Patent application title: Promoter, Promoter Control Elements, And Combinations, And Uses Thereof
Inventors:
Yu-Ping Lu (Camarillo, CA, US)
Yu-Ping Lu (Camarillo, CA, US)
Roger Pennell (Malibu, CA, US)
Jack Okamuro (Oak Park, CA, US)
Jack Okamuro (Oak Park, CA, US)
Richard Schneeberger (Carlsbad, CA, US)
Yiwen Fang (Los Angeles, CA, US)
Shing Kwok (Woodland Hills, CA, US)
Assignees:
CERES, INC.
IPC8 Class: AA01H500FI
USPC Class:
800298
Class name: Multicellular living organisms and unmodified parts thereof and related processes plant, seedling, plant seed, or plant part, per se higher plant, seedling, plant seed, or plant part (i.e., angiosperms or gymnosperms)
Publication date: 2009-04-23
Patent application number: 20090106866
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Patent application title: Promoter, Promoter Control Elements, And Combinations, And Uses Thereof
Inventors:
Richard Schneeberger
Yiwen Fang
Yu-Ping Lu
Roger Pennell
Jack Okamuro
Shing Kwok
Agents:
BIRCH STEWART KOLASCH & BIRCH
Assignees:
Ceres, Inc.
Origin: FALLS CHURCH, VA US
IPC8 Class: AA01H500FI
USPC Class:
800298
Abstract:
The present invention is directed to promoter sequences and promoter
control elements, polynucleotide constructs comprising the promoters and
control elements, and methods of identifying the promoters, control
elements, or fragments thereof. The invention further relates to the use
of the present promoters or promoter control elements to modulate
transcript levels.Claims:
1. An isolated nucleic acid molecule capable of modulating transcription
wherein the nucleic acid molecule shows at least 80% sequence identity to
a sequence set forth in Table 1, or a complement thereof.
2. The isolated nucleic acid molecule of claim 1, wherein said nucleic acid is capable of functioning as a promoter.
3. The isolated nucleic acid molecule of claim 2, wherein said nucleic acid comprises a reduced promoter nucleotide sequence having a sequence consisting of a SEQ ID NO. in Table 1 having at least one of the corresponding optional promoter fragments identified in Table 3 deleted therefrom.
4. The isolated nucleic acid molecule of claim 2, wherein said nucleic acid comprises a reduced promoter nucleotide sequence having a sequence consisting of a SEQ ID NO. in Table 1 having all of the corresponding optional promoter fragments identified in Table 3 deleted therefrom.
5. The isolated nucleic acid molecule of claim 1, wherein said nucleic acid molecule is capable of modulating transcription during the developmental times, or in response to a stimulus, or in a cell, tissue, or organ as set forth in Table 2 in the sections "Observed expression pattern" and "Expected expression pattern."
6. A vector construct comprising:a) a first nucleic acid capable of modulating transcription whereinthe nucleic acid molecule shows at least 80% sequence identity to a sequence set forth in Table 1; andb) a second nucleic acid having to be transcribed,wherein said first and second nucleic acid molecules are heterologous to each other and are operably linked together.
7. The vector construct according to claim 6, wherein said nucleic acid comprises a reduced promoter nucleotide sequence having a sequence consisting of a SEQ ID NO. in Table 1 having at least one of the corresponding optional promoter fragments identified in Table 3 deleted therefrom.
8. The vector construct according to claim 6, wherein said nucleic acid comprises a reduced promoter nucleotide sequence having a sequence consisting of a SEQ ID NO. in Table 1 having all of the corresponding optional promoter fragments identified in Table 3 deleted therefrom.
9. A host cell comprising an isolated nucleic acid molecule according to claim 1, wherein said nucleic acid molecule is flanked by exogenous sequence.
10. The host cell according to claim 9, wherein said nucleic acid comprises a reduced promoter nucleotide sequence having a sequence consisting of a SEQ ID NO. in Table 1 having at least one of the corresponding optional promoter fragments identified in Table 3 deleted therefrom.
11. The host cell according to claim 9, wherein said nucleic acid comprises a reduced promoter nucleotide sequence having a sequence consisting of a SEQ ID NO. in Table 1 having all of the corresponding optional promoter fragments identified in Table 3 deleted therefrom.
12. A host cell comprising a vector construct of claim 6.
13. A method of modulating transcription by combining, in an environment suitable for transcription:a) a first nucleic acid molecule capable of modulating transcriptionwherein the nucleic acid molecule shows at least 80% sequence identity to a sequence set forth in Table 1; andb) a second molecule to be transcribed;wherein the first and second nucleic acid molecules are heterologous to each other and operably linked together.
14. The method of claim 13, wherein said nucleic acid comprises a reduced promoter nucleotide sequence having a sequence consisting of a SEQ ID NO. in Table 1 having at least one of the corresponding optional promoter fragments identified in Table 3 deleted therefrom.
15. The method of claim 13, wherein said nucleic acid comprises a reduced promoter nucleotide sequence having a sequence consisting of a SEQ ID NO. in Table 1 having all of the corresponding optional promoter fragments identified in Table 3 deleted therefrom.
16. The method according to any one of claims 13-15, wherein said first nucleic acid molecule is capable of modulating transcription during the developmental times, or in response to a stimuli, or in a cell tissue, or organ as set forth in Table 2 wherein said first nucleic acid molecule is inserted into a plant cell and said plant cell is regenerated into a plant.
17. A plant comprising a vector construct according to claim 6.
18. A regulatory polynucleotide molecule isolated or identified from Arabidopsis thaliana, or a complement thereof, or a fragment thereof, or a cis element thereof, wherein said polynucleotide molecule functions to regulate the activity of the S-adenosylmethionine synthetase (SAMS3) gene.
19. The regulatory polynucleotide molecule of claim 18 which has the nucleotide sequence of SEQ ID NO: 52.
20. The regulatory polynucleotide molecule of claim 18 comprising a nucleic acid sequence that hybridizes under stringent conditions with SEQ ID NO: 52, or any complement thereof, or any fragment thereof, or any cis element thereof.
21. The regulatory polynucleotide molecule of claim 18, or any complement thereof, or any fragment thereof, or any cis element thereof, comprising a nucleic acid sequence wherein the nucleic acid sequence exhibits an 80% or greater identity to SEQ ID NO: 52.
22. The regulatory polynucleotide molecule of claim 18, or any complement thereof, any fragment thereof, or any cis element thereof comprising a nucleic acid sequence wherein the nucleic acid sequence exhibits a 90% or greater identity to SEQ ID NO: 52.
23. The regulatory polynucleotide molecule of claim 18, wherein said regulatory polynucleotide molecule is a promoter.
24. The promoter of claim 23, further described as the polynucleotide molecule of SEQ ID NO: 52.
25. The regulatory polynucleotide molecule of claim 18, wherein said regulatory polynucleotide molecule comprises a leader.
26. The regulatory polynucleotide molecule of claim 18, wherein said regulatory polynucleotide molecule comprises an intron.
27. A chimeric molecule comprising the regulatory polynucleotide molecule of claim 18.
28. A polynucleotide construct comprising the regulatory polynucleotide molecule of claim 18, wherein said regulatory polynucleotide molecule is operably linked to a transcribable polynucleotide molecule.
29. The polynucleotide construct of claim 28, wherein the regulatory polynucleotide molecule comprises the nucleic acid sequence of SEQ ID NO: 52.
30. The polynucleotide construct of claim 28, wherein said regulatory polynucleotide molecule comprises a polynucleotide sequence which exhibits a substantial percent sequence identity of greater than about 80% identity with the nucleic acid sequence of SEQ ID NO: 52.
31. The polynucleotide construct of claim 28, wherein said transcribable polynucleotide molecule is a gene of agronomic interest.
32. The polynucleotide construct of claim 28, wherein said transcribable polynucleotide molecule is a gene controlling the phenotype of a trait selected from the group consisting of: herbicide tolerance, insect control, modified yield, fungal disease resistance, virus resistance, nematode resistance, bacterial disease resistance, plant growth and development, starch production, modified oils production, high oil production, modified fatty acid content, high protein production, fruit ripening, enhanced animal and human nutrition, biopolymers, environmental stress resistance, pharmaceutical peptides and secretable peptides, improved processing traits, improved digestibility, enzyme production, flavor, nitrogen fixation, hybrid seed production, fiber production, and biofuel production.
33. The polynucleotide construct of claim 32, wherein said herbicide tolerance gene is selected from the group consisting of genes that encode for: phosphinothricin acetyltransferase, glyphosate resistant EPSPS, hydroxyphenyl pyruvate dehydrogenase, dalapon dehalogenase, bromoxynil resistant nitrilase, anthranilate synthase, glyphosate oxidoreductase and glyphosate-N-acetyl transferase.
34. A transgenic plant cell stably transformed with the polynucleotide construct of claim 28.
35. A transgenic plant stably transformed with the polynucleotide construct of claim 28.
36. A seed of said transgenic plant of claim 25.
37. A progeny of the plant of claim 36.
38. The transgenic plant cell of claim 34, wherein said plant cell is from a monocotyledonous plant selected from the group consisting of wheat, maize, rye, rice, corn, oat, bailey, turfgrass, sorghum, millet and sugarcane.
39. The transgenic plant of claim 35, wherein said plant is a monocotyledonous plant selected from the group consisting of wheat, maize, rye, rice, corn, oat, barley, turfgrass, sorghum, millet and sugarcane.
40. The seed of the transgenic plant of claim 39.
41. The transgenic plant cell of claim 34, wherein said plant cell is from a dicotyledonous plant selected from the group consisting of tobacco, tomato, potato, soybean, cotton, canola, sunflower and alfalfa.
42. The transgenic plant of claim 35, wherein said plant is a dicotyledonous plant selected from the group consisting of tobacco, tomato, potato, soybean, cotton, canola, sunflower and alfalfa.
43. The seed of the transgenic plant of claim 42.
44. A method of inhibiting weed growth in a field of transgenic glyphosate-tolerant crop plants comprising planting the transgenic plants transformed with an expression cassette comprising a) a regulatory element polynucleotide molecule isolated or identified from rice, active in a plant cell and operably linked to a polynucleotide molecule encoding a glyphosate tolerance gene; and b) applying glyphosate to the field at an application rate that inhibits the growth of weeds, wherein the growth and yield of the transgenic crop plant is not substantially affected by the glyphosate application.
45. A regulatory polynucleotide molecule isolated or identified from Arabidopsis thaliana, or a fragment thereof, wherein said polynucleotide molecule functions to regulate the activity of the S-adenosylmethionine synthetase (SAMS3) gene.
46. The regulatory polynucleotide molecule of claim 45 which has the nucleotide sequence of SEQ ID NO: 52.
47. The regulatory polynucleotide molecule of claim 45 comprising a nucleic acid sequence that hybridizes under stringent conditions with SEQ ID NO: 52, or any complement thereof, or any fragment thereof.
48. The regulatory polynucleotide molecule of claim 45, comprising a nucleic acid sequence wherein the nucleic acid sequence exhibits an 80% or greater identity to SEQ ID NO: 52, or a fragment thereof.
49. The regulatory polynucleotide molecule of claim 45, comprising a nucleic acid sequence wherein the nucleic acid sequence exhibits a 90% or greater identity to SEQ ID NO: 52, or a fragment thereof.
50. The regulatory polynucleotide molecule of claim 45, wherein said regulatory polynucleotide molecule is a promoter.
51. The promoter of claim 50, further described as the polynucleotide molecule of SEQ ID NO: 52.
52. The regulatory polynucleotide molecule of claim 45, wherein said regulatory polynucleotide molecule is an intron.
53. A chimeric molecule comprising the regulatory polynucleotide molecule of claim 45.
54. A polynucleotide construct comprising the regulatory polynucleotide molecule of claim 45, wherein said regulatory polynucleotide molecule is operably linked to a transcribable polynucleotide molecule.
55. The polynucleotide construct of claim 54, wherein the regulatory polynucleotide molecule comprises the nucleic acid sequence of SEQ ID NO: 52.
56. The polynucleotide construct of claim 54, wherein said regulatory polynucleotide molecule comprises a polynucleotide sequence which exhibits at least 80% sequence identity with SEQ ID NO: 52.
57. The polynucleotide construct of claim 54, wherein said transcribable polynucleotide molecule is a gene of agricultural interest.
58. The polynucleotide construct of claim 54, wherein said transcribable polynucleotide molecule is a gene controlling the phenotype of a trait selected from the group consisting of: herbicide tolerance, insect control, modified yield, fungal disease resistance, virus resistance, nematode resistance, plant growth and development, starch production, modified oils production, high oil production, fruit ripening, environmental stress resistance, and nitrogen fixation.
59. The polynucleotide construct of claim 58, wherein said herbicide tolerance gene is selected from the group consisting of genes that encode for genes that are resistant to phosphinothricin, glyphosate or bromoxynil.
60. A transgenic plant cell stably transformed with the polynucleotide construct of claim 54.
61. A transgenic plant stably transformed with the polynucleotide construct of claim 54.
62. A seed of said transgenic plant of claim 64.
63. A progeny of the plant of claim 62.
64. The transgenic plant cell of claim 60, wherein said plant cell is a plant selected from the group consisting of wheat and corn.
65. The transgenic plant of claim 61, wherein said plant is a plant selected from the group consisting of wheat and corn.
66. The seed of the transgenic plant of claim 65.
67. A polynucleotide molecule capable of modulating transcription which has the nucleotide sequence of SEQ ID NO: 52, or a fragment thereof.
68. The polynucleotide molecule of claim 67 which has the nucleotide sequence of SEQ ID NO: 52.
69. The polynucleotide molecule of claim 67 comprising a nucleic acid sequence that hybridizes tinder stringent conditions with SEQ ID NO: 52.
70. The polynucleotide molecule of claim 67, comprising a nucleic acid sequence wherein the nucleic acid sequence exhibits at least 80% sequence identity to SEQ ID NO: 52, or a fragment thereof.
71. The polynucleotide molecule of claim 67, comprising a nucleic acid sequence wherein the nucleic acid sequence exhibits at least 90% sequence identity to SEQ ID NO: 52, or any fragment thereof.
72. The polynucleotide molecule of claim 67, wherein said polynucleotide molecule is a promoter.
73. The promoter of claim 72, further described as the polynucleotide molecule of SEQ ID NO: 52.
74. A chimeric molecule comprising the polynucleotide molecule of claim 67.
75. A polynucleotide construct comprising a first polynucleotide molecule of claim 67, operably linked to a transcribable polynucleotide molecule.
76. The polynucleotide construct of claim 75, wherein the polynucleotide molecule comprises the nucleic acid sequence of SEQ ID NO: 52.
77. The polynucleotide construct of claim 75, wherein said first polynucleotide molecule comprises a polynucleotide sequence which exhibits at least 80% sequence identity with the nucleic acid sequence of SEQ ID NO: 52.
78. The polynucleotide construct of claim 75, wherein said transcribable polynucleotide molecule is a gene of agricultural interest.
79. The polynucleotide construct of claim 75, wherein said transcribable polynucleotide molecule is a gene controlling the phenotype of a trait selected from the group consisting of: herbicide tolerance, insect control, modified yield, fungal disease resistance, virus resistance, nematode resistance, plant growth and development, starch production, modified oils production, high oil production, fruit ripening, and nitrogen fixation.
80. The polynucleotide construct of claim 79, wherein said herbicide tolerance gene is selected from the group consisting of genes that encode for genes that confer resistance to phosphinothricin, glyphosate or bromoxynil.
81. A transgenic plant cell stably transformed with the polynucleotide construct of claim 75.
82. A transgenic plant stably transformed with the polynucleotide construct of claim 75.
83. A seed of said transgenic plant of claim 82.
84. A progeny of the plant of claim 82.
85. The transgenic plant cell of claim 81, wherein said plant cell is a cell selected from the group consisting of wheat and corn.
86. The transgenic plant of claim 82, wherein said plant is a plant selected from the group consisting of wheat and corn.
87. The seed of the transgenic plant of claim 86.
88. A transgenic plant cell stably transformed with the polynucleotide construct of claim 76.
89. A transgenic plant stably transformed with polynucleotide construct of claim 76.
90. The seed of the transgenic plant of claim 89.
91. A progeny of the plant of claim 89.
92. The transgenic plant cell of claim 88, wherein said plant cell is a cell selected from the group consisting of wheat and corn.
93. The transgenic plant of claim 89, wherein said plant is a plant selected from the group consisting of wheat and corn.
94. The seed of the transgenic plant of claim 93.
Description:
[0001]This Divisional Application claims priority under 35 U.S.C. §
119(e) and U.S.C. § 120 on U.S. application Ser. No. 11/603,542
filed on Nov. 22, 2006, application Ser. No. 10/981,334, filed Nov. 4,
2004, Provisional Application No. 60/518,075 filed on Nov. 6, 2003 and
Provisional Application No. 60/527,611 filed on Dec. 4, 2003, the entire
contents of which are hereby incorporated by reference.
FIELD OF THE INVENTION
[0002]The present invention relates to promoters and promoter control elements that are useful for modulating transcription of a desired polynucleotide. Such promoters and promoter control elements can be included in a polynucleotide construct, expression cassettes, vectors, or inserted into the chromosome or as an exogenous element, to modulate in vivo and in vitro transcription of a polynucleotide. Host cells, including plant cells, and organisms, such as regenerated plants therefrom, with desired traits or characteristics using polynucleotides comprising the promoters and promoter control elements of the present invention.
BACKGROUND OF THE INVENTION
[0003]This invention relates to the field of biotechnology and, in particular, to specific promoter sequences and promoter control element sequences which are useful for the transcription of polynucleotides in a host cell or transformed host organism.
