds are produced from plants that exhibit tolerance to the selection agent and that contain a single copy of the T-DNA insert.
Example 39
Evaluation Procedure
39.1 Evaluation Setup
[0868] Approximately 30 independent T0 rice transformants were generated. The primary transformants were transferred from a tissue culture chamber to a greenhouse for growing and harvest of T1 seed. Seven events, of which the T1 progeny segregated 3:1 for presence/absence of the transgene, were retained. For each of these events, approximately 10 T1 seedlings containing the transgene (hetero- and homo-zygotes) and approximately 10 T1 seedlings lacking the transgene (nullizygotes) were selected by monitoring visual marker expression. The transgenic plants and the corresponding nullizygotes were grown side-by-side at random positions. Greenhouse conditions were of shorts days (12 hours light), 28° C. in the light and 22° C. in the dark, and a relative humidity of 70%.
[0869] Four T1 events were further evaluated in the T2 generation following the same evaluation procedure as for the T1 generation but with more individuals per event. From the stage of sowing until the stage of maturity the plants were passed several times through a digital imaging cabinet. At each time point digital images (2048×1536 pixels, 16 million colours) were taken of each plant from at least 6 different angles.
39.2 Statistical Analysis: t-Test and F-Test
[0870] A two factor ANOVA (analysis of variants) was used as a statistical model for the overall evaluation of plant phenotypic characteristics. An F-test was carried out on all the parameters measured of all the plants of all the events transformed with the gene of the present invention. The F-test was carried out to check for an effect of the gene over all the transformation events and to verify for an overall effect of the gene, also known as a global gene effect. The threshold for significance for a true global gene effect was set at a 5% probability level for the F-test. A significant F-test value points to a gene effect, meaning that it is not only the mere presence or position of the gene that is causing the differences in phenotype.
[0871] To check for an effect of the genes within an event, i.e., for a line-specific effect, a t-test was performed within each event using data sets from the transgenic plants and the corresponding null plants. "Null plants" or "null segregants" or "nullizygotes" are the plants treated in the same way as the transgenic plant, but from which the transgene has segregated. Null plants can also be described as the homozygous negative transformed plants. The threshold for significance for the t-test is set at 10% probability level. The results for some events can be above or below this threshold. This is based on the hypothesis that a gene might only have an effect in certain positions in the genome, and that the occurrence of this position-dependent effect is not uncommon. This kind of gene effect is also named herein a "line effect of the gene". The p-value is obtained by comparing the t-value to the t-distribution or alternatively, by comparing the F-value to the F-distribution. The p-value then gives the probability that the null hypothesis (i.e., that there is no effect of the transgene) is correct.
Example 40
Evaluation Results
[0872] Early vigour was determined by counting the total number of pixels from aboveground plant parts discriminated from the background. This value was averaged for the pictures taken on the same time point from different angles and was converted to a physical surface value expressed in square mm by calibration. The results described below are for plants three weeks post-germination.
[0873] Early vigour (as determined by aboveground area) was seen in six out of seven events of the T1 generation, with an overall increase in aboveground area for transgenic seedlings of 22% compared to control plants. Four of these T1 events were further evaluated in the T2 generation, and all four of these events gave an increase in aboveground area for transgenic seedlings compared to control plants, with an overall increase in aboveground area for transgenic seedlings of 13% compared to control plants. The results were also shown to be statistically significant with the p-value from the F-test being 0.0007 (T2 generation) indicating that the effect seen is likely due to the transgene rather than the position of the gene or a line effect.
Example Section F
SPL15
Example 41
Identification of Sequences Related to the Nucleic Acid Sequence Used in the Methods of the Invention
[0874] Sequences (full length cDNA, ESTs or genomic) related to the nucleic acid sequence used in the methods of the present invention were identified amongst those maintained in the Entrez Nucleotides database at the National Center for Biotechnology Information using database sequence search tools, such as the Basic Local Alignment Tool (BLAST) (Altschul et al. (1990) J. Mol. Biol. 215:403-410; and Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402). The program is used to find regions of local similarity between sequences by comparing nucleic acid or polypeptide sequences to sequence databases and by calculating the statistical significance of matches. The polypeptide encoded by the nucleic acid of the present invention was used for the TBLASTN algorithm, with default settings and the filter to ignore low complexity sequences set off. The output of the analysis was viewed by pairwise comparison, and ranked according to the probability score (E-value), where the score reflect the probability that a particular alignment occurs by chance (the lower the E-value, the more significant the hit). In addition to E-values, comparisons were also scored by percentage identity. Percentage identity refers to the number of identical nucleotides (or amino acids) between the two compared nucleic acid (or polypeptide) sequences over a particular length. In some instances, the default parameters may be adjusted to modify the stringency of the search
[0875] The Table N below provides a list of nucleic acid sequences related to the nucleic acid sequence used in the methods of the present invention.
TABLE-US-00041 TABLE N nucleic acid sequences related to the nucleic acid sequence (SEQ ID NO: 234) used in the methods of the present invention, and the corresponding deduced polypeptides Nucleic acid Polypeptide Sequence NCBI accession Name SEQ ID NO SEQ ID NO length number Source organism Arath_SPL15 234 235 Full length NM_115654.1 Arabidopsis thaliana (At3g57920) Arath_SPL9 236 237 Full length AY150378 Arabidopsis thaliana (At2g42200) Aqufo_SPL 238 239 Full length contig of DR915312 Aquilegia formosa × DR949057.1 Aquilegia pubescens Goshi_SPL 240 241 Full length DT566400 Gossypium hirsutum Iponi_SPL 242 243 Full length contig of Ipomoea nil BJ576204.1 BJ556115 BJ567301 Lacsa_SPL 244 245 Full length contig of DY966949 Lactuca sativa DW119178 Maldo_SPL 246 247 Full length contig of Malus domestica CN891102.1 CO868185.1 CV523507 Medtr_SPL 248 249 Full length spliced from Medicago truncatula AC170989.2 Nicbe_SPL 250 251 Full length contig of CK284078.1 Nicotiana CK294165 bentamiana Orysa_SPL 252 253 Full length XM_483285 Oryza sativa Orysa_SPL II 254 255 Full length spliced from AC108762 Oryza sativa Soltu_SPL 256 257 Full length contig of CK246692.1 Solanum tuberosum CK254420.1 Vitvi_SPL 258 259 Full length contig of Vitis vinifera CV098277 CV092812.1 Zeama_SPL 260 261 Full length contig of EB160653 Zea mays DY235599 DV029129 Zeama_SPL 262 263 Full length contig of AJ011619 Zea mays II DV033513.1 DY532686.1 Sorpr_SPL 264 265 Partial BF422188 Sorghum propinquium Allce_SPL 266 267 Partial CF444518.1 Allium cepa Antma_SPL 268 269 Partial AMA011623 Antirrhinum majus Brana_SPL 270 271 Partial CX189447 Brassica napus Sacof_SPL 272 273 Partial contig of CA113070 Saccharum CA254724 officinarum Fesar_SPL 274 275 Partial DT706587.1 Festuca arundinacea Brara_SPL 282 283 Full length AC189445.1 Brassica rapa Glyma_SPL 284 285 Full length CX708501.1 Glycine max BG651519.1 Poptr_SPL 286 287 Full length scaff_XVI.416 Populus tremuloides Citcl_SPL 288 289 Partial DY293795 Citrus clementina Betvu_SPL 290 291 Partial BQ594361.1 Beta vulgaris Hevbr_SPL 292 293 Partial EC604947 Hevea brasiliensis
Example 42
Determination of Global Similarity and Identity Between SPL15 Transcription Factors, and their SPL DBD
[0876] Global percentages of similarity and identity between SPL15 transcription factors were determined using one of the methods available in the art, the MatGAT (Matrix Global Alignment Tool) software (BMC Bioinformatics. 2003 4:29. MatGAT: an application that generates similarity/identity matrices using protein or DNA sequences. Campanella J J, Bitincka L, Smalley J; software hosted by Ledion Bitincka). MatGAT software generates similarity/identity matrices for DNA or protein sequences without needing pre-alignment of the data. The program performs a series of pair-wise alignments using the Myers and Miller global alignment algorithm (with a gap opening penalty of 12, and a gap extension penalty of 2), calculates similarity and identity using for example Blosum 62 (for polypeptides), and then places the results in a distance matrix. Sequence similarity is shown in the bottom half of the dividing line and sequence identity is shown in the top half of the diagonal dividing line. The sequence of SEQ ID NO: 235 is indicated as number 5 in the matrix.
[0877] Parameters used in the comparison were:
[0878] Scoring matrix: Blosum62
[0879] First Gap: 12
[0880] Extending gap: 2
[0881] Results of the software analysis are shown in Table 0 for the global similarity and identity over the full length of the SPL15 transcription factor polypeptides. Percentage identity is given above the diagonal and percentage similarity is given below the diagonal. Percentage identity between the SPL15 transcription factor paralogues and orthologues ranges between 30 and 70%, reflecting the relatively low sequence identity conservation between them outside of the SPL DBD.
TABLE-US-00042 TABLE O MatGAT results for global similarity and identity over the full length of the SPL15 transcription factor polypeptides. Global similarity and identity over the full length of the SPL15 transcription factor polypeptides 1 2 3 4 5 6 7 8 9 10 11 12 13 14 1. Arath_SPL9 51.1 44.8 40.3 38.4 44 43.1 40.3 34.6 35.9 39.4 46.7 33.9 35.7 2. Arath_SPL15 63.2 39.4 39 33.7 40.1 40.3 35.1 31.3 33.7 36.5 39.6 30.5 31.9 3. Goshi_SPL 58.9 53.2 47.3 43 54.4 46.9 52 41.3 40.2 50.1 59.8 39.5 39.1 4. Iponi_SPL 53.3 55.9 61.7 43.9 50.9 46.8 56.1 33.6 37.2 56.7 53.5 36.3 38.7 5. Lacsa_SPL 52 49.4 53.2 61.3 45.8 43.5 46.4 37 38.3 47.9 48.1 37.6 39.9 6. Maldo_SPL 59.3 54.5 64.3 63.5 56.3 49.5 57.4 42 38.1 55.2 68.7 38.9 40.6 7. Medtr_SPL 55.2 57.6 60.4 64.5 58.4 65.1 46.7 37.3 39.2 46.3 55.4 37.6 39.7 8. Nicbe_SPL 58.2 51.6 63.8 66.8 56.8 70.8 62.9 36.9 40 66.6 62.1 38.2 37.5 9. Orysa_SPL 48.7 42.2 54.2 47 47.2 53.2 49.2 51.8 62.4 37.3 40.7 59.5 54.9 10. Orysa_SPL II 49.9 45.8 53.4 49.1 48.9 51.1 53.4 53.2 72.2 37.6 43.6 54.3 62.9 11. Soltu_SPL 57.3 54 64 68.5 59.4 69.6 62.9 77.6 51.8 50.4 60.7 36.9 37.9 12. Vitvi_SPL 62.8 54.9 69.2 66 59.4 80.2 67.5 73.4 53.5 56.5 73.6 42.2 40.8 13. Zeama_SPL 47.3 43.3 55.2 48.8 49.3 52 51.2 52.5 68.3 67.9 49.8 54 54.9 14. Zeama_SPL II 49.2 43.1 53.2 51.6 50.8 54.5 53.2 48.7 64.3 71.2 51.1 54.6 66.2
[0882] Results of the software analysis are shown in Table P for the global similarity and identity over the SPL DBD of the SPL15 transcription factor polypeptides. Percentage identity is given above the diagonal and percentage similarity is given below the diagonal. Percentage identity between the SPL DBD of SPL15 transcription factor paralogues and orthologues ranges between 70% and 100%.
TABLE-US-00043 TABLE P MatGAT results for global similarity and identity over the SPL DBD of the SPL15 transcription factor polypeptides. Global similarity and identity over the SPL DBD of the SPL15 transcription factor polypeptides 1 2 3 4 5 6 7 8 9 10 11 12 13 14 1. SPL\DBD\Aqufo_SPL 75.6 79.5 84.8 85.9 87.2 85.9 88.5 84.6 80.8 78.2 85.9 91 76.9 2. SPL\DBD\Arath_SPL15 82.1 78.2 83.5 82.1 78.2 76.9 78.2 79.5 71.8 74.4 79.5 79.5 71.8 3. SPL\DBD\Arath_SPL9 87.2 85.9 77.2 78.2 82.1 82.1 79.5 78.2 76.9 78.2 79.5 80.8 76.9 4. SPL\DBD\Goshi_SPL 88.6 91.1 86.1 86.1 84.8 84.8 84.8 84.8 78.5 78.5 86.1 89.9 78.5 5. SPL\DBD\Iponi_SPL 89.7 85.9 87.2 91.1 84.6 84.6 85.9 85.9 80.8 79.5 87.2 89.7 78.2 6. SPL\DBD\Lacsa_SPL 89.7 85.9 88.5 91.1 91 88.5 84.6 85.9 80.8 79.5 85.9 89.7 78.2 7. SPL\DBD\Maldo_SPL 92.3 82.1 88.5 89.9 91 91 84.6 89.7 85.9 83.3 88.5 91 80.8 8. SPL\DBD\Medtr_SPL 89.7 85.9 89.7 92.4 92.3 91 93.6 84.6 79.5 78.2 88.5 93.6 78.2 9. SPL\DBD\Nicbe_SPL 87.2 83.3 85.9 88.6 92.3 89.7 91 92.3 84.6 82.1 88.5 89.7 80.8 10. SPL\DBD\Orysa_SPL 87.2 80.8 84.6 84.8 88.5 85.9 89.7 85.9 89.7 85.9 84.6 83.3 83.3 11. SPL\DBD\Orysa_SPL II 84.6 82.1 87.2 83.5 85.9 84.6 88.5 85.9 88.5 92.3 82.1 82.1 84.6 12. SPL\DBD\Soltu_SPL 89.7 87.2 87.2 91.1 94.9 89.7 93.6 93.6 92.3 87.2 84.6 93.6 80.8 13. SPL\DBD\Vitvi_SPL 92.3 87.2 89.7 93.7 94.9 92.3 96.2 97.4 92.3 87.2 87.2 96.2 82.1 14. SPL\DBD\Zeama_SPL II 83.3 79.5 84.6 83.5 84.6 83.3 84.6 87.2 84.6 88.5 89.7 83.3 85.9
Example 43
Cloning of the Nucleic Acid Sequence Used in the Methods of the Invention
[0883] DNA manipulation: unless otherwise stated, recombinant DNA techniques are performed according to standard protocols described in (Sambrook (2001) Molecular Cloning: a laboratory manual, 3rd Edition Cold Spring Harbor Laboratory Press, CSH, New York) or in Volumes 1 and 2 of Ausubel et al. (1994), Current Protocols in Molecular Biology, Current Protocols. Standard materials and methods for plant molecular work are described in Plant Molecular Biology Labfase (1993) by R.D.D. Croy, published by BIOS Scientific Publications Ltd (UK) and Blackwell Scientific Publications (UK).
[0884] The nucleic acid sequence used in the methods of the invention was amplified by PCR using as template a custom-made Arabidopsis thaliana mixed tissues cDNA library (in pCMV Sport 6.0; Invitrogen, Paisley, UK). PCR was performed using Hifi Taq DNA polymerase in standard conditions, using 200 ng of template in a 50 μl PCR mix. The primers used were prm07277 (SEQ ID NO: 280; sense, start codon in bold, AttB1 site in lower case: 5'-ggggacaagtttgtacaaaaaagcaggcttaaacaATGGAGTTGTTAATGTGTTCG 3') and prm07278 (SEQ ID NO: 281; reverse, complementary, AttB2 site in lower case: 5' ggggaccactttgtacaagaaagctgggtTGATGAAGATCTTAAAAGGTGA 3'), which include the AttB sites for Gateway recombination. The amplified PCR fragment was purified also using standard methods. The first step of the Gateway procedure, the BP reaction, was then performed, during which the PCR fragment recombines in vivo with the pDONR201 plasmid to produce, according to the Gateway terminology, an "entry clone", pSPL15. Plasmid pDONR201 was purchased from Invitrogen, as part of the Gateway® technology.
Example 44
Expression Vector Construction
[0885] The entry clone p13075 was subsequently used in an LR reaction with p06659, a destination vector used for Oryza sativa transformation. This vector contains as functional elements within the T-DNA borders: a plant selectable marker; a screenable marker expression cassette; and a Gateway cassette intended for LR in vivo recombination with the nucleic acid sequence of interest already cloned in the entry clone. A rice HMGB promoter (SEQ ID NO: 294) for constitutive expression was located upstream of this Gateway cassette. Alternatively, the HMGB promoter represented by SEQ ID NO: 46 is equally useful. A similar construct was made with the SPL15 coding sequence under control of the constitutive GOS2 promoter (SEQ ID NO: 295).
[0886] After the LR recombination step, the resulting expression vector pHMGB::SPL15 (FIG. 26), or pGOS2::SPL15, was transformed into Agrobacterium strain LBA4044 and subsequently to Oryza sativa plants.
Example 45
Plant Transformation
Rice Transformation
[0887] The Agrobacterium containing the expression vector was used to transform Oryza sativa plants. Mature dry seeds of the rice japonica cultivar Nipponbare were dehusked. Sterilization was carried out by incubating for one minute in 70% ethanol, followed by 30 minutes in 0.2% HgCl2, followed by a 6 times 15 minutes wash with sterile distilled water. The sterile seeds were then germinated on a medium containing 2,4-D (callus induction medium). After incubation in the dark for four weeks, embryogenic, scutellum-derived calli were excised and propagated on the same medium. After two weeks, the calli were multiplied or propagated by subculture on the same medium for another 2 weeks. Embryogenic callus pieces were sub-cultured on fresh medium 3 days before co-cultivation (to boost cell division activity).
[0888] Agrobacterium strain LBA4404 containing the expression vector was used for co-cultivation. Agrobacterium was inoculated on AB medium with the appropriate antibiotics and cultured for 3 days at 28° C. The bacteria were then collected and suspended in liquid co-cultivation medium to a density (OD600) of about 1. The suspension was then transferred to a Petri dish and the calli immersed in the suspension for 15 minutes. The callus tissues were then blotted dry on a filter paper and transferred to solidified, co-cultivation medium and incubated for 3 days in the dark at 25° C. Co-cultivated calli were grown on 2,4-D-containing medium for 4 weeks in the dark at 28° C. in the presence of a selection agent. During this period, rapidly growing resistant callus islands developed. After transfer of this material to a regeneration medium and incubation in the light, the embryogenic potential was released and shoots developed in the next four to five weeks. Shoots were excised from the calli and incubated for 2 to 3 weeks on an auxin-containing medium from which they were transferred to soil. Hardened shoots were grown under high humidity and short days in a greenhouse.
[0889] Approximately 35 independent T0 rice transformants were generated for one construct. The primary transformants were transferred from a tissue culture chamber to a greenhouse. After a quantitative PCR analysis to verify copy number of the T-DNA insert, only single copy transgenic plants that exhibit tolerance to the selection agent were kept for harvest of T1 seed. Seeds were then harvested three to five months after transplanting. The method yielded single locus transformants at a rate of over 50% (Aldemita and Hodges1996, Chan et al. 1993, Hiei et al. 1994).
Corn Transformation
[0890] Transformation of maize (Zea mays) is performed with a modification of the method described by Ishida et al. (1996) Nature Biotech 14(6): 745-50. Transformation is genotype-dependent in corn and only specific genotypes are amenable to transformation and regeneration. The inbred line A188 (University of Minnesota) or hybrids with A188 as a parent are good sources of donor material for transformation, but other genotypes can be used successfully as well. Ears are harvested from corn plant approximately 11 days after pollination (DAP) when the length of the immature embryo is about 1 to 1.2 mm. Immature embryos are cocultivated with Agrobacterium tumefaciens containing the expression vector, and transgenic plants are recovered through organogenesis. Excised embryos are grown on callus induction medium, then maize regeneration medium, containing the selection agent (for example imidazolinone but various selection markers can be used). The Petri plates are incubated in the light at 25° C. for 2-3 weeks, or until shoots develop. The green shoots are transferred from each embryo to maize rooting medium and incubated at 25° C. for 2-3 weeks, until roots develop. The rooted shoots are transplanted to soil in the greenhouse. T1 seeds are produced from plants that exhibit tolerance to the selection agent and that contain a single copy of the T-DNA insert.
Wheat Transformation
[0891] Transformation of wheat is performed with the method described by Ishida et al. (1996) Nature Biotech 14(6): 745-50. The cultivar Bobwhite (available from CIMMYT, Mexico) is commonly used in transformation. Immature embryos are co-cultivated with Agrobacterium tumefaciens containing the expression vector, and transgenic plants are recovered through organogenesis. After incubation with Agrobacterium, the embryos are grown in vitro on callus induction medium, then regeneration medium, containing the selection agent (for example imidazolinone but various selection markers can be used). The Petri plates are incubated in the light at 25° C. for 2-3 weeks, or until shoots develop. The green shoots are transferred from each embryo to rooting medium and incubated at 25° C. for 2-3 weeks, until roots develop. The rooted shoots are transplanted to soil in the greenhouse. T1 seeds are produced from plants that exhibit tolerance to the selection agent and that contain a single copy of the T-DNA insert.
Soybean Transformation
[0892] Soybean is transformed according to a modification of the method described in the Texas A&M U.S. Pat. No. 5,164,310. Several commercial soybean varieties are amenable to transformation by this method. The cultivar Jack (available from the Illinois Seed foundation) is commonly used for transformation. Soybean seeds are sterilised for in vitro sowing. The hypocotyl, the radicle and one cotyledon are excised from seven-day old young seedlings. The epicotyl and the remaining cotyledon are further grown to develop axillary nodes. These axillary nodes are excised and incubated with Agrobacterium tumefaciens containing the expression vector. After the cocultivation treatment, the explants are washed and transferred to selection media. Regenerated shoots are excised and placed on a shoot elongation medium. Shoots no longer than 1 cm are placed on rooting medium until roots develop. The rooted shoots are transplanted to soil in the greenhouse. T1 seeds are produced from plants that exhibit tolerance to the selection agent and that contain a single copy of the T-DNA insert.
Rapeseed/Canola Transformation
[0893] Cotyledonary petioles and hypocotyls of 5-6 day old young seedling are used as explants for tissue culture and transformed according to Babic et al. (1998, Plant Cell Rep 17: 183-188). The commercial cultivar Westar (Agriculture Canada) is the standard variety used for transformation, but other varieties can also be used. Canola seeds are surface-sterilized for in vitro sowing. The cotyledon petiole explants with the cotyledon attached are excised from the in vitro seedlings, and inoculated with Agrobacterium (containing the expression vector) by dipping the cut end of the petiole explant into the bacterial suspension. The explants are then cultured for 2 days on MSBAP-3 medium containing 3 mg/l BAP, 3% sucrose, 0.7% Phytagar at 23° C., 16 hr light. After two days of co-cultivation with Agrobacterium, the petiole explants are transferred to MSBAP-3 medium containing 3 mg/l BAP, cefotaxime, carbenicillin, or timentin (300 mg/l) for 7 days, and then cultured on MSBAP-3 medium with cefotaxime, carbenicillin, or timentin and selection agent until shoot regeneration. When the shoots are 5-10 mm in length, they are cut and transferred to shoot elongation medium (MSBAP-0.5, containing 0.5 mg/l BAP). Shoots of about 2 cm in length are transferred to the rooting medium (MS0) for root induction. The rooted shoots are transplanted to soil in the greenhouse. T1 seeds are produced from plants that exhibit tolerance to the selection agent and that contain a single copy of the T-DNA insert.
Alfalfa Transformation
[0894] A regenerating clone of alfalfa (Medicago sativa) is transformed using the method of (McKersie et al., 1999 Plant Physiol 119: 839-847). Regeneration and transformation of alfalfa is genotype dependent and therefore a regenerating plant is required. Methods to obtain regenerating plants have been described. For example, these can be selected from the cultivar Rangelander (Agriculture Canada) or any other commercial alfalfa variety as described by Brown D C W and A Atanassov (1985. Plant Cell Tissue Organ Culture 4: 111-112). Alternatively, the RA3 variety (University of Wisconsin) has been selected for use in tissue culture (Walker et al., 1978 Am J Bot 65:654-659). Petiole explants are cocultivated with an overnight culture of Agrobacterium tumefaciens C58C1 pMP90 (McKersie et al., 1999 Plant Physiol 119: 839-847) or LBA4404 containing the expression vector. The explants are cocultivated for 3 d in the dark on SH induction medium containing 288 mg/L Pro, 53 mg/L thioproline, 4.35 g/L K2SO4, and 100 μm acetosyringinone. The explants are washed in half-strength Murashige-Skoog medium (Murashige and Skoog, 1962) and plated on the same SH induction medium without acetosyringinone but with a suitable selection agent and suitable antibiotic to inhibit Agrobacterium growth. After several weeks, somatic embryos are transferred to BOi2Y development medium containing no growth regulators, no antibiotics, and 50 g/L sucrose. Somatic embryos are subsequently germinated on half-strength Murashige-Skoog medium. Rooted seedlings were transplanted into pots and grown in a greenhouse. T1 seeds are produced from plants that exhibit tolerance to the selection agent and that contain a single copy of the T-DNA insert.
Example 46
Evaluation Procedure
46.1 Evaluation Setup
[0895] Approximately 35 independent T0 rice transformants were generated. The primary transformants were transferred from a tissue culture chamber to a greenhouse for growing and harvest of T1 seed. Seven events, of which the T1 progeny segregated 3:1 for presence/absence of the transgene, were retained. For each of these events, approximately 10 T1 seedlings containing the transgene (hetero- and homo-zygotes) and approximately 10 T1 seedlings lacking the transgene (nullizygotes) were selected by monitoring visual marker expression. The transgenic plants and the corresponding nullizygotes were grown side-by-side at random positions. Greenhouse conditions were of shorts days (12 hours light), 28° C. in the light and 22° C. in the dark, and a relative humidity of 70%.
[0896] Five T1 events were further evaluated in the T2 generation following the same evaluation procedure as for the T1 generation but with more individuals per event. From the stage of sowing until the stage of maturity the plants were passed several times through a digital imaging cabinet. At each time point digital images (2048×1536 pixels, 16 million colours) were taken of each plant from at least 6 different angles.
[0897] The early vigour is the plant (seedling) aboveground area three weeks post-germination. Increase in root biomass is expressed as an increase in total root biomass (measured as maximum biomass of roots observed during the lifespan of a plant); or as an increase in the root/shoot index (measured as the ratio between root mass and shoot mass in the period of active growth of root and shoot).
46.2 Statistical Analysis: F-Test
[0898] A two factor ANOVA (analysis of variants) was used as a statistical model for the overall evaluation of plant phenotypic characteristics. An F-test was carried out on all the parameters measured of all the plants of all the events transformed with the gene of the present invention. The F-test was carried out to check for an effect of the gene over all the transformation events and to verify for an overall effect of the gene, also known as a global gene effect. The threshold for significance for a true global gene effect was set at a 5% probability level for the F-test. A significant F-test value points to a gene effect, meaning that it is not only the mere presence or position of the gene that is causing the differences in phenotype.
[0899] To check for an effect of the genes within an event, i.e., for a line-specific effect, a t-test was performed within each event using data sets from the transgenic plants and the corresponding null plants. "Null plants" or "null segregants" or "nullizygotes" are the plants treated in the same way as the transgenic plant, but from which the transgene has segregated. Null plants can also be described as the homozygous negative transformed plants. The threshold for significance for the t-test is set at 10% probability level. The results for some events can be above or below this threshold. This is based on the hypothesis that a gene might only have an effect in certain positions in the genome, and that the occurrence of this position-dependent effect is not uncommon. This kind of gene effect is also named herein a "line effect of the gene". The p-value is obtained by comparing the t-value to the t-distribution or alternatively, by comparing the F-value to the F-distribution. The p-value then gives the probability that the null hypothesis (i.e., that there is no effect of the transgene) is correct.
Example 47
Evaluation Results
[0900] The plant aboveground area (or leafy biomass) was determined by counting the total number of pixels on the digital images from aboveground plant parts discriminated from the background. This value was averaged for the pictures taken on the same time point from the different angles and was converted to a physical surface value expressed in square mm by calibration. Experiments show that the aboveground plant area measured this way correlates with the biomass of plant parts above ground. The above ground area is the time point at which the plant had reached its maximal leafy biomass.
[0901] The mature primary panicles were harvested, counted, bagged, barcode-labeled and then dried for three days in an oven at 37° C. The panicles were then threshed and all the seeds were collected and counted. The filled husks were separated from the empty ones using an air-blowing device. The empty husks were discarded and the remaining fraction was counted again. The filled husks were weighed on an analytical balance. The number of filled seeds was determined by counting the number of filled husks that remained after the separation step. The total seed yield was measured by weighing all filled husks harvested from a plant. Total seed number per plant was measured by counting the number of husks harvested from a plant. Thousand kernel weight (TKW) is extrapolated from the number of filled seeds counted and their total weight. The harvest index (HI) in the present invention is defined as the ratio between the total seed yield and the above ground area (mm2), multiplied by a factor 106. The total number of flowers per panicle as defined in the present invention is the ratio between the total number of seeds and the number of mature primary panicles. The seed fill rate as defined in the present invention is the proportion (expressed as a %) of the number of filled seeds over the total number of seeds (or florets).
[0902] As presented in Tables Q to W, the aboveground biomass, the number of flowers per panicle, the seed yield, the total number of seeds, the number of filled seeds, the thousand kernel weight (TKW) and harvest index are increased in the transgenic plants with increased expression a nucleic acid encoding a SPL15 transcription factor polypeptide, compared to suitable control plants. Results from the T1 and the T2 generations are shown.
[0903] Table Q shows the number of transgenic events with an increase in aboveground biomass, the percentage of this increase, as well as the statistical relevance of this increase according to the F-test.
TABLE-US-00044 TABLE Q Number of transgenic events with an increase in aboveground biomass, the percentage of the increase, and P value of the F-test in T1 and T2 generation of transgenic rice with increased expression of a nucleic acid encoding an SPL15 transcription factor polypeptide. Aboveground biomass Number of events showing an increase % Difference P value of F test T1 generation 5 out of 7 5 0.0773 T2 generation 4 out of 5 7 0.0172
[0904] Table R shows the number of transgenic events with an increase in the total number of flowers per panicle, the percentage of this increase, as well as the statistical relevance of this increase according to the F-test.
TABLE-US-00045 TABLE R Number of transgenic events with an increase in flowers per panicle, the percentage of the increase, and P value of the F-test in T1 and T2 generation of transgenic rice with increased expression of a nucleic acid encoding an SPL15 transcription factor polypeptide. Flowers per panicle Number of events showing an increase % Difference P value of F test T1 generation 6 out of 7 4 0.0447 T2 generation 4 out of 5 10 0.0013
[0905] Table S shows the number of transgenic events with an increase in total seed yield (total seed weight), the percentage of this increase, as well as the statistical relevance of this increase according to the F-test.
TABLE-US-00046 TABLE S Number of transgenic events with an increase in seed yield, the percentage of the increase, and P value of the F-test in T1 and T2 generation of transgenic rice with increased expression of a nucleic acid encoding an SPL15 transcription factor polypeptide. Seed yield Number of events showing an increase % Difference P value of F test T1 generation 7 out of 7 15 0.0019 T2 generation 5 out of 5 21 0.0001
[0906] Table T shows the number of transgenic events with an increase in the total number of seeds, the percentage of this increase, as well as the statistical relevance of this increase according to the F-test.
TABLE-US-00047 TABLE T Number of transgenic events with an increase in total number of seeds, the percentage of the increase, and P value of the F-test in T1 and T2 generation of transgenic rice with increased expression of a nucleic acid encoding an SPL15 transcription factor polypeptide. Total number of seeds Number of events showing an increase % Difference P value of F test T1 generation 6 out of 7 6 0.07 T2 generation 4 out of 5 13 0.0023
[0907] Table U shows the number of transgenic events with an increase in the number of filled seeds, the percentage of this increase, as well as the statistical relevance of this increase according to the F-test.
TABLE-US-00048 TABLE U Number of transgenic events with an increase in number of filled seeds, the percentage of the increase, and P value of the F-test in T1 and T2 generation of transgenic rice with increased expression of a nucleic acid encoding an SPL15 transcription factor polypeptide. Number of filled seeds Number of events showing an increase % Difference P value of F test T1 generation 6 out of 7 14 0.0031 T2 generation 4 out of 5 19 0.0003
[0908] Table V shows the number of transgenic events with an increase in the harvest index, the percentage of this increase, as well as the statistical relevance of this increase according to the F-test.
TABLE-US-00049 TABLE V Number of transgenic events with an increase in harvest index, the percentage of the increase, and P value of the F-test in T1 and T2 generation of transgenic rice with increased expression of a nucleic acid encoding an SPL15 transcription factor polypeptide. Harvest index Number of events showing an increase % Difference P value of F test T1 generation 7 out of 7 12 0.0003 T2 generation 4 out of 5 15 0.0001
[0909] Table W shows the number of transgenic events with an increase in the thousand kernel weight (TKW), the percentage of this increase, as well as the statistical relevance of this increase according to the F-test.
TABLE-US-00050 TABLE W Number of transgenic events with an increase in thousand kernel weight (TKW), the percentage of the increase, and P value of the F-test in T1 and T2 generation of transgenic rice with increased expression of a nucleic acid encoding an SPL15 transcription factor polypeptide. Thousand kernel weight Number of events showing an increase % Difference P value of F test T1 generation 4 out of 7 2 0.0041 T2 generation 4 out of 5 1 0.0259
Example 48
Results of the Phenotypic Evaluation of the Transgenic Plants Expressing SEQ ID NO: 234 Under the Control of a Constitutive Promoter
[0910] The results of the evaluation of transgenic rice plants expressing the nucleic acid sequence useful in performing the methods of the invention, under the control of the constitutive GOS2 promoter, are presented in Table X. The percentage difference between the transgenics and the corresponding nullizygotes is also shown.
[0911] The transgenic plants expressing the nucleic acid sequence useful in performing the methods of the invention, have increased early vigour and increased TKW compared to the control plants (in this case, the nullizygotes).
TABLE-US-00051 TABLE X Results of the evaluation of T1 generation transgenic rice plants expressing the nucleic acid sequence useful in performing the methods of the invention, under the control of a strong constitutive GOS2 promoter. % Increase of the Trait best events in T1 generation Increased early vigour 17% Total seed yield (per plant) 18% Total number of filled seeds 19% Increased seed fill rate 10% Increased harvest index 15%
Sequence CWU
1
1
3011630DNAArabidopsis thaliana 1atggagaatg ggaaaagaga cagacaagac
atggaagtga ataccacacc gaggaagcct 60cgtgtactac tcgctgcaag tggaagcgtc
gctgctatca aattcggcaa tctctgccat 120tgcttcaccg aatgggcaga agtcagagcc
gtcgttacga aatcatctct acatttcctc 180gataaactct ctctcccaca agaagtgact
ctgtatactg atgaagatga atggtctagc 240tggaacaaga tcggtgatcc tgtccttcac
atcgagctta gacgttgggc tgatgtttta 300gtcattgctc ctttgtctgc taacacctta
ggcaagattg ctggtgggct ttgtgataat 360cttctgactt gcattatacg agcttgggac
tataccaaac cactgtttgt tgctccagct 420atgaatactt tgatgtggaa caatcctttc
actgaaaggc atcttttgtc tcttgatgaa 480ctgggaatca cacttattcc tcctatcaag
aagagacttg cctgtggaga ctacggtaat 540ggagctatgg ctgagccctc tcttatctat
tccactgtca gactcttctg ggagtctcag 600gctcatcagc aaaccggtgg aactagttaa
6302209PRTArabidopsis thaliana 2Met Glu
Asn Gly Lys Arg Asp Arg Gln Asp Met Glu Val Asn Thr Thr 1 5
10 15 Pro Arg Lys Pro Arg Val Leu
Leu Ala Ala Ser Gly Ser Val Ala Ala 20 25
30 Ile Lys Phe Gly Asn Leu Cys His Cys Phe Thr Glu
Trp Ala Glu Val 35 40 45
Arg Ala Val Val Thr Lys Ser Ser Leu His Phe Leu Asp Lys Leu Ser
50 55 60 Leu Pro Gln
Glu Val Thr Leu Tyr Thr Asp Glu Asp Glu Trp Ser Ser 65
70 75 80 Trp Asn Lys Ile Gly Asp Pro
Val Leu His Ile Glu Leu Arg Arg Trp 85
90 95 Ala Asp Val Leu Val Ile Ala Pro Leu Ser Ala
Asn Thr Leu Gly Lys 100 105
110 Ile Ala Gly Gly Leu Cys Asp Asn Leu Leu Thr Cys Ile Ile Arg
Ala 115 120 125 Trp
Asp Tyr Thr Lys Pro Leu Phe Val Ala Pro Ala Met Asn Thr Leu 130
135 140 Met Trp Asn Asn Pro Phe
Thr Glu Arg His Leu Leu Ser Leu Asp Glu 145 150
155 160 Leu Gly Ile Thr Leu Ile Pro Pro Ile Lys Lys
Arg Leu Ala Cys Gly 165 170
175 Asp Tyr Gly Asn Gly Ala Met Ala Glu Pro Ser Leu Ile Tyr Ser Thr
180 185 190 Val Arg
Leu Phe Trp Glu Ser Gln Ala His Gln Gln Thr Gly Gly Thr 195
200 205 Ser 337DNAArtificial
sequenceprimer prm00957 3aaaaagcagg ctcacaatgg agaatgggaa aagagac
37433DNAArtificial sequenceprimer prm00958
4agaaagctgg gttggtttta actagttcca ccg
3351243DNAOryza sativa 5aaaaccaccg agggacctga tctgcaccgg ttttgatagt
tgagggaccc gttgtgtctg 60gttttccgat cgagggacga aaatcggatt cggtgtaaag
ttaagggacc tcagatgaac 120ttattccgga gcatgattgg gaagggagga cataaggccc
atgtcgcatg tgtttggacg 180gtccagatct ccagatcact cagcaggatc ggccgcgttc
gcgtagcacc cgcggtttga 240ttcggcttcc cgcaaggcgg cggccggtgg ccgtgccgcc
gtagcttccg ccggaagcga 300gcacgccgcc gccgccgacc cggctctgcg tttgcaccgc
cttgcacgcg atacatcggg 360atagatagct actactctct ccgtttcaca atgtaaatca
ttctactatt ttccacattc 420atattgatgt taatgaatat agacatatat atctatttag
attcattaac atcaatatga 480atgtaggaaa tgctagaatg acttacattg tgaattgtga
aatggacgaa gtacctacga 540tggatggatg caggatcatg aaagaattaa tgcaagatcg
tatctgccgc atgcaaaatc 600ttactaattg cgctgcatat atgcatgaca gcctgcatgc
gggcgtgtaa gcgtgttcat 660ccattaggaa gtaaccttgt cattacttat accagtacta
catactatat agtattgatt 720tcatgagcaa atctacaaaa ctggaaagca ataaggaata
cgggactgga aaagactcaa 780cattaatcac caaatatttc gccttctcca gcagaatata
tatctctcca tcttgatcac 840tgtacacact gacagtgtac gcataaacgc agcagccagc
ttaactgtcg tctcaccgtc 900gcacactggc cttccatctc aggctagctt tctcagccac
ccatcgtaca tgtcaactcg 960gcgcgcgcac aggcacaaat tacgtacaaa acgcatgacc
aaatcaaaac caccggagaa 1020gaatcgctcc cgcgcgcggc ggcggcgcgc acgtacgaat
gcacgcacgc acgcccaacc 1080ccacgacacg atcgcgcgcg acgccggcga caccggccat
ccacccgcgc cctcacctcg 1140ccgactataa atacgtaggc atctgcttga tcttgtcatc
catctcacca ccaaaaaaaa 1200aggaaaaaaa aacaaaacac accaagccaa ataaaagcga
caa 1243634PRTArtificial sequenceconserved motif 1
6Xaa Pro Arg Xaa Leu Leu Ala Ala Xaa Gly Ser Val Ala Xaa Xaa Lys 1
5 10 15 Phe Xaa Asn Leu
Xaa Xaa Xaa Phe Xaa Xaa Trp Ala Xaa Val Xaa Ala 20
25 30 Val Xaa 798PRTArtificial
sequenceconserved motif 2 7Val Leu His Ile Glu Leu Arg Xaa Trp Ala Asp
Xaa Xaa Xaa Ile Ala 1 5 10
15 Pro Leu Ser Ala Asn Thr Leu Xaa Lys Ile Ala Gly Gly Xaa Cys Asp
20 25 30 Asn Leu
Leu Thr Cys Xaa Xaa Arg Ala Trp Asp Xaa Xaa Lys Pro Xaa 35
40 45 Phe Xaa Ala Pro Ala Met Asn
Thr Xaa Met Trp Xaa Asn Pro Phe Thr 50 55
60 Xaa Xaa His Xaa Xaa Xaa Xaa Xaa Xaa Xaa Gly Xaa
Xaa Leu Xaa Pro 65 70 75
80 Pro Xaa Xaa Lys Xaa Leu Ala Cys Gly Asp Xaa Gly Xaa Gly Ala Met
85 90 95 Xaa Glu
8171PRTArabidopsis thaliana 8Lys Pro Arg Val Leu Leu Ala Ala Ser Gly Ser
Val Ala Ala Ile Lys 1 5 10
15 Phe Gly Asn Leu Cys His Cys Phe Thr Glu Trp Ala Glu Val Arg Ala
20 25 30 Val Val
Thr Lys Ser Ser Leu His Phe Leu Asp Lys Leu Ser Leu Pro 35
40 45 Gln Glu Val Thr Leu Tyr Thr
Asp Glu Asp Glu Trp Ser Ser Trp Asn 50 55
60 Lys Ile Gly Asp Pro Val Leu His Ile Glu Leu Arg
Arg Trp Ala Asp 65 70 75
80 Val Leu Val Ile Ala Pro Leu Ser Ala Asn Thr Leu Gly Lys Ile Ala
85 90 95 Gly Gly Leu
Cys Asp Asn Leu Leu Thr Cys Ile Ile Arg Ala Trp Asp 100
105 110 Tyr Thr Lys Pro Leu Phe Val Ala
Pro Ala Met Asn Thr Leu Met Trp 115 120
125 Asn Asn Pro Phe Thr Glu Arg His Leu Leu Ser Leu Asp
Glu Leu Gly 130 135 140
Ile Thr Leu Ile Pro Pro Ile Lys Lys Arg Leu Ala Cys Gly Asp Tyr 145
150 155 160 Gly Asn Gly Ala
Met Ala Glu Pro Ser Leu Ile 165 170
9606DNAArabidopsis thaliana 9atgaatatgg aagtggatac agtaacaagg aagcctcgta
tcttactagc tgcaagtgga 60agtgtggctt caattaagtt cagtaatctc tgccattgtt
tctcagaatg ggctgaagtc 120aaagccgtcg cttcaaaatc atctctcaat ttcgttgata
aaccttctct acctcagaat 180gtgactctct atacagatga agatgaatgg tctagctgga
acaagattgg tgatcccgtt 240cttcatatcg agctcagacg ctgggctgat gttatgatca
ttgctccttt gtctgctaac 300acattagcca agattgctgg tgggttatgt gataatctat
tgacatgtat agtaagagca 360tgggattata gcaaaccgtt gtttgttgca ccggcgatga
acactttgat gtggaacaat 420cctttcacag aacggcacct tgtcttgctt gatgaacttg
gaatcaccct aattcctccc 480atcaagaaga aactggcctg tggagactac ggtaatggcg
caatggctga gccttctctg 540atttattcca ctgttagact gttctgggag tcacaagctc
gtaaacaaag agatggaacc 600agttga
60610201PRTArabidopsis thaliana 10Met Asn Met Glu
Val Asp Thr Val Thr Arg Lys Pro Arg Ile Leu Leu 1 5
10 15 Ala Ala Ser Gly Ser Val Ala Ser Ile
Lys Phe Ser Asn Leu Cys His 20 25
30 Cys Phe Ser Glu Trp Ala Glu Val Lys Ala Val Ala Ser Lys
Ser Ser 35 40 45
Leu Asn Phe Val Asp Lys Pro Ser Leu Pro Gln Asn Val Thr Leu Tyr 50
55 60 Thr Asp Glu Asp Glu
Trp Ser Ser Trp Asn Lys Ile Gly Asp Pro Val 65 70
75 80 Leu His Ile Glu Leu Arg Arg Trp Ala Asp
Val Met Ile Ile Ala Pro 85 90
95 Leu Ser Ala Asn Thr Leu Ala Lys Ile Ala Gly Gly Leu Cys Asp
Asn 100 105 110 Leu
Leu Thr Cys Ile Val Arg Ala Trp Asp Tyr Ser Lys Pro Leu Phe 115
120 125 Val Ala Pro Ala Met Asn
Thr Leu Met Trp Asn Asn Pro Phe Thr Glu 130 135
140 Arg His Leu Val Leu Leu Asp Glu Leu Gly Ile
Thr Leu Ile Pro Pro 145 150 155
160 Ile Lys Lys Lys Leu Ala Cys Gly Asp Tyr Gly Asn Gly Ala Met Ala
165 170 175 Glu Pro
Ser Leu Ile Tyr Ser Thr Val Arg Leu Phe Trp Glu Ser Gln 180
185 190 Ala Arg Lys Gln Arg Asp Gly
Thr Ser 195 200 111128DNAOryza sativa
11caaccaaagt ccaaaggcta ttctgaaccg aagtcctccc acaccaaaac ttcgaggccc
60ccgtcgcggc accctcctcc gccgccggat ctccaccgga gtacggcgcc ggccaccccc
120tcctcccccg gatcttcacc gccggtgaag tactgaggtg atgactacat cagagtcagt
180acaagaaacc ttgggattgg atttccctca tcctagcaaa cctcgggtcc tccttgctgc
240ctctggaagc gtcgctgcta taaaatttga gagcctttgc cgtagcttct cggaatgggc
300agaagtcaga gccgtcgcca ccaaggcttc attacatttt attgatagaa cgtctctgcc
360tagcaatatt attctttaca ctgatgatga tgaatggtct acctggaaga agatagggga
420tgaagttttg cacattgaac tgcgaaaatg ggcagatatc atggtgattg cgccattatc
480agctaatacc ctagctaaga ttgctggtgg tttatgtgac aacctcttga catgcatagt
540gagagcatgg gactacagca aaccactctt tgttgcccca gctatgaaca ccttcatgtg
600gaacaacccg ttcaccagtc gtcatcttga gacaatcaac ctgctaggta tatctttggt
660ccctcccatt accaaaaggc tggcctgtgg tgattatggt aatggtgcaa tggctgagcc
720ttctgtgatc gattccaccg tcaggcttgc ttgcaagaga cagccactta atacaaatag
780ttcacctgtg gttcctgccg gcagaaacct cccatctagc tgatgcggca actattctgt
840tcaagattaa actctggacc tagttttcta tggtaaagag tacttcgtgt cacaaaatga
900aatgttaagt gatgtctatg tcggccaaca tagcaccttt attggccagt tgttgtacta
960ctattagtga tatggtagga cgtggagatt ggagaaaggc tattgtccca gcactttaat
1020gttgcttttc caaatttctt gtgacataat gctaaggtgc tgatgaatat gttcatgttg
1080tagcactatt tttttctgca aatgtttgca aagactcgtg atggaatc
112812220PRTOryza sativa 12Met Thr Thr Ser Glu Ser Val Gln Glu Thr Leu
Gly Leu Asp Phe Pro 1 5 10
15 His Pro Ser Lys Pro Arg Val Leu Leu Ala Ala Ser Gly Ser Val Ala
20 25 30 Ala Ile
Lys Phe Glu Ser Leu Cys Arg Ser Phe Ser Glu Trp Ala Glu 35
40 45 Val Arg Ala Val Ala Thr Lys
Ala Ser Leu His Phe Ile Asp Arg Thr 50 55
60 Ser Leu Pro Ser Asn Ile Ile Leu Tyr Thr Asp Asp
Asp Glu Trp Ser 65 70 75
80 Thr Trp Lys Lys Ile Gly Asp Glu Val Leu His Ile Glu Leu Arg Lys
85 90 95 Trp Ala Asp
Ile Met Val Ile Ala Pro Leu Ser Ala Asn Thr Leu Ala 100
105 110 Lys Ile Ala Gly Gly Leu Cys Asp
Asn Leu Leu Thr Cys Ile Val Arg 115 120
125 Ala Trp Asp Tyr Ser Lys Pro Leu Phe Val Ala Pro Ala
Met Asn Thr 130 135 140
Phe Met Trp Asn Asn Pro Phe Thr Ser Arg His Leu Glu Thr Ile Asn 145
150 155 160 Leu Leu Gly Ile
Ser Leu Val Pro Pro Ile Thr Lys Arg Leu Ala Cys 165
170 175 Gly Asp Tyr Gly Asn Gly Ala Met Ala
Glu Pro Ser Val Ile Asp Ser 180 185
190 Thr Val Arg Leu Ala Cys Lys Arg Gln Pro Leu Asn Thr Asn
Ser Ser 195 200 205
Pro Val Val Pro Ala Gly Arg Asn Leu Pro Ser Ser 210
215 220 131164DNATriticum aestivum 13gcacgagcag
ccgcggagcc gccagagaca gacgctgccg ccgtcggccg tcctccgccc 60tccagttgag
cggttcaagc agagctgatg gctacatcag agccagtaca agagagtttg 120gtggtgcact
actcacaacc tagtaggccc cgggtcctcc ttgctgcttc aggaagtgta 180gccgctataa
aatttgagag cctttgccgt agcttctctg agtgggcgga tgtccgagct 240gtggccacca
cgccatcctt gcacttcgtt gatagatcat ctctaccaag tggcatcgtt 300ctttacactg
atgacgatga atggtctacc tggaagaaga taggagatga agtcttacac 360atcgagctgc
ggaaatgggc agacgttatg gtgatcgctc cattgtcagc aaataccctg 420gctaagatcg
ccggtgggtt atgcgacaac ctcttgacct gcatcgtgag agcgtgggac 480tacagcaaac
cgctctttgt tgccccagcc atgaacacgt taatgtggaa caacccattc 540acggagcggc
atcttcacac aatcaaccaa ctgggcatag ccttgatccc cccagttacc 600aaaaggctgg
cctgtggcga ttacgggaac ggcgcaatgg ccgaaacctc gcagatccat 660acttccgtga
ggctcgcgtg caagacgcaa ccgcacgatg cgagcagttc actcgcgggt 720cctgtcagta
ataaccggcc atctagctga tgcagcagtt ggccacttga ttgtcaagct 780taggaatttg
ttttatatgc agtgtgcatc tggagtgttg taacagattt tttttccaac 840tagtgtttgt
gtgtattgaa attggggggg aaaggctgtt gtcacaggat aacaataact 900tcctccctgc
tcagtaatta tgagtctatt cagtttgtaa ttgtcggtgg gagtacaatt 960atgctacaat
attgtttgtt tgtgtgtgtg tgggagttgg ggagtgctgc tgccacagag 1020atagcttcct
ccctgcccag ccagtaatga tagtgattat tcagtttgta gttgtcagtg 1080gaagtgtcca
gcaaactttt ctactaccct ttgatattcc tggtacaaat gttggaacga 1140ctgtcctaaa
aaaaaaaaaa aaaa
116414220PRTTriticum aestivum 14Met Ala Thr Ser Glu Pro Val Gln Glu Ser
Leu Val Val His Tyr Ser 1 5 10
15 Gln Pro Ser Arg Pro Arg Val Leu Leu Ala Ala Ser Gly Ser Val
Ala 20 25 30 Ala
Ile Lys Phe Glu Ser Leu Cys Arg Ser Phe Ser Glu Trp Ala Asp 35
40 45 Val Arg Ala Val Ala Thr
Thr Pro Ser Leu His Phe Val Asp Arg Ser 50 55
60 Ser Leu Pro Ser Gly Ile Val Leu Tyr Thr Asp
Asp Asp Glu Trp Ser 65 70 75
80 Thr Trp Lys Lys Ile Gly Asp Glu Val Leu His Ile Glu Leu Arg Lys
85 90 95 Trp Ala
Asp Val Met Val Ile Ala Pro Leu Ser Ala Asn Thr Leu Ala 100
105 110 Lys Ile Ala Gly Gly Leu Cys
Asp Asn Leu Leu Thr Cys Ile Val Arg 115 120
125 Ala Trp Asp Tyr Ser Lys Pro Leu Phe Val Ala Pro
Ala Met Asn Thr 130 135 140
Leu Met Trp Asn Asn Pro Phe Thr Glu Arg His Leu His Thr Ile Asn 145
150 155 160 Gln Leu Gly
Ile Ala Leu Ile Pro Pro Val Thr Lys Arg Leu Ala Cys 165
170 175 Gly Asp Tyr Gly Asn Gly Ala Met
Ala Glu Thr Ser Gln Ile His Thr 180 185
190 Ser Val Arg Leu Ala Cys Lys Thr Gln Pro His Asp Ala
Ser Ser Ser 195 200 205
Leu Ala Gly Pro Val Ser Asn Asn Arg Pro Ser Ser 210
215 220 151165DNAZea mays 15gcacgaggtc agatcgacct
ggactttggc ggcagctact tctagaacgc ggcggcacgg 60cagcaagcca gcaaggagaa
cgcggcggca cgggcgtgct gctacttctg cttcctctcc 120gacgcttgct gttgagcggt
tcaagcagag ctgatggcta catcagagcc agtacaagag 180agtttggtgg tgcactactc
acaacctagt aggccccggg tcctccttgc tgcttcagga 240agtgtagccg ctataaaatt
tgagagcctt tgccgtagct tctctgagtg ggcggatgtc 300cgagctgtgg ccaccacgcc
atccttgcac ttcgttgata gatcatctct accaagtggc 360atcgttcttt acactgatga
cgatgaatgg tctacctgga agaagatagg agatgaagtc 420ttacacatcg agctgcggaa
atgggcagac gttatggtga tcgctccatt gtcagcaaat 480accctggcta agatcgccgg
tgggttatgc gacaacctct tgacctgcat cgtgagagcg 540tgggactaca gcaaaccgct
ctttgttgcc ccagccatga acacgttaat gtggaacaac 600ccattcacgg agcggcatct
tcacacaatc aaccaactgg gcatagcctt gatcccccca 660gttaccaaaa ggctggcctg
tggcgattac gggaacggcg caatggccga aacctcgcag 720atccatactt ccgtgaggct
cgcgtgcaag acgcaaccgc acgatgcgag cagttcactc 780gcgggtcctg tcagtaataa
ccggccatct agctgatgca gcagttggcc acttgattgt 840caagcttagg aatttgtttt
atatgcagtg tgcatctgga gtgttgtaac agattttttt 900tccaactagt gtttgtgtgt
attgaaattg ggggggaaag gctgttgtca caggataaca 960ataacttcct ccctgctcag
taattatgag tctattcagt ttgtaattgt cggtgggagt 1020acaattatgc tacaatattg
tttgtttgtg tgtgtgtggg agttggggag tgctgctgcc 1080acagagatag cttcctccct
gcccagccag taatgatagt gattattcag tttgtaaaaa 1140aaaaaaaaaa aaaaaaaaaa
aaaaa 116516220PRTZea mays 16Met
Ala Thr Ser Glu Pro Val Gln Glu Ser Leu Val Val His Tyr Ser 1
5 10 15 Gln Pro Ser Arg Pro Arg
Val Leu Leu Ala Ala Ser Gly Ser Val Ala 20
25 30 Ala Ile Lys Phe Glu Ser Leu Cys Arg Ser
Phe Ser Glu Trp Ala Asp 35 40
45 Val Arg Ala Val Ala Thr Thr Pro Ser Leu His Phe Val Asp
Arg Ser 50 55 60
Ser Leu Pro Ser Gly Ile Val Leu Tyr Thr Asp Asp Asp Glu Trp Ser 65
70 75 80 Thr Trp Lys Lys Ile
Gly Asp Glu Val Leu His Ile Glu Leu Arg Lys 85
90 95 Trp Ala Asp Val Met Val Ile Ala Pro Leu
Ser Ala Asn Thr Leu Ala 100 105
110 Lys Ile Ala Gly Gly Leu Cys Asp Asn Leu Leu Thr Cys Ile Val
Arg 115 120 125 Ala
Trp Asp Tyr Ser Lys Pro Leu Phe Val Ala Pro Ala Met Asn Thr 130
135 140 Leu Met Trp Asn Asn Pro
Phe Thr Glu Arg His Leu His Thr Ile Asn 145 150
155 160 Gln Leu Gly Ile Ala Leu Ile Pro Pro Val Thr
Lys Arg Leu Ala Cys 165 170
175 Gly Asp Tyr Gly Asn Gly Ala Met Ala Glu Thr Ser Gln Ile His Thr
180 185 190 Ser Val
Arg Leu Ala Cys Lys Thr Gln Pro His Asp Ala Ser Ser Ser 195
200 205 Leu Ala Gly Pro Val Ser Asn
Asn Arg Pro Ser Ser 210 215 220
17631DNAPicea abiesmisc_feature(546)..(546)n is a, c, g, or t
17ggacgcgtgg gtactgtact gcatatagaa ctccggcaat gggctgatgc tatggtgatt
60gctccactat cagcaaacac gcttgctaag atagcaggtg gcctatgcga caatctactg
120acttgcataa tacgtgcatg ggactttaac aagcctctct ttgtagctcc tgcaatgaat
180actttcatgt ggaacaaccc atttactcaa cggcatttgg actctatctc ggagatggga
240gtatcactta tcccccctat aacaaagacg ttagcttgtg gtgattatgg aaatggtgca
300atgtcagaac cttcctcaat agatacaact cttaggtttt cactcgatcc ttcaattaaa
360tgaagtatct gatctccctt tctccaattc ttgttatgaa tctaacatgc ttagtactat
420ggactgtggt atagactcag attcgtagct gggctagaca gcatgaaccc agctcgtgtg
480tggctatgtt aagaaattct gatgataaac aatctcgaac ttatgattta tgtaacattt
540gttccnccat tctggacact tacaagaact gtgtggatac tcctgcttat aagaaaatgt
600gtcaatctac attgcatatc atctaatttg g
63118120PRTPicea abies 18Gly Arg Val Gly Thr Val Leu His Ile Glu Leu Arg
Gln Trp Ala Asp 1 5 10
15 Ala Met Val Ile Ala Pro Leu Ser Ala Asn Thr Leu Ala Lys Ile Ala
20 25 30 Gly Gly Leu
Cys Asp Asn Leu Leu Thr Cys Ile Ile Arg Ala Trp Asp 35
40 45 Phe Asn Lys Pro Leu Phe Val Ala
Pro Ala Met Asn Thr Phe Met Trp 50 55
60 Asn Asn Pro Phe Thr Gln Arg His Leu Asp Ser Ile Ser
Glu Met Gly 65 70 75
80 Val Ser Leu Ile Pro Pro Ile Thr Lys Thr Leu Ala Cys Gly Asp Tyr
85 90 95 Gly Asn Gly Ala
Met Ser Glu Pro Ser Ser Ile Asp Thr Thr Leu Arg 100
105 110 Phe Ser Leu Asp Pro Ser Ile Lys
115 120 19769DNABrassica napus 19gagatctgta