[0004]One of the primary goals of biotechnology is to obtain organisms, such as plants, mammals, yeast, and prokaryotes having particular desired characteristics or traits. Examples of these characteristic or traits abound and may include, for example, in plants, virus resistance, insect resistance, herbicide resistance, enhanced stability or additional nutritional value. Recent advances in genetic engineering have enabled researchers in the field to incorporate polynucleotide sequences into host cells to obtain the desired qualities in the organism of choice. This technology permits one or more polynucleotides from a source different than the organism of choice to be transcribed by the organism of choice. If desired, the transcription and/or translation of these new polynucleotides can be modulated in the organism to exhibit a desired characteristic or trait. Alternatively, new patterns of transcription and/or translation of polynucleotides endogenous to the organism can be produced. Both approaches can be used at the same time.
SUMMARY OF THE INVENTION
[0005]The present invention is directed to isolated polynucleotide sequences that comprise promoters and promoter control elements from plants, especially Arabidopsis thaliana, Glycine max, Oryza sativa, and Zea mays, and other promoters and promoter control elements functional in plants.
[0006]It is an object of the present invention to provide isolated polynucleotides that are promoter sequences. These promoter sequences comprise, for example, [0007](1) a polynucleotide having a nucleotide sequence according to Table 1 or fragment thereof; [0008](2) a polynucleotide having a nucleotide sequence having at least 80% sequence identity to sequences shown in Table 1 or fragment thereof; and [0009](3) a polynucleotide having a nucleotide sequence which hybridizes to those shown in Table 1 under a condition establishing a Tm -20° C.
[0010]It is another object of the present invention to provide isolated polynucleotides that are promoter control element sequences. These promoter control element sequences comprise, for example, [0011](1) a polynucleotide having a nucleotide sequence according to Table 1 or fragment thereof; [0012](2) a polynucleotide having a nucleotide sequence having at least 80% sequence identity to those shown in Table 1 or fragment thereof; and [0013](3) a polynucleotide having a nucleotide sequence which hybridizes to those shown in Table 1 under a condition establishing a Tm -20° C.
[0014]Promoter or promoter control element sequences of the present invention are capable of modulating preferential transcription.
[0015]In another embodiment, the present promoter control elements are capable of serving as or fulfilling the function, for example, as a core promoter, a TATA box, a polymerase binding site, an initiator site, a transcription binding site, an enhancer, an inverted repeat, a locus control region, or a scaffold/matrix attachment region.
[0016]It is yet another object of the present invention to provide a polynucleotide that includes at least a first and a second promoter control element. The first promoter control element is a promoter control element sequence as discussed above, and the second promoter control element is heterologous to the first control element. Moreover, the first and second control elements are operably linked. Such promoters may modulate transcript levels preferentially in a tissue or under particular conditions.
[0017]In another embodiment, the present isolated polynucleotide comprises a promoter or a promoter control element as described above, wherein the promoter or promoter control element is operably linked to a polynucleotide to be transcribed.
[0018]In another embodiment of the present vector, the promoter and promoter control elements of the instant invention are operably linked to a heterologous polynucleotide that is a regulatory sequence.
[0019]It is another object of the present invention to provide a host cell comprising an isolated polynucleotide or vector as described above or fragment thereof. Host cells include, for instance, bacterial, yeast, insect, mammalian, and plant. The host cell can comprise a promoter or promoter control element exogenous to the genome. Such a promoter can modulate transcription in cis- and in trans-.
[0020]In yet another embodiment, the present host cell is a plant cell capable of regenerating into a plant.
[0021]It is yet another embodiment of the present invention to provide a plant comprising an isolated polynucleotide or vector described above.
[0022]It is another object of the present invention to provide a method of modulating transcription in a sample that contains either a cell-free system of transcription or host cell. This method comprises providing a polynucleotide or vector according to the present invention as described above, and contacting the sample of the polynucleotide or vector with conditions that permit transcription.
[0023]In another embodiment of the present method, the polynucleotide or vector preferentially modulates
[0024](a) constitutive transcription,
[0025](b) stress induced transcription,
[0026](c) light induced transcription,
[0027](d) dark induced transcription,
[0028](e) leaf transcription,
[0029](f) root transcription,
[0030](g) stem or shoot transcription,
[0031](h) silique transcription,
[0032](i) callus transcription,
[0033](j) flower transcription,
[0034](k) immature bud and inflorescence specific transcription, or
[0035](l) senescing induced transcription
[0036](m) germination transcription.
Other and further objects of the present invention will be made clear or become apparent from the following description.
Brief Description of the Tables
Table 1
[0037]Table 1 identifies nucleic acid promoter sequences using the headings "SEQ ID NO" and "construct." The "SEQ ID NO" is a number that identifies the sequence of the candidate promoter used in the experiments, while the "construct" text identifies the construct used to produce a specific plant line.
Table 2
[0038]Table 2 consists of the Expression Reports and provides details for expression driven by each of the nucleic acid promoter sequences as observed in transgenic plants. The results are presented as summaries of the spatial expression, which provides information as to gross and/or specific expression in various plant organs and tissues. The observed expression pattern is also presented, which gives details of expression during different generations or different developmental stages within a generation. Additional information is provided regarding the associated gene, the GenBank reference, the source organism of the promoter, and the vector and marker genes used for the construct. The following symbols are used consistently throughout the Table:
[0039]T1: First generation transformant
[0040]T2: Second generation transformant
[0041]T3: Third generation transformant
[0042](L): low expression level
[0043](M): medium expression level
[0044](H): high expression level
Table 3
[0045]Table 3 lists the co-ordinates of nucleotides of the promoter that represent optional promoter fragments. The optional promoter fragments comprise the 5' UTR and any exon(s) of the endogenous coding region. The optional promoter fragments may also comprise any exon(s) and the 3' or 5' UTR of the gene residing upstream of the promoter (that is, 5' to the promoter). The optional promoter fragments also include any intervening sequences that are introns or sequence occurring between exons or an exon and the UTR.
[0046]The information in Table 3 can be used to generate either reduced promoter sequences or "core" promoters. A reduced promoter sequence is generated when at least one optional promoter fragment is deleted. Deletion of all optional promoter fragments generates a "core" promoter.
Table 4
[0047]Table 4 presents the results of microarray experiments that track expression of particular cDNAs under specific conditions. The column headed "cDNA_ID" provides the identifier number for the cDNA tracked in the experiment. Using Table 2, these numbers can be used to correlate the differential expression pattern observed and produced by the endogenous promoter with the isolated promoters of the invention.
[0048]The column headed "EXPT_REP_ID" provides an identifier number for the particular experiment conducted. The column "SHORT_NAME" gives a brief description of the experimental conditions or the developmental stage used. The values in the column headed "Differential" indicate whether expression of the cDNA was increased (+) or decreased (-) compared to the control.
Table 5
[0049]Table 5 links the "short name" from Table 4 with a short description of the experiment, the parameters and the utility.
FIG. 1
[0050]FIG. 1 is a schematic representation of the vector pNewBin4-HAP1-GFP. The definitions of the abbreviations used in the vector map are as follows: [0051]Ori--the origin of replication used by an E. coli host [0052]RB--sequence for the right border of the T-DNA from pMOG800 [0053]BstXI--restriction enzyme cleavage site used for cloning [0054]HAP1VP16-coding sequence for a fusion protein of the HAP1 and VP16 activation domains [0055]NOS--terminator region from the nopaline synthase gene [0056]HAP1UAS--the upstream activating sequence for HAP1 [0057]5ERGFP--the green fluorescent protein gene that has been optimized for localization to the endoplasmic reticulum [0058]OCS2--the terminator sequence from the octopine synthase 2 gene [0059]OCS--the terminator sequence from the octopine synthase gene [0060]p28716 (a.k.a 28716 short)--promoter used to drive expression of the PAT (BAR) gene [0061]PAT (BAR)-- a marker gene conferring herbicide resistance [0062]LB--sequence for the left border of the T-DNA from pMOG800 [0063]Spec--a marker gene conferring spectinomycin resistance [0064]TrfA--transcription repression factor gene [0065]RK2-OriV--origin of replication for Agrobacterium
DETAILED DESCRIPTION OF THE INVENTION
1. Definitions
[0066]Chimeric: The term "chimeric" is used to describe polynucleotides or genes, as defined supra, or constructs wherein at least two of the elements of the polynucleotide or gene or construct, such as the promoter and the polynucleotide to be transcribed and/or other regulatory sequences and/or filler sequences and/or complements thereof, are heterologous to each other.
[0067]Constitutive Promoter: Promoters referred to herein as "constitutive promoters" actively promote transcription under most, but not necessarily all, environmental conditions and states of development or cell differentiation. Examples of constitutive promoters include the cauliflower mosaic virus (CaMV) 35S transcript initiation region and the 1' or 2' promoter derived from T-DNA of Agrobacterium tumefaciens, and other transcription initiation regions from various plant genes, such as the maize ubiquitin-1 promoter, known to those of skill.
[0068]Core Promoter: This is the minimal stretch of contiguous DNA sequence that is sufficient to direct accurate initiation of transcription by the RNA polymerase II machinery (for review see: Struhl, 1987, Cell 49: 295-297; Smale, 1994, In Transcription: Mechanisms and Regulation (eds R. C. Conaway and J. W. Conaway), pp 63-81/Raven Press, Ltd., New York; Smale, 1997, Biochim. Biophys. Acta 1351: 73-88; Smale et al., 1998, Cold Spring Harb. Symp. Quant. Biol. 58: 21-31; Smale, 2001, Genes & Dev. 15: 2503-2508; Weis and Reinberg, 1992, FASEB J. 6: 3300-3309; Burke et al., 1998, Cold Spring Harb. Symp. Quant. Biol 63: 75-82). There are several sequence motifs, including the TATA box, initiator (Inr), TFIIB recognition element (BRE) and downstream core promoter element (DPE), that are commonly found in core promoters, however not all of these elements occur in all promoters and there are no universal core promoter elements (Butler and Kadonaga, 2002, Genes & Dev. 16: 2583-2592).
[0069]All of the references cited in this section are hereby incorporated by reference.
[0070]Domain: Domains are fingerprints or signatures that can be used to characterize protein families and/or parts of proteins. Such fingerprints or signatures can comprise conserved (1) primary sequence, (2) secondary structure, and/or (3) three-dimensional conformation. A similar analysis can be applied to polynucleotides. Generally, each domain has been associated with either a conserved primary sequence or a sequence motif. Generally these conserved primary sequence motifs have been correlated with specific in vitro and/or in vivo activities. A domain can be any length, including the entirety of the polynucleotide to be transcribed. Examples of domains include, without limitation, AP2, helicase, homeobox, zinc finger, etc.
[0071]Endogenous: The term "endogenous," within the context of the current invention refers to any polynucleotide, polypeptide or protein sequence which is a natural part of a cell or organisms regenerated from said cell. In the context of promoter, the term "endogenous coding region" or "endogenous cDNA" refers to the coding region that is naturally operably linked to the promoter.
[0072]Enhancer/Suppressor: An "enhancer" is a DNA regulatory element that can increase the steady state level of a transcript, usually by increasing the rate of transcription initiation. Enhancers usually exert their effect regardless of the distance, upstream or downstream location, or orientation of the enhancer relative to the start site of transcription. In contrast, a "suppressor" is a corresponding DNA regulatory element that decreases the steady state level of a transcript, again usually by affecting the rate of transcription initiation. The essential activity of enhancer and suppressor elements is to bind a protein factor(s). Such binding can be assayed, for example, by methods described below. The binding is typically in a manner that influences the steady state level of a transcript in a cell or in an in vitro transcription extract.
[0073]Exogenous: As referred to within, "exogenous" is any polynucleotide, polypeptide or protein sequence, whether chimeric or not, that is introduced into the genome of a host cell or organism regenerated from said host cell by any means other than by a sexual cross. Examples of means by which this can be accomplished are described below, and include Agrobacterium-mediated transformation (of dicots--e.g. Salomon et al. EMBO J. 3:141 (1984); Herrera-Estrella et al. EMBO J. 2:987 (1983); of monocots, representative papers are those by Escudero et al., Plant J. 10:355 (1996), Ishida et al., Nature Biotechnology 14:745 (1996), May et al., Bio/Technology 13:486 (1995)), biolistic methods (Armaleo et al., Current Genetics 17:97 1990)), electroporation, in planta techniques, and the like. Such a plant containing the exogenous nucleic acid is referred to here as a T0 for the primary transgenic plant and T1 for the first generation. The term "exogenous" as used herein is also intended to encompass inserting a naturally found element into a non-naturally found location.
[0074]All of the references cited in this section are hereby incorporated by reference.
[0075]Gene: The term "gene," as used in the context of the current invention, encompasses all regulatory and coding sequence contiguously associated with a single hereditary unit with a genetic function (see SCHEMATIC 1). Genes can include non-coding sequences that modulate the genetic function that include, but are not limited to, those that specify polyadenylation, transcriptional regulation, DNA conformation, chromatin conformation, extent and position of base methylation and binding sites of proteins that control all of these. Genes encoding proteins are comprised of "exons" (coding sequences), which may be interrupted by "introns" (non-coding sequences). In some instances complexes of a plurality of protein or nucleic acids or other molecules, or of any two of the above, may be required for a gene's function. On the other hand a gene's genetic function may require only RNA expression or protein production, or may only require binding of proteins and/or nucleic acids without associated expression. In certain cases, genes adjacent to one another may share sequence in such a way that one gene will overlap the other. A gene can be found within the genome of an organism, in an artificial chromosome, in a plasmid, in any other sort of vector, or as a separate isolated entity.
[0076]Heterologous sequences: "Heterologous sequences" are those that are not operatively linked or are not contiguous to each other in nature. For example, a promoter from corn is considered heterologous to an Arabidopsis coding region sequence. Also, a promoter from a gene encoding a growth factor from corn is considered heterologous to a sequence encoding the corn receptor for the growth factor. Regulatory element sequences, such as UTRs or 3' end termination sequences that do not originate in nature from the same gene as the coding sequence originates from, are considered heterologous to said coding sequence. Elements operatively linked in nature and contiguous to each other are not heterologous to each other.
[0077]Homologous: In the current invention, a "homologous" gene or polynucleotide or polypeptide refers to a gene or polynucleotide or polypeptide that shares sequence similarity with the gene or polynucleotide or polypeptide of interest. This similarity may be in only a fragment of the sequence and often represents a functional domain such as, examples including without limitation a DNA binding domain or a domain with tyrosine kinase activity. The functional activities of homologous polynucleotide are not necessarily the same.
[0078]Inducible Promoter: An "inducible promoter" in the context of the current invention refers to a promoter, the activity of which is influenced by certain conditions, such as light, temperature, chemical concentration, protein concentration, conditions in an organism, cell, or organelle, etc. A typical example of an inducible promoter, which can be utilized with the polynucleotides of the present invention, is PARSK1, the promoter from an Arabidopsis gene encoding a serine-threonine kinase enzyme, and which promoter is induced by dehydration, abscissic acid and sodium chloride (Wang and Goodman, Plant J. 8:37 (1995), which is hereby incorporated by reference). Examples of environmental conditions that may affect transcription by inducible promoters include anaerobic conditions, elevated temperature, the presence or absence of a nutrient or other chemical compound or the presence of light.
[0079]Modulate Transcription Level: As used herein, the phrase "modulate transcription" describes the biological activity of a promoter sequence or promoter control element. Such modulation includes, without limitation, includes up- and down-regulation of initiation of transcription, rate of transcription, and/or transcription levels.
[0080]Mutant: In the current invention, "mutant" refers to a heritable change in nucleotide sequence at a specific location. Mutant genes of the current invention may or may not have an associated identifiable phenotype.
[0081]Operable Linkage: An "operable linkage" is a linkage in which a promoter sequence or promoter control element is connected to a polynucleotide sequence (or sequences) in such a way as to place transcription of the polynucleotide sequence under the influence or control of the promoter or promoter control element. Two DNA sequences (such as a polynucleotide to be transcribed and a promoter sequence linked to the 5' end of the polynucleotide to be transcribed) are said to be operably linked if induction of promoter function results in the transcription of mRNA encoding the polynucleotide and if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frame-shift mutation, (2) interfere with the ability of the promoter sequence to direct the expression of the protein, antisense RNA or ribozyme, or (3) interfere with the ability of the DNA template to be transcribed. Thus, a promoter sequence would be operably linked to a polynucleotide sequence if the promoter was capable of effecting transcription of that polynucleotide sequence.
[0082]Optional Promoter Fragments: The phrase "optional promoter fragments" is used to refer to any sub-sequence of the promoter that is not required for driving transcription of an operationally linked coding region. These fragments comprise the 5' UTR and any exon(s) of the endogenous coding region. The optional promoter fragments may also comprise any exon(s) and the 3' or 5' UTR of the gene residing upstream of the promoter (that is, 5' to the promoter). Optional promoter fragments also include any intervening sequences that are introns or sequence that occurs between exons or an exon and the UTR.
[0083]Orthologous: "Orthologous" is a term used herein to describe a relationship between two or more polynucleotides or proteins. Two polynucleotides or proteins are "orthologous" to one another if they serve a similar function in different organisms. In general, orthologous polynucleotides or proteins will have similar catalytic functions (when they encode enzymes) or will serve similar structural functions (when they encode proteins or RNA that form part of the ultrastructure of a cell).
[0084]Percentage of sequence identity: "Percentage of sequence identity," as used herein, is determined by comparing two optimally aligned sequences over a comparison window, where the fragment of the polynucleotide or amino acid sequence in the comparison window may comprise additions or deletions (e.g., gaps or overhangs) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman Add. APL. Math. 2:482 (1981), by the homology alignment algorithm of Needleman and Wunsch J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson and Lipman Proc. Natl. Acad. Sci. (USA) 85: 2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, BLAST, PASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis.), or by inspection. Given that two sequences have been identified for comparison, GAP and BESTFIT are preferably employed to determine their optimal alignment. Typically, the default values of 5.00 for gap weight and 0.30 for gap weight length are used. All of the references discussed in this paragraph are hereby incorporated by reference.
[0085]All of the references cited in this section are hereby incorporated by reference.
[0086]Plant Promoter: A "plant promoter" is a promoter capable of initiating transcription in plant cells and can modulate transcription of a polynucleotide. Such promoters need not be of plant origin. For example, promoters derived from plant viruses, such as the CaMV35S promoter or from Agrobacterium tumefaciens such as the T-DNA promoters, can be plant promoters. A typical example of a plant promoter of plant origin is the maize ubiquitin-1 (ubi-1) promoter known to those of skill.
[0087]Plant Tissue: The term "plant tissue" includes differentiated and undifferentiated tissues or plants, including but not limited to roots, stems, shoots, cotyledons, epicotyl, hypocotyl, leaves, pollen, seeds, tumor tissue and various forms of cells in culture such as single cells, protoplast, embryos, and callus tissue. The plant tissue may be in plants or in organ, tissue or cell culture.
[0088]Preferential Transcription: "Preferential transcription" is defined as transcription that occurs in a particular pattern of cell types or developmental times or in response to specific stimuli or combination thereof. Non-limitive examples of preferential transcription include: high transcript levels of a desired sequence in root tissues; detectable transcript levels of a desired sequence in certain cell types during embryogenesis; and low transcript levels of a desired sequence under drought conditions. Such preferential transcription can be determined by measuring initiation, rate, and/or levels of transcription.
[0089]Promoter: A "promoter" is a DNA sequence that directs the transcription of a polynucleotide. Typically a promoter is located in the 5' region of a polynucleotide to be transcribed, proximal to the transcriptional start site of such polynucleotide. More typically, promoters are defined as the region upstream of the first exon; more typically, as a region upstream of the first of multiple transcription start sites; more typically, as the region downstream of the preceding gene and upstream of the first of multiple transcription start sites; more typically, the region downstream of the polyA signal and upstream of the first of multiple transcription start sites; even more typically, about 3,000 nucleotides upstream of the ATG of the first exon; even more typically, 2,000 nucleotides upstream of the first of multiple transcription start sites. The promoters of the invention comprise at least a core promoter as defined above. Frequently promoters are capable of directing transcription of genes located on each of the complementary DNA strands that are 3' to the promoter. Stated differently, many promoters exhibit bidirectionality and can direct transcription of a downstream gene when present in either orientation (i.e. 5' to 3' or 3' to 5' relative to the coding region of the gene). Additionally, the promoter may also include at least one control element such as an upstream element. Such elements include UARs and optionally, other DNA sequences that affect transcription of a polynucleotide such as a synthetic upstream element.
[0090]Promoter Control Element: The term "promoter control element" as used herein describes elements that influence the activity of the promoter. Promoter control elements include transcriptional regulatory sequence determinants such as, but not limited to, enhancers, scaffold/matrix attachment regions, TATA boxes, transcription start locus control regions, UARs, URRs, other transcription factor binding sites and inverted repeats.
[0091]Public sequence: The term "public sequence," as used in the context of the instant application, refers to any sequence that has been deposited in a publicly accessible database prior to the filing date of the present application. This term encompasses both amino acid and nucleotide sequences. Such sequences are publicly accessible, for example, on the BLAST databases on the NCBI FTP web site (accessible at ncbi.nlm.nih.gov/ftp). The database at the NCBI FTP site utilizes "gi" numbers assigned by NCBI as a unique identifier for each sequence in the databases, thereby providing a non-redundant database for sequence from various databases, including GenBank, EMBL, DBBJ, (DNA Database of Japan) and PDB (Brookhaven Protein Data Bank).
[0092]Regulatory Sequence: The term "regulatory sequence," as used in the current invention, refers to any nucleotide sequence that influences transcription or translation initiation and rate, or stability and/or mobility of a transcript or polypeptide product. Regulatory sequences include, but are not limited to, promoters, promoter control elements, protein binding sequences, 5' and 3' UTRs, transcriptional start sites, termination sequences, polyadenylation sequences, introns, certain sequences within amino acid coding sequences such as secretory signals, protease cleavage sites, etc.
[0093]Related Sequences: "Related sequences" refer to either a polypeptide or a nucleotide sequence that exhibits some degree of sequence similarity with a reference sequence.
[0094]Specific Promoters: In the context of the current invention, "specific promoters" refers to a subset of promoters that have a high preference for modulating transcript levels in a specific tissue or organ or cell and/or at a specific time during development of an organism. By "high preference" is meant at least 3-fold, preferably 5-fold, more preferably at least 10-fold still more preferably at least 20-fold, 50-fold or 100-fold increase in transcript levels under the specific condition over the transcription under any other reference condition considered. Typical examples of temporal and/or tissue or organ specific promoters of plant origin that can be used with the polynucleotides of the present invention, are: PTA29, a promoter which is capable of driving gene transcription specifically in tapetum and only during anther development (Koltonow et al., Plant Cell 2:1201 (1990); RCc2 and RCc3, promoters that direct root-specific gene transcription in rice (Xu et al., Plant Mol. Biol. 27:237 (1995); TobRB27, a root-specific promoter from tobacco (Yamamoto et al., Plant Cell 3:371 (1991)). Examples of tissue-specific promoters under developmental control include promoters that initiate transcription only in certain tissues or organs, such as root, ovule, fruit, seeds, or flowers. Other specific promoters include those from genes encoding seed storage proteins or the lipid body membrane protein, oleosin. A few root-specific promoters are noted above. See also "Preferential transcription".
[0095]All of the references cited in this section are hereby incorporated by reference.
[0096]Stringency: "Stringency" as used herein is a function of probe length, probe composition (G+C content), and salt concentration, organic solvent concentration, and temperature of hybridization or wash conditions. Stringency is typically compared by the parameter Tm, which is the temperature at which 50% of the complementary molecules in the hybridization are hybridized, in terms of a temperature differential from Tm. High stringency conditions are those providing a condition of Tm -5° C. to Tm -10° C. Medium or moderate stringency conditions are those providing Tm -20° C. to Tm -29° C. Low stringency conditions are those providing a condition of Tm -40° C. to Tm -48° C. The relationship of hybridization conditions to Tm (in ° C.) is expressed in the mathematical equation
Tm=81.5-16.6(log10[Na.sup.+])+0.41(% G+C)-(600/N) (1)
where N is the length of the probe. This equation works well for probes 14 to 70 nucleotides in length that are identical to the target sequence. The equation below for Tm of DNA-DNA hybrids is useful for probes in the range of 50 to greater than 500 nucleotides, and for conditions that include an organic solvent (formamide).
Tm=81.5+16.6 log {[Na.sup.+]/(1+0.7[Na.sup.+])}+0.41(% G+C)-500/L0.63(% formamide) (2)
where L is the length of the probe in the hybrid. (P. Tijessen, "Hybridization with Nucleic Acid Probes" in Laboratory Techniques in Biochemistry and Molecular Biology, P. C. vand der Vliet, ed., c. 1993 by Elsevier, Amsterdam.) The Tm of equation (2) is affected by the nature of the hybrid; for DNA-RNA hybrids Tm is 10-15° C. higher than calculated, for RNA-RNA hybrids Tm is 20-25° C. higher. Because the Tm decreases about 1° C. for each 1% decrease in homology when a long probe is used (Bonner et al., J. Mol. Biol. 81:123 (1973)), stringency conditions can be adjusted to favor detection of identical genes or related family members.
[0097]Equation (2) is derived assuming equilibrium and therefore, hybridizations according to the present invention are most preferably performed under conditions of probe excess and for sufficient time to achieve equilibrium. The time required to reach equilibrium can be shortened by inclusion of a hybridization accelerator such as dextran sulfate or another high volume polymer in the hybridization buffer.
[0098]Stringency can be controlled during the hybridization reaction or after hybridization has occurred by altering the salt and temperature conditions of the wash solutions used. The formulas shown above are equally valid when used to compute the stringency of a wash solution. Preferred wash solution stringencies lie within the ranges stated above; high stringency is 5-8° C. below Tm, medium or moderate stringency is 26-29° C. below Tm and low stringency is 45-48° C. below Tm.
All of the references cited in this section are hereby incorporated by reference.
[0099]Substantially free of: A composition containing A is "substantially free of" B when at least 85% by weight of the total A+B in the composition is A. Preferably, A comprises at least about 90% by weight of the total of A+B in the composition, more preferably at least about 95% or even 99% by weight. For example, a plant gene can be substantially free of other plant genes. Other examples include, but are not limited to, ligands substantially free of receptors (and vice versa), a growth factor substantially free of other growth factors and a transcription binding factor substantially free of nucleic acids.
[0100]Suppressor: See "Enhancer/Suppressor"
[0101]TATA to start: "TATA to start" shall mean the distance, in number of nucleotides, between the primary TATA motif and the start of transcription.
[0102]Transgenic plant: A "transgenic plant" is a plant having one or more plant cells that contain at least one exogenous polynucleotide introduced by recombinant nucleic acid methods.
[0103]Translational start site: In the context of the present invention, a "translational start site" is usually an ATG or AUG in a transcript, often the first ATG or AUG. A single protein encoding transcript, however, may have multiple translational start sites.
[0104]Transcription start site: "Transcription start site" is used in the current invention to describe the point at which transcription is initiated. This point is typically located about 25 nucleotides downstream from a TFIID binding site, such as a TATA box. Transcription can initiate at one or more sites within the gene, and a single polynucleotide to be transcribed may have multiple transcriptional start sites, some of which may be specific for transcription in a particular cell-type or tissue or organ. "+1" is stated relative to the transcription start site and indicates the first nucleotide in a transcript.
[0105]Upstream Activating Region (UAR): An "Upstream Activating Region" or "UAR" is a position or orientation dependent nucleic acid element that primarily directs tissue, organ, cell type, or environmental regulation of transcript level, usually by affecting the rate of transcription initiation. Corresponding DNA elements that have a transcription inhibitory effect are called herein "Upstream Repressor Regions" or "URR"s. The essential activity of these elements is to bind a protein factor. Such binding can be assayed by methods described below. The binding is typically in a manner that influences the steady state level of a transcript in a cell or in vitro transcription extract.
[0106]Untranslated region (UTR): A "UTR" is any contiguous series of nucleotide bases that is transcribed, but is not translated. A 5' UTR lies between the start site of the transcript and the translation initiation codon and includes the +1 nucleotide. A 3' UTR lies between the translation termination codon and the end of the transcript. UTRs can have particular functions such as increasing mRNA message stability or translation attenuation. Examples of 3' UTRs include, but are not limited to polyadenylation signals and transcription termination sequences.
[0107]Variant: The term "variant" is used herein to denote a polypeptide or protein or polynucleotide molecule that differs from others of its kind in some way. For example, polypeptide and protein variants can consist of changes in amino acid sequence and/or charge and/or post-translational modifications (such as glycosylation, etc). Likewise, polynucleotide variants can consist of changes that add or delete a specific UTR or exon sequence. It will be understood that there may be sequence variations within sequence or fragments used or disclosed in this application. Preferably, variants will be such that the sequences have at least 80%, preferably at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity. Variants preferably measure the primary biological function of the native polypeptide or protein or polynucleotide.
2. Introduction
[0108]The polynucleotides of the invention comprise promoters and promoter control elements that are capable of modulating transcription.
[0109]Such promoters and promoter control elements can be used in combination with native or heterologous promoter fragments, control elements or other regulatory sequences to modulate transcription and/or translation.
[0110]Specifically, promoters and control elements of the invention can be used to modulate transcription of a desired polynucleotide, which includes without limitation: [0111](a) antisense; [0112](b) ribozymes; [0113](c) coding sequences; or [0114](d) fragments thereof.The promoter also can modulate transcription in a host genome in cis- or in trans-.
[0115]In an organism, such as a plant, the promoters and promoter control elements of the instant invention are useful to produce preferential transcription which results in a desired pattern of transcript levels in a particular cells, tissues, or organs, or under particular conditions.
3. Table of Contents
[0116]The following description of the present invention is outlined in the following table of contents.
[0117]A. Identifying and Isolating Promoter Sequences of the Invention [0118](1) Cloning Methods [0119](2) Chemical Synthesis
[0120]B. Generating a "core" promoter sequence
[0121]C. Isolating Related Promoter Sequences [0122](1) Relatives Based on Nucleotide Sequence Identity [0123](2) Relatives Based on Coding Sequence Identity [0124](3) Relatives based on Common Function
[0125]D. Identifying Control Elements [0126](1) Types of Transcription Control Elements [0127](2) Those Described by the Examples [0128](3) Those Identifiable by Bioinformatics [0129](4) Those Identifiable by In Vitro and In Vivo Assays [0130](5) Non-Natural Control Elements
[0131]E. Constructing Promoters and Control Elements [0132](1) Combining Promoters and Promoter Control Elements [0133](2) Number of Promoter Control Elements [0134](3) Spacing Between Control Elements
[0135]F. Vectors [0136](1) Modification of Transcription by Promoters and Promoter Control Elements [0137](2) Polynucleotide to be Transcribed [0138](3) Other Regulatory Elements [0139](4) Other Components of Vectors
[0140]G. Insertion of Polynucleotides and Vectors Into a Host Cell [0141](1) Autonomous of the Host Genome [0142](2) Integrated into the Host Genome
[0143]H. Utility
A. Identifying and Isolating Promoter Sequences of the Invention
[0144]The promoters and promoter control elements of the present invention presented in Table 1 were identified from Arabidopsis thaliana or Oryza sativa. Additional promoter sequences encompassed by the invention can be identified as described below.
[0145](1) Cloning Methods
[0146]Isolation from genomic libraries of polynucleotides comprising the sequences of the promoters and promoter control elements of the present invention is possible using known techniques.
[0147]For example, polymerase chain reaction (PCR) can amplify the desired polynucleotides utilizing primers designed from sequences in Table 2. Polynucleotide libraries comprising genomic sequences can be constructed according to Sambrook et al., (Molecular Cloning: A Laboratory Manual, 2nd Ed. (1989) Cold Spring Harbor Press, Cold Spring Harbor, N.Y., for example.
[0148]Other procedures for isolating polynucleotides comprising the promoter sequences of the invention include, without limitation, tail-PCR, and 5' rapid amplification of cDNA ends (RACE). See, for tail-PCR, for example, Liu et al., Plant J 8(3): 457-463 (September, 1995); Liu et al., Genomics 25: 674-681 (1995); Liu et al., Nucl. Acids Res. 21(14): 3333-3334 (1993); and Zoe et al., BioTechniques 27(2): 240-248 (1999); for RACE, see, for example, PCR Protocols: A Guide to Methods and Applications, (1990) Academic Press, Inc.
[0149]All of the references cited in this section are hereby incorporated by reference.
[0150](2) Chemical Synthesis
[0151]In addition, the promoters and promoter control elements described in Table 1 can be chemically synthesized according to techniques in common use. See, for example, Beaucage et al., Tet. Lett. (1981) 22: 1859 and U.S. Pat. No. 4,668,777, both of which are hereby incorporated by reference.
[0152]Such chemical oligonucleotide synthesis can be carried out using commercially available devices, such as, Biosearch 4600 or 8600 DNA synthesizer, by Applied Biosystems, a division of Perkin-Elmer Corp., Foster City, Calif., USA; and Expedite by Perceptive Biosystems, Framingham, Mass., USA.
[0153]Synthetic RNA, including natural and/or analog building blocks, can be synthesized on the Biosearch 8600 machines, see above.
[0154]Oligonucleotides can be synthesized and then ligated together to construct the desired polynucleotide.
B. Generating Reduced and "Core" Promoter Sequences
[0155]Included in the present invention are reduced and "core" promoter sequences. The reduced promoters can be isolated from the promoters of the invention by deleting at least one 5' UTR, exon or 3' UTR sequence present in the promoter sequence that is associated with a gene or coding region located 5' to the promoter sequence or in the promoter's endogenous coding region.
[0156]Similarly, the "core" promoter sequences can be generated by deleting all 5' UTRs, exons and 3' UTRs present in the promoter sequence and the associated intervening sequences that are related to the gene or coding region 5' to the promoter region and the promoter's endogenous coding region.
[0157]This data is presented in Table 3.
C. Isolating Related Promoter Sequences
[0158]Included in the present invention are promoter and promoter control elements that are related to those described in Table 1. Such related sequence can be isolated utilizing
[0159](a) nucleotide sequence identity;
[0160](b) coding sequence identity; or
[0161](c) common function or gene products.
Relatives can include both naturally occurring promoters and non-natural promoter sequences. Non-natural related promoters include nucleotide substitutions, insertions or deletions of naturally-occurring promoter sequences that do not substantially affect transcription modulation activity. For example, the binding of relevant DNA binding proteins can still occur with the non-natural promoter sequences and promoter control elements of the present invention.
[0162]According to current knowledge, promoter sequences and promoter control elements exist as functionally important regions, such as protein binding sites, and spacer regions. These spacer regions are apparently required for proper positioning of the protein binding sites. Thus, nucleotide substitutions, insertions and deletions can be tolerated in these spacer regions to a certain degree without loss of function.
[0163]In contrast, less variation is permissible in the functionally important regions, since changes in the sequence can interfere with protein binding. Nonetheless, some variation in the functionally important regions is permissible so long as function is conserved.