agggttttca aaaagagcca tggagaacgg gaaaagagac agagaagaca 60tggaagtgca
gctctcccct agaaagcccc gcgtactcct cgcagcaacc ggaagcgtcg 120ccgccatcaa
attcggcaac ctctgccact gcttcacaga gtgggcggaa gtgagagccg 180tcgtctcgaa
atcgtctctc cacttcctcg acaagctctc tctcccacag gaagtgactc 240tctacaccga
cgaagacgag tggtcgagct ggaacaagat cggcgatccc gtgcttcaca 300tcgagctcag
acgctgggct gacgtcatgg tcatcgctcc tttgtctgct aacactttag 360ccaagatagc
tggtgggatg tgtgataatc ttctgacttg tatcataaga gcttgggatt 420atagcaaacc
gcttttcgtt gcgccggcta tgaatacttt gatgtggaac aatcctttta 480cggagaggca
tcttttgtcg cttgatgagc ttggaatcac tcttattcct ccgatcaaga 540agaggttggc
ttgtggtgac tatggtaatg gcgcgatggc tgagccgtct cttatctatt 600ccactgttag
actcttctgg gagtctcagg ctcatcagca aagtggtgga actagttaat 660accatgatgg
ttttgcactt tgcaatggtt ggtcagtgtc atagattgtt ctgtctgaaa 720tgctcgtcct
tgtatatgtt aagaacagct gtctgggttg atgtctctt
76920209PRTBrassica napus 20Met Glu Asn Gly Lys Arg Asp Arg Glu Asp Met
Glu Val Gln Leu Ser 1 5 10
15 Pro Arg Lys Pro Arg Val Leu Leu Ala Ala Thr Gly Ser Val Ala Ala
20 25 30 Ile Lys
Phe Gly Asn Leu Cys His Cys Phe Thr Glu Trp Ala Glu Val 35
40 45 Arg Ala Val Val Ser Lys Ser
Ser Leu His Phe Leu Asp Lys Leu Ser 50 55
60 Leu Pro Gln Glu Val Thr Leu Tyr Thr Asp Glu Asp
Glu Trp Ser Ser 65 70 75
80 Trp Asn Lys Ile Gly Asp Pro Val Leu His Ile Glu Leu Arg Arg Trp
85 90 95 Ala Asp Val
Met Val Ile Ala Pro Leu Ser Ala Asn Thr Leu Ala Lys 100
105 110 Ile Ala Gly Gly Met Cys Asp Asn
Leu Leu Thr Cys Ile Ile Arg Ala 115 120
125 Trp Asp Tyr Ser Lys Pro Leu Phe Val Ala Pro Ala Met
Asn Thr Leu 130 135 140
Met Trp Asn Asn Pro Phe Thr Glu Arg His Leu Leu Ser Leu Asp Glu 145
150 155 160 Leu Gly Ile Thr
Leu Ile Pro Pro Ile Lys Lys Arg Leu Ala Cys Gly 165
170 175 Asp Tyr Gly Asn Gly Ala Met Ala Glu
Pro Ser Leu Ile Tyr Ser Thr 180 185
190 Val Arg Leu Phe Trp Glu Ser Gln Ala His Gln Gln Ser Gly
Gly Thr 195 200 205
Ser 21636DNABrassica oleraceavar. alboglabra 21gacgcgtggg cggacgcgtg
gggggttttc aaaaagagcc atggagaacg ggaaaagaga 60cagagaagac atggaagtgc
agctctcccc tagaaagccc cgcgtactcc tcgcagcaac 120cggaagcgtc gccgccatca
aattcggcaa cctctgccac tgcttcacag agtgggcgga 180agtgagagcc gtcgtctcga
aatcgtctct ccacttcctc gacaagctct ctctcccaca 240ggaagtgact ctctacaccg
acgaagacga gtggtcgagc tggaacaaga tcggcgatcc 300cgtgcttcac atcgagctca
gacgctgggc tgacgtcatg gtcatcgctc ctttgtctgc 360taacacttta gccaagatag
ctggtgggat gtgtgataat cttctgactt gtatcataag 420agcttgggat tatagcaaac
cgcttttcgt tgcgccggct atgaatactt tgatgtggaa 480caatcctttt acggagaggc
atcttttgtc gcttgatgag cttggaatca ctcttattcc 540tccgatcaag aagaggttgg
cttgtggtga ctatggtaat ggcgcgatgg ctgagccgtc 600tcttatctat tccactgtta
gnctcttctg ggagtc 63622198PRTBrassica
oleraceavar. alboglabra 22Met Glu Asn Gly Lys Arg Asp Arg Glu Asp Met Glu
Val Gln Leu Ser 1 5 10
15 Pro Arg Lys Pro Arg Val Leu Leu Ala Ala Thr Gly Ser Val Ala Ala
20 25 30 Ile Lys Phe
Gly Asn Leu Cys His Cys Phe Thr Glu Trp Ala Glu Val 35
40 45 Arg Ala Val Val Ser Lys Ser Ser
Leu His Phe Leu Asp Lys Leu Ser 50 55
60 Leu Pro Gln Glu Val Thr Leu Tyr Thr Asp Glu Asp Glu
Trp Ser Ser 65 70 75
80 Trp Asn Lys Ile Gly Asp Pro Val Leu His Ile Glu Leu Arg Arg Trp
85 90 95 Ala Asp Val Met
Val Ile Ala Pro Leu Ser Ala Asn Thr Leu Ala Lys 100
105 110 Ile Ala Gly Gly Met Cys Asp Asn Leu
Leu Thr Cys Ile Ile Arg Ala 115 120
125 Trp Asp Tyr Ser Lys Pro Leu Phe Val Ala Pro Ala Met Asn
Thr Leu 130 135 140
Met Trp Asn Asn Pro Phe Thr Glu Arg His Leu Leu Ser Leu Asp Glu 145
150 155 160 Leu Gly Ile Thr Leu
Ile Pro Pro Ile Lys Lys Arg Leu Ala Cys Gly 165
170 175 Asp Tyr Gly Asn Gly Ala Met Ala Glu Pro
Ser Leu Ile Tyr Ser Thr 180 185
190 Val Xaa Leu Phe Trp Glu 195
23958DNANicotiana tabacum 23ggagctgtgt tgccgcagaa ggagcaagaa ttgttggtgc
ttcaaggata attggtgttg 60atttaattcc tagcagattt gatttagatt agggtagata
tggagacttc agagatggaa 120ccggttcaga ttaacaatgc accgaggaga cctcgcattc
tgcttgcagc aagtggaagt 180gtggctgcta tcaagtttgc cagtctatgc cgttccttta
ctgattgggc tgaagttaaa 240gcggttgcta caaaagcttc tcttcatttc atagacaaag
cttcacttcc ggaagatgtc 300attctttata ctgatgagga tgaatggtca acttggacga
agataggtga ccgtgtgcta 360cacatcgagc tccggaggtg ggctgatatt atgattattg
cccctttgtc agcaaataca 420cttgggaaga ttgctggtgg actatgtgat aacttgttaa
cctgcatcgt acgagcatgg 480gactacgata aacccctttt cgtggcacca gcaatgaata
cattgatgtg gaataatcca 540ttcacagaaa agcaccttat ggcaattgat gagcttggga
tctctctcat accaccagta 600tcaaagagac tagcctgtgg agattacggg aatggagcaa
tggctgaacc gtctctgatc 660ttccaagctg taagactcta ttatgacgca caattacgat
caggtggcag caacgtggcg 720tgatccacag gtcatgaaat ttcattcatc ggtttgtaca
agtgatagaa gttgttgaaa 780ttcaggacca ggagcccagc tgtgttattt ttctcctaaa
ttcttcacct ccctagtatc 840tttcttgtgt cctagagcta ctttcaatca aggttcatgt
tcattttagt cgataaaatg 900agaatatcac gtaactgctg ttactaatgg atgtgatcat
gatcatgaac atgaaaaa 95824207PRTNicotiana tabacum 24Met Glu Thr Ser
Glu Met Glu Pro Val Gln Ile Asn Asn Ala Pro Arg 1 5
10 15 Arg Pro Arg Ile Leu Leu Ala Ala Ser
Gly Ser Val Ala Ala Ile Lys 20 25
30 Phe Ala Ser Leu Cys Arg Ser Phe Thr Asp Trp Ala Glu Val
Lys Ala 35 40 45
Val Ala Thr Lys Ala Ser Leu His Phe Ile Asp Lys Ala Ser Leu Pro 50
55 60 Glu Asp Val Ile Leu
Tyr Thr Asp Glu Asp Glu Trp Ser Thr Trp Thr 65 70
75 80 Lys Ile Gly Asp Arg Val Leu His Ile Glu
Leu Arg Arg Trp Ala Asp 85 90
95 Ile Met Ile Ile Ala Pro Leu Ser Ala Asn Thr Leu Gly Lys Ile
Ala 100 105 110 Gly
Gly Leu Cys Asp Asn Leu Leu Thr Cys Ile Val Arg Ala Trp Asp 115
120 125 Tyr Asp Lys Pro Leu Phe
Val Ala Pro Ala Met Asn Thr Leu Met Trp 130 135
140 Asn Asn Pro Phe Thr Glu Lys His Leu Met Ala
Ile Asp Glu Leu Gly 145 150 155
160 Ile Ser Leu Ile Pro Pro Val Ser Lys Arg Leu Ala Cys Gly Asp Tyr
165 170 175 Gly Asn
Gly Ala Met Ala Glu Pro Ser Leu Ile Phe Gln Ala Val Arg 180
185 190 Leu Tyr Tyr Asp Ala Gln Leu
Arg Ser Gly Gly Ser Asn Val Ala 195 200
205 251016DNASolanum tuberosum 25gaattgatcg gcgccggact
tcgatcaccg tcggcgtttc taagccgtcg ccggaactaa 60gccggagaaa atctcaatca
agtgagctaa gcactcggcg tttcaacttc tggttataaa 120tttattaaag cctttgctcc
attaggttag gtagatacgg atcctatgac ttcagagatg 180gaaccagttc agattaatgg
tgcacctagg agacctcgta ttctgctggc agcaagtgga 240agtgtggctg caattaagtt
tgcaaatcta tgtggttgtt tttctgaatg ggcagaagtt 300aaagcagttg caacaaaacc
ttctcttcat ttcatagaca aagcttcact tccggaagat 360gccattctat atactgatga
ggaggaatgg tccacttgga agaaaattgg tgatagtgtg 420ctacacattg agctccgcag
gtgggctgat attatggtta ttgccccttt gtcagcaaac 480acacttggga agattgcagg
tggactatgt gataacttgt taacctgcat cgtacgagca 540tgggactaca ataaacccct
ttttgtggca ccagccatga atacattgat gtggaataat 600ccattcacag aacgacacct
tatggtaatt gatgagcttg gaatctctct cataccacca 660gtttctaaaa gactagcttg
tggagattat ggaaacggcg ctatggctga accttctctc 720atctactcaa ctgtaagact
cttctatgag tcacggtcac aatcaggtgg catcaacttg 780gcttgatcca cggatcatta
aattttattc gtcggtttgt acaagtggta gaaattgttg 840aaattcagga cctggaacag
tgttacttag catacaccag ttgtgtttat ttttctcctt 900aattagtgtc atgtttgtat
ccaagagctg actttcaatc aagtttcatg ttcattgtag 960tctattgact ctgaatatta
tgcaactcta tatagtaccc gctactaatt atgtat 101626206PRTSolanum
tuberosum 26Met Thr Ser Glu Met Glu Pro Val Gln Ile Asn Gly Ala Pro Arg
Arg 1 5 10 15 Pro
Arg Ile Leu Leu Ala Ala Ser Gly Ser Val Ala Ala Ile Lys Phe
20 25 30 Ala Asn Leu Cys Gly
Cys Phe Ser Glu Trp Ala Glu Val Lys Ala Val 35
40 45 Ala Thr Lys Pro Ser Leu His Phe Ile
Asp Lys Ala Ser Leu Pro Glu 50 55
60 Asp Ala Ile Leu Tyr Thr Asp Glu Glu Glu Trp Ser Thr
Trp Lys Lys 65 70 75
80 Ile Gly Asp Ser Val Leu His Ile Glu Leu Arg Arg Trp Ala Asp Ile
85 90 95 Met Val Ile Ala
Pro Leu Ser Ala Asn Thr Leu Gly Lys Ile Ala Gly 100
105 110 Gly Leu Cys Asp Asn Leu Leu Thr Cys
Ile Val Arg Ala Trp Asp Tyr 115 120
125 Asn Lys Pro Leu Phe Val Ala Pro Ala Met Asn Thr Leu Met
Trp Asn 130 135 140
Asn Pro Phe Thr Glu Arg His Leu Met Val Ile Asp Glu Leu Gly Ile 145
150 155 160 Ser Leu Ile Pro Pro
Val Ser Lys Arg Leu Ala Cys Gly Asp Tyr Gly 165
170 175 Asn Gly Ala Met Ala Glu Pro Ser Leu Ile
Tyr Ser Thr Val Arg Leu 180 185
190 Phe Tyr Glu Ser Arg Ser Gln Ser Gly Gly Ile Asn Leu Ala
195 200 205 27967DNAGlycine max
27ggttgaacag aagagattta ggcagcccga accgacgatg ttgtttttaa ctgacagcag
60cacaaagcga acactaacac taacgtgaaa cgcgttgcgt tgtcggaaaa tcaccctcac
120ctgatcactg tggaagtctc taggtgatgg ccggttcaga acctgttagg gcagagggag
180agactatggc tgtggatgct gccccaagga agccccggat tctacttgct gctagtggga
240gtgttgctgc tgtcaaattt gcaaatcttt gtcactgttt ctctgaatgg gcagatgtaa
300gagcagtttc cacaagtgca tctttgcatt tcattgatag agcagcaatg cccaaggatg
360taattctata cacggatgac aatgaatggt ctagttggaa gaaattaggt gatagcgtgc
420ttcacattga gcttcgcaaa tgggctgata tcatggtcat cgctccatta tcagcaaaca
480cccttggcaa gattgctgga gggttgtgtg acaatctact gacatgcatc gttcgagcct
540gggactacag caagccattc tttgttgcac cagccatgaa cactttgatg tggaacaatc
600ctttcactga gcggcatttc atctccattg atgagcttgg catttctctc atcccgcctg
660ttacaaagag gttagcttgt ggggattatg gcaatggtgc catggctgaa ccctctacca
720tttactcaac tgtaaggctc ttctatgagt caaaggctca gcaaggtaga gctgtggtaa
780ccttaaggtg aatgaagggg tagtcctacg cccccattca tggtagtcaa tagcactcta
840tagcaggata acggagcgca gcagccctga gttactatgg aagtcgaaat cgctgagcga
900ttttcaataa ccgctgtagc ggctgcaata gtggtctaaa tacagctttt cgggtgctac
960agcgcac
96728214PRTGlycine max 28Met Ala Gly Ser Glu Pro Val Arg Ala Glu Gly Glu
Thr Met Ala Val 1 5 10
15 Asp Ala Ala Pro Arg Lys Pro Arg Ile Leu Leu Ala Ala Ser Gly Ser
20 25 30 Val Ala Ala
Val Lys Phe Ala Asn Leu Cys His Cys Phe Ser Glu Trp 35
40 45 Ala Asp Val Arg Ala Val Ser Thr
Ser Ala Ser Leu His Phe Ile Asp 50 55
60 Arg Ala Ala Met Pro Lys Asp Val Ile Leu Tyr Thr Asp
Asp Asn Glu 65 70 75
80 Trp Ser Ser Trp Lys Lys Leu Gly Asp Ser Val Leu His Ile Glu Leu
85 90 95 Arg Lys Trp Ala
Asp Ile Met Val Ile Ala Pro Leu Ser Ala Asn Thr 100
105 110 Leu Gly Lys Ile Ala Gly Gly Leu Cys
Asp Asn Leu Leu Thr Cys Ile 115 120
125 Val Arg Ala Trp Asp Tyr Ser Lys Pro Phe Phe Val Ala Pro
Ala Met 130 135 140
Asn Thr Leu Met Trp Asn Asn Pro Phe Thr Glu Arg His Phe Ile Ser 145
150 155 160 Ile Asp Glu Leu Gly
Ile Ser Leu Ile Pro Pro Val Thr Lys Arg Leu 165
170 175 Ala Cys Gly Asp Tyr Gly Asn Gly Ala Met
Ala Glu Pro Ser Thr Ile 180 185
190 Tyr Ser Thr Val Arg Leu Phe Tyr Glu Ser Lys Ala Gln Gln Gly
Arg 195 200 205 Ala
Val Val Thr Leu Arg 210 291067DNAVitis vinifera
29ttgggagaga tgccacggcc ctcattcgct ctcgatcccc gacctcggct tcactctctc
60taattttccc gagcaattct ccgagttgag gctgatttga aagttaatga tgatgacata
120tgcagaacct ttgagtccag aagttgatgc gataccagtc aacattgctc ccagaagacc
180ccggattcta cttgctgcta gtgggagtgt agctgctatg aagtttggga atctcgtcca
240ttctttttgt gaatgggcag aagtaagagc agttgtcaca aaggcttctt tacacttcat
300tgatagagca gcactgccta aggatttata tctttacact gatgatgatg aatggtccag
360ttggacaaaa ttaggagaca gtgtgcttca cattgagctc cgcaggtggg ctgatgtcat
420ggtaatcgcc ccattatcag caaatacact tggcaagatt gccgggggac tgtgtgacaa
480cctgctgaca tgcattgtgc gagcgtggga ctacagcaag ccaatgtttg ttgcgccagc
540tatgaacacc ttcatgtgga ccaatccttt cacagaacgc catcttatga caattgatga
600acttggaatt tctcttattc cacctgtcac taaaaggctg gcttgcggag attatgggac
660tggtgcaatg gctgaacctt ttctcatcca ctcaaccgta agactcttct tggagacacg
720ggctcaatca agtagcagta atgtgcagta attgggtatg gttaatctgt ctgttggaga
780agtcaccaga gagttggctg aaatggcatg ttggaaatgc taaacgtagt tcacttggac
840cgcattgatt ttgttatgac cgtccattaa acttactacg tcttgtagat tgttggtatc
900ggtggatttg catatcctgt ttctcaatat ttctagtagc gccttttgaa tgttatgtgc
960cttgtgtatt aatggtgagg gagaatgtat cagtctccta aatttctcaa tctctgtgta
1020gacatgcttg atgatacatt cctataaata gactctgatt tctgggc
106730214PRTVitis vinifera 30Met Met Met Thr Tyr Ala Glu Pro Leu Ser Pro
Glu Val Asp Ala Ile 1 5 10
15 Pro Val Asn Ile Ala Pro Arg Arg Pro Arg Ile Leu Leu Ala Ala Ser
20 25 30 Gly Ser
Val Ala Ala Met Lys Phe Gly Asn Leu Val His Ser Phe Cys 35
40 45 Glu Trp Ala Glu Val Arg Ala
Val Val Thr Lys Ala Ser Leu His Phe 50 55
60 Ile Asp Arg Ala Ala Leu Pro Lys Asp Leu Tyr Leu
Tyr Thr Asp Asp 65 70 75
80 Asp Glu Trp Ser Ser Trp Thr Lys Leu Gly Asp Ser Val Leu His Ile
85 90 95 Glu Leu Arg
Arg Trp Ala Asp Val Met Val Ile Ala Pro Leu Ser Ala 100
105 110 Asn Thr Leu Gly Lys Ile Ala Gly
Gly Leu Cys Asp Asn Leu Leu Thr 115 120
125 Cys Ile Val Arg Ala Trp Asp Tyr Ser Lys Pro Met Phe
Val Ala Pro 130 135 140
Ala Met Asn Thr Phe Met Trp Thr Asn Pro Phe Thr Glu Arg His Leu 145
150 155 160 Met Thr Ile Asp
Glu Leu Gly Ile Ser Leu Ile Pro Pro Val Thr Lys 165
170 175 Arg Leu Ala Cys Gly Asp Tyr Gly Thr
Gly Ala Met Ala Glu Pro Phe 180 185
190 Leu Ile His Ser Thr Val Arg Leu Phe Leu Glu Thr Arg Ala
Gln Ser 195 200 205
Ser Ser Ser Asn Val Gln 210 311081DNAHordeum vulgare
31cggcacgagg ctccccaatt ccctccgccc gcggcgcgcc gctccgggca gcctcggtcg
60ccgccgccgc cgccgctgcc tccacctacc gccggccgac gacgcccttc gaacctaccg
120cctccgccgc catcttcaac cgcttatttg cctgcgctcc taccagatcg cctctgagtt
180gagggctcca agtgaagcag atggctacat cagaaccggt acagcaaagc tgggagctgg
240aatccagcag gcctcgggtc ctccttgctg cgtcagggag tgtagctgct ataaaattcg
300agagcctctg ccgtatcttc tccgagtggg cggaagtccg agctgtggcg accaagtcag
360cattgcactt tgttgacaga tcatctctgc caagcgacgt cgtcctttac actgatgatg
420atgagtggtc tacctggaca aagataggag acgaggttct gcacatagag ctgcgaaagt
480gggcagacat catggtgatc gcccccttat cagcaaacac tctggccaag atcgccggcg
540ggttatgcga caacctcctg acgtgcatta tccgagcgtg ggactacaag aagccgatct
600tcgccgcgcc agccatgaac accttcatgt ggaacaaccc attcacggcg cgccacatcg
660agaccatgaa ccaactgggc atctccctgg tcccgcccac cacgaaacgg ctggcctgcg
720gcgactacgg gaacggcgcg atggctgagc cctcgcagat ccacacgact gtgaggctcg
780cgtgcaagtc gcagacgttt ggcacgggca tttcgcccgc gcacccttcc agcggccacc
840ccgtctagcc gatgcggtga tggtcactat gttcaggtgg tctagtactc gaggtttttc
900tatgccgccc acatcatcag ggtttgagaa agtgaagggt atcaccaggt gttctgttca
960tcggtggtgt ctgtattaac cgaagtatcg tacctgtggg atatggatca gcttatttgt
1020tatgtggtag tagacgttag ggcgtagacg tagagattgg ggaattgctg ttgctccagc
1080c
108132215PRTHordeum vulgare 32Met Ala Thr Ser Glu Pro Val Gln Gln Ser Trp
Glu Leu Glu Ser Ser 1 5 10
15 Arg Pro Arg Val Leu Leu Ala Ala Ser Gly Ser Val Ala Ala Ile Lys
20 25 30 Phe Glu
Ser Leu Cys Arg Ile Phe Ser Glu Trp Ala Glu Val Arg Ala 35
40 45 Val Ala Thr Lys Ser Ala Leu
His Phe Val Asp Arg Ser Ser Leu Pro 50 55
60 Ser Asp Val Val Leu Tyr Thr Asp Asp Asp Glu Trp
Ser Thr Trp Thr 65 70 75
80 Lys Ile Gly Asp Glu Val Leu His Ile Glu Leu Arg Lys Trp Ala Asp
85 90 95 Ile Met Val
Ile Ala Pro Leu Ser Ala Asn Thr Leu Ala Lys Ile Ala 100
105 110 Gly Gly Leu Cys Asp Asn Leu Leu
Thr Cys Ile Ile Arg Ala Trp Asp 115 120
125 Tyr Lys Lys Pro Ile Phe Ala Ala Pro Ala Met Asn Thr
Phe Met Trp 130 135 140
Asn Asn Pro Phe Thr Ala Arg His Ile Glu Thr Met Asn Gln Leu Gly 145
150 155 160 Ile Ser Leu Val
Pro Pro Thr Thr Lys Arg Leu Ala Cys Gly Asp Tyr 165
170 175 Gly Asn Gly Ala Met Ala Glu Pro Ser
Gln Ile His Thr Thr Val Arg 180 185
190 Leu Ala Cys Lys Ser Gln Thr Phe Gly Thr Gly Ile Ser Pro
Ala His 195 200 205
Pro Ser Ser Gly His Pro Val 210 215
331175DNAGossypium hirsutum 33cccattgtca cctttagtgc tggtgagcat ttaagagagg
gcagagcaac ggaggaaatc 60cccttttttg tgcagtggaa gtaaaaatta cctaccatta
gaaaaggaga aggattggta 120aagtattgca gcgctccgta aaggcatttt ggtatagcaa
gtagcagcaa ttcatcaaaa 180ggcagcattt ctttttgcac caggggccct ttaaaaagct
caactctcgc gcttctttgc 240gtttcaaatc tctctgcaag tcgcaaggac atctgaagca
gaaagcccag attcctctct 300aatcttagat tccagatttg cataggttat ggcgtatcct
gagcctcaag gtgcagatag 360ggagatggtt aaggtcaatc ctaccccaag aaaaccccgg
gttttactcg ctgccagtgg 420aagtgtagct gccataaagt ttgggaatct ctgccattgt
ttctctgaat gggcagaagt 480aaaagcagtt gccacgaaag cttctttgca tttcattgac
agagcatcac ttcctaagga 540tctaaagctt tacactgatg aggaggaatg gtctagttgg
gggaaaatag gtgacagtgt 600gcttcacatt gagcttcgtc gatgggctga tattatggtc
attgccccat tgtcagcaaa 660cacacttggc aagattgctg gaggattatg tgacaatttg
ttaacttgtg tcgtacgagc 720atgggactac agcaagccaa tgtttgttgc accagctatg
aacactttca tgtggagcaa 780ccctttcaca gaaaagcatc tcatgacaat tgatgagctt
ggtatttctc tcatcccccc 840tgtctccaaa agactagctt gtggggacta tggaaacggc
gcaatggcag aaccttctct 900aatccactcg actgtaagat tattcttgga gtcacgacct
caaccaagtg actgaagatc 960tatttattcg ccatgaatta taaatactat attagttgta
tggtagccca gttgactcta 1020ggttgggtgt atgtctatta gctgtctaac aagcttattg
tacatttata gttgcatttc 1080cgagtttgct taactttgca tatcatgagg agtggtcttt
gaatactgct gaaaatttga 1140tcctgtaagc tgatgatact gatgagagtt ggcag
117534208PRTGossypium hirsutum 34Met Ala Tyr Pro
Glu Pro Gln Gly Ala Asp Arg Glu Met Val Lys Val 1 5
10 15 Asn Pro Thr Pro Arg Lys Pro Arg Val
Leu Leu Ala Ala Ser Gly Ser 20 25
30 Val Ala Ala Ile Lys Phe Gly Asn Leu Cys His Cys Phe Ser
Glu Trp 35 40 45
Ala Glu Val Lys Ala Val Ala Thr Lys Ala Ser Leu His Phe Ile Asp 50
55 60 Arg Ala Ser Leu Pro
Lys Asp Leu Lys Leu Tyr Thr Asp Glu Glu Glu 65 70
75 80 Trp Ser Ser Trp Gly Lys Ile Gly Asp Ser
Val Leu His Ile Glu Leu 85 90
95 Arg Arg Trp Ala Asp Ile Met Val Ile Ala Pro Leu Ser Ala Asn
Thr 100 105 110 Leu
Gly Lys Ile Ala Gly Gly Leu Cys Asp Asn Leu Leu Thr Cys Val 115
120 125 Val Arg Ala Trp Asp Tyr
Ser Lys Pro Met Phe Val Ala Pro Ala Met 130 135
140 Asn Thr Phe Met Trp Ser Asn Pro Phe Thr Glu
Lys His Leu Met Thr 145 150 155
160 Ile Asp Glu Leu Gly Ile Ser Leu Ile Pro Pro Val Ser Lys Arg Leu
165 170 175 Ala Cys
Gly Asp Tyr Gly Asn Gly Ala Met Ala Glu Pro Ser Leu Ile 180
185 190 His Ser Thr Val Arg Leu Phe
Leu Glu Ser Arg Pro Gln Pro Ser Asp 195 200
205 351119DNASorghum bicolor 35gcacgagggc
tctctccgct cacctccact tccccgcccc cgccccggtc tccgtccttg 60acacgggcgc
gcacccgtcc gcctccgact gacccggagc cgactcgacc ttgttcagga 120gggagggaga
gcccgaaccg cggcggggcg ggagggctcc ttttcgtttg ccccgccgga 180gggccagccg
ccccatgggc cggtcgcctg agcgcgctac gtatcagctc atcgaccggc 240ggaaagctga
gcgttagcgg agctgatggc tacatcagag ccagtacaag agaatttggc 300gatggactac
tcacaaccta gtaagcctcg ggtactcctt gctgcttcag gaagtgtagc 360cgctataaaa
tttgagaacc tttgtcgtag tttctctgag tgggcggatg tccgagccgt 420ggccaccgca
tcatctttgc actttattga tagatcatct cttccaagtg acattgttct 480ttacactgat
gatgatgagt ggtctacctg gaagaagata ggagatgaag ttttacacat 540tgagctgcgt
aaatgggcag atattatggt gattgctccg ttatcagcaa ataccctggc 600taagatcgcc
ggtgggttat gtgacaacct cttaacatgc atcgtgagag cgtgggacta 660cagcaaaccg
ctctttgttg ccccagctat gaacacatta atgtggaaca acccattcac 720agagcgtcat
cttcaaacga tcaaccaact gggcataatc ttgatccccc cagttaccaa 780aaggttggct
tgtggcgatc acgggaatgg tgcaatggct gaaacctcgc agatctatac 840ttctgtgagg
cttgcatgga agacgcaacc acatgatgca agcagttcac ttgtggttcc 900tgtcagtaat
aaccgcccat ctagctggtg tttgacctct taattaagag cttaggaatt 960tgttttatct
gcagtgtgca tcttgatgtt agaaatgttg taccaatttt ttttaaccag 1020tgttttagtg
tgtattgtga tgagaatgtg gggaaaggaa ggttgtcagt tgtcacggtg 1080ataaattact
gccagctcag tatcgatagt gaggatgag
111936225PRTSorghum bicolor 36Met Ala Thr Ser Glu Pro Val Gln Glu Asn Leu
Ala Met Asp Tyr Ser 1 5 10
15 Gln Pro Ser Lys Pro Arg Val Leu Leu Ala Ala Ser Gly Ser Val Ala
20 25 30 Ala Ile
Lys Phe Glu Asn Leu Cys Arg Ser Phe Ser Glu Trp Ala Asp 35
40 45 Val Arg Ala Val Ala Thr Ala
Ser Ser Leu His Phe Ile Asp Arg Ser 50 55
60 Ser Leu Pro Ser Asp Ile Val Leu Tyr Thr Asp Asp
Asp Glu Trp Ser 65 70 75
80 Thr Trp Lys Lys Ile Gly Asp Glu Val Leu His Ile Glu Leu Arg Lys
85 90 95 Trp Ala Asp
Ile Met Val Ile Ala Pro Leu Ser Ala Asn Thr Leu Ala 100
105 110 Lys Ile Ala Gly Gly Leu Cys Asp
Asn Leu Leu Thr Cys Ile Val Arg 115 120
125 Ala Trp Asp Tyr Ser Lys Pro Leu Phe Val Ala Pro Ala
Met Asn Thr 130 135 140
Leu Met Trp Asn Asn Pro Phe Thr Glu Arg His Leu Gln Thr Ile Asn 145
150 155 160 Gln Leu Gly Ile
Ile Leu Ile Pro Pro Val Thr Lys Arg Leu Ala Cys 165
170 175 Gly Asp His Gly Asn Gly Ala Met Ala
Glu Thr Ser Gln Ile Tyr Thr 180 185
190 Ser Val Arg Leu Ala Trp Lys Thr Gln Pro His Asp Ala Ser
Ser Ser 195 200 205
Leu Val Val Pro Val Ser Asn Asn Arg Pro Ser Ser Trp Cys Leu Thr 210
215 220 Ser 225
37975DNALycopersicon esculentum 37ctacaattga tcggcgccgg actttgatca
ccgtcggcgt ttctaagccg ttgccggact 60atgccggagc aaatctcaat caagtgagct
aaccactctg tgtttcaact tctggttata 120aattttttaa agcctttgct ccgttaggtt
aggtagatac ggatcctatg acttcagaga 180tggaaccagt tcagattaat ggtgcaccta
ggagacctcg tattctgctg gcagcaagtg 240gaagtgtggc ttcaattaag tttgctaatc
tatgtcgttg tttttctgaa tgggcagaag 300ttaaagcagt tgcaacgaaa ccttctcttc
atttcataga caaagcttcg cttccggaag 360atgtcattct ttatactgat gaggaggaat
ggtccacttg gtgcagattg gtgatagtgt 420gctacacatt gagctccgca gatgggctga
tattatggtt attgcccctt tgtcagcaaa 480cacacttggg aagattgcag gtggactatg
tgataacttg ttaacctgca tcgtacgagc 540atgggactac aataaacccc tttttgtggc
accagccatg aatacattga tgtggaataa 600tccattcaca gaacgacacc ttatggtaat
tgatgagctt ggaatctctc tcataccccc 660agtttcaaaa agactagcat gtggagatta
tggaaatggc gctatggctg aaccttctct 720catttactca actgtaagac tcttttatga
gtcacggtca caatcaggtg gcatcaactt 780ggcttgattc gcagatcatt aaattttatt
catcggtttg tacaagtggt aaaaattgtt 840gaaattcagg acctggaaca gtgttactta
gcatacacca gttgggttta tttttctcct 900aaattcttca cctctttaac ttgtgtcatg
tttgtatcca agcggtaact ttcaatcaag 960tttcatgttc attgt
97538206PRTLycopersicon
esculentumUNSURE(74)..(74)Xaa can be any naturally occurring amino acid
38Met Thr Ser Glu Met Glu Pro Val Gln Ile Asn Gly Ala Pro Arg Arg 1
5 10 15 Pro Arg Ile Leu
Leu Ala Ala Ser Gly Ser Val Ala Ser Ile Lys Phe 20
25 30 Ala Asn Leu Cys Arg Cys Phe Ser Glu
Trp Ala Glu Val Lys Ala Val 35 40
45 Ala Thr Lys Pro Ser Leu His Phe Ile Asp Lys Ala Ser Leu
Pro Glu 50 55 60
Asp Val Ile Leu Tyr Thr Asp Glu Glu Xaa Met Val His Leu Val Gln 65
70 75 80 Ile Gly Asp Ser Val
Leu His Ile Glu Leu Arg Arg Trp Ala Asp Ile 85
90 95 Met Val Ile Ala Pro Leu Ser Ala Asn Thr
Leu Gly Lys Ile Ala Gly 100 105
110 Gly Leu Cys Asp Asn Leu Leu Thr Cys Ile Val Arg Ala Trp Asp
Tyr 115 120 125 Asn
Lys Pro Leu Phe Val Ala Pro Ala Met Asn Thr Leu Met Trp Asn 130
135 140 Asn Pro Phe Thr Glu Arg
His Leu Met Val Ile Asp Glu Leu Gly Ile 145 150
155 160 Ser Leu Ile Pro Pro Val Ser Lys Arg Leu Ala
Cys Gly Asp Tyr Gly 165 170
175 Asn Gly Ala Met Ala Glu Pro Ser Leu Ile Tyr Ser Thr Val Arg Leu
180 185 190 Phe Tyr
Glu Ser Arg Ser Gln Ser Gly Gly Ile Asn Leu Ala 195
200 205 391395DNAArabidopsis thaliana 39atggcgaaaa
cagctcttac tcctcccgcg tctggttccg aagttccaag atccggtaca 60cctggagatg
cgtctggcaa caaacctcaa acggatgcca ccggcgtctc agctactgat 120actgcttctc
agaaacgcgg tcgtggtcga ccgccaaagg ctaaatctga ctcttcccaa 180atcggtgccg
tttctgcgaa ggcgagtact aaaccaagtg gtcgtccgaa aagaaacgta 240gctcaggctg
ttccttctac gtctgtggcg gcggctgtga agaaacgtgg tagggcaaag 300aggtcgactg
taacggcggc tgtggttact actgctactg gagagggttc tagaaaacga 360gggaggccaa
agaaagatga cgtggcggct gcaactgttc cagcagaaac tgtggtggct 420ccagctaaga
gacgtggaag gaaacctact gtcgaagtag ctgcacagcc tgtgcgcagg 480actaggaagg
tattcgggtt ttctatgcat gaacaaaaga gcacttcagt ggcaccggta 540gctgcaaacg
tcggagatct caagaaaaga accgcactct tacaaaagaa agtgaaggaa 600gctgcagcta
agttgaaaca agcagtaaca gcaattgacg aggtccagaa gttagcggat 660ggaatattga
ccagcgatga cgtcgacttc tctgttttgt tttcaaactt aaagaccctc 720gcggcatttg
atttcgattt ccgattaggg ttcctcgcag atcctccttc ctcggctata 780gagaagaaga
tgaatatgga agtggataca gtaacaagga agcctcgtat cttactagct 840gcaagtggaa
gtgtggcttc aattaagttc agtaatctct gccattgttt ctcagaatgg 900gctgaagtca
aagccgtcgc ttcaaaatca tctctcaatt tcgttgataa accttctcta 960cctcagaatg
tgactctcta tacagatgaa gatgaatggt ctagctggaa caagattggt 1020gatcccgttc
ttcatatcga gctcagacgc tgggctgatg ttatgatcat tgctcctttg 1080tctgctaaca
cattagccaa gattgctggt gggttatgtg ataatctatt gacatgtata 1140gtaagagcat
gggattatag caaaccgttg tttgttgcac cggcgatgaa cactttgatg 1200tggaacaatc
ctttcacaga acggcacctt gtcttgcttg atgaacttgg aatcacccta 1260attcctccca
tcaagaagaa actggcctgt ggagactacg gtaatggcgc aatggctgag 1320ccttctctga
tttattccac tgttagactg ttctgggagt cacaagctcg taaacaaaga 1380gatggaacca
gttga
139540464PRTArabidopsis thaliana 40Met Ala Lys Thr Ala Leu Thr Pro Pro
Ala Ser Gly Ser Glu Val Pro 1 5 10
15 Arg Ser Gly Thr Pro Gly Asp Ala Ser Gly Asn Lys Pro Gln
Thr Asp 20 25 30
Ala Thr Gly Val Ser Ala Thr Asp Thr Ala Ser Gln Lys Arg Gly Arg
35 40 45 Gly Arg Pro Pro
Lys Ala Lys Ser Asp Ser Ser Gln Ile Gly Ala Val 50
55 60 Ser Ala Lys Ala Ser Thr Lys Pro
Ser Gly Arg Pro Lys Arg Asn Val 65 70
75 80 Ala Gln Ala Val Pro Ser Thr Ser Val Ala Ala Ala
Val Lys Lys Arg 85 90
95 Gly Arg Ala Lys Arg Ser Thr Val Thr Ala Ala Val Val Thr Thr Ala
100 105 110 Thr Gly Glu
Gly Ser Arg Lys Arg Gly Arg Pro Lys Lys Asp Asp Val 115
120 125 Ala Ala Ala Thr Val Pro Ala Glu
Thr Val Val Ala Pro Ala Lys Arg 130 135
140 Arg Gly Arg Lys Pro Thr Val Glu Val Ala Ala Gln Pro
Val Arg Arg 145 150 155
160 Thr Arg Lys Val Phe Gly Phe Ser Met His Glu Gln Lys Ser Thr Ser
165 170 175 Val Ala Pro Val
Ala Ala Asn Val Gly Asp Leu Lys Lys Arg Thr Ala 180
185 190 Leu Leu Gln Lys Lys Val Lys Glu Ala
Ala Ala Lys Leu Lys Gln Ala 195 200
205 Val Thr Ala Ile Asp Glu Val Gln Lys Leu Ala Asp Gly Ile
Leu Thr 210 215 220
Ser Asp Asp Val Asp Phe Ser Val Leu Phe Ser Asn Leu Lys Thr Leu 225
230 235 240 Ala Ala Phe Asp Phe
Asp Phe Arg Leu Gly Phe Leu Ala Asp Pro Pro 245
250 255 Ser Ser Ala Ile Glu Lys Lys Met Asn Met
Glu Val Asp Thr Val Thr 260 265
270 Arg Lys Pro Arg Ile Leu Leu Ala Ala Ser Gly Ser Val Ala Ser
Ile 275 280 285 Lys
Phe Ser Asn Leu Cys His Cys Phe Ser Glu Trp Ala Glu Val Lys 290
295 300 Ala Val Ala Ser Lys Ser
Ser Leu Asn Phe Val Asp Lys Pro Ser Leu 305 310
315 320 Pro Gln Asn Val Thr Leu Tyr Thr Asp Glu Asp
Glu Trp Ser Ser Trp 325 330
335 Asn Lys Ile Gly Asp Pro Val Leu His Ile Glu Leu Arg Arg Trp Ala
340 345 350 Asp Val
Met Ile Ile Ala Pro Leu Ser Ala Asn Thr Leu Ala Lys Ile 355
360 365 Ala Gly Gly Leu Cys Asp Asn
Leu Leu Thr Cys Ile Val Arg Ala Trp 370 375
380 Asp Tyr Ser Lys Pro Leu Phe Val Ala Pro Ala Met
Asn Thr Leu Met 385 390 395
400 Trp Asn Asn Pro Phe Thr Glu Arg His Leu Val Leu Leu Asp Glu Leu
405 410 415 Gly Ile Thr
Leu Ile Pro Pro Ile Lys Lys Lys Leu Ala Cys Gly Asp 420
425 430 Tyr Gly Asn Gly Ala Met Ala Glu
Pro Ser Leu Ile Tyr Ser Thr Val 435 440
445 Arg Leu Phe Trp Glu Ser Gln Ala Arg Lys Gln Arg Asp
Gly Thr Ser 450 455 460
411182DNAPinus sp. 41atattgaaac tcatttttaa acacttattc tgcactataa
gaagcagcag gcccccagaa 60atatgagctt catacacaca acgggggagt aatcagcacc
atcaaagggg ggatttgcgg 120caattgtgag atcaagtgga ggaagctgga atagatctaa
atctaggccc ttgaggagaa 180tgctctccta atggtggttt taaaaatcaa tactctagtg
caaaacatgg atgcaacaaa 240ttctcaacct gatgaacctg gaagggatgc taattcatct
cagaaaccac gaatcattct 300agcagccagc gggagtgtgg cagcaataaa atttggaatt
cttgcccatt gtctatgtca 360atgggcagaa gtcaaagcag tggtcacaaa atctgctttg
catttcattg acaagatgtc 420tcttccggct aatgttaagc tctacactga tgaaaatgaa
tggtctagct ggagcaaaat 480aggtgatact gtactgcata tagaactccg gcaatgggct
gatgctatgg tgattgctcc 540actctcagca aacacgcttg ctaagatagc aggtggccta
tgcgacaatc tacttacttg 600catagtacgt gcatgggact ttaacaagcc tctctttgta
gctcctgcaa tgaatacttt 660catgtggaac aatccattta ctcaacggca tttggactca
atctcagagc tcggactatc 720acttatcccc ccaataacaa agaagttagc ttgtggtgat
tatggaaatg gtgcaatggc 780tgaaccttct acgatagata caactcttag gttttcactt
gatccttcaa ttgtatgaag 840tgtctgatgt cccgttcaac aattcttgtt atgaatctaa
catgcttagt acaatggact 900atggtataga cccagatagt gtttcttggt agctgggtta
gacagcatga actcagcttg 960tgtgtggtga agttaagttc tgatgataaa caatcatgaa
cttgtgattt tatgtaacat 1020ttgctcattt attctggaca cttacaagaa ctgtgtggat
actcctgctt ataagagaat 1080gtgtcaaact actatgcaaa tcatctaatt tggaacttga
tgatgtaaga ggagtatgca 1140ctttattttc ttccctgtga taattattaa tctctatgat
tg 118242215PRTPinus sp. 42Met Val Val Leu Lys Ile
Asn Thr Leu Val Gln Asn Met Asp Ala Thr 1 5
10 15 Asn Ser Gln Pro Asp Glu Pro Gly Arg Asp Ala
Asn Ser Ser Gln Lys 20 25
30 Pro Arg Ile Ile Leu Ala Ala Ser Gly Ser Val Ala Ala Ile Lys
Phe 35 40 45 Gly
Ile Leu Ala His Cys Leu Cys Gln Trp Ala Glu Val Lys Ala Val 50
55 60 Val Thr Lys Ser Ala Leu
His Phe Ile Asp Lys Met Ser Leu Pro Ala 65 70
75 80 Asn Val Lys Leu Tyr Thr Asp Glu Asn Glu Trp
Ser Ser Trp Ser Lys 85 90
95 Ile Gly Asp Thr Val Leu His Ile Glu Leu Arg Gln Trp Ala Asp Ala
100 105 110 Met Val
Ile Ala Pro Leu Ser Ala Asn Thr Leu Ala Lys Ile Ala Gly 115
120 125 Gly Leu Cys Asp Asn Leu Leu
Thr Cys Ile Val Arg Ala Trp Asp Phe 130 135
140 Asn Lys Pro Leu Phe Val Ala Pro Ala Met Asn Thr
Phe Met Trp Asn 145 150 155
160 Asn Pro Phe Thr Gln Arg His Leu Asp Ser Ile Ser Glu Leu Gly Leu
165 170 175 Ser Leu Ile
Pro Pro Ile Thr Lys Lys Leu Ala Cys Gly Asp Tyr Gly 180
185 190 Asn Gly Ala Met Ala Glu Pro Ser
Thr Ile Asp Thr Thr Leu Arg Phe 195 200
205 Ser Leu Asp Pro Ser Ile Val 210
215 43804DNAOryza sativa 43atggggcggg ggaaggtgca gctgaagcgg atagagaaca
agatcaacag gcaggtgacg 60ttctccaaga ggaggaatgg attgctgaag aaggcgcacg
agatctccgt cctctgcgac 120gccgaggtcg ccgccatcgt cttctccccc aagggcaagc
tctacgagta cgccactgac 180tccaggatgg acaaaatcct tgaacgttat gagcgctatt
catatgctga aaaggctctt 240atttcagctg aatccgagag tgagggaaat tggtgccatg
aatacaggaa acttaaggca 300aagattgaga ccatacaaaa atgtcacaaa cacctcatgg
gagaggatct agaatccctg 360aatctcaaag aactccaaca gctagagcag cagctggaga
gttcattgaa gcacataata 420tcaagaaaga gccaccttat gcttgagtcc atttccgagc
tgcagaaaaa ggagaggtca 480ctgcaggagg agaacaaggc tctgcagaag gaactggtgg
agaggcagaa gaatgtgagg 540ggccagcagc aagtagggca gtgggaccaa acccaggtcc
aggcccaggc ccaagcccaa 600ccccaagccc agacaagctc ctcctcctcc tccatgctga
gggatcagca ggcacttctt 660ccaccacaaa atatctgcta cccgccggtg atgatgggcg
agagaaatga tgcggcggcg 720gcggcggcgg tggcggcgca gggccaggtg caactccgca
tcggaggtct tccgccatgg 780atgctgagcc acctcaatgc ttaa
80444267PRTOryza sativa 44Met Gly Arg Gly Lys Val
Gln Leu Lys Arg Ile Glu Asn Lys Ile Asn 1 5
10 15 Arg Gln Val Thr Phe Ser Lys Arg Arg Asn Gly
Leu Leu Lys Lys Ala 20 25
30 His Glu Ile Ser Val Leu Cys Asp Ala Glu Val Ala Ala Ile Val
Phe 35 40 45 Ser
Pro Lys Gly Lys Leu Tyr Glu Tyr Ala Thr Asp Ser Arg Met Asp 50
55 60 Lys Ile Leu Glu Arg Tyr
Glu Arg Tyr Ser Tyr Ala Glu Lys Ala Leu 65 70
75 80 Ile Ser Ala Glu Ser Glu Ser Glu Gly Asn Trp
Cys His Glu Tyr Arg 85 90
95 Lys Leu Lys Ala Lys Ile Glu Thr Ile Gln Lys Cys His Lys His Leu
100 105 110 Met Gly
Glu Asp Leu Glu Ser Leu Asn Leu Lys Glu Leu Gln Gln Leu 115
120 125 Glu Gln Gln Leu Glu Ser Ser
Leu Lys His Ile Ile Ser Arg Lys Ser 130 135
140 His Leu Met Leu Glu Ser Ile Ser Glu Leu Gln Lys
Lys Glu Arg Ser 145 150 155
160 Leu Gln Glu Glu Asn Lys Ala Leu Gln Lys Glu Leu Val Glu Arg Gln
165 170 175 Lys Asn Val
Arg Gly Gln Gln Gln Val Gly Gln Trp Asp Gln Thr Gln 180
185 190 Val Gln Ala Gln Ala Gln Ala Gln
Pro Gln Ala Gln Thr Ser Ser Ser 195 200
205 Ser Ser Ser Met Leu Arg Asp Gln Gln Ala Leu Leu Pro
Pro Gln Asn 210 215 220
Ile Cys Tyr Pro Pro Val Met Met Gly Glu Arg Asn Asp Ala Ala Ala 225
230 235 240 Ala Ala Ala Val
Ala Ala Gln Gly Gln Val Gln Leu Arg Ile Gly Gly 245
250 255 Leu Pro Pro Trp Met Leu Ser His Leu
Asn Ala 260 265 4552DNAArtificial
sequenceprimer 45ggggacaagt ttgtacaaaa aagcaggctt aaacaatggg gcgggggaag
gt 524647DNAArtificial sequenceprimer 46ggggaccact ttgtacaaga
aagctgggtt tggccgacga cgacgac 47472193DNAOryza sativa
47aatccgaaaa gtttctgcac cgttttcacc ccctaactaa caatataggg aacgtgtgct
60aaatataaaa tgagacctta tatatgtagc gctgataact agaactatgc aagaaaaact
120catccaccta ctttagtggc aatcgggcta aataaaaaag agtcgctaca ctagtttcgt
180tttccttagt aattaagtgg gaaaatgaaa tcattattgc ttagaatata cgttcacatc
240tctgtcatga agttaaatta ttcgaggtag ccataattgt catcaaactc ttcttgaata
300aaaaaatctt tctagctgaa ctcaatgggt aaagagagag atttttttta aaaaaataga
360atgaagatat tctgaacgta ttggcaaaga tttaaacata taattatata attttatagt
420ttgtgcattc gtcatatcgc acatcattaa ggacatgtct tactccatcc caatttttat
480ttagtaatta aagacaattg acttattttt attatttatc ttttttcgat tagatgcaag
540gtacttacgc acacactttg tgctcatgtg catgtgtgag tgcacctcct caatacacgt
600tcaactagca acacatctct aatatcactc gcctatttaa tacatttagg tagcaatatc
660tgaattcaag cactccacca tcaccagacc acttttaata atatctaaaa tacaaaaaat
720aattttacag aatagcatga aaagtatgaa acgaactatt taggtttttc acatacaaaa
780aaaaaaagaa ttttgctcgt gcgcgagcgc caatctccca tattgggcac acaggcaaca
840acagagtggc tgcccacaga acaacccaca aaaaacgatg atctaacgga ggacagcaag
900tccgcaacaa ccttttaaca gcaggctttg cggccaggag agaggaggag aggcaaagaa
960aaccaagcat cctcctcctc ccatctataa attcctcccc ccttttcccc tctctatata
1020ggaggcatcc aagccaagaa gagggagagc accaaggaca cgcgactagc agaagccgag
1080cgaccgcctt cttcgatcca tatcttccgg tcgagttctt ggtcgatctc ttccctcctc
1140cacctcctcc tcacagggta tgtgcccttc ggttgttctt ggatttattg ttctaggttg
1200tgtagtacgg gcgttgatgt taggaaaggg gatctgtatc tgtgatgatt cctgttcttg
1260gatttgggat agaggggttc ttgatgttgc atgttatcgg ttcggtttga ttagtagtat
1320ggttttcaat cgtctggaga gctctatgga aatgaaatgg tttagggtac ggaatcttgc
1380gattttgtga gtaccttttg tttgaggtaa aatcagagca ccggtgattt tgcttggtgt
1440aataaaagta cggttgtttg gtcctcgatt ctggtagtga tgcttctcga tttgacgaag
1500ctatcctttg tttattccct attgaacaaa aataatccaa ctttgaagac ggtcccgttg
1560atgagattga atgattgatt cttaagcctg tccaaaattt cgcagctggc ttgtttagat
1620acagtagtcc ccatcacgaa attcatggaa acagttataa tcctcaggaa caggggattc
1680cctgttcttc cgatttgctt tagtcccaga attttttttc ccaaatatct taaaaagtca
1740ctttctggtt cagttcaatg aattgattgc tacaaataat gcttttatag cgttatccta
1800gctgtagttc agttaatagg taatacccct atagtttagt caggagaaga acttatccga
1860tttctgatct ccatttttaa ttatatgaaa tgaactgtag cataagcagt attcatttgg
1920attatttttt ttattagctc tcaccccttc attattctga gctgaaagtc tggcatgaac
1980tgtcctcaat tttgttttca aattcacatc gattatctat gcattatcct cttgtatcta
2040cctgtagaag tttctttttg gttattcctt gactgcttga ttacagaaag aaatttatga
2100agctgtaatc gggatagtta tactgcttgt tcttatgatt catttccttt gtgcagttct
2160tggtgtagct tgccactttc accagcaaag ttc
21934842PRTArtificial sequencemotif 48Leu Xaa Lys Lys Ala Xaa Glu Ile Ser
Xaa Leu Xaa Asp Ala Glu Xaa 1 5 10
15 Xaa Xaa Xaa Xaa Phe Ser Xaa Lys Gly Lys Leu Tyr Glu Xaa
Xaa Xaa 20 25 30
Xaa Ser Xaa Met Xaa Xaa Ile Leu Xaa Arg 35 40
4919PRTArtificial sequencemotif 49Lys Leu Lys Xaa Xaa Xaa Glu Xaa
Xaa Xaa Xaa Xaa Xaa Xaa His Leu 1 5 10
15 Met Gly Glu 5011PRTArtificial sequencemotif 50Gln
Xaa Gln Thr Ser Xaa Xaa Xaa Xaa Xaa Xaa 1 5
10 517PRTArtificial sequencemotif 51Xaa Xaa Xaa Trp Met Xaa Xaa 1
5 52735DNAHordeum vulgare 52atggggcgcg ggaaggtgca
gctgaagcgg atcgagaaca agatcaaccg ccaggtcacc 60ttctccaagc gccgctcggg
gctgctcaag aaggcgcacg agatctccgt gctctgcgac 120gccgaggtcg gcctcatcat
cttctccacc aagggaaagc tctacgagtt ctccaccgag 180tcatgtatgg acaaaattct
tgaacggtat gagcgctact cttatgcaga aaaggttctc 240gtttcaagtg aatctgaaat
tcagggaaac tggtgtcacg aatataggaa actgaaggcg 300aaggttgaga caatacagaa
atgtcaaaag catctcatgg gagaggatct tgaatctttg 360aatctcaagg agttgcagca
actggagcag cagctggaaa gctcactgaa acatatcaga 420gccaggaaga accaacttat
gcacgaatcc atttctgagc ttcagaagaa ggagaggtca 480ctgcaggagg agaataaagt
tctccagaag gaacttgtgg agaagcagaa ggcccaggcg 540gcgcagcaag atcaaactca
gcctcaaacc agctcttctt cttcttcctt catgatgagg 600gatgctcccc ctgtcgcaga
taccagcaat cacccagcgg cggcaggcga gagggcagag 660gatgtggcag tgcagcctca
ggtcccactc cggacggcgc ttccactgtg gatggtgagc 720cacatcaacg gctga
73553244PRTHordeum vulgare
53Met Gly Arg Gly Lys Val Gln Leu Lys Arg Ile Glu Asn Lys Ile Asn 1
5 10 15 Arg Gln Val Thr
Phe Ser Lys Arg Arg Ser Gly Leu Leu Lys Lys Ala 20
25 30 His Glu Ile Ser Val Leu Cys Asp Ala
Glu Val Gly Leu Ile Ile Phe 35 40
45 Ser Thr Lys Gly Lys Leu Tyr Glu Phe Ser Thr Glu Ser Cys
Met Asp 50 55 60
Lys Ile Leu Glu Arg Tyr Glu Arg Tyr Ser Tyr Ala Glu Lys Val Leu 65
70 75 80 Val Ser Ser Glu Ser
Glu Ile Gln Gly Asn Trp Cys His Glu Tyr Arg 85
90 95 Lys Leu Lys Ala Lys Val Glu Thr Ile Gln
Lys Cys Gln Lys His Leu 100 105
110 Met Gly Glu Asp Leu Glu Ser Leu Asn Leu Lys Glu Leu Gln Gln
Leu 115 120 125 Glu
Gln Gln Leu Glu Ser Ser Leu Lys His Ile Arg Ala Arg Lys Asn 130
135 140 Gln Leu Met His Glu Ser
Ile Ser Glu Leu Gln Lys Lys Glu Arg Ser 145 150
155 160 Leu Gln Glu Glu Asn Lys Val Leu Gln Lys Glu
Leu Val Glu Lys Gln 165 170
175 Lys Ala Gln Ala Ala Gln Gln Asp Gln Thr Gln Pro Gln Thr Ser Ser
180 185 190 Ser Ser
Ser Ser Phe Met Met Arg Asp Ala Pro Pro Val Ala Asp Thr 195
200 205 Ser Asn His Pro Ala Ala Ala
Gly Glu Arg Ala Glu Asp Val Ala Val 210 215
220 Gln Pro Gln Val Pro Leu Arg Thr Ala Leu Pro Leu
Trp Met Val Ser 225 230 235
240 His Ile Asn Gly 541196DNAHordeum vulgare 54ctctcccctc ccacttcacc
caaccacctg acagccatgg ctccgccacc tcgcctccgc 60ccgcgcctct gagagtagcc
gtcgcggtcg ctcgctcgct cgctcgctgc tgccggtgtt 120ggcccggtcc tcgagcggag
atggggcgca ggaaggtgca gctgaagcgg atcgagaaca 180agatcaaccg ccaggtcacc
ttctccaagc gccgctcggg gctgctcaag aaggcgcacg 240agatctccgt gctctacgac
gccgaggtcg gcctcatcat cttctccacc aagggaaagc 300tctacgagtt ctccaccgag
tcatgtatgg acaaaattct tgaacggtat gagcgctact 360cttatgcaga aaaggttctc
gtttcaagtg aatctgaaat tcagggaaac tggtgtcacg 420aatataggaa actgaaggcg
aaggttgaga caatacagaa atgtcaaaag catctcatgg 480gagaggatct tgaatctttg
aatctcaagg agttgcagca actggagcag cagctggaaa 540gctcactgaa acatatcaga
gccaggaaga accaacttat gcacgaatcc atttctgagc 600ttcagaagaa ggagaggtca
ctgcaggagg agaataaagt tctccagaag gaacttgtgg 660agaagcagaa ggcccaggcg
gcgcagcaag atcaaactca gcctcaaacc agctcttctt 720cttcttcctt catgatgagg
gatgctcccc ctgtcgcaga taccagcaat cacccagcgg 780cggcaggcga gagggcagag
gatgtggcag tgcagcctca ggtcccactc cggacggcgc 840ttccactgtg gatggtgagc
cacatcaacg gctgaagggc ttccagccca tgtaagcgta 900ctattcagta cgagtaacaa
gttgcagcgg ccagcctggt gtatcatgcg gttgcgaaca 960tgctaacccc atggagggga
gaggaaaaga aatcagagta aagcagcaag ctgcaggaat 1020gtgtatattt cacttcgtcc
acctcagttt cctttccacc tgggctgaga tggctgtacg 1080agtaatctac catgtaattt
atatgtagca tgagtgacga attttcaact ttcgatgata 1140tccgttgctc ctgggtgttg
tttctgtgaa ttaacctatc gaatatgagc gttgtg 119655244PRTHordeum vulgare
55Met Gly Arg Arg Lys Val Gln Leu Lys Arg Ile Glu Asn Lys Ile Asn 1
5 10 15 Arg Gln Val Thr
Phe Ser Lys Arg Arg Ser Gly Leu Leu Lys Lys Ala 20
25 30 His Glu Ile Ser Val Leu Tyr Asp Ala
Glu Val Gly Leu Ile Ile Phe 35 40
45 Ser Thr Lys Gly Lys Leu Tyr Glu Phe Ser Thr Glu Ser Cys
Met Asp 50 55 60
Lys Ile Leu Glu Arg Tyr Glu Arg Tyr Ser Tyr Ala Glu Lys Val Leu 65
70 75 80 Val Ser Ser Glu Ser
Glu Ile Gln Gly Asn Trp Cys His Glu Tyr Arg 85
90 95 Lys Leu Lys Ala Lys Val Glu Thr Ile Gln
Lys Cys Gln Lys His Leu 100 105
110 Met Gly Glu Asp Leu Glu Ser Leu Asn Leu Lys Glu Leu Gln Gln
Leu 115 120 125 Glu
Gln Gln Leu Glu Ser Ser Leu Lys His Ile Arg Ala Arg Lys Asn 130
135 140 Gln Leu Met His Glu Ser
Ile Ser Glu Leu Gln Lys Lys Glu Arg Ser 145 150
155 160 Leu Gln Glu Glu Asn Lys Val Leu Gln Lys Glu
Leu Val Glu Lys Gln 165 170
175 Lys Ala Gln Ala Ala Gln Gln Asp Gln Thr Gln Pro Gln Thr Ser Ser
180 185 190 Ser Ser
Ser Ser Phe Met Met Arg Asp Ala Pro Pro Val Ala Asp Thr 195
200 205 Ser Asn His Pro Ala Ala Ala
Gly Glu Arg Ala Glu Asp Val Ala Val 210 215
220 Gln Pro Gln Val Pro Leu Arg Thr Ala Leu Pro Leu
Trp Met Val Ser 225 230 235
240 His Ile Asn Gly 561161DNATriticum aestivum 56ggcacgagct ctctctctct
ctctctctct ctctctctct ctctctcctc gtgccgaatt 60cggcacgagc ggagatgggg
cgcgggaagg tgcagctgaa gcggatcgag aacaagatca 120accggcaggt gaccttctcc
aagcgccgct cggggctgct caagaaggcg cacgagatct 180ccgtgctctg cgacgccgag
gtcggcctca tcatcttctc caccaaggga aagctctacg 240agttctccac cgagtcatgt
atggacaaaa ttcttgaacg gtatgagcgc tactcttatg 300cagaaaaggt tctcgtttca
agtgaatctg aaattcaggg aaactggtgt cacgaatata 360ggaaactgaa ggcgaaggtt
gagacaatac agaaatgtca aaagcatctg atgggagagg 420atcttgaatc tttgaatctc
aaggagttgc agcaactgga gcagcagctg gaaagctcac 480tgaaacatat cagatccagg
aagaaccaac ttatgcacga atccatttct gagcttcaga 540agaaggagag gtcactgcag
gaggagaata aagttctcca gaaggaactc gtcgagaagc 600agaaggccca ggcggcgcaa
caagatcaga ctcagcctca aacaagctct tcttcttctt 660ccttcatgat gagggatgct
ccccctgccg cagctaccag cattcatcca gcggcggcag 720gcgagagggc aggggatgcg
gcagtgcagc cgcaggcccc accccggacg gggcttccac 780tgtggatggt gagccacatc
aacggctgaa gggcttccag cccatataag cgtactattc 840agtagagagt aacaagttgc
accggccagt ctggtgtatg ttgcggttgc tagcacgcct 900gaccccttgg aggggaaagg
aaaagaaatc agagtaaagt agcaagctgc agcgatgtgt 960atatttcact ttgtccaccc
cagtttccct cccagctggg ctcaatttac catgtaatct 1020atatgtagct tgagtgatga
attttcaagt ttccatgata cccgtctcta gtgggatgtt 1080gtttatgtga attaacctat
caaatatgag cattgtgtat attgtgattc ttgaaaataa 1140ataaatcagg atctttgtct t
116157244PRTTriticum aestivum
57Met Gly Arg Gly Lys Val Gln Leu Lys Arg Ile Glu Asn Lys Ile Asn 1
5 10 15 Arg Gln Val Thr
Phe Ser Lys Arg Arg Ser Gly Leu Leu Lys Lys Ala 20
25 30 His Glu Ile Ser Val Leu Cys Asp Ala
Glu Val Gly Leu Ile Ile Phe 35 40
45 Ser Thr Lys Gly Lys Leu Tyr Glu Phe Ser Thr Glu Ser Cys
Met Asp 50 55 60
Lys Ile Leu Glu Arg Tyr Glu Arg Tyr Ser Tyr Ala Glu Lys Val Leu 65
70 75 80 Val Ser Ser Glu Ser
Glu Ile Gln Gly Asn Trp Cys His Glu Tyr Arg 85
90 95 Lys Leu Lys Ala Lys Val Glu Thr Ile Gln
Lys Cys Gln Lys His Leu 100 105
110 Met Gly Glu Asp Leu Glu Ser Leu Asn Leu Lys Glu Leu Gln Gln
Leu 115 120 125 Glu
Gln Gln Leu Glu Ser Ser Leu Lys His Ile Arg Ser Arg Lys Asn 130
135 140 Gln Leu Met His Glu Ser
Ile Ser Glu Leu Gln Lys Lys Glu Arg Ser 145 150
155 160 Leu Gln Glu Glu Asn Lys Val Leu Gln Lys Glu
Leu Val Glu Lys Gln 165 170
175 Lys Ala Gln Ala Ala Gln Gln Asp Gln Thr Gln Pro Gln Thr Ser Ser
180 185 190 Ser Ser
Ser Ser Phe Met Met Arg Asp Ala Pro Pro Ala Ala Ala Thr 195
200 205 Ser Ile His Pro Ala Ala Ala
Gly Glu Arg Ala Gly Asp Ala Ala Val 210 215
220 Gln Pro Gln Ala Pro Pro Arg Thr Gly Leu Pro Leu
Trp Met Val Ser 225 230 235
240 His Ile Asn Gly 581210DNATriticum aestivum 58tccctctcct ccctctcttc
cgcctcaccc aaccacctga cagccatggc tccgcccccc 60cgcccccgcc tgcgcctgtc
ggagtagccg tcgcggtctg ccggtgttgg aggcttgggg 120tgtagggttg gccccgttct
ccagcggaga tggggcgcgg gaaggtgcag ctgaagcgga 180tcgagaacaa gatcaaccgg
caggtgacct tctccaagcg ccgctcgggg ctgctcaaga 240aggcgcacga gatctccgtg
ctctgcgacg ccgaggtcgg cctcatcatc ttctccacca 300agggaaagct ctacgagttc
tccaccgagt catgtatgga caaaattctt gaacggtatg 360agcgctactc ttatgcagaa
aaggttctcg tttcaagtga atctgaaatt cagggaaact 420ggtgtcacga atataggaaa
ctgaaggcga aggttgagac aatacagaaa tgtcaaaagc 480atctgatggg agaggatctt
gaatctttga atctcaagga gttgcagcaa ctggagcagc 540agctggaaag ctcactgaaa
catatcagat ccaggaagaa ccaacttatg cacgaatcca 600tttctgagct tcagaagaag
gagaggtcac tgcaggagga gaataaagtt ctccagaagg 660aactcgtcga gaagcagaag
gcccaggcgg cgcaacaaga tcagactcag cctcaaacaa 720gctcttcttc ttcttccttc
atgatgaggg atgctccccc tgccgcaact accagcattc 780atccagcggc atcaggagag
agggcagagg atgcggcagt gcagccgcag gccccacccc 840ggacggggct tccactgtgg
atggttagcc acatcaacgg ctgaagggct tccagcccat 900ataagcgtac tattcagtag
agagtaacaa gttgcaccgg ccagcctggt gtatgttgcg 960gttgctagca tgcctgaccc
cttggagggg aaaggaaaag aaatcagagt aaagtagcaa 1020gctgcagtga tgtgtatatt
tcactttgtc cacctcagtt tccctcccag ctgggctcaa 1080tttaccatgt aatctatatg
tagcttgagt gatgaatttt caagtttcca tgatacccgt 1140ctcgagcggg tgttgtttat
gtgaattaac ctatcaaata tgagcattgt gtaaaaaaaa 1200aaaaaaaaaa
121059244PRTTriticum aestivum
59Met Gly Arg Gly Lys Val Gln Leu Lys Arg Ile Glu Asn Lys Ile Asn 1
5 10 15 Arg Gln Val Thr
Phe Ser Lys Arg Arg Ser Gly Leu Leu Lys Lys Ala 20
25 30 His Glu Ile Ser Val Leu Cys Asp Ala
Glu Val Gly Leu Ile Ile Phe 35 40
45 Ser Thr Lys Gly Lys Leu Tyr Glu Phe Ser Thr Glu Ser Cys
Met Asp 50 55 60
Lys Ile Leu Glu Arg Tyr Glu Arg Tyr Ser Tyr Ala Glu Lys Val Leu 65
70 75 80 Val Ser Ser Glu Ser
Glu Ile Gln Gly Asn Trp Cys His Glu Tyr Arg 85
90 95 Lys Leu Lys Ala Lys Val Glu Thr Ile Gln
Lys Cys Gln Lys His Leu 100 105
110 Met Gly Glu Asp Leu Glu Ser Leu Asn Leu Lys Glu Leu Gln Gln
Leu 115 120 125 Glu
Gln Gln Leu Glu Ser Ser Leu Lys His Ile Arg Ser Arg Lys Asn 130
135 140 Gln Leu Met His Glu Ser
Ile Ser Glu Leu Gln Lys Lys Glu Arg Ser 145 150
155 160 Leu Gln Glu Glu Asn Lys Val Leu Gln Lys Glu
Leu Val Glu Lys Gln 165 170
175 Lys Ala Gln Ala Ala Gln Gln Asp Gln Thr Gln Pro Gln Thr Ser Ser
180 185 190 Ser Ser
Ser Ser Phe Met Met Arg Asp Ala Pro Pro Ala Ala Thr Thr 195
200 205 Ser Ile His Pro Ala Ala Ser
Gly Glu Arg Ala Glu Asp Ala Ala Val 210 215
220 Gln Pro Gln Ala Pro Pro Arg Thr Gly Leu Pro Leu
Trp Met Val Ser 225 230 235
240 His Ile Asn Gly 60735DNATriticum monococcum 60atggggcgcg ggaaggtgca
gctgaagcgg atcgagaaca agatcaaccg gcaggtgacc 60ttctccaagc gccgctcggg
gcttctcaag aaggcgcacg agatctccgt gctctgcgac 120gccgaggtcg gcctcatcat
cttctccacc aagggaaagc tctacgagtt ctccaccgag 180tcatgtatgg acaaaattct
tgaacggtat gagcgctatt cttatgcaga aaaggttctc 240gtttcaagtg aatctgaaat
tcagggaaac tggtgtcacg aatataggaa actgaaggcg 300aaggttgaga caatacagaa
atgtcaaaaa catctcatgg gagaggatct tgaatctttg 360aatctcaagg agttgcagca
actggagcag cagctggaaa gctcactgaa acatatcaga 420tccaggaaga accaacttat
gcacgaatcc atttctgagc tgcagaagaa ggagaggtca 480ctgcaggagg agaataaagt
tctccagaag gaactcgtgg agaagcagaa ggcccatgcg 540gcgcagcaag atcaaactca
gcctcaaacc agctcttctt cttcttcctt catgctgagg 600gatgctcccc ctgccgcaaa
taccagcatt catccagcgg cggcaggcga gagggcagag 660gatgcggcag tgcagccgca
ggccccaccc cggacggggc ttccaccgtg gatggtgagc 720cacatcaacg ggtga
73561244PRTTriticum
monococcum 61Met Gly Arg Gly Lys Val Gln Leu Lys Arg Ile Glu Asn Lys Ile
Asn 1 5 10 15 Arg
Gln Val Thr Phe Ser Lys Arg Arg Ser Gly Leu Leu Lys Lys Ala
20 25 30 His Glu Ile Ser Val
Leu Cys Asp Ala Glu Val Gly Leu Ile Ile Phe 35
40 45 Ser Thr Lys Gly Lys Leu Tyr Glu Phe
Ser Thr Glu Ser Cys Met Asp 50 55
60 Lys Ile Leu Glu Arg Tyr Glu Arg Tyr Ser Tyr Ala Glu
Lys Val Leu 65 70 75
80 Val Ser Ser Glu Ser Glu Ile Gln Gly Asn Trp Cys His Glu Tyr Arg
85 90 95 Lys Leu Lys Ala
Lys Val Glu Thr Ile Gln Lys Cys Gln Lys His Leu 100
105 110 Met Gly Glu Asp Leu Glu Ser Leu Asn
Leu Lys Glu Leu Gln Gln Leu 115 120
125 Glu Gln Gln Leu Glu Ser Ser Leu Lys His Ile Arg Ser Arg
Lys Asn 130 135 140
Gln Leu Met His Glu Ser Ile Ser Glu Leu Gln Lys Lys Glu Arg Ser 145
150 155 160 Leu Gln Glu Glu Asn
Lys Val Leu Gln Lys Glu Leu Val Glu Lys Gln 165
170 175 Lys Ala His Ala Ala Gln Gln Asp Gln Thr
Gln Pro Gln Thr Ser Ser 180 185
190 Ser Ser Ser Ser Phe Met Leu Arg Asp Ala Pro Pro Ala Ala Asn
Thr 195 200 205 Ser
Ile His Pro Ala Ala Ala Gly Glu Arg Ala Glu Asp Ala Ala Val 210
215 220 Gln Pro Gln Ala Pro Pro
Arg Thr Gly Leu Pro Pro Trp Met Val Ser 225 230
235 240 His Ile Asn Gly 62735DNATriticum aestivum
62atggggcggg ggaaggtgca gctgaagcgg atcgagaaca agatcaaccg gcaggtgacc
60ttctccaagc gccgctcggg gcttctcaag aaggcgcacg agatctccgt gctctgcgac
120gccgaggtcg gcctcatcat cttctccacc aagggaaagc tctacgagtt ctccaccgag
180tcatgtatgg acaaaattct tgaacggtat gagcgctatt cttatgcaga aaaggttctc
240gtttcaagtg aatctgaaat tcagggaaac tggtgtcacg aatataggaa actgaaggcg
300aaggttgaga caatacagaa atgtcaaaag catctcatgg gagaggatct tgaatctttg
360aatctcaagg agttgcagca actggagcag cagctggaaa gctcactgaa acatatcaga
420tccaggaaga accaacttat gcacgaatcc atttctgagc ttcagaagaa ggagaggtca
480ctgcaggagg agaataaagt tctccagaag gaactcgtgg agaagcagaa ggcccatgcg
540gcgcagcaag atcaaactca gcctcaaacc agctcttcat cttcttcctt catgctgagg
600gatgctcccc ctgccgcaaa taccagcatt catccagcgg caacaggcga gagggcagag
660gatgcggcag tgcagccgca ggccccaccc cggacggggc ttccaccgtg gatggtgagc
720cacatcaacg ggtga
73563244PRTTriticum aestivum 63Met Gly Arg Gly Lys Val Gln Leu Lys Arg
Ile Glu Asn Lys Ile Asn 1 5 10
15 Arg Gln Val Thr Phe Ser Lys Arg Arg Ser Gly Leu Leu Lys Lys
Ala 20 25 30 His
Glu Ile Ser Val Leu Cys Asp Ala Glu Val Gly Leu Ile Ile Phe 35
40 45 Ser Thr Lys Gly Lys Leu
Tyr Glu Phe Ser Thr Glu Ser Cys Met Asp 50 55
60 Lys Ile Leu Glu Arg Tyr Glu Arg Tyr Ser Tyr
Ala Glu Lys Val Leu 65 70 75
80 Val Ser Ser Glu Ser Glu Ile Gln Gly Asn Trp Cys His Glu Tyr Arg
85 90 95 Lys Leu
Lys Ala Lys Val Glu Thr Ile Gln Lys Cys Gln Lys His Leu 100
105 110 Met Gly Glu Asp Leu Glu Ser
Leu Asn Leu Lys Glu Leu Gln Gln Leu 115 120
125 Glu Gln Gln Leu Glu Ser Ser Leu Lys His Ile Arg
Ser Arg Lys Asn 130 135 140
Gln Leu Met His Glu Ser Ile Ser Glu Leu Gln Lys Lys Glu Arg Ser 145
150 155 160 Leu Gln Glu
Glu Asn Lys Val Leu Gln Lys Glu Leu Val Glu Lys Gln 165
170 175 Lys Ala His Ala Ala Gln Gln Asp
Gln Thr Gln Pro Gln Thr Ser Ser 180 185
190 Ser Ser Ser Ser Phe Met Leu Arg Asp Ala Pro Pro Ala
Ala Asn Thr 195 200 205
Ser Ile His Pro Ala Ala Thr Gly Glu Arg Ala Glu Asp Ala Ala Val 210
215 220 Gln Pro Gln Ala
Pro Pro Arg Thr Gly Leu Pro Pro Trp Met Val Ser 225 230
235 240 His Ile Asn Gly 64738DNALolium
perenne 64atggggcgcg gcaaggtgca gctcaagcgg atcgagaaca agatcaaccg
ccaggtcacc 60ttctccaagc gccgctcggg gctgctcaag aaggcgcacg agatctccgt
gctctgcgac 120gccgaggtcg ggctcatcat cttctccacc aagggaaagc tctacgagtt
cgcaaccgac 180tcatgtatgg acaaaattct tgagcggtat gagcgctact cctatgcaga
gaaagtgctc 240atttcaaccg aatctgaaat tcagggaaac tggtgtcatg aatataggaa
actgaaggcg 300aaggttgaga caatacagag atgtcaaaag catctaatgg gagaggatct
tgaatcattg 360aatctcaagg agttgcagca actagagcag cagctggaaa gttcactgaa
acatattaga 420gccagaaaga accagcttat gcacgaatcc atatctgagc ttcaaaagaa
ggagaggtca 480ctgcaggagg agaataaaat tctccagaag gaactcatag agaagcagaa
ggcccacacg 540cagcaagcgc agtgggagca aactcagccc caaaccagct cttcctcctc
ctcctttatg 600atgggggaag ctaccccagc aacaaattgc agtaatcccc cagcagcggc
cagcgacaga 660gcagaggatg cgacggggca gccttcagct cgcacggtgc ttccaccatg
gatggtgagt 720cacatcaaca atggctga
73865245PRTLolium perenne 65Met Gly Arg Gly Lys Val Gln Leu
Lys Arg Ile Glu Asn Lys Ile Asn 1 5 10
15 Arg Gln Val Thr Phe Ser Lys Arg Arg Ser Gly Leu Leu
Lys Lys Ala 20 25 30
His Glu Ile Ser Val Leu Cys Asp Ala Glu Val Gly Leu Ile Ile Phe
35 40 45 Ser Thr Lys Gly
Lys Leu Tyr Glu Phe Ala Thr Asp Ser Cys Met Asp 50
55 60 Lys Ile Leu Glu Arg Tyr Glu Arg
Tyr Ser Tyr Ala Glu Lys Val Leu 65 70
75 80 Ile Ser Thr Glu Ser Glu Ile Gln Gly Asn Trp Cys
His Glu Tyr Arg 85 90
95 Lys Leu Lys Ala Lys Val Glu Thr Ile Gln Arg Cys Gln Lys His Leu
100 105 110 Met Gly Glu
Asp Leu Glu Ser Leu Asn Leu Lys Glu Leu Gln Gln Leu 115
120 125 Glu Gln Gln Leu Glu Ser Ser Leu
Lys His Ile Arg Ala Arg Lys Asn 130 135
140 Gln Leu Met His Glu Ser Ile Ser Glu Leu Gln Lys Lys
Glu Arg Ser 145 150 155
160 Leu Gln Glu Glu Asn Lys Ile Leu Gln Lys Glu Leu Ile Glu Lys Gln
165 170 175 Lys Ala His Thr
Gln Gln Ala Gln Trp Glu Gln Thr Gln Pro Gln Thr 180
185 190 Ser Ser Ser Ser Ser Ser Phe Met Met
Gly Glu Ala Thr Pro Ala Thr 195 200
205 Asn Cys Ser Asn Pro Pro Ala Ala Ala Ser Asp Arg Ala Glu
Asp Ala 210 215 220
Thr Gly Gln Pro Ser Ala Arg Thr Val Leu Pro Pro Trp Met Val Ser 225
230 235 240 His Ile Asn Asn Gly
245 66738DNALolium temulentum 66atggggcgcg gcaaggtgca
gctcaagcgg atcgagaaca agatcaaccg ccaggtcacc 60ttctccaagc gccgctcagg
cctgctcaag aaggcgcacg agatctccgt gctctgcgac 120gcagaggtcg ggctcatcat
cttctccacc aagggaaagc tctacgagtt cgccaccgac 180tcatgtatgg acaaaattct
tgagcggtat gagcgctact cctatgcaga gaaagtgctc 240atttcaactg aatctgaaat
tcagggaaac tggtgtcatg aatataggaa actgaaggcg 300aaggttgaga caatacagag
atgtcaaaag catctaatgg gagaggatct tgaatcattg 360aatctcaagg agttgcagca
actagagcag cagctggaaa gttcactgaa acatattaga 420tccagaaaga gccagcttat
gcacgaatcc atatctgagc ttcaaaagaa ggagaggtca 480ctgcaagagg agaataaaat
tctccagaag gaactcatag agaagcagaa ggcccacacg 540cagcaagcgc agttggagca
aactcagccc caaaccagct cttcctcctc ctcctttatg 600atgggggaag ctaccccagc
aacaaatcgc agtaatcccc cagcagcggc cagcgacaga 660gcagaggatg cgacggggca
gcctccagct cgcacggtgc ttccaccatg gatggtgagt 720cacctcaaca atggctga
73867245PRTLolium temulentum
67Met Gly Arg Gly Lys Val Gln Leu Lys Arg Ile Glu Asn Lys Ile Asn 1
5 10 15 Arg Gln Val Thr
Phe Ser Lys Arg Arg Ser Gly Leu Leu Lys Lys Ala 20
25 30 His Glu Ile Ser Val Leu Cys Asp Ala
Glu Val Gly Leu Ile Ile Phe 35 40
45 Ser Thr Lys Gly Lys Leu Tyr Glu Phe Ala Thr Asp Ser Cys
Met Asp 50 55 60
Lys Ile Leu Glu Arg Tyr Glu Arg Tyr Ser Tyr Ala Glu Lys Val Leu 65
70 75 80 Ile Ser Thr Glu Ser
Glu Ile Gln Gly Asn Trp Cys His Glu Tyr Arg 85
90 95 Lys Leu Lys Ala Lys Val Glu Thr Ile Gln
Arg Cys Gln Lys His Leu 100 105
110 Met Gly Glu Asp Leu Glu Ser Leu Asn Leu Lys Glu Leu Gln Gln
Leu 115 120 125 Glu
Gln Gln Leu Glu Ser Ser Leu Lys His Ile Arg Ser Arg Lys Ser 130
135 140 Gln Leu Met His Glu Ser
Ile Ser Glu Leu Gln Lys Lys Glu Arg Ser 145 150
155 160 Leu Gln Glu Glu Asn Lys Ile Leu Gln Lys Glu
Leu Ile Glu Lys Gln 165 170
175 Lys Ala His Thr Gln Gln Ala Gln Leu Glu Gln Thr Gln Pro Gln Thr
180 185 190 Ser Ser
Ser Ser Ser Ser Phe Met Met Gly Glu Ala Thr Pro Ala Thr 195
200 205 Asn Arg Ser Asn Pro Pro Ala
Ala Ala Ser Asp Arg Ala Glu Asp Ala 210 215
220 Thr Gly Gln Pro Pro Ala Arg Thr Val Leu Pro Pro
Trp Met Val Ser 225 230 235
240 His Leu Asn Asn Gly 245 68738DNAZea mays
68atggggcgcg ggaaggtgca gctgaagcgg atcgagaaca agatcaaccg ccaggtgacc
60ttctccaagc gccgctcggg gctgctcaag aaggcgcacg agatctccgt gctctgcgac
120gccgaggtcg cgctcatcat cttctccacc aaagggaagc tctacgagta ttccaccgat
180tcatgtatgg acaaaattct tgaccggtac gagcgctact cctatgcaga aaaggttctt
240atttcagcag aatctgaaac tcagggcaat tggtgccacg agtatagaaa actaaaggcg
300aaggtcgaga caatacaaaa atgtcaaaag cacctcatgg gagaggatct tgaaacgttg
360aatctcaaag agcttcagca actagagcag cagctggaga gttcactgaa acatatcaga
420accaggaaga accaacttat gctcgagtca atttcggagc tccaacggaa ggagaagtcg
480ctgcaggagg agaacaaggt tctgcagaag gagctcgcgg agaagcagaa agcccagcgg
540aagcaagtgc aatggggcca aacccaacag cagaccagtt cgtcttcctc gtgcttcgtg
600ataagggaag ctgccccaac aacaaatatc agcatttttc ctgtggcagc aggcgggagg
660ttggtggaag gtgcagcagc gcagccacag gctcgcgttg gactaccacc atggatgctt
720agccacctga gcagctga
73869245PRTZea mays 69Met Gly Arg Gly Lys Val Gln Leu Lys Arg Ile Glu Asn
Lys Ile Asn 1 5 10 15
Arg Gln Val Thr Phe Ser Lys Arg Arg Ser Gly Leu Leu Lys Lys Ala
20 25 30 His Glu Ile Ser
Val Leu Cys Asp Ala Glu Val Ala Leu Ile Ile Phe 35
40 45 Ser Thr Lys Gly Lys Leu Tyr Glu Tyr
Ser Thr Asp Ser Cys Met Asp 50 55
60 Lys Ile Leu Asp Arg Tyr Glu Arg Tyr Ser Tyr Ala Glu
Lys Val Leu 65 70 75
80 Ile Ser Ala Glu Ser Glu Thr Gln Gly Asn Trp Cys His Glu Tyr Arg
85 90 95 Lys Leu Lys Ala
Lys Val Glu Thr Ile Gln Lys Cys Gln Lys His Leu 100
105 110 Met Gly Glu Asp Leu Glu Thr Leu Asn
Leu Lys Glu Leu Gln Gln Leu 115 120
125 Glu Gln Gln Leu Glu Ser Ser Leu Lys His Ile Arg Thr Arg
Lys Asn 130 135 140
Gln Leu Met Leu Glu Ser Ile Ser Glu Leu Gln Arg Lys Glu Lys Ser 145
150 155 160 Leu Gln Glu Glu Asn
Lys Val Leu Gln Lys Glu Leu Ala Glu Lys Gln 165
170 175 Lys Ala Gln Arg Lys Gln Val Gln Trp Gly
Gln Thr Gln Gln Gln Thr 180 185
190 Ser Ser Ser Ser Ser Cys Phe Val Ile Arg Glu Ala Ala Pro Thr
Thr 195 200 205 Asn
Ile Ser Ile Phe Pro Val Ala Ala Gly Gly Arg Leu Val Glu Gly 210
215 220 Ala Ala Ala Gln Pro Gln
Ala Arg Val Gly Leu Pro Pro Trp Met Leu 225 230
235 240 Ser His Leu Ser Ser 245
70738DNAZea mays 70atggggcgcg ggaaggtgca gctgaagcgg atcgagaaca agatcaaccg
ccaggtgaca 60ttctccaagc gccgctcggg gctactcaag aaggcgcacg agatctccgt
gctctgcgac 120gccgaggtcg cgctcatcat cttctccacc aagggcaagc tctacgagta
ctctaccgat 180tcatgtatgg acaaaattct tgaacggtat gagcgctact cctatgcaga
aaaggttctc 240atttccgcag aatatgaaac tcagggcaat tggtgccatg aatatagaaa
actaaaggcg 300aaggtcgaga caatacagaa atgtcaaaag cacctcatgg gagaggatct
tgaaactttg 360aatctcaaag agcttcagca actagagcag cagctggaga gttcactgaa
acatatcaga 420acaaggaaga gccagcttat ggtcgagtca atttcagcgc tccaacggaa
ggagaagtca 480ctgcaggagg agaacaaggt tctgcagaag gagctcgcgg agaagcagaa
agaccagcgg 540cagcaagtgc aacgggacca aactcaacag cagaccagtt cgtcttccac
gtccttcatg 600ttaagggaag ctgccccaac aacaaatgtc agcatcttcc ctgtggcagc
aggcgggagg 660gtggtggaag gggcagcagc gcagccgcag gctcgcgttg gactgccacc
atggatgctt 720agccatctga gctgctga
73871245PRTZea mays 71Met Gly Arg Gly Lys Val Gln Leu Lys Arg
Ile Glu Asn Lys Ile Asn 1 5 10
15 Arg Gln Val Thr Phe Ser Lys Arg Arg Ser Gly Leu Leu Lys Lys
Ala 20 25 30 His
Glu Ile Ser Val Leu Cys Asp Ala Glu Val Ala Leu Ile Ile Phe 35
40 45 Ser Thr Lys Gly Lys Leu
Tyr Glu Tyr Ser Thr Asp Ser Cys Met Asp 50 55
60 Lys Ile Leu Glu Arg Tyr Glu Arg Tyr Ser Tyr
Ala Glu Lys Val Leu 65 70 75
80 Ile Ser Ala Glu Tyr Glu Thr Gln Gly Asn Trp Cys His Glu Tyr Arg
85 90 95 Lys Leu
Lys Ala Lys Val Glu Thr Ile Gln Lys Cys Gln Lys His Leu 100
105 110 Met Gly Glu Asp Leu Glu Thr
Leu Asn Leu Lys Glu Leu Gln Gln Leu 115 120
125 Glu Gln Gln Leu Glu Ser Ser Leu Lys His Ile Arg
Thr Arg Lys Ser 130 135 140
Gln Leu Met Val Glu Ser Ile Ser Ala Leu Gln Arg Lys Glu Lys Ser 145
150 155 160 Leu Gln Glu
Glu Asn Lys Val Leu Gln Lys Glu Leu Ala Glu Lys Gln 165
170 175 Lys Asp Gln Arg Gln Gln Val Gln
Arg Asp Gln Thr Gln Gln Gln Thr 180 185
190 Ser Ser Ser Ser Thr Ser Phe Met Leu Arg Glu Ala Ala
Pro Thr Thr 195 200 205
Asn Val Ser Ile Phe Pro Val Ala Ala Gly Gly Arg Val Val Glu Gly 210
215 220 Ala Ala Ala Gln
Pro Gln Ala Arg Val Gly Leu Pro Pro Trp Met Leu 225 230
235 240 Ser His Leu Ser Cys
245 72762DNAOryza sativa 72atggggcggg gcaaggtgca gctgaagcgg atcgagaaca
agatcaaccg gcaggtgacc 60ttctccaagc gcaggtcggg gctgctcaag aaggcgaatg
agatctccgt gctctgcgac 120gccgaggtcg cgctcatcat cttctccacc aagggcaagc
tctacgagta cgccaccgac 180tcatgtatgg acaaaatcct tgaacgttat gagcgctact
cctatgcaga aaaggtcctt 240atttcagctg aatctgacac tcagggcaac tggtgccacg
aatataggaa actgaaggct 300aaggttgaga caatacagaa atgtcaaaag cacctcatgg
gagaggatct tgaatctttg 360aatctcaaag agctgcagca gctggagcag cagctggaaa
attcgttgaa acatatcaga 420tccagaaaga gccaactaat gctcgagtcc attaacgagc
ttcaacggaa ggaaaagtca 480ctgcaggagg agaataaggt cctacagaaa gaaaaccctt
gctccttcct acagctggtg 540gagaagcaga aagtccagaa gcaacaagtg caatgggacc
agacacaacc tcaaacaagt 600tcctcatcat cctccttcat gatgagggaa gcccttccaa
caactaatat cagtaactac 660cctgcagcag ctggcgaaag gatagaggat gtagcagcag
ggcagccaca gcatgttcgc 720attgggctgc caccatggat gctgagccac atcaacggct
aa 76273253PRTOryza sativa 73Met Gly Arg Gly Lys
Val Gln Leu Lys Arg Ile Glu Asn Lys Ile Asn 1 5
10 15 Arg Gln Val Thr Phe Ser Lys Arg Arg Ser
Gly Leu Leu Lys Lys Ala 20 25
30 Asn Glu Ile Ser Val Leu Cys Asp Ala Glu Val Ala Leu Ile Ile
Phe 35 40 45 Ser
Thr Lys Gly Lys Leu Tyr Glu Tyr Ala Thr Asp Ser Cys Met Asp 50
55 60 Lys Ile Leu Glu Arg Tyr
Glu Arg Tyr Ser Tyr Ala Glu Lys Val Leu 65 70
75 80 Ile Ser Ala Glu Ser Asp Thr Gln Gly Asn Trp
Cys His Glu Tyr Arg 85 90
95 Lys Leu Lys Ala Lys Val Glu Thr Ile Gln Lys Cys Gln Lys His Leu
100 105 110 Met Gly
Glu Asp Leu Glu Ser Leu Asn Leu Lys Glu Leu Gln Gln Leu 115
120 125 Glu Gln Gln Leu Glu Asn Ser
Leu Lys His Ile Arg Ser Arg Lys Ser 130 135
140 Gln Leu Met Leu Glu Ser Ile Asn Glu Leu Gln Arg
Lys Glu Lys Ser 145 150 155
160 Leu Gln Glu Glu Asn Lys Val Leu Gln Lys Glu Asn Pro Cys Ser Phe
165 170 175 Leu Gln Leu
Val Glu Lys Gln Lys Val Gln Lys Gln Gln Val Gln Trp 180
185 190 Asp Gln Thr Gln Pro Gln Thr Ser
Ser Ser Ser Ser Ser Phe Met Met 195 200
205 Arg Glu Ala Leu Pro Thr Thr Asn Ile Ser Asn Tyr Pro
Ala Ala Ala 210 215 220
Gly Glu Arg Ile Glu Asp Val Ala Ala Gly Gln Pro Gln His Val Arg 225
230 235 240 Ile Gly Leu Pro
Pro Trp Met Leu Ser His Ile Asn Gly 245
250 74741DNAOryza sativa 74atggggcggg gcaaggtgca gctgaagcgg
atcgagaaca agatcaaccg gcaggtgacc 60ttctccaagc gcaggtcaaa actgctcaag
aaggcgaatg agatctccgt gctctgcgac 120gccgaggtcg cgctcatcat cttctccacc
aagggcaagc tctacgagta cgccaccgac 180tcatgtatgg acaaaatcct tgaacgttat
gagcgctact cctatgcaga aaaggtcctt 240atttcagctg aatctgacac tcagggcaac
tggtgccacg aatataggaa actgaaggct 300aaggttgaga caatacagaa atgtcaaaag
cacctcatgg gagaggatct tgaatctttg 360aatctcaaag agctgcagca gctggagcag
cagctggaaa attcgttgaa acatatcaga 420tccagaaaga gccaactaat gctcgagtcc
attaacgagc ttcaacggaa ggaaaagtca 480ctgcaggagg agaataaggt cctacagaaa
gaactggtgg agaagcagaa agtccagaag 540caacaagtgc aatgggacca gacacaacct
caaacaagtt cctcatcatc ctccttcatg 600atgagggaag cccttccaac aactaatatc
agtaactacc ctgcagcagc tggcgaaagg 660atagaggatg tagcagcagg gcagccacag
catgaacgca ttgggctgcc accatggatg 720ctgagccaca tcaacggcta a
74175246PRTOryza sativa 75Met Gly Arg
Gly Lys Val Gln Leu Lys Arg Ile Glu Asn Lys Ile Asn 1 5
10 15 Arg Gln Val Thr Phe Ser Lys Arg
Arg Ser Lys Leu Leu Lys Lys Ala 20 25
30 Asn Glu Ile Ser Val Leu Cys Asp Ala Glu Val Ala Leu
Ile Ile Phe 35 40 45
Ser Thr Lys Gly Lys Leu Tyr Glu Tyr Ala Thr Asp Ser Cys Met Asp 50
55 60 Lys Ile Leu Glu
Arg Tyr Glu Arg Tyr Ser Tyr Ala Glu Lys Val Leu 65 70
75 80 Ile Ser Ala Glu Ser Asp Thr Gln Gly
Asn Trp Cys His Glu Tyr Arg 85 90
95 Lys Leu Lys Ala Lys Val Glu Thr Ile Gln Lys Cys Gln Lys
His Leu 100 105 110
Met Gly Glu Asp Leu Glu Ser Leu Asn Leu Lys Glu Leu Gln Gln Leu
115 120 125 Glu Gln Gln Leu
Glu Asn Ser Leu Lys His Ile Arg Ser Arg Lys Ser 130
135 140 Gln Leu Met Leu Glu Ser Ile Asn
Glu Leu Gln Arg Lys Glu Lys Ser 145 150
155 160 Leu Gln Glu Glu Asn Lys Val Leu Gln Lys Glu Leu
Val Glu Lys Gln 165 170
175 Lys Val Gln Lys Gln Gln Val Gln Trp Asp Gln Thr Gln Pro Gln Thr
180 185 190 Ser Ser Ser
Ser Ser Ser Phe Met Met Arg Glu Ala Leu Pro Thr Thr 195
200 205 Asn Ile Ser Asn Tyr Pro Ala Ala
Ala Gly Glu Arg Ile Glu Asp Val 210 215
220 Ala Ala Gly Gln Pro Gln His Glu Arg Ile Gly Leu Pro
Pro Trp Met 225 230 235
240 Leu Ser His Ile Asn Gly 245 76735DNADendrocalamus
latiflorus 76atggggcgcg ggaaggtgca gctgaagcgg atcgagaaca agatcaaccg
gcaagtgact 60ttctccaagc gccggtcggg gctgctcaag aaggcgcatg agatctccgt
cctctgcgac 120gccgaggtcg gccttatcat cttctccacc aagggcaagc tctacgagta
cgccaccgac 180tcatgtatgg acaaaattct tgaacggtac gagcgttact cctatgcaga
aaaggttctt 240atttcagccg aatctgaaac tcagggcaac tggtgtcacg aatataggaa
actgaaggcg 300aaggttgaga cgatacagaa atgtcaaaag cacctcatgg gagaggatcc
tgaatctttg 360aatctcaagg agctgcagca actcgagcag cagctggaaa gttcagtgaa
acatatcaga 420tccagaaaga gccagcttat gctcgagtcc atttccgagc ttcaaaagaa
ggagaagtca 480ctgcaggagg agaacaaggt tctgcagaag gaactcgtgg agaagcagca
ggtccataaa 540cggttagtgc aatgggacca aactcagccg caaactagtt cctcttcctc
gtccttcatg 600atgagggaag ctctcccaac aacaaatatc agtatttacg ctgcggcagc
cggcgagagg 660gcagaggacg cagcagggca gcctcagatt cacattgggc tgccgccatg
gatggtgagc 720cacatcaacg gctaa
73577244PRTDendrocalamus latiflorus 77Met Gly Arg Gly Lys Val
Gln Leu Lys Arg Ile Glu Asn Lys Ile Asn 1 5
10 15 Arg Gln Val Thr Phe Ser Lys Arg Arg Ser Gly
Leu Leu Lys Lys Ala 20 25
30 His Glu Ile Ser Val Leu Cys Asp Ala Glu Val Gly Leu Ile Ile
Phe 35 40 45 Ser
Thr Lys Gly Lys Leu Tyr Glu Tyr Ala Thr Asp Ser Cys Met Asp 50
55 60 Lys Ile Leu Glu Arg Tyr
Glu Arg Tyr Ser Tyr Ala Glu Lys Val Leu 65 70
75 80 Ile Ser Ala Glu Ser Glu Thr Gln Gly Asn Trp
Cys His Glu Tyr Arg 85 90
95 Lys Leu Lys Ala Lys Val Glu Thr Ile Gln Lys Cys Gln Lys His Leu
100 105 110 Met Gly
Glu Asp Pro Glu Ser Leu Asn Leu Lys Glu Leu Gln Gln Leu 115
120 125 Glu Gln Gln Leu Glu Ser Ser
Val Lys His Ile Arg Ser Arg Lys Ser 130 135
140 Gln Leu Met Leu Glu Ser Ile Ser Glu Leu Gln Lys
Lys Glu Lys Ser 145 150 155
160 Leu Gln Glu Glu Asn Lys Val Leu Gln Lys Glu Leu Val Glu Lys Gln
165 170 175 Gln Val His
Lys Arg Leu Val Gln Trp Asp Gln Thr Gln Pro Gln Thr 180
185 190 Ser Ser Ser Ser Ser Ser Phe Met
Met Arg Glu Ala Leu Pro Thr Thr 195 200
205 Asn Ile Ser Ile Tyr Ala Ala Ala Ala Gly Glu Arg Ala
Glu Asp Ala 210 215 220
Ala Gly Gln Pro Gln Ile His Ile Gly Leu Pro Pro Trp Met Val Ser 225
230 235 240 His Ile Asn Gly
78735DNADendrocalamus latiflorus 78atggggcgcg ggaaggtgca gctgaagcgg
atcgagaaca agatcaaccg gcaagtgact 60ttctccaagc gccggtcggg gctgctcaag
aaggcgcatg agatctccgt cctctgcgac 120gccgaggtcg gccttatcat cttctccacc
aagggcaagc tctacgagta cgccaccgac 180tcatgtatgg acaaaattct tgaacggtac
gagcgttact cctatgcaga aaaggttctt 240atttcagccg aatctgaaac tcagggcaac
tggtgtcacg aatataggaa actgaaggcg 300aaggttgaga cgatacagaa atgtcaaaag
cacctcatgg gagaggatct tgaatctttg 360aatctcaagg agctgcagca actcgagcag
cagctggaaa gttcagtgaa acatatcaga 420tccagaaaga gccagcttat gctcgagtcc
atttccgagc ttcaaaagaa ggagaagtca 480ctgcaggagg agaacaaggt tctgcagaag
gaactcgtgg agaagcagca ggtccataaa 540cggttagtgc aatgggacca aactcagccg
caaactagtt cctcttcctc gtccttcatg 600atgagggaag ctctcccaac aacaaatatc
agtatttacg ctgcggcagc cggcgagagg 660gcagaggacg cagcagggca gcctcagatt
cacattgggc tgccgccatg gatggtgagc 720cacatcaacg gctaa
73579244PRTDendrocalamus latiflorus
79Met Gly Arg Gly Lys Val Gln Leu Lys Arg Ile Glu Asn Lys Ile Asn 1
5 10 15 Arg Gln Val Thr
Phe Ser Lys Arg Arg Ser Gly Leu Leu Lys Lys Ala 20
25 30 His Glu Ile Ser Val Leu Cys Asp Ala
Glu Val Gly Leu Ile Ile Phe 35 40
45 Ser Thr Lys Gly Lys Leu Tyr Glu Tyr Ala Thr Asp Ser Cys
Met Asp 50 55 60
Lys Ile Leu Glu Arg Tyr Glu Arg Tyr Ser Tyr Ala Glu Lys Val Leu 65
70 75 80 Ile Ser Ala Glu Ser
Glu Thr Gln Gly Asn Trp Cys His Glu Tyr Arg 85
90 95 Lys Leu Lys Ala Lys Val Glu Thr Ile Gln
Lys Cys Gln Lys His Leu 100 105
110 Met Gly Glu Asp Leu Glu Ser Leu Asn Leu Lys Glu Leu Gln Gln
Leu 115 120 125 Glu
Gln Gln Leu Glu Ser Ser Val Lys His Ile Arg Ser Arg Lys Ser 130
135 140 Gln Leu Met Leu Glu Ser
Ile Ser Glu Leu Gln Lys Lys Glu Lys Ser 145 150
155 160 Leu Gln Glu Glu Asn Lys Val Leu Gln Lys Glu
Leu Val Glu Lys Gln 165 170
175 Gln Val His Lys Arg Leu Val Gln Trp Asp Gln Thr Gln Pro Gln Thr
180 185 190 Ser Ser
Ser Ser Ser Ser Phe Met Met Arg Glu Ala Leu Pro Thr Thr 195
200 205 Asn Ile Ser Ile Tyr Ala Ala
Ala Ala Gly Glu Arg Ala Glu Asp Ala 210 215
220 Ala Gly Gln Pro Gln Ile His Ile Gly Leu Pro Pro
Trp Met Val Ser 225 230 235
240 His Ile Asn Gly 80813DNAZea mays 80atggggcgcg gcaaggtgca gctgaagcgg
atagagaaca agataaaccg gcaggtgacc 60ttctccaagc gccggaacgg gctgctgaag
aaggcgcacg agatctccgt cctctgcgac 120gccgaggtcg ccgtcatcgt cttctccccc
aagggcaagc tctacgagta cgcctccgac 180tcccgcatgg acaaaattct agaacgttat
gagcgatatt cctatgctga aaaggctctt 240atttcagctg aatctgaaag tgagggaaat
tggtgccacg aatacaggaa actgaaggcc 300aaaattgaga ccatacaaag atgccacaag
cacctgatgg gagaggatct agagtctttg 360aatccaaaag agctccaaca actagagcag
cagctggaga gctcactgaa gcacatcaga 420tcaagaaaga gccaccttat ggccgagtca
atttctgagc tacagaagaa ggagaggtca 480ctgcaggagg agaacaagat tctacagaag
gaactttcag agaggcagaa ggcggtcgct 540agccggcagc agcagcagca gcaggtgcag
tgggaccagc agacacaggt ccaggtccag 600acaagctcat cgtcttcttc cttcatgatg
aggcaggatc agcagggact gccacctcca 660caaaacatct gcttcccgcc gttgagcatc
ggagagagag gcgaagaggt ggctgcggcg 720gcgcagcagc agctgcctcc tccggggcag
gcgcaaccac agctccgcat cgcaggtctg 780ccgccatgga tgctgagcca cctcaatgca
taa 81381270PRTZea mays 81Met Gly Arg Gly
Lys Val Gln Leu Lys Arg Ile Glu Asn Lys Ile Asn 1 5
10 15 Arg Gln Val Thr Phe Ser Lys Arg Arg
Asn Gly Leu Leu Lys Lys Ala 20 25
30 His Glu Ile Ser Val Leu Cys Asp Ala Glu Val Ala Val Ile
Val Phe 35 40 45
Ser Pro Lys Gly Lys Leu Tyr Glu Tyr Ala Ser Asp Ser Arg Met Asp 50
55 60 Lys Ile Leu Glu Arg
Tyr Glu Arg Tyr Ser Tyr Ala Glu Lys Ala Leu 65 70
75 80 Ile Ser Ala Glu Ser Glu Ser Glu Gly Asn
Trp Cys His Glu Tyr Arg 85 90
95 Lys Leu Lys Ala Lys Ile Glu Thr Ile Gln Arg Cys His Lys His
Leu 100 105 110 Met
Gly Glu Asp Leu Glu Ser Leu Asn Pro Lys Glu Leu Gln Gln Leu 115
120 125 Glu Gln Gln Leu Glu Ser
Ser Leu Lys His Ile Arg Ser Arg Lys Ser 130 135
140 His Leu Met Ala Glu Ser Ile Ser Glu Leu Gln
Lys Lys Glu Arg Ser 145 150 155
160 Leu Gln Glu Glu Asn Lys Ile Leu Gln Lys Glu Leu Ser Glu Arg Gln
165 170 175 Lys Ala
Val Ala Ser Arg Gln Gln Gln Gln Gln Gln Val Gln Trp Asp 180
185 190 Gln Gln Thr Gln Val Gln Val
Gln Thr Ser Ser Ser Ser Ser Ser Phe 195 200
205 Met Met Arg Gln Asp Gln Gln Gly Leu Pro Pro Pro
Gln Asn Ile Cys 210 215 220
Phe Pro Pro Leu Ser Ile Gly Glu Arg Gly Glu Glu Val Ala Ala Ala 225
230 235 240 Ala Gln Gln
Gln Leu Pro Pro Pro Gly Gln Ala Gln Pro Gln Leu Arg 245
250 255 Ile Ala Gly Leu Pro Pro Trp Met
Leu Ser His Leu Asn Ala 260 265
270 82689DNASorghum bicolor 82gcaaggtgca gctcaagcgg atagagaaca
agataaaccg gcaggtgacc ttctccaagc 60gccgcaacgg gctgctcaag aaggcgcacg
agatctccgt cctctgcgac gccgaggtcg 120ccgtcatcgt cttctccccc aagggcaagc
tctatgagta cgccaccgac tcccgcatgg 180acaaaattct cgaacgttat gagcgatatt
cctatgctga aaaggctctt atttcagctg 240aatctgaaag tgagggaaac tggtgccacg
aatacaggaa actgaaggcc aaaattgaga 300ccattcaaaa atgccacaag cacctgatgg
gagaggatct agagtctttg aatcccaaag 360agctccaaca actagagcag cagctggaga
gctcactgaa gcacatcaga tcaagaaaga 420gccaccttat ggctgagtct atttctgaac
tacagaagaa ggagaggtca ctgcaggagg 480agaacaaggc tctacagaag gaacttgcgg
agaggcagaa ggcggccgcg agcaggcagc 540agcagcaagg tgcagtggga ccagcagaca
cagacccagg cccagacaag ctcatcatcg 600tcctccttca tgatgaggca ggatcagcag
ggtctgccgc ctccacaaaa catatgcttc 660ccgccgctga taatcggaga gagaggtga
68983228PRTSorghum bicolor 83Lys Val
Gln Leu Lys Arg Ile Glu Asn Lys Ile Asn Arg Gln Val Thr 1 5
10 15 Phe Ser Lys Arg Arg Asn Gly
Leu Leu Lys Lys Ala His Glu Ile Ser 20 25
30 Val Leu Cys Asp Ala Glu Val Ala Val Ile Val Phe
Ser Pro Lys Gly 35 40 45
Lys Leu Tyr Glu Tyr Ala Thr Asp Ser Arg Met Asp Lys Ile Leu Glu
50 55 60 Arg Tyr Glu
Arg Tyr Ser Tyr Ala Glu Lys Ala Leu Ile Ser Ala Glu 65
70 75 80 Ser Glu Ser Glu Gly Asn Trp
Cys His Glu Tyr Arg Lys Leu Lys Ala 85
90 95 Lys Ile Glu Thr Ile Gln Lys Cys His Lys His
Leu Met Gly Glu Asp 100 105
110 Leu Glu Ser Leu Asn Pro Lys Glu Leu Gln Gln Leu Glu Gln Gln
Leu 115 120 125 Glu
Ser Ser Leu Lys His Ile Arg Ser Arg Lys Ser His Leu Met Ala 130
135 140 Glu Ser Ile Ser Glu Leu
Gln Lys Lys Glu Arg Ser Leu Gln Glu Glu 145 150
155 160 Asn Lys Ala Leu Gln Lys Glu Leu Ala Glu Arg
Gln Lys Ala Ala Ala 165 170
175 Ser Arg Gln Gln Gln Gln Gly Ala Val Gly Pro Ala Asp Thr Asp Pro
180 185 190 Gly Pro
Asp Lys Leu Ile Ile Val Leu Leu His Asp Glu Ala Gly Ser 195
200 205 Ala Gly Ser Ala Ala Ser Thr
Lys His Met Leu Pro Ala Ala Asp Asn 210 215
220 Arg Arg Glu Arg 225 84822DNAZea
mays 84atggggcgcg gcaaggtaca gctgaagcgg atagagaaca agataaaccg gcaggtgacc
60ttctccaagc gccggaacgg cctgctcaag aaggcgcacg agatctccgt cctctgcgat
120gccgaggtcg ccgtcatcgt cttctccccc aagggcaagc tctacgagta cgccaccgac
180tcccgcatgg acaaaattct tgaacgctat gagcgatatt cctatgctga aaaggctctt
240atttcagctg aatctgaaag tgagggaaat tggtgccacg aatacaggaa actgaaggcc
300aaaattgaga ccatacaaaa atgccacaag cacctgatgg gagaggatct agagtctttg
360aatcccaaag agctccagca actagagcag cagctggata gctcactgaa gcacatcaga
420tcaaggaaga gccaccttat ggccgagtct atttctgagc tacagaagaa ggagaggtca
480ctgcaggagg agaacaaggc tctgcagaag gaacttgcgg agaggcagaa ggccgtcgcg
540agccggcagc agcagcaaca gcagcaggtg cagtgggacc agcagacaca tgcccaggcc
600cagacaagct catcatcgtc ctccttcatg atgaggcagg atcagcaggg actgccgcct
660ccacacaaca tctgcttccc gccgttgaca atgggagata gaggtgaaga gctggctgcg
720gcggcggcgg cgcagcagca gcagccactg ccggggcagg cgcaaccgca gctccgcatc
780gcaggtctgc caccatggat gctgagccac ctcaatgcat aa
82285273PRTZea mays 85Met Gly Arg Gly Lys Val Gln Leu Lys Arg Ile Glu Asn
Lys Ile Asn 1 5 10 15
Arg Gln Val Thr Phe Ser Lys Arg Arg Asn Gly Leu Leu Lys Lys Ala
20 25 30 His Glu Ile Ser
Val Leu Cys Asp Ala Glu Val Ala Val Ile Val Phe 35
40 45 Ser Pro Lys Gly Lys Leu Tyr Glu Tyr
Ala Thr Asp Ser Arg Met Asp 50 55
60 Lys Ile Leu Glu Arg Tyr Glu Arg Tyr Ser Tyr Ala Glu
Lys Ala Leu 65 70 75
80 Ile Ser Ala Glu Ser Glu Ser Glu Gly Asn Trp Cys His Glu Tyr Arg
85 90 95 Lys Leu Lys Ala
Lys Ile Glu Thr Ile Gln Lys Cys His Lys His Leu 100
105 110 Met Gly Glu Asp Leu Glu Ser Leu Asn
Pro Lys Glu Leu Gln Gln Leu 115 120
125 Glu Gln Gln Leu Asp Ser Ser Leu Lys His Ile Arg Ser Arg
Lys Ser 130 135 140
His Leu Met Ala Glu Ser Ile Ser Glu Leu Gln Lys Lys Glu Arg Ser 145
150 155 160 Leu Gln Glu Glu Asn
Lys Ala Leu Gln Lys Glu Leu Ala Glu Arg Gln 165
170 175 Lys Ala Val Ala Ser Arg Gln Gln Gln Gln
Gln Gln Gln Val Gln Trp 180 185
190 Asp Gln Gln Thr His Ala Gln Ala Gln Thr Ser Ser Ser Ser Ser
Ser 195 200 205 Phe
Met Met Arg Gln Asp Gln Gln Gly Leu Pro Pro Pro His Asn Ile 210
215 220 Cys Phe Pro Pro Leu Thr
Met Gly Asp Arg Gly Glu Glu Leu Ala Ala 225 230
235 240 Ala Ala Ala Ala Gln Gln Gln Gln Pro Leu Pro
Gly Gln Ala Gln Pro 245 250
255 Gln Leu Arg Ile Ala Gly Leu Pro Pro Trp Met Leu Ser His Leu Asn
260 265 270 Ala
86786DNALolium temulentum 86atgggtcgcg gcaaggtgca gctgaagcgg atagagaaca
agataaaccg tcaggtgaca 60ttctccaagc gccgcaacgg gctactcaag aaggcgcacg
agatctccgt cctctgcgac 120gccgaggtcg ccgtcgtcgt cttctccccg aaagggaagc
tctatgagta cgccactgac 180tccagcatgg acaaaattct tgaacgttat gaacgctact
cttatgctga aaaggctttg 240atttcagctg aatctgaaag tgagggaaat tggtgccatg
aatacaggaa gctgaaggcg 300aagattgaga ctatacaaaa atgtcacaag cacctcatgg
gggaggatct ggagtgtcta 360aacctgaaag agctccaaca actagagcag cagctggaga
gttcattgaa gcacatcaga 420tcgagaaaga gccaccttat gatggagtcc atttctgagc
tacagaagaa ggagcggtca 480ctccaggagg agaacaaggc tctacagaag gaactggtgg
agaggcagaa ggcggccagg 540cagcagcagc aagagcagtg ggaccgtcag acccaaacac
aacaagccca aaaccaacct 600caggcccaga cgagctcatc atcttcctcc ttcatgatga
gggatcagca ggcccatgct 660caacaaaaca tctgttaccc gctggtgaca atgggtggag
aggctgtggc cgcggcgcca 720gggcagcagg ggcagcttcg catcggaggc ctgccaccat
ggatgctgag ccacctcaac 780gcttga
78687261PRTLolium temulentum 87Met Gly Arg Gly Lys
Val Gln Leu Lys Arg Ile Glu Asn Lys Ile Asn 1 5
10 15 Arg Gln Val Thr Phe Ser Lys Arg Arg Asn
Gly Leu Leu Lys Lys Ala 20 25
30 His Glu Ile Ser Val Leu Cys Asp Ala Glu Val Ala Val Val Val
Phe 35 40 45 Ser
Pro Lys Gly Lys Leu Tyr Glu Tyr Ala Thr Asp Ser Ser Met Asp 50
55 60 Lys Ile Leu Glu Arg Tyr
Glu Arg Tyr Ser Tyr Ala Glu Lys Ala Leu 65 70
75 80 Ile Ser Ala Glu Ser Glu Ser Glu Gly Asn Trp
Cys His Glu Tyr Arg 85 90
95 Lys Leu Lys Ala Lys Ile Glu Thr Ile Gln Lys Cys His Lys His Leu
100 105 110 Met Gly
Glu Asp Leu Glu Cys Leu Asn Leu Lys Glu Leu Gln Gln Leu 115
120 125 Glu Gln Gln Leu Glu Ser Ser
Leu Lys His Ile Arg Ser Arg Lys Ser 130 135
140 His Leu Met Met Glu Ser Ile Ser Glu Leu Gln Lys
Lys Glu Arg Ser 145 150 155
160 Leu Gln Glu Glu Asn Lys Ala Leu Gln Lys Glu Leu Val Glu Arg Gln
165 170 175 Lys Ala Ala
Arg Gln Gln Gln Gln Glu Gln Trp Asp Arg Gln Thr Gln 180
185 190 Thr Gln Gln Ala Gln Asn Gln Pro
Gln Ala Gln Thr Ser Ser Ser Ser 195 200
205 Ser Ser Phe Met Met Arg Asp Gln Gln Ala His Ala Gln
Gln Asn Ile 210 215 220
Cys Tyr Pro Leu Val Thr Met Gly Gly Glu Ala Val Ala Ala Ala Pro 225
230 235 240 Gly Gln Gln Gly
Gln Leu Arg Ile Gly Gly Leu Pro Pro Trp Met Leu 245
250 255 Ser His Leu Asn Ala 260
88786DNALolium perenne 88atgggtcgcg gcaaggtgca gctgaagcgg atagagaaca
agataaaccg tcaggtgacc 60ttctccaagc gccgcaacgg gctactcaag aaggcgcacg
agatctccgt cctctgcgac 120gccgaggtcg ccgtcgtcgt cttctccccg aaagggaagc
tctatgagta cgccactgac 180tccagcatgg acaaaattct tgaacgttat gaacgctact
cttatgctga aaaggctttg 240atttcagctg aatctgaaag tgagggaaat tggtgccatg
aatacaggaa actgaaggcg 300aagattgaga ctatacaaaa atgtcacaag cacctcatgg
gggaggatct ggagtgtcta 360aacctgaaag agctccaaca actagagcag cagctggaga
gttcattgaa gcacatcaga 420tcgagaaaga gccaccttat gatggagtcc atttctgagc
tacagaagaa ggagcggtca 480ctccaggagg agaacaaggc tctacagaag gaactggtgg
agaggcagaa ggcggccagg 540cagcagcagc aagagcagtg ggaccgtcag acccaaacac
aacaagccca aaaccaacct 600caggcccaga cgagctcatc atcttcctcc ttcatgatga
gggatcagca ggcccatgct 660caacaaaaca tctgttaccc gccggtgaca atgggtggag
aggctgtggc cgcggcgcca 720gggcagcagg ggcagcttcg catcggaggc ctgccaccat
ggatgctgag ccacctcaac 780gcttga
78689261PRTLolium perenne 89Met Gly Arg Gly Lys
Val Gln Leu Lys Arg Ile Glu Asn Lys Ile Asn 1 5
10 15 Arg Gln Val Thr Phe Ser Lys Arg Arg Asn
Gly Leu Leu Lys Lys Ala 20 25
30 His Glu Ile Ser Val Leu Cys Asp Ala Glu Val Ala Val Val Val
Phe 35 40 45 Ser
Pro Lys Gly Lys Leu Tyr Glu Tyr Ala Thr Asp Ser Ser Met Asp 50
55 60 Lys Ile Leu Glu Arg Tyr
Glu Arg Tyr Ser Tyr Ala Glu Lys Ala Leu 65 70
75 80 Ile Ser Ala Glu Ser Glu Ser Glu Gly Asn Trp
Cys His Glu Tyr Arg 85 90
95 Lys Leu Lys Ala Lys Ile Glu Thr Ile Gln Lys Cys His Lys His Leu
100 105 110 Met Gly
Glu Asp Leu Glu Cys Leu Asn Leu Lys Glu Leu Gln Gln Leu 115
120 125 Glu Gln Gln Leu Glu Ser Ser
Leu Lys His Ile Arg Ser Arg Lys Ser 130 135
140 His Leu Met Met Glu Ser Ile Ser Glu Leu Gln Lys
Lys Glu Arg Ser 145 150 155
160 Leu Gln Glu Glu Asn Lys Ala Leu Gln Lys Glu Leu Val Glu Arg Gln
165 170 175 Lys Ala Ala
Arg Gln Gln Gln Gln Glu Gln Trp Asp Arg Gln Thr Gln 180
185 190 Thr Gln Gln Ala Gln Asn Gln Pro
Gln Ala Gln Thr Ser Ser Ser Ser 195 200
205 Ser Ser Phe Met Met Arg Asp Gln Gln Ala His Ala Gln
Gln Asn Ile 210 215 220
Cys Tyr Pro Pro Val Thr Met Gly Gly Glu Ala Val Ala Ala Ala Pro 225
230 235 240 Gly Gln Gln Gly
Gln Leu Arg Ile Gly Gly Leu Pro Pro Trp Met Leu 245
250 255 Ser His Leu Asn Ala 260
90831DNAHordeum vulgare 90atgggtcgcg gtaaggtgca gctgaagcgg atagagaaca
agataaatcg gcaggtgacc 60ttctccaagc gccgcaacgg gctcctgaag aaggcgcacg
agatctccgt cctctgtgac 120gcagaggtcg ccgtcatcgt cttctccccc aaaggcaagc
tctatgagta cgccaccgac 180tccagcatgg acaaaattct tgaacgttat gagcgctact
cttatgctga aaaggctctt 240atttcagctg aatctgaaag tgaggggaat tggtgtcatg
aatacaggaa acttaaggcg 300aagattgaga ccatacagaa gtgtcacaag cacctcatgg
gagaggatct ggattctctg 360aacctcaaag aactccaaca actggagcag cagctggaga
gttcattgaa gcacatcaga 420tcgagaaaga gccatcttat gatggagtcc atttctgagc
tacagaagaa ggagaggtca 480ctgcaggagg agaacaaggc cctacagaag gaactggtgg
agaggcagaa ggcggccagc 540aggcagcagc agctgcagca gcagcaacaa caacaacaaa
tgcaatggga gcaccaagcc 600cagacccaaa cccataccca tactcaaaac cagccccaag
cccagactag ctcatcatct 660tcctctttca tgatgaggga tcagcaggcc catgcccctc
aacagaacat ttgtagctac 720ccaccggtga cgatgggtgg ggaggcgacg gcggcggcgg
cggcgccgga gcagcaggct 780cagcttcgca tatgcctacc gccatggatg ctgagccacc
tcaacgcttg a 83191276PRTHordeum vulgare 91Met Gly Arg Gly Lys
Val Gln Leu Lys Arg Ile Glu Asn Lys Ile Asn 1 5
10 15 Arg Gln Val Thr Phe Ser Lys Arg Arg Asn
Gly Leu Leu Lys Lys Ala 20 25
30 His Glu Ile Ser Val Leu Cys Asp Ala Glu Val Ala Val Ile Val
Phe 35 40 45 Ser
Pro Lys Gly Lys Leu Tyr Glu Tyr Ala Thr Asp Ser Ser Met Asp 50
55 60 Lys Ile Leu Glu Arg Tyr
Glu Arg Tyr Ser Tyr Ala Glu Lys Ala Leu 65 70
75 80 Ile Ser Ala Glu Ser Glu Ser Glu Gly Asn Trp
Cys His Glu Tyr Arg 85 90
95 Lys Leu Lys Ala Lys Ile Glu Thr Ile Gln Lys Cys His Lys His Leu
100 105 110 Met Gly
Glu Asp Leu Asp Ser Leu Asn Leu Lys Glu Leu Gln Gln Leu 115
120 125 Glu Gln Gln Leu Glu Ser Ser
Leu Lys His Ile Arg Ser Arg Lys Ser 130 135
140 His Leu Met Met Glu Ser Ile Ser Glu Leu Gln Lys
Lys Glu Arg Ser 145 150 155
160 Leu Gln Glu Glu Asn Lys Ala Leu Gln Lys Glu Leu Val Glu Arg Gln
165 170 175 Lys Ala Ala
Ser Arg Gln Gln Gln Leu Gln Gln Gln Gln Gln Gln Gln 180
185 190 Gln Met Gln Trp Glu His Gln Ala
Gln Thr Gln Thr His Thr His Thr 195 200
205 Gln Asn Gln Pro Gln Ala Gln Thr Ser Ser Ser Ser Ser
Ser Phe Met 210 215 220
Met Arg Asp Gln Gln Ala His Ala Pro Gln Gln Asn Ile Cys Ser Tyr 225
230 235 240 Pro Pro Val Thr
Met Gly Gly Glu Ala Thr Ala Ala Ala Ala Ala Pro 245
250 255 Glu Gln Gln Ala Gln Leu Arg Ile Cys
Leu Pro Pro Trp Met Leu Ser 260 265
270 His Leu Asn Ala 275 92804DNAOryza sativa
92atggggcggg ggaaggtgca gctgaagcgg atagagaact cgatgaaccg gcaggtgacg
60ttctccaaga ggaggaatgg attgctgaag aaggcgcacg agatctccgt cctctgcgac
120gccgaggtcg ccgccatcgt cttctccccc aagggcaagc tctacgagta cgccactgac
180tccaggatgg acaaaatcct tgaacgttat gagcgctatt catatgctga aaaggctctt
240atttcagctg aatctgagag tgagggaaat tggtgccatg aatacaggaa gcttaaggca
300aagattgaga ccatacaaaa atgtcacaaa cacctcatgg gagaggatct agaatccctg
360aatctcaaag aactccaaca gctagagcag cagctggaga gttcattgaa gcacataaga
420tcaagaaaga gccaccttat gcttgagtcc atttccgagc tgcagaaaaa ggagaggtca
480ctgcaggagg agaacaaggc tctgcagaag gaactggtgg agaggcagaa gaatgtgagg
540ggccagcagc aagtagggca gtgggaccaa acccaggtcc aggcccaggc ccaagcccaa
600ccccaagccc agacaagctt cttcttcttc ttcatgctga gggatcagca ggcacttctt
660tcaccacaaa atatctgcta cccgccggtg atgatgggcc agagaaatga tgcggcggcg
720cggcggcggt ggcggcccaa ggccaggtgc aacttccgca ttggaggctt tccgccatgg
780atgctgagca ccttcaaggc ttaa
80493267PRTOryza sativa 93Met Gly Arg Gly Lys Val Gln Leu Lys Arg Ile Glu
Asn Ser Met Asn 1 5 10
15 Arg Gln Val Thr Phe Ser Lys Arg Arg Asn Gly Leu Leu Lys Lys Ala
20 25 30 His Glu Ile
Ser Val Leu Cys Asp Ala Glu Val Ala Ala Ile Val Phe 35
40 45 Ser Pro Lys Gly Lys Leu Tyr Glu
Tyr Ala Thr Asp Ser Arg Met Asp 50 55
60 Lys Ile Leu Glu Arg Tyr Glu Arg Tyr Ser Tyr Ala Glu
Lys Ala Leu 65 70 75
80 Ile Ser Ala Glu Ser Glu Ser Glu Gly Asn Trp Cys His Glu Tyr Arg
85 90 95 Lys Leu Lys Ala
Lys Ile Glu Thr Ile Gln Lys Cys His Lys His Leu 100
105 110 Met Gly Glu Asp Leu Glu Ser Leu Asn
Leu Lys Glu Leu Gln Gln Leu 115 120
125 Glu Gln Gln Leu Glu Ser Ser Leu Lys His Ile Arg Ser Arg
Lys Ser 130 135 140
His Leu Met Leu Glu Ser Ile Ser Glu Leu Gln Lys Lys Glu Arg Ser 145
150 155 160 Leu Gln Glu Glu Asn
Lys Ala Leu Gln Lys Glu Leu Val Glu Arg Gln 165
170 175 Lys Asn Val Arg Gly Gln Gln Gln Val Gly
Gln Trp Asp Gln Thr Gln 180 185
190 Val Gln Ala Gln Ala Gln Ala Gln Pro Gln Ala Gln Thr Ser Phe
Phe 195 200 205 Phe
Phe Phe Met Leu Arg Asp Gln Gln Ala Leu Leu Ser Pro Gln Asn 210
215 220 Ile Cys Tyr Pro Pro Val
Met Met Gly Gln Arg Asn Asp Ala Ala Ala 225 230
235 240 Arg Arg Arg Trp Arg Pro Lys Ala Arg Cys Asn
Phe Arg Ile Gly Gly 245 250
255 Phe Pro Pro Trp Met Leu Ser Thr Phe Lys Ala 260
265 94804DNAOryza sativa 94atggggcggg ggaaggtgca
gctgaagcgg atagagaaca agatcaacag gcaggtgacg 60ttctccaaga ggaggaatgg
attgctgaag aaggcgcacg agatctccgt cctctgcgac 120gccgaggtcg ccgccatcgt
cttctccccc aagggcaagc tctacgagta cgccactgac 180tccaggatgg acaaaatcct
tgaacgttat gagcgctatt catatgctga aaaggctctt 240atttcagctg aatccgagag
tgagggaaat tggtgccatg aatacaggaa acttaaggca 300aagattgaga ccatacaaaa
atgtcacaaa cacctcatgg gagaggatca tgaatccctg 360aatctcaaag aactccaaca
gctagagcag cagctggaga gttcattgaa gcacataata 420tcaagaaaga gccaccttat
gcttgagtcc atttccgagc tgcagaaaaa ggagaggtca 480ctgcaggagg agaacaaggc
tctgcagaag gaactggtgg agaggcagaa gaatgtgagg 540ggccagcagc aagtagggca
gtgggaccaa acccaggtcc aggcccaggc ccaagcccaa 600ccccaagccc agacaagctc
ctcctcctcc tccatgctga gggatcagca ggcacttctt 660ccaccacaaa atatctgcta
cccgccggtg atgatgggcg agagaaatga tgcggcggcg 720gcggcggcgg tggcggcgca
gggccaggtg caactccgca tcggaggtct tccgccatgg 780atgctgagcc acctcaatgc
ttaa 80495267PRTOryza sativa
95Met Gly Arg Gly Lys Val Gln Leu Lys Arg Ile Glu Asn Lys Ile Asn 1
5 10 15 Arg Gln Val Thr
Phe Ser Lys Arg Arg Asn Gly Leu Leu Lys Lys Ala 20
25 30 His Glu Ile Ser Val Leu Cys Asp Ala
Glu Val Ala Ala Ile Val Phe 35 40
45 Ser Pro Lys Gly Lys Leu Tyr Glu Tyr Ala Thr Asp Ser Arg
Met Asp 50 55 60
Lys Ile Leu Glu Arg Tyr Glu Arg Tyr Ser Tyr Ala Glu Lys Ala Leu 65
70 75 80 Ile Ser Ala Glu Ser
Glu Ser Glu Gly Asn Trp Cys His Glu Tyr Arg 85
90 95 Lys Leu Lys Ala Lys Ile Glu Thr Ile Gln
Lys Cys His Lys His Leu 100 105
110 Met Gly Glu Asp His Glu Ser Leu Asn Leu Lys Glu Leu Gln Gln
Leu 115 120 125 Glu
Gln Gln Leu Glu Ser Ser Leu Lys His Ile Ile Ser Arg Lys Ser 130
135 140 His Leu Met Leu Glu Ser
Ile Ser Glu Leu Gln Lys Lys Glu Arg Ser 145 150
155 160 Leu Gln Glu Glu Asn Lys Ala Leu Gln Lys Glu
Leu Val Glu Arg Gln 165 170
175 Lys Asn Val Arg Gly Gln Gln Gln Val Gly Gln Trp Asp Gln Thr Gln
180 185 190 Val Gln
Ala Gln Ala Gln Ala Gln Pro Gln Ala Gln Thr Ser Ser Ser 195
200 205 Ser Ser Ser Met Leu Arg Asp
Gln Gln Ala Leu Leu Pro Pro Gln Asn 210 215
220 Ile Cys Tyr Pro Pro Val Met Met Gly Glu Arg Asn
Asp Ala Ala Ala 225 230 235
240 Ala Ala Ala Val Ala Ala Gln Gly Gln Val Gln Leu Arg Ile Gly Gly
245 250 255 Leu Pro Pro
Trp Met Leu Ser His Leu Asn Ala 260 265
96668DNATradescantia virginiana 96gggctggtga agaaggctca tgagatctcd
atkytrtgtg atgcygaggt tgcgcttatt 60attttctcta ctaaaggcaa actatacgag
tatgccactg attccaaaat ggaaaatatc 120cttgaacgct atgaacgtta ctcatatgct
gagaaggctt taacttcatc agatcctgaa 180ttacagggaa attggtgcca agagtatgtt
aaacttaagg ctaaggttga ggccttacat 240aaaagccaaa ggcatcttat gggagagcaa
ctagaagcgt tggatctcaa agaattgcag 300caactagagc atcaacttga aggttctttg
aggcttgtca ggtcaagaaa gactcaaatg 360atgttggact ccatttccga acttcagagg
aaggaaaagt ctctggaaga gcaaaacaag 420aacctagaga aggagatttt ggagaagcag
aaagaaaagg ctctggcaca ccaagctcac 480tgggaacagc agaatcagcc actacaaagc
actaattcgc ctccaaggcc cttcgtgatt 540gcagaaactc atccaacact aaacattgga
aatttccaag gtagaacaaa taccgtccat 600gcagaagaaa gtctgcagcg tcagatgagg
atcagcagca gcctactgcc mycmtggatg 660mttcacma
66897222PRTTradescantia
virginianaVARIANT(11)..(11)Xaa can be any naturally occurring amino acid
97Gly Leu Val Lys Lys Ala His Glu Ile Ser Xaa Leu Cys Asp Ala Glu 1
5 10 15 Val Ala Leu Ile
Ile Phe Ser Thr Lys Gly Lys Leu Tyr Glu Tyr Ala 20
25 30 Thr Asp Ser Lys Met Glu Asn Ile Leu
Glu Arg Tyr Glu Arg Tyr Ser 35 40
45 Tyr Ala Glu Lys Ala Leu Thr Ser Ser Asp Pro Glu Leu Gln
Gly Asn 50 55 60
Trp Cys Gln Glu Tyr Val Lys Leu Lys Ala Lys Val Glu Ala Leu His 65
70 75 80 Lys Ser Gln Arg His
Leu Met Gly Glu Gln Leu Glu Ala Leu Asp Leu 85
90 95 Lys Glu Leu Gln Gln Leu Glu His Gln Leu
Glu Gly Ser Leu Arg Leu 100 105
110 Val Arg Ser Arg Lys Thr Gln Met Met Leu Asp Ser Ile Ser Glu
Leu 115 120 125 Gln
Arg Lys Glu Lys Ser Leu Glu Glu Gln Asn Lys Asn Leu Glu Lys 130
135 140 Glu Ile Leu Glu Lys Gln
Lys Glu Lys Ala Leu Ala His Gln Ala His 145 150
155 160 Trp Glu Gln Gln Asn Gln Pro Leu Gln Ser Thr
Asn Ser Pro Pro Arg 165 170
175 Pro Phe Val Ile Ala Glu Thr His Pro Thr Leu Asn Ile Gly Asn Phe
180 185 190 Gln Gly
Arg Thr Asn Thr Val His Ala Glu Glu Ser Leu Gln Arg Gln 195
200 205 Met Arg Ile Ser Ser Ser Leu
Leu Pro Xaa Trp Met Xaa His 210 215
220 98723DNATradescantia virginiana 98gtgcagctga aacggatgga
gaacaagatt aacaggcagg tgacgttttc taaacgtcga 60ggagggctgc tgaagaaagc
tcatgagatc tctattctat gtgatgctga gattgctctt 120attattttct ctactaaagg
gaagctctat gagtatgcca ccaattccaa aatggacaat 180attcttgaac gctatgagcg
ttactcatat gctgaaaagg ctctaacttc atcagatcct 240gatatacagg gaaattggtg
ccaagagtat gctaaactta agtctaaggt tgaggcttta 300tgtaaaagcc aaaggcatct
tatgggagag cagcttgaaa cattgaatct caaagaattg 360cagcaactag agcaacagct
cgaaggttct ctaaagcatg tcaggtcaag aaagactcaa 420gttatgctgg actctatttc
tgaacttcag aggaaggaaa agtcactaga ggagcaaaac 480aagaacctag agaaggagat
tttggagaag cagaaaatca aggctcttgc acagcaggct 540cactgggaac accagaatca
accagcacca aggggttcac ctcctaggcc atttgtgatt 600gcagagtctc atccgacact
aaatattgga catttccaag gcaggacaaa tgcagtcgaa 660gcagaagaaa atcagcagcc
tcakatgaga atttgcagta gcctcctgcc cccctggatg 720ctt
72399241PRTTradescantia
virginianaUNSURE(228)..(228)Xaa can be any naturally occurring amino acid
99Val Gln Leu Lys Arg Met Glu Asn Lys Ile Asn Arg Gln Val Thr Phe 1
5 10 15 Ser Lys Arg Arg
Gly Gly Leu Leu Lys Lys Ala His Glu Ile Ser Ile 20
25 30 Leu Cys Asp Ala Glu Ile Ala Leu Ile
Ile Phe Ser Thr Lys Gly Lys 35 40
45 Leu Tyr Glu Tyr Ala Thr Asn Ser Lys Met Asp Asn Ile Leu
Glu Arg 50 55 60
Tyr Glu Arg Tyr Ser Tyr Ala Glu Lys Ala Leu Thr Ser Ser Asp Pro 65
70 75 80 Asp Ile Gln Gly Asn
Trp Cys Gln Glu Tyr Ala Lys Leu Lys Ser Lys 85
90 95 Val Glu Ala Leu Cys Lys Ser Gln Arg His
Leu Met Gly Glu Gln Leu 100 105
110 Glu Thr Leu Asn Leu Lys Glu Leu Gln Gln Leu Glu Gln Gln Leu
Glu 115 120 125 Gly
Ser Leu Lys His Val Arg Ser Arg Lys Thr Gln Val Met Leu Asp 130
135 140 Ser Ile Ser Glu Leu Gln
Arg Lys Glu Lys Ser Leu Glu Glu Gln Asn 145 150
155 160 Lys Asn Leu Glu Lys Glu Ile Leu Glu Lys Gln
Lys Ile Lys Ala Leu 165 170
175 Ala Gln Gln Ala His Trp Glu His Gln Asn Gln Pro Ala Pro Arg Gly
180 185 190 Ser Pro
Pro Arg Pro Phe Val Ile Ala Glu Ser His Pro Thr Leu Asn 195
200 205 Ile Gly His Phe Gln Gly Arg
Thr Asn Ala Val Glu Ala Glu Glu Asn 210 215
220 Gln Gln Pro Xaa Met Arg Ile Cys Ser Ser Leu Leu
Pro Pro Trp Met 225 230 235
240 Leu 100599DNATradescantia virginiana 100gggctkgtga agaaagctca
tgagatctcg gtactttgtg atgctgagct tgctcttatt 60atcttctctc ccaaaggcaa
gctctatgag tatgccaccg attccaaaat ggaaattatt 120cttgaacgct atgaacgtta
cacctacgct gaaaaagctt taattgcatc agatcctgat 180gtacagggaa actggtgtca
tgagtacatt aagcttaaag ctaaatttga ggccttgaat 240aaaagccaga ggcatcttat
gggagaacaa ctagatacgt tgaaccaaaa ggaattgctg 300caactagaga ctaagcttga
aggttctctg aaaaacgtca ggtcaagaaa gactcaactt 360atgttggatt ccatttctga
gcttcaagaa aagggaaagt cactccagga gcaaaacacc 420tgcctagaaa aggagatttt
gggaaaacag aaagacaagg ctcccaaaca gcatgttcag 480tgggaaaaac agaatcaacc
accacctacc tcttctgcgc caatgccatt cctcattggt 540gatattcacc caacccctaa
tatcagaaat ttccaaggca gaacagtagc tgatgcaga 599101199PRTTradescantia
virginiana 101Gly Leu Val Lys Lys Ala His Glu Ile Ser Val Leu Cys Asp Ala
Glu 1 5 10 15 Leu
Ala Leu Ile Ile Phe Ser Pro Lys Gly Lys Leu Tyr Glu Tyr Ala
20 25 30 Thr Asp Ser Lys Met
Glu Ile Ile Leu Glu Arg Tyr Glu Arg Tyr Thr 35
40 45 Tyr Ala Glu Lys Ala Leu Ile Ala Ser
Asp Pro Asp Val Gln Gly Asn 50 55
60 Trp Cys His Glu Tyr Ile Lys Leu Lys Ala Lys Phe Glu
Ala Leu Asn 65 70 75
80 Lys Ser Gln Arg His Leu Met Gly Glu Gln Leu Asp Thr Leu Asn Gln
85 90 95 Lys Glu Leu Leu
Gln Leu Glu Thr Lys Leu Glu Gly Ser Leu Lys Asn 100
105 110 Val Arg Ser Arg Lys Thr Gln Leu Met
Leu Asp Ser Ile Ser Glu Leu 115 120
125 Gln Glu Lys Gly Lys Ser Leu Gln Glu Gln Asn Thr Cys Leu
Glu Lys 130 135 140
Glu Ile Leu Gly Lys Gln Lys Asp Lys Ala Pro Lys Gln His Val Gln 145
150 155 160 Trp Glu Lys Gln Asn
Gln Pro Pro Pro Thr Ser Ser Ala Pro Met Pro 165
170 175 Phe Leu Ile Gly Asp Ile His Pro Thr Pro
Asn Ile Arg Asn Phe Gln 180 185
190 Gly Arg Thr Val Ala Asp Ala 195
102753DNAElaeis guineensis 102atggggagag ggagggtgca gctgaggcgg atcgagaaca
agataaaccg gcaggtgacg 60ttctcgaagc gccggtcggg gctcctgaag aaagcccacg
agatctccgt cctctgcgac 120gccgaggtcg ccctcatcat cttctcgacc aagggcaagc
tctacgagta cgccaccgac 180tcctgcatgg aaaggattct tgaacgctat gaacgttaca
cctatgcaga aaaagcacta 240atttcatctg gacccgaatt gcagggtaac tggtgccatg
aatttggcaa actcaaagct 300aaggttgagg ctttacaaaa aagccaaagg catctcatgg
gtgagcaact tgagcccttg 360aatctcaaag aactccagca actagagcaa cagcttgaaa
gttctttaaa gcatataaga 420accagaaagt gccaactcat gtttgaatcc atctctgagc
ttcaaaaaaa ggaaaagtca 480ctgcaggagc agaacaagat gctggagaag gagctcatgg
agaagcagaa ggtgaaggca 540ctaaaccagc aggcaccttg ggagcagcaa ggcccgccgc
agacaagctc atcatcccca 600acctccttcc tgatcggaga ctctctcccc accctgaata
ttgggacata ccaatgtagc 660ggaaatgaac atggggagga agcagcacaa ccccaggttc
gtataggaaa cagcctgtta 720ccaccttgga tgcttagcca cttgaacggg tag
753103250PRTElaeis guineensis 103Met Gly Arg Gly
Arg Val Gln Leu Arg Arg Ile Glu Asn Lys Ile Asn 1 5
10 15 Arg Gln Val Thr Phe Ser Lys Arg Arg
Ser Gly Leu Leu Lys Lys Ala 20 25
30 His Glu Ile Ser Val Leu Cys Asp Ala Glu Val Ala Leu Ile
Ile Phe 35 40 45
Ser Thr Lys Gly Lys Leu Tyr Glu Tyr Ala Thr Asp Ser Cys Met Glu 50
55 60 Arg Ile Leu Glu Arg
Tyr Glu Arg Tyr Thr Tyr Ala Glu Lys Ala Leu 65 70
75 80 Ile Ser Ser Gly Pro Glu Leu Gln Gly Asn
Trp Cys His Glu Phe Gly 85 90
95 Lys Leu Lys Ala Lys Val Glu Ala Leu Gln Lys Ser Gln Arg His
Leu 100 105 110 Met
Gly Glu Gln Leu Glu Pro Leu Asn Leu Lys Glu Leu Gln Gln Leu 115
120 125 Glu Gln Gln Leu Glu Ser
Ser Leu Lys His Ile Arg Thr Arg Lys Cys 130 135
140 Gln Leu Met Phe Glu Ser Ile Ser Glu Leu Gln
Lys Lys Glu Lys Ser 145 150 155
160 Leu Gln Glu Gln Asn Lys Met Leu Glu Lys Glu Leu Met Glu Lys Gln
165 170 175 Lys Val
Lys Ala Leu Asn Gln Gln Ala Pro Trp Glu Gln Gln Gly Pro 180
185 190 Pro Gln Thr Ser Ser Ser Ser
Pro Thr Ser Phe Leu Ile Gly Asp Ser 195 200
205 Leu Pro Thr Leu Asn Ile Gly Thr Tyr Gln Cys Ser
Gly Asn Glu His 210 215 220
Gly Glu Glu Ala Ala Gln Pro Gln Val Arg Ile Gly Asn Ser Leu Leu 225
230 235 240 Pro Pro Trp
Met Leu Ser His Leu Asn Gly 245 250
104699DNAAllium sp. 104gtgcaattga agaggatgga aaacaagatt aatagacaag
tgaccttctc aaaaagaaga 60aatggtttgt tgaagaaagc tcatgagatt tcwgtgcttt
gtgatgcaga agttgcactt 120attgttttct ctgctaaagg aaaactctat gaatattcaa
ctgattcaag tatggaaaaa 180attctggaga ggtatgaacg ttattgcttt gcggagaaat
catcaacaat gagtgacatt 240gactcccagg aggattggag ccttgaatat cacaaactga
aggctaaggt tgagagttta 300aacaacaggc aaaggcatct tatgggagag caacttgaat
ctctgagtct tcgagaaatt 360ggacagcttg agcaacaact tgagaattct ctcaaaactg
ttcggacgcg caagagccaa 420gaattgttaa gttctatttc agagcttcag gacaaggaga
aaactttgcg agatgagaac 480aaagctttag aaaatgagct tatgaaaagg gccagggcaa
aagctattct ggaacaacaa 540gcacgatgga agcatcataa tcataaacaa caggataatc
ttcataatcc aaatatcaac 600attggaaatt accaaacaag gaacaatgag ggaggagttg
agccagcaac ggatgttcaa 660gtacgtgttg ttagaaattt gttgccccac tggatgctt
699105233PRTAllium sp. 105Val Gln Leu Lys Arg Met
Glu Asn Lys Ile Asn Arg Gln Val Thr Phe 1 5
10 15 Ser Lys Arg Arg Asn Gly Leu Leu Lys Lys Ala
His Glu Ile Ser Val 20 25
30 Leu Cys Asp Ala Glu Val Ala Leu Ile Val Phe Ser Ala Lys Gly
Lys 35 40 45 Leu
Tyr Glu Tyr Ser Thr Asp Ser Ser Met Glu Lys Ile Leu Glu Arg 50
55 60 Tyr Glu Arg Tyr Cys Phe
Ala Glu Lys Ser Ser Thr Met Ser Asp Ile 65 70
75 80 Asp Ser Gln Glu Asp Trp Ser Leu Glu Tyr His
Lys Leu Lys Ala Lys 85 90
95 Val Glu Ser Leu Asn Asn Arg Gln Arg His Leu Met Gly Glu Gln Leu
100 105 110 Glu Ser
Leu Ser Leu Arg Glu Ile Gly Gln Leu Glu Gln Gln Leu Glu 115
120 125 Asn Ser Leu Lys Thr Val Arg
Thr Arg Lys Ser Gln Glu Leu Leu Ser 130 135
140 Ser Ile Ser Glu Leu Gln Asp Lys Glu Lys Thr Leu
Arg Asp Glu Asn 145 150 155
160 Lys Ala Leu Glu Asn Glu Leu Met Lys Arg Ala Arg Ala Lys Ala Ile
165 170 175 Leu Glu Gln
Gln Ala Arg Trp Lys His His Asn His Lys Gln Gln Asp 180
185 190 Asn Leu His Asn Pro Asn Ile Asn
Ile Gly Asn Tyr Gln Thr Arg Asn 195 200
205 Asn Glu Gly Gly Val Glu Pro Ala Thr Asp Val Gln Val
Arg Val Val 210 215 220
Arg Asn Leu Leu Pro His Trp Met Leu 225 230
106744DNADendrobium grex Madame Thong-IN 106atgggtcgtg gcagggtgca
gctgaagcga atcgagaata aaataaaccg gcaggtgacg 60ttctcgaagc ggagatctgg
tttgcttaag aaggcgcacg agatctccgt gctctgtgac 120gctgaagttg ctctgatcgt
tttttccaat aagggaaagc tttatgagta ttccaccgat 180tccagcatgg agaaaattct
tgaacggtat gagcgttatt catatgctga aagagcatta 240ttttccaatg aggccaaccc
ccaggctgat tggcgccttg aatataataa actgaaggca 300agggttgaaa gcttacagaa
gagccaaagg caccttatgg gggagcaact tgactccttg 360agcattaaag aactccaacg
tctagagcaa cagcttgaaa gttccttgaa gtttatacga 420tccagaaaga cacagctcat
actacattca atttccgagc tacaaaagat ggaaaaaata 480ttgctggagc aaaacaagac
cttagagaag gagattatag ctaaagagaa agccaaagct 540ttggtgcagc atgccccatg
ggagaagcaa aaccagtccc aatatagctc tgcactcccg 600cctgtgattt cggattctgt
cccaactccc accagcagaa cgtttcaagc cagagccaat 660gaagaagaat cacctcagcc
acagttaaga gtaagcaaca ctctgctgcc cccatggatg 720ctcagtcata tgaatggaca
ataa 744107247PRTDendrobium
grex Madame Thong-IN 107Met Gly Arg Gly Arg Val Gln Leu Lys Arg Ile Glu
Asn Lys Ile Asn 1 5 10
15 Arg Gln Val Thr Phe Ser Lys Arg Arg Ser Gly Leu Leu Lys Lys Ala
20 25 30 His Glu Ile
Ser Val Leu Cys Asp Ala Glu Val Ala Leu Ile Val Phe 35
40 45 Ser Asn Lys Gly Lys Leu Tyr Glu
Tyr Ser Thr Asp Ser Ser Met Glu 50 55
60 Lys Ile Leu Glu Arg Tyr Glu Arg Tyr Ser Tyr Ala Glu
Arg Ala Leu 65 70 75
80 Phe Ser Asn Glu Ala Asn Pro Gln Ala Asp Trp Arg Leu Glu Tyr Asn
85 90 95 Lys Leu Lys Ala
Arg Val Glu Ser Leu Gln Lys Ser Gln Arg His Leu 100
105 110 Met Gly Glu Gln Leu Asp Ser Leu Ser
Ile Lys Glu Leu Gln Arg Leu 115 120
125 Glu Gln Gln Leu Glu Ser Ser Leu Lys Phe Ile Arg Ser Arg
Lys Thr 130 135 140
Gln Leu Ile Leu His Ser Ile Ser Glu Leu Gln Lys Met Glu Lys Ile 145
150 155 160 Leu Leu Glu Gln Asn
Lys Thr Leu Glu Lys Glu Ile Ile Ala Lys Glu 165
170 175 Lys Ala Lys Ala Leu Val Gln His Ala Pro
Trp Glu Lys Gln Asn Gln 180 185
190 Ser Gln Tyr Ser Ser Ala Leu Pro Pro Val Ile Ser Asp Ser Val
Pro 195 200 205 Thr
Pro Thr Ser Arg Thr Phe Gln Ala Arg Ala Asn Glu Glu Glu Ser 210
215 220 Pro Gln Pro Gln Leu Arg
Val Ser Asn Thr Leu Leu Pro Pro Trp Met 225 230
235 240 Leu Ser His Met Asn Gly Gln
245 1082194DNAOryza sativa 108aatccgaaaa gtttctgcac cgttttcacc
ccctaactaa caatataggg aacgtgtgct 60aaatataaaa tgagacctta tatatgtagc
gctgataact agaactatgc aagaaaaact 120catccaccta ctttagtggc aatcgggcta
aataaaaaag agtcgctaca ctagtttcgt 180tttccttagt aattaagtgg gaaaatgaaa
tcattattgc ttagaatata cgttcacatc 240tctgtcatga agttaaatta ttcgaggtag
ccataattgt catcaaactc ttcttgaata 300aaaaaatctt tctagctgaa ctcaatgggt
aaagagagag atttttttta aaaaaataga 360atgaagatat tctgaacgta ttggcaaaga
tttaaacata taattatata attttatagt 420ttgtgcattc gtcatatcgc acatcattaa
ggacatgtct tactccatcc caatttttat 480ttagtaatta aagacaattg acttattttt
attatttatc ttttttcgat tagatgcaag 540gtacttacgc acacactttg tgctcatgtg
catgtgtgag tgcacctcct caatacacgt 600tcaactagca acacatctct aatatcactc
gcctatttaa tacatttagg tagcaatatc 660tgaattcaag cactccacca tcaccagacc
acttttaata atatctaaaa tacaaaaaat 720aattttacag aatagcatga aaagtatgaa
acgaactatt taggtttttc acatacaaaa 780aaaaaaagaa ttttgctcgt gcgcgagcgc
caatctccca tattgggcac acaggcaaca 840acagagtggc tgcccacaga acaacccaca
aaaaacgatg atctaacgga ggacagcaag 900tccgcaacaa ccttttaaca gcaggctttg
cggccaggag agaggaggag aggcaaagaa 960aaccaagcat cctccttctc ccatctataa
attcctcccc ccttttcccc tctctatata 1020ggaggcatcc aagccaagaa gagggagagc
accaaggaca cgcgactagc agaagccgag 1080cgaccgcctt ctcgatccat atcttccggt
cgagttcttg gtcgatctct tccctcctcc 1140acctcctcct cacagggtat gtgcctccct
tcggttgttc ttggatttat tgttctaggt 1200tgtgtagtac gggcgttgat gttaggaaag
gggatctgta tctgtgatga ttcctgttct 1260tggatttggg atagaggggt tcttgatgtt
gcatgttatc ggttcggttt gattagtagt 1320atggttttca atcgtctgga gagctctatg
gaaatgaaat ggtttaggga tcggaatctt 1380gcgattttgt gagtaccttt tgtttgaggt
aaaatcagag caccggtgat tttgcttggt 1440gtaataaagt acggttgttt ggtcctcgat
tctggtagtg atgcttctcg atttgacgaa 1500gctatccttt gtttattccc tattgaacaa
aaataatcca actttgaaga cggtcccgtt 1560gatgagattg aatgattgat tcttaagcct
gtccaaaatt tcgcagctgg cttgtttaga 1620tacagtagtc cccatcacga aattcatgga
aacagttata atcctcagga acaggggatt 1680ccctgttctt ccgatttgct ttagtcccag
aatttttttt cccaaatatc ttaaaaagtc 1740actttctggt tcagttcaat gaattgattg
ctacaaataa tgcttttata gcgttatcct 1800agctgtagtt cagttaatag gtaatacccc
tatagtttag tcaggagaag aacttatccg 1860atttctgatc tccattttta attatatgaa
atgaactgta gcataagcag tattcatttg 1920gattattttt tttattagct ctcacccctt
cattattctg agctgaaagt ctggcatgaa 1980ctgtcctcaa ttttgttttc aaattcacat
cgattatcta tgcattatcc tcttgtatct 2040acctgtagaa gtttcttttt ggttattcct
tgactgcttg attacagaaa gaaatttatg 2100aagctgtaat cgggatagtt atactgcttg
ttcttatgat tcatttcctt tgtgcagttc 2160ttggtgtagc ttgccacttt caccagcaaa
gttc 2194109804DNAOryza sativa
109atggggcggg ggaaggtgca gctgaagcgg atagagaaca agatcaacag gcaggtgacg
60ttctccaaga ggaggaatgg attgctgaag aaggcgcacg agatctccgt cctctgcgac
120gccgaggtcg ccgccatcgt cttctccccc aagggcaagc tctacgagta cgccactgac
180tccaggatgg acaaaatcct tgaacgttat gagcgctatt catatgctga aaaggctctt
240atttcagctg aatccgagag tgagggaaat tggtgccatg aatacaggaa acttaaggca
300aagattgaga ccatacaaaa atgtcacaaa cacctcatgg gagaggatct agaatccctg
360aatctcaaag aactccaaca gctagagcag cagctggaga gttcattgaa gcacataata
420tcaagaaaga gccaccttat gcttgagtcc atttccgagc tgcagaaaaa ggagaggtca
480ctgcaggagg agaacaaggc tctgcagaag gaactggtgg agaggcagaa gaatgtgagg
540ggccagcagc aagtagggca gtgggaccaa acccaggtcc aggcccaggc ccaagcccaa
600ccccaagccc agacaagctc ctcctcctcc tccatgctga gggatcagca ggcacttctt
660ccaccacaaa atatctgcta cccgccggtg atgatgggcg agagaaatga tgcggcggcg
720gcggcggcgg tggcggcgca gggccaggtg caactccgca tcggaggtct tccgccatgg
780atgctgagcc acctcaatgc ttaa
804110267PRTOryza sativa 110Met Gly Arg Gly Lys Val Gln Leu Lys Arg Ile
Glu Asn Lys Ile Asn 1 5 10
15 Arg Gln Val Thr Phe Ser Lys Arg Arg Asn Gly Leu Leu Lys Lys Ala
20 25 30 His Glu
Ile Ser Val Leu Cys Asp Ala Glu Val Ala Ala Ile Val Phe 35
40 45 Ser Pro Lys Gly Lys Leu Tyr
Glu Tyr Ala Thr Asp Ser Arg Met Asp 50 55
60 Lys Ile Leu Glu Arg Tyr Glu Arg Tyr Ser Tyr Ala
Glu Lys Ala Leu 65 70 75
80 Ile Ser Ala Glu Ser Glu Ser Glu Gly Asn Trp Cys His Glu Tyr Arg
85 90 95 Lys Leu Lys
Ala Lys Ile Glu Thr Ile Gln Lys Cys His Lys His Leu 100
105 110 Met Gly Glu Asp Leu Glu Ser Leu
Asn Leu Lys Glu Leu Gln Gln Leu 115 120
125 Glu Gln Gln Leu Glu Ser Ser Leu Lys His Ile Ile Ser
Arg Lys Ser 130 135 140
His Leu Met Leu Glu Ser Ile Ser Glu Leu Gln Lys Lys Glu Arg Ser 145
150 155 160 Leu Gln Glu Glu
Asn Lys Ala Leu Gln Lys Glu Leu Val Glu Arg Gln 165
170 175 Lys Asn Val Arg Gly Gln Gln Gln Val
Gly Gln Trp Asp Gln Thr Gln 180 185
190 Val Gln Ala Gln Ala Gln Ala Gln Pro Gln Ala Gln Thr Ser
Ser Ser 195 200 205
Ser Ser Ser Met Leu Arg Asp Gln Gln Ala Leu Leu Pro Pro Gln Asn 210
215 220 Ile Cys Tyr Pro Pro
Val Met Met Gly Glu Arg Asn Asp Ala Ala Ala 225 230
235 240 Ala Ala Ala Val Ala Ala Gln Gly Gln Val
Gln Leu Arg Ile Gly Gly 245 250
255 Leu Pro Pro Trp Met Leu Ser His Leu Asn Ala 260
265 11152DNAArtificial sequenceprimer
111ggggacaagt ttgtacaaaa aagcaggctt aaacaatggg gcgggggaag gt
5211247DNAArtificial sequenceprimer 112ggggaccact ttgtacaaga aagctgggtt
tggccgacga cgacgac 471132193DNAOryza sativa
113aatccgaaaa gtttctgcac cgttttcacc ccctaactaa caatataggg aacgtgtgct
60aaatataaaa tgagacctta tatatgtagc gctgataact agaactatgc aagaaaaact
120catccaccta ctttagtggc aatcgggcta aataaaaaag agtcgctaca ctagtttcgt
180tttccttagt aattaagtgg gaaaatgaaa tcattattgc ttagaatata cgttcacatc
240tctgtcatga agttaaatta ttcgaggtag ccataattgt catcaaactc ttcttgaata
300aaaaaatctt tctagctgaa ctcaatgggt aaagagagag atttttttta aaaaaataga
360atgaagatat tctgaacgta ttggcaaaga tttaaacata taattatata attttatagt
420ttgtgcattc gtcatatcgc acatcattaa ggacatgtct tactccatcc caatttttat
480ttagtaatta aagacaattg acttattttt attatttatc ttttttcgat tagatgcaag
540gtacttacgc acacactttg tgctcatgtg catgtgtgag tgcacctcct caatacacgt
600tcaactagca acacatctct aatatcactc gcctatttaa tacatttagg tagcaatatc
660tgaattcaag cactccacca tcaccagacc acttttaata atatctaaaa tacaaaaaat
720aattttacag aatagcatga aaagtatgaa acgaactatt taggtttttc acatacaaaa
780aaaaaaagaa ttttgctcgt gcgcgagcgc caatctccca tattgggcac acaggcaaca
840acagagtggc tgcccacaga acaacccaca aaaaacgatg atctaacgga ggacagcaag
900tccgcaacaa ccttttaaca gcaggctttg cggccaggag agaggaggag aggcaaagaa
960aaccaagcat cctcctcctc ccatctataa attcctcccc ccttttcccc tctctatata
1020ggaggcatcc aagccaagaa gagggagagc accaaggaca cgcgactagc agaagccgag
1080cgaccgcctt cttcgatcca tatcttccgg tcgagttctt ggtcgatctc ttccctcctc
1140cacctcctcc tcacagggta tgtgcccttc ggttgttctt ggatttattg ttctaggttg
1200tgtagtacgg gcgttgatgt taggaaaggg gatctgtatc tgtgatgatt cctgttcttg
1260gatttgggat agaggggttc ttgatgttgc atgttatcgg ttcggtttga ttagtagtat
1320ggttttcaat cgtctggaga gctctatgga aatgaaatgg tttagggtac ggaatcttgc
1380gattttgtga gtaccttttg tttgaggtaa aatcagagca ccggtgattt tgcttggtgt
1440aataaaagta cggttgtttg gtcctcgatt ctggtagtga tgcttctcga tttgacgaag
1500ctatcctttg tttattccct attgaacaaa aataatccaa ctttgaagac ggtcccgttg
1560atgagattga atgattgatt cttaagcctg tccaaaattt cgcagctggc ttgtttagat
1620acagtagtcc ccatcacgaa attcatggaa acagttataa tcctcaggaa caggggattc
1680cctgttcttc cgatttgctt tagtcccaga attttttttc ccaaatatct taaaaagtca
1740ctttctggtt cagttcaatg aattgattgc tacaaataat gcttttatag cgttatccta
1800gctgtagttc agttaatagg taatacccct atagtttagt caggagaaga acttatccga
1860tttctgatct ccatttttaa ttatatgaaa tgaactgtag cataagcagt attcatttgg
1920attatttttt ttattagctc tcaccccttc attattctga gctgaaagtc tggcatgaac
1980tgtcctcaat tttgttttca aattcacatc gattatctat gcattatcct cttgtatcta
2040cctgtagaag tttctttttg gttattcctt gactgcttga ttacagaaag aaatttatga
2100agctgtaatc gggatagtta tactgcttgt tcttatgatt catttccttt gtgcagttct
2160tggtgtagct tgccactttc accagcaaag ttc
219311442PRTArtificial sequencemotif 114Leu Xaa Lys Lys Ala Xaa Glu Ile
Ser Xaa Leu Xaa Asp Ala Glu Xaa 1 5 10
15 Xaa Xaa Xaa Xaa Phe Ser Xaa Lys Gly Lys Leu Tyr Glu
Xaa Xaa Xaa 20 25 30
Xaa Ser Xaa Met Xaa Xaa Ile Leu Xaa Arg 35 40
11519PRTArtificial sequencemotif 115Lys Leu Lys Xaa Xaa Xaa Glu
Xaa Xaa Xaa Xaa Xaa Xaa Xaa His Leu 1 5
10 15 Met Gly Glu 11611PRTArtificial sequencemotif
116Gln Xaa Gln Thr Ser Xaa Xaa Xaa Xaa Xaa Xaa 1 5
10 1177PRTArtificial sequencemotif 117Xaa Xaa Xaa Trp Met
Xaa Xaa1 5 118735DNAHordeum vulgare 118atggggcgcg
ggaaggtgca gctgaagcgg atcgagaaca agatcaaccg ccaggtcacc 60ttctccaagc
gccgctcggg gctgctcaag aaggcgcacg agatctccgt gctctgcgac 120gccgaggtcg
gcctcatcat cttctccacc aagggaaagc tctacgagtt ctccaccgag 180tcatgtatgg
acaaaattct tgaacggtat gagcgctact cttatgcaga aaaggttctc 240gtttcaagtg
aatctgaaat tcagggaaac tggtgtcacg aatataggaa actgaaggcg 300aaggttgaga
caatacagaa atgtcaaaag catctcatgg gagaggatct tgaatctttg 360aatctcaagg
agttgcagca actggagcag cagctggaaa gctcactgaa acatatcaga 420gccaggaaga
accaacttat gcacgaatcc atttctgagc ttcagaagaa ggagaggtca 480ctgcaggagg
agaataaagt tctccagaag gaacttgtgg agaagcagaa ggcccaggcg 540gcgcagcaag
atcaaactca gcctcaaacc agctcttctt cttcttcctt catgatgagg 600gatgctcccc
ctgtcgcaga taccagcaat cacccagcgg cggcaggcga gagggcagag 660gatgtggcag
tgcagcctca ggtcccactc cggacggcgc ttccactgtg gatggtgagc 720cacatcaacg
gctga
735119244PRTHordeum vulgare 119Met Gly Arg Gly Lys Val Gln Leu Lys Arg
Ile Glu Asn Lys Ile Asn 1 5 10
15 Arg Gln Val Thr Phe Ser Lys Arg Arg Ser Gly Leu Leu Lys Lys
Ala 20 25 30 His
Glu Ile Ser Val Leu Cys Asp Ala Glu Val Gly Leu Ile Ile Phe 35
40 45 Ser Thr Lys Gly Lys Leu
Tyr Glu Phe Ser Thr Glu Ser Cys Met Asp 50 55
60 Lys Ile Leu Glu Arg Tyr Glu Arg Tyr Ser Tyr
Ala Glu Lys Val Leu 65 70 75
80 Val Ser Ser Glu Ser Glu Ile Gln Gly Asn Trp Cys His Glu Tyr Arg
85 90 95 Lys Leu
Lys Ala Lys Val Glu Thr Ile Gln Lys Cys Gln Lys His Leu 100
105 110 Met Gly Glu Asp Leu Glu Ser
Leu Asn Leu Lys Glu Leu Gln Gln Leu 115 120
125 Glu Gln Gln Leu Glu Ser Ser Leu Lys His Ile Arg
Ala Arg Lys Asn 130 135 140
Gln Leu Met His Glu Ser Ile Ser Glu Leu Gln Lys Lys Glu Arg Ser 145
150 155 160 Leu Gln Glu
Glu Asn Lys Val Leu Gln Lys Glu Leu Val Glu Lys Gln 165
170 175 Lys Ala Gln Ala Ala Gln Gln Asp
Gln Thr Gln Pro Gln Thr Ser Ser 180 185
190 Ser Ser Ser Ser Phe Met Met Arg Asp Ala Pro Pro Val
Ala Asp Thr 195 200 205
Ser Asn His Pro Ala Ala Ala Gly Glu Arg Ala Glu Asp Val Ala Val 210
215 220 Gln Pro Gln Val
Pro Leu Arg Thr Ala Leu Pro Leu Trp Met Val Ser 225 230
235 240 His Ile Asn Gly 1201196DNAHordeum
vulgare 120ctctcccctc ccacttcacc caaccacctg acagccatgg ctccgccacc
tcgcctccgc 60ccgcgcctct gagagtagcc gtcgcggtcg ctcgctcgct cgctcgctgc
tgccggtgtt 120ggcccggtcc tcgagcggag atggggcgca ggaaggtgca gctgaagcgg
atcgagaaca 180agatcaaccg ccaggtcacc ttctccaagc gccgctcggg gctgctcaag
aaggcgcacg 240agatctccgt gctctacgac gccgaggtcg gcctcatcat cttctccacc
aagggaaagc 300tctacgagtt ctccaccgag tcatgtatgg acaaaattct tgaacggtat
gagcgctact 360cttatgcaga aaaggttctc gtttcaagtg aatctgaaat tcagggaaac
tggtgtcacg 420aatataggaa actgaaggcg aaggttgaga caatacagaa atgtcaaaag
catctcatgg 480gagaggatct tgaatctttg aatctcaagg agttgcagca actggagcag
cagctggaaa 540gctcactgaa acatatcaga gccaggaaga accaacttat gcacgaatcc
atttctgagc 600ttcagaagaa ggagaggtca ctgcaggagg agaataaagt tctccagaag
gaacttgtgg 660agaagcagaa ggcccaggcg gcgcagcaag atcaaactca gcctcaaacc
agctcttctt 720cttcttcctt catgatgagg gatgctcccc ctgtcgcaga taccagcaat
cacccagcgg 780cggcaggcga gagggcagag gatgtggcag tgcagcctca ggtcccactc
cggacggcgc 840ttccactgtg gatggtgagc cacatcaacg gctgaagggc ttccagccca
tgtaagcgta 900ctattcagta cgagtaacaa gttgcagcgg ccagcctggt gtatcatgcg
gttgcgaaca 960tgctaacccc atggagggga gaggaaaaga aatcagagta aagcagcaag
ctgcaggaat 1020gtgtatattt cacttcgtcc acctcagttt cctttccacc tgggctgaga
tggctgtacg 1080agtaatctac catgtaattt atatgtagca tgagtgacga attttcaact
ttcgatgata 1140tccgttgctc ctgggtgttg tttctgtgaa ttaacctatc gaatatgagc