[0164]The effects of substitutions, insertions and deletions to the promoter sequences or promoter control elements may be to increase or decrease the binding of relevant DNA binding proteins to modulate transcript levels of a polynucleotide to be transcribed. Effects may include tissue-specific or condition-specific modulation of transcript levels of the polypeptide to be transcribed. Polynucleotides representing changes to the nucleotide sequence of the DNA-protein contact region by insertion of additional nucleotides, changes to identity of relevant nucleotides, including use of chemically-modified bases, or deletion of one or more nucleotides are considered encompassed by the present invention.
[0165](1) Relatives Based on Nucleotide Sequence Identity
[0166]Included in the present invention are promoters exhibiting nucleotide sequence identity to those described in Table 1.
DEFINITION
[0167]Typically, such related promoters exhibit at least 80% sequence identity, preferably at least 85%, more preferably at least 90%, and most preferably at least 95%, even more preferably, at least 96%, at least 97%, at least 98% or at least 99% sequence identity compared to those shown in Table 1. Such sequence identity can be calculated by the algorithms and computers programs described above.
[0168]Usually, such sequence identity is exhibited in an alignment region that is at least 75% of the length of a sequence shown in Table 1 or corresponding full-length sequence; more usually at least 80%; more usually, at least 85%, more usually at least 90%, and most usually at least 95%, even more usually, at least 96%, at least 97%, at least 98% or at least 99% of the length of a sequence shown in Table 1.
[0169]The percentage of the alignment length is calculated by counting the number of residues of the sequence in region of strongest alignment, e.g., a continuous region of the sequence that contains the greatest number of residues that are identical to the residues between two sequences that are being aligned. The number of residues in the region of strongest alignment is divided by the total residue length of a sequence in Table 1.
[0170]These related promoters may exhibit similar preferential transcription as those promoters described in Table 1.
[0171]Construction of Polynucleotides
[0172]Naturally occurring promoters that exhibit nucleotide sequence identity to those shown in Table 1 can be isolated using the techniques as described above. More specifically, such related promoters can be identified by varying stringencies, as defined above, in typical hybridization procedures such as Southern blots or probing of polynucleotide libraries, for example.
[0173]Non-natural promoter variants of those shown in Table 1 can be constructed using cloning methods that incorporate the desired nucleotide variation. See, for example, Ho, S. N., et al. Gene 77:51-59 1989, describing a procedure site directed mutagenesis using PCR, which is hereby incorporated by reference.
[0174]Any related promoter showing sequence identity to those shown in Table 1 can be chemically synthesized as described above.
[0175]Also, the present invention includes non-natural promoters that exhibit the above-sequence identity to those in Table 1
[0176]The promoters and promoter control elements of the present invention may also be synthesized with 5' or 3' extensions, to facilitate additional manipulation, for instance.
[0177]The present invention also includes reduced promoter sequences. These sequences have at least one of the optional promoter fragments deleted.
[0178]Core promoter sequences are another embodiment of the present invention. The core promoter sequences have all of the optional promoter fragments deleted.
[0179]Testing of Polynucleotides
[0180]Polynucleotides of the invention were tested for activity by cloning the sequence into an appropriate vector, transforming plants with the construct and assaying for marker gene expression. Recombinant DNA constructs were prepared which comprise the polynucleotide sequences of the invention inserted into a vector suitable for transformation of plant cells. The construct can be made using standard recombinant DNA techniques (Sambrook et al. 1989) and can be introduced to the species of interest by Agrobacterium-mediated transformation or by other means of transformation as referenced below.
[0181]The vector backbone can be any of those typical in the art such as plasmids, viruses, artificial chromosomes, BACs, YACs and PACs and vectors of the sort described by [0182](a) BAC: Shizuya et al., Proc. Natl. Acad. Sci. USA 89: 8794-8797 (1992); Hamilton et al., Proc. Natl. Acad. Sci. USA 93: 9975-9979 (1996); [0183](b) YAC: Burke et al., Science 236:806-812 (1987); [0184](c) PAC: Sternberg N. et al., Proc Natl Acad Sci USA. January; 87(1):103-7 (1990); [0185](d) Bacteria-Yeast Shuttle Vectors: Bradshaw et al., Nucl Acids Res 23: 4850-4856 (1995); [0186](e) Lambda Phage Vectors: Replacement Vector, e.g., Frischauf et al., J. Mol Biol 170: 827-842 (1983); or Insertion vector, e.g., Huynh et al., In: Glover N M (ed) DNA Cloning: A practical Approach, Vol. 1 Oxford: IRL Press (1985); T-DNA gene fusion vectors: Walden et al., Mol Cell Biol 1: 175-194 (1990); and [0187](g) Plasmid vectors: Sambrook et al., infra.
[0188]Typically, the construct comprises a vector containing a sequence of the present invention operationally linked to any marker gene. The polynucleotide was identified as a promoter by the expression of the marker gene. Although many marker genes can be used, Green Fluorescent Protein (GFP) is preferred. The vector may also comprise a marker gene that confers a selectable phenotype on plant cells. The marker may encode biocide resistance, particularly antibiotic resistance, such as resistance to kanamycin, G418, bleomycin, hygromycin, or herbicide resistance, such as resistance to chlorosulfuron or phosphinothricin. Vectors can also include origins of replication, scaffold attachment regions (SARs), markers, homologous sequences, introns, etc.
[0189]All of the references cited in this section are hereby incorporated by reference.
[0190]Promoter Control Elements of the Invention
[0191]The promoter control elements of the present invention include those that comprise a sequence shown in Table 1 and fragments thereof. The size of the fragments of Table 1 can range from 5 bases to 10 kilobases (kb). Typically, the fragment size is no smaller than 8 bases; more typically, no smaller than 12; more typically, no smaller than 15 bases; more typically, no smaller than 20 bases; more typically, no smaller than 25 bases; even more typically, no more than any one of the following: 30, 35, 40 or 50 bases.
[0192]Usually, the fragment size in no larger than 5 kb bases; more usually, no larger than 2 kb; more usually, no larger than 1 kb; more usually, no larger than 800 bases; more usually, no larger than 500 bases; even more usually, no more than any one of the following: 250, 200, 150 or 100 bases.
[0193]Relatives Based on Nucleotide Sequence Identity
[0194]Included in the present invention are promoter control elements exhibiting nucleotide sequence identity to those described in Table 1 of fragments thereof.
[0195]Typically, such related promoters exhibit at least 80% sequence identity, preferably at least 85%, more preferably at least 90%, and most preferably at least 95%, even more preferably, at least 96%, at least 97%, at least 98% or at least 99% sequence identity compared to those shown in Table 1. Such sequence identity can be calculated by the algorithms and computers programs described above.
[0196]Promoter Control Element Configuration
[0197]A common configuration of the promoter control elements in RNA polymerase II promoters is shown below:
##STR00001##
For more description, see, for example, "Models for prediction and recognition of eukaryotic promoters", T. Werner, Mammalian Genome, 10, 168-175 (1999).
[0198]Promoters are generally modular in nature. Promoters can consist of a basal promoter which functions as a site for assembly of a transcription complex comprising an RNA polymerase, for example RNA polymerase II. A typical transcription complex will include additional factors such as TFIIB, TFIID, and TFIIE. Of these, TFIID appears to be the only one to bind DNA directly. The promoter might also contain one or more promoter control elements such as the elements discussed above. These additional control elements may function as binding sites for additional transcription factors that have the function of modulating the level of transcription with respect to tissue specificity and of transcriptional responses to particular environmental or nutritional factors, and the like.
[0199]One type of promoter control element is a polynucleotide sequence representing a binding site for proteins. Typically, within a particular functional module, protein binding sites constitute regions of 5 to 60, preferably 10 to 30, more preferably 10 to 20 nucleotides. Within such binding sites, there are typically 2 to 6 nucleotides which specifically contact amino acids of the nucleic acid binding protein.
[0200]The protein binding sites are usually separated from each other by 10 to several hundred nucleotides, typically by 15 to 150 nucleotides, often by 20 to 50 nucleotides.
[0201]Further, protein binding sites in promoter control elements often display dyad symmetry in their sequence. Such elements can bind several different proteins, and/or a plurality of sites can bind the same protein. Both types of elements may be combined in a region of 50 to 1,000 base pairs.
[0202]Binding sites for any specific factor have been known to occur almost anywhere in a promoter. For example, functional AP-1 binding sites can be located far upstream, as in the rat bone sialoprotein gene, where an AP-1 site located about 900 nucleotides upstream of the transcription start site suppresses expression. Yamauchi et al., Matrix Biol., 15, 119-130 (1996). Alternatively, an AP-1 site located close to the transcription start site plays an important role in the expression of Moloney murine leukemia virus. Sap et al., Nature, 340, 242-244, (1989).
[0203]All of the references cited in this section are hereby incorporated by reference.
[0204](2) Those Identifiable by Bioinformatics
[0205]Promoter control elements from the promoters of the instant invention can be identified utilizing bioinformatic or computer driven techniques.
[0206]One method uses a computer program AlignACE to identify regulatory motifs in genes that exhibit common preferential transcription across a number of time points. The program identifies common sequence motifs in such genes. See, Roth et al., Nature Biotechnol. 16: 949-945 (1998); Tavazoie et al., Nat Genet 1999 July; 22(3):281-5;
[0207]Genomatix, also makes available a GEMS Launcher program and other programs to identify promoter control elements and configuration of such elements. Genomatix is located in Munich, Germany.
[0208]Other references also describe detection of promoter modules by models independent of overall nucleotide sequence similarity. See, for instance, Klingenhoff et al., Bioinformatics 15, 180-186 (1999).
[0209]Protein binding sites of promoters can be identified as reported in "Computer-assisted prediction, classification, and delimination of protein binding sites in nucleic acids", Frech, et al., Nucleic Acids Research, Vol. 21, No. 7, 1655-1664, 1993.
[0210]Other programs used to identify protein binding sites include, for example, Signal Scan, Prestridge et al., Comput. Appl. Biosci. 12: 157-160 (1996); Matrix Search, Chen et al., Comput. Appl. Biosci. 11: 563-566 (1995), available as part of Signal Scan 4.0; Matlnspector, Ghosh et al., Nucl. Acid Res. 21: 3117-3118 (1993) available on the internet, Conslnspector, Frech et al., Nucl. Acids Res. 21: 1655-1664 (1993), available at on the internet; TFSearch; and TESS.
[0211]Frech et al., "Software for the analysis of DNA sequence elements of transcription", Bioinformatics & Sequence Analysis, Vol. 13, no. 1, 89-97 (1997) is a review of different software for analysis of promoter control elements. This paper also reports the usefulness of matrix-based approaches to yield more specific results.
[0212]For other procedures, see, Fickett et al., Curr. Op. Biotechnol. 11: 19-24 (2000); and Quandt et al., Nucleic Acids Res., 23, 4878-4884 (1995).
[0213]All of the references cited in this section are hereby incorporated by reference.
[0214](3) Those Identifiable by In-Vitro and In-Vivo Assays
[0215]Promoter control elements also can be identified with in-vitro assays, such as transcription detection methods; and with in-vivo assays, such as enhancer trapping protocols.
[0216]In-Vitro Assays
[0217]Examples of in-vitro assays include detection of binding of protein factors that bind promoter control elements. Fragments of the instant promoters can be used to identify the location of promoter control elements. Another option for obtaining a promoter control element with desired properties is to modify known promoter sequences. This is based on the fact that the function of a promoter is dependent on the interplay of regulatory proteins that bind to specific, discrete nucleotide sequences in the promoter, termed motifs. Such interplay subsequently affects the general transcription machinery and regulates transcription efficiency. These proteins are positive regulators or negative regulators (repressors), and one protein can have a dual role depending on the context (Johnson, P. F. and McKnight, S. L. Annu. Rev. Biochem. 58:799-839 (1989)).
[0218]One type of in-vitro assay utilizes a known DNA binding factor to isolate DNA fragments that bind. If a fragment or promoter variant does not bind, then a promoter control element has been removed or disrupted. For specific assays, see, for instance, B. Luo et al., J. Mol. Biol. 266:470 (1997), S. Chusacultanachai et al., J. Biol. Chem. 274:23591 (1999), D. Fabbro et al., Biochem. Biophys. Res. Comm. 213:781 (1995)).
[0219]Alternatively, a fragment of DNA suspected of conferring a particular pattern of specificity can be examined for activity in binding transcription factors involved in that specificity by methods such as DNA footprinting (e.g. D. J. Cousins et al., Immunology 99:101 (2000); V. Kolla et al., Biochem. Biophys. Res. Comm. 266:5 (1999)) or "mobility-shift" assays (E. D. Fabiani et al., J. Biochem. 347:147 (2000); N. Sugiura et al., J. Biochem 347:155 (2000)) or fluorescence polarization (e.g. Royer et al., U.S. Pat. No. 5,445,935). Both mobility shift and DNA footprinting assays can also be used to identify portions of large DNA fragments that are bound by proteins in unpurified transcription extracts prepared from tissues or organs of interest.
[0220]Cell-free transcription extracts can be prepared and used to directly assay in a reconstitutable system (Narayan et al., Biochemistry 39:818 (2000)).
[0221]All of the references cited in this section are hereby incorporated by reference.
[0222]In-Vivo Assays
[0223]Promoter control elements can be identified with reporter genes in in-vivo assays with the use of fragments of the instant promoters or variants of the instant promoter polynucleotides.
[0224]For example, various fragments can be inserted into a vector, comprising a basal or "core" promoter, for example, operably linked to a reporter sequence, which, when transcribed, can produce a detectable label. Examples of reporter genes include those encoding luciferase, green fluorescent protein, GUS, neo, cat and bar. Alternatively, reporter sequence can be detected utilizing AFLP and microarray techniques.
[0225]In promoter probe vector systems, genomic DNA fragments are inserted upstream of the coding sequence of a reporter gene that is expressed only when the cloned fragment contains DNA having transcription modulation activity (Neve, R. L. et al., Nature 277:324-325 (1979)). Control elements are disrupted when fragments or variants lacking any transcription modulation activity. Probe vectors have been designed for assaying transcription modulation in E. coli (An, G. et al., J. Bact. 140:400-407 (1979)) and other bacterial hosts (Band, L. et al., Gene 26:313-315 (1983); Achen, M. G., Gene 45:45-49 (1986)), yeast (Goodey, A. R. et al., Mol. Gen. Genet. 204:505-511 (1986)) and mammalian cells (Pater, M. M. et al., J. Mol. App. Gen. 2:363-371 (1984)).
[0226]A different design of a promoter/control element trap includes packaging into retroviruses for more efficient delivery into cells. One type of retroviral enhancer trap was described by von Melchner et al. (Genes Dev. 1992; U.S. Pat. No. 5,364,783). The basic design of this vector includes a reporter protein coding sequence engineered into the U3 portion of the 3' LTR. No splice acceptor consensus sequences are included, limiting its utility to work as an enhancer trap only. A different approach to a gene trap using retroviral vectors was pursued by Friedrich and Soriano (Genes Dev. 1991), who engineered a lacZ-neo fusion protein linked to a splicing acceptor. LacZ-neo fusion protein expression from trapped loci allows not only for drug selection, but also for visualization of β-galatactosidase expression using the chromogenic substrate, X-gal.
[0227]A general review of tools for identifying transcriptional regulatory regions of genomic DNA is provided by J. W. Fickett et al. (Curr. Opn. Biotechnol. 11:19 (2000).
[0228]All of the references cited in this section are hereby incorporated by reference.
[0229](4) Non-Natural Control Elements
[0230]Non-natural control elements can be constructed by inserting, deleting or substituting nucleotides into the promoter control elements described above. Such control elements are capable of transcription modulation that can be determined using any of the assays described above.
D. Constructing Promoters with Control Elements
[0231](1) Combining Promoters and Promoter Control Elements
[0232]The promoter polynucleotides and promoter control elements of the present invention, both naturally occurring and synthetic, can be combined with each other to produce the desired preferential transcription. Also, the polynucleotides of the invention can be combined with other known sequences to obtain other useful promoters to modulate, for example, tissue transcription specific or transcription specific to certain conditions. Such preferential transcription can be determined using the techniques or assays described above.
[0233]Fragments, variants, as well as full-length sequences those shown in Table 1 and relatives are useful alone or in combination.
[0234]The location and relation of promoter control elements within a promoter can affect the ability of the promoter to modulate transcription. The order and spacing of control elements is a factor when constructing promoters.
[0235](2) Number of Promoter Control Elements
[0236]Promoters can contain any number of control elements. For example, a promoter can contain multiple transcription binding sites or other control elements. One element may confer tissue or organ specificity; another element may limit transcription to specific time periods, etc. Typically, promoters will contain at least a basal or core promoter as described above. Any additional element can be included as desired. For example, a fragment comprising a basal or "core" promoter can be fused with another fragment with any number of additional control elements.
[0237](3) Spacing Between Control Elements
[0238]Spacing between control elements or the configuration or control elements can be determined or optimized to permit the desired protein-polynucleotide or polynucleotide interactions to occur.
[0239]For example, if two transcription factors bind to a promoter simultaneously or relatively close in time, the binding sites are spaced to allow each factor to bind without steric hinderance. The spacing between two such hybridizing control elements can be as small as a profile of a protein bound to a control element. In some cases, two protein binding sites can be adjacent to each other when the proteins bind at different times during the transcription process.
[0240]Further, when two control elements hybridize the spacing between such elements will be sufficient to allow the promoter polynucleotide to hairpin or loop to permit the two elements to bind. The spacing between two such hybridizing control elements can be as small as a t-RNA loop, to as large as 10 kb.
[0241]Typically, the spacing is no smaller than 5 bases; more typically, no smaller than 8; more typically, no smaller than 15 bases; more typically, no smaller than 20 bases; more typically, no smaller than 25 bases; even more typically, no more than one of the following: 30, 35, 40 or 50 bases.