gttgtg 1196121244PRTHordeum vulgare 121Met Gly Arg Arg Lys Val Gln
Leu Lys Arg Ile Glu Asn Lys Ile Asn 1 5
10 15 Arg Gln Val Thr Phe Ser Lys Arg Arg Ser Gly
Leu Leu Lys Lys Ala 20 25
30 His Glu Ile Ser Val Leu Tyr Asp Ala Glu Val Gly Leu Ile Ile
Phe 35 40 45 Ser
Thr Lys Gly Lys Leu Tyr Glu Phe Ser Thr Glu Ser Cys Met Asp 50
55 60 Lys Ile Leu Glu Arg Tyr
Glu Arg Tyr Ser Tyr Ala Glu Lys Val Leu 65 70
75 80 Val Ser Ser Glu Ser Glu Ile Gln Gly Asn Trp
Cys His Glu Tyr Arg 85 90
95 Lys Leu Lys Ala Lys Val Glu Thr Ile Gln Lys Cys Gln Lys His Leu
100 105 110 Met Gly
Glu Asp Leu Glu Ser Leu Asn Leu Lys Glu Leu Gln Gln Leu 115
120 125 Glu Gln Gln Leu Glu Ser Ser
Leu Lys His Ile Arg Ala Arg Lys Asn 130 135
140 Gln Leu Met His Glu Ser Ile Ser Glu Leu Gln Lys
Lys Glu Arg Ser 145 150 155
160 Leu Gln Glu Glu Asn Lys Val Leu Gln Lys Glu Leu Val Glu Lys Gln
165 170 175 Lys Ala Gln
Ala Ala Gln Gln Asp Gln Thr Gln Pro Gln Thr Ser Ser 180
185 190 Ser Ser Ser Ser Phe Met Met Arg
Asp Ala Pro Pro Val Ala Asp Thr 195 200
205 Ser Asn His Pro Ala Ala Ala Gly Glu Arg Ala Glu Asp
Val Ala Val 210 215 220
Gln Pro Gln Val Pro Leu Arg Thr Ala Leu Pro Leu Trp Met Val Ser 225
230 235 240 His Ile Asn Gly
1221161DNATriticum aestivum 122ggcacgagct ctctctctct ctctctctct
ctctctctct ctctctcctc gtgccgaatt 60cggcacgagc ggagatgggg cgcgggaagg
tgcagctgaa gcggatcgag aacaagatca 120accggcaggt gaccttctcc aagcgccgct
cggggctgct caagaaggcg cacgagatct 180ccgtgctctg cgacgccgag gtcggcctca
tcatcttctc caccaaggga aagctctacg 240agttctccac cgagtcatgt atggacaaaa
ttcttgaacg gtatgagcgc tactcttatg 300cagaaaaggt tctcgtttca agtgaatctg
aaattcaggg aaactggtgt cacgaatata 360ggaaactgaa ggcgaaggtt gagacaatac
agaaatgtca aaagcatctg atgggagagg 420atcttgaatc tttgaatctc aaggagttgc
agcaactgga gcagcagctg gaaagctcac 480tgaaacatat cagatccagg aagaaccaac
ttatgcacga atccatttct gagcttcaga 540agaaggagag gtcactgcag gaggagaata
aagttctcca gaaggaactc gtcgagaagc 600agaaggccca ggcggcgcaa caagatcaga
ctcagcctca aacaagctct tcttcttctt 660ccttcatgat gagggatgct ccccctgccg
cagctaccag cattcatcca gcggcggcag 720gcgagagggc aggggatgcg gcagtgcagc
cgcaggcccc accccggacg gggcttccac 780tgtggatggt gagccacatc aacggctgaa
gggcttccag cccatataag cgtactattc 840agtagagagt aacaagttgc accggccagt
ctggtgtatg ttgcggttgc tagcacgcct 900gaccccttgg aggggaaagg aaaagaaatc
agagtaaagt agcaagctgc agcgatgtgt 960atatttcact ttgtccaccc cagtttccct
cccagctggg ctcaatttac catgtaatct 1020atatgtagct tgagtgatga attttcaagt
ttccatgata cccgtctcta gtgggatgtt 1080gtttatgtga attaacctat caaatatgag
cattgtgtat attgtgattc ttgaaaataa 1140ataaatcagg atctttgtct t
1161123244PRTTriticum aestivum 123Met
Gly Arg Gly Lys Val Gln Leu Lys Arg Ile Glu Asn Lys Ile Asn 1
5 10 15 Arg Gln Val Thr Phe Ser
Lys Arg Arg Ser Gly Leu Leu Lys Lys Ala 20
25 30 His Glu Ile Ser Val Leu Cys Asp Ala Glu
Val Gly Leu Ile Ile Phe 35 40
45 Ser Thr Lys Gly Lys Leu Tyr Glu Phe Ser Thr Glu Ser Cys
Met Asp 50 55 60
Lys Ile Leu Glu Arg Tyr Glu Arg Tyr Ser Tyr Ala Glu Lys Val Leu 65
70 75 80 Val Ser Ser Glu Ser
Glu Ile Gln Gly Asn Trp Cys His Glu Tyr Arg 85
90 95 Lys Leu Lys Ala Lys Val Glu Thr Ile Gln
Lys Cys Gln Lys His Leu 100 105
110 Met Gly Glu Asp Leu Glu Ser Leu Asn Leu Lys Glu Leu Gln Gln
Leu 115 120 125 Glu
Gln Gln Leu Glu Ser Ser Leu Lys His Ile Arg Ser Arg Lys Asn 130
135 140 Gln Leu Met His Glu Ser
Ile Ser Glu Leu Gln Lys Lys Glu Arg Ser 145 150
155 160 Leu Gln Glu Glu Asn Lys Val Leu Gln Lys Glu
Leu Val Glu Lys Gln 165 170
175 Lys Ala Gln Ala Ala Gln Gln Asp Gln Thr Gln Pro Gln Thr Ser Ser
180 185 190 Ser Ser
Ser Ser Phe Met Met Arg Asp Ala Pro Pro Ala Ala Ala Thr 195
200 205 Ser Ile His Pro Ala Ala Ala
Gly Glu Arg Ala Gly Asp Ala Ala Val 210 215
220 Gln Pro Gln Ala Pro Pro Arg Thr Gly Leu Pro Leu
Trp Met Val Ser 225 230 235
240 His Ile Asn Gly 1241210DNATriticum aestivum 124tccctctcct
ccctctcttc cgcctcaccc aaccacctga cagccatggc tccgcccccc 60cgcccccgcc
tgcgcctgtc ggagtagccg tcgcggtctg ccggtgttgg aggcttgggg 120tgtagggttg
gccccgttct ccagcggaga tggggcgcgg gaaggtgcag ctgaagcgga 180tcgagaacaa
gatcaaccgg caggtgacct tctccaagcg ccgctcgggg ctgctcaaga 240aggcgcacga
gatctccgtg ctctgcgacg ccgaggtcgg cctcatcatc ttctccacca 300agggaaagct
ctacgagttc tccaccgagt catgtatgga caaaattctt gaacggtatg 360agcgctactc
ttatgcagaa aaggttctcg tttcaagtga atctgaaatt cagggaaact 420ggtgtcacga
atataggaaa ctgaaggcga aggttgagac aatacagaaa tgtcaaaagc 480atctgatggg
agaggatctt gaatctttga atctcaagga gttgcagcaa ctggagcagc 540agctggaaag
ctcactgaaa catatcagat ccaggaagaa ccaacttatg cacgaatcca 600tttctgagct
tcagaagaag gagaggtcac tgcaggagga gaataaagtt ctccagaagg 660aactcgtcga
gaagcagaag gcccaggcgg cgcaacaaga tcagactcag cctcaaacaa 720gctcttcttc
ttcttccttc atgatgaggg atgctccccc tgccgcaact accagcattc 780atccagcggc
atcaggagag agggcagagg atgcggcagt gcagccgcag gccccacccc 840ggacggggct
tccactgtgg atggttagcc acatcaacgg ctgaagggct tccagcccat 900ataagcgtac
tattcagtag agagtaacaa gttgcaccgg ccagcctggt gtatgttgcg 960gttgctagca
tgcctgaccc cttggagggg aaaggaaaag aaatcagagt aaagtagcaa 1020gctgcagtga
tgtgtatatt tcactttgtc cacctcagtt tccctcccag ctgggctcaa 1080tttaccatgt
aatctatatg tagcttgagt gatgaatttt caagtttcca tgatacccgt 1140ctcgagcggg
tgttgtttat gtgaattaac ctatcaaata tgagcattgt gtaaaaaaaa 1200aaaaaaaaaa
1210125244PRTTriticum aestivum 125Met Gly Arg Gly Lys Val Gln Leu Lys Arg
Ile Glu Asn Lys Ile Asn 1 5 10
15 Arg Gln Val Thr Phe Ser Lys Arg Arg Ser Gly Leu Leu Lys Lys
Ala 20 25 30 His
Glu Ile Ser Val Leu Cys Asp Ala Glu Val Gly Leu Ile Ile Phe 35
40 45 Ser Thr Lys Gly Lys Leu
Tyr Glu Phe Ser Thr Glu Ser Cys Met Asp 50 55
60 Lys Ile Leu Glu Arg Tyr Glu Arg Tyr Ser Tyr
Ala Glu Lys Val Leu 65 70 75
80 Val Ser Ser Glu Ser Glu Ile Gln Gly Asn Trp Cys His Glu Tyr Arg
85 90 95 Lys Leu
Lys Ala Lys Val Glu Thr Ile Gln Lys Cys Gln Lys His Leu 100
105 110 Met Gly Glu Asp Leu Glu Ser
Leu Asn Leu Lys Glu Leu Gln Gln Leu 115 120
125 Glu Gln Gln Leu Glu Ser Ser Leu Lys His Ile Arg
Ser Arg Lys Asn 130 135 140
Gln Leu Met His Glu Ser Ile Ser Glu Leu Gln Lys Lys Glu Arg Ser 145
150 155 160 Leu Gln Glu
Glu Asn Lys Val Leu Gln Lys Glu Leu Val Glu Lys Gln 165
170 175 Lys Ala Gln Ala Ala Gln Gln Asp
Gln Thr Gln Pro Gln Thr Ser Ser 180 185
190 Ser Ser Ser Ser Phe Met Met Arg Asp Ala Pro Pro Ala
Ala Thr Thr 195 200 205
Ser Ile His Pro Ala Ala Ser Gly Glu Arg Ala Glu Asp Ala Ala Val 210
215 220 Gln Pro Gln Ala
Pro Pro Arg Thr Gly Leu Pro Leu Trp Met Val Ser 225 230
235 240 His Ile Asn Gly 126735DNATriticum
monococcum 126atggggcgcg ggaaggtgca gctgaagcgg atcgagaaca agatcaaccg
gcaggtgacc 60ttctccaagc gccgctcggg gcttctcaag aaggcgcacg agatctccgt
gctctgcgac 120gccgaggtcg gcctcatcat cttctccacc aagggaaagc tctacgagtt
ctccaccgag 180tcatgtatgg acaaaattct tgaacggtat gagcgctatt cttatgcaga
aaaggttctc 240gtttcaagtg aatctgaaat tcagggaaac tggtgtcacg aatataggaa
actgaaggcg 300aaggttgaga caatacagaa atgtcaaaaa catctcatgg gagaggatct
tgaatctttg 360aatctcaagg agttgcagca actggagcag cagctggaaa gctcactgaa
acatatcaga 420tccaggaaga accaacttat gcacgaatcc atttctgagc tgcagaagaa
ggagaggtca 480ctgcaggagg agaataaagt tctccagaag gaactcgtgg agaagcagaa
ggcccatgcg 540gcgcagcaag atcaaactca gcctcaaacc agctcttctt cttcttcctt
catgctgagg 600gatgctcccc ctgccgcaaa taccagcatt catccagcgg cggcaggcga
gagggcagag 660gatgcggcag tgcagccgca ggccccaccc cggacggggc ttccaccgtg
gatggtgagc 720cacatcaacg ggtga
735127244PRTTriticum monococcum 127Met Gly Arg Gly Lys Val
Gln Leu Lys Arg Ile Glu Asn Lys Ile Asn 1 5
10 15 Arg Gln Val Thr Phe Ser Lys Arg Arg Ser Gly
Leu Leu Lys Lys Ala 20 25
30 His Glu Ile Ser Val Leu Cys Asp Ala Glu Val Gly Leu Ile Ile
Phe 35 40 45 Ser
Thr Lys Gly Lys Leu Tyr Glu Phe Ser Thr Glu Ser Cys Met Asp 50
55 60 Lys Ile Leu Glu Arg Tyr
Glu Arg Tyr Ser Tyr Ala Glu Lys Val Leu 65 70
75 80 Val Ser Ser Glu Ser Glu Ile Gln Gly Asn Trp
Cys His Glu Tyr Arg 85 90
95 Lys Leu Lys Ala Lys Val Glu Thr Ile Gln Lys Cys Gln Lys His Leu
100 105 110 Met Gly
Glu Asp Leu Glu Ser Leu Asn Leu Lys Glu Leu Gln Gln Leu 115
120 125 Glu Gln Gln Leu Glu Ser Ser
Leu Lys His Ile Arg Ser Arg Lys Asn 130 135
140 Gln Leu Met His Glu Ser Ile Ser Glu Leu Gln Lys
Lys Glu Arg Ser 145 150 155
160 Leu Gln Glu Glu Asn Lys Val Leu Gln Lys Glu Leu Val Glu Lys Gln
165 170 175 Lys Ala His
Ala Ala Gln Gln Asp Gln Thr Gln Pro Gln Thr Ser Ser 180
185 190 Ser Ser Ser Ser Phe Met Leu Arg
Asp Ala Pro Pro Ala Ala Asn Thr 195 200
205 Ser Ile His Pro Ala Ala Ala Gly Glu Arg Ala Glu Asp
Ala Ala Val 210 215 220
Gln Pro Gln Ala Pro Pro Arg Thr Gly Leu Pro Pro Trp Met Val Ser 225
230 235 240 His Ile Asn Gly
128735DNATriticum aestivum 128atggggcggg ggaaggtgca gctgaagcgg atcgagaaca
agatcaaccg gcaggtgacc 60ttctccaagc gccgctcggg gcttctcaag aaggcgcacg
agatctccgt gctctgcgac 120gccgaggtcg gcctcatcat cttctccacc aagggaaagc
tctacgagtt ctccaccgag 180tcatgtatgg acaaaattct tgaacggtat gagcgctatt
cttatgcaga aaaggttctc 240gtttcaagtg aatctgaaat tcagggaaac tggtgtcacg
aatataggaa actgaaggcg 300aaggttgaga caatacagaa atgtcaaaag catctcatgg
gagaggatct tgaatctttg 360aatctcaagg agttgcagca actggagcag cagctggaaa
gctcactgaa acatatcaga 420tccaggaaga accaacttat gcacgaatcc atttctgagc
ttcagaagaa ggagaggtca 480ctgcaggagg agaataaagt tctccagaag gaactcgtgg
agaagcagaa ggcccatgcg 540gcgcagcaag atcaaactca gcctcaaacc agctcttcat
cttcttcctt catgctgagg 600gatgctcccc ctgccgcaaa taccagcatt catccagcgg
caacaggcga gagggcagag 660gatgcggcag tgcagccgca ggccccaccc cggacggggc
ttccaccgtg gatggtgagc 720cacatcaacg ggtga
735129244PRTTriticum aestivum 129Met Gly Arg Gly
Lys Val Gln Leu Lys Arg Ile Glu Asn Lys Ile Asn 1 5
10 15 Arg Gln Val Thr Phe Ser Lys Arg Arg
Ser Gly Leu Leu Lys Lys Ala 20 25
30 His Glu Ile Ser Val Leu Cys Asp Ala Glu Val Gly Leu Ile
Ile Phe 35 40 45
Ser Thr Lys Gly Lys Leu Tyr Glu Phe Ser Thr Glu Ser Cys Met Asp 50
55 60 Lys Ile Leu Glu Arg
Tyr Glu Arg Tyr Ser Tyr Ala Glu Lys Val Leu 65 70
75 80 Val Ser Ser Glu Ser Glu Ile Gln Gly Asn
Trp Cys His Glu Tyr Arg 85 90
95 Lys Leu Lys Ala Lys Val Glu Thr Ile Gln Lys Cys Gln Lys His
Leu 100 105 110 Met
Gly Glu Asp Leu Glu Ser Leu Asn Leu Lys Glu Leu Gln Gln Leu 115
120 125 Glu Gln Gln Leu Glu Ser
Ser Leu Lys His Ile Arg Ser Arg Lys Asn 130 135
140 Gln Leu Met His Glu Ser Ile Ser Glu Leu Gln
Lys Lys Glu Arg Ser 145 150 155
160 Leu Gln Glu Glu Asn Lys Val Leu Gln Lys Glu Leu Val Glu Lys Gln
165 170 175 Lys Ala
His Ala Ala Gln Gln Asp Gln Thr Gln Pro Gln Thr Ser Ser 180
185 190 Ser Ser Ser Ser Phe Met Leu
Arg Asp Ala Pro Pro Ala Ala Asn Thr 195 200
205 Ser Ile His Pro Ala Ala Thr Gly Glu Arg Ala Glu
Asp Ala Ala Val 210 215 220
Gln Pro Gln Ala Pro Pro Arg Thr Gly Leu Pro Pro Trp Met Val Ser 225
230 235 240 His Ile Asn
Gly 130738DNALolium perenne 130atggggcgcg gcaaggtgca gctcaagcgg
atcgagaaca agatcaaccg ccaggtcacc 60ttctccaagc gccgctcggg gctgctcaag
aaggcgcacg agatctccgt gctctgcgac 120gccgaggtcg ggctcatcat cttctccacc
aagggaaagc tctacgagtt cgcaaccgac 180tcatgtatgg acaaaattct tgagcggtat
gagcgctact cctatgcaga gaaagtgctc 240atttcaaccg aatctgaaat tcagggaaac
tggtgtcatg aatataggaa actgaaggcg 300aaggttgaga caatacagag atgtcaaaag
catctaatgg gagaggatct tgaatcattg 360aatctcaagg agttgcagca actagagcag
cagctggaaa gttcactgaa acatattaga 420gccagaaaga accagcttat gcacgaatcc
atatctgagc ttcaaaagaa ggagaggtca 480ctgcaggagg agaataaaat tctccagaag
gaactcatag agaagcagaa ggcccacacg 540cagcaagcgc agtgggagca aactcagccc
caaaccagct cttcctcctc ctcctttatg 600atgggggaag ctaccccagc aacaaattgc
agtaatcccc cagcagcggc cagcgacaga 660gcagaggatg cgacggggca gccttcagct
cgcacggtgc ttccaccatg gatggtgagt 720cacatcaaca atggctga
738131245PRTLolium perenne 131Met Gly
Arg Gly Lys Val Gln Leu Lys Arg Ile Glu Asn Lys Ile Asn 1 5
10 15 Arg Gln Val Thr Phe Ser Lys
Arg Arg Ser Gly Leu Leu Lys Lys Ala 20 25
30 His Glu Ile Ser Val Leu Cys Asp Ala Glu Val Gly
Leu Ile Ile Phe 35 40 45
Ser Thr Lys Gly Lys Leu Tyr Glu Phe Ala Thr Asp Ser Cys Met Asp
50 55 60 Lys Ile Leu
Glu Arg Tyr Glu Arg Tyr Ser Tyr Ala Glu Lys Val Leu 65
70 75 80 Ile Ser Thr Glu Ser Glu Ile
Gln Gly Asn Trp Cys His Glu Tyr Arg 85
90 95 Lys Leu Lys Ala Lys Val Glu Thr Ile Gln Arg
Cys Gln Lys His Leu 100 105
110 Met Gly Glu Asp Leu Glu Ser Leu Asn Leu Lys Glu Leu Gln Gln
Leu 115 120 125 Glu
Gln Gln Leu Glu Ser Ser Leu Lys His Ile Arg Ala Arg Lys Asn 130
135 140 Gln Leu Met His Glu Ser
Ile Ser Glu Leu Gln Lys Lys Glu Arg Ser 145 150
155 160 Leu Gln Glu Glu Asn Lys Ile Leu Gln Lys Glu
Leu Ile Glu Lys Gln 165 170
175 Lys Ala His Thr Gln Gln Ala Gln Trp Glu Gln Thr Gln Pro Gln Thr
180 185 190 Ser Ser
Ser Ser Ser Ser Phe Met Met Gly Glu Ala Thr Pro Ala Thr 195
200 205 Asn Cys Ser Asn Pro Pro Ala
Ala Ala Ser Asp Arg Ala Glu Asp Ala 210 215
220 Thr Gly Gln Pro Ser Ala Arg Thr Val Leu Pro Pro
Trp Met Val Ser 225 230 235
240 His Ile Asn Asn Gly 245 132738DNALolium temulentum
132atggggcgcg gcaaggtgca gctcaagcgg atcgagaaca agatcaaccg ccaggtcacc
60ttctccaagc gccgctcagg cctgctcaag aaggcgcacg agatctccgt gctctgcgac
120gcagaggtcg ggctcatcat cttctccacc aagggaaagc tctacgagtt cgccaccgac
180tcatgtatgg acaaaattct tgagcggtat gagcgctact cctatgcaga gaaagtgctc
240atttcaactg aatctgaaat tcagggaaac tggtgtcatg aatataggaa actgaaggcg
300aaggttgaga caatacagag atgtcaaaag catctaatgg gagaggatct tgaatcattg
360aatctcaagg agttgcagca actagagcag cagctggaaa gttcactgaa acatattaga
420tccagaaaga gccagcttat gcacgaatcc atatctgagc ttcaaaagaa ggagaggtca
480ctgcaagagg agaataaaat tctccagaag gaactcatag agaagcagaa ggcccacacg
540cagcaagcgc agttggagca aactcagccc caaaccagct cttcctcctc ctcctttatg
600atgggggaag ctaccccagc aacaaatcgc agtaatcccc cagcagcggc cagcgacaga
660gcagaggatg cgacggggca gcctccagct cgcacggtgc ttccaccatg gatggtgagt
720cacctcaaca atggctga
738133245PRTLolium temulentum 133Met Gly Arg Gly Lys Val Gln Leu Lys Arg
Ile Glu Asn Lys Ile Asn 1 5 10
15 Arg Gln Val Thr Phe Ser Lys Arg Arg Ser Gly Leu Leu Lys Lys
Ala 20 25 30 His
Glu Ile Ser Val Leu Cys Asp Ala Glu Val Gly Leu Ile Ile Phe 35
40 45 Ser Thr Lys Gly Lys Leu
Tyr Glu Phe Ala Thr Asp Ser Cys Met Asp 50 55
60 Lys Ile Leu Glu Arg Tyr Glu Arg Tyr Ser Tyr
Ala Glu Lys Val Leu 65 70 75
80 Ile Ser Thr Glu Ser Glu Ile Gln Gly Asn Trp Cys His Glu Tyr Arg
85 90 95 Lys Leu
Lys Ala Lys Val Glu Thr Ile Gln Arg Cys Gln Lys His Leu 100
105 110 Met Gly Glu Asp Leu Glu Ser
Leu Asn Leu Lys Glu Leu Gln Gln Leu 115 120
125 Glu Gln Gln Leu Glu Ser Ser Leu Lys His Ile Arg
Ser Arg Lys Ser 130 135 140
Gln Leu Met His Glu Ser Ile Ser Glu Leu Gln Lys Lys Glu Arg Ser 145
150 155 160 Leu Gln Glu
Glu Asn Lys Ile Leu Gln Lys Glu Leu Ile Glu Lys Gln 165
170 175 Lys Ala His Thr Gln Gln Ala Gln
Leu Glu Gln Thr Gln Pro Gln Thr 180 185
190 Ser Ser Ser Ser Ser Ser Phe Met Met Gly Glu Ala Thr
Pro Ala Thr 195 200 205
Asn Arg Ser Asn Pro Pro Ala Ala Ala Ser Asp Arg Ala Glu Asp Ala 210
215 220 Thr Gly Gln Pro
Pro Ala Arg Thr Val Leu Pro Pro Trp Met Val Ser 225 230
235 240 His Leu Asn Asn Gly
245 134738DNAZea mays 134atggggcgcg ggaaggtgca gctgaagcgg atcgagaaca
agatcaaccg ccaggtgacc 60ttctccaagc gccgctcggg gctgctcaag aaggcgcacg
agatctccgt gctctgcgac 120gccgaggtcg cgctcatcat cttctccacc aaagggaagc
tctacgagta ttccaccgat 180tcatgtatgg acaaaattct tgaccggtac gagcgctact
cctatgcaga aaaggttctt 240atttcagcag aatctgaaac tcagggcaat tggtgccacg
agtatagaaa actaaaggcg 300aaggtcgaga caatacaaaa atgtcaaaag cacctcatgg
gagaggatct tgaaacgttg 360aatctcaaag agcttcagca actagagcag cagctggaga
gttcactgaa acatatcaga 420accaggaaga accaacttat gctcgagtca atttcggagc
tccaacggaa ggagaagtcg 480ctgcaggagg agaacaaggt tctgcagaag gagctcgcgg
agaagcagaa agcccagcgg 540aagcaagtgc aatggggcca aacccaacag cagaccagtt
cgtcttcctc gtgcttcgtg 600ataagggaag ctgccccaac aacaaatatc agcatttttc
ctgtggcagc aggcgggagg 660ttggtggaag gtgcagcagc gcagccacag gctcgcgttg
gactaccacc atggatgctt 720agccacctga gcagctga
738135245PRTZea mays 135Met Gly Arg Gly Lys Val
Gln Leu Lys Arg Ile Glu Asn Lys Ile Asn 1 5
10 15 Arg Gln Val Thr Phe Ser Lys Arg Arg Ser Gly
Leu Leu Lys Lys Ala 20 25
30 His Glu Ile Ser Val Leu Cys Asp Ala Glu Val Ala Leu Ile Ile
Phe 35 40 45 Ser
Thr Lys Gly Lys Leu Tyr Glu Tyr Ser Thr Asp Ser Cys Met Asp 50
55 60 Lys Ile Leu Asp Arg Tyr
Glu Arg Tyr Ser Tyr Ala Glu Lys Val Leu 65 70
75 80 Ile Ser Ala Glu Ser Glu Thr Gln Gly Asn Trp
Cys His Glu Tyr Arg 85 90
95 Lys Leu Lys Ala Lys Val Glu Thr Ile Gln Lys Cys Gln Lys His
Leu 100 105 110 Met
Gly Glu Asp Leu Glu Thr Leu Asn Leu Lys Glu Leu Gln Gln Leu 115
120 125 Glu Gln Gln Leu Glu Ser
Ser Leu Lys His Ile Arg Thr Arg Lys Asn 130 135
140 Gln Leu Met Leu Glu Ser Ile Ser Glu Leu Gln
Arg Lys Glu Lys Ser 145 150 155
160 Leu Gln Glu Glu Asn Lys Val Leu Gln Lys Glu Leu Ala Glu Lys Gln
165 170 175 Lys Ala
Gln Arg Lys Gln Val Gln Trp Gly Gln Thr Gln Gln Gln Thr 180
185 190 Ser Ser Ser Ser Ser Cys Phe
Val Ile Arg Glu Ala Ala Pro Thr Thr 195 200
205 Asn Ile Ser Ile Phe Pro Val Ala Ala Gly Gly Arg
Leu Val Glu Gly 210 215 220
Ala Ala Ala Gln Pro Gln Ala Arg Val Gly Leu Pro Pro Trp Met Leu 225
230 235 240 Ser His Leu
Ser Ser 245 136738DNAZea mays 136atggggcgcg ggaaggtgca
gctgaagcgg atcgagaaca agatcaaccg ccaggtgaca 60ttctccaagc gccgctcggg
gctactcaag aaggcgcacg agatctccgt gctctgcgac 120gccgaggtcg cgctcatcat
cttctccacc aagggcaagc tctacgagta ctctaccgat 180tcatgtatgg acaaaattct
tgaacggtat gagcgctact cctatgcaga aaaggttctc 240atttccgcag aatatgaaac
tcagggcaat tggtgccatg aatatagaaa actaaaggcg 300aaggtcgaga caatacagaa
atgtcaaaag cacctcatgg gagaggatct tgaaactttg 360aatctcaaag agcttcagca
actagagcag cagctggaga gttcactgaa acatatcaga 420acaaggaaga gccagcttat
ggtcgagtca atttcagcgc tccaacggaa ggagaagtca 480ctgcaggagg agaacaaggt
tctgcagaag gagctcgcgg agaagcagaa agaccagcgg 540cagcaagtgc aacgggacca
aactcaacag cagaccagtt cgtcttccac gtccttcatg 600ttaagggaag ctgccccaac
aacaaatgtc agcatcttcc ctgtggcagc aggcgggagg 660gtggtggaag gggcagcagc
gcagccgcag gctcgcgttg gactgccacc atggatgctt 720agccatctga gctgctga
738137245PRTZea mays 137Met
Gly Arg Gly Lys Val Gln Leu Lys Arg Ile Glu Asn Lys Ile Asn 1
5 10 15 Arg Gln Val Thr Phe Ser
Lys Arg Arg Ser Gly Leu Leu Lys Lys Ala 20
25 30 His Glu Ile Ser Val Leu Cys Asp Ala Glu
Val Ala Leu Ile Ile Phe 35 40
45 Ser Thr Lys Gly Lys Leu Tyr Glu Tyr Ser Thr Asp Ser Cys
Met Asp 50 55 60
Lys Ile Leu Glu Arg Tyr Glu Arg Tyr Ser Tyr Ala Glu Lys Val Leu 65
70 75 80 Ile Ser Ala Glu Tyr
Glu Thr Gln Gly Asn Trp Cys His Glu Tyr Arg 85
90 95 Lys Leu Lys Ala Lys Val Glu Thr Ile Gln
Lys Cys Gln Lys His Leu 100 105
110 Met Gly Glu Asp Leu Glu Thr Leu Asn Leu Lys Glu Leu Gln Gln
Leu 115 120 125 Glu
Gln Gln Leu Glu Ser Ser Leu Lys His Ile Arg Thr Arg Lys Ser 130
135 140 Gln Leu Met Val Glu Ser
Ile Ser Ala Leu Gln Arg Lys Glu Lys Ser 145 150
155 160 Leu Gln Glu Glu Asn Lys Val Leu Gln Lys Glu
Leu Ala Glu Lys Gln 165 170
175 Lys Asp Gln Arg Gln Gln Val Gln Arg Asp Gln Thr Gln Gln Gln Thr
180 185 190 Ser Ser
Ser Ser Thr Ser Phe Met Leu Arg Glu Ala Ala Pro Thr Thr 195
200 205 Asn Val Ser Ile Phe Pro Val
Ala Ala Gly Gly Arg Val Val Glu Gly 210 215
220 Ala Ala Ala Gln Pro Gln Ala Arg Val Gly Leu Pro
Pro Trp Met Leu 225 230 235
240 Ser His Leu Ser Cys 245 138762DNAOryza sativa
138atggggcggg gcaaggtgca gctgaagcgg atcgagaaca agatcaaccg gcaggtgacc
60ttctccaagc gcaggtcggg gctgctcaag aaggcgaatg agatctccgt gctctgcgac
120gccgaggtcg cgctcatcat cttctccacc aagggcaagc tctacgagta cgccaccgac
180tcatgtatgg acaaaatcct tgaacgttat gagcgctact cctatgcaga aaaggtcctt
240atttcagctg aatctgacac tcagggcaac tggtgccacg aatataggaa actgaaggct
300aaggttgaga caatacagaa atgtcaaaag cacctcatgg gagaggatct tgaatctttg
360aatctcaaag agctgcagca gctggagcag cagctggaaa attcgttgaa acatatcaga
420tccagaaaga gccaactaat gctcgagtcc attaacgagc ttcaacggaa ggaaaagtca
480ctgcaggagg agaataaggt cctacagaaa gaaaaccctt gctccttcct acagctggtg
540gagaagcaga aagtccagaa gcaacaagtg caatgggacc agacacaacc tcaaacaagt
600tcctcatcat cctccttcat gatgagggaa gcccttccaa caactaatat cagtaactac
660cctgcagcag ctggcgaaag gatagaggat gtagcagcag ggcagccaca gcatgttcgc
720attgggctgc caccatggat gctgagccac atcaacggct aa
762139253PRTOryza sativa 139Met Gly Arg Gly Lys Val Gln Leu Lys Arg Ile
Glu Asn Lys Ile Asn 1 5 10
15 Arg Gln Val Thr Phe Ser Lys Arg Arg Ser Gly Leu Leu Lys Lys Ala
20 25 30 Asn Glu
Ile Ser Val Leu Cys Asp Ala Glu Val Ala Leu Ile Ile Phe 35
40 45 Ser Thr Lys Gly Lys Leu Tyr
Glu Tyr Ala Thr Asp Ser Cys Met Asp 50 55
60 Lys Ile Leu Glu Arg Tyr Glu Arg Tyr Ser Tyr Ala
Glu Lys Val Leu 65 70 75
80 Ile Ser Ala Glu Ser Asp Thr Gln Gly Asn Trp Cys His Glu Tyr Arg
85 90 95 Lys Leu Lys
Ala Lys Val Glu Thr Ile Gln Lys Cys Gln Lys His Leu 100
105 110 Met Gly Glu Asp Leu Glu Ser Leu
Asn Leu Lys Glu Leu Gln Gln Leu 115 120
125 Glu Gln Gln Leu Glu Asn Ser Leu Lys His Ile Arg Ser
Arg Lys Ser 130 135 140
Gln Leu Met Leu Glu Ser Ile Asn Glu Leu Gln Arg Lys Glu Lys Ser 145
150 155 160 Leu Gln Glu Glu
Asn Lys Val Leu Gln Lys Glu Asn Pro Cys Ser Phe 165
170 175 Leu Gln Leu Val Glu Lys Gln Lys Val
Gln Lys Gln Gln Val Gln Trp 180 185
190 Asp Gln Thr Gln Pro Gln Thr Ser Ser Ser Ser Ser Ser Phe
Met Met 195 200 205
Arg Glu Ala Leu Pro Thr Thr Asn Ile Ser Asn Tyr Pro Ala Ala Ala 210
215 220 Gly Glu Arg Ile Glu
Asp Val Ala Ala Gly Gln Pro Gln His Val Arg 225 230
235 240 Ile Gly Leu Pro Pro Trp Met Leu Ser His
Ile Asn Gly 245 250
140741DNAOryza sativa 140atggggcggg gcaaggtgca gctgaagcgg atcgagaaca
agatcaaccg gcaggtgacc 60ttctccaagc gcaggtcaaa actgctcaag aaggcgaatg
agatctccgt gctctgcgac 120gccgaggtcg cgctcatcat cttctccacc aagggcaagc
tctacgagta cgccaccgac 180tcatgtatgg acaaaatcct tgaacgttat gagcgctact
cctatgcaga aaaggtcctt 240atttcagctg aatctgacac tcagggcaac tggtgccacg
aatataggaa actgaaggct 300aaggttgaga caatacagaa atgtcaaaag cacctcatgg
gagaggatct tgaatctttg 360aatctcaaag agctgcagca gctggagcag cagctggaaa
attcgttgaa acatatcaga 420tccagaaaga gccaactaat gctcgagtcc attaacgagc
ttcaacggaa ggaaaagtca 480ctgcaggagg agaataaggt cctacagaaa gaactggtgg
agaagcagaa agtccagaag 540caacaagtgc aatgggacca gacacaacct caaacaagtt
cctcatcatc ctccttcatg 600atgagggaag cccttccaac aactaatatc agtaactacc
ctgcagcagc tggcgaaagg 660atagaggatg tagcagcagg gcagccacag catgaacgca
ttgggctgcc accatggatg 720ctgagccaca tcaacggcta a
741141246PRTOryza sativa 141Met Gly Arg Gly Lys
Val Gln Leu Lys Arg Ile Glu Asn Lys Ile Asn 1 5
10 15 Arg Gln Val Thr Phe Ser Lys Arg Arg Ser
Lys Leu Leu Lys Lys Ala 20 25
30 Asn Glu Ile Ser Val Leu Cys Asp Ala Glu Val Ala Leu Ile Ile
Phe 35 40 45 Ser
Thr Lys Gly Lys Leu Tyr Glu Tyr Ala Thr Asp Ser Cys Met Asp 50
55 60 Lys Ile Leu Glu Arg Tyr
Glu Arg Tyr Ser Tyr Ala Glu Lys Val Leu 65 70
75 80 Ile Ser Ala Glu Ser Asp Thr Gln Gly Asn Trp
Cys His Glu Tyr Arg 85 90
95 Lys Leu Lys Ala Lys Val Glu Thr Ile Gln Lys Cys Gln Lys His Leu
100 105 110 Met Gly
Glu Asp Leu Glu Ser Leu Asn Leu Lys Glu Leu Gln Gln Leu 115
120 125 Glu Gln Gln Leu Glu Asn Ser
Leu Lys His Ile Arg Ser Arg Lys Ser 130 135
140 Gln Leu Met Leu Glu Ser Ile Asn Glu Leu Gln Arg
Lys Glu Lys Ser 145 150 155
160 Leu Gln Glu Glu Asn Lys Val Leu Gln Lys Glu Leu Val Glu Lys Gln
165 170 175 Lys Val Gln
Lys Gln Gln Val Gln Trp Asp Gln Thr Gln Pro Gln Thr 180
185 190 Ser Ser Ser Ser Ser Ser Phe Met
Met Arg Glu Ala Leu Pro Thr Thr 195 200
205 Asn Ile Ser Asn Tyr Pro Ala Ala Ala Gly Glu Arg Ile
Glu Asp Val 210 215 220
Ala Ala Gly Gln Pro Gln His Glu Arg Ile Gly Leu Pro Pro Trp Met 225
230 235 240 Leu Ser His Ile
Asn Gly 245 142735DNADendrocalamus latiflorus
142atggggcgcg ggaaggtgca gctgaagcgg atcgagaaca agatcaaccg gcaagtgact
60ttctccaagc gccggtcggg gctgctcaag aaggcgcatg agatctccgt cctctgcgac
120gccgaggtcg gccttatcat cttctccacc aagggcaagc tctacgagta cgccaccgac
180tcatgtatgg acaaaattct tgaacggtac gagcgttact cctatgcaga aaaggttctt
240atttcagccg aatctgaaac tcagggcaac tggtgtcacg aatataggaa actgaaggcg
300aaggttgaga cgatacagaa atgtcaaaag cacctcatgg gagaggatcc tgaatctttg
360aatctcaagg agctgcagca actcgagcag cagctggaaa gttcagtgaa acatatcaga
420tccagaaaga gccagcttat gctcgagtcc atttccgagc ttcaaaagaa ggagaagtca
480ctgcaggagg agaacaaggt tctgcagaag gaactcgtgg agaagcagca ggtccataaa
540cggttagtgc aatgggacca aactcagccg caaactagtt cctcttcctc gtccttcatg
600atgagggaag ctctcccaac aacaaatatc agtatttacg ctgcggcagc cggcgagagg
660gcagaggacg cagcagggca gcctcagatt cacattgggc tgccgccatg gatggtgagc
720cacatcaacg gctaa
735143244PRTDendrocalamus latiflorus 143Met Gly Arg Gly Lys Val Gln Leu
Lys Arg Ile Glu Asn Lys Ile Asn 1 5 10
15 Arg Gln Val Thr Phe Ser Lys Arg Arg Ser Gly Leu Leu
Lys Lys Ala 20 25 30
His Glu Ile Ser Val Leu Cys Asp Ala Glu Val Gly Leu Ile Ile Phe
35 40 45 Ser Thr Lys Gly
Lys Leu Tyr Glu Tyr Ala Thr Asp Ser Cys Met Asp 50
55 60 Lys Ile Leu Glu Arg Tyr Glu Arg
Tyr Ser Tyr Ala Glu Lys Val Leu 65 70
75 80 Ile Ser Ala Glu Ser Glu Thr Gln Gly Asn Trp Cys
His Glu Tyr Arg 85 90
95 Lys Leu Lys Ala Lys Val Glu Thr Ile Gln Lys Cys Gln Lys His Leu
100 105 110 Met Gly Glu
Asp Pro Glu Ser Leu Asn Leu Lys Glu Leu Gln Gln Leu 115
120 125 Glu Gln Gln Leu Glu Ser Ser Val
Lys His Ile Arg Ser Arg Lys Ser 130 135
140 Gln Leu Met Leu Glu Ser Ile Ser Glu Leu Gln Lys Lys
Glu Lys Ser 145 150 155
160 Leu Gln Glu Glu Asn Lys Val Leu Gln Lys Glu Leu Val Glu Lys Gln
165 170 175 Gln Val His Lys
Arg Leu Val Gln Trp Asp Gln Thr Gln Pro Gln Thr 180
185 190 Ser Ser Ser Ser Ser Ser Phe Met Met
Arg Glu Ala Leu Pro Thr Thr 195 200
205 Asn Ile Ser Ile Tyr Ala Ala Ala Ala Gly Glu Arg Ala Glu
Asp Ala 210 215 220
Ala Gly Gln Pro Gln Ile His Ile Gly Leu Pro Pro Trp Met Val Ser 225
230 235 240 His Ile Asn Gly
144735DNADendrocalamus latiflorus 144atggggcgcg ggaaggtgca gctgaagcgg
atcgagaaca agatcaaccg gcaagtgact 60ttctccaagc gccggtcggg gctgctcaag
aaggcgcatg agatctccgt cctctgcgac 120gccgaggtcg gccttatcat cttctccacc
aagggcaagc tctacgagta cgccaccgac 180tcatgtatgg acaaaattct tgaacggtac
gagcgttact cctatgcaga aaaggttctt 240atttcagccg aatctgaaac tcagggcaac
tggtgtcacg aatataggaa actgaaggcg 300aaggttgaga cgatacagaa atgtcaaaag
cacctcatgg gagaggatct tgaatctttg 360aatctcaagg agctgcagca actcgagcag
cagctggaaa gttcagtgaa acatatcaga 420tccagaaaga gccagcttat gctcgagtcc
atttccgagc ttcaaaagaa ggagaagtca 480ctgcaggagg agaacaaggt tctgcagaag
gaactcgtgg agaagcagca ggtccataaa 540cggttagtgc aatgggacca aactcagccg
caaactagtt cctcttcctc gtccttcatg 600atgagggaag ctctcccaac aacaaatatc
agtatttacg ctgcggcagc cggcgagagg 660gcagaggacg cagcagggca gcctcagatt
cacattgggc tgccgccatg gatggtgagc 720cacatcaacg gctaa
735145244PRTDendrocalamus latiflorus
145Met Gly Arg Gly Lys Val Gln Leu Lys Arg Ile Glu Asn Lys Ile Asn 1
5 10 15 Arg Gln Val Thr
Phe Ser Lys Arg Arg Ser Gly Leu Leu Lys Lys Ala 20
25 30 His Glu Ile Ser Val Leu Cys Asp Ala
Glu Val Gly Leu Ile Ile Phe 35 40
45 Ser Thr Lys Gly Lys Leu Tyr Glu Tyr Ala Thr Asp Ser Cys
Met Asp 50 55 60
Lys Ile Leu Glu Arg Tyr Glu Arg Tyr Ser Tyr Ala Glu Lys Val Leu 65
70 75 80 Ile Ser Ala Glu Ser
Glu Thr Gln Gly Asn Trp Cys His Glu Tyr Arg 85
90 95 Lys Leu Lys Ala Lys Val Glu Thr Ile Gln
Lys Cys Gln Lys His Leu 100 105
110 Met Gly Glu Asp Leu Glu Ser Leu Asn Leu Lys Glu Leu Gln Gln
Leu 115 120 125 Glu
Gln Gln Leu Glu Ser Ser Val Lys His Ile Arg Ser Arg Lys Ser 130
135 140 Gln Leu Met Leu Glu Ser
Ile Ser Glu Leu Gln Lys Lys Glu Lys Ser 145 150
155 160 Leu Gln Glu Glu Asn Lys Val Leu Gln Lys Glu
Leu Val Glu Lys Gln 165 170
175 Gln Val His Lys Arg Leu Val Gln Trp Asp Gln Thr Gln Pro Gln Thr
180 185 190 Ser Ser
Ser Ser Ser Ser Phe Met Met Arg Glu Ala Leu Pro Thr Thr 195
200 205 Asn Ile Ser Ile Tyr Ala Ala
Ala Ala Gly Glu Arg Ala Glu Asp Ala 210 215
220 Ala Gly Gln Pro Gln Ile His Ile Gly Leu Pro Pro
Trp Met Val Ser 225 230 235
240 His Ile Asn Gly 146813DNAZea mays 146atggggcgcg gcaaggtgca
gctgaagcgg atagagaaca agataaaccg gcaggtgacc 60ttctccaagc gccggaacgg
gctgctgaag aaggcgcacg agatctccgt cctctgcgac 120gccgaggtcg ccgtcatcgt
cttctccccc aagggcaagc tctacgagta cgcctccgac 180tcccgcatgg acaaaattct
agaacgttat gagcgatatt cctatgctga aaaggctctt 240atttcagctg aatctgaaag
tgagggaaat tggtgccacg aatacaggaa actgaaggcc 300aaaattgaga ccatacaaag
atgccacaag cacctgatgg gagaggatct agagtctttg 360aatccaaaag agctccaaca
actagagcag cagctggaga gctcactgaa gcacatcaga 420tcaagaaaga gccaccttat
ggccgagtca atttctgagc tacagaagaa ggagaggtca 480ctgcaggagg agaacaagat
tctacagaag gaactttcag agaggcagaa ggcggtcgct 540agccggcagc agcagcagca
gcaggtgcag tgggaccagc agacacaggt ccaggtccag 600acaagctcat cgtcttcttc
cttcatgatg aggcaggatc agcagggact gccacctcca 660caaaacatct gcttcccgcc
gttgagcatc ggagagagag gcgaagaggt ggctgcggcg 720gcgcagcagc agctgcctcc
tccggggcag gcgcaaccac agctccgcat cgcaggtctg 780ccgccatgga tgctgagcca
cctcaatgca taa 813147270PRTZea mays
147Met Gly Arg Gly Lys Val Gln Leu Lys Arg Ile Glu Asn Lys Ile Asn 1
5 10 15 Arg Gln Val Thr
Phe Ser Lys Arg Arg Asn Gly Leu Leu Lys Lys Ala 20
25 30 His Glu Ile Ser Val Leu Cys Asp Ala
Glu Val Ala Val Ile Val Phe 35 40
45 Ser Pro Lys Gly Lys Leu Tyr Glu Tyr Ala Ser Asp Ser Arg
Met Asp 50 55 60
Lys Ile Leu Glu Arg Tyr Glu Arg Tyr Ser Tyr Ala Glu Lys Ala Leu 65
70 75 80 Ile Ser Ala Glu Ser
Glu Ser Glu Gly Asn Trp Cys His Glu Tyr Arg 85
90 95 Lys Leu Lys Ala Lys Ile Glu Thr Ile Gln
Arg Cys His Lys His Leu 100 105
110 Met Gly Glu Asp Leu Glu Ser Leu Asn Pro Lys Glu Leu Gln Gln
Leu 115 120 125 Glu
Gln Gln Leu Glu Ser Ser Leu Lys His Ile Arg Ser Arg Lys Ser 130
135 140 His Leu Met Ala Glu Ser
Ile Ser Glu Leu Gln Lys Lys Glu Arg Ser 145 150
155 160 Leu Gln Glu Glu Asn Lys Ile Leu Gln Lys Glu
Leu Ser Glu Arg Gln 165 170
175 Lys Ala Val Ala Ser Arg Gln Gln Gln Gln Gln Gln Val Gln Trp Asp
180 185 190 Gln Gln
Thr Gln Val Gln Val Gln Thr Ser Ser Ser Ser Ser Ser Phe 195
200 205 Met Met Arg Gln Asp Gln Gln
Gly Leu Pro Pro Pro Gln Asn Ile Cys 210 215
220 Phe Pro Pro Leu Ser Ile Gly Glu Arg Gly Glu Glu
Val Ala Ala Ala 225 230 235
240 Ala Gln Gln Gln Leu Pro Pro Pro Gly Gln Ala Gln Pro Gln Leu Arg
245 250 255 Ile Ala Gly
Leu Pro Pro Trp Met Leu Ser His Leu Asn Ala 260
265 270 148689DNASorghum bicolor 148gcaaggtgca
gctcaagcgg atagagaaca agataaaccg gcaggtgacc ttctccaagc 60gccgcaacgg
gctgctcaag aaggcgcacg agatctccgt cctctgcgac gccgaggtcg 120ccgtcatcgt
cttctccccc aagggcaagc tctatgagta cgccaccgac tcccgcatgg 180acaaaattct
cgaacgttat gagcgatatt cctatgctga aaaggctctt atttcagctg 240aatctgaaag
tgagggaaac tggtgccacg aatacaggaa actgaaggcc aaaattgaga 300ccattcaaaa
atgccacaag cacctgatgg gagaggatct agagtctttg aatcccaaag 360agctccaaca
actagagcag cagctggaga gctcactgaa gcacatcaga tcaagaaaga 420gccaccttat
ggctgagtct atttctgaac tacagaagaa ggagaggtca ctgcaggagg 480agaacaaggc
tctacagaag gaacttgcgg agaggcagaa ggcggccgcg agcaggcagc 540agcagcaagg
tgcagtggga ccagcagaca cagacccagg cccagacaag ctcatcatcg 600tcctccttca
tgatgaggca ggatcagcag ggtctgccgc ctccacaaaa catatgcttc 660ccgccgctga
taatcggaga gagaggtga
689149228PRTSorghum bicolor 149Lys Val Gln Leu Lys Arg Ile Glu Asn Lys
Ile Asn Arg Gln Val Thr 1 5 10
15 Phe Ser Lys Arg Arg Asn Gly Leu Leu Lys Lys Ala His Glu Ile
Ser 20 25 30 Val
Leu Cys Asp Ala Glu Val Ala Val Ile Val Phe Ser Pro Lys Gly 35
40 45 Lys Leu Tyr Glu Tyr Ala
Thr Asp Ser Arg Met Asp Lys Ile Leu Glu 50 55
60 Arg Tyr Glu Arg Tyr Ser Tyr Ala Glu Lys Ala
Leu Ile Ser Ala Glu 65 70 75
80 Ser Glu Ser Glu Gly Asn Trp Cys His Glu Tyr Arg Lys Leu Lys Ala
85 90 95 Lys Ile
Glu Thr Ile Gln Lys Cys His Lys His Leu Met Gly Glu Asp 100
105 110 Leu Glu Ser Leu Asn Pro Lys
Glu Leu Gln Gln Leu Glu Gln Gln Leu 115 120
125 Glu Ser Ser Leu Lys His Ile Arg Ser Arg Lys Ser
His Leu Met Ala 130 135 140
Glu Ser Ile Ser Glu Leu Gln Lys Lys Glu Arg Ser Leu Gln Glu Glu 145
150 155 160 Asn Lys Ala
Leu Gln Lys Glu Leu Ala Glu Arg Gln Lys Ala Ala Ala 165
170 175 Ser Arg Gln Gln Gln Gln Gly Ala
Val Gly Pro Ala Asp Thr Asp Pro 180 185
190 Gly Pro Asp Lys Leu Ile Ile Val Leu Leu His Asp Glu
Ala Gly Ser 195 200 205
Ala Gly Ser Ala Ala Ser Thr Lys His Met Leu Pro Ala Ala Asp Asn 210
215 220 Arg Arg Glu Arg
225 150822DNAZea mays 150atggggcgcg gcaaggtaca gctgaagcgg
atagagaaca agataaaccg gcaggtgacc 60ttctccaagc gccggaacgg cctgctcaag
aaggcgcacg agatctccgt cctctgcgat 120gccgaggtcg ccgtcatcgt cttctccccc
aagggcaagc tctacgagta cgccaccgac 180tcccgcatgg acaaaattct tgaacgctat
gagcgatatt cctatgctga aaaggctctt 240atttcagctg aatctgaaag tgagggaaat
tggtgccacg aatacaggaa actgaaggcc 300aaaattgaga ccatacaaaa atgccacaag
cacctgatgg gagaggatct agagtctttg 360aatcccaaag agctccagca actagagcag
cagctggata gctcactgaa gcacatcaga 420tcaaggaaga gccaccttat ggccgagtct
atttctgagc tacagaagaa ggagaggtca 480ctgcaggagg agaacaaggc tctgcagaag
gaacttgcgg agaggcagaa ggccgtcgcg 540agccggcagc agcagcaaca gcagcaggtg
cagtgggacc agcagacaca tgcccaggcc 600cagacaagct catcatcgtc ctccttcatg
atgaggcagg atcagcaggg actgccgcct 660ccacacaaca tctgcttccc gccgttgaca
atgggagata gaggtgaaga gctggctgcg 720gcggcggcgg cgcagcagca gcagccactg
ccggggcagg cgcaaccgca gctccgcatc 780gcaggtctgc caccatggat gctgagccac
ctcaatgcat aa 822151273PRTZea mays 151Met Gly Arg
Gly Lys Val Gln Leu Lys Arg Ile Glu Asn Lys Ile Asn 1 5
10 15 Arg Gln Val Thr Phe Ser Lys Arg
Arg Asn Gly Leu Leu Lys Lys Ala 20 25
30 His Glu Ile Ser Val Leu Cys Asp Ala Glu Val Ala Val
Ile Val Phe 35 40 45
Ser Pro Lys Gly Lys Leu Tyr Glu Tyr Ala Thr Asp Ser Arg Met Asp 50
55 60 Lys Ile Leu Glu
Arg Tyr Glu Arg Tyr Ser Tyr Ala Glu Lys Ala Leu 65 70
75 80 Ile Ser Ala Glu Ser Glu Ser Glu Gly
Asn Trp Cys His Glu Tyr Arg 85 90
95 Lys Leu Lys Ala Lys Ile Glu Thr Ile Gln Lys Cys His Lys
His Leu 100 105 110
Met Gly Glu Asp Leu Glu Ser Leu Asn Pro Lys Glu Leu Gln Gln Leu
115 120 125 Glu Gln Gln Leu
Asp Ser Ser Leu Lys His Ile Arg Ser Arg Lys Ser 130
135 140 His Leu Met Ala Glu Ser Ile Ser
Glu Leu Gln Lys Lys Glu Arg Ser 145 150
155 160 Leu Gln Glu Glu Asn Lys Ala Leu Gln Lys Glu Leu
Ala Glu Arg Gln 165 170
175 Lys Ala Val Ala Ser Arg Gln Gln Gln Gln Gln Gln Gln Val Gln Trp
180 185 190 Asp Gln Gln
Thr His Ala Gln Ala Gln Thr Ser Ser Ser Ser Ser Ser 195
200 205 Phe Met Met Arg Gln Asp Gln Gln
Gly Leu Pro Pro Pro His Asn Ile 210 215
220 Cys Phe Pro Pro Leu Thr Met Gly Asp Arg Gly Glu Glu
Leu Ala Ala 225 230 235
240 Ala Ala Ala Ala Gln Gln Gln Gln Pro Leu Pro Gly Gln Ala Gln Pro
245 250 255 Gln Leu Arg Ile
Ala Gly Leu Pro Pro Trp Met Leu Ser His Leu Asn 260
265 270 Ala 152786DNALolium temulentum
152atgggtcgcg gcaaggtgca gctgaagcgg atagagaaca agataaaccg tcaggtgaca
60ttctccaagc gccgcaacgg gctactcaag aaggcgcacg agatctccgt cctctgcgac
120gccgaggtcg ccgtcgtcgt cttctccccg aaagggaagc tctatgagta cgccactgac
180tccagcatgg acaaaattct tgaacgttat gaacgctact cttatgctga aaaggctttg
240atttcagctg aatctgaaag tgagggaaat tggtgccatg aatacaggaa gctgaaggcg
300aagattgaga ctatacaaaa atgtcacaag cacctcatgg gggaggatct ggagtgtcta
360aacctgaaag agctccaaca actagagcag cagctggaga gttcattgaa gcacatcaga
420tcgagaaaga gccaccttat gatggagtcc atttctgagc tacagaagaa ggagcggtca
480ctccaggagg agaacaaggc tctacagaag gaactggtgg agaggcagaa ggcggccagg
540cagcagcagc aagagcagtg ggaccgtcag acccaaacac aacaagccca aaaccaacct
600caggcccaga cgagctcatc atcttcctcc ttcatgatga gggatcagca ggcccatgct
660caacaaaaca tctgttaccc gctggtgaca atgggtggag aggctgtggc cgcggcgcca
720gggcagcagg ggcagcttcg catcggaggc ctgccaccat ggatgctgag ccacctcaac
780gcttga
786153261PRTLolium temulentum 153Met Gly Arg Gly Lys Val Gln Leu Lys Arg
Ile Glu Asn Lys Ile Asn 1 5 10
15 Arg Gln Val Thr Phe Ser Lys Arg Arg Asn Gly Leu Leu Lys Lys
Ala 20 25 30 His
Glu Ile Ser Val Leu Cys Asp Ala Glu Val Ala Val Val Val Phe 35
40 45 Ser Pro Lys Gly Lys Leu
Tyr Glu Tyr Ala Thr Asp Ser Ser Met Asp 50 55
60 Lys Ile Leu Glu Arg Tyr Glu Arg Tyr Ser Tyr
Ala Glu Lys Ala Leu 65 70 75
80 Ile Ser Ala Glu Ser Glu Ser Glu Gly Asn Trp Cys His Glu Tyr Arg
85 90 95 Lys Leu
Lys Ala Lys Ile Glu Thr Ile Gln Lys Cys His Lys His Leu 100
105 110 Met Gly Glu Asp Leu Glu Cys
Leu Asn Leu Lys Glu Leu Gln Gln Leu 115 120
125 Glu Gln Gln Leu Glu Ser Ser Leu Lys His Ile Arg
Ser Arg Lys Ser 130 135 140
His Leu Met Met Glu Ser Ile Ser Glu Leu Gln Lys Lys Glu Arg Ser 145
150 155 160 Leu Gln Glu
Glu Asn Lys Ala Leu Gln Lys Glu Leu Val Glu Arg Gln 165
170 175 Lys Ala Ala Arg Gln Gln Gln Gln
Glu Gln Trp Asp Arg Gln Thr Gln 180 185
190 Thr Gln Gln Ala Gln Asn Gln Pro Gln Ala Gln Thr Ser
Ser Ser Ser 195 200 205
Ser Ser Phe Met Met Arg Asp Gln Gln Ala His Ala Gln Gln Asn Ile 210
215 220 Cys Tyr Pro Leu
Val Thr Met Gly Gly Glu Ala Val Ala Ala Ala Pro 225 230
235 240 Gly Gln Gln Gly Gln Leu Arg Ile Gly
Gly Leu Pro Pro Trp Met Leu 245 250
255 Ser His Leu Asn Ala 260 154786DNALolium
perenne 154atgggtcgcg gcaaggtgca gctgaagcgg atagagaaca agataaaccg
tcaggtgacc 60ttctccaagc gccgcaacgg gctactcaag aaggcgcacg agatctccgt
cctctgcgac 120gccgaggtcg ccgtcgtcgt cttctccccg aaagggaagc tctatgagta
cgccactgac 180tccagcatgg acaaaattct tgaacgttat gaacgctact cttatgctga
aaaggctttg 240atttcagctg aatctgaaag tgagggaaat tggtgccatg aatacaggaa
actgaaggcg 300aagattgaga ctatacaaaa atgtcacaag cacctcatgg gggaggatct
ggagtgtcta 360aacctgaaag agctccaaca actagagcag cagctggaga gttcattgaa
gcacatcaga 420tcgagaaaga gccaccttat gatggagtcc atttctgagc tacagaagaa
ggagcggtca 480ctccaggagg agaacaaggc tctacagaag gaactggtgg agaggcagaa
ggcggccagg 540cagcagcagc aagagcagtg ggaccgtcag acccaaacac aacaagccca
aaaccaacct 600caggcccaga cgagctcatc atcttcctcc ttcatgatga gggatcagca
ggcccatgct 660caacaaaaca tctgttaccc gccggtgaca atgggtggag aggctgtggc
cgcggcgcca 720gggcagcagg ggcagcttcg catcggaggc ctgccaccat ggatgctgag
ccacctcaac 780gcttga
786155261PRTLolium perenne 155Met Gly Arg Gly Lys Val Gln Leu
Lys Arg Ile Glu Asn Lys Ile Asn 1 5 10
15 Arg Gln Val Thr Phe Ser Lys Arg Arg Asn Gly Leu Leu
Lys Lys Ala 20 25 30
His Glu Ile Ser Val Leu Cys Asp Ala Glu Val Ala Val Val Val Phe
35 40 45 Ser Pro Lys Gly
Lys Leu Tyr Glu Tyr Ala Thr Asp Ser Ser Met Asp 50
55 60 Lys Ile Leu Glu Arg Tyr Glu Arg
Tyr Ser Tyr Ala Glu Lys Ala Leu 65 70
75 80 Ile Ser Ala Glu Ser Glu Ser Glu Gly Asn Trp Cys
His Glu Tyr Arg 85 90
95 Lys Leu Lys Ala Lys Ile Glu Thr Ile Gln Lys Cys His Lys His Leu
100 105 110 Met Gly Glu
Asp Leu Glu Cys Leu Asn Leu Lys Glu Leu Gln Gln Leu 115
120 125 Glu Gln Gln Leu Glu Ser Ser Leu
Lys His Ile Arg Ser Arg Lys Ser 130 135
140 His Leu Met Met Glu Ser Ile Ser Glu Leu Gln Lys Lys
Glu Arg Ser 145 150 155
160 Leu Gln Glu Glu Asn Lys Ala Leu Gln Lys Glu Leu Val Glu Arg Gln
165 170 175 Lys Ala Ala Arg
Gln Gln Gln Gln Glu Gln Trp Asp Arg Gln Thr Gln 180
185 190 Thr Gln Gln Ala Gln Asn Gln Pro Gln
Ala Gln Thr Ser Ser Ser Ser 195 200
205 Ser Ser Phe Met Met Arg Asp Gln Gln Ala His Ala Gln Gln
Asn Ile 210 215 220
Cys Tyr Pro Pro Val Thr Met Gly Gly Glu Ala Val Ala Ala Ala Pro 225
230 235 240 Gly Gln Gln Gly Gln
Leu Arg Ile Gly Gly Leu Pro Pro Trp Met Leu 245
250 255 Ser His Leu Asn Ala 260
156831DNAHordeum vulgare 156atgggtcgcg gtaaggtgca gctgaagcgg atagagaaca
agataaatcg gcaggtgacc 60ttctccaagc gccgcaacgg gctcctgaag aaggcgcacg
agatctccgt cctctgtgac 120gcagaggtcg ccgtcatcgt cttctccccc aaaggcaagc
tctatgagta cgccaccgac 180tccagcatgg acaaaattct tgaacgttat gagcgctact
cttatgctga aaaggctctt 240atttcagctg aatctgaaag tgaggggaat tggtgtcatg
aatacaggaa acttaaggcg 300aagattgaga ccatacagaa gtgtcacaag cacctcatgg
gagaggatct ggattctctg 360aacctcaaag aactccaaca actggagcag cagctggaga
gttcattgaa gcacatcaga 420tcgagaaaga gccatcttat gatggagtcc atttctgagc
tacagaagaa ggagaggtca 480ctgcaggagg agaacaaggc cctacagaag gaactggtgg
agaggcagaa ggcggccagc 540aggcagcagc agctgcagca gcagcaacaa caacaacaaa
tgcaatggga gcaccaagcc 600cagacccaaa cccataccca tactcaaaac cagccccaag
cccagactag ctcatcatct 660tcctctttca tgatgaggga tcagcaggcc catgcccctc
aacagaacat ttgtagctac 720ccaccggtga cgatgggtgg ggaggcgacg gcggcggcgg
cggcgccgga gcagcaggct 780cagcttcgca tatgcctacc gccatggatg ctgagccacc
tcaacgcttg a 831157276PRTHordeum vulgare 157Met Gly Arg Gly
Lys Val Gln Leu Lys Arg Ile Glu Asn Lys Ile Asn 1 5
10 15 Arg Gln Val Thr Phe Ser Lys Arg Arg
Asn Gly Leu Leu Lys Lys Ala 20 25
30 His Glu Ile Ser Val Leu Cys Asp Ala Glu Val Ala Val Ile
Val Phe 35 40 45
Ser Pro Lys Gly Lys Leu Tyr Glu Tyr Ala Thr Asp Ser Ser Met Asp 50
55 60 Lys Ile Leu Glu Arg
Tyr Glu Arg Tyr Ser Tyr Ala Glu Lys Ala Leu 65 70
75 80 Ile Ser Ala Glu Ser Glu Ser Glu Gly Asn
Trp Cys His Glu Tyr Arg 85 90
95 Lys Leu Lys Ala Lys Ile Glu Thr Ile Gln Lys Cys His Lys His
Leu 100 105 110 Met
Gly Glu Asp Leu Asp Ser Leu Asn Leu Lys Glu Leu Gln Gln Leu 115
120 125 Glu Gln Gln Leu Glu Ser
Ser Leu Lys His Ile Arg Ser Arg Lys Ser 130 135
140 His Leu Met Met Glu Ser Ile Ser Glu Leu Gln
Lys Lys Glu Arg Ser 145 150 155
160 Leu Gln Glu Glu Asn Lys Ala Leu Gln Lys Glu Leu Val Glu Arg Gln
165 170 175 Lys Ala
Ala Ser Arg Gln Gln Gln Leu Gln Gln Gln Gln Gln Gln Gln 180
185 190 Gln Met Gln Trp Glu His Gln
Ala Gln Thr Gln Thr His Thr His Thr 195 200
205 Gln Asn Gln Pro Gln Ala Gln Thr Ser Ser Ser Ser
Ser Ser Phe Met 210 215 220
Met Arg Asp Gln Gln Ala His Ala Pro Gln Gln Asn Ile Cys Ser Tyr 225
230 235 240 Pro Pro Val
Thr Met Gly Gly Glu Ala Thr Ala Ala Ala Ala Ala Pro 245
250 255 Glu Gln Gln Ala Gln Leu Arg Ile
Cys Leu Pro Pro Trp Met Leu Ser 260 265
270 His Leu Asn Ala 275 158804DNAOryza
sativa 158atggggcggg ggaaggtgca gctgaagcgg atagagaact cgatgaaccg
gcaggtgacg 60ttctccaaga ggaggaatgg attgctgaag aaggcgcacg agatctccgt
cctctgcgac 120gccgaggtcg ccgccatcgt cttctccccc aagggcaagc tctacgagta
cgccactgac 180tccaggatgg acaaaatcct tgaacgttat gagcgctatt catatgctga
aaaggctctt 240atttcagctg aatctgagag tgagggaaat tggtgccatg aatacaggaa
gcttaaggca 300aagattgaga ccatacaaaa atgtcacaaa cacctcatgg gagaggatct
agaatccctg 360aatctcaaag aactccaaca gctagagcag cagctggaga gttcattgaa
gcacataaga 420tcaagaaaga gccaccttat gcttgagtcc atttccgagc tgcagaaaaa
ggagaggtca 480ctgcaggagg agaacaaggc tctgcagaag gaactggtgg agaggcagaa
gaatgtgagg 540ggccagcagc aagtagggca gtgggaccaa acccaggtcc aggcccaggc
ccaagcccaa 600ccccaagccc agacaagctt cttcttcttc ttcatgctga gggatcagca
ggcacttctt 660tcaccacaaa atatctgcta cccgccggtg atgatgggcc agagaaatga
tgcggcggcg 720cggcggcggt ggcggcccaa ggccaggtgc aacttccgca ttggaggctt
tccgccatgg 780atgctgagca ccttcaaggc ttaa
804159267PRTOryza sativa 159Met Gly Arg Gly Lys Val Gln Leu
Lys Arg Ile Glu Asn Ser Met Asn 1 5 10
15 Arg Gln Val Thr Phe Ser Lys Arg Arg Asn Gly Leu Leu
Lys Lys Ala 20 25 30
His Glu Ile Ser Val Leu Cys Asp Ala Glu Val Ala Ala Ile Val Phe
35 40 45 Ser Pro Lys Gly
Lys Leu Tyr Glu Tyr Ala Thr Asp Ser Arg Met Asp 50
55 60 Lys Ile Leu Glu Arg Tyr Glu Arg
Tyr Ser Tyr Ala Glu Lys Ala Leu 65 70
75 80 Ile Ser Ala Glu Ser Glu Ser Glu Gly Asn Trp Cys
His Glu Tyr Arg 85 90
95 Lys Leu Lys Ala Lys Ile Glu Thr Ile Gln Lys Cys His Lys His Leu
100 105 110 Met Gly Glu
Asp Leu Glu Ser Leu Asn Leu Lys Glu Leu Gln Gln Leu 115
120 125 Glu Gln Gln Leu Glu Ser Ser Leu
Lys His Ile Arg Ser Arg Lys Ser 130 135
140 His Leu Met Leu Glu Ser Ile Ser Glu Leu Gln Lys Lys
Glu Arg Ser 145 150 155
160 Leu Gln Glu Glu Asn Lys Ala Leu Gln Lys Glu Leu Val Glu Arg Gln
165 170 175 Lys Asn Val Arg
Gly Gln Gln Gln Val Gly Gln Trp Asp Gln Thr Gln 180
185 190 Val Gln Ala Gln Ala Gln Ala Gln Pro
Gln Ala Gln Thr Ser Phe Phe 195 200
205 Phe Phe Phe Met Leu Arg Asp Gln Gln Ala Leu Leu Ser Pro
Gln Asn 210 215 220
Ile Cys Tyr Pro Pro Val Met Met Gly Gln Arg Asn Asp Ala Ala Ala 225
230 235 240 Arg Arg Arg Trp Arg
Pro Lys Ala Arg Cys Asn Phe Arg Ile Gly Gly 245
250 255 Phe Pro Pro Trp Met Leu Ser Thr Phe Lys
Ala 260 265 160804DNAOryza sativa
160atggggcggg ggaaggtgca gctgaagcgg atagagaaca agatcaacag gcaggtgacg
60ttctccaaga ggaggaatgg attgctgaag aaggcgcacg agatctccgt cctctgcgac
120gccgaggtcg ccgccatcgt cttctccccc aagggcaagc tctacgagta cgccactgac
180tccaggatgg acaaaatcct tgaacgttat gagcgctatt catatgctga aaaggctctt
240atttcagctg aatccgagag tgagggaaat tggtgccatg aatacaggaa acttaaggca
300aagattgaga ccatacaaaa atgtcacaaa cacctcatgg gagaggatca tgaatccctg
360aatctcaaag aactccaaca gctagagcag cagctggaga gttcattgaa gcacataata
420tcaagaaaga gccaccttat gcttgagtcc atttccgagc tgcagaaaaa ggagaggtca
480ctgcaggagg agaacaaggc tctgcagaag gaactggtgg agaggcagaa gaatgtgagg
540ggccagcagc aagtagggca gtgggaccaa acccaggtcc aggcccaggc ccaagcccaa
600ccccaagccc agacaagctc ctcctcctcc tccatgctga gggatcagca ggcacttctt
660ccaccacaaa atatctgcta cccgccggtg atgatgggcg agagaaatga tgcggcggcg
720gcggcggcgg tggcggcgca gggccaggtg caactccgca tcggaggtct tccgccatgg
780atgctgagcc acctcaatgc ttaa
804161267PRTOryza sativa 161Met Gly Arg Gly Lys Val Gln Leu Lys Arg Ile
Glu Asn Lys Ile Asn 1 5 10
15 Arg Gln Val Thr Phe Ser Lys Arg Arg Asn Gly Leu Leu Lys Lys Ala
20 25 30 His Glu
Ile Ser Val Leu Cys Asp Ala Glu Val Ala Ala Ile Val Phe 35
40 45 Ser Pro Lys Gly Lys Leu Tyr
Glu Tyr Ala Thr Asp Ser Arg Met Asp 50 55
60 Lys Ile Leu Glu Arg Tyr Glu Arg Tyr Ser Tyr
Ala Glu Lys Ala Leu 65 70 75
80 Ile Ser Ala Glu Ser Glu Ser Glu Gly Asn Trp Cys His Glu Tyr Arg
85 90 95 Lys Leu
Lys Ala Lys Ile Glu Thr Ile Gln Lys Cys His Lys His Leu 100
105 110 Met Gly Glu Asp His Glu Ser
Leu Asn Leu Lys Glu Leu Gln Gln Leu 115 120
125 Glu Gln Gln Leu Glu Ser Ser Leu Lys His Ile Ile
Ser Arg Lys Ser 130 135 140
His Leu Met Leu Glu Ser Ile Ser Glu Leu Gln Lys Lys Glu Arg Ser 145
150 155 160 Leu Gln Glu
Glu Asn Lys Ala Leu Gln Lys Glu Leu Val Glu Arg Gln 165
170 175 Lys Asn Val Arg Gly Gln Gln Gln
Val Gly Gln Trp Asp Gln Thr Gln 180 185
190 Val Gln Ala Gln Ala Gln Ala Gln Pro Gln Ala Gln Thr
Ser Ser Ser 195 200 205
Ser Ser Ser Met Leu Arg Asp Gln Gln Ala Leu Leu Pro Pro Gln Asn 210
215 220 Ile Cys Tyr Pro
Pro Val Met Met Gly Glu Arg Asn Asp Ala Ala Ala 225 230
235 240 Ala Ala Ala Val Ala Ala Gln Gly Gln
Val Gln Leu Arg Ile Gly Gly 245 250
255 Leu Pro Pro Trp Met Leu Ser His Leu Asn Ala
260 265 162668DNATradescantia virginiana
162gggctggtga agaaggctca tgagatctcd atkytrtgtg atgcygaggt tgcgcttatt
60attttctcta ctaaaggcaa actatacgag tatgccactg attccaaaat ggaaaatatc
120cttgaacgct atgaacgtta ctcatatgct gagaaggctt taacttcatc agatcctgaa
180ttacagggaa attggtgcca agagtatgtt aaacttaagg ctaaggttga ggccttacat
240aaaagccaaa ggcatcttat gggagagcaa ctagaagcgt tggatctcaa agaattgcag
300caactagagc atcaacttga aggttctttg aggcttgtca ggtcaagaaa gactcaaatg
360atgttggact ccatttccga acttcagagg aaggaaaagt ctctggaaga gcaaaacaag
420aacctagaga aggagatttt ggagaagcag aaagaaaagg ctctggcaca ccaagctcac
480tgggaacagc agaatcagcc actacaaagc actaattcgc ctccaaggcc cttcgtgatt
540gcagaaactc atccaacact aaacattgga aatttccaag gtagaacaaa taccgtccat
600gcagaagaaa gtctgcagcg tcagatgagg atcagcagca gcctactgcc mycmtggatg
660mttcacma
668163222PRTTradescantia virginianaUNSURE(11)..(11)Xaa can be any
naturally occurring amino acid 163Gly Leu Val Lys Lys Ala His Glu Ile Ser
Xaa Leu Cys Asp Ala Glu 1 5 10
15 Val Ala Leu Ile Ile Phe Ser Thr Lys Gly Lys Leu Tyr Glu Tyr
Ala 20 25 30 Thr
Asp Ser Lys Met Glu Asn Ile Leu Glu Arg Tyr Glu Arg Tyr Ser 35
40 45 Tyr Ala Glu Lys Ala Leu
Thr Ser Ser Asp Pro Glu Leu Gln Gly Asn 50 55
60 Trp Cys Gln Glu Tyr Val Lys Leu Lys Ala Lys
Val Glu Ala Leu His 65 70 75
80 Lys Ser Gln Arg His Leu Met Gly Glu Gln Leu Glu Ala Leu Asp Leu
85 90 95 Lys Glu
Leu Gln Gln Leu Glu His Gln Leu Glu Gly Ser Leu Arg Leu 100
105 110 Val Arg Ser Arg Lys Thr Gln
Met Met Leu Asp Ser Ile Ser Glu Leu 115 120
125 Gln Arg Lys Glu Lys Ser Leu Glu Glu Gln Asn Lys
Asn Leu Glu Lys 130 135 140
Glu Ile Leu Glu Lys Gln Lys Glu Lys Ala Leu Ala His Gln Ala His 145
150 155 160 Trp Glu Gln
Gln Asn Gln Pro Leu Gln Ser Thr Asn Ser Pro Pro Arg 165
170 175 Pro Phe Val Ile Ala Glu Thr His
Pro Thr Leu Asn Ile Gly Asn Phe 180 185
190 Gln Gly Arg Thr Asn Thr Val His Ala Glu Glu Ser Leu
Gln Arg Gln 195 200 205
Met Arg Ile Ser Ser Ser Leu Leu Pro Xaa Trp Met Xaa His 210
215 220 164723DNATradescantia virginiana
164gtgcagctga aacggatgga gaacaagatt aacaggcagg tgacgttttc taaacgtcga
60ggagggctgc tgaagaaagc tcatgagatc tctattctat gtgatgctga gattgctctt
120attattttct ctactaaagg gaagctctat gagtatgcca ccaattccaa aatggacaat
180attcttgaac gctatgagcg ttactcatat gctgaaaagg ctctaacttc atcagatcct
240gatatacagg gaaattggtg ccaagagtat gctaaactta agtctaaggt tgaggcttta
300tgtaaaagcc aaaggcatct tatgggagag cagcttgaaa cattgaatct caaagaattg
360cagcaactag agcaacagct cgaaggttct ctaaagcatg tcaggtcaag aaagactcaa
420gttatgctgg actctatttc tgaacttcag aggaaggaaa agtcactaga ggagcaaaac
480aagaacctag agaaggagat tttggagaag cagaaaatca aggctcttgc acagcaggct
540cactgggaac accagaatca accagcacca aggggttcac ctcctaggcc atttgtgatt
600gcagagtctc atccgacact aaatattgga catttccaag gcaggacaaa tgcagtcgaa
660gcagaagaaa atcagcagcc tcakatgaga atttgcagta gcctcctgcc cccctggatg
720ctt
723165241PRTTradescantia virginianaUNSURE(228)..(228)Xaa can be any
naturally occurring amino acid 165Val Gln Leu Lys Arg Met Glu Asn Lys Ile
Asn Arg Gln Val Thr Phe 1 5 10
15 Ser Lys Arg Arg Gly Gly Leu Leu Lys Lys Ala His Glu Ile Ser
Ile 20 25 30 Leu
Cys Asp Ala Glu Ile Ala Leu Ile Ile Phe Ser Thr Lys Gly Lys 35
40 45 Leu Tyr Glu Tyr Ala Thr
Asn Ser Lys Met Asp Asn Ile Leu Glu Arg 50 55
60 Tyr Glu Arg Tyr Ser Tyr Ala Glu Lys Ala Leu
Thr Ser Ser Asp Pro 65 70 75
80 Asp Ile Gln Gly Asn Trp Cys Gln Glu Tyr Ala Lys Leu Lys Ser Lys
85 90 95 Val Glu
Ala Leu Cys Lys Ser Gln Arg His Leu Met Gly Glu Gln Leu 100
105 110 Glu Thr Leu Asn Leu Lys Glu
Leu Gln Gln Leu Glu Gln Gln Leu Glu 115 120
125 Gly Ser Leu Lys His Val Arg Ser Arg Lys Thr Gln
Val Met Leu Asp 130 135 140
Ser Ile Ser Glu Leu Gln Arg Lys Glu Lys Ser Leu Glu Glu Gln Asn 145
150 155 160 Lys Asn Leu
Glu Lys Glu Ile Leu Glu Lys Gln Lys Ile Lys Ala Leu 165
170 175 Ala Gln Gln Ala His Trp Glu His
Gln Asn Gln Pro Ala Pro Arg Gly 180 185
190 Ser Pro Pro Arg Pro Phe Val Ile Ala Glu Ser His Pro
Thr Leu Asn 195 200 205
Ile Gly His Phe Gln Gly Arg Thr Asn Ala Val Glu Ala Glu Glu Asn 210
215 220 Gln Gln Pro Xaa
Met Arg Ile Cys Ser Ser Leu Leu Pro Pro Trp Met 225 230
235 240 Leu 166599DNATradescantia
virginiana 166gggctkgtga agaaagctca tgagatctcg gtactttgtg atgctgagct
tgctcttatt 60atcttctctc ccaaaggcaa gctctatgag tatgccaccg attccaaaat
ggaaattatt 120cttgaacgct atgaacgtta cacctacgct gaaaaagctt taattgcatc
agatcctgat 180gtacagggaa actggtgtca tgagtacatt aagcttaaag ctaaatttga
ggccttgaat 240aaaagccaga ggcatcttat gggagaacaa ctagatacgt tgaaccaaaa
ggaattgctg 300caactagaga ctaagcttga aggttctctg aaaaacgtca ggtcaagaaa
gactcaactt 360atgttggatt ccatttctga gcttcaagaa aagggaaagt cactccagga
gcaaaacacc 420tgcctagaaa aggagatttt gggaaaacag aaagacaagg ctcccaaaca
gcatgttcag 480tgggaaaaac agaatcaacc accacctacc tcttctgcgc caatgccatt
cctcattggt 540gatattcacc caacccctaa tatcagaaat ttccaaggca gaacagtagc
tgatgcaga 599167199PRTTradescantia virginiana 167Gly Leu Val Lys Lys
Ala His Glu Ile Ser Val Leu Cys Asp Ala Glu 1 5
10 15 Leu Ala Leu Ile Ile Phe Ser Pro Lys Gly
Lys Leu Tyr Glu Tyr Ala 20 25
30 Thr Asp Ser Lys Met Glu Ile Ile Leu Glu Arg Tyr Glu Arg Tyr
Thr 35 40 45 Tyr
Ala Glu Lys Ala Leu Ile Ala Ser Asp Pro Asp Val Gln Gly Asn 50
55 60 Trp Cys His Glu Tyr Ile
Lys Leu Lys Ala Lys Phe Glu Ala Leu Asn 65 70
75 80 Lys Ser Gln Arg His Leu Met Gly Glu Gln Leu
Asp Thr Leu Asn Gln 85 90
95 Lys Glu Leu Leu Gln Leu Glu Thr Lys Leu Glu Gly Ser Leu Lys Asn
100 105 110 Val Arg
Ser Arg Lys Thr Gln Leu Met Leu Asp Ser Ile Ser Glu Leu 115
120 125 Gln Glu Lys Gly Lys Ser Leu
Gln Glu Gln Asn Thr Cys Leu Glu Lys 130 135
140 Glu Ile Leu Gly Lys Gln Lys Asp Lys Ala Pro Lys
Gln His Val Gln 145 150 155
160 Trp Glu Lys Gln Asn Gln Pro Pro Pro Thr Ser Ser Ala Pro Met Pro
165 170 175 Phe Leu Ile
Gly Asp Ile His Pro Thr Pro Asn Ile Arg Asn Phe Gln 180
185 190 Gly Arg Thr Val Ala Asp Ala
195 168753DNAElaeis guineensis 168atggggagag
ggagggtgca gctgaggcgg atcgagaaca agataaaccg gcaggtgacg 60ttctcgaagc
gccggtcggg gctcctgaag aaagcccacg agatctccgt cctctgcgac 120gccgaggtcg
ccctcatcat cttctcgacc aagggcaagc tctacgagta cgccaccgac 180tcctgcatgg
aaaggattct tgaacgctat gaacgttaca cctatgcaga aaaagcacta 240atttcatctg
gacccgaatt gcagggtaac tggtgccatg aatttggcaa actcaaagct 300aaggttgagg
ctttacaaaa aagccaaagg catctcatgg gtgagcaact tgagcccttg 360aatctcaaag
aactccagca actagagcaa cagcttgaaa gttctttaaa gcatataaga 420accagaaagt
gccaactcat gtttgaatcc atctctgagc ttcaaaaaaa ggaaaagtca 480ctgcaggagc
agaacaagat gctggagaag gagctcatgg agaagcagaa ggtgaaggca 540ctaaaccagc
aggcaccttg ggagcagcaa ggcccgccgc agacaagctc atcatcccca 600acctccttcc
tgatcggaga ctctctcccc accctgaata ttgggacata ccaatgtagc 660ggaaatgaac
atggggagga agcagcacaa ccccaggttc gtataggaaa cagcctgtta 720ccaccttgga
tgcttagcca cttgaacggg tag
753169250PRTElaeis guineensis 169Met Gly Arg Gly Arg Val Gln Leu Arg Arg
Ile Glu Asn Lys Ile Asn 1 5 10
15 Arg Gln Val Thr Phe Ser Lys Arg Arg Ser Gly Leu Leu Lys Lys
Ala 20 25 30 His
Glu Ile Ser Val Leu Cys Asp Ala Glu Val Ala Leu Ile Ile Phe 35
40 45 Ser Thr Lys Gly Lys Leu
Tyr Glu Tyr Ala Thr Asp Ser Cys Met Glu 50 55
60 Arg Ile Leu Glu Arg Tyr Glu Arg Tyr Thr Tyr
Ala Glu Lys Ala Leu 65 70 75
80 Ile Ser Ser Gly Pro Glu Leu Gln Gly Asn Trp Cys His Glu Phe Gly
85 90 95 Lys Leu
Lys Ala Lys Val Glu Ala Leu Gln Lys Ser Gln Arg His Leu 100
105 110 Met Gly Glu Gln Leu Glu Pro
Leu Asn Leu Lys Glu Leu Gln Gln Leu 115 120
125 Glu Gln Gln Leu Glu Ser Ser Leu Lys His Ile Arg
Thr Arg Lys Cys 130 135 140
Gln Leu Met Phe Glu Ser Ile Ser Glu Leu Gln Lys Lys Glu Lys Ser 145
150 155 160 Leu Gln Glu
Gln Asn Lys Met Leu Glu Lys Glu Leu Met Glu Lys Gln 165
170 175 Lys Val Lys Ala Leu Asn Gln Gln
Ala Pro Trp Glu Gln Gln Gly Pro 180 185
190 Pro Gln Thr Ser Ser Ser Ser Pro Thr Ser Phe Leu Ile
Gly Asp Ser 195 200 205
Leu Pro Thr Leu Asn Ile Gly Thr Tyr Gln Cys Ser Gly Asn Glu His 210
215 220 Gly Glu Glu Ala
Ala Gln Pro Gln Val Arg Ile Gly Asn Ser Leu Leu 225 230
235 240 Pro Pro Trp Met Leu Ser His Leu Asn
Gly 245 250 170699DNAAllium sp.
170gtgcaattga agaggatgga aaacaagatt aatagacaag tgaccttctc aaaaagaaga
60aatggtttgt tgaagaaagc tcatgagatt tcwgtgcttt gtgatgcaga agttgcactt
120attgttttct ctgctaaagg aaaactctat gaatattcaa ctgattcaag tatggaaaaa
180attctggaga ggtatgaacg ttattgcttt gcggagaaat catcaacaat gagtgacatt
240gactcccagg aggattggag ccttgaatat cacaaactga aggctaaggt tgagagttta
300aacaacaggc aaaggcatct tatgggagag caacttgaat ctctgagtct tcgagaaatt
360ggacagcttg agcaacaact tgagaattct ctcaaaactg ttcggacgcg caagagccaa
420gaattgttaa gttctatttc agagcttcag gacaaggaga aaactttgcg agatgagaac
480aaagctttag aaaatgagct tatgaaaagg gccagggcaa aagctattct ggaacaacaa
540gcacgatgga agcatcataa tcataaacaa caggataatc ttcataatcc aaatatcaac
600attggaaatt accaaacaag gaacaatgag ggaggagttg agccagcaac ggatgttcaa
660gtacgtgttg ttagaaattt gttgccccac tggatgctt
699171233PRTAllium sp. 171Val Gln Leu Lys Arg Met Glu Asn Lys Ile Asn Arg
Gln Val Thr Phe 1 5 10
15 Ser Lys Arg Arg Asn Gly Leu Leu Lys Lys Ala His Glu Ile Ser Val
20 25 30 Leu Cys Asp
Ala Glu Val Ala Leu Ile Val Phe Ser Ala Lys Gly Lys 35
40 45 Leu Tyr Glu Tyr Ser Thr Asp Ser
Ser Met Glu Lys Ile Leu Glu Arg 50 55
60 Tyr Glu Arg Tyr Cys Phe Ala Glu Lys Ser Ser Thr Met
Ser Asp Ile 65 70 75
80 Asp Ser Gln Glu Asp Trp Ser Leu Glu Tyr His Lys Leu Lys Ala Lys
85 90 95 Val Glu Ser Leu
Asn Asn Arg Gln Arg His Leu Met Gly Glu Gln Leu 100
105 110 Glu Ser Leu Ser Leu Arg Glu Ile Gly
Gln Leu Glu Gln Gln Leu Glu 115 120
125 Asn Ser Leu Lys Thr Val Arg Thr Arg Lys Ser Gln Glu Leu
Leu Ser 130 135 140
Ser Ile Ser Glu Leu Gln Asp Lys Glu Lys Thr Leu Arg Asp Glu Asn 145
150 155 160 Lys Ala Leu Glu Asn
Glu Leu Met Lys Arg Ala Arg Ala Lys Ala Ile 165
170 175 Leu Glu Gln Gln Ala Arg Trp Lys His His
Asn His Lys Gln Gln Asp 180 185
190 Asn Leu His Asn Pro Asn Ile Asn Ile Gly Asn Tyr Gln Thr Arg
Asn 195 200 205 Asn
Glu Gly Gly Val Glu Pro Ala Thr Asp Val Gln Val Arg Val Val 210
215 220 Arg Asn Leu Leu Pro His
Trp Met Leu 225 230 172744DNADendrobium grex
Madame Thong-IN 172atgggtcgtg gcagggtgca gctgaagcga atcgagaata aaataaaccg
gcaggtgacg 60ttctcgaagc ggagatctgg tttgcttaag aaggcgcacg agatctccgt
gctctgtgac 120gctgaagttg ctctgatcgt tttttccaat aagggaaagc tttatgagta
ttccaccgat 180tccagcatgg agaaaattct tgaacggtat gagcgttatt catatgctga
aagagcatta 240ttttccaatg aggccaaccc ccaggctgat tggcgccttg aatataataa
actgaaggca 300agggttgaaa gcttacagaa gagccaaagg caccttatgg gggagcaact
tgactccttg 360agcattaaag aactccaacg tctagagcaa cagcttgaaa gttccttgaa
gtttatacga 420tccagaaaga cacagctcat actacattca atttccgagc tacaaaagat
ggaaaaaata 480ttgctggagc aaaacaagac cttagagaag gagattatag ctaaagagaa
agccaaagct 540ttggtgcagc atgccccatg ggagaagcaa aaccagtccc aatatagctc
tgcactcccg 600cctgtgattt cggattctgt cccaactccc accagcagaa cgtttcaagc
cagagccaat 660gaagaagaat cacctcagcc acagttaaga gtaagcaaca ctctgctgcc
cccatggatg 720ctcagtcata tgaatggaca ataa
744173247PRTDendrobium grex Madame Thong-IN 173Met Gly Arg
Gly Arg Val Gln Leu Lys Arg Ile Glu Asn Lys Ile Asn 1 5
10 15 Arg Gln Val Thr Phe Ser Lys Arg
Arg Ser Gly Leu Leu Lys Lys Ala 20 25
30 His Glu Ile Ser Val Leu Cys Asp Ala Glu Val Ala Leu
Ile Val Phe 35 40 45
Ser Asn Lys Gly Lys Leu Tyr Glu Tyr Ser Thr Asp Ser Ser Met Glu 50
55 60 Lys Ile Leu Glu
Arg Tyr Glu Arg Tyr Ser Tyr Ala Glu Arg Ala Leu 65 70
75 80 Phe Ser Asn Glu Ala Asn Pro Gln Ala
Asp Trp Arg Leu Glu Tyr Asn 85 90
95 Lys Leu Lys Ala Arg Val Glu Ser Leu Gln Lys Ser Gln Arg
His Leu 100 105 110
Met Gly Glu Gln Leu Asp Ser Leu Ser Ile Lys Glu Leu Gln Arg Leu
115 120 125 Glu Gln Gln Leu
Glu Ser Ser Leu Lys Phe Ile Arg Ser Arg Lys Thr 130
135 140 Gln Leu Ile Leu His Ser Ile Ser
Glu Leu Gln Lys Met Glu Lys Ile 145 150
155 160 Leu Leu Glu Gln Asn Lys Thr Leu Glu Lys Glu Ile
Ile Ala Lys Glu 165 170
175 Lys Ala Lys Ala Leu Val Gln His Ala Pro Trp Glu Lys Gln Asn Gln
180 185 190 Ser Gln Tyr
Ser Ser Ala Leu Pro Pro Val Ile Ser Asp Ser Val Pro 195
200 205 Thr Pro Thr Ser Arg Thr Phe Gln
Ala Arg Ala Asn Glu Glu Glu Ser 210 215
220 Pro Gln Pro Gln Leu Arg Val Ser Asn Thr Leu Leu Pro
Pro Trp Met 225 230 235
240 Leu Ser His Met Asn Gly Gln 245
1742194DNAOryza sativa 174aatccgaaaa gtttctgcac cgttttcacc ccctaactaa
caatataggg aacgtgtgct 60aaatataaaa tgagacctta tatatgtagc gctgataact
agaactatgc aagaaaaact 120catccaccta ctttagtggc aatcgggcta aataaaaaag
agtcgctaca ctagtttcgt 180tttccttagt aattaagtgg gaaaatgaaa tcattattgc
ttagaatata cgttcacatc 240tctgtcatga agttaaatta ttcgaggtag ccataattgt
catcaaactc ttcttgaata 300aaaaaatctt tctagctgaa ctcaatgggt aaagagagag
atttttttta aaaaaataga 360atgaagatat tctgaacgta ttggcaaaga tttaaacata
taattatata attttatagt 420ttgtgcattc gtcatatcgc acatcattaa ggacatgtct
tactccatcc caatttttat 480ttagtaatta aagacaattg acttattttt attatttatc
ttttttcgat tagatgcaag 540gtacttacgc acacactttg tgctcatgtg catgtgtgag
tgcacctcct caatacacgt 600tcaactagca acacatctct aatatcactc gcctatttaa
tacatttagg tagcaatatc 660tgaattcaag cactccacca tcaccagacc acttttaata
atatctaaaa tacaaaaaat 720aattttacag aatagcatga aaagtatgaa acgaactatt
taggtttttc acatacaaaa 780aaaaaaagaa ttttgctcgt gcgcgagcgc caatctccca
tattgggcac acaggcaaca 840acagagtggc tgcccacaga acaacccaca aaaaacgatg
atctaacgga ggacagcaag 900tccgcaacaa ccttttaaca gcaggctttg cggccaggag
agaggaggag aggcaaagaa 960aaccaagcat cctccttctc ccatctataa attcctcccc
ccttttcccc tctctatata 1020ggaggcatcc aagccaagaa gagggagagc accaaggaca
cgcgactagc agaagccgag 1080cgaccgcctt ctcgatccat atcttccggt cgagttcttg
gtcgatctct tccctcctcc 1140acctcctcct cacagggtat gtgcctccct tcggttgttc
ttggatttat tgttctaggt 1200tgtgtagtac gggcgttgat gttaggaaag gggatctgta
tctgtgatga ttcctgttct 1260tggatttggg atagaggggt tcttgatgtt gcatgttatc
ggttcggttt gattagtagt 1320atggttttca atcgtctgga gagctctatg gaaatgaaat
ggtttaggga tcggaatctt 1380gcgattttgt gagtaccttt tgtttgaggt aaaatcagag
caccggtgat tttgcttggt 1440gtaataaagt acggttgttt ggtcctcgat tctggtagtg
atgcttctcg atttgacgaa 1500gctatccttt gtttattccc tattgaacaa aaataatcca
actttgaaga cggtcccgtt 1560gatgagattg aatgattgat tcttaagcct gtccaaaatt
tcgcagctgg cttgtttaga 1620tacagtagtc cccatcacga aattcatgga aacagttata
atcctcagga acaggggatt 1680ccctgttctt ccgatttgct ttagtcccag aatttttttt
cccaaatatc ttaaaaagtc 1740actttctggt tcagttcaat gaattgattg ctacaaataa
tgcttttata gcgttatcct 1800agctgtagtt cagttaatag gtaatacccc tatagtttag
tcaggagaag aacttatccg 1860atttctgatc tccattttta attatatgaa atgaactgta
gcataagcag tattcatttg 1920gattattttt tttattagct ctcacccctt cattattctg
agctgaaagt ctggcatgaa 1980ctgtcctcaa ttttgttttc aaattcacat cgattatcta
tgcattatcc tcttgtatct 2040acctgtagaa gtttcttttt ggttattcct tgactgcttg
attacagaaa gaaatttatg 2100aagctgtaat cgggatagtt atactgcttg ttcttatgat
tcatttcctt tgtgcagttc 2160ttggtgtagc ttgccacttt caccagcaaa gttc
21941751725DNAArabidopsis thaliana 175atgaattcta
acaactggct tggctttcct ctttcaccga acaactcttc tttgcctcct 60catgaataca
accttggctt ggtcagcgac catatggaca acccttttca aacacaagag 120tggaatatga
tcaatccaca cggtggagga ggagatgaag gaggagaggt tccaaaagtg 180gccgattttc
tcggtgtgag caaaccggac gaaaaccaat ccaaccacct agtagcttac 240aacgactcag
actactactt ccataccaat agcttgatgc ctagcgtcca atcaaacgat 300gtcgttgtag
cagcttgtga ctccaatact cctaacaaca gtagctatca tgagcttcaa 360gagagtgctc
acaatctaca gtcacttact ttgtccatgg ggaccaccgc tggtaataat 420gttgtagaca
aagcttcacc atccgagacc accggggata acgctagcgg tggagcacta 480gccgttgttg
agacggccac gccaagacgt gcattggaca ctttcggaca acgaacctcg 540atctatcgtg
gtgtcacaag acatcgatgg actggtcgat atgaggctca tctatgggat 600aatagttgta
gaagggaagg ccagtctagg aaaggaagac aagtttactt gggtggatat 660gataaagaag
ataaagcagc aagatcatat gatctagctg cacttaagta ctggggtccc 720tcaactacta
ctaatttccc cattacaaac tacgagaaag aagtagagga aatgaagcac 780atgacgaggc
aagagttcgt ggctgccatt agaaggaaaa gtagtggatt ttcgagaggc 840gcttcgatgt
atcgaggagt tacaaggcac caccaacatg gaagatggca agcaaggatc 900ggccgagtcg
ccgggaacaa agacctctac ttgggaactt ttagcactga ggaagaagca 960gcagaagctt
acgatatagc tgcaataaag tttagaggac ttaatgcagt gaccaacttc 1020gagatcaacc
ggtacgacgt gaaagccatt ctagagagta gcactcttcc catcggagga 1080ggcgcagcta
aacggctcaa agaagctcaa gctcttgagt cttcaaggaa acgcgaggcg 1140gagatgatag
cccttggttc aagtttccag tacggtggtg gctcgagcac aggctctggc 1200tccacctcat
caagacttca gcttcaacct taccctctaa gcattcaaca accattagag 1260ccttttctat
ctcttcagaa caatgacatc tctcattaca acaacaacaa tgctcacgat 1320tcctcctctt
ttaatcacca tagctatatc cagacacaac ttcatctcca ccaacagacc 1380aacaattact
tgcagcaaca gtcgagccag aactctcagc agctctacaa tgcgtatctt 1440catagcaatc
cggctctgct tcatggactt gtctctacct ctatcgttga caacaataat 1500aacaatggag
gctctagtgg gagctacaac actgcagcat ttcttgggaa ccacggtatt 1560ggtattgggt
ccagctcgac tgttggatcg accgaggagt ttccaaccgt taaaacagat 1620tacgatatgc
cttccagtga tggaaccgga gggtatagtg gttggaccag tgagtctgtt 1680caggggtcaa
accctggtgg tgttttcact atgtggaatg agtaa
1725176574PRTArabidopsis thaliana 176Met Asn Ser Asn Asn Trp Leu Gly Phe
Pro Leu Ser Pro Asn Asn Ser 1 5 10
15 Ser Leu Pro Pro His Glu Tyr Asn Leu Gly Leu Val Ser Asp
His Met 20 25 30
Asp Asn Pro Phe Gln Thr Gln Glu Trp Asn Met Ile Asn Pro His Gly
35 40 45 Gly Gly Gly Asp
Glu Gly Gly Glu Val Pro Lys Val Ala Asp Phe Leu 50
55 60 Gly Val Ser Lys Pro Asp Glu Asn
Gln Ser Asn His Leu Val Ala Tyr 65 70
75 80 Asn Asp Ser Asp Tyr Tyr Phe His Thr Asn Ser Leu
Met Pro Ser Val 85 90
95 Gln Ser Asn Asp Val Val Val Ala Ala Cys Asp Ser Asn Thr Pro Asn
100 105 110 Asn Ser Ser
Tyr His Glu Leu Gln Glu Ser Ala His Asn Leu Gln Ser 115
120 125 Leu Thr Leu Ser Met Gly Thr Thr
Ala Gly Asn Asn Val Val Asp Lys 130 135
140 Ala Ser Pro Ser Glu Thr Thr Gly Asp Asn Ala Ser Gly
Gly Ala Leu 145 150 155
160 Ala Val Val Glu Thr Ala Thr Pro Arg Arg Ala Leu Asp Thr Phe Gly
165 170 175 Gln Arg Thr Ser
Ile Tyr Arg Gly Val Thr Arg His Arg Trp Thr Gly 180
185 190 Arg Tyr Glu Ala His Leu Trp Asp Asn
Ser Cys Arg Arg Glu Gly Gln 195 200
205 Ser Arg Lys Gly Arg Gln Val Tyr Leu Gly Gly Tyr Asp Lys
Glu Asp 210 215 220
Lys Ala Ala Arg Ser Tyr Asp Leu Ala Ala Leu Lys Tyr Trp Gly Pro 225
230 235 240 Ser Thr Thr Thr Asn
Phe Pro Ile Thr Asn Tyr Glu Lys Glu Val Glu 245
250 255 Glu Met Lys His Met Thr Arg Gln Glu Phe
Val Ala Ala Ile Arg Arg 260 265
270 Lys Ser Ser Gly Phe Ser Arg Gly Ala Ser Met Tyr Arg Gly Val
Thr 275 280 285 Arg
His His Gln His Gly Arg Trp Gln Ala Arg Ile Gly Arg Val Ala 290
295 300 Gly Asn Lys Asp Leu Tyr
Leu Gly Thr Phe Ser Thr Glu Glu Glu Ala 305 310
315 320 Ala Glu Ala Tyr Asp Ile Ala Ala Ile Lys Phe
Arg Gly Leu Asn Ala 325 330
335 Val Thr Asn Phe Glu Ile Asn Arg Tyr Asp Val Lys Ala Ile Leu Glu
340 345 350 Ser Ser
Thr Leu Pro Ile Gly Gly Gly Ala Ala Lys Arg Leu Lys Glu 355
360 365 Ala Gln Ala Leu Glu Ser Ser
Arg Lys Arg Glu Ala Glu Met Ile Ala 370 375
380 Leu Gly Ser Ser Phe Gln Tyr Gly Gly Gly Ser Ser
Thr Gly Ser Gly 385 390 395
400 Ser Thr Ser Ser Arg Leu Gln Leu Gln Pro Tyr Pro Leu Ser Ile Gln
405 410 415 Gln Pro Leu
Glu Pro Phe Leu Ser Leu Gln Asn Asn Asp Ile Ser His 420
425 430 Tyr Asn Asn Asn Asn Ala His Asp
Ser Ser Ser Phe Asn His His Ser 435 440
445 Tyr Ile Gln Thr Gln Leu His Leu His Gln Gln Thr Asn
Asn Tyr Leu 450 455 460
Gln Gln Gln Ser Ser Gln Asn Ser Gln Gln Leu Tyr Asn Ala Tyr Leu 465
470 475 480 His Ser Asn Pro
Ala Leu Leu His Gly Leu Val Ser Thr Ser Ile Val 485
490 495 Asp Asn Asn Asn Asn Asn Gly Gly Ser
Ser Gly Ser Tyr Asn Thr Ala 500 505
510 Ala Phe Leu Gly Asn His Gly Ile Gly Ile Gly Ser Ser Ser
Thr Val 515 520 525
Gly Ser Thr Glu Glu Phe Pro Thr Val Lys Thr Asp Tyr Asp Met Pro 530
535 540 Ser Ser Asp Gly Thr
Gly Gly Tyr Ser Gly Trp Thr Ser Glu Ser Val 545 550
555 560 Gln Gly Ser Asn Pro Gly Gly Val Phe Thr
Met Trp Asn Glu 565 570
1771707DNAArabidopsis thaliana 177atgaattcta acaactggct cgcgttccct
ctatcaccaa ctcactcttc tttgccgcct 60cacattcact cttcacaaaa ttctcatttc
aatctaggtt tggtcaacga caatatcgac 120aacccttttc aaaaccaagg atggaatatg
atcaatccac atggtggagg cggcgaaggt 180ggagaggttc caaaagtggc tgatttctta
ggagtgagca aatcggggga tcatcacacc 240gatcacaacc tcgtacctta taacgacatt
catcaaacca acgcctccga ctactacttt 300caaaccaata gcttgttacc tacagtcgtc
acttgtgcct ctaatgctcc taataattat 360gagcttcaag agagtgcaca caatttgcaa
tctctcactc tctctatggg aagtactgga 420gctgccgctg cagaagtcgc cactgtgaaa
gcctcgccgg ctgagactag tgccgataat 480agtagcagca ctaccaacac aagcggagga
gccatcgttg aggctacacc gagacggact 540ttggaaactt ttggacaacg aacctctatc
tatcgtggag ttacaagaca tagatggacc 600ggtagatatg aagctcatct ttgggataat
agctgtagaa gagaaggaca atcaaggaaa 660ggaagacaag tctacttagg tgggtatgac
aaagaagaga aagcagccag agcatatgat 720ctagctgcac ttaaatattg gggtccctct
actactacca actttccgat aactaactac 780gagaaggaag tagaggagat gaaaaacatg
acgagacaag agtttgtggc ttctataaga 840aggaagagta gcggattctc gcgtggtgca
tccatgtatc gtggagtaac aaggcatcat 900caacatggaa gatggcaagc aaggatcggc
cgagttgctg gaaacaaaga tctctacttg 960ggaacattca gcacggagga agaagcagca
gaagcttatg acatagctgc gataaagttt 1020cgaggtctaa acgcggttac aaactttgag
ataaatcggt atgatgtgaa agccatcctg 1080gagagcaaca cacttcctat aggaggtggt
gcggctaaac ggctcaaaga agctcaagct 1140ctagaatcat caagaaaacg agaggaaatg
atagccctcg gatcaaattt ccatcaatat 1200ggtgcagcga gcggctcgag ctctgttgct
tccagctcta ggcttcagct tcaaccttac 1260cctctaagca ttcaacaacc ttttgagcat
cttcatcatc atcagccttt acttactcta 1320cagaacaaca acgatatctc tcagtatcat
gattccttta gttacattca gacgcagctt 1380catcttcacc aacaacaaac caacaattac
ttgcagtctt ctagtcacac ttcacagctc 1440tacaatgctt atcttcagag taaccctggt
ctgcttcatg gatttgtctc tgataataac 1500aacacttcag ggtttcttgg aaacaatggg
attggtattg ggtcaagctc taccgttgga 1560tcatcggctg aggaagagtt tccagccgtg
aaagtcgatt acgatatgcc tccttccggt 1620ggagctacag ggtatggagg atggaatagt
ggagagtctg ctcaaggatc gaatccagga 1680ggtgttttca cgatgtggaa tgaataa
1707178568PRTArabidopsis thaliana 178Met
Asn Ser Asn Asn Trp Leu Ala Phe Pro Leu Ser Pro Thr His Ser 1
5 10 15 Ser Leu Pro Pro His Ile
His Ser Ser Gln Asn Ser His Phe Asn Leu 20
25 30 Gly Leu Val Asn Asp Asn Ile Asp Asn Pro
Phe Gln Asn Gln Gly Trp 35 40
45 Asn Met Ile Asn Pro His Gly Gly Gly Gly Glu Gly Gly Glu
Val Pro 50 55 60
Lys Val Ala Asp Phe Leu Gly Val Ser Lys Ser Gly Asp His His Thr 65
70 75 80 Asp His Asn Leu Val
Pro Tyr Asn Asp Ile His Gln Thr Asn Ala Ser 85
90 95 Asp Tyr Tyr Phe Gln Thr Asn Ser Leu Leu
Pro Thr Val Val Thr Cys 100 105
110 Ala Ser Asn Ala Pro Asn Asn Tyr Glu Leu Gln Glu Ser Ala His
Asn 115 120 125 Leu
Gln Ser Leu Thr Leu Ser Met Gly Ser Thr Gly Ala Ala Ala Ala 130
135 140 Glu Val Ala Thr Val Lys
Ala Ser Pro Ala Glu Thr Ser Ala Asp Asn 145 150
155 160 Ser Ser Ser Thr Thr Asn Thr Ser Gly Gly Ala
Ile Val Glu Ala Thr 165 170
175 Pro Arg Arg Thr Leu Glu Thr Phe Gly Gln Arg Thr Ser Ile Tyr Arg
180 185 190 Gly Val
Thr Arg His Arg Trp Thr Gly Arg Tyr Glu Ala His Leu Trp 195
200 205 Asp Asn Ser Cys Arg Arg Glu
Gly Gln Ser Arg Lys Gly Arg Gln Val 210 215
220 Tyr Leu Gly Gly Tyr Asp Lys Glu Glu Lys Ala Ala
Arg Ala Tyr Asp 225 230 235
240 Leu Ala Ala Leu Lys Tyr Trp Gly Pro Ser Thr Thr Thr Asn Phe Pro
245 250 255 Ile Thr Asn
Tyr Glu Lys Glu Val Glu Glu Met Lys Asn Met Thr Arg 260
265 270 Gln Glu Phe Val Ala Ser Ile Arg
Arg Lys Ser Ser Gly Phe Ser Arg 275 280
285 Gly Ala Ser Met Tyr Arg Gly Val Thr Arg His His Gln
His Gly Arg 290 295 300
Trp Gln Ala Arg Ile Gly Arg Val Ala Gly Asn Lys Asp Leu Tyr Leu 305
310 315 320 Gly Thr Phe Ser
Thr Glu Glu Glu Ala Ala Glu Ala Tyr Asp Ile Ala 325
330 335 Ala Ile Lys Phe Arg Gly Leu Asn Ala
Val Thr Asn Phe Glu Ile Asn 340 345
350 Arg Tyr Asp Val Lys Ala Ile Leu Glu Ser Asn Thr Leu Pro
Ile Gly 355 360 365
Gly Gly Ala Ala Lys Arg Leu Lys Glu Ala Gln Ala Leu Glu Ser Ser 370
375 380 Arg Lys Arg Glu Glu
Met Ile Ala Leu Gly Ser Asn Phe His Gln Tyr 385 390
395 400 Gly Ala Ala Ser Gly Ser Ser Ser Val Ala
Ser Ser Ser Arg Leu Gln 405 410
415 Leu Gln Pro Tyr Pro Leu Ser Ile Gln Gln Pro Phe Glu His Leu
His 420 425 430 His
His Gln Pro Leu Leu Thr Leu Gln Asn Asn Asn Asp Ile Ser Gln 435
440 445 Tyr His Asp Ser Phe Ser
Tyr Ile Gln Thr Gln Leu His Leu His Gln 450 455
460 Gln Gln Thr Asn Asn Tyr Leu Gln Ser Ser Ser
His Thr Ser Gln Leu 465 470 475
480 Tyr Asn Ala Tyr Leu Gln Ser Asn Pro Gly Leu Leu His Gly Phe Val
485 490 495 Ser Asp
Asn Asn Asn Thr Ser Gly Phe Leu Gly Asn Asn Gly Ile Gly 500
505 510 Ile Gly Ser Ser Ser Thr Val
Gly Ser Ser Ala Glu Glu Glu Phe Pro 515 520
525 Ala Val Lys Val Asp Tyr Asp Met Pro Pro Ser Gly
Gly Ala Thr Gly 530 535 540
Tyr Gly Gly Trp Asn Ser Gly Glu Ser Ala Gln Gly Ser Asn Pro Gly 545
550 555 560 Gly Val Phe
Thr Met Trp Asn Glu 565 1791662DNAGlycine max
179atgaacaaca actggctttc gttccctctt tctcctactc attcttcctt accagctcat
60gatcttcaag caactcaata tcatcaattt tcccttgggt tagtgaacga gaacatggat
120aaccctttcc aaaatcatga ttggaatctg attaacaccc atagtagcaa cgaaattcca
180aaagtggctg attttctagg agtgagcaag tctgaaaatc agtcagacct tgcagcctta
240aacgaaattc attcaaatga ttcagattat ctgttcacaa acaacagtct ggtgcctatg
300caaaaccctg tgttggacac acctagcaat gagtatcaag aaaatgctaa tagtaatttg
360caatcattga cattatccat gggaagtggt aaggattcaa catgtgaaac cagtggtgaa
420aatagcacaa acactactgt tgaagttgca cctagaagaa ctttggatac attcgggcag
480agaacatcca tatatcgtgg agtaactcga catagatgga ctggaaggta tgaagctcat
540ctttgggata atagctgtag aagggaaggc caatcaagaa aaggacgcca agtttatttg
600ggtggatatg ataaagaaga gaaagcagct agagcttatg atttagctgc actgaagtac
660tgggggacat ccaccactac caactttcca attagcaact atgagaagga attggatgaa
720atgaaacaca tgacgagaca agaatttgtt gccgccatta gaaggaaaag cagtggtttc
780tccaggggtg catcaatgta tcgtggagtt acaaggcatc accaacacgg aagatggcaa
840gcaaggattg gcagagttgc aggaaacaaa gatctttact tgggaacttt cagtactgag
900gaagaggctg cagaagcata cgacatagca gcgataaagt tcagaggtct caacgctgtc
960acaaactttg acatgagccg ctacgacgtg aaagccattc ttgaaagcaa cactctccca
1020ataggaggag gcgctgcaaa gcgtctgaaa gaagctcaag ctctagaatc ttcgagaaaa
1080cgcgaagaga tgattgcact aggctcatct tccacgttcc aatacggaac ctcagcaagc
1140tcttctaggc ttcacgctta ccctctaatg cagcaccacc accagttcga gcaacctcaa
1200cctctgctaa ctcttcaaaa ccacgacata agttcttctc acttctctca ccagcaagac
1260cctttgcatc atcagggtta catccaaacg cagcttcagt tgcaccagca gagtggcgct
1320tcttcttata gctttcagaa taatgctcag ttctacaatg gttaccttca gaaccaccct
1380gcattgcttc agggaatgat gaacatgggg tcttcttctt cttcctcatc tgtgttggag
1440aataataata gtaacaataa taataataat gttggtgggt ttgtgggaag tgggtttggt
1500atggcttcga atgcaacggc ggggaacacg gtggggacag cggaggagtt agggctggtg
1560aaggtggact atgacatgcc ggctggaggt tacggtggct ggtcggcggc ggactccatg
1620cagacgtcaa atggtggggt gttcacaatg tggaatgatt aa
1662180553PRTGlycine max 180Met Asn Asn Asn Trp Leu Ser Phe Pro Leu Ser
Pro Thr His Ser Ser 1 5 10
15 Leu Pro Ala His Asp Leu Gln Ala Thr Gln Tyr His Gln Phe Ser Leu
20 25 30 Gly Leu
Val Asn Glu Asn Met Asp Asn Pro Phe Gln Asn His Asp Trp 35
40 45 Asn Leu Ile Asn Thr His Ser
Ser Asn Glu Ile Pro Lys Val Ala Asp 50 55
60 Phe Leu Gly Val Ser Lys Ser Glu Asn Gln Ser Asp
Leu Ala Ala Leu 65 70 75
80 Asn Glu Ile His Ser Asn Asp Ser Asp Tyr Leu Phe Thr Asn Asn Ser
85 90 95 Leu Val Pro
Met Gln Asn Pro Val Leu Asp Thr Pro Ser Asn Glu Tyr 100
105 110 Gln Glu Asn Ala Asn Ser Asn Leu
Gln Ser Leu Thr Leu Ser Met Gly 115 120
125 Ser Gly Lys Asp Ser Thr Cys Glu Thr Ser Gly Glu Asn
Ser Thr Asn 130 135 140
Thr Thr Val Glu Val Ala Pro Arg Arg Thr Leu Asp Thr Phe Gly Gln 145
150 155 160 Arg Thr Ser Ile
Tyr Arg Gly Val Thr Arg His Arg Trp Thr Gly Arg 165
170 175 Tyr Glu Ala His Leu Trp Asp Asn Ser
Cys Arg Arg Glu Gly Gln Ser 180 185
190 Arg Lys Gly Arg Gln Val Tyr Leu Gly Gly Tyr Asp Lys Glu
Glu Lys 195 200 205
Ala Ala Arg Ala Tyr Asp Leu Ala Ala Leu Lys Tyr Trp Gly Thr Ser 210
215 220 Thr Thr Thr Asn Phe
Pro Ile Ser Asn Tyr Glu Lys Glu Leu Asp Glu 225 230
235 240 Met Lys His Met Thr Arg Gln Glu Phe Val
Ala Ala Ile Arg Arg Lys 245 250
255 Ser Ser Gly Phe Ser Arg Gly Ala Ser Met Tyr Arg Gly Val Thr
Arg 260 265 270 His
His Gln His Gly Arg Trp Gln Ala Arg Ile Gly Arg Val Ala Gly 275
280 285 Asn Lys Asp Leu Tyr Leu
Gly Thr Phe Ser Thr Glu Glu Glu Ala Ala 290 295
300 Glu Ala Tyr Asp Ile Ala Ala Ile Lys Phe Arg
Gly Leu Asn Ala Val 305 310 315
320 Thr Asn Phe Asp Met Ser Arg Tyr Asp Val Lys Ala Ile Leu Glu Ser
325 330 335 Asn Thr
Leu Pro Ile Gly Gly Gly Ala Ala Lys Arg Leu Lys Glu Ala 340
345 350 Gln Ala Leu Glu Ser Ser Arg
Lys Arg Glu Glu Met Ile Ala Leu Gly 355 360
365 Ser Ser Ser Thr Phe Gln Tyr Gly Thr Ser Ala Ser
Ser Ser Arg Leu 370 375 380
His Ala Tyr Pro Leu Met Gln His His His Gln Phe Glu Gln Pro Gln 385
390 395 400 Pro Leu Leu
Thr Leu Gln Asn His Asp Ile Ser Ser Ser His Phe Ser 405
410 415 His Gln Gln Asp Pro Leu His His
Gln Gly Tyr Ile Gln Thr Gln Leu 420 425
430 Gln Leu His Gln Gln Ser Gly Ala Ser Ser Tyr Ser Phe
Gln Asn Asn 435 440 445
Ala Gln Phe Tyr Asn Gly Tyr Leu Gln Asn His Pro Ala Leu Leu Gln 450
455 460 Gly Met Met Asn
Met Gly Ser Ser Ser Ser Ser Ser Ser Val Leu Glu 465 470
475 480 Asn Asn Asn Ser Asn Asn Asn Asn Asn
Asn Val Gly Gly Phe Val Gly 485 490
495 Ser Gly Phe Gly Met Ala Ser Asn Ala Thr Ala Gly Asn Thr
Val Gly 500 505 510
Thr Ala Glu Glu Leu Gly Leu Val Lys Val Asp Tyr Asp Met Pro Ala
515 520 525 Gly Gly Tyr Gly
Gly Trp Ser Ala Ala Asp Ser Met Gln Thr Ser Asn 530
535 540 Gly Gly Val Phe Thr Met Trp Asn
Asp 545 550 1811674DNAGlycine max
181atgaacaaca actggctttc gttccctctt tctcctactc attcttcctt accagctcat
60gatcttcaag caactcaata tcatcaattt tcccttgggt tagtgaacga gaacatggat
120aaccctttcc aaaatcatga ttggaatctg attaacaccc atagtagcaa cgaaattcca
180aaagtggctg attttctagg agtgagcaag tctgaaaatc agtcagacct tgcagcctta
240aacgaaattc attcaaatga ttcagattat ctgttcacaa acaacagtct ggtgcctatg
300caaaaccctg tgttggacac acctagcaat gagtatcaag aaaatgctaa tagtaatttg
360caatcattga cattatccat gggaagtggt aaggattcaa catgtgaaac cagtggtgaa
420aatagcacaa acactactgt tgaagttgca cctagaagaa ctttggatac attcgggcag
480agaacatcca tatatcgtgg agtaactcga catagatgga ctggaaggta tgaagctcat
540ctttgggata atagctgtag aagggaaggc caatcaagaa aaggacgcca agtttatttg
600ggtggatatg ataaagaaga gaaagcagct agggcttatg atttagctgc actgaagtac
660tgggggacat ccaccactac caactttcca attagtaact atgagaagga attggatgaa
720atgaaacaca tgacgcgaca agaatttgtt gctgccatta gaaggaaaag cagtggtttc
780tccaggggtg catcaatgta tcgtggagtt acaaggcatc accaacacgg aagatggcaa
840gcaagaattg gcagagttgc aggaaacaaa gatctttact tgggaacttt cagtactgaa
900gaagaggctg ctgaagcata cgacatagct gcgataaagt tcagaggtct caacgctgtc
960acaaactttg acatgagccg ctacgacgtg aaagccatcc ttgaaagcaa cactctccca
1020ataggaggag gagctgcaaa gcgtctgaaa gaagctcaag ctctagaatc ttcgagaaag
1080cgcgaagaga tgattgcact aggatcatcc acattccaat atggaaccac aagctctaat
1140tctaggctac atgcttaccc tctaatgcag caccaccacc agtttgaaca acctcaacct
1200ctgctaactt tgcaaaacca tgatatcagt tctcacttct ctcaccagca agaccctttg
1260catcagggtt acatccaaac gcagcttcag ttgcaccagc agcagagtgg tggttcttct
1320tcttatagct ttcagaataa taatataaat aatgctcagt tctataatgg ttataatctt
1380cagaaccacc ctgcattgct tcagggaatg attaacatgg ggtcttcatc ttcttcatct
1440gtgttggaga ataataatag taccaataat aatgttggtg ggtttgtggg aagtgggttt
1500ggtatggctt ctaatgcaac gtcggggaac acggtgggga cggcggagga gctagggctg
1560gtgaaggtgg actatgacat gccgactggt ggttacggtg gatggtcggc ggcggcggcg
1620gcggagtcca tgcagacgtc gaatagtggg gtgttcacaa tgtggaatga ctga
1674182557PRTGlycine max 182Met Asn Asn Asn Trp Leu Ser Phe Pro Leu Ser
Pro Thr His Ser Ser 1 5 10
15 Leu Pro Ala His Asp Leu Gln Ala Thr Gln Tyr His Gln Phe Ser Leu
20 25 30 Gly Leu
Val Asn Glu Asn Met Asp Asn Pro Phe Gln Asn His Asp Trp 35
40 45 Asn Leu Ile Asn Thr His Ser
Ser Asn Glu Ile Pro Lys Val Ala Asp 50 55
60 Phe Leu Gly Val Ser Lys Ser Glu Asn Gln Ser Asp
Leu Ala Ala Leu 65 70 75
80 Asn Glu Ile His Ser Asn Asp Ser Asp Tyr Leu Phe Thr Asn Asn Ser
85 90 95 Leu Val Pro
Met Gln Asn Pro Val Leu Asp Thr Pro Ser Asn Glu Tyr 100
105 110 Gln Glu Asn Ala Asn Ser Asn Leu
Gln Ser Leu Thr Leu Ser Met Gly 115 120
125 Ser Gly Lys Asp Ser Thr Cys Glu Thr Ser Gly Glu Asn
Ser Thr Asn 130 135 140
Thr Thr Val Glu Val Ala Pro Arg Arg Thr Leu Asp Thr Phe Gly Gln 145
150 155 160 Arg Thr Ser Ile
Tyr Arg Gly Val Thr Arg His Arg Trp Thr Gly Arg 165
170 175 Tyr Glu Ala His Leu Trp Asp Asn Ser
Cys Arg Arg Glu Gly Gln Ser 180 185
190 Arg Lys Gly Arg Gln Val Tyr Leu Gly Gly Tyr Asp Lys Glu
Glu Lys 195 200 205
Ala Ala Arg Ala Tyr Asp Leu Ala Ala Leu Lys Tyr Trp Gly Thr Ser 210
215 220 Thr Thr Thr Asn Phe
Pro Ile Ser Asn Tyr Glu Lys Glu Leu Asp Glu 225 230
235 240 Met Lys His Met Thr Arg Gln Glu Phe Val
Ala Ala Ile Arg Arg Lys 245 250
255 Ser Ser Gly Phe Ser Arg Gly Ala Ser Met Tyr Arg Gly Val Thr
Arg 260 265 270 His
His Gln His Gly Arg Trp Gln Ala Arg Ile Gly Arg Val Ala Gly 275
280 285 Asn Lys Asp Leu Tyr Leu
Gly Thr Phe Ser Thr Glu Glu Glu Ala Ala 290 295
300 Glu Ala Tyr Asp Ile Ala Ala Ile Lys Phe Arg
Gly Leu Asn Ala Val 305 310 315
320 Thr Asn Phe Asp Met Ser Arg Tyr Asp Val Lys Ala Ile Leu Glu Ser
325 330 335 Asn Thr
Leu Pro Ile Gly Gly Gly Ala Ala Lys Arg Leu Lys Glu Ala 340
345 350 Gln Ala Leu Glu Ser Ser Arg
Lys Arg Glu Glu Met Ile Ala Leu Gly 355 360
365 Ser Ser Thr Phe Gln Tyr Gly Thr Thr Ser Ser Asn
Ser Arg Leu His 370 375 380
Ala Tyr Pro Leu Met Gln His His His Gln Phe Glu Gln Pro Gln Pro 385
390 395 400 Leu Leu Thr
Leu Gln Asn His Asp Ile Ser Ser His Phe Ser His Gln 405
410 415 Gln Asp Pro Leu His Gln Gly Tyr
Ile Gln Thr Gln Leu Gln Leu His 420 425
430 Gln Gln Gln Ser Gly Gly Ser Ser Ser Tyr Ser Phe Gln
Asn Asn Asn 435 440 445
Ile Asn Asn Ala Gln Phe Tyr Asn Gly Tyr Asn Leu Gln Asn His Pro 450
455 460 Ala Leu Leu Gln
Gly Met Ile Asn Met Gly Ser Ser Ser Ser Ser Ser 465 470
475 480 Val Leu Glu Asn Asn Asn Ser Thr Asn
Asn Asn Val Gly Gly Phe Val 485 490
495 Gly Ser Gly Phe Gly Met Ala Ser Asn Ala Thr Ser Gly Asn
Thr Val 500 505 510
Gly Thr Ala Glu Glu Leu Gly Leu Val Lys Val Asp Tyr Asp Met Pro
515 520 525 Thr Gly Gly Tyr
Gly Gly Trp Ser Ala Ala Ala Ala Ala Glu Ser Met 530
535 540 Gln Thr Ser Asn Ser Gly Val Phe
Thr Met Trp Asn Asp 545 550 555
1831632DNAMedicago trunculata 183atgaacaata actggctttc attccctctc
tcaccttctc attcttcctt accttctaat 60gatcttcaag caactcaata tcatcacttt
cctcttggat tagtcaatga caacatggaa 120aaccctttcc aaaatcatga ttggaatctg
atgaacacac acaacagcaa tgaagttcca 180aaggttgcgg attttctcgg tgtatgcaag
tctgaaaatc actcagatct tgctacaccg 240aacgaaattc aatctaatga ttcagattat
ctgtttacaa ataacaatac tctcatgcca 300atgcaaaacc aaatggttac aacatgcacc
aatgagtatc aagaaaaggc tagtaatagt 360aatttgcagt ctttgacatt atccatggga
agtggtaaag attcaacatg tgaaactagt 420ggtgaaaata gtacaaacac tgttgaagtt
gctgttccta aaagaacttc agagacattt 480ggacaaagaa cttcgatata tcgcggtgta
acaaaacata gatggactgg aaggtatgaa 540gctcaccttt gggataacag ctgtagaagg
gaaggtcagt cgagaaaagg ccgccaaggt 600ggatatgata aagaagagaa agctgctagg
tcttatgatt tagctgcact taagtactgg 660gggacatcca ccactaccaa ctttccagtt
agcaactatg agaaggaaat agatgaaatg 720aagcacatga caagacaaga atttgttgcc
tctattagaa ggaaaagcag cggtttctct 780aggggtgcat caatgtaccg tggagttaca
aggcatcacc aacatggaag atggcaagca 840aggattggca gagttgcagg aaataaagat
ctatacttgg gaactttcag cactgaagaa 900gaggctgcag aagcatacga catagcagca
ataaaattca gaggactcaa cgctgtaaca 960aactttgaca tgactcgtta cgacgtgaaa
gccattctcg aaagcaacac actgccaatt 1020ggaggaggag ctgcaaaaag actaaaagaa
gcacaagctc tagaaacttc gagaaaacgc 1080gaagaaatgc ttgcacttaa ctcatcatct
ttccaatatg gaacatcaag ctctagtaac 1140actagactcc aaccctaccc tctcatgcaa
tatcatcacc aatttgaaca acctcaacca 1200ttgctaacat tacaaaacaa ccatgaaagc
ttgaattctc aacaattctc tcaacaccaa 1260ggtggtggtt atttccaaac acagcttgag
ttgtgtcaac aacaaaacca acaaccatct 1320cagaatagta acataggttc attctacaat
ggttattatc agaatcatcc tggtttgttt 1380cagatgaata atataggatc ttcttcttca
tcttcggtga tgggaaataa tggtggtggt 1440tctagtggga tttatagtaa tagtggaggg
ttaattagta ataatgctgt tgaggaattt 1500gtgccggtta aggttgatta tgacatgcaa
ggtgatggaa gtggttttgg cggctggtcg 1560gcggcaggag agaacatgca gactgctgat
ttgtttacaa tgtggaatga ctatgagaca 1620agagagaatt ag
1632184557PRTMedicago trunculata 184Met
Asn Asn Asn Trp Leu Ser Phe Pro Leu Ser Pro Thr His Ser Ser 1
5 10 15 Leu Pro Ala His Asp Leu
Gln Ala Thr Gln Tyr His Gln Phe Ser Leu 20
25 30 Gly Leu Val Asn Glu Asn Met Asp Asn Pro
Phe Gln Asn His Asp Trp 35 40
45 Asn Leu Ile Asn Thr His Ser Ser Asn Glu Ile Pro Lys Val
Ala Asp 50 55 60
Phe Leu Gly Val Ser Lys Ser Glu Asn Gln Ser Asp Leu Ala Ala Leu 65
70 75 80 Asn Glu Ile His Ser
Asn Asp Ser Asp Tyr Leu Phe Thr Asn Asn Ser 85
90 95 Leu Val Pro Met Gln Asn Pro Val Leu Asp
Thr Pro Ser Asn Glu Tyr 100 105
110 Gln Glu Asn Ala Asn Ser Asn Leu Gln Ser Leu Thr Leu Ser Met
Gly 115 120 125 Ser
Gly Lys Asp Ser Thr Cys Glu Thr Ser Gly Glu Asn Ser Thr Asn 130
135 140 Thr Thr Val Glu Val Ala
Pro Arg Arg Thr Leu Asp Thr Phe Gly Gln 145 150
155 160 Arg Thr Ser Ile Tyr Arg Gly Val Thr Arg His
Arg Trp Thr Gly Arg 165 170
175 Tyr Glu Ala His Leu Trp Asp Asn Ser Cys Arg Arg Glu Gly Gln Ser
180 185 190 Arg Lys
Gly Arg Gln Val Tyr Leu Gly Gly Tyr Asp Lys Glu Glu Lys 195
200 205 Ala Ala Arg Ala Tyr Asp Leu
Ala Ala Leu Lys Tyr Trp Gly Thr Ser 210 215
220 Thr Thr Thr Asn Phe Pro Ile Ser Asn Tyr Glu Lys
Glu Leu Asp Glu 225 230 235
240 Met Lys His Met Thr Arg Gln Glu Phe Val Ala Ala Ile Arg Arg Lys
245 250 255 Ser Ser Gly
Phe Ser Arg Gly Ala Ser Met Tyr Arg Gly Val Thr Arg 260
265 270 His His Gln His Gly Arg Trp Gln
Ala Arg Ile Gly Arg Val Ala Gly 275 280
285 Asn Lys Asp Leu Tyr Leu Gly Thr Phe Ser Thr Glu Glu
Glu Ala Ala 290 295 300
Glu Ala Tyr Asp Ile Ala Ala Ile Lys Phe Arg Gly Leu Asn Ala Val 305
310 315 320 Thr Asn Phe Asp
Met Ser Arg Tyr Asp Val Lys Ala Ile Leu Glu Ser 325
330 335 Asn Thr Leu Pro Ile Gly Gly Gly Ala
Ala Lys Arg Leu Lys Glu Ala 340 345
350 Gln Ala Leu Glu Ser Ser Arg Lys Arg Glu Glu Met Ile Ala
Leu Gly 355 360 365
Ser Ser Thr Phe Gln Tyr Gly Thr Thr Ser Ser Asn Ser Arg Leu His 370
375 380 Ala Tyr Pro Leu Met
Gln His His His Gln Phe Glu Gln Pro Gln Pro 385 390
395 400 Leu Leu Thr Leu Gln Asn His Asp Ile Ser
Ser His Phe Ser His Gln 405 410
415 Gln Asp Pro Leu His Gln Gly Tyr Ile Gln Thr Gln Leu Gln Leu
His 420 425 430 Gln
Gln Gln Ser Gly Gly Ser Ser Ser Tyr Ser Phe Gln Asn Asn Asn 435
440 445 Ile Asn Asn Ala Gln Phe
Tyr Asn Gly Tyr Asn Leu Gln Asn His Pro 450 455
460 Ala Leu Leu Gln Gly Met Ile Asn Met Gly Ser
Ser Ser Ser Ser Ser 465 470 475
480 Val Leu Glu Asn Asn Asn Ser Thr Asn Asn Asn Val Gly Gly Phe Val
485 490 495 Gly Ser
Gly Phe Gly Met Ala Ser Asn Ala Thr Ser Gly Asn Thr Val 500
505 510 Gly Thr Ala Glu Glu Leu Gly
Leu Val Lys Val Asp Tyr Asp Met Pro 515 520
525 Thr Gly Gly Tyr Gly Gly Trp Ser Ala Ala Ala Ala
Ala Glu Ser Met 530 535 540
Gln Thr Ser Asn Ser Gly Val Phe Thr Met Trp Asn Asp 545
550 555 1852079DNAOryza sativa 185atggccacca
tgaacaactg gctggccttc tccctctccc cgcaggatca gctcccgccg 60tctcagacca
actccactct catctccgcc gccgccacca ccaccaccgc cggcgactcc 120tccaccggcg
acgtctgctt caacatcccc caagattgga gcatgagggg atcggagctc 180tcggcgctcg
tcgccgagcc gaagctggag gacttcctcg gcggcatctc cttctcggag 240cagcagcatc
atcacggcgg caagggcggc gtgatcccga gcagcgccgc cgcttgctac 300gcgagctccg
gcagcagcgt cggctacctg taccctcctc caagctcatc ctcgctccag 360ttcgccgact
ccgtcatggt ggccacctcc tcgcccgtcg tcgcccacga cggcgtcagc 420ggcggcggca
tggtgagcgc cgccgccgcc gcggcggcca gtggcaacgg cggcattggc 480ctgtccatga
tcaagaactg gctccggagc cagccggcgc cgcagccggc gcaggcgctg 540tctctgtcca
tgaacatggc ggggacgacg acggcgcagg gcggcggcgc catggcgctc 600ctcgccggcg
caggggagcg aggccggacg acgcccgcgt cagagagcct gtccacgtcg 660gcgcacggag
cgacgacggc gacgatggct ggtggtcgca aggagattaa cgaggaaggc 720agcggcagcg
ccggcgccgt ggttgccgtc ggctcggagt caggcggcag cggcgccgtg 780gtggaggccg
gcgcggcggc ggcggcggcg aggaagtccg tcgacacgtt cggccagaga 840acatcgatct
accgcggcgt gacaaggcat agatggacag ggaggtatga ggctcatctt 900tgggacaaca
gctgcagaag agagggccaa actcgcaagg gtcgtcaagg tggttatgac 960aaagaggaaa
aagctgctag agcttatgat ttggctgctc tcaaatactg gggcccgacg 1020acgacgacaa
attttccggt aaataactat gaaaaggagc tggaggagat gaagcacatg 1080acaaggcagg
agttcgtagc ctctttgaga aggaagagca gtggtttctc cagaggtgca 1140tccatttacc
gtggagtaac taggcatcac cagcatggga gatggcaagc aaggatagga 1200agagttgcag
ggaacaagga cctctacttg ggcaccttca gcacgcagga ggaggcggcg 1260gaggcgtacg
acatcgcggc gatcaagttc cgggggctca acgccgtcac caacttcgac 1320atgagccgct
acgacgtcaa gagcatcctc gacagcgctg ccctccccgt cggcaccgcc 1380gccaagcgcc
tcaaggacgc cgaggccgcc gccgcctacg acgtcggccg catcgcctcg 1440cacctcggcg
gcgacggcgc ctacgccgcg cattacggcc accaccacca ctcggccgcc 1500gccgcctggc
cgaccatcgc gttccaggcg gcggcggcgc cgccgccgca cgccgccggg 1560ctttaccacc
cgtacgcgca gccgctgcgt gggtggtgca agcaggagca ggaccacgcc 1620gtgatcgcgg
cggcgcacag cctgcaggat ctccaccacc tcaacctcgg cgccgccgcc 1680gccgcgcatg
acttcttctc gcaggcgatg cagcagcagc acggcctcgg cagcatcgac 1740aacgcgtcgc
tcgagcacag caccggctcc aactccgtcg tctacaacgg cgacaatggc 1800ggcggaggcg
gcggctacat catggcgccg atgagcgccg tgtcggccac ggccaccgcg 1860gtggcgagca
gccacgatca cggcggcgac ggcgggaagc aggtgcagat ggggtacgac 1920agctacctcg
tcggcgcaga cgcctacggc ggcggcggcg ccgggaggat gccatcctgg 1980gcgatgacgc
cggcgtcggc gccggccgcc acgagcagca gcgacatgac cggagtctgc 2040catggcgcac
agctcttcag cgtctggaac gacacataa
2079186692PRTOryza sativa 186Met Ala Thr Met Asn Asn Trp Leu Ala Phe Ser
Leu Ser Pro Gln Asp 1 5 10
15 Gln Leu Pro Pro Ser Gln Thr Asn Ser Thr Leu Ile Ser Ala Ala Ala
20 25 30 Thr Thr
Thr Thr Ala Gly Asp Ser Ser Thr Gly Asp Val Cys Phe Asn 35
40 45 Ile Pro Gln Asp Trp Ser Met
Arg Gly Ser Glu Leu Ser Ala Leu Val 50 55
60 Ala Glu Pro Lys Leu Glu Asp Phe Leu Gly Gly Ile
Ser Phe Ser Glu 65 70 75
80 Gln Gln His His His Gly Gly Lys Gly Gly Val Ile Pro Ser Ser Ala
85 90 95 Ala Ala Cys
Tyr Ala Ser Ser Gly Ser Ser Val Gly Tyr Leu Tyr Pro 100
105 110 Pro Pro Ser Ser Ser Ser Leu Gln
Phe Ala Asp Ser Val Met Val Ala 115 120
125 Thr Ser Ser Pro Val Val Ala His Asp Gly Val Ser Gly
Gly Gly Met 130 135 140
Val Ser Ala Ala Ala Ala Ala Ala Ala Ser Gly Asn Gly Gly Ile Gly 145
150 155 160 Leu Ser Met Ile
Lys Asn Trp Leu Arg Ser Gln Pro Ala Pro Gln Pro 165
170 175 Ala Gln Ala Leu Ser Leu Ser Met Asn
Met Ala Gly Thr Thr Thr Ala 180 185
190 Gln Gly Gly Gly Ala Met Ala Leu Leu Ala Gly Ala Gly Glu
Arg Gly 195 200 205
Arg Thr Thr Pro Ala Ser Glu Ser Leu Ser Thr Ser Ala His Gly Ala 210
215 220 Thr Thr Ala Thr Met
Ala Gly Gly Arg Lys Glu Ile Asn Glu Glu Gly 225 230
235 240 Ser Gly Ser Ala Gly Ala Val Val Ala Val
Gly Ser Glu Ser Gly Gly 245 250
255 Ser Gly Ala Val Val Glu Ala Gly Ala Ala Ala Ala Ala Ala Arg
Lys 260 265 270 Ser
Val Asp Thr Phe Gly Gln Arg Thr Ser Ile Tyr Arg Gly Val Thr 275
280 285 Arg His Arg Trp Thr Gly
Arg Tyr Glu Ala His Leu Trp Asp Asn Ser 290 295
300 Cys Arg Arg Glu Gly Gln Thr Arg Lys Gly Arg
Gln Gly Gly Tyr Asp 305 310 315
320 Lys Glu Glu Lys Ala Ala Arg Ala Tyr Asp Leu Ala Ala Leu Lys Tyr
325 330 335 Trp Gly
Pro Thr Thr Thr Thr Asn Phe Pro Val Asn Asn Tyr Glu Lys 340
345 350 Glu Leu Glu Glu Met Lys His
Met Thr Arg Gln Glu Phe Val Ala Ser 355 360
365 Leu Arg Arg Lys Ser Ser Gly Phe Ser Arg Gly Ala
Ser Ile Tyr Arg 370 375 380
Gly Val Thr Arg His His Gln His Gly Arg Trp Gln Ala Arg Ile Gly 385
390 395 400 Arg Val Ala
Gly Asn Lys Asp Leu Tyr Leu Gly Thr Phe Ser Thr Gln 405
410 415 Glu Glu Ala Ala Glu Ala Tyr Asp
Ile Ala Ala Ile Lys Phe Arg Gly 420 425
430 Leu Asn Ala Val Thr Asn Phe Asp Met Ser Arg Tyr Asp
Val Lys Ser 435 440 445
Ile Leu Asp Ser Ala Ala Leu Pro Val Gly Thr Ala Ala Lys Arg Leu 450
455 460 Lys Asp Ala Glu
Ala Ala Ala Ala Tyr Asp Val Gly Arg Ile Ala Ser 465 470
475 480 His Leu Gly Gly Asp Gly Ala Tyr Ala
Ala His Tyr Gly His His His 485 490
495 His Ser Ala Ala Ala Ala Trp Pro Thr Ile Ala Phe Gln Ala
Ala Ala 500 505 510
Ala Pro Pro Pro His Ala Ala Gly Leu Tyr His Pro Tyr Ala Gln Pro
515 520 525 Leu Arg Gly Trp
Cys Lys Gln Glu Gln Asp His Ala Val Ile Ala Ala 530
535 540 Ala His Ser Leu Gln Asp Leu His
His Leu Asn Leu Gly Ala Ala Ala 545 550
555 560 Ala Ala His Asp Phe Phe Ser Gln Ala Met Gln Gln
Gln His Gly Leu 565 570
575 Gly Ser Ile Asp Asn Ala Ser Leu Glu His Ser Thr Gly Ser Asn Ser
580 585 590 Val Val Tyr
Asn Gly Asp Asn Gly Gly Gly Gly Gly Gly Tyr Ile Met 595
600 605 Ala Pro Met Ser Ala Val Ser Ala
Thr Ala Thr Ala Val Ala Ser Ser 610 615
620 His Asp His Gly Gly Asp Gly Gly Lys Gln Val Gln Met
Gly Tyr Asp 625 630 635
640 Ser Tyr Leu Val Gly Ala Asp Ala Tyr Gly Gly Gly Gly Ala Gly Arg
645 650 655 Met Pro Ser Trp
Ala Met Thr Pro Ala Ser Ala Pro Ala Ala Thr Ser 660
665 670 Ser Ser Asp Met Thr Gly Val Cys His
Gly Ala Gln Leu Phe Ser Val 675 680
685 Trp Asn Asp Thr 690 1872133DNAZea mays
187atggccactg tgaacaactg gctcgctttc tccctctccc cgcaggagct gccgccctcc
60cagacgacgg actccacact catctcggcc gccaccgccg accatgtctc cggcgatgtc
120tgcttcaaca tcccccaaga ttggagcatg aggggatcag agctttcggc gctcgtcgcg
180gagccgaagc tggaggactt cctcggcggc atctccttct ccgagcagca tcacaaggcc
240aactgcaaca tgatacccag cactagcagc acagtttgct acgcgagctc aggtgctagc
300accggctacc atcaccagct gtaccaccag cccaccagct cagcgctcca cttcgcggac
360tccgtaatgg tggcctcctc ggccggtgtc cacgacggcg gtgccatgct cagcgcggcc
420gccgctaacg gtgtcgctgg cgctgccagt gccaacggcg gcggcatcgg gctgtccatg
480attaagaact ggctgcggag ccaaccggcg cccatgcagc cgagggtggc ggcggctgag
540ggcgcgcagg ggctctcttt gtccatgaac atggcgggga cgacccaagg cgctgctggc
600atgccacttc tcgctggaga gcgcgcacgg gcgcccgaga gtgtatcgac gtcagcacag
660ggtggagccg tcgtcgtcac ggcgccgaag gaggatagcg gtggcagcgg tgttgccggc
720gctctagtag ccgtgagcac ggacacgggt ggcagcggcg gcgcgtcggc tgacaacacg
780gcaaggaaga cggtggacac gttcgggcag cgcacgtcga tttaccgtgg cgtgacaagg
840catagatgga ctgggagata tgaggcacat ctttgggata acagttgcag aagggaaggg
900caaactcgta agggtcgtca agtctattta ggtggctatg ataaagagga gaaagctgct
960agggcttatg atcttgctgc tctgaagtac tggggtgcca caacaacaac aaattttcca
1020gtgagtaact acgaaaagga gctcgaggac atgaagcaca tgacaaggca ggagtttgta
1080gcgtctctga gaaggaagag cagtggtttc tccagaggtg catccattta caggggagtg
1140actaggcatc accaacatgg aagatggcaa gcacggattg gacgagttgc agggaacaag
1200gatctttact tgggcacctt cagcacccag gaggaggcag cggaggcgta cgacatcgcg
1260gcgatcaagt tccgcggcct caacgccgtc accaacttcg acatgagccg ctacgacgtg
1320aagagcatcc tggacagcag cgccctcccc atcggcagcg ccgccaagcg cctcaaggag
1380gccgaggccg cagcgtccgc gcagcaccac cacgccggcg tggtgagcta cgacgtcggc
1440cgcatcgcct cgcagctcgg cgacggcgga gccctggcgg cggcgtacgg cgcgcactac
1500cacggcgccg cctggccgac catcgcgttc cagccgggcg ccgccagcac aggcctgtac
1560cacccgtacg cgcagcagcc aatgcgcggc ggcgggtggt gcaagcagga gcaggaccac
1620gcggtgatcg cggccgcgca cagcctgcag gacctccacc acctgaacct gggcgcggcc
1680ggcgcgcacg actttttctc ggcagggcag caggccgccg ccgctgcgat gcacggcctg
1740ggtagcatcg acagtgcgtc gctcgagcac agcaccggct ccaactccgt cgtctacaac
1800ggcggggtcg gcgacagcaa cggcgccagc gccgtcggcg gcagtggcgg tggctacatg
1860atgccgatga gcgctgccgg agcaaccact acatcggcaa tggtgagcca cgagcaggtg
1920catgcacggg cctacgacga agccaagcag gctgctcaga tggggtacga gagctacctg
1980gtgaacgcgg agaacaatgg tggcggaagg atgtctgcat gggggactgt cgtgtctgca
2040gccgcggcgg cagcagcaag cagcaacgac aacatggccg ccgacgtcgg ccatggcggc
2100gcgcagctct tcagtgtctg gaacgacact taa
2133188710PRTZea mays 188Met Ala Thr Val Asn Asn Trp Leu Ala Phe Ser Leu
Ser Pro Gln Glu 1 5 10
15 Leu Pro Pro Ser Gln Thr Thr Asp Ser Thr Leu Ile Ser Ala Ala Thr
20 25 30 Ala Asp His
Val Ser Gly Asp Val Cys Phe Asn Ile Pro Gln Asp Trp 35
40 45 Ser Met Arg Gly Ser Glu Leu Ser
Ala Leu Val Ala Glu Pro Lys Leu 50 55
60 Glu Asp Phe Leu Gly Gly Ile Ser Phe Ser Glu Gln His
His Lys Ala 65 70 75
80 Asn Cys Asn Met Ile Pro Ser Thr Ser Ser Thr Val Cys Tyr Ala Ser
85 90 95 Ser Gly Ala Ser
Thr Gly Tyr His His Gln Leu Tyr His Gln Pro Thr 100
105 110 Ser Ser Ala Leu His Phe Ala Asp Ser
Val Met Val Ala Ser Ser Ala 115 120
125 Gly Val His Asp Gly Gly Ala Met Leu Ser Ala Ala Ala Ala
Asn Gly 130 135 140
Val Ala Gly Ala Ala Ser Ala Asn Gly Gly Gly Ile Gly Leu Ser Met 145
150 155 160 Ile Lys Asn Trp Leu
Arg Ser Gln Pro Ala Pro Met Gln Pro Arg Val 165
170 175 Ala Ala Ala Glu Gly Ala Gln Gly Leu Ser
Leu Ser Met Asn Met Ala 180 185
190 Gly Thr Thr Gln Gly Ala Ala Gly Met Pro Leu Leu Ala Gly Glu
Arg 195 200 205 Ala
Arg Ala Pro Glu Ser Val Ser Thr Ser Ala Gln Gly Gly Ala Val 210
215 220 Val Val Thr Ala Pro Lys
Glu Asp Ser Gly Gly Ser Gly Val Ala Gly 225 230
235 240 Ala Leu Val Ala Val Ser Thr Asp Thr Gly Gly
Ser Gly Gly Ala Ser 245 250
255 Ala Asp Asn Thr Ala Arg Lys Thr Val Asp Thr Phe Gly Gln Arg Thr
260 265 270 Ser Ile
Tyr Arg Gly Val Thr Arg His Arg Trp Thr Gly Arg Tyr Glu 275
280 285 Ala His Leu Trp Asp Asn Ser
Cys Arg Arg Glu Gly Gln Thr Arg Lys 290 295
300 Gly Arg Gln Val Tyr Leu Gly Gly Tyr Asp Lys Glu
Glu Lys Ala Ala 305 310 315
320 Arg Ala Tyr Asp Leu Ala Ala Leu Lys Tyr Trp Gly Ala Thr Thr Thr
325 330 335 Thr Asn Phe
Pro Val Ser Asn Tyr Glu Lys Glu Leu Glu Asp Met Lys 340
345 350 His Met Thr Arg Gln Glu Phe Val
Ala Ser Leu Arg Arg Lys Ser Ser 355 360
365 Gly Phe Ser Arg Gly Ala Ser Ile Tyr Arg Gly Val Thr
Arg His His 370 375 380
Gln His Gly Arg Trp Gln Ala Arg Ile Gly Arg Val Ala Gly Asn Lys 385
390 395 400 Asp Leu Tyr Leu
Gly Thr Phe Ser Thr Gln Glu Glu Ala Ala Glu Ala 405
410 415 Tyr Asp Ile Ala Ala Ile Lys Phe Arg
Gly Leu Asn Ala Val Thr Asn 420 425
430 Phe Asp Met Ser Arg Tyr Asp Val Lys Ser Ile Leu Asp Ser
Ser Ala 435 440 445
Leu Pro Ile Gly Ser Ala Ala Lys Arg Leu Lys Glu Ala Glu Ala Ala 450
455 460 Ala Ser Ala Gln His
His His Ala Gly Val Val Ser Tyr Asp Val Gly 465 470
475 480 Arg Ile Ala Ser Gln Leu Gly Asp Gly Gly
Ala Leu Ala Ala Ala Tyr 485 490
495 Gly Ala His Tyr His Gly Ala Ala Trp Pro Thr Ile Ala Phe Gln
Pro 500 505 510 Gly
Ala Ala Ser Thr Gly Leu Tyr His Pro Tyr Ala Gln Gln Pro Met 515
520 525 Arg Gly Gly Gly Trp Cys
Lys Gln Glu Gln Asp His Ala Val Ile Ala 530 535
540 Ala Ala His Ser Leu Gln Asp Leu His His Leu
Asn Leu Gly Ala Ala 545 550 555
560 Gly Ala His Asp Phe Phe Ser Ala Gly Gln Gln Ala Ala Ala Ala Ala
565 570 575 Met His
Gly Leu Gly Ser Ile Asp Ser Ala Ser Leu Glu His Ser Thr 580
585 590 Gly Ser Asn Ser Val Val Tyr
Asn Gly Gly Val Gly Asp Ser Asn Gly 595 600
605 Ala Ser Ala Val Gly Gly Ser Gly Gly Gly Tyr Met
Met Pro Met Ser 610 615 620
Ala Ala Gly Ala Thr Thr Thr Ser Ala Met Val Ser His Glu Gln Val 625
630 635 640 His Ala Arg
Ala Tyr Asp Glu Ala Lys Gln Ala Ala Gln Met Gly Tyr 645
650 655 Glu Ser Tyr Leu Val Asn Ala Glu
Asn Asn Gly Gly Gly Arg Met Ser 660 665
670 Ala Trp Gly Thr Val Val Ser Ala Ala Ala Ala Ala Ala
Ala Ser Ser 675 680 685
Asn Asp Asn Met Ala Ala Asp Val Gly His Gly Gly Ala Gln Leu Phe 690
695 700 Ser Val Trp Asn
Asp Thr 705 710 1891374DNALotus corniculatus
189agatctctct ctgcagtgca caatccagtg gtaagcacat gtagtaatgg taatgagtct
60caagaaaatg gtaatggtaa tttgcaatcg ttgacattat ccatgggaag tggtaaggat
120tcaacatgtg aaaccagtgg tgacactagt accaacacta tcgaagctgt gcctagaaga
180accttggaga catttgggca aagaacatct atatatcgag gtgtaacaag acatagatgg
240accggaaggt atgaagctca cctttgggac aatagctgta gaagggaagg acagtcaagg
300aaaggtcgcc aaggtggata tgataaagaa gacaaagcag ctagggccta tgatttagct
360gcccttaagt actgggggac atccactact accaactttc cggttagcaa ctatgagaag
420gaagtggatg acatgaagca tatgacaaga caagaatttg tggcttccat tagaaggaaa
480agcagtggtt tctcgagggg tgcttcaatg tatcgtggag ttacaaggca tcatcaacat
540gggaggtggc aagcaaggat tggaagagtt gcaggaaaca aagatcttta cttgggaact
600ttcggtactg aggaagaggc ggcagaagct tacgacatag ctgcgataaa gttcagaggc
660cttaacgcca tcaccaactt tgacatgaac cgttacgatg tgaaagccat tctagagagc
720aacaccctcc caatcggagg aggagcttca aaaaggctaa aagaaactca agctctggaa
780tcttcaagaa aacgtgaaga tcagatgatt gcactcggct caacatttca tcaatacgga
840attgcaaccc catcaagctc tacaaggcca caaccttacc cgctaaacct aatgcatcat
900cacaatcagt ttgaacaaca gcctcaacca tttctaacct tacaaaacca tgacattaat
960tctcaatact cccatcatca gcaggaccct tcgtttcagc agagttacat tcaaacacag
1020cttcagttgc agttgaatca acacggtggt ggtggttcta gttatgctca agaaacagct
1080cctcatcaga acagtgagtt ctataatggt ggaaattatt accttcagaa ccttcaggga
1140atgatgatga acaatagtat ggggtcttgt tcttcatcgt ctgtgttgga gaatgatcat
1200aatgctgctg ctggtggggc ttcttttgtg ggtcccgcag cggaggaact tgggttggtt
1260aaggttgatt atgacatgga tgctgctgct ggcggtggtt atggtggttg gtcagcggcg
1320gagtccatgc acacgtcggc ggctggtggt ttgtttacta tgtggaatga gtga
1374190457PRTLotus corniculatus 190Arg Ser Leu Ser Ala Val His Asn Pro