[0242]Usually, the fragment size in no larger than 5 kb bases; more usually, no larger than 2 kb; more usually, no larger than 1 kb; more usually, no larger than 800 bases; more usually, no larger than 500 bases; even more usually, no more than one of the following: 250, 200, 150 or 100 bases.
[0243]Such spacing between promoter control elements can be determined using the techniques and assays described above.
[0244](4) Other Promoters
[0245]The following are promoters that are induced under stress conditions and can be combined with those of the present invention: ldh1 (oxygen stress; tomato; see Germain and Ricard, 1997, Plant Mol Biol 35:949-54), GPx and CAT (oxygen stress; mouse; see Franco et al., 1999, Free Radic Biol Med 27:1122-32), ci7 (cold stress; potato; see Kirch et al., 1997, Plant Mol Biol. 33:897-909), Bz2 (heavy metals; maize; see Marrs and Walbot, 1997, Plant Physiol 113:93-102), HSP32 (hyperthermia; rat; see Raju and Maines, 1994, Biochim Biophys Acta 1217:273-80); MAPKAPK-2 (heat shock; Drosophila; see Larochelle and Suter, 1995, Gene 163:209-14).
[0246]In addition, the following examples of promoters are induced by the presence or absence of light can be used in combination with those of the present invention: Topoisomerase II (pea; see Reddy et al., 1999, Plant Mol Biol 41:125-37), chalcone synthase (soybean; see Wingender et al., 1989, Mol Gen Genet 218:315-22), mdm2 gene (human tumor; see Saucedo et al., 1998, Cell Growth Differ 9:119-30), Clock and BMAL1 (rat; see Namihira et al., 1999, Neurosci Lett 271:1-4), PHYA (Arabidopsis; see Canton and Quail, 1999, Plant Physiol 121:1207-16), PRB-1b (tobacco; see Sessa et al., 1995, Plant Mol Biol 28:537-47) and Ypr10 (common bean; see Walter et al., 1996, Eur J Biochem 239:281-93).
[0247]The promoters and control elements of the following genes can be used in combination with the present invention to confer tissue specificity: MipB (iceplant; Yamada et al., 1995, Plant Cell 7:1129-42) and SUCS (root nodules; broadbean; Kuster et al., 1993, Mol Plant Microbe Interact 6:507-14) for roots, OsSUT1 (rice; Hirose et al., 1997, Plant Cell Physiol 38:1389-96) for leaves, Msg (soybean; Stomvik et al., 1999, Plant Mol Biol 41:217-31) for siliques, cell (Arabidopsis; Shani et al., 1997, Plant Mol Biol 34(6):837-42) and ACT11 (Arabidopsis; Huang et al., 1997, Plant Mol Biol 33:125-39) for inflorescence.
[0248]Still other promoters are affected by hormones or participate in specific physiological processes, which can be used in combination with those of present invention. Some examples are the ACC synthase gene that is induced differently by ethylene and brassinosteroids (mung bean; Yi et al., 1999, Plant Mol Biol 41:443-54), the TAPG1 gene that is active during abscission (tomato; Kalaitzis et al., 1995, Plant Mol Biol 28:647-56), and the 1-aminocyclopropane-1-carboxylate synthase gene (carnation; Jones et al., 1995, Plant Mol Biol 28:505-12) and the CP-2/cathepsin L gene (rat; Kim and Wright, 1997, Biol Reprod 57:1467-77), both active during senescence.
[0249]All of the references cited in this section are hereby incorporated by reference.
E. Vectors
[0250]Vectors are a useful component of the present invention. In particular, the present promoters and/or promoter control elements may be delivered to a system such as a cell by way of a vector. For the purposes of this invention, such delivery may range from simply introducing the promoter or promoter control element by itself randomly into a cell to integration of a cloning vector containing the present promoter or promoter control element. Thus, a vector need not be limited to a DNA molecule such as a plasmid, cosmid or bacterial phage that has the capability of replicating autonomously in a host cell. All other manner of delivery of the promoters and promoter control elements of the invention are envisioned. The various T-DNA vector types are a preferred vector for use with the present invention. Many useful vectors are commercially available.
[0251]It may also be useful to attach a marker sequence to the present promoter and promoter control element in order to determine activity of such sequences. Marker sequences typically include genes that provide antibiotic resistance, such as tetracycline resistance, hygromycin resistance or ampicillin resistance, or provide herbicide resistance. Specific selectable marker genes may be used to confer resistance to herbicides such as glyphosate, glufosinate or broxynil (Comai et al., Nature 317: 741-744 (1985); Gordon-Kamm et al., Plant Cell 2: 603-618 (1990); and Stalker et al., Science 242: 419-423 (1988)). Other marker genes exist which provide hormone responsiveness.
[0252]All of the references cited in this section are hereby incorporated by reference.
[0253](1) Modification of Transcription by Promoters and Promoter Control Elements
[0254]The promoter or promoter control element of the present invention may be operably linked to a polynucleotide to be transcribed. In this manner, the promoter or promoter control element may modify transcription by modulate transcript levels of that polynucleotide when inserted into a genome.
[0255]However, prior to insertion into a genome, the promoter or promoter control element need not be linked, operably or otherwise, to a polynucleotide to be transcribed. For example, the promoter or promoter control element may be inserted alone into the genome in front of a polynucleotide already present in the genome. In this manner, the promoter or promoter control element may modulate the transcription of a polynucleotide that was already present in the genome. This polynucleotide may be native to the genome or inserted at an earlier time.
[0256]Alternatively, the promoter or promoter control element may be inserted into a genome alone to modulate transcription. See, for example, Vaucheret, H et al. (1998) Plant J 16: 651-659, which is hereby incorporated by reference. Rather, the promoter or promoter control element may be simply inserted into a genome or maintained extrachromosomally as a way to divert transcription resources of the system to itself. This approach may be used to down-regulate the transcript levels of a group of polynucleotide(s).
[0257](2) Polynucleotide to be Transcribed
[0258]The nature of the polynucleotide to be transcribed is not limited. Specifically, the polynucleotide may include sequences that will have activity as RNA as well as sequences that result in a polypeptide product. These sequences may include, but are not limited to antisense sequences, ribozyme sequences, spliceosomes, amino acid coding sequences, and fragments thereof.
[0259]Specific coding sequences may include, but are not limited to endogenous proteins or fragments thereof, or heterologous proteins including marker genes or fragments thereof.
[0260]Promoters and control elements of the present invention are useful for modulating metabolic or catabolic processes. Such processes include, but are not limited to, secondary product metabolism, amino acid synthesis, seed protein storage, oil development, pest defense and nitrogen usage. Some examples of genes, transcripts and peptides or polypeptides participating in these processes, which can be modulated by the present invention: are tryptophan decarboxylase (tdc) and strictosidine synthase (str1), dihydrodipicolinate synthase (DHDPS) and aspartate kinase (AK), 2S albumin and alpha-, beta-, and gamma-zeins, ricinoleate and 3-ketoacyl-ACP synthase (KAS), Bacillus thuringiensis (Bt) insecticidal protein, cowpea trypsin inhibitor (CpTI), asparagine synthetase and nitrite reductase. Alternatively, expression constructs can be used to inhibit expression of these peptides and polypeptides by incorporating the promoters in constructs for antisense use, co-suppression use or for the production of dominant negative mutations.
[0261](3) Other Regulatory Elements
[0262]As explained above, several types of regulatory elements exist concerning transcription regulation. Each of these regulatory elements may be combined with the present vector if desired.
[0263](4) Other Components of Vectors
[0264]Translation of eukaryotic mRNA is often initiated at the codon that encodes the first methionine. Thus, when constructing a recombinant polynucleotide according to the present invention for expressing a protein product, it is preferable to ensure that the linkage between the 3' portion, preferably including the TATA box, of the promoter and the polynucleotide to be transcribed, or a functional derivative thereof, does not contain any intervening codons which are capable of encoding a methionine.
[0265]The vector of the present invention may contain additional components. For example, an origin of replication allows for replication of the vector in a host cell. Additionally, homologous sequences flanking a specific sequence allows for specific recombination of the specific sequence at a desired location in the target genome. T-DNA sequences also allow for insertion of a specific sequence randomly into a target genome.
[0266]The vector may also be provided with a plurality of restriction sites for insertion of a polynucleotide to be transcribed as well as the promoter and/or promoter control elements of the present invention. The vector may additionally contain selectable marker genes. The vector may also contain a transcriptional and translational initiation region, and a transcriptional and translational termination region functional in the host cell. The termination region may be native with the transcriptional initiation region, may be native with the polynucleotide to be transcribed, or may be derived from another source. Convenient termination regions are available from the Ti-plasmid of A. tumefaciens, such as the octopine synthase and nopaline synthase termination regions. See also, Guerineau et al., (199 1) Mol. Gen. Genet. 262:141-144; Proudfoot (199 1) Cell 64:671-674; Sanfacon et al. (199 1) Genes Dev. 5:141-149; Mogen et al. (1990) Plant Cell 2:1261-1272; Munroe et al. (1990) Gene 91:151-158; Ballas et al. 1989) Nucleic Acids Res. 17:7891-7903; Joshi et al. (1987) Nucleic Acid Res. 15:9627-9639.
[0267]Where appropriate, the polynucleotide to be transcribed may be optimized for increased expression in a certain host cell. For example, the polynucleotide can be synthesized using preferred codons for improved transcription and translation. See U.S. Pat. Nos. 5,380,831, 5,436,391; see also and Murray et al., (1989) Nucleic Acids Res. 17:477-498.
[0268]Additional sequence modifications include elimination of sequences encoding spurious polyadenylation signals, exon intron splice site signals, transposon-like repeats, and other such sequences well characterized as deleterious to expression. The G-C content of the polynucleotide may be adjusted to levels average for a given cellular host, as calculated by reference to known genes expressed in the host cell. The polynucleotide sequence may be modified to avoid hairpin secondary mRNA structures.
[0269]A general description of expression vectors and reporter genes can be found in Gruber, et al., "Vectors for Plant Transformation, in Methods in Plant Molecular Biology & Biotechnology" in Glich et al., (Eds. pp. 89-119, CRC Press, 1993). Moreover GUS expression vectors and GUS gene cassettes are available from Clonetech Laboratories, Inc., Palo Alto, Calif., while luciferase expression vectors and luciferase gene cassettes are available from Promega Corp. (Madison, Wis.). GFP vectors are available from Aurora Biosciences.
[0270]All of the references cited in this section are hereby incorporated by reference.
F. Polynucleotide Insertion into a Host Cell
[0271]The polynucleotides according to the present invention can be inserted into a host cell. A host cell includes but is not limited to a plant, mammalian, insect, yeast, and prokaryotic cell, preferably a plant cell.
[0272]The method of insertion into the host cell genome is chosen based on convenience. For example, the insertion into the host cell genome may either be accomplished by vectors that integrate into the host cell genome or by vectors which exist independent of the host cell genome.
[0273](1) Polynucleotides Autonomous of the Host Genome
[0274]The polynucleotides of the present invention can exist autonomously or independent of the host cell genome. Vectors of these types are known in the art and include, for example, certain type of non-integrating viral vectors, autonomously replicating plasmids, artificial chromosomes, and the like.
[0275]Additionally, in some cases transient expression of a polynucleotide may be desired.
[0276](2) Polynucleotides Integrated into the Host Genome
[0277]The promoter sequences, promoter control elements or vectors of the present invention may be transformed into host cells. These transformations may be into protoplasts or intact tissues or isolated cells. Preferably expression vectors are introduced into intact tissue. General methods of culturing plant tissues are provided for example by Maki et al. "Procedures for Introducing Foreign DNA into Plants" in Methods in Plant Molecular Biology & Biotechnology, Glich et al. (Eds. pp. 67-88 CRC Press, 1993); and by Phillips et al. "Cell-Tissue Culture and In-Vitro Manipulation" in Corn & Corn Improvement, 3rd Edition 10 Sprague et al. (Eds. pp. 345-387) American Society of Agronomy Inc. et al. 1988.
[0278]Methods of introducing polynucleotides into plant tissue include the direct infection or co-cultivation of plant cell with Agrobacterium tumefaciens, Horsch et al., Science, 227:1229 (1985). Descriptions of Agrobacterium vector systems and methods for Agrobacterium-mediated gene transfer provided by Gruber et al. supra.
[0279]Alternatively, polynucleotides are introduced into plant cells or other plant tissues using a direct gene transfer method such as microprojectile-mediated delivery, DNA injection, electroporation and the like. More preferably polynucleotides are introduced into plant tissues using the microprojectile media delivery with the biolistic device. See, for example, Tomes et al., "Direct DNA transfer into intact plant cells via microprojectile bombardment" In: Gamborg and Phillips (Eds.) Plant Cell, Tissue and Organ Culture: Fundamental Methods, Springer Verlag, Berlin (1995).
[0280]In another embodiment of the current invention, expression constructs can be used for gene expression in callus culture for the purpose of expressing marker genes encoding peptides or polypeptides that allow identification of transformed plants. Here, a promoter that is operatively linked to a polynucleotide to be transcribed is transformed into plant cells and the transformed tissue is then placed on callus-inducing media. If the transformation is conducted with leaf discs, for example, callus will initiate along the cut edges. Once callus growth has initiated, callus cells can be transferred to callus shoot-inducing or callus root-inducing media. Gene expression will occur in the callus cells developing on the appropriate media: callus root-inducing promoters will be activated on callus root-inducing media, etc. Examples of such peptides or polypeptides useful as transformation markers include, but are not limited to barstar, glyphosate, chloramphenicol acetyltransferase (CAT), kanamycin, spectinomycin, streptomycin or other antibiotic resistance enzymes, green fluorescent protein (GFP), and β-glucuronidase (GUS), etc. Some of the exemplary promoters of Table 1 will also be capable of sustaining expression in some tissues or organs after the initiation or completion of regeneration. Examples of these tissues or organs are somatic embryos, cotyledon, hypocotyl, epicotyl, leaf, stems, roots, flowers and seed.
[0281]Integration into the host cell genome also can be accomplished by methods known in the art, for example, by the homologous sequences or T-DNA discussed above or using the cre-lox system (A. C. Vergunst et al., Plant Mol. Biol. 38:393 (1998)).
[0282]All of the references cited in this section are hereby incorporated by reference.
G. Utility
[0283]Common Uses
[0284]In yet another embodiment, the promoters of the present invention can be used to further understand developmental mechanisms. For example, promoters that are specifically induced during callus formation, somatic embryo formation, shoot formation or root formation can be used to explore the effects of overexpression, repression or ectopic expression of target genes, or for isolation of trans-acting factors.
[0285]The vectors of the invention can be used not only for expression of coding regions but may also be used in exon-trap cloning, or promoter trap procedures to detect differential gene expression in various tissues, K. Lindsey et al., 1993 "Tagging Genomic Sequences That Direct Transgene Expression by Activation of a Promoter Trap in Plants", Transgenic Research 2:3347. D. Auch & Reth, et al., "Exon Trap Cloning: Using PCR to Rapidly Detect and Clone Exons from Genomic DNA Fragments", Nucleic Acids Research, Vol. 18, No. 22, p. 674.
[0286]Entrapment vectors, first described for use in bacteria (Casadaban and Cohen, 1979, Proc. Nat. Aca. Sci. U.S.A., 76: 4530; Casadaban et al., 1980, J. Bacteriol., 143: 971) permit selection of insertional events that lie within coding sequences. Entrapment vectors can be introduced into pluripotent ES cells in culture and then passed into the germline via chimeras (Gossler et al., 1989, Science, 244: 463; Skarnes, 1990, Biotechnology, 8: 827). Promoter or gene trap vectors often contain a reporter gene, e.g., lacZ, lacking its own promoter and/or splice acceptor sequence upstream. That is, promoter gene traps contain a reporter gene with a splice site but no promoter. If the vector lands in a gene and is spliced into the gene product, then the reporter gene is expressed.
[0287]Recently, the isolation of preferentially-induced genes has been made possible with the use of sophisticated promoter traps (e.g. IVET) that are based on conditional auxotrophy complementation or drug resistance. In one IVET approach, various bacterial genome fragments are placed in front of a necessary metabolic gene coupled to a reporter gene. The DNA constructs are inserted into a bacterial strain otherwise lacking the metabolic gene, and the resulting bacteria are used to infect the host organism. Only bacteria expressing the metabolic gene survive in the host organism; consequently, inactive constructs can be eliminated by harvesting only bacteria that survive for some minimum period in the host. At the same time, constitutively active constructs can be eliminated by screening only bacteria that do not express the reporter gene under laboratory conditions. The bacteria selected by such a method contain constructs that are selectively induced only during infection of the host. The IVET approach can be modified for use in plants to identify genes induced in either the bacteria or the plant cells upon pathogen infection or root colonization. For information on IVET see the articles by Mahan et al. in Science 259:686-688 (1993), Mahan et al. in PNAS USA 92:669-673 (1995), Heithoff et al. in PNAS USA 94:934-939 (1997), and Wang et al. in PNAS USA. 93:10434 (1996).
[0288]All of the references cited in this section are hereby incorporated by reference.
[0289]Constitutive Transcription
[0290]Use of promoters and control elements providing constitutive transcription is desired for modulation of transcription in most cells of an organism under most environmental conditions. In a plant, for example, constitutive transcription is useful for modulating genes involved in defense, pest resistance, herbicide resistance, etc.
[0291]Constitutive up-regulation and transcription down-regulation is useful for these applications. For instance, genes, transcripts, and/or polypeptides that increase defense, pest and herbicide resistance may require constitutive up-regulation of transcription. In contrast, constitutive transcriptional down-regulation may be desired to inhibit those genes, transcripts, and/or polypeptides that lower defense, pest and herbicide resistance.