Val Val Ser Thr Cys Ser Asn 1 5 10
15 Gly Asn Glu Ser Gln Glu Asn Gly Asn Gly Asn Leu Gln Ser
Leu Thr 20 25 30
Leu Ser Met Gly Ser Gly Lys Asp Ser Thr Cys Glu Thr Ser Gly Asp
35 40 45 Thr Ser Thr Asn
Thr Ile Glu Ala Val Pro Arg Arg Thr Leu Glu Thr 50
55 60 Phe Gly Gln Arg Thr Ser Ile Tyr
Arg Gly Val Thr Arg His Arg Trp 65 70
75 80 Thr Gly Arg Tyr Glu Ala His Leu Trp Asp Asn Ser
Cys Arg Arg Glu 85 90
95 Gly Gln Ser Arg Lys Gly Arg Gln Gly Gly Tyr Asp Lys Glu Asp Lys
100 105 110 Ala Ala Arg
Ala Tyr Asp Leu Ala Ala Leu Lys Tyr Trp Gly Thr Ser 115
120 125 Thr Thr Thr Asn Phe Pro Val Ser
Asn Tyr Glu Lys Glu Val Asp Asp 130 135
140 Met Lys His Met Thr Arg Gln Glu Phe Val Ala Ser Ile
Arg Arg Lys 145 150 155
160 Ser Ser Gly Phe Ser Arg Gly Ala Ser Met Tyr Arg Gly Val Thr Arg
165 170 175 His His Gln His
Gly Arg Trp Gln Ala Arg Ile Gly Arg Val Ala Gly 180
185 190 Asn Lys Asp Leu Tyr Leu Gly Thr Phe
Gly Thr Glu Glu Glu Ala Ala 195 200
205 Glu Ala Tyr Asp Ile Ala Ala Ile Lys Phe Arg Gly Leu Asn
Ala Ile 210 215 220
Thr Asn Phe Asp Met Asn Arg Tyr Asp Val Lys Ala Ile Leu Glu Ser 225
230 235 240 Asn Thr Leu Pro Ile
Gly Gly Gly Ala Ser Lys Arg Leu Lys Glu Thr 245
250 255 Gln Ala Leu Glu Ser Ser Arg Lys Arg Glu
Asp Gln Met Ile Ala Leu 260 265
270 Gly Ser Thr Phe His Gln Tyr Gly Ile Ala Thr Pro Ser Ser Ser
Thr 275 280 285 Arg
Pro Gln Pro Tyr Pro Leu Asn Leu Met His His His Asn Gln Phe 290
295 300 Glu Gln Gln Pro Gln Pro
Phe Leu Thr Leu Gln Asn His Asp Ile Asn 305 310
315 320 Ser Gln Tyr Ser His His Gln Gln Asp Pro Ser
Phe Gln Gln Ser Tyr 325 330
335 Ile Gln Thr Gln Leu Gln Leu Gln Leu Asn Gln His Gly Gly Gly Gly
340 345 350 Ser Ser
Tyr Ala Gln Glu Thr Ala Pro His Gln Asn Ser Glu Phe Tyr 355
360 365 Asn Gly Gly Asn Tyr Tyr Leu
Gln Asn Leu Gln Gly Met Met Met Asn 370 375
380 Asn Ser Met Gly Ser Cys Ser Ser Ser Ser Val Leu
Glu Asn Asp His 385 390 395
400 Asn Ala Ala Ala Gly Gly Ala Ser Phe Val Gly Pro Ala Ala Glu Glu
405 410 415 Leu Gly Leu
Val Lys Val Asp Tyr Asp Met Asp Ala Ala Ala Gly Gly 420
425 430 Gly Tyr Gly Gly Trp Ser Ala Ala
Glu Ser Met His Thr Ser Ala Ala 435 440
445 Gly Gly Leu Phe Thr Met Trp Asn Glu 450
455 191202PRTArtificial sequenceAP2 domain of Arath_PLT1
and Arath_PLT2 191Pro Arg Arg Xaa Leu Xaa Thr Phe Gly Gln Arg Thr Ser Ile
Tyr Arg 1 5 10 15
Gly Val Thr Arg His Arg Trp Thr Gly Arg Tyr Glu Ala His Leu Trp
20 25 30 Asp Asn Ser Cys Arg
Arg Glu Gly Gln Ser Arg Lys Gly Arg Gln Val 35
40 45 Tyr Leu Gly Gly Tyr Asp Lys Glu Xaa
Lys Ala Ala Arg Xaa Tyr Asp 50 55
60 Leu Ala Ala Leu Lys Tyr Trp Gly Pro Ser Thr Thr Thr
Asn Phe Pro 65 70 75
80 Ile Thr Asn Tyr Glu Lys Glu Val Glu Glu Met Lys Xaa Met Thr Arg
85 90 95 Gln Glu Phe Val
Ala Xaa Ile Arg Arg Lys Ser Ser Gly Phe Ser Arg 100
105 110 Gly Ala Ser Met Tyr Arg Gly Val Thr
Arg His His Gln His Gly Arg 115 120
125 Trp Gln Ala Arg Ile Gly Arg Val Ala Gly Asn Lys Asp Leu
Tyr Leu 130 135 140
Gly Thr Phe Ser Thr Glu Glu Glu Ala Ala Glu Ala Tyr Asp Ile Ala 145
150 155 160 Ala Ile Lys Phe Arg
Gly Leu Asn Ala Val Thr Asn Phe Glu Ile Asn 165
170 175 Arg Tyr Asp Val Lys Ala Ile Leu Glu Ser
Xaa Thr Leu Pro Ile Gly 180 185
190 Gly Gly Ala Ala Lys Arg Leu Lys Glu Ala 195
200 1928PRTArtificial sequencemotif 1 192Pro Lys Xaa Xaa
Asp Phe Leu Gly 1 5 1937PRTArtificial
sequencemotif 2 193Xaa Phe Xaa Xaa Trp Asn Xaa 1 5
1942193DNAOryza sativa 194aatccgaaaa gtttctgcac cgttttcacc ccctaactaa
caatataggg aacgtgtgct 60aaatataaaa tgagacctta tatatgtagc gctgataact
agaactatgc aagaaaaact 120catccaccta ctttagtggc aatcgggcta aataaaaaag
agtcgctaca ctagtttcgt 180tttccttagt aattaagtgg gaaaatgaaa tcattattgc
ttagaatata cgttcacatc 240tctgtcatga agttaaatta ttcgaggtag ccataattgt
catcaaactc ttcttgaata 300aaaaaatctt tctagctgaa ctcaatgggt aaagagagag
atttttttta aaaaaataga 360atgaagatat tctgaacgta ttggcaaaga tttaaacata
taattatata attttatagt 420ttgtgcattc gtcatatcgc acatcattaa ggacatgtct
tactccatcc caatttttat 480ttagtaatta aagacaattg acttattttt attatttatc
ttttttcgat tagatgcaag 540gtacttacgc acacactttg tgctcatgtg catgtgtgag
tgcacctcct caatacacgt 600tcaactagca acacatctct aatatcactc gcctatttaa
tacatttagg tagcaatatc 660tgaattcaag cactccacca tcaccagacc acttttaata
atatctaaaa tacaaaaaat 720aattttacag aatagcatga aaagtatgaa acgaactatt
taggtttttc acatacaaaa 780aaaaaaagaa ttttgctcgt gcgcgagcgc caatctccca
tattgggcac acaggcaaca 840acagagtggc tgcccacaga acaacccaca aaaaacgatg
atctaacgga ggacagcaag 900tccgcaacaa ccttttaaca gcaggctttg cggccaggag
agaggaggag aggcaaagaa 960aaccaagcat cctcctcctc ccatctataa attcctcccc
ccttttcccc tctctatata 1020ggaggcatcc aagccaagaa gagggagagc accaaggaca
cgcgactagc agaagccgag 1080cgaccgcctt cttcgatcca tatcttccgg tcgagttctt
ggtcgatctc ttccctcctc 1140cacctcctcc tcacagggta tgtgcccttc ggttgttctt
ggatttattg ttctaggttg 1200tgtagtacgg gcgttgatgt taggaaaggg gatctgtatc
tgtgatgatt cctgttcttg 1260gatttgggat agaggggttc ttgatgttgc atgttatcgg
ttcggtttga ttagtagtat 1320ggttttcaat cgtctggaga gctctatgga aatgaaatgg
tttagggtac ggaatcttgc 1380gattttgtga gtaccttttg tttgaggtaa aatcagagca
ccggtgattt tgcttggtgt 1440aataaaagta cggttgtttg gtcctcgatt ctggtagtga
tgcttctcga tttgacgaag 1500ctatcctttg tttattccct attgaacaaa aataatccaa
ctttgaagac ggtcccgttg 1560atgagattga atgattgatt cttaagcctg tccaaaattt
cgcagctggc ttgtttagat 1620acagtagtcc ccatcacgaa attcatggaa acagttataa
tcctcaggaa caggggattc 1680cctgttcttc cgatttgctt tagtcccaga attttttttc
ccaaatatct taaaaagtca 1740ctttctggtt cagttcaatg aattgattgc tacaaataat
gcttttatag cgttatccta 1800gctgtagttc agttaatagg taatacccct atagtttagt
caggagaaga acttatccga 1860tttctgatct ccatttttaa ttatatgaaa tgaactgtag
cataagcagt attcatttgg 1920attatttttt ttattagctc tcaccccttc attattctga
gctgaaagtc tggcatgaac 1980tgtcctcaat tttgttttca aattcacatc gattatctat
gcattatcct cttgtatcta 2040cctgtagaag tttctttttg gttattcctt gactgcttga
ttacagaaag aaatttatga 2100agctgtaatc gggatagtta tactgcttgt tcttatgatt
catttccttt gtgcagttct 2160tggtgtagct tgccactttc accagcaaag ttc
219319554DNAArtificial sequenceprimer PRM08180
195ggggacaagt ttgtacaaaa aagcaggctt aaacaatgat caatccacac ggtg
5419650DNAArtificial sequenceprimer PRM08181 196ggggaccact ttgtacaaga
aagctgggtt ccttgtttac tcattccaca 5019756DNAArtificial
sequenceprimer PRM08182 197ggggacaagt ttgtacaaaa aagcaggctt aaacaatgaa
ttctaacaac tggctc 5619850DNAArtificial sequenceprimer PRM08183
198ggggaccact ttgtacaaga aagctgggtt catcttttat tcattccaca
501991638DNAPopulus tremuloides 199atgaattcta acaactggct ttcatttcct
ctttctccta ctcatccttc cttgcctgct 60catctacatg catcccaccc tcatcaattc
tctctagggt tagtcaatga taatatggaa 120aacccatttc aaactcaaga gtggagtctt
cttaacactc atcaaggcaa caatgaagtg 180ccaaaggttg cagactttct tggtgtgagc
aaatctgaga atcaatcaga tcttgtagcc 240ttcaatgaaa ttcaagctaa tgaatctgac
tatctctttt caaacaatag tctagtacca 300gtccaaaatg ctgttgtagg cgccaataat
acctttgagt ttcaagaaaa tgctagcaat 360ttgcagtcat taacattgtc tatgggcagt
gctagtggta aaggttctac atgtgaaccc 420agtggggata atagcactaa tactgttgaa
gctgctgcac caagaagaac tttggataca 480tttgggcaaa gaacatccat atatcgtggt
gtaacaaggc atcgatggac aggaaggtat 540gaagctcatt tatgggataa tagttgcaga
agagaaggtc aatctaggaa aggaagacag 600ggtggctatg acaaagaaga aaaggcagct
agggcttatg atcttgctgc acttaagtac 660tggggaacat ccaccactac caattttcca
atcagcaact acgagaaaga aatagaggaa 720atgaagcaca tgaccaggca agaatttgtg
gcctccatta gaaggaagag tagtggcttc 780tctaggggtg catccatgta tcgtggagtt
acaaggcatc accagcatgg tagatggcaa 840gcaaggatag gcagagttgc aggaaacaaa
gatctctact tgggaacttt tagcactgag 900gaggaggctg cagaagctta tgacatagca
gcaataaagt ttagagggct taatgcagtg 960actaactttg acatgaatcg atatgatgtg
aagagcattc ttgaaagcaa tactttgcca 1020attggaggag gggcagccaa acggctaaag
gaggctcaag caattgaatc atcacgaaaa 1080agagaagaaa tgattgctct tggctcaagt
tttccatatg gatcaacttc aagctctagc 1140aggctacaag cttaccctct aatgcagaca
ccatttgagc aacctcaacc tttacttact 1200ctacaaaatc aagacatttc tcagtacact
caggattcct catcattcca ccaaaatttc 1260cttcaaacac agcttcattt gcaccagcaa
tctacagggt ctaatttcct gcataaccaa 1320tcaaaccaaa accctcaata ttacaatagt
tatatccaaa acaatccagc tttacttcat 1380ggattgtgga acatgggttc ttcatcatct
gtaatggaga ataatggaag ttctagtggg 1440agctatagta ctggaggtta tctgggaaat
gggctgggaa tggcttccaa ttcaacaggg 1500tctaatgcag taggatcagc cgaggaactt
gcacttgtca aagttgatta tgatatgcct 1560tctagtggct atggaagctg gtctggggac
tcagtccagg gatccaatcc aggtgttttc 1620actatgtgga atgagtga
1638200545PRTPopulus tremuloides 200Met
Asn Ser Asn Asn Trp Leu Ser Phe Pro Leu Ser Pro Thr His Pro 1
5 10 15 Ser Leu Pro Ala His Leu
His Ala Ser His Pro His Gln Phe Ser Leu 20
25 30 Gly Leu Val Asn Asp Asn Met Glu Asn Pro
Phe Gln Thr Gln Glu Trp 35 40
45 Ser Leu Leu Asn Thr His Gln Gly Asn Asn Glu Val Pro Lys
Val Ala 50 55 60
Asp Phe Leu Gly Val Ser Lys Ser Glu Asn Gln Ser Asp Leu Val Ala 65
70 75 80 Phe Asn Glu Ile Gln
Ala Asn Glu Ser Asp Tyr Leu Phe Ser Asn Asn 85
90 95 Ser Leu Val Pro Val Gln Asn Ala Val Val
Gly Ala Asn Asn Thr Phe 100 105
110 Glu Phe Gln Glu Asn Ala Ser Asn Leu Gln Ser Leu Thr Leu Ser
Met 115 120 125 Gly
Ser Ala Ser Gly Lys Gly Ser Thr Cys Glu Pro Ser Gly Asp Asn 130
135 140 Ser Thr Asn Thr Val Glu
Ala Ala Ala Pro Arg Arg Thr Leu Asp Thr 145 150
155 160 Phe Gly Gln Arg Thr Ser Ile Tyr Arg Gly Val
Thr Arg His Arg Trp 165 170
175 Thr Gly Arg Tyr Glu Ala His Leu Trp Asp Asn Ser Cys Arg Arg Glu
180 185 190 Gly Gln
Ser Arg Lys Gly Arg Gln Gly Gly Tyr Asp Lys Glu Glu Lys 195
200 205 Ala Ala Arg Ala Tyr Asp Leu
Ala Ala Leu Lys Tyr Trp Gly Thr Ser 210 215
220 Thr Thr Thr Asn Phe Pro Ile Ser Asn Tyr Glu Lys
Glu Ile Glu Glu 225 230 235
240 Met Lys His Met Thr Arg Gln Glu Phe Val Ala Ser Ile Arg Arg Lys
245 250 255 Ser Ser Gly
Phe Ser Arg Gly Ala Ser Met Tyr Arg Gly Val Thr Arg 260
265 270 His His Gln His Gly Arg Trp Gln
Ala Arg Ile Gly Arg Val Ala Gly 275 280
285 Asn Lys Asp Leu Tyr Leu Gly Thr Phe Ser Thr Glu Glu
Glu Ala Ala 290 295 300
Glu Ala Tyr Asp Ile Ala Ala Ile Lys Phe Arg Gly Leu Asn Ala Val 305
310 315 320 Thr Asn Phe Asp
Met Asn Arg Tyr Asp Val Lys Ser Ile Leu Glu Ser 325
330 335 Asn Thr Leu Pro Ile Gly Gly Gly Ala
Ala Lys Arg Leu Lys Glu Ala 340 345
350 Gln Ala Ile Glu Ser Ser Arg Lys Arg Glu Glu Met Ile Ala
Leu Gly 355 360 365
Ser Ser Phe Pro Tyr Gly Ser Thr Ser Ser Ser Ser Arg Leu Gln Ala 370
375 380 Tyr Pro Leu Met Gln
Thr Pro Phe Glu Gln Pro Gln Pro Leu Leu Thr 385 390
395 400 Leu Gln Asn Gln Asp Ile Ser Gln Tyr Thr
Gln Asp Ser Ser Ser Phe 405 410
415 His Gln Asn Phe Leu Gln Thr Gln Leu His Leu His Gln Gln Ser
Thr 420 425 430 Gly
Ser Asn Phe Leu His Asn Gln Ser Asn Gln Asn Pro Gln Tyr Tyr 435
440 445 Asn Ser Tyr Ile Gln Asn
Asn Pro Ala Leu Leu His Gly Leu Trp Asn 450 455
460 Met Gly Ser Ser Ser Ser Val Met Glu Asn Asn
Gly Ser Ser Ser Gly 465 470 475
480 Ser Tyr Ser Thr Gly Gly Tyr Leu Gly Asn Gly Leu Gly Met Ala Ser
485 490 495 Asn Ser
Thr Gly Ser Asn Ala Val Gly Ser Ala Glu Glu Leu Ala Leu 500
505 510 Val Lys Val Asp Tyr Asp Met
Pro Ser Ser Gly Tyr Gly Ser Trp Ser 515 520
525 Gly Asp Ser Val Gln Gly Ser Asn Pro Gly Val Phe
Thr Met Trp Asn 530 535 540
Glu 545 2011515DNAPopulus tremuloides 201atgttttttt ttttgtttcc
tttagagtgg agtcttctta acactcaagg caacaatgaa 60gtgccaaagg ttgcagattt
tcttggtgta agtaaatctg agaatcaatc agatctcgta 120gccttcaatg aaattcaagc
cagtgattct gagtatctct tttcaagcaa tagtctgttg 180ccggtccaaa atgctgtggt
agccgccagt actaactacg aatttcaaga aaatcctagc 240aatttgcagt cattaacatt
gtctatgggc agtgctagtg gtaagggttc taaatgtgaa 300accagtggtg ataatagtac
taattctgtc gaagctgctg ctccaagaag gactttggat 360acatttggtc aaagaacatc
catctatcgt ggtgtaacaa ggcatcgatg gacaggaagg 420tatgaagctc atttatggga
taatagttgc agaagagaag gtcaatccag gaaaggaaga 480caaggtggct atgacaaaga
agacaaggct gctagggctt atgatcttgc tgcacttaag 540tactggggaa catcgaccac
taccaatttt cctatcagca actatgagaa agaactagag 600gacatgaaga acatgaccag
acaagaattt gtggcctcca ttagaaggaa gagtagtggc 660ttctctaggg gtgcatccat
gtatcgtgga gtcacaaggc atcaccaaca tggaagatgg 720caagcaagaa ttggtagagt
tgcaggaaac aaagatctct acttgggaac ttttagcact 780gaggaggagg ctgcagaagc
ttatgacata gcagcaataa agtttagagg gcttaatgca 840gtgactaatt ttgacatgaa
tcgatatgat gtgaaaagca ttcttgagag caatagtttg 900ccaattggag gaggggcagc
caaaaggcta aaggaggctc aagcaatcga atcgtcacaa 960aaacgagaag aaatgattgc
tcttggatca agttatccat atggatcaac ttcaagctct 1020agtcgacaac aagcttactc
tctaatgcag aaaccatttg agcaacctca acctttactt 1080actctacaaa atcaagacat
ttctcagtac actcaagatt cttcatttca gcaaaattac 1140cttcaaacac agcttcattt
gcaccagcta tctgcagggt ctaatttcct gcataataac 1200caatcaagcc aaaatcctca
gtattacaac agctatatcc aaaacaatcc cactttgctt 1260catggattgt ggaacatggg
ttcttcatca tctctaatgg agaataatgg cagttctagt 1320gggagttata gtactgtcgg
ttatctggga aatgggttgg gaatggctac caattcaaca 1380gggtctaatg cagtagctga
ggaacttcca cttgttaaga tagattatga tatgccttct 1440ggtggctatg gaagttggtc
tggggaatca gttcagggat ccaacccagg tgtttttaca 1500atgtggaatg agtga
1515202504PRTPopulus
tremuloides 202Met Phe Phe Phe Leu Phe Pro Leu Glu Trp Ser Leu Leu Asn
Thr Gln 1 5 10 15
Gly Asn Asn Glu Val Pro Lys Val Ala Asp Phe Leu Gly Val Ser Lys
20 25 30 Ser Glu Asn Gln Ser
Asp Leu Val Ala Phe Asn Glu Ile Gln Ala Ser 35
40 45 Asp Ser Glu Tyr Leu Phe Ser Ser Asn
Ser Leu Leu Pro Val Gln Asn 50 55
60 Ala Val Val Ala Ala Ser Thr Asn Tyr Glu Phe Gln Glu
Asn Pro Ser 65 70 75
80 Asn Leu Gln Ser Leu Thr Leu Ser Met Gly Ser Ala Ser Gly Lys Gly
85 90 95 Ser Lys Cys Glu
Thr Ser Gly Asp Asn Ser Thr Asn Ser Val Glu Ala 100
105 110 Ala Ala Pro Arg Arg Thr Leu Asp Thr
Phe Gly Gln Arg Thr Ser Ile 115 120
125 Tyr Arg Gly Val Thr Arg His Arg Trp Thr Gly Arg Tyr Glu
Ala His 130 135 140
Leu Trp Asp Asn Ser Cys Arg Arg Glu Gly Gln Ser Arg Lys Gly Arg 145
150 155 160 Gln Gly Gly Tyr Asp
Lys Glu Asp Lys Ala Ala Arg Ala Tyr Asp Leu 165
170 175 Ala Ala Leu Lys Tyr Trp Gly Thr Ser Thr
Thr Thr Asn Phe Pro Ile 180 185
190 Ser Asn Tyr Glu Lys Glu Leu Glu Asp Met Lys Asn Met Thr Arg
Gln 195 200 205 Glu
Phe Val Ala Ser Ile Arg Arg Lys Ser Ser Gly Phe Ser Arg Gly 210
215 220 Ala Ser Met Tyr Arg Gly
Val Thr Arg His His Gln His Gly Arg Trp 225 230
235 240 Gln Ala Arg Ile Gly Arg Val Ala Gly Asn Lys
Asp Leu Tyr Leu Gly 245 250
255 Thr Phe Ser Thr Glu Glu Glu Ala Ala Glu Ala Tyr Asp Ile Ala Ala
260 265 270 Ile Lys
Phe Arg Gly Leu Asn Ala Val Thr Asn Phe Asp Met Asn Arg 275
280 285 Tyr Asp Val Lys Ser Ile Leu
Glu Ser Asn Ser Leu Pro Ile Gly Gly 290 295
300 Gly Ala Ala Lys Arg Leu Lys Glu Ala Gln Ala Ile
Glu Ser Ser Gln 305 310 315
320 Lys Arg Glu Glu Met Ile Ala Leu Gly Ser Ser Tyr Pro Tyr Gly Ser
325 330 335 Thr Ser Ser
Ser Ser Arg Gln Gln Ala Tyr Ser Leu Met Gln Lys Pro 340
345 350 Phe Glu Gln Pro Gln Pro Leu Leu
Thr Leu Gln Asn Gln Asp Ile Ser 355 360
365 Gln Tyr Thr Gln Asp Ser Ser Phe Gln Gln Asn Tyr Leu
Gln Thr Gln 370 375 380
Leu His Leu His Gln Leu Ser Ala Gly Ser Asn Phe Leu His Asn Asn 385
390 395 400 Gln Ser Ser Gln
Asn Pro Gln Tyr Tyr Asn Ser Tyr Ile Gln Asn Asn 405
410 415 Pro Thr Leu Leu His Gly Leu Trp Asn
Met Gly Ser Ser Ser Ser Leu 420 425
430 Met Glu Asn Asn Gly Ser Ser Ser Gly Ser Tyr Ser Thr Val
Gly Tyr 435 440 445
Leu Gly Asn Gly Leu Gly Met Ala Thr Asn Ser Thr Gly Ser Asn Ala 450
455 460 Val Ala Glu Glu Leu
Pro Leu Val Lys Ile Asp Tyr Asp Met Pro Ser 465 470
475 480 Gly Gly Tyr Gly Ser Trp Ser Gly Glu Ser
Val Gln Gly Ser Asn Pro 485 490
495 Gly Val Phe Thr Met Trp Asn Glu 500
203785DNAVitis vinifera 203tcttgataaa tcccaaaact gactgtgtag
gtactgagga ggaagctgca gaagcctatg 60atattgcagc aataaagttc agaggcctta
atgcagtgac caactttgac atgaatcgat 120acgatgtgaa gagcattctt gaaagcaaca
ctcttccgat aggtggggga gcggccaagc 180ggctaaagga ggctcaagca attgaatcat
caagaaaacg ggaagaaatg atagccctag 240gttcaagttt ccaatatggg agctcgagct
ctagcaggtt acagacatat cctctaatgc 300agcagcagtt tgagcaacct cagcctttac
taacattaca gaaccaagaa ccattactaa 360ctttgcaaaa ccctgaaatt tctcagtacc
cccaagactc ccagtttcac caaaactaca 420tccaaactca gttgcagttg caccagcaat
ctgggtcgta cctgaaccat tcaagccaaa 480gtcctcagtt ctacaacagt tacctccaca
acaacccggc tcttcttcat gggctgatga 540gtatgggctc ttcttcatct gtcatggaga
ataatgggag ttctagtggg agttacaatg 600gaggmtactt caataatgga cttggggttg
cttcgaattc tacggtggct agtgcagtag 660gatcagcaga ggagcttccc ctcatcaagg
ttgattacga tatgccggcc gcaggctatg 720gcagctggtc aggtgactca gttcagggac
agaatgctgg agtttttaca atgtggaatg 780actga
785204260PRTVitis vinifera 204Leu Ile
Asn Pro Lys Thr Asp Cys Val Gly Thr Glu Glu Glu Ala Ala 1 5
10 15 Glu Ala Tyr Asp Ile Ala Ala
Ile Lys Phe Arg Gly Leu Asn Ala Val 20 25
30 Thr Asn Phe Asp Met Asn Arg Tyr Asp Val Lys Ser
Ile Leu Glu Ser 35 40 45
Asn Thr Leu Pro Ile Gly Gly Gly Ala Ala Lys Arg Leu Lys Glu Ala
50 55 60 Gln Ala Ile
Glu Ser Ser Arg Lys Arg Glu Glu Met Ile Ala Leu Gly 65
70 75 80 Ser Ser Phe Gln Tyr Gly Ser
Ser Ser Ser Ser Arg Leu Gln Thr Tyr 85
90 95 Pro Leu Met Gln Gln Gln Phe Glu Gln Pro Gln
Pro Leu Leu Thr Leu 100 105
110 Gln Asn Gln Glu Pro Leu Leu Thr Leu Gln Asn Pro Glu Ile Ser
Gln 115 120 125 Tyr
Pro Gln Asp Ser Gln Phe His Gln Asn Tyr Ile Gln Thr Gln Leu 130
135 140 Gln Leu His Gln Gln Ser
Gly Ser Tyr Leu Asn His Ser Ser Gln Ser 145 150
155 160 Pro Gln Phe Tyr Asn Ser Tyr Leu His Asn Asn
Pro Ala Leu Leu His 165 170
175 Gly Leu Met Ser Met Gly Ser Ser Ser Ser Val Met Glu Asn Asn Gly
180 185 190 Ser Ser
Ser Gly Ser Tyr Asn Gly Gly Tyr Phe Asn Asn Gly Leu Gly 195
200 205 Val Ala Ser Asn Ser Thr Val
Ala Ser Ala Val Gly Ser Ala Glu Glu 210 215
220 Leu Pro Leu Ile Lys Val Asp Tyr Asp Met Pro Ala
Ala Gly Tyr Gly 225 230 235
240 Ser Trp Ser Gly Asp Ser Val Gln Gly Gln Asn Ala Gly Val Phe Thr
245 250 255 Met Trp Asn
Asp 260 205150DNAbrassica napus 205ggaacatcgg ccgaggaaga
gtttcccacg gttaaagttg attacgatat gcctccttta 60ggtggagcca cagggtgtga
acgatggact aatggagaga atggtcaggg gtcaaatcca 120ggaggtgtct tcacaatgtg
gaatgaataa 15020649PRTBrassica napus
206Gly Thr Ser Ala Glu Glu Glu Phe Pro Thr Val Lys Val Asp Tyr Asp 1
5 10 15 Met Pro Pro Leu
Gly Gly Ala Thr Gly Cys Glu Arg Trp Thr Asn Gly 20
25 30 Glu Asn Gly Gln Gly Ser Asn Pro Gly
Gly Val Phe Thr Met Trp Asn 35 40
45 Glu 207162DNAPhaseolus coccineus 207tcgaatgcat
cgtcgggtaa tgcggtgggc acggcggagg aacttggatt ggtgaaagtt 60gactatgaca
tgccggccgg aggttacggt ggttggtcgg cggcggcggc ggaatccatg 120cagacgtcaa
atggtggggt gttcacaatg tggaatgagt ga
16220853PRTPhaseolus coccineus 208Ser Asn Ala Ser Ser Gly Asn Ala Val Gly
Thr Ala Glu Glu Leu Gly 1 5 10
15 Leu Val Lys Val Asp Tyr Asp Met Pro Ala Gly Gly Tyr Gly Gly
Trp 20 25 30 Ser
Ala Ala Ala Ala Glu Ser Met Gln Thr Ser Asn Gly Gly Val Phe 35
40 45 Thr Met Trp Asn Glu
50 2097PRTArtificial sequenceMotif 2 209Xaa Phe Xaa Xaa Trp
Asn Xaa 1 5 2102194DNAOryza sativa 210aatccgaaaa
gtttctgcac cgttttcacc ccctaactaa caatataggg aacgtgtgct 60aaatataaaa
tgagacctta tatatgtagc gctgataact agaactatgc aagaaaaact 120catccaccta
ctttagtggc aatcgggcta aataaaaaag agtcgctaca ctagtttcgt 180tttccttagt
aattaagtgg gaaaatgaaa tcattattgc ttagaatata cgttcacatc 240tctgtcatga
agttaaatta ttcgaggtag ccataattgt catcaaactc ttcttgaata 300aaaaaatctt
tctagctgaa ctcaatgggt aaagagagag atttttttta aaaaaataga 360atgaagatat
tctgaacgta ttggcaaaga tttaaacata taattatata attttatagt 420ttgtgcattc
gtcatatcgc acatcattaa ggacatgtct tactccatcc caatttttat 480ttagtaatta
aagacaattg acttattttt attatttatc ttttttcgat tagatgcaag 540gtacttacgc
acacactttg tgctcatgtg catgtgtgag tgcacctcct caatacacgt 600tcaactagca
acacatctct aatatcactc gcctatttaa tacatttagg tagcaatatc 660tgaattcaag
cactccacca tcaccagacc acttttaata atatctaaaa tacaaaaaat 720aattttacag
aatagcatga aaagtatgaa acgaactatt taggtttttc acatacaaaa 780aaaaaaagaa
ttttgctcgt gcgcgagcgc caatctccca tattgggcac acaggcaaca 840acagagtggc
tgcccacaga acaacccaca aaaaacgatg atctaacgga ggacagcaag 900tccgcaacaa
ccttttaaca gcaggctttg cggccaggag agaggaggag aggcaaagaa 960aaccaagcat
cctccttctc ccatctataa attcctcccc ccttttcccc tctctatata 1020ggaggcatcc
aagccaagaa gagggagagc accaaggaca cgcgactagc agaagccgag 1080cgaccgcctt
ctcgatccat atcttccggt cgagttcttg gtcgatctct tccctcctcc 1140acctcctcct
cacagggtat gtgcctccct tcggttgttc ttggatttat tgttctaggt 1200tgtgtagtac
gggcgttgat gttaggaaag gggatctgta tctgtgatga ttcctgttct 1260tggatttggg
atagaggggt tcttgatgtt gcatgttatc ggttcggttt gattagtagt 1320atggttttca
atcgtctgga gagctctatg gaaatgaaat ggtttaggga tcggaatctt 1380gcgattttgt
gagtaccttt tgtttgaggt aaaatcagag caccggtgat tttgcttggt 1440gtaataaagt
acggttgttt ggtcctcgat tctggtagtg atgcttctcg atttgacgaa 1500gctatccttt
gtttattccc tattgaacaa aaataatcca actttgaaga cggtcccgtt 1560gatgagattg
aatgattgat tcttaagcct gtccaaaatt tcgcagctgg cttgtttaga 1620tacagtagtc
cccatcacga aattcatgga aacagttata atcctcagga acaggggatt 1680ccctgttctt
ccgatttgct ttagtcccag aatttttttt cccaaatatc ttaaaaagtc 1740actttctggt
tcagttcaat gaattgattg ctacaaataa tgcttttata gcgttatcct 1800agctgtagtt
cagttaatag gtaatacccc tatagtttag tcaggagaag aacttatccg 1860atttctgatc
tccattttta attatatgaa atgaactgta gcataagcag tattcatttg 1920gattattttt
tttattagct ctcacccctt cattattctg agctgaaagt ctggcatgaa 1980ctgtcctcaa
ttttgttttc aaattcacat cgattatcta tgcattatcc tcttgtatct 2040acctgtagaa
gtttcttttt ggttattcct tgactgcttg attacagaaa gaaatttatg 2100aagctgtaat
cgggatagtt atactgcttg ttcttatgat tcatttcctt tgtgcagttc 2160ttggtgtagc
ttgccacttt caccagcaaa gttc
21942111245DNAOryza sativa 211cttgttgttg atctgtgccc ccaagaagaa taacactcta
ctcttacttg ttggaaaaaa 60atagtattag caaccacgca tatgcaaatt ttaatgcagt
aataataaga gatggatcga 120tcgttttcca gctcttgtat atgtgactgg ccctgcttta
tgtgtgtagt gttaatttca 180gctttagcag tacgtgatta gtgatggaca ataattgtcg
cagacgtatc tatcaattgc 240tcctgttgtg tgatgcttta actgttggaa tcaaagttgc
gttgcctttg ttgttatgag 300gaggaatata tatgttgggg caggaaaaga atggaggaga
gatcgttctc catatcctta 360tcatcggcct cgtcactgct cgcagtttaa ctttttggtg
atgcgagcga tggtcagcca 420tatatatact cccatgctgc atgctagtaa tcaatatacg
ccttgtaaaa gtaaacgatc 480gtctagtaat tgcaatatca taggggtagc cattgacaga
gatctacata gatagagggg 540gaacaagaat tgacactcca cagatgctcc actcattcac
ctttactaat ttatatcttt 600tgatgtttga tcgatcgatc gatccgtccg tcggtgtctc
gacgaataaa aactgcaaat 660cgaactgtat gtatataata tagcgtcgta aattaaatta
aattaaatcg aactgaatac 720tacatgtcga agcaagaatt agttcaacta aaagatttag
tttttccggt tgcaatatct 780gtgaaattaa ttgaagaaat taagaagaaa actggagaga
tatatatatg gatgagacaa 840aatgagataa gacgcatgat ggtccctcgg atgatgtcgt
ccgttcctta tttccattcc 900atggcagctg ctatcgctat ctagtgcgcg cggcatctcc
aatcccatcc attctagtgg 960tcgatctagc tactactgag tattgttttt tcttcttttt
actactgttg attattctgc 1020aactgcagtt agatgcttgc tactcctaca tcgatctctc
tcgcgcgggc gtatgcattg 1080cattcactac tgatgatccg tgggtgtagt gtgggtggct
ataaataggg cagggtgcgg 1140ttgccattgc tcctcaggcc agcaactgag aagctccata
caagtaagca gcagctagtt 1200gccgacaagg ccagagaagg aagaagaagc tctcatcatc
atcac 1245212675DNAOryza sativa 212atgaagagca
ggaagaacag cacgacgagc acaaaagcag caggcagctg ccacaccagc 60agcagcggag
gaggaggagg cggcggcaac tgctatagca gcagcagtag caagatggag 120cgcaaggatg
tggagaagaa tcggcgcctc cacatgaagg gtctctgcct caagctctcc 180tccctcatcc
ccgccgccgc tccccgccgc catcaccacc actactccac ctcctcctcc 240tcatcgccgc
cctcctccac caaggaggct gtgacgcagc tggatcacct ggagcaggcg 300gcggcgtaca
tcaagcagct caaggggagg atcgacgagc tgaagaagag gaagcagcag 360gcggcggcac
tcaccaccag caccagcaat ggcggcggcg gcgggatgcc ggtggtggag 420gtgcggtgcc
aggatgggac gctggacgtg gtggtggtga gcgaggcgat cagggaggag 480agggagaggg
cggtgcggct gcacgaggtg atcggcgtgc tggaggaaga aggcgcggag 540gtggtgaacg
ccagcttctc cgtcgtcggc gacaagatct tctacactct ccactcccag 600gcgctctgct
ccaggatcgg cctcgacgcc tccagggtct cccacaggct gcgcaacctc 660ctcctccaat
attaa
675213224PRTOryza sativa 213Met Lys Ser Arg Lys Asn Ser Thr Thr Ser Thr
Lys Ala Ala Gly Ser 1 5 10
15 Cys His Thr Ser Ser Ser Gly Gly Gly Gly Gly Gly Gly Asn Cys Tyr
20 25 30 Ser Ser
Ser Ser Ser Lys Met Glu Arg Lys Asp Val Glu Lys Asn Arg 35
40 45 Arg Leu His Met Lys Gly Leu
Cys Leu Lys Leu Ser Ser Leu Ile Pro 50 55
60 Ala Ala Ala Pro Arg Arg His His His His Tyr Ser
Thr Ser Ser Ser 65 70 75
80 Ser Ser Pro Pro Ser Ser Thr Lys Glu Ala Val Thr Gln Leu Asp His
85 90 95 Leu Glu Gln
Ala Ala Ala Tyr Ile Lys Gln Leu Lys Gly Arg Ile Asp 100
105 110 Glu Leu Lys Lys Arg Lys Gln Gln
Ala Ala Ala Leu Thr Thr Ser Thr 115 120
125 Ser Asn Gly Gly Gly Gly Gly Met Pro Val Val Glu Val
Arg Cys Gln 130 135 140
Asp Gly Thr Leu Asp Val Val Val Val Ser Glu Ala Ile Arg Glu Glu 145
150 155 160 Arg Glu Arg Ala
Val Arg Leu His Glu Val Ile Gly Val Leu Glu Glu 165
170 175 Glu Gly Ala Glu Val Val Asn Ala Ser
Phe Ser Val Val Gly Asp Lys 180 185
190 Ile Phe Tyr Thr Leu His Ser Gln Ala Leu Cys Ser Arg Ile
Gly Leu 195 200 205
Asp Ala Ser Arg Val Ser His Arg Leu Arg Asn Leu Leu Leu Gln Tyr 210
215 220 214696DNAOryza
sativa 214atggatcagg agaaggcgga cggcggcggc aggaggagga ggagcagggc
gacgagtagc 60agcggcagcg gcgcgagcag cacggcggcg gcggcggcgg cggagaggaa
ggagatggag 120cgcaggaggc ggcaggacat gaagggcctc tgcgtcaagc tcgcctctct
catccccaaa 180gaacactgct ccatgtccaa gatgcaggcg gcgtctagga cccagctggg
cagcctggac 240gaagcggcgg cctacatcaa gaagctcaag gaaagggtgg acgagctgca
ccacaagagg 300agcatgatga gtatcacatc atcgcgctgc cgctcaggag gaggaggagg
accagctgct 360gctgctggcc agtcgacgag cggcggcggc ggcggggaag aagaagaaga
agatatgacg 420aggacgacgg cggcggcggc ggtggtggag gtgcggcagc acgtgcagga
ggggtcgctg 480atcagcttgg acgtggtgct gatctgcagc gcagcgaggc cggtcaagtt
ccacgacgtc 540atcaccgtcc tcgaggaaga aggcgccgac atcatctccg ccaacttctc
cctcgccgcc 600cacaatttct actacaccat ctactccagg gcctttagct caagaattgg
catagaggct 660tcgaggattt ctgagagact acgggcattg gtatga
696215231PRTOryza sativa 215Met Asp Gln Glu Lys Ala Asp Gly
Gly Gly Arg Arg Arg Arg Ser Arg 1 5 10
15 Ala Thr Ser Ser Ser Gly Ser Gly Ala Ser Ser Thr Ala
Ala Ala Ala 20 25 30
Ala Ala Glu Arg Lys Glu Met Glu Arg Arg Arg Arg Gln Asp Met Lys
35 40 45 Gly Leu Cys Val
Lys Leu Ala Ser Leu Ile Pro Lys Glu His Cys Ser 50
55 60 Met Ser Lys Met Gln Ala Ala Ser
Arg Thr Gln Leu Gly Ser Leu Asp 65 70
75 80 Glu Ala Ala Ala Tyr Ile Lys Lys Leu Lys Glu Arg
Val Asp Glu Leu 85 90
95 His His Lys Arg Ser Met Met Ser Ile Thr Ser Ser Arg Cys Arg Ser
100 105 110 Gly Gly Gly
Gly Gly Pro Ala Ala Ala Ala Gly Gln Ser Thr Ser Gly 115
120 125 Gly Gly Gly Gly Glu Glu Glu Glu
Glu Asp Met Thr Arg Thr Thr Ala 130 135
140 Ala Ala Ala Val Val Glu Val Arg Gln His Val Gln Glu
Gly Ser Leu 145 150 155
160 Ile Ser Leu Asp Val Val Leu Ile Cys Ser Ala Ala Arg Pro Val Lys
165 170 175 Phe His Asp Val
Ile Thr Val Leu Glu Glu Glu Gly Ala Asp Ile Ile 180
185 190 Ser Ala Asn Phe Ser Leu Ala Ala His
Asn Phe Tyr Tyr Thr Ile Tyr 195 200
205 Ser Arg Ala Phe Ser Ser Arg Ile Gly Ile Glu Ala Ser Arg
Ile Ser 210 215 220
Glu Arg Leu Arg Ala Leu Val 225 230 216633DNAOryza
sativa 216atggagatcc cgccgccgtc agctggtggc ggaggcggcg gcggcaagcc
cgaccggaag 60acgacggagc gcatccgccg cgagcagatg aacaagctct actcccacct
cgactccctc 120gtccgctccg ctccccccac agttaattcc attccttcac attcaaattc
aaattcaaaa 180taccatcaaa gaaaattacg gattctcggt ggggcggcgg cggcgacgac
gaggccggac 240aggttggggg tggcggcgga gtacataagg cagacgcagg agagggtgga
catgctgagg 300gagaagaaga gggagctcac cggcggcggc ggcggcggct cctcgtcgtc
gtccggcgcc 360ggggcggcca cggccgccgc gccggaggtg gaggtgcagc acctggggtc
cggcctgcac 420gccatcctct tcaccggcgc gccgcccacc gacggcgcct ccttccaccg
cgccgtccgc 480gccgtcgagg acgccggcgg ccaggtgcag aacgcgcact tctccgtcgc
cggcgccaag 540gccgtctaca ccatccacgc catgattgga gatggatatg gaggcattga
gagggtggtg 600cagagattaa aggaagcaat acggagcaac taa
633217210PRTOryza sativa 217Met Glu Ile Pro Pro Pro Ser Ala
Gly Gly Gly Gly Gly Gly Gly Lys 1 5 10
15 Pro Asp Arg Lys Thr Thr Glu Arg Ile Arg Arg Glu Gln
Met Asn Lys 20 25 30
Leu Tyr Ser His Leu Asp Ser Leu Val Arg Ser Ala Pro Pro Thr Val
35 40 45 Asn Ser Ile Pro
Ser His Ser Asn Ser Asn Ser Lys Tyr His Gln Arg 50
55 60 Lys Leu Arg Ile Leu Gly Gly Ala
Ala Ala Ala Thr Thr Arg Pro Asp 65 70
75 80 Arg Leu Gly Val Ala Ala Glu Tyr Ile Arg Gln Thr
Gln Glu Arg Val 85 90
95 Asp Met Leu Arg Glu Lys Lys Arg Glu Leu Thr Gly Gly Gly Gly Gly
100 105 110 Gly Ser Ser
Ser Ser Ser Gly Ala Gly Ala Ala Thr Ala Ala Ala Pro 115
120 125 Glu Val Glu Val Gln His Leu Gly
Ser Gly Leu His Ala Ile Leu Phe 130 135
140 Thr Gly Ala Pro Pro Thr Asp Gly Ala Ser Phe His Arg
Ala Val Arg 145 150 155
160 Ala Val Glu Asp Ala Gly Gly Gln Val Gln Asn Ala His Phe Ser Val
165 170 175 Ala Gly Ala Lys
Ala Val Tyr Thr Ile His Ala Met Ile Gly Asp Gly 180
185 190 Tyr Gly Gly Ile Glu Arg Val Val Gln
Arg Leu Lys Glu Ala Ile Arg 195 200
205 Ser Asn 210 218546DNAArabidopsis thaliana
218atggggagag caagagaaat aggagaagga aactcatcgt cgttaaggga acaacgaaac
60ctcagagaga aggatcgaag gatgcgcatg aaacatctct tctctatact ttcttctcat
120gtttctccca ctcgcaagtt accagtgcct caccttatag atcaagcgac atcatacatg
180atccaattga aagagaatgt aaattatttg aaagagaaga aaaggacatt gttacaagga
240gaactcggga atctctacga agggtcgttt cttctaccca aactcagtat tcgttcgcgg
300gattcgacca tagaaatgaa tctgatcatg gatctaaaca tgaaaagagt aatgttacac
360gagcttgtga gtatttttga agaagaagga gctcaagtaa tgagtgctaa tcttcagaac
420ttgaatgata ggaccactta cacaatcata gcccaggcta tcattagtcg gattggcatt
480gatccatcaa ggatagaaga gagagtacgg aaaatcatct atggatatat atattttgaa
540gcatga
546219188PRTArabidopsis thaliana 219Met Gly Arg Ala Arg Glu Ile Gly Glu
Gly Asn Ser Ser Ser Leu Arg 1 5 10
15 Glu Gln Arg Asn Leu Arg Glu Lys Asp Arg Arg Met Arg Met
Lys His 20 25 30
Leu Phe Ser Ile Leu Ser Ser His Val Ser Pro Thr Arg Lys Leu Pro
35 40 45 Val Pro His Leu
Ile Asp Gln Ala Thr Ser Tyr Met Ile Gln Leu Lys 50
55 60 Glu Asn Val Asn Tyr Leu Lys Glu
Lys Lys Arg Thr Leu Leu Gln Gly 65 70
75 80 Glu Leu Gly Asn Leu Tyr Glu Gly Ser Phe Leu Leu
Pro Lys Leu Ser 85 90
95 Ile Arg Ser Arg Asp Ser Thr Ile Glu Met Asn Leu Ile Met Asp Leu
100 105 110 Asn Met Lys
Arg Val Met Leu His Glu Leu Val Ser Ile Phe Glu Glu 115
120 125 Glu Gly Ala Gln Val Met Ser Ala
Asn Leu Gln Asn Leu Asn Asp Arg 130 135
140 Thr Thr Tyr Thr Ile Ile Ala Gln Val Pro His Pro His
Ala Tyr Ala 145 150 155
160 Ile Ile Ser Arg Ile Gly Ile Asp Pro Ser Arg Ile Glu Glu Arg Val
165 170 175 Arg Lys Ile Ile
Tyr Gly Tyr Ile Tyr Phe Glu Ala 180 185
220525DNAArabidopsis thaliana 220atggaaagag cgagagaaat aggagaagga
agcgcatcgt cattacggga acaacgaaac 60ctcagagaga aagaacgacg aatgcgcatg
aaacatctct tctccatact ctcttctcat 120gtttctccca ctcgtaggtt gccagtgcct
caacttatag atcaagcggt atcatacatg 180atccaactga aagagaaggt aaactatttg
aatgagatga aaaggagaat gttaggagga 240gaagtcaaaa atcgctctga agggtcgtct
cttctgccaa aactcagtat tcgttcactg 300gattcgatca tagaaatgaa tctggttatg
gatctaaaca tgaaaggagt aatgttacac 360aagcttgtga gtgtttttga agaagaagga
gctcaagtga tgagtgctaa tcttcagaac 420ttgaatgata ggacctttta tacaatcata
gcccaggcta tcatatgtcg gatcgggatt 480gatccatcaa ggatagaaga gagattaagg
gatataatct catga 525221174PRTArabidopsis thaliana
221Met Glu Arg Ala Arg Glu Ile Gly Glu Gly Ser Ala Ser Ser Leu Arg 1
5 10 15 Glu Gln Arg Asn
Leu Arg Glu Lys Glu Arg Arg Met Arg Met Lys His 20
25 30 Leu Phe Ser Ile Leu Ser Ser His Val
Ser Pro Thr Arg Arg Leu Pro 35 40
45 Val Pro Gln Leu Ile Asp Gln Ala Val Ser Tyr Met Ile Gln
Leu Lys 50 55 60
Glu Lys Val Asn Tyr Leu Asn Glu Met Lys Arg Arg Met Leu Gly Gly 65
70 75 80 Glu Val Lys Asn Arg
Ser Glu Gly Ser Ser Leu Leu Pro Lys Leu Ser 85
90 95 Ile Arg Ser Leu Asp Ser Ile Ile Glu Met
Asn Leu Val Met Asp Leu 100 105
110 Asn Met Lys Gly Val Met Leu His Lys Leu Val Ser Val Phe Glu
Glu 115 120 125 Glu
Gly Ala Gln Val Met Ser Ala Asn Leu Gln Asn Leu Asn Asp Arg 130
135 140 Thr Phe Tyr Thr Ile Ile
Ala Gln Ala Ile Ile Cys Arg Ile Gly Ile 145 150
155 160 Asp Pro Ser Arg Ile Glu Glu Arg Leu Arg Asp
Ile Ile Ser 165 170
222369DNAArabidopsis thaliana 222atgatccaat tgaaagagaa tgtaaattat
ttgaaagaga agaaaaggac attgttacaa 60ggagaactcg ggaatctcta cgaagggtcg
tttcttctac ccaaactcag tattcgttcg 120cgggattcga ccatagaaat gaatctgatc
atggatctaa acatgaaaag agtaatgtta 180cacgagcttg tgagtatttt tgaagaagaa
ggagctcaag taatgagtgc taatcttcag 240aacttgaatg ataggaccac ttacacaatc
atagcccagg ctatcattag tcggattggc 300attgatccat caaggataga agagagagta
cggaaaatca tctatggata tatatatttt 360gaagcatga
369223122PRTArabidopsis thaliana 223Met
Ile Gln Leu Lys Glu Asn Val Asn Tyr Leu Lys Glu Lys Lys Arg 1
5 10 15 Thr Leu Leu Gln Gly Glu
Leu Gly Asn Leu Tyr Glu Gly Ser Phe Leu 20
25 30 Leu Pro Lys Leu Ser Ile Arg Ser Arg Asp
Ser Thr Ile Glu Met Asn 35 40
45 Leu Ile Met Asp Leu Asn Met Lys Arg Val Met Leu His Glu
Leu Val 50 55 60
Ser Ile Phe Glu Glu Glu Gly Ala Gln Val Met Ser Ala Asn Leu Gln 65
70 75 80 Asn Leu Asn Asp Arg
Thr Thr Tyr Thr Ile Ile Ala Gln Ala Ile Ile 85
90 95 Ser Arg Ile Gly Ile Asp Pro Ser Arg Ile
Glu Glu Arg Val Arg Lys 100 105
110 Ile Ile Tyr Gly Tyr Ile Tyr Phe Glu Ala 115
120 224573DNAArabidopsis thaliana 224atggagccga
gccattcaaa cacaggtcaa tcaagatctg tagatcgcaa aacggttgag 60aaaaatagaa
ggatgcaaat gaagtctctc tactcagaac tcatctctct tcttcctcat 120cattcttcta
cggagccttt aacactacct gatcagctag atgaagctgc aaactacatc 180aagaagctac
aagtgaacgt ggagaaaaag agagaaagga aaaggaacct cgttgcgact 240acaactttgg
agaaactgaa ttccgtcgga tcttcatcgg tttcgtcgag cgtcgatgtc 300tccgtgccaa
gaaagctgcc aaaaatcgag attcaagaaa ctggttccat ttttcacatc 360tttcttgtga
caagcttgga acacaagttt atgttttgtg agatcattcg tgttctcacc 420gaggaattag
gagctgagat cactcatgct ggatactcca ttgttgatga tgctgtcttc 480cacacccttc
actgcaaggt ggaagaacac gattatggag ctaggagtca aattcctgaa 540agactggaga
agattgtgaa cagtgttcac taa
573225190PRTArabidopsis thaliana 225Met Glu Pro Ser His Ser Asn Thr Gly
Gln Ser Arg Ser Val Asp Arg 1 5 10
15 Lys Thr Val Glu Lys Asn Arg Arg Met Gln Met Lys Ser Leu
Tyr Ser 20 25 30
Glu Leu Ile Ser Leu Leu Pro His His Ser Ser Thr Glu Pro Leu Thr
35 40 45 Leu Pro Asp Gln
Leu Asp Glu Ala Ala Asn Tyr Ile Lys Lys Leu Gln 50
55 60 Val Asn Val Glu Lys Lys Arg Glu
Arg Lys Arg Asn Leu Val Ala Thr 65 70
75 80 Thr Thr Leu Glu Lys Leu Asn Ser Val Gly Ser Ser
Ser Val Ser Ser 85 90
95 Ser Val Asp Val Ser Val Pro Arg Lys Leu Pro Lys Ile Glu Ile Gln
100 105 110 Glu Thr Gly
Ser Ile Phe His Ile Phe Leu Val Thr Ser Leu Glu His 115
120 125 Lys Phe Met Phe Cys Glu Ile Ile
Arg Val Leu Thr Glu Glu Leu Gly 130 135
140 Ala Glu Ile Thr His Ala Gly Tyr Ser Ile Val Asp Asp
Ala Val Phe 145 150 155
160 His Thr Leu His Cys Lys Val Glu Glu His Asp Tyr Gly Ala Arg Ser
165 170 175 Gln Ile Pro Glu
Arg Leu Glu Lys Ile Val Asn Ser Val His 180
185 190 226423DNAMedicago truncatula 226atgacatctc
taacaaagga ggcgatttca gtgccggatc agctaaagga agcaacaaac 60tacataaaga
aattgcagat caacctggag aaaatgaagg aaaagaagaa ttttctacta 120ggaattcaaa
ggccaaatgt gaatttgaat agaaaccaga agatgggatt aaagtctcca 180aaaattaaga
tacaacaaat tggtttagtc ttagaggttg ttctaataac tggattggag 240tctcagtttt
tgttcagcga aacctttcga gttcttcatg aagaaggagt tgatattgtt 300aatgctagtt
ataaggtcaa tgaagattct gttttccatt caatacactg ccaggtagga 360gaatttggca
atgaagctgc aagaatatct gagagattga agaagtttat gcaagactat 420tag
423227140PRTMedicago truncatula 227Met Thr Ser Leu Thr Lys Glu Ala Ile
Ser Val Pro Asp Gln Leu Lys 1 5 10
15 Glu Ala Thr Asn Tyr Ile Lys Lys Leu Gln Ile Asn Leu Glu
Lys Met 20 25 30
Lys Glu Lys Lys Asn Phe Leu Leu Gly Ile Gln Arg Pro Asn Val Asn
35 40 45 Leu Asn Arg Asn
Gln Lys Met Gly Leu Lys Ser Pro Lys Ile Lys Ile 50
55 60 Gln Gln Ile Gly Leu Val Leu Glu
Val Val Leu Ile Thr Gly Leu Glu 65 70
75 80 Ser Gln Phe Leu Phe Ser Glu Thr Phe Arg Val Leu
His Glu Glu Gly 85 90
95 Val Asp Ile Val Asn Ala Ser Tyr Lys Val Asn Glu Asp Ser Val Phe
100 105 110 His Ser Ile
His Cys Gln Val Gly Glu Phe Gly Asn Glu Ala Ala Arg 115
120 125 Ile Ser Glu Arg Leu Lys Lys Phe
Met Gln Asp Tyr 130 135 140
228882DNAHordeum vulgare 228atgaatgcct gcgctctgaa ctcgatggag ccggtgaagg
cgaagccggc gaggggcggg 60aagaggagca gggagagcgg cggcacggcg gtggtgctgc
tggagaagaa ggagtcggag 120aaggagagga ggaagcgcat gaaggcgctc tgcgagaagc
tcgcatccct catcccaagg 180gaacactgct gctccaccac tgatacaatg acccagctag
gcagcctgga tgtgggggca 240tcgtatatca agaagctgaa ggagagggtc gatgagctac
aacgtaggat gacctctgcg 300cagaccttgg ataccttcag aggagacact agcatcccaa
cgcccactac caccactacc 360acgaaaagcg ttgtagggtc gctggaagaa gagaaagctc
gggaggcatc ggcacccgta 420ttgcaggtgc ggcaacacga cgattcaagc atggaggtga
gattgatatg ctgcatgaag 480aggccgatca agctccatga ggtgatcacc atccatgagg
aagaaggtgc tgagatcatc 540aacgccaatc actctgttgc tggccaaaaa atgttctaca
ctatacactc tcgggcctct 600agctcgagaa ttggcataga tgttccaagg gtttctgaac
gactgcgagc attgctccaa 660ctttattcgc atgaaaatca ggcaccgtcg tgcatgcaca
acaatcaagt cctcaggtac 720aatgatgggg ccaacgcgac tcctcccaag gagctcgtcg
gcaacaatga tataggtgga 780accaacaagg tggcaaacca cgagtgtgag tcttgggttg
agcaagacca agtgatgtgg 840agttccctgc ttgcgtcaat atcaccagag ttgctcgagt
ag 882229293PRTHordeum vulgare 229Met Asn Ala Cys
Ala Leu Asn Ser Met Glu Pro Val Lys Ala Lys Pro 1 5
10 15 Ala Arg Gly Gly Lys Arg Ser Arg Glu
Ser Gly Gly Thr Ala Val Val 20 25
30 Leu Leu Glu Lys Lys Glu Ser Glu Lys Glu Arg Arg Lys Arg
Met Lys 35 40 45
Ala Leu Cys Glu Lys Leu Ala Ser Leu Ile Pro Arg Glu His Cys Cys 50
55 60 Ser Thr Thr Asp Thr
Met Thr Gln Leu Gly Ser Leu Asp Val Gly Ala 65 70
75 80 Ser Tyr Ile Lys Lys Leu Lys Glu Arg Val
Asp Glu Leu Gln Arg Arg 85 90
95 Met Thr Ser Ala Gln Thr Leu Asp Thr Phe Arg Gly Asp Thr Ser
Ile 100 105 110 Pro
Thr Pro Thr Thr Thr Thr Thr Thr Lys Ser Val Val Gly Ser Leu 115
120 125 Glu Glu Glu Lys Ala Arg
Glu Ala Ser Ala Pro Val Leu Gln Val Arg 130 135
140 Gln His Asp Asp Ser Ser Met Glu Val Arg Leu
Ile Cys Cys Met Lys 145 150 155
160 Arg Pro Ile Lys Leu His Glu Val Ile Thr Ile His Glu Glu Glu Gly
165 170 175 Ala Glu
Ile Ile Asn Ala Asn His Ser Val Ala Gly Gln Lys Met Phe 180
185 190 Tyr Thr Ile His Ser Arg Ala
Ser Ser Ser Arg Ile Gly Ile Asp Val 195 200
205 Pro Arg Val Ser Glu Arg Leu Arg Ala Leu Leu Gln
Leu Tyr Ser His 210 215 220
Glu Asn Gln Ala Pro Ser Cys Met His Asn Asn Gln Val Leu Arg Tyr 225
230 235 240 Asn Asp Gly
Ala Asn Ala Thr Pro Pro Lys Glu Leu Val Gly Asn Asn 245
250 255 Asp Ile Gly Gly Thr Asn Lys Val
Ala Asn His Glu Cys Glu Ser Trp 260 265
270 Val Glu Gln Asp Gln Val Met Trp Ser Ser Leu Leu Ala
Ser Ile Ser 275 280 285
Pro Glu Leu Leu Glu 290 2302193DNAOryza sativa
230aatccgaaaa gtttctgcac cgttttcacc ccctaactaa caatataggg aacgtgtgct
60aaatataaaa tgagacctta tatatgtagc gctgataact agaactatgc aagaaaaact
120catccaccta ctttagtggc aatcgggcta aataaaaaag agtcgctaca ctagtttcgt
180tttccttagt aattaagtgg gaaaatgaaa tcattattgc ttagaatata cgttcacatc
240tctgtcatga agttaaatta ttcgaggtag ccataattgt catcaaactc ttcttgaata
300aaaaaatctt tctagctgaa ctcaatgggt aaagagagag atttttttta aaaaaataga
360atgaagatat tctgaacgta ttggcaaaga tttaaacata taattatata attttatagt
420ttgtgcattc gtcatatcgc acatcattaa ggacatgtct tactccatcc caatttttat
480ttagtaatta aagacaattg acttattttt attatttatc ttttttcgat tagatgcaag
540gtacttacgc acacactttg tgctcatgtg catgtgtgag tgcacctcct caatacacgt
600tcaactagca acacatctct aatatcactc gcctatttaa tacatttagg tagcaatatc
660tgaattcaag cactccacca tcaccagacc acttttaata atatctaaaa tacaaaaaat
720aattttacag aatagcatga aaagtatgaa acgaactatt taggtttttc acatacaaaa
780aaaaaaagaa ttttgctcgt gcgcgagcgc caatctccca tattgggcac acaggcaaca
840acagagtggc tgcccacaga acaacccaca aaaaacgatg atctaacgga ggacagcaag
900tccgcaacaa ccttttaaca gcaggctttg cggccaggag agaggaggag aggcaaagaa
960aaccaagcat cctcctcctc ccatctataa attcctcccc ccttttcccc tctctatata
1020ggaggcatcc aagccaagaa gagggagagc accaaggaca cgcgactagc agaagccgag
1080cgaccgcctt cttcgatcca tatcttccgg tcgagttctt ggtcgatctc ttccctcctc
1140cacctcctcc tcacagggta tgtgcccttc ggttgttctt ggatttattg ttctaggttg
1200tgtagtacgg gcgttgatgt taggaaaggg gatctgtatc tgtgatgatt cctgttcttg
1260gatttgggat agaggggttc ttgatgttgc atgttatcgg ttcggtttga ttagtagtat
1320ggttttcaat cgtctggaga gctctatgga aatgaaatgg tttagggtac ggaatcttgc
1380gattttgtga gtaccttttg tttgaggtaa aatcagagca ccggtgattt tgcttggtgt
1440aataaaagta cggttgtttg gtcctcgatt ctggtagtga tgcttctcga tttgacgaag
1500ctatcctttg tttattccct attgaacaaa aataatccaa ctttgaagac ggtcccgttg
1560atgagattga atgattgatt cttaagcctg tccaaaattt cgcagctggc ttgtttagat
1620acagtagtcc ccatcacgaa attcatggaa acagttataa tcctcaggaa caggggattc
1680cctgttcttc cgatttgctt tagtcccaga attttttttc ccaaatatct taaaaagtca
1740ctttctggtt cagttcaatg aattgattgc tacaaataat gcttttatag cgttatccta
1800gctgtagttc agttaatagg taatacccct atagtttagt caggagaaga acttatccga
1860tttctgatct ccatttttaa ttatatgaaa tgaactgtag cataagcagt attcatttgg
1920attatttttt ttattagctc tcaccccttc attattctga gctgaaagtc tggcatgaac
1980tgtcctcaat tttgttttca aattcacatc gattatctat gcattatcct cttgtatcta
2040cctgtagaag tttctttttg gttattcctt gactgcttga ttacagaaag aaatttatga
2100agctgtaatc gggatagtta tactgcttgt tcttatgatt catttccttt gtgcagttct
2160tggtgtagct tgccactttc accagcaaag ttc
219323156DNAArtificial sequenceprimer prm06808 231ggggacaagt ttgtacaaaa
aagcaggctt aaacaatgaa gagcaggaag aacagc 5623250DNAArtificial
sequenceprimer prm06809 232ggggaccact ttgtacaaga aagctgggtg cagagtgaaa
gagtggtgtg 502332194DNAOryza sativa 233aatccgaaaa
gtttctgcac cgttttcacc ccctaactaa caatataggg aacgtgtgct 60aaatataaaa
tgagacctta tatatgtagc gctgataact agaactatgc aagaaaaact 120catccaccta
ctttagtggc aatcgggcta aataaaaaag agtcgctaca ctagtttcgt 180tttccttagt
aattaagtgg gaaaatgaaa tcattattgc ttagaatata cgttcacatc 240tctgtcatga
agttaaatta ttcgaggtag ccataattgt catcaaactc ttcttgaata 300aaaaaatctt
tctagctgaa ctcaatgggt aaagagagag atttttttta aaaaaataga 360atgaagatat
tctgaacgta ttggcaaaga tttaaacata taattatata attttatagt 420ttgtgcattc
gtcatatcgc acatcattaa ggacatgtct tactccatcc caatttttat 480ttagtaatta
aagacaattg acttattttt attatttatc ttttttcgat tagatgcaag 540gtacttacgc
acacactttg tgctcatgtg catgtgtgag tgcacctcct caatacacgt 600tcaactagca
acacatctct aatatcactc gcctatttaa tacatttagg tagcaatatc 660tgaattcaag
cactccacca tcaccagacc acttttaata atatctaaaa tacaaaaaat 720aattttacag
aatagcatga aaagtatgaa acgaactatt taggtttttc acatacaaaa 780aaaaaaagaa
ttttgctcgt gcgcgagcgc caatctccca tattgggcac acaggcaaca 840acagagtggc
tgcccacaga acaacccaca aaaaacgatg atctaacgga ggacagcaag 900tccgcaacaa
ccttttaaca gcaggctttg cggccaggag agaggaggag aggcaaagaa 960aaccaagcat
cctccttctc ccatctataa attcctcccc ccttttcccc tctctatata 1020ggaggcatcc
aagccaagaa gagggagagc accaaggaca cgcgactagc agaagccgag 1080cgaccgcctt
ctcgatccat atcttccggt cgagttcttg gtcgatctct tccctcctcc 1140acctcctcct
cacagggtat gtgcctccct tcggttgttc ttggatttat tgttctaggt 1200tgtgtagtac
gggcgttgat gttaggaaag gggatctgta tctgtgatga ttcctgttct 1260tggatttggg
atagaggggt tcttgatgtt gcatgttatc ggttcggttt gattagtagt 1320atggttttca
atcgtctgga gagctctatg gaaatgaaat ggtttaggga tcggaatctt 1380gcgattttgt
gagtaccttt tgtttgaggt aaaatcagag caccggtgat tttgcttggt 1440gtaataaagt
acggttgttt ggtcctcgat tctggtagtg atgcttctcg atttgacgaa 1500gctatccttt
gtttattccc tattgaacaa aaataatcca actttgaaga cggtcccgtt 1560gatgagattg
aatgattgat tcttaagcct gtccaaaatt tcgcagctgg cttgtttaga 1620tacagtagtc
cccatcacga aattcatgga aacagttata atcctcagga acaggggatt 1680ccctgttctt
ccgatttgct ttagtcccag aatttttttt cccaaatatc ttaaaaagtc 1740actttctggt
tcagttcaat gaattgattg ctacaaataa tgcttttata gcgttatcct 1800agctgtagtt
cagttaatag gtaatacccc tatagtttag tcaggagaag aacttatccg 1860atttctgatc
tccattttta attatatgaa atgaactgta gcataagcag tattcatttg 1920gattattttt
tttattagct ctcacccctt cattattctg agctgaaagt ctggcatgaa 1980ctgtcctcaa
ttttgttttc aaattcacat cgattatcta tgcattatcc tcttgtatct 2040acctgtagaa
gtttcttttt ggttattcct tgactgcttg attacagaaa gaaatttatg 2100aagctgtaat
cgggatagtt atactgcttg ttcttatgat tcatttcctt tgtgcagttc 2160ttggtgtagc
ttgccacttt caccagcaaa gttc
21942341065DNAArabidopsis thaliana 234atggagttgt taatgtgttc gggtcaggcc
gagtcaggtg gttcctcttc caccgagtct 60tcttcactca gtggtggact caggtttggt
cagaagatct acttcgagga tggatccgga 120tccagaagca agaaccgggt caataccgtt
cgtaagtcgt ctaccacggc gaggtgccaa 180gtggaaggtt gtagaatgga tctaagcaat
gttaaagctt attactcgag acacaaagtt 240tgttgcattc actctaaatc atctaaagtc
attgtctctg gtcttcatca aaggttttgt 300caacaatgta gcaggtttca ccagctttct
gagtttgact tggagaaaag aagttgtcgc 360agaagactcg cttgtcataa cgaacgacga
agaaaaccac aacccacaac ggctcttttc 420acttctcatt actctcgaat cgctccatct
ctttacggaa accccaatgc tgcaatgatt 480aaaagcgttt tgggagatcc tactgcgtgg