[0292]Typically, promoter or control elements that provide constitutive transcription produce transcription levels that are statistically similar in many tissues and environmental conditions observed.
[0293]Calculation of P-value from the different observed transcript levels is one means of determining whether a promoter or control element is providing constitutive up-regulation. P-value is the probability that the difference of transcript levels is not statistically significant. The higher the P-value, the more likely the difference of transcript levels is not significant. One formula used to calculate P-value is as follows:
∫ Φ ( x ) x , integrated from a to ∞ , where Φ ( x ) is a normal distribution ; ##EQU00001## where a = Sx - μ σ ( all Samples except Sx ) ; ##EQU00001.2## where Sx = the intensity of the sample of interest ##EQU00001.3## where μ = is the average of the intensities of all samples except Sx , = ( S 1 Sn ) - Sx n - 1 ##EQU00001.4##
[0294]where σ(S1 . . . S11, not including Sx)=the standard deviation of all sample intensities except Sx.
The P-value from the formula ranges from 1.0 to 0.0.
[0295]Usually, each P-value of the transcript levels observed in a majority of cells, tissues, or organs under various environmental conditions produced by the promoter or control element is greater than 10-8; more usually, greater than 10-7; even more usually, greater than 10-6; even more usually, greater than 10-5 or 10-4.
[0296]For up-regulation of transcription, promoter and control elements produce transcript levels that are above background of the assay.
[0297]Stress Induced Preferential Transcription
[0298]Promoters and control elements providing modulation of transcription under oxidative, drought, oxygen, wound, and methyl jasmonate stress are particularly useful for producing host cells or organisms that are more resistant to biotic and abiotic stresses. In a plant, for example, modulation of genes, transcripts, and/or polypeptides in response to oxidative stress can protect cells against damage caused by oxidative agents, such as hydrogen peroxide and other free radicals.
[0299]Drought induction of genes, transcripts, and/or polypeptides are useful to increase the viability of a plant, for example, when water is a limiting factor. In contrast, genes, transcripts, and/or polypeptides induced during oxygen stress can help the flood tolerance of a plant.
[0300]The promoters and control elements of the present invention can modulate stresses similar to those described in, for example, stress conditions are VuPLD1 (drought stress; Cowpea; see Pham-Thi et al., 1999, Plant Mol Biol 1257-65), pyruvate decarboxylase (oxygen stress; rice; see Rivosal et al., 1997, Plant Physiol 114(3): 1021-29), chromoplast specific carotenoid gene (oxidative stress; capsicum; see Bouvier et al., 1998, J Biol Chem 273: 30651-59).
[0301]Promoters and control elements providing preferential transcription during wounding or induced by methyl jasmonate can produce a defense response in host cells or organisms. In a plant, for example, preferential modulation of genes, transcripts, and/or polypeptides under such conditions is useful to induce a defense response to mechanical wounding, pest or pathogen attack or treatment with certain chemicals.
[0302]Promoters and control elements of the present invention also can trigger a response similar to those described for cf9 (viral pathogen; tomato; see O'Donnell et al., 1998, Plant J 14(1): 137-42), hepatocyte growth factor activator inhibitor type 1 (HAI-1), which enhances tissue regeneration (tissue injury; human; Koono et al., 1999, J Histochem Cytochem 47: 673-82), copper amine oxidase (CuAO), induced during ontogenesis and wound healing (wounding; chick-pea; Rea et al., 1998, FEBS Ltr 437: 177-82), proteinase inhibitor II (wounding; potato; see Pena-Cortes et al., 1988, Planta 174: 84-89), protease inhibitor II (methyl jasmonate; tomato; see Farmer and Ryan, 1990, Proc Natl Acad Sci USA 87: 7713-7716), two vegetative storage protein genes VspA and VspB (wounding, jasmonic acid, and water deficit; soybean; see Mason and Mullet, 1990, Plant Cell 2: 569-579).
[0303]Up-regulation and transcription down-regulation are useful for these applications. For instance, genes, transcripts, and/or polypeptides that increase oxidative, flood, or drought tolerance may require up-regulation of transcription. In contrast, transcriptional down-regulation may be desired to inhibit those genes, transcripts, and/or polypeptides that lower such tolerance.
[0304]Typically, promoter or control elements, which provide preferential transcription in wounding or under methyl jasmonate induction, produce transcript levels that are statistically significant as compared to cell types, organs or tissues under other conditions.
[0305]For preferential up-regulation of transcription, promoter and control elements produce transcript levels that are above background of the assay.
[0306]All of the references cited in this section are hereby incorporated by reference.
[0307]Light Induced Preferential Transcription
[0308]Promoters and control elements providing preferential transcription when induced by light exposure can be utilized to modulate growth, metabolism, and development; to increase drought tolerance; and decrease damage from light stress for host cells or organisms. In a plant, for example, modulation of genes, transcripts, and/or polypeptides in response to light is useful [0309](1) to increase the photosynthetic rate; [0310](2) to increase storage of certain molecules in leaves or green parts only, e.g., silage with high protein or starch content; [0311](3) to modulate production of exogenous compositions in green tissue, e.g., certain feed enzymes; [0312](4) to induce growth or development, such as fruit development and maturity, during extended exposure to light; [0313](5) to modulate guard cells to control the size of stomata in leaves to prevent water loss, or [0314](6) to induce accumulation of beta-carotene to help plants cope with light induced stress.The promoters and control elements of the present invention also can trigger responses similar to those described in: abscisic acid insensitive3 (ABI3) (dark-grown Arabidopsis seedlings, see Rohde et al., 2000, Plant Cell 12: 35-52), asparagine synthetase (pea root nodules, see Tsai and Coruzzi, 1990, EMBO J 9: 323-32), mdm2 gene (human tumor; see Saucedo et al., 1998, Cell Growth Differ 9: 119-30).
[0315]Up-regulation and transcription down-regulation are useful for these applications. For instance, genes, transcripts, and/or polypeptides that increase drought or light tolerance may require up-regulation of transcription. In contrast, transcriptional down-regulation may be desired to inhibit those genes, transcripts, and/or polypeptides that lower such tolerance.
[0316]Typically, promoter or control elements, which provide preferential transcription in cells, tissues or organs exposed to light, produce transcript levels that are statistically significant as compared to cells, tissues, or organs under decreased light exposure (intensity or length of time).
[0317]For preferential up-regulation of transcription, promoter and control elements produce transcript levels that are above background of the assay.
[0318]All of the references cited in this section are hereby incorporated by reference.
[0319]Dark Induced Preferential Transcription
[0320]Promoters and control elements providing preferential transcription when induced by dark or decreased light intensity or decreased light exposure time can be utilized to time growth, metabolism, and development, to modulate photosynthesis capabilities for host cells or organisms. In a plant, for example, modulation of genes, transcripts, and/or polypeptides in response to dark is useful, for example, [0321](1) to induce growth or development, such as fruit development and maturity, despite lack of light; [0322](2) to modulate genes, transcripts, and/or polypeptide active at night or on cloudy days; or [0323](3) to preserve the plastid ultra structure present at the onset of darkness.The present promoters and control elements can also trigger response similar to those described in the section above.
[0324]Up-regulation and transcription down-regulation is useful for these applications. For instance, genes, transcripts, and/or polypeptides that increase growth and development may require up-regulation of transcription. In contrast, transcriptional down-regulation may be desired to inhibit those genes, transcripts, and/or polypeptides that modulate photosynthesis capabilities.
[0325]Typically, promoter or control elements, which provide preferential transcription under exposure to dark or decrease light intensity or decrease exposure time, produce transcript levels that are statistically significant.
[0326]For preferential up-regulation of transcription, promoter and control elements produce transcript levels that are above background of the assay.
[0327]Leaf Preferential Transcription
[0328]Promoters and control elements providing preferential transcription in a leaf can modulate growth, metabolism, and development or modulate energy and nutrient utilization in host cells or organisms. In a plant, for example, preferential modulation of genes, transcripts, and/or polypeptide in a leaf, is useful, for example,
[0329](1) to modulate leaf size, shape, and development;
[0330](2) to modulate the number of leaves; or
[0331](3) to modulate energy or nutrient usage in relation to other organs and tissues
[0332]Up-regulation and transcription down-regulation is useful for these applications. For instance, genes, transcripts, and/or polypeptides that increase growth, for example, may require up-regulation of transcription. In contrast, transcriptional down-regulation may be desired to inhibit energy usage in a leaf to be directed to the fruit instead, for instance.
[0333]Typically, promoter or control elements, which provide preferential transcription in the cells, tissues, or organs of a leaf, produce transcript levels that are statistically significant as compared to other cells, organs or tissues.
[0334]For preferential up-regulation of transcription, promoter and control elements produce transcript levels that are above background of the assay.
[0335]Root Preferential Transcription
[0336]Promoters and control elements providing preferential transcription in a root can modulate growth, metabolism, development, nutrient uptake, nitrogen fixation, or modulate energy and nutrient utilization in host cells or organisms. In a plant, for example, preferential modulation of genes, transcripts, and/or in a leaf, is useful
[0337](1) to modulate root size, shape, and development;
[0338](2) to modulate the number of roots, or root hairs;
[0339](3) to modulate mineral, fertilizer, or water uptake;
[0340](4) to modulate transport of nutrients; or
[0341](4) to modulate energy or nutrient usage in relation to other organs and tissues.
[0342]Up-regulation and transcription down-regulation is useful for these applications. For instance, genes, transcripts, and/or polypeptides that increase growth, for example, may require up-regulation of transcription. In contrast, transcriptional down-regulation may be desired to inhibit nutrient usage in a root to be directed to the leaf instead, for instance.
[0343]Typically, promoter or control elements, which provide preferential transcription in cells, tissues, or organs of a root, produce transcript levels that are statistically significant as compared to other cells, organs or tissues.
[0344]For preferential up-regulation of transcription, promoter and control elements produce transcript levels that are above background of the assay.
[0345]Stem/Shoot Preferential Transcription
[0346]Promoters and control elements providing preferential transcription in a stem or shoot can modulate growth, metabolism, and development or modulate energy and nutrient utilization in host cells or organisms. In a plant, for example, preferential modulation of genes, transcripts, and/or polypeptide in a stem or shoot, is useful, for example,
[0347](1) to modulate stem/shoot size, shape, and development; or
[0348](2) to modulate energy or nutrient usage in relation to other organs and tissues
[0349]Up-regulation and transcription down-regulation is useful for these applications. For instance, genes, transcripts, and/or polypeptides that increase growth, for example, may require up-regulation of transcription. In contrast, transcriptional down-regulation may be desired to inhibit energy usage in a stem/shoot to be directed to the fruit instead, for instance.
[0350]Typically, promoter or control elements, which provide preferential transcription in the cells, tissues, or organs of a stem or shoot, produce transcript levels that are statistically significant as compared to other cells, organs or tissues. [0351]For preferential up-regulation of transcription, promoter and control elements produce transcript levels that are above background of the assay.
[0352]Fruit and Seed Preferential Transcription
[0353]Promoters and control elements providing preferential transcription in a silique or fruit can time growth, development, or maturity; or modulate fertility; or modulate energy and nutrient utilization in host cells or organisms. In a plant, for example, preferential modulation of genes, transcripts, and/or polypeptides in a fruit, is useful [0354](1) to modulate fruit size, shape, development, and maturity; [0355](2) to modulate the number of fruit or seeds; [0356](3) to modulate seed shattering; [0357](4) to modulate components of seeds, such as, storage molecules, starch, protein, oil, vitamins, anti-nutritional components, such as phytic acid; [0358](5) to modulate seed and/or seedling vigor or viability; [0359](6) to incorporate exogenous compositions into a seed, such as lysine rich proteins; [0360](7) to permit similar fruit maturity timing for early and late blooming flowers; or [0361](8) to modulate energy or nutrient usage in relation to other organs and tissues.
[0362]Up-regulation and transcription down-regulation is useful for these applications. For instance, genes, transcripts, and/or polypeptides that increase growth, for example, may require up-regulation of transcription. In contrast, transcriptional down-regulation may be desired to inhibit late fruit maturity, for instance.
[0363]Typically, promoter or control elements, which provide preferential transcription in the cells, tissues, or organs of siliques or fruits, produce transcript levels that are statistically significant as compared to other cells, organs or tissues.
[0364]For preferential up-regulation of transcription, promoter and control elements produce transcript levels that are above background of the assay.
[0365]Callus Preferential Transcription
[0366]Promoters and control elements providing preferential transcription in a callus can be useful to modulating transcription in dedifferentiated host cells. In a plant transformation, for example, preferential modulation of genes, transcripts, in callus is useful to modulate transcription of a marker gene, which can facilitate selection of cells that are transformed with exogenous polynucleotides.
[0367]Up-regulation and transcription down-regulation is useful for these applications. For instance, genes, transcripts, and/or polypeptides that increase marker gene detectability, for example, may require up-regulation of transcription. In contrast, transcriptional down-regulation may be desired to increase the ability of the calluses to later differentiate, for instance.
[0368]Typically, promoter or control elements, which provide preferential transcription in callus, produce transcript levels that are statistically significant as compared to other cell types, tissues, or organs. Calculation of P-value from the different observed transcript levels is one means of determining whether a promoter or control element is providing such preferential transcription.
[0369]Usually, each P-value of the transcript levels observed in callus as compared to, at least one other cell type, tissue or organ, is less than 10-4; more usually, less than 10-5; even more usually, less than 10-6; even more usually, less than 10-7 or 10-8.
[0370]For preferential up-regulation of transcription, promoter and control elements produce transcript levels that are above background of the assay.
[0371]Flower Specific Transcription
[0372]Promoters and control elements providing preferential transcription in flowers can modulate pigmentation; or modulate fertility in host cells or organisms. In a plant, for example, preferential modulation of genes, transcripts, and/or polypeptides in a flower, is useful,
[0373](1) to modulate petal color; or
[0374](2) to modulate the fertility of pistil and/or stamen.
[0375]Up-regulation and transcription down-regulation is useful for these applications. For instance, genes, transcripts, and/or polypeptides that increase pigmentation, for example, may require up-regulation of transcription. In contrast, transcriptional down-regulation may be desired to inhibit fertility, for instance.
[0376]Typically, promoter or control elements, which provide preferential transcription in flowers, produce transcript levels that are statistically significant as compared to other cells, organs or tissues.
[0377]For preferential up-regulation of transcription, promoter and control elements produce transcript levels that are above background of the assay.
[0378]Immature Bud and Inflorescence Preferential Transcription
[0379]Promoters and control elements providing preferential transcription in a immature bud or inflorescence can time growth, development, or maturity; or modulate fertility or viability in host cells or organisms. In a plant, for example, preferential modulation of genes, transcripts, and/or polypeptide in a fruit, is useful,
[0380](1) to modulate embryo development, size, and maturity;
[0381](2) to modulate endosperm development, size, and composition;
[0382](3) to modulate the number of seeds and fruits; or
[0383](4) to modulate seed development and viability.
[0384]Up-regulation and transcription down-regulation is useful for these applications. For instance, genes, transcripts, and/or polypeptides that increase growth, for example, may require up-regulation of transcription. In contrast, transcriptional down-regulation may be desired to decrease endosperm size, for instance.
[0385]Typically, promoter or control elements, which provide preferential transcription in immature buds and inflorescences, produce transcript levels that are statistically significant as compared to other cell types, organs or tissues.
[0386]For preferential up-regulation of transcription, promoter and control elements produce transcript levels that are above background of the assay.
[0387]Senescence Preferential Transcription
[0388]Promoters and control elements providing preferential transcription during senescencing can be used to modulate cell degeneration, nutrient mobilization, and scavenging of free radicals in host cells or organisms. Other types of responses that can be modulated include, for example, senescence associated genes (SAG) that encode enzymes thought to be involved in cell degeneration and nutrient mobilization (arabidopsis; see Hensel et al. 1993. Plant Cell 5: 553-64), and the CP-2/cathepsin L gene (rat; Kim and Wright. 1997. Biol Reprod 57: 1467-77), both induced during senescence.
[0389]In a plant, for example, preferential modulation of genes, transcripts, and/or polypeptides during senescence is useful to modulate fruit ripening.
[0390]Up-regulation and transcription down-regulation is useful for these applications. For instance, genes, transcripts, and/or polypeptides that increase scavenging of free radicals, for example, may require up-regulation of transcription. In contrast, transcriptional down-regulation may be desired to inhibit cell degeneration, for instance.
[0391]Typically, promoter or control elements, which provide preferential transcription in cells, tissues, or organs during senescence, produce transcript levels that are statistically significant as compared to other conditions.
[0392]For preferential up-regulation of transcription, promoter and control elements produce transcript levels that are above background of the assay.
[0393]Germination Preferential Transcription
[0394]Promoters and control elements providing preferential transcription in a germinating seed can time growth, development, or maturity; or modulate viability in host cells or organisms. In a plant, for example, preferential modulation of genes, transcripts, and/or polypeptide in a germinating seed, is useful,
[0395](1) to modulate the emergence of they hypocotyls, cotyledons and radical; or
[0396](2) to modulate shoot and primary root growth and development;
[0397]Up-regulation and transcription down-regulation is useful for these applications. For instance, genes, transcripts, and/or polypeptides that increase growth, for example, may require up-regulation of transcription. In contrast, transcriptional down-regulation may be desired to decrease endosperm size, for instance.
[0398]Typically, promoter or control elements, which provide preferential transcription in a germinating seed, produce transcript levels that are statistically significant as compared to other cell types, organs or tissues.
[0399]For preferential up-regulation of transcription, promoter and control elements produce transcript levels that are above background of the assay.
Microarray Analysis
[0400]A major way that a cell controls its response to internal or external stimuli is by regulating the rate of transcription of specific genes. For example, the differentiation of cells during organogenesis into forms characteristic of the organ is associated with the selective activation and repression of large numbers of genes. Thus, specific organs, tissues and cells are functionally distinct due to the different populations of mRNAs and protein products they possess. Internal signals program the selective activation and repression programs. For example, internally synthesized hormones produce such signals. The level of hormone can be raised by increasing the level of transcription of genes encoding proteins concerned with hormone synthesis.
[0401]To measure how a cell reacts to internal and/or external stimuli, individual mRNA levels can be measured and used as an indicator for the extent of transcription of the gene. Cells can be exposed to a stimulus, and mRNA can be isolated and assayed at different time points after stimulation. The mRNA from the stimulated cells can be compared to control cells that were not stimulated. The mRNA levels that are higher in the stimulated cell versus the control indicate a stimulus-specific response of the cell. The same is true of mRNA levels that are lower in stimulated cells versus the control condition.
[0402]Similar studies can be performed with cells taken from an organism with a defined mutation in their genome as compared with cells without the mutation. Altered mRNA levels in the mutated cells indicate how the mutation causes transcriptional changes. These transcriptional changes are associated with the phenotype that the mutated cells exhibit that is different from the phenotype exhibited by the control cells.
[0403]Applicants have utilized microarray techniques to measure the levels of mRNAs in cells from plants transformed with a construct containing the promoter or control elements of the present invention together with their endogenous cDNA sequences. In general, transformants with the constructs were grown to an appropriate stage, and tissue samples were prepared for the microarray differential expression analysis. In this manner it is possible to determine the differential expression for the cDNAs under the control of the endogenous promoter under various conditions.
Microarray Experimental Procedures and Results
Procedures
1. Sample Tissue Preparation
[0404]Tissue samples for each of the expression analysis experiments were prepared as follows:
[0405](a) Roots
[0406]Seeds of Arabidopsis thaliana (Ws) were sterilized in full strength bleach for less than 5 min., washed more than 3 times in sterile distilled deionized water and plated on MS agar plates. The plates were placed at 4° C. for 3 nights and then placed vertically into a growth chamber having 16 hr light/8 hr dark cycles, 23° C., 70% relative humidity and ˜11,000 LUX. After 2 weeks, the roots were cut from the agar, flash frozen in liquid nitrogen and stored at -80° C.
[0407](b) Rosette Leaves, Stems, and Siliques
[0408]Arabidopsis thaliana (Ws) seed was vernalized at 4° C. for 3 days before sowing in Metro-mix soil type 350. Flats were placed in a growth chamber having 16 hr light/8 hr dark, 80% relative humidity, 23° C. and 13,000 LUX for germination and growth. After 3 weeks, rosette leaves, stems, and siliques were harvested, flash frozen in liquid nitrogen and stored at -80° C. until use. After 4 weeks, siliques (<5 mm, 5-10 mm and >10 mm) were harvested, flash frozen in liquid nitrogen and stored at -80° C. until use. 5 week old whole plants (used as controls) were harvested, flash frozen in liquid nitrogen and kept at -80° C. until RNA was isolated.
[0409](c) Germination
[0410]Arabidopsis thaliana seeds (ecotype Ws) were sterilized in bleach and rinsed with sterile water. The seeds were placed in 100 mm petri plates containing soaked autoclaved filter paper. Plates were foil-wrapped and left at 4° C. for 3 nights to vernalize. After cold treatment, the foil was removed and plates were placed into a growth chamber having 16 hr light/8 hr dark cycles, 23° C., 70% relative humidity and ˜11,000 lux. Seeds were collected 1 d, 2 d, 3 d and 4 d later, flash frozen in liquid nitrogen and stored at -80° C. until RNA was isolated.
[0411](d) Abscissic Acid (ABA)
[0412]Seeds of Arabidopsis thaliana (ecotype Wassilewskija) were sown in trays and left at 4° C. for 4 days to vernalize. They were then transferred to a growth chamber having grown 16 hr light/8 hr dark, 13,000 LUX, 70% humidity, and 20° C. and watered twice a week with 1 L of 1× Hoagland's solution. Approximately 1,000 14 day old plants were spayed with 200-250 mls of 100 μM ABA in a 0.02% solution of the detergent Silwet L-77. Whole seedlings, including roots, were harvested within a 15 to 20 minute time period at 1 hr and 6 hr after treatment, flash-frozen in liquid nitrogen and stored at -80° C.
[0413]Seeds of maize hybrid 35A (Pioneer) were sown in water-moistened sand in flats (10 rows, 5-6 seed/row) and covered with clear, plastic lids before being placed in a growth chamber having 16 hr light (25° C.)/8 hr dark (20° C.), 75% relative humidity and 13,000-14,000 LUX. Covered flats were watered every three days for 7 days. Seedlings were carefully removed from the sand and placed in 1-liter beakers with 100 μM ABA for treatment. Control plants were treated with water. After 6 hr and 24 hr, aerial and root tissues were separated and flash frozen in liquid nitrogen prior to storage at -80° C.
[0414](e) Brassinosteroid Responsive
[0415]Two separate experiments were performed, one with epi-brassinolide and one with the brassinosteroid biosynthetic inhibitor brassinazole. In the epi-brassinolide experiments, seeds of wild-type Arabidopsis thaliana (ecotype Wassilewskij a) and the brassinosteroid biosynthetic mutant dwf4-1 were sown in trays and left at 4° C. for 4 days to vernalize. They were then transferred to a growth chamber having 16 hr light/8 hr dark, 11,000 LUX, 70% humidity and 22° C. temperature. Four week old plants were spayed with a 1 μM solution of epi-brassinolide and shoot parts (unopened floral primordia and shoot apical meristems) harvested three hours later. Tissue was flash-frozen in liquid nitrogen and stored at -80° C. In the brassinazole experiments, seeds of wild-type Arabidopsis thaliana (ecotype Wassilewskija) were grown as described above. Four week old plants were spayed with a 1 μM solution of brassinazole and shoot parts (unopened floral primordia and shoot apical meristems) harvested three hours later. Tissue was flash-frozen in liquid nitrogen and stored at -80° C.
[0416]In addition to the spray experiments, tissue was prepared from two different mutants; (1) a dwf4-1 knock out mutant and (2) a mutant overexpressing the dwf4-1 gene.
[0417]Seeds of wild-type Arabidopsis thaliana (ecotype Wassilewskija) and of the dwf4-1 knock out and over-expressor mutants were sown in trays and left at 4° C. for 4 days to vernalize. They were then transferred to a growth chamber having 16 hr light/8 hr dark, 11,000 LUX, 70% humidity and 22° C. temperature. Tissue from shoot parts (unopened floral primordia and shoot apical meristems) was flash-frozen in liquid nitrogen and stored at -80° C.
[0418]Another experiment was completed with seeds of Arabidopsis thaliana (ecotype Wassilewskija) were sown in trays and left at 4° C. for 4 days to vernalize. They were then transferred to a growth chamber. Plants were grown under long-day (16 hr light: 8 hr. dark) conditions, 13,000 LUX light intensity, 70% humidity, 20° C. temperature and watered twice a week with 1 L 1× Hoagland's solution (recipe recited in Feldmann et al., (1987) Mol. Gen. Genet. 208: 1-9, hereby incorporated by reference) and described as complete nutrient solution). Approximately 1,000 14 day old plants were spayed with 200-250 mls of 0.1 μM Epi-Brassinolite in 0.02% solution of the detergent Silwet L-77. At 1 hr. and 6 hrs. after treatment aerial tissues were harvested within a 15 to 20 minute time period and flash-frozen in liquid nitrogen.
[0419]Seeds of maize hybrid 35A (Pioneer) were sown in water-moistened sand in flats (10 rows, 5-6 seed/row) and covered with clear, plastic lids before being placed in a growth chamber having 16 hr light (25° C.)/8 hr dark (20° C.), 75% relative humidity and 13,000-14,000 LUX. Covered flats were watered every three days for 7 days. Seedlings were carefully removed from the sand and placed in 1-liter beakers with 0.1 μM epi-brassinolide for treatment. Control plants were treated with distilled deionized water. After 24 hr, aerial and root tissues were separated and flash frozen in liquid nitrogen prior to storage at -80° C.
[0420](f) Nitrogen: High to Low
[0421]Wild type Arabidopsis thaliana seeds (ecotype Ws) were surface sterilized with 30% Clorox, 0.1% Triton X-100 for 5 minutes. Seeds were then rinsed with 4-5 exchanges of sterile double distilled deionized water. Seeds were vernalized at 4° C. for 2-4 days in darkness. After cold treatment, seeds were plated on modified 1×MS media (without NH4NO3 or KNO3), 0.5% sucrose, 0.5 g/L MES pH5.7, 1% phytagar and supplemented with KNO3 to a final concentration of 60 mM (high nitrate modified 1×MS media). Plates were then grown for 7 days in a Percival growth chamber at 22° C. with 16 hr. light/8 hr dark.
[0422]Germinated seedlings were then transferred to a sterile flask containing 50 mL of high nitrate modified 1×MS liquid media. Seedlings were grown with mild shaking for 3 additional days at 22° C. in 16 hr. light/8 hr dark (in a Percival growth chamber) on the high nitrate modified 1×MS liquid media.
[0423]After three days of growth on high nitrate modified 1×MS liquid media, seedlings were transferred either to a new sterile flask containing 50 mL of high nitrate modified 1×MS liquid media or to low nitrate modified 1×MS liquid media (containing 20 μM KNO3). Seedlings were grown in these media conditions with mild shaking at 22° C. in 16 hr light/8 hr dark for the appropriate time points and whole seedlings harvested for total RNA isolation via the Trizol method (LifeTech.). The time points used for the microarray experiments were 10 min. and 1 hour time points for both the high and low nitrate modified 1×MS media.
[0424]Alternatively, seeds that were surface sterilized in 30% bleach containing 0.1% Triton X-100 and further rinsed in sterile water, were planted on MS agar, (0.5% sucrose) plates containing 50 mM KNO3 (potassium nitrate). The seedlings were grown under constant light (3500 LUX) at 22° C. After 12 days, seedlings were transferred to MS agar plates containing either 1 mM KNO3 or 50 mM KNO3. Seedlings transferred to agar plates containing 50 mM KNO3 were treated as controls in the experiment. Seedlings transferred to plates with 1 mM KNO3 were rinsed thoroughly with sterile MS solution containing 1 mM KNO3. There were ten plates per transfer. Root tissue was collected and frozen in 15 mL Falcon tubes at various time points which included 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, 9 hours, 12 hours, 16 hours, and 24 hours.
[0425]Maize 35A19 Pioneer hybrid seeds were sown on flats containing sand and grown in a Conviron growth chamber at 25° C., 16 hr light/8 hr dark, ˜13,000 LUX and 80% relative humidity. Plants were watered every three days with double distilled deionized water. Germinated seedlings are allowed to grow for 10 days and were watered with high nitrate modified 1×MS liquid media (see above). On day 11, young corn seedlings were removed from the sand (with their roots intact) and rinsed briefly in high nitrate modified 1×MS liquid media. The equivalent of half a flat of seedlings were then submerged (up to their roots) in a beaker containing either 500 mL of high or low nitrate modified 1×MS liquid media (see above for details).
[0426]At appropriate time points, seedlings were removed from their respective liquid media, the roots separated from the shoots and each tissue type flash frozen in liquid nitrogen and stored at -80° C. This was repeated for each time point. Total RNA was isolated using the Trizol method (see above) with root tissues only.
[0427]Corn root tissues isolated at the 4 hr and 16 hr time points were used for the microarray experiments. Both the high and low nitrate modified 1×MS media were used.
[0428](g) Nitrogen: Low to High
[0429]Arabidopsis thaliana ecotype Ws seeds were sown on flats containing 4 L of a 1:2 mixture of Grace Zonolite vermiculite and soil. Flats were watered with 3 L of water and vernalized at 4° C. for five days. Flats were placed in a Conviron growth chamber having 16 hr light/8 hr dark at 20° C., 80% humidity and 17,450 LUX. Flats were watered with approximately 1.5 L of water every four days. Mature, bolting plants (24 days after germination) were bottom treated with 2 L of either a control (100 mM mannitol pH 5.5) or an experimental (50 mM ammonium nitrate, pH 5.5) solution. Roots, leaves and siliques were harvested separately 30, 120 and 240 minutes after treatment, flash frozen in liquid nitrogen and stored at -80° C.
[0430]Hybrid maize seed (Pioneer hybrid 35A19) were aerated overnight in deionized water. Thirty seeds were plated in each flat, which contained 4 liters of Grace zonolite vermiculite. Two liters of water were bottom fed and flats were kept in a Conviron growth chamber with 16 hr light/8 hr dark at 20° C. and 80% humidity. Flats were watered with 1 L of tap water every three days. Five day old seedlings were treated as described above with 2 L of either a control (100 mM mannitol pH 6.5) solution or 1 L of an experimental (50 mM ammonium nitrate, pH 6.8) solution. Fifteen shoots per time point per treatment were harvested 10, 90 and 180 minutes after treatment, flash frozen in liquid nitrogen and stored at -80° C.
[0431]Alternatively, seeds of Arabidopsis thaliana (ecotype Wassilewskija) were left at 4° C. for 3 days to vernalize. They were then sown on vermiculite in a growth chamber having 16 hours light/8 hours dark, 12,000-14,000 LUX, 70% humidity, and 20° C. They were bottom-watered with tap water, twice weekly. Twenty-four days old plants were sprayed with either water (control) or 0.6% ammonium nitrate at 4 μL/cm2 of tray surface. Total shoots and some primary roots were cleaned of vermiculite, flash-frozen in liquid nitrogen and stored at -80° C.
[0432](h) Methyl Jasmonate
[0433]Seeds of Arabidopsis thaliana (ecotype Wassilewskija) were sown in trays and left at 4° C. for 4 days to vernalize before being transferred to a growth chamber having 16 hr light/8 hr. dark, 13,000 LUX, 70% humidity, 20° C. temperature and watered twice a week with 1 L of a 1× Hoagland's solution. Approximately 1,000 14 day old plants were spayed with 200-250 mls of 0.001% methyl jasmonate in a 0.02% solution of the detergent Silwet L-77. At 1 hr and 6 hrs after treatment, whole seedlings, including roots, were harvested within a 15 to 20 minute time period, flash-frozen in liquid nitrogen and stored at -80° C.
[0434]Seeds of maize hybrid 35A (Pioneer) were sown in water-moistened sand in flats (10 rows, 5-6 seed/row) and covered with clear, plastic lids before being placed in a growth chamber having 16 hr light (25° C.)/8 hr dark (20° C.), 75% relative humidity and 13,000-14,000 LUX. Covered flats were watered every three days for 7 days. Seedlings were carefully removed from the sand and placed in 1-liter beakers with 0.001% methyl jasmonate for treatment. Control plants were treated with water. After 24 hr, aerial and root tissues were separated and flash frozen in liquid nitrogen prior to storage at -80° C.
[0435](i) Salicylic Acid
[0436]Seeds of Arabidopsis thaliana (ecotype Wassilewskija) were sown in trays and left at 4° C. for 4 days to vernalize before being transferred to a growth chamber having 16 hr light/8 hr. dark, 13,000 LUX, 70% humidity, 20° C. temperature and watered twice a week with 1 L of a 1× Hoagland's solution. Approximately 1,000 14 day old plants were spayed with 200-250 mls of 5 mM salicylic acid (solubilized in 70% ethanol) in a 0.02% solution of the detergent Silwet L-77. At 1 hr and 6 hrs after treatment, whole seedlings, including roots, were harvested within a 15 to 20 minute time period flash-frozen in liquid nitrogen and stored at -80° C.
[0437]Alternatively, seeds of wild-type Arabidopsis thaliana (ecotype Columbia) and mutant CS3726 were sown in soil type 200 mixed with osmocote fertilizer and Marathon insecticide and left at 4° C. for 3 days to vernalize. Flats were incubated at room temperature with continuous light. Sixteen days post germination plants were sprayed with 2 mM SA, 0.02% SilwettL-77 or control solution (0.02% SilwettL-77. Aerial parts or flowers were harvested 1 hr, 4 hr, 6 hr, 24 hr and 3 weeks post-treatment flash frozen and stored at -80° C.
[0438]Seeds of maize hybrid 35A (Pioneer) were sown in water-moistened sand in flats (10 rows, 5-6 seed/row) and covered with clear, plastic lids before being placed in a growth chamber having 16 hr light (25° C.)/8 hr dark (20° C.), 75% relative humidity and 13,000-14,000 LUX. Covered flats were watered every three days for 7 days. Seedlings were carefully removed from the sand and placed in 1-liter beakers with 2 mM SA for treatment. Control plants were treated with water. After 12 hr and 24 hr, aerial and root tissues were separated and flash frozen in liquid nitrogen prior to storage at -80° C.
[0439](j) Drought Stress
[0440]Seeds of Arabidopsis thaliana (Wassilewskija) were sown in pots and left at 4° C. for three days to vernalize before being transferred to a growth chamber having 16 hr light/8 hr dark, 150,000-160,000 LUX, 20° C. and 70% humidity. After 14 days, aerial tissues were cut and left to dry on 3MM Whattman paper in a Petri-plate for 1 hour and 6 hours. Aerial tissues exposed for 1 hour and 6 hours to 3 MM Whattman paper wetted with 1× Hoagland's solution served as controls. Tissues were harvested, flash-frozen in liquid nitrogen and stored at -80° C.