tcaaccgcaa gatcagtgat gcagcggcct 540ggaccgtggc agattaatcc agttagggaa
acccatccac acatgaatgt tttatcacat 600ggaagctcaa gctttactac atgtccagag
atgataaaca acaatagcac agattcaagc 660tgtgctctct ctcttctgtc aaactcatac
ccaattcatc agcagcaact tcagacacca 720acaaatacat ggcgaccatc ttctggtttc
gactcgatga tctcattctc cgataaggtt 780acaatggctc agccaccgcc catttcaacc
catcagccgc ccatctcaac acatcagcag 840tacctcagcc aaacttggga agtcatcgcg
ggcgaaaaga gcaattcaca ttatatgtct 900cctgtgagtc aaatctcgga gccagcagat
ttccagataa gcaatggcac cacaatgggt 960ggatttgagc tgtatcttca tcagcaggtt
ctgaagcaat acatggaacc cgagaacaca 1020agagcttatg actcctctcc tcaacatttc
aattggtctc tttga 1065235354PRTArabidopsis thaliana
235Met Glu Leu Leu Met Cys Ser Gly Gln Ala Glu Ser Gly Gly Ser Ser 1
5 10 15 Ser Thr Glu Ser
Ser Ser Leu Ser Gly Gly Leu Arg Phe Gly Gln Lys 20
25 30 Ile Tyr Phe Glu Asp Gly Ser Gly Ser
Arg Ser Lys Asn Arg Val Asn 35 40
45 Thr Val Arg Lys Ser Ser Thr Thr Ala Arg Cys Gln Val Glu
Gly Cys 50 55 60
Arg Met Asp Leu Ser Asn Val Lys Ala Tyr Tyr Ser Arg His Lys Val 65
70 75 80 Cys Cys Ile His Ser
Lys Ser Ser Lys Val Ile Val Ser Gly Leu His 85
90 95 Gln Arg Phe Cys Gln Gln Cys Ser Arg Phe
His Gln Leu Ser Glu Phe 100 105
110 Asp Leu Glu Lys Arg Ser Cys Arg Arg Arg Leu Ala Cys His Asn
Glu 115 120 125 Arg
Arg Arg Lys Pro Gln Pro Thr Thr Ala Leu Phe Thr Ser His Tyr 130
135 140 Ser Arg Ile Ala Pro Ser
Leu Tyr Gly Asn Pro Asn Ala Ala Met Ile 145 150
155 160 Lys Ser Val Leu Gly Asp Pro Thr Ala Trp Ser
Thr Ala Arg Ser Val 165 170
175 Met Gln Arg Pro Gly Pro Trp Gln Ile Asn Pro Val Arg Glu Thr His
180 185 190 Pro His
Met Asn Val Leu Ser His Gly Ser Ser Ser Phe Thr Thr Cys 195
200 205 Pro Glu Met Ile Asn Asn Asn
Ser Thr Asp Ser Ser Cys Ala Leu Ser 210 215
220 Leu Leu Ser Asn Ser Tyr Pro Ile His Gln Gln Gln
Leu Gln Thr Pro 225 230 235
240 Thr Asn Thr Trp Arg Pro Ser Ser Gly Phe Asp Ser Met Ile Ser Phe
245 250 255 Ser Asp Lys
Val Thr Met Ala Gln Pro Pro Pro Ile Ser Thr His Gln 260
265 270 Pro Pro Ile Ser Thr His Gln Gln
Tyr Leu Ser Gln Thr Trp Glu Val 275 280
285 Ile Ala Gly Glu Lys Ser Asn Ser His Tyr Met Ser Pro
Val Ser Gln 290 295 300
Ile Ser Glu Pro Ala Asp Phe Gln Ile Ser Asn Gly Thr Thr Met Gly 305
310 315 320 Gly Phe Glu Leu
Tyr Leu His Gln Gln Val Leu Lys Gln Tyr Met Glu 325
330 335 Pro Glu Asn Thr Arg Ala Tyr Asp Ser
Ser Pro Gln His Phe Asn Trp 340 345
350 Ser Leu 2361128DNAArabidopsis thaliana 236atggagatgg
gttccaactc gggtccgggt catggtccgg gtcaggcaga gtcgggtggt 60tcctccactg
agtcatcctc tttcagtgga gggctcatgt ttggccagaa gatctacttc 120gaggacggtg
gtggtggatc cgggtcttct tcctcaggtg gtcgttcaaa cagacgtgtc 180cgtggaggcg
ggtcgggtca gtcgggtcag ataccaaggt gccaagtgga aggttgtggg 240atggatctaa
ccaatgcaaa aggttattac tcgagacacc gagtttgtgg agtgcactct 300aaaacaccta
aagtcactgt ggctggtatc gaacagaggt tttgtcaaca gtgcagcagg 360tttcatcagc
ttccggaatt tgacctagag aaaaggagtt gccgcaggag actcgctggt 420cataatgagc
gacgaaggaa gccacagcct gcgtctctct ctgtgttagc ttctcgttac 480gggaggatcg
caccttcgct ttacgaaaat ggtgatgctg gaatgaatgg aagctttctt 540gggaaccaag
agataggatg gccaagttca agaacattgg atacaagagt gatgaggcgg 600ccagtgtcgt
caccgtcatg gcagatcaat ccaatgaatg tatttagtca aggttcagtt 660ggtggaggag
ggacaagctt ctcatctcca gagattatgg acactaaact agagagctac 720aagggaattg
gcgactcaaa ctgtgctctc tctcttctgt caaatccaca tcaaccacat 780gacaacaaca
acaacaacaa caacaacaac aacaacaaca acaatacatg gcgagcttct 840tcaggttttg
gcccgatgac ggttacaatg gctcaaccac cacctgcacc tagccagcat 900cagtatctga
acccgccttg ggtattcaag gacaatgata atgatatgtc tcctgttttg 960aatttaggtc
gatacaccga gccagataat tgtcagataa gtagtggcac ggcaatgggt 1020gagttcgagt
tatctgatca ccatcatcaa agtaggagac agtacatgga agatgagaac 1080acaagggctt
atgactcttc ttctcaccat accaactggt ccctctga
1128237375PRTArabidopsis thaliana 237Met Glu Met Gly Ser Asn Ser Gly Pro
Gly His Gly Pro Gly Gln Ala 1 5 10
15 Glu Ser Gly Gly Ser Ser Thr Glu Ser Ser Ser Phe Ser Gly
Gly Leu 20 25 30
Met Phe Gly Gln Lys Ile Tyr Phe Glu Asp Gly Gly Gly Gly Ser Gly
35 40 45 Ser Ser Ser Ser
Gly Gly Arg Ser Asn Arg Arg Val Arg Gly Gly Gly 50
55 60 Ser Gly Gln Ser Gly Gln Ile Pro
Arg Cys Gln Val Glu Gly Cys Gly 65 70
75 80 Met Asp Leu Thr Asn Ala Lys Gly Tyr Tyr Ser Arg
His Arg Val Cys 85 90
95 Gly Val His Ser Lys Thr Pro Lys Val Thr Val Ala Gly Ile Glu Gln
100 105 110 Arg Phe Cys
Gln Gln Cys Ser Arg Phe His Gln Leu Pro Glu Phe Asp 115
120 125 Leu Glu Lys Arg Ser Cys Arg Arg
Arg Leu Ala Gly His Asn Glu Arg 130 135
140 Arg Arg Lys Pro Gln Pro Ala Ser Leu Ser Val Leu Ala
Ser Arg Tyr 145 150 155
160 Gly Arg Ile Ala Pro Ser Leu Tyr Glu Asn Gly Asp Ala Gly Met Asn
165 170 175 Gly Ser Phe Leu
Gly Asn Gln Glu Ile Gly Trp Pro Ser Ser Arg Thr 180
185 190 Leu Asp Thr Arg Val Met Arg Arg Pro
Val Ser Ser Pro Ser Trp Gln 195 200
205 Ile Asn Pro Met Asn Val Phe Ser Gln Gly Ser Val Gly Gly
Gly Gly 210 215 220
Thr Ser Phe Ser Ser Pro Glu Ile Met Asp Thr Lys Leu Glu Ser Tyr 225
230 235 240 Lys Gly Ile Gly Asp
Ser Asn Cys Ala Leu Ser Leu Leu Ser Asn Pro 245
250 255 His Gln Pro His Asp Asn Asn Asn Asn Asn
Asn Asn Asn Asn Asn Asn 260 265
270 Asn Asn Asn Thr Trp Arg Ala Ser Ser Gly Phe Gly Pro Met Thr
Val 275 280 285 Thr
Met Ala Gln Pro Pro Pro Ala Pro Ser Gln His Gln Tyr Leu Asn 290
295 300 Pro Pro Trp Val Phe Lys
Asp Asn Asp Asn Asp Met Ser Pro Val Leu 305 310
315 320 Asn Leu Gly Arg Tyr Thr Glu Pro Asp Asn Cys
Gln Ile Ser Ser Gly 325 330
335 Thr Ala Met Gly Glu Phe Glu Leu Ser Asp His His His Gln Ser Arg
340 345 350 Arg Gln
Tyr Met Glu Asp Glu Asn Thr Arg Ala Tyr Asp Ser Ser Ser 355
360 365 His His Thr Asn Trp Ser Leu
370 375 2381149DNAAquilegia formosa x Aquilegia
pubescens 238atggaaatgg gttccagctc tttcgctggt ggtgggggta agggtgcttc
tggttcatct 60gattcttcac tgaatggttt gaaatttggg aagaaaatct actttgaaga
tgtgggtatt 120ggagttttag gcaagtcaag tgttgggtct ccgtcaattt tggtttctga
agctcggttg 180ccaccggctt cgtcggtgaa gaagggtaga ggggttttgc aaggacaacc
acctagatgt 240tctgttgaag gttgtaagct tgatcttact gatgctaagc cttattactc
aaggcacaaa 300gtctgtggta tgcactctaa atctcctaaa gtaattgttg gtggtttgga
gcagaggttt 360tgccagcagt gtagcagatt tcatctactc tgtgaatttg accaaggcaa
gcgaagctgt 420cgtagacgtc tagctggcca caatgagcgt cgaagaaaac cacaacctgg
atcaatattt 480tcaccgcgct atggtcgtgt gtcaccatct ttccaagaaa atagcaccag
aggaggaggt 540tttctaatgg acttcacagc gtacccaagg ctggcaggaa gggatgcatg
gcaaacagta 600aaagccggca attgggcaga tggaaaccaa acctctccta ttaagaagct
tctcccacat 660caatggcaag gcaattcaga gaatcctcct cctattgtct attctcaggc
acctcacccg 720tatctgcaag gtgtggctag tggatcaatt ttttccagta caagaatacc
ttcagccgag 780tgttttgctg gtgtctctga ctccagctgt gctctctctc ttctgtcaaa
tcaaccatgg 840aaccccagaa accagacttc cgatttggag gtgaataaca tagtgaacgg
tgaaggggta 900tccatggcaa gatctatagc acctcatagt gctgtagtca ccaacttcac
gaacaacaca 960tggaatttta agggcaacag tgaagttagt ggcagttccc atgaaattcc
acgtcaggtg 1020tcgcagccag tcaccaatca tttctccagt gagctcgatc tagctcagca
ggggaacagg 1080cagtacatgg agatccggca ttcaagggat tttggttctt ccactcacca
gatgcactgg 1140tctctttga
1149239382PRTAquilegia formosa x Aquilegia pubescens 239Met
Glu Met Gly Ser Ser Ser Phe Ala Gly Gly Gly Gly Lys Gly Ala 1
5 10 15 Ser Gly Ser Ser Asp Ser
Ser Leu Asn Gly Leu Lys Phe Gly Lys Lys 20
25 30 Ile Tyr Phe Glu Asp Val Gly Ile Gly Val
Leu Gly Lys Ser Ser Val 35 40
45 Gly Ser Pro Ser Ile Leu Val Ser Glu Ala Arg Leu Pro Pro
Ala Ser 50 55 60
Ser Val Lys Lys Gly Arg Gly Val Leu Gln Gly Gln Pro Pro Arg Cys 65
70 75 80 Ser Val Glu Gly Cys
Lys Leu Asp Leu Thr Asp Ala Lys Pro Tyr Tyr 85
90 95 Ser Arg His Lys Val Cys Gly Met His Ser
Lys Ser Pro Lys Val Ile 100 105
110 Val Gly Gly Leu Glu Gln Arg Phe Cys Gln Gln Cys Ser Arg Phe
His 115 120 125 Leu
Leu Cys Glu Phe Asp Gln Gly Lys Arg Ser Cys Arg Arg Arg Leu 130
135 140 Ala Gly His Asn Glu Arg
Arg Arg Lys Pro Gln Pro Gly Ser Ile Phe 145 150
155 160 Ser Pro Arg Tyr Gly Arg Val Ser Pro Ser Phe
Gln Glu Asn Ser Thr 165 170
175 Arg Gly Gly Gly Phe Leu Met Asp Phe Thr Ala Tyr Pro Arg Leu Ala
180 185 190 Gly Arg
Asp Ala Trp Gln Thr Val Lys Ala Gly Asn Trp Ala Asp Gly 195
200 205 Asn Gln Thr Ser Pro Ile Lys
Lys Leu Leu Pro His Gln Trp Gln Gly 210 215
220 Asn Ser Glu Asn Pro Pro Pro Ile Val Tyr Ser Gln
Ala Pro His Pro 225 230 235
240 Tyr Leu Gln Gly Val Ala Ser Gly Ser Ile Phe Ser Ser Thr Arg Ile
245 250 255 Pro Ser Ala
Glu Cys Phe Ala Gly Val Ser Asp Ser Ser Cys Ala Leu 260
265 270 Ser Leu Leu Ser Asn Gln Pro Trp
Asn Pro Arg Asn Gln Thr Ser Asp 275 280
285 Leu Glu Val Asn Asn Ile Val Asn Gly Glu Gly Val Ser
Met Ala Arg 290 295 300
Ser Ile Ala Pro His Ser Ala Val Val Thr Asn Phe Thr Asn Asn Thr 305
310 315 320 Trp Asn Phe Lys
Gly Asn Ser Glu Val Ser Gly Ser Ser His Glu Ile 325
330 335 Pro Arg Gln Val Ser Gln Pro Val Thr
Asn His Phe Ser Ser Glu Leu 340 345
350 Asp Leu Ala Gln Gln Gly Asn Arg Gln Tyr Met Glu Ile Arg
His Ser 355 360 365
Arg Asp Phe Gly Ser Ser Thr His Gln Met His Trp Ser Leu 370
375 380 2401170DNAGossypium hirsutum
240atggaaatgg gttcgggctc tttgactgag tcgggcggtt ctcccaccaa ctcttccgcc
60gagtcactca acggcttgaa gttcggtaaa aagatctact ttgaggatac agccgctgtc
120gccgccgccg ctggtggtaa aagtgttggt ggtggtgcaa acatagggac tccatccaag
180tccggtccag catcttcaag cttagccggg tcgtgtagga aagccagggt tgatggaatg
240gcgcaagggg ttctgccttc taggtgtcaa gtagaagggt gtaaagtgga tctgagtgat
300gctaaggctt actattcaag gcataaggtt tgttgtatgc actctaagtc atctaaagtc
360attgttgctg gtctcgagca aagattttgt cagcagtgta gcagatttca tcagctttct
420gaatttgaca aagggaaacg gagttgtcgt agacggcttg caggtcacaa tgagcgacgc
480aggaaaccac cacctggatc attattttcc tctccttatg gccggctttc ttcctctatt
540attgaaaatg gcagtagagg tggaagcttt atagtggatt tctcagcata cccaaggctt
600tcaggaaggg atgcatggcc agcagctcga tcgttagaat gcataactgg aaatcgaagc
660acagccactg gaagctcatt ttcacatcca cggcaaaaca actccagcaa acctcctcat
720gaccatttct tgcaaggttc accatgtggg actagtttct ccagcactgg aatttctcca
780ggagaatgct tcacagggtc tggtgactca agctgtgctc tctctcttct gtcaaatcaa
840ccgtggggct ccaggaacca ggctttgaat ttttctgtaa atggcgtgat aagtactgaa
900gggtcctctg cggcacaacc aacaacgctt catggtgcag ttgtgaaccc ttattcaaat
960gcctctttgg atttcaatgg cagtgacact gttcgcagtt ctcacaagat gctgccacac
1020ctagatttgg gtcaaatccc agaccctgtt aactgtcaat tctctagtga ccttgagttg
1080tctcaacaaa gctggaggtc atatatcgaa catgagcagt ccggggcagc ctatgacgac
1140tccatgcagc atatccactg gacgctctaa
1170241389PRTGossypium hirsutum 241Met Glu Met Gly Ser Gly Ser Leu Thr
Glu Ser Gly Gly Ser Pro Thr 1 5 10
15 Asn Ser Ser Ala Glu Ser Leu Asn Gly Leu Lys Phe Gly Lys
Lys Ile 20 25 30
Tyr Phe Glu Asp Thr Ala Ala Val Ala Ala Ala Ala Gly Gly Lys Ser
35 40 45 Val Gly Gly Gly
Ala Asn Ile Gly Thr Pro Ser Lys Ser Gly Pro Ala 50
55 60 Ser Ser Ser Leu Ala Gly Ser Cys
Arg Lys Ala Arg Val Asp Gly Met 65 70
75 80 Ala Gln Gly Val Leu Pro Ser Arg Cys Gln Val Glu
Gly Cys Lys Val 85 90
95 Asp Leu Ser Asp Ala Lys Ala Tyr Tyr Ser Arg His Lys Val Cys Cys
100 105 110 Met His Ser
Lys Ser Ser Lys Val Ile Val Ala Gly Leu Glu Gln Arg 115
120 125 Phe Cys Gln Gln Cys Ser Arg Phe
His Gln Leu Ser Glu Phe Asp Lys 130 135
140 Gly Lys Arg Ser Cys Arg Arg Arg Leu Ala Gly His Asn
Glu Arg Arg 145 150 155
160 Arg Lys Pro Pro Pro Gly Ser Leu Phe Ser Ser Pro Tyr Gly Arg Leu
165 170 175 Ser Ser Ser Ile
Ile Glu Asn Gly Ser Arg Gly Gly Ser Phe Ile Val 180
185 190 Asp Phe Ser Ala Tyr Pro Arg Leu Ser
Gly Arg Asp Ala Trp Pro Ala 195 200
205 Ala Arg Ser Leu Glu Cys Ile Thr Gly Asn Arg Ser Thr Ala
Thr Gly 210 215 220
Ser Ser Phe Ser His Pro Arg Gln Asn Asn Ser Ser Lys Pro Pro His 225
230 235 240 Asp His Phe Leu Gln
Gly Ser Pro Cys Gly Thr Ser Phe Ser Ser Thr 245
250 255 Gly Ile Ser Pro Gly Glu Cys Phe Thr Gly
Ser Gly Asp Ser Ser Cys 260 265
270 Ala Leu Ser Leu Leu Ser Asn Gln Pro Trp Gly Ser Arg Asn Gln
Ala 275 280 285 Leu
Asn Phe Ser Val Asn Gly Val Ile Ser Thr Glu Gly Ser Ser Ala 290
295 300 Ala Gln Pro Thr Thr Leu
His Gly Ala Val Val Asn Pro Tyr Ser Asn 305 310
315 320 Ala Ser Leu Asp Phe Asn Gly Ser Asp Thr Val
Arg Ser Ser His Lys 325 330
335 Met Leu Pro His Leu Asp Leu Gly Gln Ile Pro Asp Pro Val Asn Cys
340 345 350 Gln Phe
Ser Ser Asp Leu Glu Leu Ser Gln Gln Ser Trp Arg Ser Tyr 355
360 365 Ile Glu His Glu Gln Ser Gly
Ala Ala Tyr Asp Asp Ser Met Gln His 370 375
380 Ile His Trp Thr Leu 385
2421017DNAIpomoea nil 242atggaactgg gttcttcttc ttcttctacc tctacctccg
ccgactcctc atccgacggc 60ttgaagttcg gcaaaaaagt ctactttgaa gatgcgggtg
gtgggagtgg tgggtcgtcg 120ctgccggcca agagagggag gagcgcggtg gcgcaaggcg
gacagccacc caggtgccag 180gtggaagggt gcaaggcaga tctgagtgaa tttaaggctt
attactcaaa gcataaagtt 240tgtggtatgc actccaagtc tcctaaggtc attgtttctg
ggcttgaaca gagattctgc 300cagcagtgca gcaggtttca tcaattgagt gaatttgatc
aagtaaaaag gagctgccgt 360aggcgtttgg ctggtcataa tgagcgtcgt aggaagcccc
cacttggatc catattgtcc 420acacattatg ggactctttc ttcttcaatg tttggaaaca
atggccactt tgtgatggat 480ttcgcctcat acccatatcc gggtggtaag ggctcatggc
ggccaagtac caatggcgtg 540gggaactttc ttcaacaacc atggcagaga aactctgaag
atcccccacc caagcttctt 600ttgctaggtt cggatgctgc tgctagggct acttatccca
gtccttgtgg agaatacttc 660aatggggtct cggattccac ccgtgctctc tctcttctgt
caaactcaaa tcagccctgg 720ggctcgagaa accaacaacc ctctggtctc ggggttaata
gcttacttaa cactgatgga 780acgcttgctg ttcatccatc cggttcccat gctgccgtta
tcaatgaatt ttcttcaagt 840ccatggggtt ttaaaggcaa tcaagccact accagctcag
ataagatcct tcctgatagt 900cactattctg gtgagctcga gatgatggct catcaacaaa
ctggacgagc atacatggga 960atggagtact cgacgggtta tgattcttct gtccagaatg
tgcactggac tctctga 1017243338PRTIpomoea nil 243Met Glu Leu Gly Ser
Ser Ser Ser Ser Thr Ser Thr Ser Ala Asp Ser 1 5
10 15 Ser Ser Asp Gly Leu Lys Phe Gly Lys Lys
Val Tyr Phe Glu Asp Ala 20 25
30 Gly Gly Gly Ser Gly Gly Ser Ser Leu Pro Ala Lys Arg Gly Arg
Ser 35 40 45 Ala
Val Ala Gln Gly Gly Gln Pro Pro Arg Cys Gln Val Glu Gly Cys 50
55 60 Lys Ala Asp Leu Ser Glu
Phe Lys Ala Tyr Tyr Ser Lys His Lys Val 65 70
75 80 Cys Gly Met His Ser Lys Ser Pro Lys Val Ile
Val Ser Gly Leu Glu 85 90
95 Gln Arg Phe Cys Gln Gln Cys Ser Arg Phe His Gln Leu Ser Glu Phe
100 105 110 Asp Gln
Val Lys Arg Ser Cys Arg Arg Arg Leu Ala Gly His Asn Glu 115
120 125 Arg Arg Arg Lys Pro Pro Leu
Gly Ser Ile Leu Ser Thr His Tyr Gly 130 135
140 Thr Leu Ser Ser Ser Met Phe Gly Asn Asn Gly His
Phe Val Met Asp 145 150 155
160 Phe Ala Ser Tyr Pro Tyr Pro Gly Gly Lys Gly Ser Trp Arg Pro Ser
165 170 175 Thr Asn Gly
Val Gly Asn Phe Leu Gln Gln Pro Trp Gln Arg Asn Ser 180
185 190 Glu Asp Pro Pro Pro Lys Leu Leu
Leu Leu Gly Ser Asp Ala Ala Ala 195 200
205 Arg Ala Thr Tyr Pro Ser Pro Cys Gly Glu Tyr Phe Asn
Gly Val Ser 210 215 220
Asp Ser Thr Arg Ala Leu Ser Leu Leu Ser Asn Ser Asn Gln Pro Trp 225
230 235 240 Gly Ser Arg Asn
Gln Gln Pro Ser Gly Leu Gly Val Asn Ser Leu Leu 245
250 255 Asn Thr Asp Gly Thr Leu Ala Val His
Pro Ser Gly Ser His Ala Ala 260 265
270 Val Ile Asn Glu Phe Ser Ser Ser Pro Trp Gly Phe Lys Gly
Asn Gln 275 280 285
Ala Thr Thr Ser Ser Asp Lys Ile Leu Pro Asp Ser His Tyr Ser Gly 290
295 300 Glu Leu Glu Met Met
Ala His Gln Gln Thr Gly Arg Ala Tyr Met Gly 305 310
315 320 Met Glu Tyr Ser Thr Gly Tyr Asp Ser Ser
Val Gln Asn Val His Trp 325 330
335 Thr Leu 2441035DNALactuca sativa 244atggagatgg gtggttcgag
tggttcttcg gagtcgcaac tgctcaaaat tggtttgcaa 60ttcgggaaag aaatctattt
tgaggatgtg ggagttggag ctcaggttaa atccgatgat 120gggttgtctc cggcgagcgg
cggcgatgct gctggagggc cgcagaagaa agggagaact 180gctggtgggg tggtgagtgg
ttttggacaa cagcaaccac cgaggtgtca ggtggaaggt 240tgtaatctgg atctgagtga
tgctaaaagt tactattcaa ggcacaaagt ttgtggtgct 300cattcgaaaa cggctaaggt
cattgttaat ggccttgaac agagattctg ccaacagtgc 360agcaggttcc atcaactacc
agagtttgac cagggaaaaa gaagctgcag gagacgattg 420gctgggcaca atgaacgtag
aagaaagcca tctctgctat ccactcgcta tggaactgtc 480tcctcctcaa tctttgaaaa
caatgggaat tctggaggct ttctaatgga cttttcgtca 540tgctcaagag gaaggattca
gtggccagga acaagggcgg caccaccgcc tcgagccgcc 600atcgacctcc caattgccgg
agaaaagttc ccaccgcttc catggcaaag caacctggat 660aatccacctc cttatgttcc
accaggaggg tgttttaatg gagtccatga ggactccaac 720tgtgctctct ctcttctgtc
aaatcactca tctggctcaa ggaaccaatc cctgagccat 780gagtactata tcaaccctga
agctggtgca tatgtgcacc agcttcatca accaacaacc 840ggaaccggag ctgggttcgg
aaccatggtt gaggtcaccg ccactggctg ggtatacgag 900acccatgatg cccatttggg
tttgggtcac gtcccacagt ctggtggtgg ctactctggt 960gaggttggac ttggtccaca
tggtggggga aggcgatatg actcatctgt tgaccacatt 1020gactggtcac tttga
1035245344PRTLactuca sativa
245Met Glu Met Gly Gly Ser Ser Gly Ser Ser Glu Ser Gln Leu Leu Lys 1
5 10 15 Ile Gly Leu Gln
Phe Gly Lys Glu Ile Tyr Phe Glu Asp Val Gly Val 20
25 30 Gly Ala Gln Val Lys Ser Asp Asp Gly
Leu Ser Pro Ala Ser Gly Gly 35 40
45 Asp Ala Ala Gly Gly Pro Gln Lys Lys Gly Arg Thr Ala Gly
Gly Val 50 55 60
Val Ser Gly Phe Gly Gln Gln Gln Pro Pro Arg Cys Gln Val Glu Gly 65
70 75 80 Cys Asn Leu Asp Leu
Ser Asp Ala Lys Ser Tyr Tyr Ser Arg His Lys 85
90 95 Val Cys Gly Ala His Ser Lys Thr Ala Lys
Val Ile Val Asn Gly Leu 100 105
110 Glu Gln Arg Phe Cys Gln Gln Cys Ser Arg Phe His Gln Leu Pro
Glu 115 120 125 Phe
Asp Gln Gly Lys Arg Ser Cys Arg Arg Arg Leu Ala Gly His Asn 130
135 140 Glu Arg Arg Arg Lys Pro
Ser Leu Leu Ser Thr Arg Tyr Gly Thr Val 145 150
155 160 Ser Ser Ser Ile Phe Glu Asn Asn Gly Asn Ser
Gly Gly Phe Leu Met 165 170
175 Asp Phe Ser Ser Cys Ser Arg Gly Arg Ile Gln Trp Pro Gly Thr Arg
180 185 190 Ala Ala
Pro Pro Pro Arg Ala Ala Ile Asp Leu Pro Ile Ala Gly Glu 195
200 205 Lys Phe Pro Pro Leu Pro Trp
Gln Ser Asn Leu Asp Asn Pro Pro Pro 210 215
220 Tyr Val Pro Pro Gly Gly Cys Phe Asn Gly Val His
Glu Asp Ser Asn 225 230 235
240 Cys Ala Leu Ser Leu Leu Ser Asn His Ser Ser Gly Ser Arg Asn Gln
245 250 255 Ser Leu Ser
His Glu Tyr Tyr Ile Asn Pro Glu Ala Gly Ala Tyr Val 260
265 270 His Gln Leu His Gln Pro Thr Thr
Gly Thr Gly Ala Gly Phe Gly Thr 275 280
285 Met Val Glu Val Thr Ala Thr Gly Trp Val Tyr Glu Thr
His Asp Ala 290 295 300
His Leu Gly Leu Gly His Val Pro Gln Ser Gly Gly Gly Tyr Ser Gly 305
310 315 320 Glu Val Gly Leu
Gly Pro His Gly Gly Gly Arg Arg Tyr Asp Ser Ser 325
330 335 Val Asp His Ile Asp Trp Ser Leu
340 2461137DNAMalus domestica 246atggaaatgg
gctcgagttc taagaccgag tcagcgagct cttcctcctc ttcgccgccc 60aactcctccg
ctgagtcact caacggcttg aaattcggcc ggaaaatcta ctttgaggat 120gggggttttg
gagctctgca caaatcatca tgcgggtctg ctgctgggtc ttcctccgcc 180ggggctacgc
cgccaaagaa gcaaaggggc ggcggaaatt tgggtcagcc gccgcggtgt 240caggtggagg
gctgcgaggt agatctgagt ggtgccaaag cttactattc caggcacaaa 300gtctgtggct
tgcactctaa aactcccact gtcattgttg ctggtcttga acagaggttt 360tgccaacagt
gtagcaggtt tcatttactt cctgaatttg atcaaggaaa acgtagttgt 420cgtagacgct
tggctgggca taatgagcgt cgtagaaaac cacctccagg atccatactg 480tctacgcgtg
gcagactttc ttcgtctctc tacgaaaaca gcagcagaat tggaagcttt 540ctgatggact
tcactgcata cccaaggttt tctgggaggg atacatggac aacaagaacc 600tctgagcgag
cacctgttaa tcaaaatgcc aatgacgcag ggaagtttct acaacagccg 660tggcagagca
actctgatat ttctacatcc ggcttttacc tacaaggttc agcaggcggg 720actagttatc
ctggtcctgg aattcctcca ggagaatgtg tcacagtagt cactgactca 780agctgtgctc
tctctcttct gtcaaatcag ccatggggct ctcgaaaccg agtattgggt 840gctgggatga
attccttgat gaacactcaa ggggtacctg tggctcaacc agtccctcat 900tctgcaacct
ccaatcactt tccgaccact tcgtggggtt tcaaaggaaa tgaaaatggt 960agcagctcac
acgggatgct tccagatctg ggtctcggtc aaatctcgca gccgcttagc 1020agtcagtact
ctggtgtgct ggagctgtct caacagggta ggaggcagca acacatggaa 1080ctcggacaca
ccaggggcta tgactccacc agtcagcaga tgcactggtc actttaa
1137247378PRTMalus domestica 247Met Glu Met Gly Ser Ser Ser Lys Thr Glu
Ser Ala Ser Ser Ser Ser 1 5 10
15 Ser Ser Pro Pro Asn Ser Ser Ala Glu Ser Leu Asn Gly Leu Lys
Phe 20 25 30 Gly
Arg Lys Ile Tyr Phe Glu Asp Gly Gly Phe Gly Ala Leu His Lys 35
40 45 Ser Ser Cys Gly Ser Ala
Ala Gly Ser Ser Ser Ala Gly Ala Thr Pro 50 55
60 Pro Lys Lys Gln Arg Gly Gly Gly Asn Leu Gly
Gln Pro Pro Arg Cys 65 70 75
80 Gln Val Glu Gly Cys Glu Val Asp Leu Ser Gly Ala Lys Ala Tyr Tyr
85 90 95 Ser Arg
His Lys Val Cys Gly Leu His Ser Lys Thr Pro Thr Val Ile 100
105 110 Val Ala Gly Leu Glu Gln Arg
Phe Cys Gln Gln Cys Ser Arg Phe His 115 120
125 Leu Leu Pro Glu Phe Asp Gln Gly Lys Arg Ser Cys
Arg Arg Arg Leu 130 135 140
Ala Gly His Asn Glu Arg Arg Arg Lys Pro Pro Pro Gly Ser Ile Leu 145
150 155 160 Ser Thr Arg
Gly Arg Leu Ser Ser Ser Leu Tyr Glu Asn Ser Ser Arg 165
170 175 Ile Gly Ser Phe Leu Met Asp Phe
Thr Ala Tyr Pro Arg Phe Ser Gly 180 185
190 Arg Asp Thr Trp Thr Thr Arg Thr Ser Glu Arg Ala Pro
Val Asn Gln 195 200 205
Asn Ala Asn Asp Ala Gly Lys Phe Leu Gln Gln Pro Trp Gln Ser Asn 210
215 220 Ser Asp Ile Ser
Thr Ser Gly Phe Tyr Leu Gln Gly Ser Ala Gly Gly 225 230
235 240 Thr Ser Tyr Pro Gly Pro Gly Ile Pro
Pro Gly Glu Cys Val Thr Val 245 250
255 Val Thr Asp Ser Ser Cys Ala Leu Ser Leu Leu Ser Asn Gln
Pro Trp 260 265 270
Gly Ser Arg Asn Arg Val Leu Gly Ala Gly Met Asn Ser Leu Met Asn
275 280 285 Thr Gln Gly Val
Pro Val Ala Gln Pro Val Pro His Ser Ala Thr Ser 290
295 300 Asn His Phe Pro Thr Thr Ser Trp
Gly Phe Lys Gly Asn Glu Asn Gly 305 310
315 320 Ser Ser Ser His Gly Met Leu Pro Asp Leu Gly Leu
Gly Gln Ile Ser 325 330
335 Gln Pro Leu Ser Ser Gln Tyr Ser Gly Val Leu Glu Leu Ser Gln Gln
340 345 350 Gly Arg Arg
Gln Gln His Met Glu Leu Gly His Thr Arg Gly Tyr Asp 355
360 365 Ser Thr Ser Gln Gln Met His Trp
Ser Leu 370 375 2481014DNAMedicago
trunculata 248atggattcag gaggcaactc ttcttcagaa gagtcctcac ttaatggctt
gaaatttggc 60caacgaatct atttcgaaga tacagctctt actgctgctt ctgctgctgc
tgctagtact 120accattgctg ctggttctcc ttcttcttct ggttcaaaga aaggaagagg
tgggtcagtt 180caacattctc aaccacctag gtgtcaagtt gaaggatgta aactagatct
gactgatgct 240aaagcttact attctagaca caaagtttgt agcatgcact ctaaatcccc
tactgttact 300gtttctggtc ttcaacaaag gttttgtcaa caatgtagca gatttcatca
gcttgctgag 360tttgatcaag gaaaaagaag ttgtcggaga cgactagctg gtcataatga
gcgtcgcaga 420aagcccccac ccagctctct cttaacctca cgttttgcca ggctttcttc
gtctgttttt 480ggtaacagcg acagaggtgg cagctttttg atggaatttg cttcaaatcc
gaaacatagt 540ctgaggaatt cacccggaaa tcaaaccaca gcaatcggtt ggccttggcc
ggggaacacg 600gagtcgccat ctagcaacct tttcttgcaa ggttcggtgg gtgggacaag
cttccctggt 660gccaggcatc ctcccgagga aacttacact ggagtcacag attcaaactg
tgctctctct 720cttctgtcaa atcaaacatg gggttctcaa aacacagaac caagtcctgg
attgaataac 780atgctgaatt tcaacgggac acccatgaca caacttggta catcttctca
tggtgtagcc 840atgcatcaaa ttccaaacaa ttacgaggtt gtccctgatc ttggtcgggg
tcacattttg 900catcctcttg gtagccaaca ctctggcgag cttgatctgt tgcagcaggg
aaggaggcat 960tatatggatg tagaacattc cagggcctat gaatcttctc agtggtcact
gtaa 1014249337PRTMedicago trunculata 249Met Asp Ser Gly Gly Asn
Ser Ser Ser Glu Glu Ser Ser Leu Asn Gly 1 5
10 15 Leu Lys Phe Gly Gln Arg Ile Tyr Phe Glu Asp
Thr Ala Leu Thr Ala 20 25
30 Ala Ser Ala Ala Ala Ala Ser Thr Thr Ile Ala Ala Gly Ser Pro
Ser 35 40 45 Ser
Ser Gly Ser Lys Lys Gly Arg Gly Gly Ser Val Gln His Ser Gln 50
55 60 Pro Pro Arg Cys Gln Val
Glu Gly Cys Lys Leu Asp Leu Thr Asp Ala 65 70
75 80 Lys Ala Tyr Tyr Ser Arg His Lys Val Cys Ser
Met His Ser Lys Ser 85 90
95 Pro Thr Val Thr Val Ser Gly Leu Gln Gln Arg Phe Cys Gln Gln Cys
100 105 110 Ser Arg
Phe His Gln Leu Ala Glu Phe Asp Gln Gly Lys Arg Ser Cys 115
120 125 Arg Arg Arg Leu Ala Gly His
Asn Glu Arg Arg Arg Lys Pro Pro Pro 130 135
140 Ser Ser Leu Leu Thr Ser Arg Phe Ala Arg Leu Ser
Ser Ser Val Phe 145 150 155
160 Gly Asn Ser Asp Arg Gly Gly Ser Phe Leu Met Glu Phe Ala Ser Asn
165 170 175 Pro Lys His
Ser Leu Arg Asn Ser Pro Gly Asn Gln Thr Thr Ala Ile 180
185 190 Gly Trp Pro Trp Pro Gly Asn Thr
Glu Ser Pro Ser Ser Asn Leu Phe 195 200
205 Leu Gln Gly Ser Val Gly Gly Thr Ser Phe Pro Gly Ala
Arg His Pro 210 215 220
Pro Glu Glu Thr Tyr Thr Gly Val Thr Asp Ser Asn Cys Ala Leu Ser 225
230 235 240 Leu Leu Ser Asn
Gln Thr Trp Gly Ser Gln Asn Thr Glu Pro Ser Pro 245
250 255 Gly Leu Asn Asn Met Leu Asn Phe Asn
Gly Thr Pro Met Thr Gln Leu 260 265
270 Gly Thr Ser Ser His Gly Val Ala Met His Gln Ile Pro Asn
Asn Tyr 275 280 285
Glu Val Val Pro Asp Leu Gly Arg Gly His Ile Leu His Pro Leu Gly 290
295 300 Ser Gln His Ser Gly
Glu Leu Asp Leu Leu Gln Gln Gly Arg Arg His 305 310
315 320 Tyr Met Asp Val Glu His Ser Arg Ala Tyr
Glu Ser Ser Gln Trp Ser 325 330
335 Leu 2501143DNANicotiana bentamiana 250atggaacttc
tggcttctgc ttcttcttct acttctacta attctacttc ccctgactct 60cctcccaaca
ctttaaaatt tggtcaaaaa atctactttg agcatgttgg acttcagcac 120ctcaaatcag
caactgggtc gtcgtcgtca tcgccgccgg tcatcggaac tccggcgccg 180gcgatgtcca
agaaaggaag agggggtggt gtagttcaag gtcggcaacc acctaggtgt 240caagttgaag
ggtgtgaagc agatctgagt gatgttaagg cttactattc aaggcacaaa 300gtctgtgcta
cacattctaa gtctcctgtg gtcattgttg ctggtcttga acaaagattt 360tgtcaacagt
gtagcaggtt tcatcggttg ccagaatttg accaagggaa acgcagttgc 420cgcaggcgcc
tagcaggcca taatgagcgt cggaggaaac ctccacctgg atctcttttg 480tctaatcgct
atggaagtct ttcttcatca atatttgaaa acaatggcag atctggaagt 540tttctggttg
acttcactgc atatccgaat ctcactggag gtgcatggcc aaatactaga 600tcatctgatc
gaggatggga taatcaatcc actgcgtcag ggaagcttct ccaaagtcat 660tggctgaaca
gttctgaaaa tccgacatcc gaccttgttc tgcaaggttc agttgctagg 720ggtgccaatt
attctggtcc tggtattatt ccttccggaa actgcttctc tggagtctca 780gattccaatg
gtgctctctc tcttctgtca aatgagccat ggggctcgag gaaccaatcc 840tctagcctcg
gggttaacgg cttggtcaac actgatggcg gacataccgt tcacccatcg 900ggttcccatg
ctgctcctgt caatcactac tcaggccctc tatggggatt taaaggaaat 960gaagctagta
gcagttcaca tgcaatacct cctgatctcg ggctgggtca catttctcaa 1020catgctgtca
atcagtactc tggtgagcct gggatggctc agcacagtgg aagacagtac 1080atgggactgg
agcattcaaa gggttataat tcttctgttc agaatgtgca ctggacactt 1140tga
1143251380PRTNicotiana bentamiana 251Met Glu Leu Leu Ala Ser Ala Ser Ser
Ser Thr Ser Thr Asn Ser Thr 1 5 10
15 Ser Pro Asp Ser Pro Pro Asn Thr Leu Lys Phe Gly Gln Lys
Ile Tyr 20 25 30
Phe Glu His Val Gly Leu Gln His Leu Lys Ser Ala Thr Gly Ser Ser
35 40 45 Ser Ser Ser Pro
Pro Val Ile Gly Thr Pro Ala Pro Ala Met Ser Lys 50
55 60 Lys Gly Arg Gly Gly Gly Val Val
Gln Gly Arg Gln Pro Pro Arg Cys 65 70
75 80 Gln Val Glu Gly Cys Glu Ala Asp Leu Ser Asp Val
Lys Ala Tyr Tyr 85 90
95 Ser Arg His Lys Val Cys Ala Thr His Ser Lys Ser Pro Val Val Ile
100 105 110 Val Ala Gly
Leu Glu Gln Arg Phe Cys Gln Gln Cys Ser Arg Phe His 115
120 125 Arg Leu Pro Glu Phe Asp Gln Gly
Lys Arg Ser Cys Arg Arg Arg Leu 130 135
140 Ala Gly His Asn Glu Arg Arg Arg Lys Pro Pro Pro Gly
Ser Leu Leu 145 150 155
160 Ser Asn Arg Tyr Gly Ser Leu Ser Ser Ser Ile Phe Glu Asn Asn Gly
165 170 175 Arg Ser Gly Ser
Phe Leu Val Asp Phe Thr Ala Tyr Pro Asn Leu Thr 180
185 190 Gly Gly Ala Trp Pro Asn Thr Arg Ser
Ser Asp Arg Gly Trp Asp Asn 195 200
205 Gln Ser Thr Ala Ser Gly Lys Leu Leu Gln Ser His Trp Leu
Asn Ser 210 215 220
Ser Glu Asn Pro Thr Ser Asp Leu Val Leu Gln Gly Ser Val Ala Arg 225
230 235 240 Gly Ala Asn Tyr Ser
Gly Pro Gly Ile Ile Pro Ser Gly Asn Cys Phe 245
250 255 Ser Gly Val Ser Asp Ser Asn Gly Ala Leu
Ser Leu Leu Ser Asn Glu 260 265
270 Pro Trp Gly Ser Arg Asn Gln Ser Ser Ser Leu Gly Val Asn Gly
Leu 275 280 285 Val
Asn Thr Asp Gly Gly His Thr Val His Pro Ser Gly Ser His Ala 290
295 300 Ala Pro Val Asn His Tyr
Ser Gly Pro Leu Trp Gly Phe Lys Gly Asn 305 310
315 320 Glu Ala Ser Ser Ser Ser His Ala Ile Pro Pro
Asp Leu Gly Leu Gly 325 330
335 His Ile Ser Gln His Ala Val Asn Gln Tyr Ser Gly Glu Pro Gly Met
340 345 350 Ala Gln
His Ser Gly Arg Gln Tyr Met Gly Leu Glu His Ser Lys Gly 355
360 365 Tyr Asn Ser Ser Val Gln Asn
Val His Trp Thr Leu 370 375 380
2521254DNAOryza sativa 252atggagatgg ccagtggagg aggcgccgcc gccgccgccg
gcggcggagt aggcggcagc 60ggcggcggtg gtggtggagg ggacgagcac cgccagctgc
acggtctcaa gttcggcaag 120aagatctact tcgaggacgc cgccgcggca gcaggcggcg
gcggcactgg cagtggcagt 180ggcagcgcga gcgccgcgcc gccgtcctcg tcttccaagg
cggcgggtgg tggacgcggc 240ggagggggca agaacaaggg gaagggcgtg gccgcggcgg
cgccaccgcc gccgccgccg 300ccgccgcggt gccaggtgga ggggtgcggc gcggatctga
gcgggatcaa gaactactac 360tgccgccaca aggtgtgctt catgcattcc aaggctcccc
gcgtcgtcgt cgccggcctc 420gagcagcgct tctgccagca gtgcagcagg ttccacctgc
tgcctgaatt tgaccaagga 480aaacgcagct gccgcagacg ccttgcaggt cataatgagc
gccggaggag gccgcaaacc 540cctttggcat cacgctacgg tcgactagct gcatctgttg
gtgagcatcg caggttcaga 600agctttacgt tggatttctc ctacccaagg gttccaagca
gcgtaaggaa tgcatggcca 660gcaattcaac caggcgatcg gatctccggt ggtatccagt
ggcacaggaa cgtagctcct 720catggtcact ctagtgcagt ggcgggatat ggtgccaaca
catacagcgg ccaaggtagc 780tcttcttcag ggccaccggt gttcgctggc ccaaatctcc
ctccaggtgg atgtctcgca 840ggggtcggtg ccgccaccga ctcgagctgt gctctctctc
ttctgtcaac ccagccatgg 900gatactacta cccacagtgc cgctgccagc cacaaccagg
ctgcagccat gtccactacc 960accagctttg atggcaatcc tgtggcaccc tccgccatgg
cgggtagcta catggcacca 1020agcccctgga caggttctcg gggccatgag ggtggtggtc
ggagcgtggc gcaccagcta 1080ccacatgaag tctcacttga tgaggtgcac cctggtccta
gccatcatgc ccacttctcc 1140ggtgagcttg agcttgctct gcaggggaac ggtccagccc
cagcaccacg catcgatcct 1200gggtccggca gcaccttcga ccaaaccagc aacacgatgg
attggtctct gtag 1254253417PRTOryza sativa 253Met Glu Met Ala Ser
Gly Gly Gly Ala Ala Ala Ala Ala Gly Gly Gly 1 5
10 15 Val Gly Gly Ser Gly Gly Gly Gly Gly Gly
Gly Asp Glu His Arg Gln 20 25
30 Leu His Gly Leu Lys Phe Gly Lys Lys Ile Tyr Phe Glu Asp Ala
Ala 35 40 45 Ala
Ala Ala Gly Gly Gly Gly Thr Gly Ser Gly Ser Gly Ser Ala Ser 50
55 60 Ala Ala Pro Pro Ser Ser
Ser Ser Lys Ala Ala Gly Gly Gly Arg Gly 65 70
75 80 Gly Gly Gly Lys Asn Lys Gly Lys Gly Val Ala
Ala Ala Ala Pro Pro 85 90
95 Pro Pro Pro Pro Pro Pro Arg Cys Gln Val Glu Gly Cys Gly Ala Asp
100 105 110 Leu Ser
Gly Ile Lys Asn Tyr Tyr Cys Arg His Lys Val Cys Phe Met 115
120 125 His Ser Lys Ala Pro Arg Val
Val Val Ala Gly Leu Glu Gln Arg Phe 130 135
140 Cys Gln Gln Cys Ser Arg Phe His Leu Leu Pro Glu
Phe Asp Gln Gly 145 150 155
160 Lys Arg Ser Cys Arg Arg Arg Leu Ala Gly His Asn Glu Arg Arg Arg
165 170 175 Arg Pro Gln
Thr Pro Leu Ala Ser Arg Tyr Gly Arg Leu Ala Ala Ser 180
185 190 Val Gly Glu His Arg Arg Phe Arg
Ser Phe Thr Leu Asp Phe Ser Tyr 195 200
205 Pro Arg Val Pro Ser Ser Val Arg Asn Ala Trp Pro Ala
Ile Gln Pro 210 215 220
Gly Asp Arg Ile Ser Gly Gly Ile Gln Trp His Arg Asn Val Ala Pro 225
230 235 240 His Gly His Ser
Ser Ala Val Ala Gly Tyr Gly Ala Asn Thr Tyr Ser 245
250 255 Gly Gln Gly Ser Ser Ser Ser Gly Pro
Pro Val Phe Ala Gly Pro Asn 260 265
270 Leu Pro Pro Gly Gly Cys Leu Ala Gly Val Gly Ala Ala Thr
Asp Ser 275 280 285
Ser Cys Ala Leu Ser Leu Leu Ser Thr Gln Pro Trp Asp Thr Thr Thr 290
295 300 His Ser Ala Ala Ala
Ser His Asn Gln Ala Ala Ala Met Ser Thr Thr 305 310
315 320 Thr Ser Phe Asp Gly Asn Pro Val Ala Pro
Ser Ala Met Ala Gly Ser 325 330
335 Tyr Met Ala Pro Ser Pro Trp Thr Gly Ser Arg Gly His Glu Gly
Gly 340 345 350 Gly
Arg Ser Val Ala His Gln Leu Pro His Glu Val Ser Leu Asp Glu 355
360 365 Val His Pro Gly Pro Ser
His His Ala His Phe Ser Gly Glu Leu Glu 370 375
380 Leu Ala Leu Gln Gly Asn Gly Pro Ala Pro Ala
Pro Arg Ile Asp Pro 385 390 395
400 Gly Ser Gly Ser Thr Phe Asp Gln Thr Ser Asn Thr Met Asp Trp Ser
405 410 415 Leu
2541182DNAOryza sativa 254atggcgaccg gcggcagcgg cggcggcggc ggaggtggag
gtggtggtga cgatgtccac 60gggctcaagt tcggcaagaa gatctacttc gagcaggacg
cggcggcgtc ggcgtcggcg 120gcggcggtgg agtcgtcgtc gacgtcgtcg ggcggaggcg
gcaagaaggg gaagggcgtg 180gcggcggcgg cggcgccccc gccgccgctg ccgccgaggt
gccaggtgga gggttgcggc 240gtggatctga gcggcgtcaa gccgtactac tgccgccaca
aggtgtgcta catgcacgcc 300aaggagccca tcgtcgtcgt cgccggcctc gagcagcgct
tctgccaaca gtgcagcagg 360ttccaccaat tacctgaatt tgatcaagaa aaaaaaagct
gccgcagacg ccttgcaggt 420cacaatgaac gccggaggaa gccgacacct ggacctcttt
cttctcgcta tggccggctt 480gctgcatcct ttcatgaaga gccaggcagg tccagaagct
ttgtggtaga tttctcatac 540ccaagggttc caagcagtgt gagggatgcg tggcctgcta
ttcagcccag cgatcgcatg 600tccggttcaa tccagtggca agggggccat gaactccatc
ctcaccgcag cgcagttgcg 660ggatacagtg atcaccatgc gttcagcagc catggtggct
cagcggctgg ggcaccaatg 720ctccaccacc cagcctttga gctcacctca ggtggatgtc
tcgcgggagt cgccaccgac 780tccagctgtg ctctctctct tctgtcaact cagccatggg
atactaccca aagcaccagc 840agccacaacc ggtccccgcc aatgtcgtca acggccagcg
ccttcggagg cggcaacaac 900ccggtgtcgc cctcggtcat ggcaagcaac tacatggcgg
cgagccccgg ctggaacagc 960tccagccggg gccatgacgg cgccaggaac gtgcacctgc
cgccaccgca cggggttgtg 1020ctgaacgagg tccctccggg ctctgtccac cacggccatt
tctccggcga gctcgagctc 1080gcactgcagg gaggtgcccc gtccaaccgg ccggaagccg
agcatggctc cggcagcggc 1140gccttcagcc actccaccaa tgccatgaac tggtctctgt
ag 1182255393PRTOryza sativa 255Met Ala Thr Gly Gly
Ser Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 1 5
10 15 Asp Asp Val His Gly Leu Lys Phe Gly Lys
Lys Ile Tyr Phe Glu Gln 20 25
30 Asp Ala Ala Ala Ser Ala Ser Ala Ala Ala Val Glu Ser Ser Ser
Thr 35 40 45 Ser
Ser Gly Gly Gly Gly Lys Lys Gly Lys Gly Val Ala Ala Ala Ala 50
55 60 Ala Pro Pro Pro Pro Leu
Pro Pro Arg Cys Gln Val Glu Gly Cys Gly 65 70
75 80 Val Asp Leu Ser Gly Val Lys Pro Tyr Tyr Cys
Arg His Lys Val Cys 85 90
95 Tyr Met His Ala Lys Glu Pro Ile Val Val Val Ala Gly Leu Glu Gln
100 105 110 Arg Phe
Cys Gln Gln Cys Ser Arg Phe His Gln Leu Pro Glu Phe Asp 115
120 125 Gln Glu Lys Lys Ser Cys Arg
Arg Arg Leu Ala Gly His Asn Glu Arg 130 135
140 Arg Arg Lys Pro Thr Pro Gly Pro Leu Ser Ser Arg
Tyr Gly Arg Leu 145 150 155
160 Ala Ala Ser Phe His Glu Glu Pro Gly Arg Ser Arg Ser Phe Val Val
165 170 175 Asp Phe Ser
Tyr Pro Arg Val Pro Ser Ser Val Arg Asp Ala Trp Pro 180
185 190 Ala Ile Gln Pro Ser Asp Arg Met
Ser Gly Ser Ile Gln Trp Gln Gly 195 200
205 Gly His Glu Leu His Pro His Arg Ser Ala Val Ala Gly
Tyr Ser Asp 210 215 220
His His Ala Phe Ser Ser His Gly Gly Ser Ala Ala Gly Ala Pro Met 225
230 235 240 Leu His His Pro
Ala Phe Glu Leu Thr Ser Gly Gly Cys Leu Ala Gly 245
250 255 Val Ala Thr Asp Ser Ser Cys Ala Leu
Ser Leu Leu Ser Thr Gln Pro 260 265
270 Trp Asp Thr Thr Gln Ser Thr Ser Ser His Asn Arg Ser Pro
Pro Met 275 280 285
Ser Ser Thr Ala Ser Ala Phe Gly Gly Gly Asn Asn Pro Val Ser Pro 290
295 300 Ser Val Met Ala Ser
Asn Tyr Met Ala Ala Ser Pro Gly Trp Asn Ser 305 310
315 320 Ser Ser Arg Gly His Asp Gly Ala Arg Asn
Val His Leu Pro Pro Pro 325 330
335 His Gly Val Val Leu Asn Glu Val Pro Pro Gly Ser Val His His
Gly 340 345 350 His
Phe Ser Gly Glu Leu Glu Leu Ala Leu Gln Gly Gly Ala Pro Ser 355
360 365 Asn Arg Pro Glu Ala Glu
His Gly Ser Gly Ser Gly Ala Phe Ser His 370 375
380 Ser Thr Asn Ala Met Asn Trp Ser Leu 385
390 2561119DNASolanum tuberosum 256atggaactgg
gttcagtttc ttcttctggt aattcaagct catctgattc tttgaatggt 60ttgaagtttg
gtaagaaaat ctactttgga aatgtgggtg ttggagttca ggtcaagaat 120ggaagtgggt
cgtcgccggt gaccggagat gggaacatgc caccggctcc ggcgacgact 180aagaggggga
ggggtgggtt ggtgcagggt ggtcatccac ctaggtgtca agttgaaggt 240tgtcaggcag
atctgagtga tgctaaggct tactattcaa ggcataaagt ttgtggtatg 300cactctaagt
ctcctactgt tgttgttgct ggtcttgaac agaggttttg ccaacagtgt 360agcaggttcc
atcaattaac tgaattcgac caggggaaaa ggagttgccg caggagactg 420gcatgccata
atgagcgtcg taggaagcct ccatctggat ctcttttctc tacacactac 480gggaatcttt
cttcatcaat atttgaaaat aatagcagca gatccggaag ctttctggtc 540gacttcagct
cacaccaaaa tgtcaatgat agttcatggc caaatactcg agcatctgaa 600caaggatggg
atcatcaatc atcagggaag ttccttcaac gtccttggct gaataactct 660gaaaatgctg
ccagtgagct tgttttgcaa ggttcagcta ccaggaccag ttatcctagt 720gttccttctg
gagactattt tcctggagtc tcagattcaa gtggtgctct ctctcttctg 780tcaaatcggt
cctggggatc aaggaatcga tctccaagtc ttggggttaa cagccaagtt 840cacattgatg
gggtacacac cattcaacct tcaggttctc atggtgcacc taccaatcac 900ttctcaagcc
cttcattgag ttttaaagga aatgaagcta gcagcagttc acatgagatg 960cctcctgatc
tcggtttggg tcaaatgtta caagcttctg ataatccata ctgtggcgag 1020cttgggatgg
ctcagcatgg tgatggacga caatacatgg aactggacca gtccaagggt 1080tatcatcctt
ctgttcagaa tgtgcactgg actctttga
1119257372PRTSolanum tuberosum 257Met Glu Leu Gly Ser Val Ser Ser Ser Gly
Asn Ser Ser Ser Ser Asp 1 5 10
15 Ser Leu Asn Gly Leu Lys Phe Gly Lys Lys Ile Tyr Phe Gly Asn
Val 20 25 30 Gly
Val Gly Val Gln Val Lys Asn Gly Ser Gly Ser Ser Pro Val Thr 35
40 45 Gly Asp Gly Asn Met
Pro Pro Ala Pro Ala Thr Thr Lys Arg Gly Arg 50 55
60 Gly Gly Leu Val Gln Gly Gly His Pro Pro
Arg Cys Gln Val Glu Gly 65 70 75
80 Cys Gln Ala Asp Leu Ser Asp Ala Lys Ala Tyr Tyr Ser Arg His
Lys 85 90 95 Val
Cys Gly Met His Ser Lys Ser Pro Thr Val Val Val Ala Gly Leu
100 105 110 Glu Gln Arg Phe Cys
Gln Gln Cys Ser Arg Phe His Gln Leu Thr Glu 115
120 125 Phe Asp Gln Gly Lys Arg Ser Cys Arg
Arg Arg Leu Ala Cys His Asn 130 135
140 Glu Arg Arg Arg Lys Pro Pro Ser Gly Ser Leu Phe Ser
Thr His Tyr 145 150 155
160 Gly Asn Leu Ser Ser Ser Ile Phe Glu Asn Asn Ser Ser Arg Ser Gly
165 170 175 Ser Phe Leu Val
Asp Phe Ser Ser His Gln Asn Val Asn Asp Ser Ser 180
185 190 Trp Pro Asn Thr Arg Ala Ser Glu Gln
Gly Trp Asp His Gln Ser Ser 195 200
205 Gly Lys Phe Leu Gln Arg Pro Trp Leu Asn Asn Ser Glu Asn
Ala Ala 210 215 220
Ser Glu Leu Val Leu Gln Gly Ser Ala Thr Arg Thr Ser Tyr Pro Ser 225
230 235 240 Val Pro Ser Gly Asp
Tyr Phe Pro Gly Val Ser Asp Ser Ser Gly Ala 245
250 255 Leu Ser Leu Leu Ser Asn Arg Ser Trp Gly
Ser Arg Asn Arg Ser Pro 260 265
270 Ser Leu Gly Val Asn Ser Gln Val His Ile Asp Gly Val His Thr
Ile 275 280 285 Gln
Pro Ser Gly Ser His Gly Ala Pro Thr Asn His Phe Ser Ser Pro 290
295 300 Ser Leu Ser Phe Lys Gly
Asn Glu Ala Ser Ser Ser Ser His Glu Met 305 310
315 320 Pro Pro Asp Leu Gly Leu Gly Gln Met Leu Gln
Ala Ser Asp Asn Pro 325 330
335 Tyr Cys Gly Glu Leu Gly Met Ala Gln His Gly Asp Gly Arg Gln Tyr
340 345 350 Met Glu
Leu Asp Gln Ser Lys Gly Tyr His Pro Ser Val Gln Asn Val 355
360 365 His Trp Thr Leu 370
2581140DNAVitis vinifera 258atggaaaggg gttcgagctc tttgaccgtt
tccagctctt cggccaactc gtctgagtcg 60ctcaacgggt tgaaatttgg gcagaagata
tattttgaag atttgggcgt tggagctccg 120gccaaatcgg gaaccggctc ctcctcctcc
tcctctgccg ccggctccgg tggtcgccca 180cctccggcgc cgccaaagaa ggtaagaggt
agtggggttg ttcagggagg ccaaccaccg 240aggtgtcaag ttgaagggtg taaactagat
ctgagtgatg ccaaagctta ctattcaagg 300cataaagtgt gtggtatgca ttcgaagtct
ccaacggtca ttgttgcggg ccttgagcag 360aggttttgcc agcagtgtag cagatttcat
cagcttgccg aatttgacca aggaaaacga 420agttgtcgta ggcgcctggc tggtcataat
gagcgtcgca ggaagccacc acctggatct 480ttattgtcct cacgctatgg gcgactttct
tcatccattt ttgaaaacag cagcagggtg 540ggaggaggct ttctgatgga ctttgctgca
tacccaaggc atcccgagag ggatacttgg 600ccaactacaa gagcatctga tcgggtacct
ggaaatcaaa ccactgcgat gggaaggttt 660cttccacatc catggcagag caactctgag
aatcctctct ttctgcaagg ttcagccggc 720gggaccagct ttcatggtcc tggaattcct
tcaggagaat gtttcacagg ggcctccgac 780tcaagctgtg ctctctctct tctgtcaaat
cagccatgga gctccaggaa tcgagcatct 840ggtcttggag caaacagctt catgaatcct
gaaggggcat ccatggcgca acccacagct 900cctcatagtg cagctatcaa tcacttccca
agcacctcgt gggatttcaa gggcaatgaa 960ggtagtagca gttcgcagga gatgccacct
gatcttggtc ttggtcaaat ttcacagcct 1020attaatagcc agttctcagg tgggggcgag
ttgccccaac agagtggaag gcaatacatg 1080gaactcgagc actccagggc ttatgacact
tccactcagc agatgcactg gtcactttag 1140259379PRTVitis vinifera 259Met Glu
Arg Gly Ser Ser Ser Leu Thr Val Ser Ser Ser Ser Ala Asn 1 5
10 15 Ser Ser Glu Ser Leu Asn Gly
Leu Lys Phe Gly Gln Lys Ile Tyr Phe 20 25
30 Glu Asp Leu Gly Val Gly Ala Pro Ala Lys Ser Gly
Thr Gly Ser Ser 35 40 45
Ser Ser Ser Ser Ala Ala Gly Ser Gly Gly Arg Pro Pro Pro Ala Pro
50 55 60 Pro Lys Lys
Val Arg Gly Ser Gly Val Val Gln Gly Gly Gln Pro Pro 65
70 75 80 Arg Cys Gln Val Glu Gly Cys
Lys Leu Asp Leu Ser Asp Ala Lys Ala 85
90 95 Tyr Tyr Ser Arg His Lys Val Cys Gly Met His
Ser Lys Ser Pro Thr 100 105
110 Val Ile Val Ala Gly Leu Glu Gln Arg Phe Cys Gln Gln Cys Ser
Arg 115 120 125 Phe
His Gln Leu Ala Glu Phe Asp Gln Gly Lys Arg Ser Cys Arg Arg 130
135 140 Arg Leu Ala Gly His Asn
Glu Arg Arg Arg Lys Pro Pro Pro Gly Ser 145 150
155 160 Leu Leu Ser Ser Arg Tyr Gly Arg Leu Ser Ser
Ser Ile Phe Glu Asn 165 170
175 Ser Ser Arg Val Gly Gly Gly Phe Leu Met Asp Phe Ala Ala Tyr Pro
180 185 190 Arg His
Pro Glu Arg Asp Thr Trp Pro Thr Thr Arg Ala Ser Asp Arg 195
200 205 Val Pro Gly Asn Gln Thr Thr
Ala Met Gly Arg Phe Leu Pro His Pro 210 215
220 Trp Gln Ser Asn Ser Glu Asn Pro Leu Phe Leu Gln
Gly Ser Ala Gly 225 230 235
240 Gly Thr Ser Phe His Gly Pro Gly Ile Pro Ser Gly Glu Cys Phe Thr
245 250 255 Gly Ala Ser
Asp Ser Ser Cys Ala Leu Ser Leu Leu Ser Asn Gln Pro 260
265 270 Trp Ser Ser Arg Asn Arg Ala Ser
Gly Leu Gly Ala Asn Ser Phe Met 275 280
285 Asn Pro Glu Gly Ala Ser Met Ala Gln Pro Thr Ala Pro
His Ser Ala 290 295 300
Ala Ile Asn His Phe Pro Ser Thr Ser Trp Asp Phe Lys Gly Asn Glu 305
310 315 320 Gly Ser Ser Ser
Ser Gln Glu Met Pro Pro Asp Leu Gly Leu Gly Gln 325
330 335 Ile Ser Gln Pro Ile Asn Ser Gln Phe
Ser Gly Gly Gly Glu Leu Pro 340 345
350 Gln Gln Ser Gly Arg Gln Tyr Met Glu Leu Glu His Ser Arg
Ala Tyr 355 360 365
Asp Thr Ser Thr Gln Gln Met His Trp Ser Leu 370 375
2601209DNAZea mays 260atggagtccg gcggtggcgg ggacgaccag
ctgcacggcc tcaagttcgg caagaagatc 60tacttcgagg acgccgccgg ctccagcagc
ggcagcagca gcggcggtgg cagcgcgccc 120gcgcctccag cgacgcagca gccgtcgccg
ccggccgctt cgcctagggc cccggccggc 180ggcggcagga ggggcagggc cgcggccggc
ggcgcgggcc cctcgacggc gcccgcgccc 240gcgcgctgcc aggtcgacgg ctgcaacgtt
gacctcaccg acgtcaagcc cgcctactac 300tgccgccaca aggtgtgcaa aatgcactcc
aaggagcccc gcgtcctcgt caacggcctc 360gagcagcgct tctgccagca gtgcagcagg
ttccaccagc tgcctgaatt cgaccagcta 420aagaagagct gccgcaaacg cctcgcaggc
cacaacgagc gccggaggag gccgccgcct 480ggaccccttg cgtcacgata cggccgtcac
gctgcgtcgc tcggcgagcc cggcaggctc 540agaagcttca tgctggattt ctcgtacccg
agggtctcaa gcgccatgag gggtgggttt 600cccgcggtga gggccggtgg tgaaagggtg
cctggcggga tccagtggca agcgggcttg 660gatcctcgtc accaccaagg cgcggtcgcg
ggatacggcg cccactatgg gagcgagggt 720ggtagctcgt cgtcggcgag gccgccggtg
ttccctggcc cggagctgcc cccaggtgga 780tgccttgcag gagtccccgc ggactccagc
tgtgctctct ctcttctgtc aactcagcca 840tgggatgctg cccacagcca cagccacagc
cacgctgcgc caacagcggg tttcgacggc 900ggcagccctg tggcgccctc cctcatggcg
gcgagtagct acatcgcgcc gagcccctgg 960accgagaccg actcctgggg ccacgaaggc
gggcggagcg tgcctcagct gccacctgac 1020gacgtccccc tcggcgaggt gcactccggc
tcgagcagcc accacggcca gttctcaggt 1080gagctcgagc tcgccctgca gggaaacagg
ccagcgccag ggtcggcggc accgccagcg 1140ccgcgcaata atcagggctc cgcgggcacg
ttcgaccagg ctggcaacac gatggactgg 1200tcgctctag
1209261402PRTZea mays 261Met Glu Ser Gly
Gly Gly Gly Asp Asp Gln Leu His Gly Leu Lys Phe 1 5
10 15 Gly Lys Lys Ile Tyr Phe Glu Asp Ala
Ala Gly Ser Ser Ser Gly Ser 20 25
30 Ser Ser Gly Gly Gly Ser Ala Pro Ala Pro Pro Ala Thr Gln
Gln Pro 35 40 45
Ser Pro Pro Ala Ala Ser Pro Arg Ala Pro Ala Gly Gly Gly Arg Arg 50
55 60 Gly Arg Ala Ala Ala
Gly Gly Ala Gly Pro Ser Thr Ala Pro Ala Pro 65 70
75 80 Ala Arg Cys Gln Val Asp Gly Cys Asn Val
Asp Leu Thr Asp Val Lys 85 90
95 Pro Ala Tyr Tyr Cys Arg His Lys Val Cys Lys Met His Ser Lys
Glu 100 105 110 Pro
Arg Val Leu Val Asn Gly Leu Glu Gln Arg Phe Cys Gln Gln Cys 115
120 125 Ser Arg Phe His Gln Leu
Pro Glu Phe Asp Gln Leu Lys Lys Ser Cys 130 135
140 Arg Lys Arg Leu Ala Gly His Asn Glu Arg Arg
Arg Arg Pro Pro Pro 145 150 155
160 Gly Pro Leu Ala Ser Arg Tyr Gly Arg His Ala Ala Ser Leu Gly Glu
165 170 175 Pro Gly
Arg Leu Arg Ser Phe Met Leu Asp Phe Ser Tyr Pro Arg Val 180
185 190 Ser Ser Ala Met Arg Gly Gly
Phe Pro Ala Val Arg Ala Gly Gly Glu 195 200
205 Arg Val Pro Gly Gly Ile Gln Trp Gln Ala Gly Leu
Asp Pro Arg His 210 215 220
His Gln Gly Ala Val Ala Gly Tyr Gly Ala His Tyr Gly Ser Glu Gly 225
230 235 240 Gly Ser Ser
Ser Ser Ala Arg Pro Pro Val Phe Pro Gly Pro Glu Leu 245
250 255 Pro Pro Gly Gly Cys Leu Ala Gly
Val Pro Ala Asp Ser Ser Cys Ala 260 265
270 Leu Ser Leu Leu Ser Thr Gln Pro Trp Asp Ala Ala His
Ser His Ser 275 280 285
His Ser His Ala Ala Pro Thr Ala Gly Phe Asp Gly Gly Ser Pro Val 290
295 300 Ala Pro Ser Leu
Met Ala Ala Ser Ser Tyr Ile Ala Pro Ser Pro Trp 305 310
315 320 Thr Glu Thr Asp Ser Trp Gly His Glu
Gly Gly Arg Ser Val Pro Gln 325 330
335 Leu Pro Pro Asp Asp Val Pro Leu Gly Glu Val His Ser Gly
Ser Ser 340 345 350
Ser His His Gly Gln Phe Ser Gly Glu Leu Glu Leu Ala Leu Gln Gly
355 360 365 Asn Arg Pro Ala
Pro Gly Ser Ala Ala Pro Pro Ala Pro Arg Asn Asn 370
375 380 Gln Gly Ser Ala Gly Thr Phe Asp
Gln Ala Gly Asn Thr Met Asp Trp 385 390
395 400 Ser Leu 2621137DNAZea mays 262atggcgaccg
gcggcggcag cagcaggagc gacgacgtgc gcgggctcaa gtttggcaag 60aagatctact
tcgagcagga cggcgggagc gggagcgggg cgggggcggt gggcggcagg 120aaggggaagg
gcgtggccac cggtggcgcg aggccggcgt ccgccgcctc cgcagcccag 180ccgccgaggt
gccaggtgga cgggtgcggc gtggatctga gcgccgtcaa gcagtactac 240tgccggcaca
aggtgtgcaa catgcactcc aaggagccgc gcgtcttcgt cgccggcatc 300gagcagcgct
tctgccaaca gtgcagcagg ttccaccagc tacatgaatt tgaccaaggg 360aaacgtagct
gccgccgccg cctcatcggt cacaacgagc gccggaggaa gccaccacct 420ggacctctca
cttcacgata tggccggctc gctgcatcac ttcaagagcc tggcaggttc 480agaagcttcc
tgctcgactt ctcgtaccca agggttccaa gcagcgtgag ggatgcgtgg 540ccaggaatcc
agcacggtgg cgacaggatg ctgggcaccg tccagtggca tgggcaccaa 600gaacctcctc
acccacaccg cagtgcagct gctggctatg gcaaccatgc tgcatacaac 660tgccatggcg
gcttggtagc aggcggggcc ccaatgctct cctctgccgc ctttgagctc 720ccgcctggcg
gatgtgtcgc gggagttgcc gccgactcca gctgtgctct ctctcttctg 780tcaactcagc
catgggacac gacctcccac gaccaccggt ccccagcaat gcccgcggcc 840ggcgccttcg
acggcacccc ggtggcgccg tccgtcatgg cgagcagcta cgcggcgtcg 900agcgcctgga
cgggctcgcg ggaccccgct gccgacggcg ccaggaacgc gcagcgtctc 960gacgatgctc
tgcacctggt ccacccaggc tccgcggcgg tccacttctc cggcgagctc 1020gagctcgccc
tgcagggaag cggcgggccg ccacacctgc cgcgcgtcga ccatggcggc 1080tccggcggcg
gcaccttcaa ccattccacc accagcgcga tgaactggtc cctgtag 1137263378PRTZea
mays 263Met Ala Thr Gly Gly Gly Ser Ser Arg Ser Asp Asp Val Arg Gly Leu 1
5 10 15 Lys Phe Gly
Lys Lys Ile Tyr Phe Glu Gln Asp Gly Gly Ser Gly Ser 20
25 30 Gly Ala Gly Ala Val Gly Gly Arg
Lys Gly Lys Gly Val Ala Thr Gly 35 40
45 Gly Ala Arg Pro Ala Ser Ala Ala Ser Ala Ala Gln Pro
Pro Arg Cys 50 55 60
Gln Val Asp Gly Cys Gly Val Asp Leu Ser Ala Val Lys Gln Tyr Tyr 65
70 75 80 Cys Arg His Lys
Val Cys Asn Met His Ser Lys Glu Pro Arg Val Phe 85
90 95 Val Ala Gly Ile Glu Gln Arg Phe Cys
Gln Gln Cys Ser Arg Phe His 100 105
110 Gln Leu His Glu Phe Asp Gln Gly Lys Arg Ser Cys Arg Arg
Arg Leu 115 120 125
Ile Gly His Asn Glu Arg Arg Arg Lys Pro Pro Pro Gly Pro Leu Thr 130
135 140 Ser Arg Tyr Gly Arg
Leu Ala Ala Ser Leu Gln Glu Pro Gly Arg Phe 145 150
155 160 Arg Ser Phe Leu Leu Asp Phe Ser Tyr Pro
Arg Val Pro Ser Ser Val 165 170
175 Arg Asp Ala Trp Pro Gly Ile Gln His Gly Gly Asp Arg Met Leu
Gly 180 185 190 Thr
Val Gln Trp His Gly His Gln Glu Pro Pro His Pro His Arg Ser 195
200 205 Ala Ala Ala Gly Tyr Gly
Asn His Ala Ala Tyr Asn Cys His Gly Gly 210 215
220 Leu Val Ala Gly Gly Ala Pro Met Leu Ser Ser
Ala Ala Phe Glu Leu 225 230 235
240 Pro Pro Gly Gly Cys Val Ala Gly Val Ala Ala Asp Ser Ser Cys Ala
245 250 255 Leu Ser
Leu Leu Ser Thr Gln Pro Trp Asp Thr Thr Ser His Asp His 260
265 270 Arg Ser Pro Ala Met Pro Ala
Ala Gly Ala Phe Asp Gly Thr Pro Val 275 280
285 Ala Pro Ser Val Met Ala Ser Ser Tyr Ala Ala Ser
Ser Ala Trp Thr 290 295 300
Gly Ser Arg Asp Pro Ala Ala Asp Gly Ala Arg Asn Ala Gln Arg Leu 305
310 315 320 Asp Asp Ala
Leu His Leu Val His Pro Gly Ser Ala Ala Val His Phe 325
330 335 Ser Gly Glu Leu Glu Leu Ala Leu
Gln Gly Ser Gly Gly Pro Pro His 340 345
350 Leu Pro Arg Val Asp His Gly Gly Ser Gly Gly Gly Thr
Phe Asn His 355 360 365
Ser Thr Thr Ser Ala Met Asn Trp Ser Leu 370 375
264403DNASorghum propinquum 264atggagtccg gtggcgccag tggcggcggc
ggcggggacg accagctgca cggcctcaag 60ttcggcaaga agatctactt cgaggacgcc
gccgcggtcg gctccagcag cggtggcggt 120ggcggtggca gtggcagtgc tagcgcgacc
cccgcgcctc cagcgacgca gcagccgtcc 180ccgccgcagg ccgcttcgcc cagggcagcc
agcggcggcg gcggcaggag gggcagggcc 240gcgggcggcc cctccccggc gcccgcgccc
gcgcgctgcc aggtcgacgg ctgcaacgtg 300gacctcaccg acgtcaagcc ctactactgc
cgccacaagg tctgcaaaat gcactccaag 360gagccccgcg tcgtcgtcaa cggcctcgag
cagcgcttct gcc 403265134PRTSorghum propinquum 265Met
Glu Ser Gly Gly Ala Ser Gly Gly Gly Gly Gly Asp Asp Gln Leu 1
5 10 15 His Gly Leu Lys Phe Gly
Lys Lys Ile Tyr Phe Glu Asp Ala Ala Ala 20
25 30 Val Gly Ser Ser Ser Gly Gly Gly Gly Gly
Gly Ser Gly Ser Ala Ser 35 40
45 Ala Thr Pro Ala Pro Pro Ala Thr Gln Gln Pro Ser Pro Pro
Gln Ala 50 55 60
Ala Ser Pro Arg Ala Ala Ser Gly Gly Gly Gly Arg Arg Gly Arg Ala 65
70 75 80 Ala Gly Gly Pro Ser
Pro Ala Pro Ala Pro Ala Arg Cys Gln Val Asp 85
90 95 Gly Cys Asn Val Asp Leu Thr Asp Val Lys
Pro Tyr Tyr Cys Arg His 100 105
110 Lys Val Cys Lys Met His Ser Lys Glu Pro Arg Val Val Val Asn
Gly 115 120 125 Leu
Glu Gln Arg Phe Cys 130 266732DNAAllium cepa
266atggaaatgg gttcgagctc tgagccaatt tctgactctc tcgatgggct cacattcggc
60gaaaaaattt actttgaaga ttccactacc agtactgcaa gtactactag caatacaatt
120gctgctcctc ccaaaaaggg aaaatcttta gcttcttcgt ccactaatat caactctaat
180cagcaacaaa taagcatacc tagatgccag gttgaaggat gcaaagttga tttgactgga
240gctaaagcct attactgcag gcataaggtt tgcggagttc attcaaaatc accaaaagtt
300gtggttgctg gaattgaaca gaggttttgt cagcagtgca gcaggttcca ccagttgcaa
360gaatttgacc aaggaaaaag aagctgccgc agacgcctag ctggacacaa tgagcgtcgt
420agaaagccgc ctccaggccc ttcccgttat ggaaggatgt catcatctgt ctatgatggc
480agtaacagat tcagaggatt cctgatggac ttcagttacc cgaggcccat acagcctcct
540ccaggagatc atctatggcc tagactgtcc aacattccat catacccagc aaacccaaac
600actgcatcta acattcatgt tgctcaaccg tacatgcatg caatgaatca ctacagttcc
660gagcttccac acaggaatac atggtctggc gtctgcgact tgagctgtgc tctctctctt
720ctgtcactca ac
732267244PRTAllium cepa 267Met Glu Met Gly Ser Ser Ser Glu Pro Ile Ser
Asp Ser Leu Asp Gly 1 5 10
15 Leu Thr Phe Gly Glu Lys Ile Tyr Phe Glu Asp Ser Thr Thr Ser Thr
20 25 30 Ala Ser
Thr Thr Ser Asn Thr Ile Ala Ala Pro Pro Lys Lys Gly Lys 35
40 45 Ser Leu Ala Ser Ser Ser Thr
Asn Ile Asn Ser Asn Gln Gln Gln Ile 50 55
60 Ser Ile Pro Arg Cys Gln Val Glu Gly Cys Lys Val
Asp Leu Thr Gly 65 70 75
80 Ala Lys Ala Tyr Tyr Cys Arg His Lys Val Cys Gly Val His Ser Lys
85 90 95 Ser Pro Lys
Val Val Val Ala Gly Ile Glu Gln Arg Phe Cys Gln Gln 100
105 110 Cys Ser Arg Phe His Gln Leu Gln
Glu Phe Asp Gln Gly Lys Arg Ser 115 120
125 Cys Arg Arg Arg Leu Ala Gly His Asn Glu Arg Arg Arg
Lys Pro Pro 130 135 140
Pro Gly Pro Ser Arg Tyr Gly Arg Met Ser Ser Ser Val Tyr Asp Gly 145
150 155 160 Ser Asn Arg Phe
Arg Gly Phe Leu Met Asp Phe Ser Tyr Pro Arg Pro 165
170 175 Ile Gln Pro Pro Pro Gly Asp His Leu
Trp Pro Arg Leu Ser Asn Ile 180 185
190 Pro Ser Tyr Pro Ala Asn Pro Asn Thr Ala Ser Asn Ile His
Val Ala 195 200 205
Gln Pro Tyr Met His Ala Met Asn His Tyr Ser Ser Glu Leu Pro His 210
215 220 Arg Asn Thr Trp Ser
Gly Val Cys Asp Leu Ser Cys Ala Leu Ser Leu 225 230
235 240 Leu Ser Leu Asn 268969DNAAntirrhinum
majus 268tcagctgctg gtggagcaga ggaatctctc aatgggttga agtttggcaa
gaaaatatac 60tttgaggagg ctaaggcaaa gaaagggaag agtaccggtg gggtggttag
gtgccaggtg 120gaggggtgtg aggtagatct gagtgatgct aaggcttact atttgagaca
caaagtttgt 180agtatgcatt caaagtctcc aaaggtcatt gttgctggaa tagaacaaag
gttttgccag 240cagtgcagca ggttccatca attgcctgaa tttgaccaag gaaaacggag
ttgccgcaga 300cgccttgcag gccacaacga acgtcggagg aagccatctc caggatctat
gatgtctcct 360tactatggaa gtctttctcc aaccttattt gataaccaaa atagaactgg
aggctttttg 420atggacttca gcacttaccc aaatctcgct gggaaagatt catggccaaa
tacaataccc 480gaacgaggat tgggaggtcc agcaagtcca tggcagagcg acatgcaaaa
tcctgtacct 540gagtttttgc gaggtacaac aaataggcca agtttttctg gtcttggagt
atcttccgaa 600gaatgtttta gcggagtctc taattccagc actgctctct ctcttctgtc
aaatcagtcc 660tggggctcca gaaactcgaa caattttctt ggtaccaatg gaaacgggcc
aaccatagtt 720cagccgtcta ttaaccctgg tgccacaatt ggacagttta cctgtccctc
ttggggtttt 780ggaggcaacc cagctgataa cacctcccat gatatgcctc ccaatctgaa
tttaggacaa 840ttttctcact ccagtaacag tcactatact ggagagcctg gggtagtcca
actgagccac 900ggacaattcc aggacctcga tcactcaaga ggctatgatt cttccgttca
ggacatgcat 960tggtcactt
969269323PRTAntirrhinum majus 269Ser Ala Ala Gly Gly Ala Glu
Glu Ser Leu Asn Gly Leu Lys Phe Gly 1 5
10 15 Lys Lys Ile Tyr Phe Glu Glu Ala Lys Ala Lys
Lys Gly Lys Ser Thr 20 25
30 Gly Gly Val Val Arg Cys Gln Val Glu Gly Cys Glu Val Asp Leu
Ser 35 40 45 Asp
Ala Lys Ala Tyr Tyr Leu Arg His Lys Val Cys Ser Met His Ser 50
55 60 Lys Ser Pro Lys Val Ile
Val Ala Gly Ile Glu Gln Arg Phe Cys Gln 65 70
75 80 Gln Cys Ser Arg Phe His Gln Leu Pro Glu Phe
Asp Gln Gly Lys Arg 85 90
95 Ser Cys Arg Arg Arg Leu Ala Gly His Asn Glu Arg Arg Arg Lys Pro
100 105 110 Ser Pro
Gly Ser Met Met Ser Pro Tyr Tyr Gly Ser Leu Ser Pro Thr 115
120 125 Leu Phe Asp Asn Gln Asn Arg
Thr Gly Gly Phe Leu Met Asp Phe Ser 130 135
140 Thr Tyr Pro Asn Leu Ala Gly Lys Asp Ser Trp Pro
Asn Thr Ile Pro 145 150 155
160 Glu Arg Gly Leu Gly Gly Pro Ala Ser Pro Trp Gln Ser Asp Met Gln
165 170 175 Asn Pro Val
Pro Glu Phe Leu Arg Gly Thr Thr Asn Arg Pro Ser Phe 180
185 190 Ser Gly Leu Gly Val Ser Ser Glu
Glu Cys Phe Ser Gly Val Ser Asn 195 200
205 Ser Ser Thr Ala Leu Ser Leu Leu Ser Asn Gln Ser Trp
Gly Ser Arg 210 215 220
Asn Ser Asn Asn Phe Leu Gly Thr Asn Gly Asn Gly Pro Thr Ile Val 225
230 235 240 Gln Pro Ser Ile
Asn Pro Gly Ala Thr Ile Gly Gln Phe Thr Cys Pro 245
250 255 Ser Trp Gly Phe Gly Gly Asn Pro Ala
Asp Asn Thr Ser His Asp Met 260 265
270 Pro Pro Asn Leu Asn Leu Gly Gln Phe Ser His Ser Ser Asn
Ser His 275 280 285
Tyr Thr Gly Glu Pro Gly Val Val Gln Leu Ser His Gly Gln Phe Gln 290
295 300 Asp Leu Asp His Ser
Arg Gly Tyr Asp Ser Ser Val Gln Asp Met His 305 310
315 320 Trp Ser Leu 270633DNABrassica napus
270gacttggaga aaagaagttg tagaagacgt ctagcttgtc ataacgaaag aagaagaaag
60ccccaagcaa caacagcagc tcttttggct tctggttact ctagaatcgc tccatctctt
120tacggaagcg ttttgggaga tcctacaacg tggtcaaccg caagatctgt gatgggacgg
180tccgcaccgt gggatagcca tcaactgatg aacgttttgt cacagggaag ttcaaggttt
240agtataacat acccagagat ggtgaacaat aatagcacag actcaagctg tgctctctct
300cttctgtcaa actcaaacac aactcagcag cagcagcaga catcaaccaa tgcttacttg
360atggacgcag aaagggttac aatggctaag tcaccgcctg tttcagtaca caatcagtac
420tcgaaacaaa cctgggagtt catgtcaggc gaaaagagca attggccttg tgtgtcgtcc
480cctgttttgg gactgagaca aatctctgag ccagatgatg acctccagtt cctgatgagc
540aatggcacca caatgggtgg attcgagctg aacctacagc aggagcaggt tctgaggcaa
600tactcttcta ctcaaaattt tacttggcct ctt
633271211PRTBrassica napus 271Asp Leu Glu Lys Arg Ser Cys Arg Arg Arg Leu
Ala Cys His Asn Glu 1 5 10
15 Arg Arg Arg Lys Pro Gln Ala Thr Thr Ala Ala Leu Leu Ala Ser Gly
20 25 30 Tyr Ser
Arg Ile Ala Pro Ser Leu Tyr Gly Ser Val Leu Gly Asp Pro 35
40 45 Thr Thr Trp Ser Thr Ala Arg
Ser Val Met Gly Arg Ser Ala Pro Trp 50 55
60 Asp Ser His Gln Leu Met Asn Val Leu Ser Gln Gly
Ser Ser Arg Phe 65 70 75
80 Ser Ile Thr Tyr Pro Glu Met Val Asn Asn Asn Ser Thr Asp Ser Ser
85 90 95 Cys Ala Leu
Ser Leu Leu Ser Asn Ser Asn Thr Thr Gln Gln Gln Gln 100
105 110 Gln Thr Ser Thr Asn Ala Tyr Leu
Met Asp Ala Glu Arg Val Thr Met 115 120
125 Ala Lys Ser Pro Pro Val Ser Val His Asn Gln Tyr Ser
Lys Gln Thr 130 135 140
Trp Glu Phe Met Ser Gly Glu Lys Ser Asn Trp Pro Cys Val Ser Ser 145
150 155 160 Pro Val Leu Gly
Leu Arg Gln Ile Ser Glu Pro Asp Asp Asp Leu Gln 165
170 175 Phe Leu Met Ser Asn Gly Thr Thr Met
Gly Gly Phe Glu Leu Asn Leu 180 185
190 Gln Gln Glu Gln Val Leu Arg Gln Tyr Ser Ser Thr Gln Asn
Phe Thr 195 200 205
Trp Pro Leu 210 272852DNASaccharum officinarum 272ctcgtcgtca
acggcctcga gcagcgcttc tgccagcagt gcagcaggtt ccaccagctg 60cctgaatttg
accaactaaa gaaaagctgc cgcagacgtc ttgcaggcca caatgaacgc 120cggaggaggc
cacctcctgg acctcttgca tcacgatatg gtcgccttgc tgcatcattt 180ggtgagcccg
gcaggttccg aagctttatg ttggatttct catacccaag ggttccaggc 240accatgaggg
atgggtttcc ggcagttcga cctggcgaaa gggtgcctgg tagtatccag 300tggcaagcgg
gcttagatcc tcatcatcat caaagcgcgg tcgcaggata cggtgcccac 360tcatatggga
gccagggtag ctcgtcgtcg tcaaggccac cggtgttccc tggtccagag 420ctccccccag
gtggatgtct tgcaggagtc ccctcggact ctagctgtgc tctctctctt 480ctgtcaactc
agccatggga tactacccac agcgccggcc acagccatgc tggatcaatg 540cctgcaacag
caggttttga cggcaaccct gtggcaccct ccctcatggc gagtagctac 600attgcgccaa
gcccctggac tgactcccgg ggccatgaag gcgggcggaa cgtgcctcag 660ttgccacctg
acgtccccct cagcgaggtg cactctggct caagcagcca tcacggccag 720ttctcaggtg
agctcgagct tgccctgcag ggaaacaggc cagcaccagg gtcagcgcca 780gcgccgcgca
atgatcaggg ctccacgggc acgttcgacc agtccggcaa cacaatggac 840tggtcgctct
ag
852273283PRTSaccharum officinarum 273Leu Val Val Asn Gly Leu Glu Gln Arg
Phe Cys Gln Gln Cys Ser Arg 1 5 10
15 Phe His Gln Leu Pro Glu Phe Asp Gln Leu Lys Lys Ser Cys
Arg Arg 20 25 30
Arg Leu Ala Gly His Asn Glu Arg Arg Arg Arg Pro Pro Pro Gly Pro
35 40 45 Leu Ala Ser Arg
Tyr Gly Arg Leu Ala Ala Ser Phe Gly Glu Pro Gly 50
55 60 Arg Phe Arg Ser Phe Met Leu Asp
Phe Ser Tyr Pro Arg Val Pro Gly 65 70
75 80 Thr Met Arg Asp Gly Phe Pro Ala Val Arg Pro Gly
Glu Arg Val Pro 85 90
95 Gly Ser Ile Gln Trp Gln Ala Gly Leu Asp Pro His His His Gln Ser
100 105 110 Ala Val Ala
Gly Tyr Gly Ala His Ser Tyr Gly Ser Gln Gly Ser Ser 115
120 125 Ser Ser Ser Arg Pro Pro Val Phe
Pro Gly Pro Glu Leu Pro Pro Gly 130 135
140 Gly Cys Leu Ala Gly Val Pro Ser Asp Ser Ser Cys Ala
Leu Ser Leu 145 150 155
160 Leu Ser Thr Gln Pro Trp Asp Thr Thr His Ser Ala Gly His Ser His
165 170 175 Ala Gly Ser Met
Pro Ala Thr Ala Gly Phe Asp Gly Asn Pro Val Ala 180
185 190 Pro Ser Leu Met Ala Ser Ser Tyr Ile
Ala Pro Ser Pro Trp Thr Asp 195 200
205 Ser Arg Gly His Glu Gly Gly Arg Asn Val Pro Gln Leu Pro
Pro Asp 210 215 220
Val Pro Leu Ser Glu Val His Ser Gly Ser Ser Ser His His Gly Gln 225
230 235 240 Phe Ser Gly Glu Leu
Glu Leu Ala Leu Gln Gly Asn Arg Pro Ala Pro 245
250 255 Gly Ser Ala Pro Ala Pro Arg Asn Asp Gln
Gly Ser Thr Gly Thr Phe 260 265
270 Asp Gln Ser Gly Asn Thr Met Asp Trp Ser Leu 275
280 274647DNAFestuca arundinacea 274ggcacgaggg
tggatctgag cggctccaag acctactact gccgccacaa ggtctgctcc 60atgcactcca
aggcgccccg cgtcgtcgtc gccggcctcg agcagcgctt ctgccagcag 120tgcagcaggt
tccaccagtt gcctgaattt gacaatggaa aacgcagctg ccgcagacgt 180ctcgcaggtc
acaatgaacg ccgtaggaag ccgcctcctg gccctctggc gtcacgctat 240ggccgactcg
ctgcatcctt tgaagaaccg ggcaggtaca gaagctttct gttagatttc 300tcctacccaa
gggttccgag cagcgtgcgg gatgcttggc ctgcagttcg accaggctac 360cgtatgccca
gtgaaatcca gtggcaaggg aacctagacc tgcgtcctca cacgggttat 420ggcccacatg
catatggcag ccacggcttc cccggtccag agctccctcc aggcgggtgt 480ctcacagggg
tcgccaccga ctccagctgt gctctctctc ttctgtcaac tcagccatgg 540gataccacca
cccacggtgc cagccacgac catcggtctg cggccatgtc cgcggccgcg 600ggcttcgacg
gcagccctgc ggcagtgtca ccctccatca tggcgag
647275215PRTFestuca arundinacea 275Gly Thr Arg Val Asp Leu Ser Gly Ser
Lys Thr Tyr Tyr Cys Arg His 1 5 10
15 Lys Val Cys Ser Met His Ser Lys Ala Pro Arg Val Val Val
Ala Gly 20 25 30
Leu Glu Gln Arg Phe Cys Gln Gln Cys Ser Arg Phe His Gln Leu Pro
35 40 45 Glu Phe Asp Asn
Gly Lys Arg Ser Cys Arg Arg Arg Leu Ala Gly His 50
55 60 Asn Glu Arg Arg Arg Lys Pro Pro
Pro Gly Pro Leu Ala Ser Arg Tyr 65 70
75 80 Gly Arg Leu Ala Ala Ser Phe Glu Glu Pro Gly Arg
Tyr Arg Ser Phe 85 90
95 Leu Leu Asp Phe Ser Tyr Pro Arg Val Pro Ser Ser Val Arg Asp Ala
100 105 110 Trp Pro Ala
Val Arg Pro Gly Tyr Arg Met Pro Ser Glu Ile Gln Trp 115
120 125 Gln Gly Asn Leu Asp Leu Arg Pro
His Thr Gly Tyr Gly Pro His Ala 130 135
140 Tyr Gly Ser His Gly Phe Pro Gly Pro Glu Leu Pro Pro
Gly Gly Cys 145 150 155
160 Leu Thr Gly Val Ala Thr Asp Ser Ser Cys Ala Leu Ser Leu Leu Ser
165 170 175 Thr Gln Pro Trp
Asp Thr Thr Thr His Gly Ala Ser His Asp His Arg 180
185 190 Ser Ala Ala Met Ser Ala Ala Ala Gly
Phe Asp Gly Ser Pro Ala Ala 195 200
205 Val Ser Pro Ser Ile Met Ala 210 215
27611PRTArtificial sequencemotif 1 276Xaa Leu Xaa Phe Gly Xaa Xaa Xaa Tyr
Phe Xaa 1 5 10 27778PRTArtificial
sequenceSPL DNA-binding domain (DBD) of Arath_SPL15 transcription
factor 277Thr Ala Arg Cys Gln Val Glu Gly Cys Arg Met Asp Leu Ser Asn Val
1 5 10 15 Lys Ala
Tyr Tyr Ser Arg His Lys Val Cys Cys Ile His Ser Lys Ser 20
25 30 Ser Lys Val Ile Val Ser Gly
Leu His Gln Arg Phe Cys Gln Gln Cys 35 40
45 Ser Arg Phe His Gln Leu Ser Glu Phe Asp Leu Glu
Lys Arg Ser Cys 50 55 60
Arg Arg Arg Leu Ala Cys His Asn Glu Arg Arg Arg Lys Pro 65
70 75 27811PRTArtificial
sequencemotif 2 278Asp Ser Xaa Xaa Ala Leu Ser Leu Leu Ser Xaa 1
5 10 2791130DNAOryza sativa 279catgcggcta
atgtagatgc tcactgcgct agtagtaagg tactccagta cattatggaa 60tatacaaagc
tgtaatactc gtatcagcaa gagagaggca cacaagttgt agcagtagca 120caggattaga
aaaacgggac gacaaatagt aatggaaaaa caaaaaaaaa caaggaaaca 180catggcaata
taaatggaga aatcacaaga ggaacagaat ccgggcaata cgctgcgaaa 240gtactcgtac
gtaaaaaaaa gaggcgcatt catgtgtgga cagcgtgcag cagaagcagg 300gatttgaaac
cactcaaatc caccactgca aaccttcaaa cgaggccatg gtttgaagca 360tagaaagcac
aggtaagaag cacaacgccc tcgctctcca ccctcccacc caatcgcgac 420gcacctcgcg
gatcggtgac gtggcctcgc cccccaaaaa tatcccgcgg cgtgaagctg 480acaccccggg
cccacccacc tgtcacgttg gcacatgttg gttatggttc ccggccgcac 540caaaatatca
acgcggcgcg gcccaaaatt tccaaaatcc cgcccaagcc cctggcgcgt 600gccgctcttc
cacccaggtc cctctcgtaa tccataatgg cgtgtgtacc ctcggctggt 660tgtacgtggg
cgggttaccc tgggggtgtg ggtggatgac gggtgggccc ggaggaggtc 720cggccccgcg
cgtcatcgcg gggcggggtg tagcgggtgc gaaaaggagg cgatcggtac 780gaaaattcaa
attaggaggt ggggggcggg gcccttggag aataagcgga atcgcagata 840tgcccctgac
ttggcttggc tcctcttctt cttatccctt gtcctcgcaa ccccgcttcc 900ttctctcctc
tcctcttctc ttctcttctc tggtggtgtg ggtgtgtccc tgtctcccct 960ctccttcctc
ctctcctttc ccctcctctc ttcccccctc tcacaagaga gagagcgcca 1020gactctcccc
aggtgaggtg agaccagtct ttttgctcga ttcgacgcgc ctttcacgcc 1080gcctcgcgcg
gatctgaccg cttccctcgc ccttctcgca ggattcagcc
113028056DNAArtificial sequenceprimer prm07277 280ggggacaagt ttgtacaaaa
aagcaggctt aaacaatgga gttgttaatg tgttcg 5628151DNAArtificial
sequenceprimer prm07278 281ggggaccact ttgtacaaga aagctgggtt gatgaagatc
ttaaaaggtg a 512821110DNABrassica rapa 282atggataggg
gttccaactc gggtcttggt cttggtccag gccagacaga gtcgggtggt 60tcatccactg
agtcatcctc tttaagtgga gggctcatgt ttggccagag gatctacttc 120gaggacgctg
gaggtggaac cgggtcttct tcctccggcg ggtcaaacag aagggtacgt 180ggaagcgggt
cgggtccttc gggtcagata ccaaggtgtc aagtggaagg ttgtggaatg 240gatctaacca
atgcaaaggg ttattacacg aggcatagag tttgtggaat gcactctaaa 300acaccaaaag
tcattgtcgc tggtatagaa caaaggtttt gtcaacagtg cagcaggttt 360catcagcttc
cggaatttga cctagagaaa aggagttgcc gtaggagact cgctggtcat 420aatgagagac
gaaggaagcc acagcctgcg tctctatctg tgttgtcttc tcgttatggg 480aggatcactc
cttctctata cggaaatggt gaaactacaa tgaatgggag ctttcttggt 540tcccaagaaa
tgggttggaa tagtgcaaga acgttggata caagagtgat gagacggcca 600ccgtcgtggc
agatcaatcc tatgaatgtg tttagtcatg gatcagtaag tggaggagga 660ggaggaggga
taagcttctc atctccagag attatggaca ctaaaccaga gagctacaag 720ggaattggca
gcgactcaaa ctgtgctctc tctcttctgt caaacccaca tcagccacat 780gacaacaaca
acagcaacaa cacatggaga acttcttcgg gttttggtcc gatgacggtt 840acaatggctc
agccaccacc tgcacctagc cagcagcatc agtatctgaa cccgccttgg 900gtattcaagg
acgatgataa tagctgtccg aatgatatgt ctcctgtttt gaatcttggt 960cggttcaccg
agacagagat aagcggtgga acgactttgg gtgagttcga gttatctgac 1020catcatcatc
agaataggag gcagtacatg gaaagtgaga acacaagggc ttatggctct 1080tcttcacacc
ataacaactg gtctctctga
1110283369PRTBrassica rapa 283Met Asp Arg Gly Ser Asn Ser Gly Leu Gly Leu
Gly Pro Gly Gln Thr 1 5 10
15 Glu Ser Gly Gly Ser Ser Thr Glu Ser Ser Ser Leu Ser Gly Gly Leu
20 25 30 Met Phe
Gly Gln Arg Ile Tyr Phe Glu Asp Ala Gly Gly Gly Thr Gly 35
40 45 Ser Ser Ser Ser Gly Gly Ser
Asn Arg Arg Val Arg Gly Ser Gly Ser 50 55
60 Gly Pro Ser Gly Gln Ile Pro Arg Cys Gln Val Glu
Gly Cys Gly Met 65 70 75
80 Asp Leu Thr Asn Ala Lys Gly Tyr Tyr Thr Arg His Arg Val Cys Gly
85 90 95 Met His Ser
Lys Thr Pro Lys Val Ile Val Ala Gly Ile Glu Gln Arg 100
105 110 Phe Cys Gln Gln Cys Ser Arg Phe
His Gln Leu Pro Glu Phe Asp Leu 115 120
125 Glu Lys Arg Ser Cys Arg Arg Arg Leu Ala Gly His Asn
Glu Arg Arg 130 135 140
Arg Lys Pro Gln Pro Ala Ser Leu Ser Val Leu Ser Ser Arg Tyr Gly 145
150 155 160 Arg Ile Thr Pro
Ser Leu Tyr Gly Asn Gly Glu Thr Thr Met Asn Gly 165
170 175 Ser Phe Leu Gly Ser Gln Glu Met Gly
Trp Asn Ser Ala Arg Thr Leu 180 185
190 Asp Thr Arg Val Met Arg Arg Pro Pro Ser Trp Gln Ile Asn
Pro Met 195 200 205
Asn Val Phe Ser His Gly Ser Val Ser Gly Gly Gly Gly Gly Gly Ile 210
215 220 Ser Phe Ser Ser Pro
Glu Ile Met Asp Thr Lys Pro Glu Ser Tyr Lys 225 230
235 240 Gly Ile Gly Ser Asp Ser Asn Cys Ala Leu
Ser Leu Leu Ser Asn Pro 245 250
255 His Gln Pro His Asp Asn Asn Asn Ser Asn Asn Thr Trp Arg Thr
Ser 260 265 270 Ser
Gly Phe Gly Pro Met Thr Val Thr Met Ala Gln Pro Pro Pro Ala 275
280 285 Pro Ser Gln Gln His Gln
Tyr Leu Asn Pro Pro Trp Val Phe Lys Asp 290 295
300 Asp Asp Asn Ser Cys Pro Asn Asp Met Ser Pro
Val Leu Asn Leu Gly 305 310 315
320 Arg Phe Thr Glu Thr Glu Ile Ser Gly Gly Thr Thr Leu Gly Glu Phe
325 330 335 Glu Leu
Ser Asp His His His Gln Asn Arg Arg Gln Tyr Met Glu Ser 340
345 350 Glu Asn Thr Arg Ala Tyr Gly
Ser Ser Ser His His Asn Asn Trp Ser 355 360
365 Leu 2841080DNAGlycine max 284atggcttcag
acgccaaact ctcctcttct gagtccctca acggtttgaa attcggccaa 60aaaatctatt
ttgaggatgt tggtcttgct actccagcca cctctcttac ttcttcttct 120tcttcttctt
ctgctgctac tgttacttct tcttcttctt caaggaaagg aagaggtggg 180tctgttcaac
cagctcaacc tcccaggtgt caagttgaag ggtgcaaagt agatctgagt 240gatgcaaaag
cttactattc tagacacaag gtctgtggca tgcactctaa atccccttca 300gtcattgttg
ctggtcttca acaaaggttt tgtcaacagt gtagcaggtt tcatcagctt 360cctgagtttg
atcaaggaaa aagaagttgc cgtaggcgac tagctggcca taatgaacgt 420cggagaaagc
ccccaacaag ctccctctta acctctcgct atgccagact ttcttcgtct 480gcttttgata
atagtggcag aggtagcaac tttctgatgg aattgacttc atacccaaag 540cttagtctga
gaaattcact tccaactcct agatcatctg agctagctcc tggaaatcaa 600acctccacac
ttagctggaa tggcaactca gagacatcat ctgacctttt cttgcaaggt 660tcggtgggtg
ggacaagctt cgccagcccg ggacatcctc caggggaaag ttacattggg 720gtcaccgaca
cgagctgtgc tctctctctt ctgtcaaatc aaacatgggg ttctagaaac 780acagcaccaa
gtcttgggtt gagtaacatg ataaatttca acgggacacc cttgacacaa 840cttgctgcat
catctcatgg tgcatcaatc catcaacttc caaatacctc gtggtttttc 900aagggcattg
attctggtaa ctgttcgccc gaggtggtcc ctgatctagg tctcggtcag 960atttcacagc
ctctcaatag ccaacttcat ggtgagctgg acctgtccca acagggcagg 1020aggcattata
tggatctaga acagtccagg gcatatgaat ctgctcattg gtcactttaa
1080285359PRTGlycine max 285Met Ala Ser Asp Ala Lys Leu Ser Ser Ser Glu
Ser Leu Asn Gly Leu 1 5 10
15 Lys Phe Gly Gln Lys Ile Tyr Phe Glu Asp Val Gly Leu Ala Thr Pro
20 25 30 Ala Thr
Ser Leu Thr Ser Ser Ser Ser Ser Ser Ser Ala Ala Thr Val 35
40 45 Thr Ser Ser Ser Ser Ser Arg
Lys Gly Arg Gly Gly Ser Val Gln Pro 50 55
60 Ala Gln Pro Pro Arg Cys Gln Val Glu Gly Cys Lys
Val Asp Leu Ser 65 70 75
80 Asp Ala Lys Ala Tyr Tyr Ser Arg His Lys Val Cys Gly Met His Ser
85 90 95 Lys Ser Pro
Ser Val Ile Val Ala Gly Leu Gln Gln Arg Phe Cys Gln 100
105 110 Gln Cys Ser Arg Phe His Gln Leu
Pro Glu Phe Asp Gln Gly Lys Arg 115 120
125 Ser Cys Arg Arg Arg Leu Ala Gly His Asn Glu Arg Arg
Arg Lys Pro 130 135 140
Pro Thr Ser Ser Leu Leu Thr Ser Arg Tyr Ala Arg Leu Ser Ser Ser 145
150 155 160 Ala Phe Asp Asn
Ser Gly Arg Gly Ser Asn Phe Leu Met Glu Leu Thr 165
170 175 Ser Tyr Pro Lys Leu Ser Leu Arg Asn
Ser Leu Pro Thr Pro Arg Ser 180 185
190 Ser Glu Leu Ala Pro Gly Asn Gln Thr Ser Thr Leu Ser Trp
Asn Gly 195 200 205
Asn Ser Glu Thr Ser Ser Asp Leu Phe Leu Gln Gly Ser Val Gly Gly 210
215 220 Thr Ser Phe Ala Ser
Pro Gly His Pro Pro Gly Glu Ser Tyr Ile Gly 225 230
235 240 Val Thr Asp Thr Ser Cys Ala Leu Ser Leu
Leu Ser Asn Gln Thr Trp 245 250
255 Gly Ser Arg Asn Thr Ala Pro Ser Leu Gly Leu Ser Asn Met Ile
Asn 260 265 270 Phe
Asn Gly Thr Pro Leu Thr Gln Leu Ala Ala Ser Ser His Gly Ala 275
280 285 Ser Ile His Gln Leu Pro
Asn Thr Ser Trp Phe Phe Lys Gly Ile Asp 290 295
300 Ser Gly Asn Cys Ser Pro Glu Val Val Pro Asp
Leu Gly Leu Gly Gln 305 310 315
320 Ile Ser Gln Pro Leu Asn Ser Gln Leu His Gly Glu Leu Asp Leu Ser
325 330 335 Gln Gln
Gly Arg Arg His Tyr Met Asp Leu Glu Gln Ser Arg Ala Tyr 340
345 350 Glu Ser Ala His Trp Ser Leu
355 2861128DNAPopulus tremuloides 286atggaaatgg
attcaggctc cctaaccgag tcagctactt ccaatgcaac ttctccgcca 60gctgagtctg
ttaatggatt gaaatttggt aagaagattt actttgagga tcacgtgggg 120gtcggtgctc
cggctaagag cggaactggg tcatcctcat ccggttccgg gtcagggtca 180tctaggaagg
ctcaaggtgg acagcaccag cagccaccaa ggtgtcaagt tgaagggtgc 240aaagtagatc
tgagtgatgc taagacttac tattcaaggc acaaagtttg tagtatgcac 300tccaagtctc
ctagagttat tgttgctggt ttggtgcaaa gattttgcca gcaatgtagc 360agatttcatc
tacttcctga atttgaccaa ggaaaacgaa gttgccgcag gcgcctagct 420ggccataatg
agcgacggag gaagccacca tctggatcgg tgttgtccgc tcgccatggc 480cgattctctc
cctctttgtt tgataatagc agcagagctg gaggccttct tgtggacttt 540agtgcatatc
caaggcatac tgggagagat ggatggcctg cagcaaggtc ttctgagctt 600acccccggga
atgatactgc tgccacagga aggtctatat ctcatatgtg gcagataagc 660tcccagaatc
ctccatccaa cctttgcttg caaggctcaa ctggcgggac tggccttttc 720agttcaggaa
ttcctccggg agaatgcttc acaggagttg ctgtttcaga ctcgagctgt 780gctctctctc
ttctgtcaaa tcaaccatgg ggctccacaa accgagcatc aagtcttgcg 840gtgaatgact
tgtttagtgc cgaagaggca cccgtggttc aatcaacagc tcaccatggt 900gcggctgtca
atcagtatcc aatcccttgg agcttcaaga gcaatgaagg aagtaacagt 960tcacatgaga
tgtgccctga tctaggtctg ggtcaaattt caatgcctct caacagtcaa 1020cttgctggtc
agctcgagca gtctcaacag aataggaggc aatacatgga cctcgagcat 1080tccagggctt
atgactcttc aacccagcac atccactggt cactttaa
1128287375PRTPopulus tremuloides 287Met Glu Met Asp Ser Gly Ser Leu Thr
Glu Ser Ala Thr Ser Asn Ala 1 5 10
15 Thr Ser Pro Pro Ala Glu Ser Val Asn Gly Leu Lys Phe Gly
Lys Lys 20 25 30
Ile Tyr Phe Glu Asp His Val Gly Val Gly Ala Pro Ala Lys Ser Gly
35 40 45 Thr Gly Ser Ser
Ser Ser Gly Ser Gly Ser Gly Ser Ser Arg Lys Ala 50
55 60 Gln Gly Gly Gln His Gln Gln Pro
Pro Arg Cys Gln Val Glu Gly Cys 65 70
75 80 Lys Val Asp Leu Ser Asp Ala Lys Thr Tyr Tyr Ser
Arg His Lys Val 85 90
95 Cys Ser Met His Ser Lys Ser Pro Arg Val Ile Val Ala Gly Leu Val
100 105 110 Gln Arg Phe
Cys Gln Gln Cys Ser Arg Phe His Leu Leu Pro Glu Phe 115
120 125 Asp Gln Gly Lys Arg Ser Cys Arg
Arg Arg Leu Ala Gly His Asn Glu 130 135
140 Arg Arg Arg Lys Pro Pro Ser Gly Ser Val Leu Ser Ala
Arg His Gly 145 150 155
160 Arg Phe Ser Pro Ser Leu Phe Asp Asn Ser Ser Arg Ala Gly Gly Leu
165 170 175 Leu Val Asp Phe
Ser Ala Tyr Pro Arg His Thr Gly Arg Asp Gly Trp 180
185 190 Pro Ala Ala Arg Ser Ser Glu Leu Thr
Pro Gly Asn Asp Thr Ala Ala 195 200
205 Thr Gly Arg Ser Ile Ser His Met Trp Gln Ile Ser Ser Gln
Asn Pro 210 215 220
Pro Ser Asn Leu Cys Leu Gln Gly Ser Thr Gly Gly Thr Gly Leu Phe 225
230 235 240 Ser Ser Gly Ile Pro
Pro Gly Glu Cys Phe Thr Gly Val Ala Val Ser 245
250 255 Asp Ser Ser Cys Ala Leu Ser Leu Leu Ser
Asn Gln Pro Trp Gly Ser 260 265
270 Thr Asn Arg Ala Ser Ser Leu Ala Val Asn Asp Leu Phe Ser Ala
Glu 275 280 285 Glu
Ala Pro Val Val Gln Ser Thr Ala His His Gly Ala Ala Val Asn 290
295 300 Gln Tyr Pro Ile Pro Trp
Ser Phe Lys Ser Asn Glu Gly Ser Asn Ser 305 310
315 320 Ser His Glu Met Cys Pro Asp Leu Gly Leu Gly
Gln Ile Ser Met Pro 325 330
335 Leu Asn Ser Gln Leu Ala Gly Gln Leu Glu Gln Ser Gln Gln Asn Arg
340 345 350 Arg Gln
Tyr Met Asp Leu Glu His Ser Arg Ala Tyr Asp Ser Ser Thr 355
360 365 Gln His Ile His Trp Ser Leu
370 375 2881059DNACitrus clementina 288atggaactgg
gttcagacta tttggctgaa tcaggtggtg gctccggctc cggctcaggc 60tccggcttat
catccgctga gccatcactt aatggtttga agtttggcaa aaaaatctat 120tttgaggatg
tcggtactgc cggagctcca tttccaggat ctgggtcatc atctgggtcc 180gggtcagggt
caggttcagg gtcagggagg aaggtgaggg gtgttggcgg tggtatggtt 240actagtgggc
agcagccacc aaggtgccaa gtggaggggt gtaaagttga tctgagtgat 300gccaaagctt
actattcaag gcacaaagtt tgtggcatgc attcaaagtc tcctgttgtc 360actgttgccg
gccttgagca gaggttttgc cagcaatgta gcagatttca tcagcttccg 420gagtttgacc
aaggaaaacg aagttgccgc aggcgcctgg caggccataa tgagcgccgg 480aggaagccaa
cttctggacc atttttgggc actcgttatg gcaggctctc ttcctctgtc 540attgagaaca
gcagccaagg tggaggattt ctgatcgact tcagtgcata tcagatggtt 600ggtgggaggg
atggatggcc agtgacaagt gtctccaagc aggtatctgg aaatcaaacc 660actgtcacag
caaggcatct tcctcagcca ctatggcaaa accactctca ggatcctcca 720cctgatcgtt
accttcagtg ttcaacagct gggactggtt tctctggtcc tggaattcct 780tgtggaggat
gcttcacagg agttgctgac tcaaactgtg ctctctctct tctgtcaaat 840caaccatggg
gctctaagaa cccgacaccg ggtcatggag tgggtgactt aatgcatgcc 900cataccaaat
ccgtcactca accagtatcg ccccatggag cagctattaa tcaatatcca 960aacatgtcat
ggggggttca agggcaatgc aacctggtag cagttccaca ccaaatggcc 1020ccccaaatgg
ggtttgggtt caacattccg gcccattaa
1059289352PRTCitrus clementina 289Met Glu Leu Gly Ser Asp Tyr Leu Ala Glu
Ser Gly Gly Gly Ser Gly 1 5 10
15 Ser Gly Ser Gly Ser Gly Leu Ser Ser Ala Glu Pro Ser Leu Asn
Gly 20 25 30 Leu
Lys Phe Gly Lys Lys Ile Tyr Phe Glu Asp Val Gly Thr Ala Gly 35
40 45 Ala Pro Phe Pro Gly Ser
Gly Ser Ser Ser Gly Ser Gly Ser Gly Ser 50 55
60 Gly Ser Gly Ser Gly Arg Lys Val Arg Gly Val
Gly Gly Gly Met Val 65 70 75
80 Thr Ser Gly Gln Gln Pro Pro Arg Cys Gln Val Glu Gly Cys Lys Val
85 90 95 Asp Leu
Ser Asp Ala Lys Ala Tyr Tyr Ser Arg His Lys Val Cys Gly 100
105 110 Met His Ser Lys Ser Pro Val
Val Thr Val Ala Gly Leu Glu Gln Arg 115 120
125 Phe Cys Gln Gln Cys Ser Arg Phe His Gln Leu Pro
Glu Phe Asp Gln 130 135 140
Gly Lys Arg Ser Cys Arg Arg Arg Leu Ala Gly His Asn Glu Arg Arg 145
150 155 160 Arg Lys Pro
Thr Ser Gly Pro Phe Leu Gly Thr Arg Tyr Gly Arg Leu 165
170 175 Ser Ser Ser Val Ile Glu Asn Ser
Ser Gln Gly Gly Gly Phe Leu Ile 180 185
190 Asp Phe Ser Ala Tyr Gln Met Val Gly Gly Arg Asp Gly
Trp Pro Val 195 200 205
Thr Ser Val Ser Lys Gln Val Ser Gly Asn Gln Thr Thr Val Thr Ala 210
215 220 Arg His Leu Pro
Gln Pro Leu Trp Gln Asn His Ser Gln Asp Pro Pro 225 230
235 240 Pro Asp Arg Tyr Leu Gln Cys Ser Thr
Ala Gly Thr Gly Phe Ser Gly 245 250
255 Pro Gly Ile Pro Cys Gly Gly Cys Phe Thr Gly Val Ala Asp
Ser Asn 260 265 270
Cys Ala Leu Ser Leu Leu Ser Asn Gln Pro Trp Gly Ser Lys Asn Pro
275 280 285 Thr Pro Gly His
Gly Val Gly Asp Leu Met His Ala His Thr Lys Ser 290
295 300 Val Thr Gln Pro Val Ser Pro His
Gly Ala Ala Ile Asn Gln Tyr Pro 305 310
315 320 Asn Met Ser Trp Gly Val Gln Gly Gln Cys Asn Leu
Val Ala Val Pro 325 330
335 His Gln Met Ala Pro Gln Met Gly Phe Gly Phe Asn Ile Pro Ala His
340 345 350
290565DNABeta vulgaris 290atggatacgg gttcaaatta tccgaccgtt aaggggtcat
catcaacctc ttcatcatct 60gggttgtcgg attctttaaa tgggctgaaa tttgggcaaa
aaatatactt tgaagatgtg 120ggtggtgttg gaacttccgg caagtcttcc gtcgccggaa
gtggtggtgc tccggcgaaa 180agggccggaa aaggggtggt gcaaagtggg caaccaccaa
ggtgtcaagt agaagggtgt 240aagatagatc ttagtgatgc taaaacttat tattctaggc
ataaagtttg tggtatgcac 300tctaaatctt ctgttgttat tgttgctggt cttgagcaac
gtttttgcca gcagtgcagc 360agatttcatc ggcttcctga gtttgaccaa gggaaacgaa
gttgtcgcag acgccttgct 420ggtcataatg agcgtcgaag aaaaccacca cctgggtctt
tgttatcatc acgtttggga 480cgtctctctt catccctttt tggtgataac accggcggaa
gtggtggatt cttattggac 540ttctcttcgt atccacggca ttctg
565291188PRTBeta vulgaris 291Met Asp Thr Gly Ser
Asn Tyr Pro Thr Val Lys Gly Ser Ser Ser Thr 1 5
10 15 Ser Ser Ser Ser Gly Leu Ser Asp Ser Leu
Asn Gly Leu Lys Phe Gly 20 25
30 Gln Lys Ile Tyr Phe Glu Asp Val Gly Gly Val Gly Thr Ser Gly
Lys 35 40 45 Ser
Ser Val Ala Gly Ser Gly Gly Ala Pro Ala Lys Arg Ala Gly Lys 50
55 60 Gly Val Val Gln Ser Gly
Gln Pro Pro Arg Cys Gln Val Glu Gly Cys 65 70
75 80 Lys Ile Asp Leu Ser Asp Ala Lys Thr Tyr Tyr
Ser Arg His Lys Val 85 90
95 Cys Gly Met His Ser Lys Ser Ser Val Val Ile Val Ala Gly Leu Glu
100 105 110 Gln Arg
Phe Cys Gln Gln Cys Ser Arg Phe His Arg Leu Pro Glu Phe 115
120 125 Asp Gln Gly Lys Arg Ser Cys
Arg Arg Arg Leu Ala Gly His Asn Glu 130 135
140 Arg Arg Arg Lys Pro Pro Pro Gly Ser Leu Leu Ser
Ser Arg Leu Gly 145 150 155
160 Arg Leu Ser Ser Ser Leu Phe Gly Asp Asn Thr Gly Gly Ser Gly Gly
165 170 175 Phe Leu Leu
Asp Phe Ser Ser Tyr Pro Arg His Ser 180 185
292536DNAHevea brasiliensis 292atggaaatgg gttcgggctc tttgacagag
tcaggtacct ccaacgccac ttctccacct 60gctgagtcaa taaatgggtt gaaatttggc
caaaaaatct attttgagaa tgcgggggct 120aagactccgg ccaaatctgc acccgggtct
tcatcttccg ggtccggggc cccgtccagg 180aaggttcacg gtgggcagca gcagcagcca
cccaggtgtc aagttgaggg atgtaaagtg 240gatctgagtg atgctaaggc ttattattcg
aggcacaaag tttgtggtat gcactctaag 300tctcctaagg tcattgttgc tggtttggag
caaagatttt gccagcagtg tagtagattt 360catcagcttc ctgaatttga ccaaggaaaa
cgaagttgcc gcagacgcct agctggtcat 420aatgaacggc ggaggaagcc accaactgga
tcagtgctgt catctcgcta taacagactt 480ttttcaacaa tttttgataa cagcagccga
gctgggggca ttcttgtgga tttcag 536293177PRTHevea brasiliensis 293Met
Glu Met Gly Ser Gly Ser Leu Thr Glu Ser Gly Thr Ser Asn Ala 1
5 10 15 Thr Ser Pro Pro Ala Glu
Ser Ile Asn Gly Leu Lys Phe Gly Gln Lys 20
25 30 Ile Tyr Phe Glu Asn Ala Gly Ala Lys Thr
Pro Ala Lys Ser Ala Pro 35 40
45 Gly Ser Ser Ser Ser Gly Ser Gly Ala Pro Ser Arg Lys Val
His Gly 50 55 60
Gly Gln Gln Gln Gln Pro Pro Arg Cys Gln Val Glu Gly Cys Lys Val 65
70 75 80 Asp Leu Ser Asp Ala
Lys Ala Tyr Tyr Ser Arg His Lys Val Cys Gly 85
90 95 Met His Ser Lys Ser Pro Lys Val Ile Val
Ala Gly Leu Glu Gln Arg 100 105
110 Phe Cys Gln Gln Cys Ser Arg Phe His Gln Leu Pro Glu Phe Asp
Gln 115 120 125 Gly
Lys Arg Ser Cys Arg Arg Arg Leu Ala Gly His Asn Glu Arg Arg 130
135 140 Arg Lys Pro Pro Thr Gly
Ser Val Leu Ser Ser Arg Tyr Asn Arg Leu 145 150
155 160 Phe Ser Thr Ile Phe Asp Asn Ser Ser Arg Ala
Gly Gly Ile Leu Val 165 170
175 Asp 2941130DNAOryza sativa 294catgcggcta atgtagatgc tcactgcgct
agtagtaagg tactccagta cattatggaa 60tatacaaagc tgtaatactc gtatcagcaa
gagagaggca cacaagttgt agcagtagca 120caggattaga aaaacgggac gacaaatagt
aatggaaaaa caaaaaaaaa caaggaaaca 180catggcaata taaatggaga aatcacaaga
ggaacagaat ccgggcaata cgctgcgaaa 240gtactcgtac gtaaaaaaaa gaggcgcatt
catgtgtgga cagcgtgcag cagaagcagg 300gatttgaaac cactcaaatc caccactgca
aaccttcaaa cgaggccatg gtttgaagca 360tagaaagcac aggtaagaag cacaacgccc
tcgctctcca ccctcccacc caatcgcgac 420gcacctcgcg gatcggtgac gtggcctcgc
cccccaaaaa tatcccgcgg cgtgaagctg 480acaccccggg cccacccacc tgtcacgttg
gcacatgttg gttatggttc ccggccgcac 540caaaatatca acgcggcgcg gcccaaaatt
tccaaaatcc cgcccaagcc cctggcgcgt 600gccgctcttc cacccaggtc cctctcgtaa
tccataatgg cgtgtgtacc ctcggctggt 660tgtacgtggg cgggttaccc tgggggtgtg
ggtggatgac gggtgggccc ggaggaggtc 720cggccccgcg cgtcatcgcg gggcggggtg
tagcgggtgc gaaaaggagg cgatcggtac 780gaaaattcaa attaggaggt ggggggcggg
gcccttggag aataagcgga atcgcagata 840tgcccctgac ttggcttggc tcctcttctt
cttatccctt gtcctcgcaa ccccgcttcc 900ttctctcctc tcctcttctc ttctcttctc
tggtggtgtg ggtgtgtccc tgtctcccct 960ctccttcctc ctctcctttc ccctcctctc
ttcccccctc tcacaagaga gagagcgcca 1020gactctcccc aggtgaggtg agaccagtct
ttttgctcga ttcgacgcgc ctttcacgcc 1080gcctcgcgcg gatctgaccg cttccctcgg
ccttctcgca ggattcagcc 11302952194DNAOryza sativa
295aatccgaaaa gtttctgcac cgttttcacc ccctaactaa caatataggg aacgtgtgct
60aaatataaaa tgagacctta tatatgtagc gctgataact agaactatgc aagaaaaact
120catccaccta ctttagtggc aatcgggcta aataaaaaag agtcgctaca ctagtttcgt
180tttccttagt aattaagtgg gaaaatgaaa tcattattgc ttagaatata cgttcacatc
240tctgtcatga agttaaatta ttcgaggtag ccataattgt catcaaactc ttcttgaata
300aaaaaatctt tctagctgaa ctcaatgggt aaagagagag atttttttta aaaaaataga
360atgaagatat tctgaacgta ttggcaaaga tttaaacata taattatata attttatagt
420ttgtgcattc gtcatatcgc acatcattaa ggacatgtct tactccatcc caatttttat
480ttagtaatta aagacaattg acttattttt attatttatc ttttttcgat tagatgcaag
540gtacttacgc acacactttg tgctcatgtg catgtgtgag tgcacctcct caatacacgt
600tcaactagca acacatctct aatatcactc gcctatttaa tacatttagg tagcaatatc
660tgaattcaag cactccacca tcaccagacc acttttaata atatctaaaa tacaaaaaat
720aattttacag aatagcatga aaagtatgaa acgaactatt taggtttttc acatacaaaa
780aaaaaaagaa ttttgctcgt gcgcgagcgc caatctccca tattgggcac acaggcaaca
840acagagtggc tgcccacaga acaacccaca aaaaacgatg atctaacgga ggacagcaag
900tccgcaacaa ccttttaaca gcaggctttg cggccaggag agaggaggag aggcaaagaa
960aaccaagcat cctccttctc ccatctataa attcctcccc ccttttcccc tctctatata
1020ggaggcatcc aagccaagaa gagggagagc accaaggaca cgcgactagc agaagccgag
1080cgaccgcctt ctcgatccat atcttccggt cgagttcttg gtcgatctct tccctcctcc
1140acctcctcct cacagggtat gtgcctccct tcggttgttc ttggatttat tgttctaggt
1200tgtgtagtac gggcgttgat gttaggaaag gggatctgta tctgtgatga ttcctgttct
1260tggatttggg atagaggggt tcttgatgtt gcatgttatc ggttcggttt gattagtagt
1320atggttttca atcgtctgga gagctctatg gaaatgaaat ggtttaggga tcggaatctt
1380gcgattttgt gagtaccttt tgtttgaggt aaaatcagag caccggtgat tttgcttggt
1440gtaataaagt acggttgttt ggtcctcgat tctggtagtg atgcttctcg atttgacgaa
1500gctatccttt gtttattccc tattgaacaa aaataatcca actttgaaga cggtcccgtt
1560gatgagattg aatgattgat tcttaagcct gtccaaaatt tcgcagctgg cttgtttaga
1620tacagtagtc cccatcacga aattcatgga aacagttata atcctcagga acaggggatt
1680ccctgttctt ccgatttgct ttagtcccag aatttttttt cccaaatatc ttaaaaagtc
1740actttctggt tcagttcaat gaattgattg ctacaaataa tgcttttata gcgttatcct
1800agctgtagtt cagttaatag gtaatacccc tatagtttag tcaggagaag aacttatccg
1860atttctgatc tccattttta attatatgaa atgaactgta gcataagcag tattcatttg
1920gattattttt tttattagct ctcacccctt cattattctg agctgaaagt ctggcatgaa
1980ctgtcctcaa ttttgttttc aaattcacat cgattatcta tgcattatcc tcttgtatct
2040acctgtagaa gtttcttttt ggttattcct tgactgcttg attacagaaa gaaatttatg
2100aagctgtaat cgggatagtt atactgcttg ttcttatgat tcatttcctt tgtgcagttc
2160ttggtgtagc ttgccacttt caccagcaaa gttc
21942961149DNAZea maysmisc_feature(157)..(157)n is a, c, g, or t
296atgcacaact gcaaagaaac cagcaagaat gcgccggcgg tttatagttc cccgctactg
60caccgctctc tgctgcaggc tgcagctttg actaccaact catacagtac tagtactata
120ctagagtact actacgccat tagcacggtg gttgcantag gccgtggtct ggaggtctct
180ctcagctcag acgaggatcg tcggagcaag cagcagcgcg tgcagcctct gggagaccct
240ggcggcgtcg acgccgatcc tggggctgag cgcctgcgag tggacggtgt agaatatctt
300gtccccgatg acggagaagc tggcgctgac cacctcggcg ccctcctgct ccagcacggt
360gatgacctcg tgcagcttga agggcctggc ggcctcgctg atgagcacca cgtccagcgt
420cccgtcctgg caccgcacct cgatgaccgg catgcgcacg cctccgccgc cgccggtgga
480cccggccgcc gccgttgcag cctccgtggc cgcgccgccg ccgttgcagc agcccgcctt
540ccgctgcttc agcgcctcga tccgctcctt gagctgcttg atgtacgccg ccgcgctgtc
600cagctggtcc agctgcgtca ccgcgtcctg cttgttgccg ggattggagg acaccgccgc
660cgacgcggcg tcggagagga gggaggcgtg cgtggcggcg gcggcggcgg cgggagggat
720gagggaggag agcttgaggc agaggccctt catgtgcagc ctccggttct tctccacgtc
780cttcctctcc agcttgcacc ccccgccgct gctgtgcgcg ttcctctccc ccgcggcgca
840ggcgcccgag ctgcccccgc tgctctgcct ccggctcttc atctcgatcg accngaccgg
900tctgcagatc tctccttggc tgttctcgtc gctgctcctg ccgggcgtga agcngcgcag
960cctttggttg ggacttggga gggacaagtt gcaacagcac ccacgggcca cggcacggag
1020ggggcgaaag gcaaggaggc caggacaaga cgagagaaat acaggccggg ggagatggct
1080ccgtggcgcg tacgtgtgtc tacctgcatg ttggttgatc cgattgcatc tgctgtaacc
1140atatattaa
1149297382PRTZea maysUNSURE(53)..(53)Xaa can be any naturally occurring
amino acid 297Met His Asn Cys Lys Glu Thr Ser Lys Asn Ala Pro Ala Val Tyr
Ser 1 5 10 15 Ser
Pro Leu Leu His Arg Ser Leu Leu Gln Ala Ala Ala Leu Thr Thr
20 25 30 Asn Ser Tyr Ser Thr
Ser Thr Ile Leu Glu Tyr Tyr Tyr Ala Ile Ser 35
40 45 Thr Val Val Ala Xaa Gly Arg Gly Leu
Glu Val Ser Leu Ser Ser Asp 50 55
60 Glu Asp Arg Arg Ser Lys Gln Gln Arg Val Gln Pro Leu
Gly Asp Pro 65 70 75
80 Gly Gly Val Asp Ala Asp Pro Gly Ala Glu Arg Leu Arg Val Asp Gly
85 90 95 Val Glu Tyr Leu
Val Pro Asp Asp Gly Glu Ala Gly Ala Asp His Leu 100
105 110 Gly Ala Leu Leu Leu Gln His Gly Asp
Asp Leu Val Gln Leu Glu Gly 115 120
125 Pro Gly Gly Leu Ala Asp Glu His His Val Gln Arg Pro Val
Leu Ala 130 135 140
Pro His Leu Asp Asp Arg His Ala His Ala Ser Ala Ala Ala Gly Gly 145
150 155 160 Pro Gly Arg Arg Arg
Cys Ser Leu Arg Gly Arg Ala Ala Ala Val Ala 165
170 175 Ala Ala Arg Leu Pro Leu Leu Gln Arg Leu
Asp Pro Leu Leu Glu Leu 180 185
190 Leu Asp Val Arg Arg Arg Ala Val Gln Leu Val Gln Leu Arg His
Arg 195 200 205 Val
Leu Leu Val Ala Gly Ile Gly Gly His Arg Arg Arg Arg Gly Val 210
215 220 Gly Glu Glu Gly Gly Val
Arg Gly Gly Gly Gly Gly Gly Gly Arg Asp 225 230
235 240 Glu Gly Gly Glu Leu Glu Ala Glu Ala Leu His
Val Gln Pro Pro Val 245 250
255 Leu Leu His Val Leu Pro Leu Gln Leu Ala Pro Pro Ala Ala Ala Val
260 265 270 Arg Val
Pro Leu Pro Arg Gly Ala Gly Ala Arg Ala Ala Pro Ala Ala 275
280 285 Leu Pro Pro Ala Leu His Leu
Asp Arg Pro Asp Arg Ser Ala Asp Leu 290 295
300 Ser Leu Ala Val Leu Val Ala Ala Pro Ala Gly Arg
Glu Ala Ala Gln 305 310 315
320 Pro Leu Val Gly Thr Trp Glu Gly Gln Val Ala Thr Ala Pro Thr Gly
325 330 335 His Gly Thr
Glu Gly Ala Lys Gly Lys Glu Ala Arg Thr Arg Arg Glu 340
345 350 Lys Tyr Arg Pro Gly Glu Met Ala
Pro Trp Arg Val Arg Val Ser Thr 355 360
365 Cys Met Leu Val Asp Pro Ile Ala Ser Ala Val Thr Ile
Tyr 370 375 380 298660DNAZea
mays 298atggagatga gagcgaagaa gagcagcaga agcagcacga gcaacaccag cggcagcacg
60acgaccacgg cggtggagag gaaggagatc gagaggagga ggaggcagca gatgaagagc
120ctctgcgcca agctcgcctc cctcatcccg aaagaacact actcctccaa ggatgctatg
180acccagctgg gtagcctaga cgaggcagcc acgtacataa agagactcaa ggagagggtg
240gaggagctgc ggcacaagag cgcctctgca cggctcttgg ccgctggcag tggcacgaga
300cgaggcggag gaggaggagg cgcctccaca tcgtcggcag cgacgaccac ggcgagcggt
360ggcgcaggat catctgaaga agccggccgg cgggaggacg acatgccgcc ggcggtggta
420gaggttcggc agcacaatga cgggtcaagc ctggacgtgg tgctcatcag cagcgcggcg
480cgacccttca agctgcacga ggtggtcacc gtgctggagg aagaaggcgc cgagaccgac
540aacgccaacc tctccgtcgc cggcaccaaa atcttctaca ccatccactg caaggccttt
600tgcccaagaa tcggtataga tgtttcaaga gtttctgaaa gattaagagc attgggatga
660299219PRTZea mays 299Met Glu Met Arg Ala Lys Lys Ser Ser Arg Ser Ser
Thr Ser Asn Thr 1 5 10
15 Ser Gly Ser Thr Thr Thr Thr Ala Val Glu Arg Lys Glu Ile Glu Arg
20 25 30 Arg Arg Arg
Gln Gln Met Lys Ser Leu Cys Ala Lys Leu Ala Ser Leu 35
40 45 Ile Pro Lys Glu His Tyr Ser Ser
Lys Asp Ala Met Thr Gln Leu Gly 50 55
60 Ser Leu Asp Glu Ala Ala Thr Tyr Ile Lys Arg Leu Lys
Glu Arg Val 65 70 75
80 Glu Glu Leu Arg His Lys Ser Ala Ser Ala Arg Leu Leu Ala Ala Gly
85 90 95 Ser Gly Thr Arg
Arg Gly Gly Gly Gly Gly Gly Ala Ser Thr Ser Ser 100
105 110 Ala Ala Thr Thr Thr Ala Ser Gly Gly
Ala Gly Ser Ser Glu Glu Ala 115 120
125 Gly Arg Arg Glu Asp Asp Met Pro Pro Ala Val Val Glu Val
Arg Gln 130 135 140
His Asn Asp Gly Ser Ser Leu Asp Val Val Leu Ile Ser Ser Ala Ala 145
150 155 160 Arg Pro Phe Lys Leu
His Glu Val Val Thr Val Leu Glu Glu Glu Gly 165
170 175 Ala Glu Thr Asp Asn Ala Asn Leu Ser Val
Ala Gly Thr Lys Ile Phe 180 185
190 Tyr Thr Ile His Cys Lys Ala Phe Cys Pro Arg Ile Gly Ile Asp
Val 195 200 205 Ser
Arg Val Ser Glu Arg Leu Arg Ala Leu Gly 210 215
300669DNAGlycine max 300atgggtcaac aacaacaggg aagtcaacct
tctccaacca aagtcgaaag aaagattgta 60gagaaaaaca ggagaagcca gatgaagaac
ctctattccg aactcaactc tcttctccct 120acccgtaatc ccaaggaagc gatgtcactg
cctgatcaaa tagatgaagc aatcaactat 180atcaaaagcc tagagacaaa agtgaagctg
gagcaggaga agaaagaaag gttaaaggaa 240aggaagagaa ctcgtggtgg ctgttcgagt
tcttctgaag cacaaggaag cctgaaatcg 300ccaaatatcc agattcacga aacgggaaat
ttgcttgaag tcattctaac atgtggggtc 360gatagccagt tcatgttctg tgaaattatt
cgaattttgc atgaagagaa cgtcgaggtc 420atcaatgcca attcttcaat ggtcggagat
ttagtgattc atgttgtgca cggggaggtt 480gagccatcta tctatcaatt cggagcgacc
aaagtgagtg agaagctgaa atggtttatg 540aacggatcct tcagtgatgt ggaaatggag
cctgaattaa tgtggaattt taaaattgat 600gctactgagc cgtgggggct tctagatgat
cttacactgg acaatgtctt accaccaaat 660actttgtaa
669301222PRTGlycine max 301Met Gly Gln
Gln Gln Gln Gly Ser Gln Pro Ser Pro Thr Lys Val Glu 1 5
10 15 Arg Lys Ile Val Glu Lys Asn Arg
Arg Ser Gln Met Lys Asn Leu Tyr 20 25
30 Ser Glu Leu Asn Ser Leu Leu Pro Thr Arg Asn Pro Lys
Glu Ala Met 35 40 45
Ser Leu Pro Asp Gln Ile Asp Glu Ala Ile Asn Tyr Ile Lys Ser Leu 50
55 60 Glu Thr Lys Val
Lys Leu Glu Gln Glu Lys Lys Glu Arg Leu Lys Glu 65 70
75 80 Arg Lys Arg Thr Arg Gly Gly Cys Ser
Ser Ser Ser Glu Ala Gln Gly 85 90
95 Ser Leu Lys Ser Pro Asn Ile Gln Ile His Glu Thr Gly Asn
Leu Leu 100 105 110
Glu Val Ile Leu Thr Cys Gly Val Asp Ser Gln Phe Met Phe Cys Glu
115 120 125 Ile Ile Arg Ile
Leu His Glu Glu Asn Val Glu Val Ile Asn Ala Asn 130
135 140 Ser Ser Met Val Gly Asp Leu Val
Ile His Val Val His Gly Glu Val 145 150
155 160 Glu Pro Ser Ile Tyr Gln Phe Gly Ala Thr Lys Val
Ser Glu Lys Leu 165 170
175 Lys Trp Phe Met Asn Gly Ser Phe Ser Asp Val Glu Met Glu Pro Glu
180 185 190 Leu Met Trp
Asn Phe Lys Ile Asp Ala Thr Glu Pro Trp Gly Leu Leu 195
200 205 Asp Asp Leu Thr Leu Asp Asn Val
Leu Pro Pro Asn Thr Leu 210 215 220
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