[0441]Alternatively, Arabidopsis thaliana (Ws) seed was vernalized at 4° C. for 3 days before sowing in Metromix soil type 350. Flats were placed in a growth chamber with 23° C., 16 hr light/8 hr. dark, 80% relative humidity, ˜13,000 LUX for germination and growth. Plants were watered with 1-1.5 L of water every four days. Watering was stopped 16 days after germination for the treated samples, but continued for the control samples. Rosette leaves and stems, flowers and siliques were harvested 2 d, 3 d, 4 d, 5 d, 6 d and 7 d after watering was stopped. Tissue was flash frozen in liquid nitrogen and kept at -80° C. until RNA was isolated. Flowers and siliques were also harvested on day 8 from plants that had undergone a 7 d drought treatment followed by 1 day of watering. Control plants (whole plants) were harvested after 5 weeks, flash frozen in liquid nitrogen and stored as above.
[0442]Seeds of maize hybrid 35A (Pioneer) were sown in water-moistened sand in flats (10 rows, 5-6 seed/row) and covered with clear, plastic lids before being placed in a growth chamber having 16 hr light (25° C.)/8 hr dark (20° C.), 75% relative humidity and 13,000-14,000 LUX. Covered flats were watered every three days for 7 days. Seedlings were carefully removed from the sand and placed in empty 1-liter beakers at room temperature for treatment. Control plants were placed in water. After 1 hr, 6 hr, 12 hr and 24 hr aerial and root tissues were separated and flash frozen in liquid nitrogen prior to storage at -80° C.
[0443](k) Osmotic Stress
[0444]Seeds of Arabidopsis thaliana (Wassilewskija) were sown in trays and left at 4° C. for three days to vernalize before being transferred to a growth chamber having 16 hr light/8 hr dark, 12,000-14,000 LUX, 20° C., and 70% humidity. After 14 days, the aerial tissues were cut and placed on 3 MM Whattman paper in a Petri-plate wetted with 20% PEG (polyethylene glycol-Mr 8,000) in 1× Hoagland's solution. Aerial tissues on 3 MM Whattman paper containing 1×Hoagland's solution alone served as the control. Aerial tissues were harvested at 1 hour and 6 hours after treatment, flash-frozen in liquid nitrogen and stored at -80° C.
[0445]Seeds of maize hybrid 35A (Pioneer) were sown in water-moistened sand in flats (10 rows, 5-6 seed/row) and covered with clear, plastic lids before being placed in a growth chamber having 16 hr light (25° C.)/8 hr dark (20° C.), 75% relative humidity and 13,000-14,000 LUX. Covered flats were watered every three days for 7 days. Seedlings were carefully removed from the sand and placed in 1-liter beakers with 10% PEG (polyethylene glycol-Mr 8,000) for treatment. Control plants were treated with water. After 1 hr and 6 hr aerial and root tissues were separated and flash frozen in liquid nitrogen prior to storage at -80° C.
[0446]Seeds of maize hybrid 35A (Pioneer) were sown in water-moistened sand in flats (10 rows, 5-6 seed/row) and covered with clear, plastic lids before being placed in a growth chamber having 16 hr light (25° C.)/8 hr dark (20° C.), 75% relative humidity and 13,000-14,000 LUX. Covered flats were watered every three days for 7 days. Seedlings were carefully removed from the sand and placed in 1-liter beakers with 150 mM NaCl for treatment. Control plants were treated with water. After 1 hr, 6 hr, and 24 hr aerial and root tissues were separated and flash frozen in liquid nitrogen prior to storage at -80° C.
[0447](1) Heat Shock Treatment
[0448]Seeds of Arabidopsis Thaliana (Wassilewskija) were sown in trays and left at 4° C. for three days to vernalize before being transferred to a growth chamber with 16 hr light/8 hr dark, 12,000-14,000 Lux, 70% humidity and 20° C., fourteen day old plants were transferred to a 42° C. growth chamber and aerial tissues were harvested 1 hr and 6 hr after transfer. Control plants were left at 20° C. and aerial tissues were harvested. Tissues were flash-frozen in liquid nitrogen and stored at -80° C.
[0449]Seeds of maize hybrid 35A (Pioneer) were sown in water-moistened sand in flats (10 rows, 5-6 seed/row) and covered with clear, plastic lids before being placed in a growth chamber having 16 hr light (25° C.)/8 hr dark (20° C.), 75% relative humidity and 13,000-14,000 LUX. Covered flats were watered every three days for 7 days. Seedlings were carefully removed from the sand and placed in 1-liter beakers containing 42° C., water for treatment. Control plants were treated with water at 25° C. After 1 hr and 6 hr aerial and root tissues were separated and flash frozen in liquid nitrogen prior to storage at -80° C.
[0450](m) Cold Shock Treatment
[0451]Seeds of Arabidopsis thaliana (Wassilewskija) were sown in trays and left at 4° C. for three days to vernalize before being transferred to a growth chamber having 16 hr light/8 hr dark, 12,000-14,000 LUX, 20° C. and 70% humidity. Fourteen day old plants were transferred to a 4° C. dark growth chamber and aerial tissues were harvested 1 hour and 6 hours later. Control plants were maintained at 20° C. and covered with foil to avoid exposure to light. Tissues were flash-frozen in liquid nitrogen and stored at -80° C.
[0452]Seeds of maize hybrid 35A (Pioneer) were sown in water-moistened sand in flats (10 rows, 5-6 seed/row) and covered with clear, plastic lids before being placed in a growth chamber having 16 hr light (25° C.)/8 hr dark (20° C.), 75% relative humidity and 13,000-14,000 LUX. Covered flats were watered every three days for 7 days. Seedlings were carefully removed from the sand and placed in 1-liter beakers containing 4° C., water for treatment. Control plants were treated with water at 25° C. After 1 hr and 6 hr aerial and root tissues were separated and flash frozen in liquid nitrogen prior to storage at -80° C.
[0453](n) Arabidopsis Seeds
[0454]Fruits (Pod+Seed) 0-5 mm
[0455]Seeds of Arabidopsis thaliana (ecotype Wassilewskija) were sown in pots and left at 4° C. for two to three days to vernalize. They were then transferred to a growth chamber. Plants were grown under long-day (16 hr light: 8 hr dark) conditions, 7000-8000 LUX light intensity, 70% humidity, and 22° C. temperature. 3-4 siliques (fruits) bearing developing seeds were selected from at least 3 plants and were hand-dissected to determine what developmental stage(s) were represented by the enclosed embryos. Description of the stages of Arabidopsis embryogenesis used in this determination were summarized by Bowman (1994). Silique lengths were then determined and used as an approximate determinant for embryonic stage. Siliques 0-5 mm in length containing post fertilization through pre-heart stage [0-72 hours after fertilization (HAF)] embryos were harvested and flash frozen in liquid nitrogen.
[0456]Fruits (Pod+Seed) 5-10 mm
[0457]Seeds of Arabidopsis thaliana (ecotype Wassilewskija) were sown in pots and left at 4° C. for two to three days to vernalize. They were then transferred to a growth chamber. Plants were grown under long-day (16 hr light: 8 hr dark) conditions, 7000-8000 LUX light intensity, 70% humidity, and 22° C. temperature. 3-4 siliques (fruits) bearing developing seeds were selected from at least 3 plants and were hand-dissected to determine what developmental stage(s) were represented by the enclosed embryos. Description of the stages of Arabidopsis embryogenesis used in this determination were summarized by Bowman (1994). Silique lengths were then determined and used as an approximate determinant for embryonic stage. Siliques 5-10 mm in length containing heart- through early upturned-U-stage [72-120 hours after fertilization (HAF)] embryos were harvested and flash frozen in liquid nitrogen.
[0458]Fruits (Pod+Seed)>10 mm
[0459]Seeds of Arabidopsis thaliana (ecotype Wassilewskija) were sown in pots and left at 4° C. for two to three days to vernalize. They were then transferred to a growth chamber. Plants were grown under long-day (16 hr light: 8 hr dark) conditions, 7000-8000 LUX light intensity, 70% humidity, and 22° C. temperature. 3-4 siliques (fruits) bearing developing seeds were selected from at least 3 plants and were hand-dissected to determine what developmental stage(s) were represented by the enclosed embryos. Description of the stages of Arabidopsis embryogenesis used in this determination were summarized by Bowman (1994). Silique lengths were then determined and used as an approximate determinant for embryonic stage. Siliques>10 mm in length containing green, late upturned-U-stage [>120 hours after fertilization (HAF)-9 days after flowering (DAF)] embryos were harvested and flash frozen in liquid nitrogen.
[0460]Green Pods 5-10 mm (Control Tissue for Samples 72-74)
[0461]Seeds of Arabidopsis thaliana (ecotype Wassilewskija) were sown in pots and left at 4° C. for two to three days to vernalize. They were then transferred to a growth chamber. Plants were grown under long-day (16 hr light: 8 hr dark) conditions, 7000-8000 LUX light intensity, 70% humidity, and 22° C. temperature. 3-4 siliques (fruits) bearing developing seeds were selected from at least 3 plants and were hand-dissected to determine what developmental stage(s) were represented by the enclosed embryos. Description of the stages of Arabidopsis embryogenesis used in this determination were summarized by Bowman (1994). Silique lengths were then determined and used as an approximate determinant for embryonic stage. Green siliques 5-10 mm in length containing developing seeds 72-120 hours after fertilization (HAF)] were opened and the seeds removed. The remaining tissues (green pods minus seed) were harvested and flash frozen in liquid nitrogen.
[0462]Green Seeds from Fruits>10 mm
[0463]Seeds of Arabidopsis thaliana (ecotype Wassilewskija) were sown in pots and left at 4° C. for two to three days to vernalize. They were then transferred to a growth chamber. Plants were grown under long-day (16 hr light: 8 hr dark) conditions, 7000-8000 LUX light intensity, 70% humidity, and 22° C. temperature. 3-4 siliques (fruits) bearing developing seeds were selected from at least 3 plants and were hand-dissected to determine what developmental stage(s) were represented by the enclosed embryos. Description of the stages of Arabidopsis embryogenesis used in this determination were summarized by Bowman (1994). Silique lengths were then determined and used as an approximate determinant for embryonic stage. Green siliques>10 mm in length containing developing seeds up to 9 days after flowering (DAF)] were opened and the seeds removed and harvested and flash frozen in liquid nitrogen.
[0464]Brown Seeds from Fruits>10 mm
[0465]Seeds of Arabidopsis thaliana (ecotype Wassilewskija) were sown in pots and left at 4° C. for two to three days to vernalize. They were then transferred to a growth chamber. Plants were grown under long-day (16 hr light: 8 hr dark) conditions, 7000-8000 LUX light intensity, 70% humidity, and 22° C. temperature. 3-4 siliques (fruits) bearing developing seeds were selected from at least 3 plants and were hand-dissected to determine what developmental stage(s) were represented by the enclosed embryos. Description of the stages of Arabidopsis embryogenesis used in this determination were summarized by Bowman (1994). Silique lengths were then determined and used as an approximate determinant for embryonic stage. Yellowing siliques>10 mm in length containing brown, desiccating seeds >11 days after flowering (DAF)] were opened and the seeds removed and harvested and flash frozen in liquid nitrogen.
[0466]Green/Brown Seeds from Fruits>10 mm
[0467]Seeds of Arabidopsis thaliana (ecotype Wassilewskija) were sown in pots and left at 4° C. for two to three days to vernalize. They were then transferred to a growth chamber. Plants were grown under long-day (16 hr light: 8 hr dark) conditions, 7000-8000 LUX light intensity, 70% humidity, and 22° C. temperature. 3-4 siliques (fruits) bearing developing seeds were selected from at least 3 plants and were hand-dissected to determine what developmental stage(s) were represented by the enclosed embryos. Description of the stages of Arabidopsis embryogenesis used in this determination were summarized by Bowman (1994). Silique lengths were then determined and used as an approximate determinant for embryonic stage. Green siliques>10 mm in length containing both green and brown seeds >9 days after flowering (DAF)] were opened and the seeds removed and harvested and flash frozen in liquid nitrogen.
[0468]Mature Seeds (24 Hours after Imbibition)
[0469]Mature dry seeds of Arabidopsis thaliana (ecotype Wassilewskija) were sown onto moistened filter paper and left at 4° C. for two to three days to vernalize. Imbibed seeds were then transferred to a growth chamber [16 hr light: 8 hr dark conditions, 7000-8000 LUX light intensity, 70% humidity, and 22° C. temperature], the emerging seedlings harvested after 48 hours and flash frozen in liquid nitrogen.
[0470]Mature Seeds (Dry)
[0471]Seeds of Arabidopsis thaliana (ecotype Wassilewskija) were sown in pots and left at 4° C. for two to three days to vernalize. They were then transferred to a growth chamber. Plants were grown under long-day (16 hr light: 8 hr dark) conditions, 7000-8000 LUX light intensity, 70% humidity, and 22° C. temperature and taken to maturity. Mature dry seeds are collected, dried for one week at 28° C., and vernalized for one week at 4° C. before used as a source of RNA.
(o) Herbicide Treatment
[0472]Arabidopsis thaliana (Ws) seeds were sterilized for 5 min. with 30% bleach, 50 μl Triton in a total volume of 50 ml. Seeds were vernalized at 4° C. for 3 days before being plated onto GM agar plates at a density of about 144 seeds per plate. Plates were incubated in a Percival growth chamber having 16 hr light/8 hr dark, 80% relative humidity, 22° C. and 11,000 LUX for 14 days.
[0473]Plates were sprayed (˜0.5 mls/plate) with water, Finale (1.128 g/L), Glean (1.88 g/L), RoundUp (0.01 g/L) or Trimec (0.08 g/L). Tissue was collected and flash frozen in liquid nitrogen at the following time points: 0, 1, 2, 4, 8, 12 and 24 hours. Frozen tissue was stored at -80° C. prior to RNA isolation.
[0474](p) Root Tips
[0475]Seeds of Arabidopsis thaliana (ecotype Ws) were placed on MS plates and vernalized at 4° C. for 3 days before being placed in a 25° C. growth chamber having 16 hr light/8 hr dark, 70% relative humidity and about 3 W/m2. After 6 days, young seedlings were transferred to flasks containing B5 liquid medium, 1% sucrose and 0.05 mg/l indole-3-butyric acid. Flasks were incubated at room temperature with 100 rpm agitation. Media was replaced weekly. After three weeks, roots were harvested and incubated for 1 hr with 2% pectinase, 0.2% cellulase, pH 7 before straining through a #80 (Sigma) sieve. The root body material remaining on the sieve (used as the control) was flash frozen and stored at -80° C. until use. The material that passed through the #80 sieve was strained through a #200 (Sigma) sieve and the material remaining on the sieve (root tips) was flash frozen and stored at -80° C. until use. Approximately 10 mg of root tips were collected from one flask of root culture.
[0476]Seeds of maize hybrid 35A (Pioneer) were sown in water-moistened sand in flats (10 rows, 5-6 seed/row) and covered with clear, plastic lids before being placed in a growth chamber having 16 hr light (25° C.)/8 hr dark (20° C.), 75% relative humidity and 13,000-14,000 LUX. Covered flats were watered every three days for 8 days. Seedlings were carefully removed from the sand and the root tips (˜2 mm long) were removed and flash frozen in liquid nitrogen prior to storage at -80° C. The tissues above the root tips (˜1 cm long) were cut, treated as above and used as control tissue.
[0477](q) Imbibed Seed
[0478]Seeds of maize hybrid 35A (Pioneer) were sown in water-moistened sand in covered flats (10 rows, 5-6 seed/row) and covered with clear, plastic lids before being placed in a growth chamber having 16 hr light (25° C.)/8 hr dark (20° C.), 75% relative humidity and 13,000-14,000 LUX. One day after sowing, whole seeds were flash frozen in liquid nitrogen prior to storage at -80° C. Two days after sowing, embryos and endosperm were isolated and flash frozen in liquid nitrogen prior to storage at -80° C. On days 3-6, aerial tissues, roots and endosperm were isolated and flash frozen in liquid nitrogen prior to storage at -80° C.
[0479](r) Flowers (Green, White or Buds)
[0480]Approximately 10 μl of Arabidopsis thaliana seeds (ecotype Ws) were sown on 350 soil (containing 0.03% marathon) and vernalized at 4 C for 3 days. Plants were then grown at room temperature under fluorescent lighting until flowering. Flowers were harvested after 28 days in three different categories. Buds that had not opened at all and were completely green were categorized as "flower buds" (also referred to as green buds by the investigator). Buds that had started to open, with white petals emerging slightly were categorized as "green flowers" (also referred to as white buds by the investigator). Flowers that had opened mostly (with no silique elongation) with white petals completely visible were categorized as "white flowers" (also referred to as open flowers by the investigator). Buds and flowers were harvested with forceps, flash frozen in liquid nitrogen and stored at -80 C until RNA was isolated.
[0481]s) Ovules
[0482]Seeds of Arabidopsis thaliana heterozygous for pistillata (pi) [ecotype Landsberg erecta (Ler)] were sown in pots and left at 4° C. for two to three days to vernalize. They were then transferred to a growth chamber. Plants were grown under long-day (16 hr light: 8 hr dark) conditions, 7000-8000 LUX light intensity, 76% humidity, and 24° C. temperature. Inflorescences were harvested from seedlings about 40 days old. The inflorescences were cut into small pieces and incubated in the following enzyme solution (pH 5) at room temperature for 0.5-1 hr.: 0.2% pectolyase Y-23, 0.04% pectinase, 5 mM MES, 3% Sucrose and MS salts (1900 mg/l KNO3, 1650 mg/l NH4NO3, 370 mg/l MgSO4.7H2O, 170 mg/l KH2PO4, 440 mg/l CaCl2. 2H2O, 6.2 mg/l H2BO3, 15.6 mg/l MnSO4.4H2O, 8.6 mg/l ZnSO4. 7H2O, 0.25 mg/l NaMoO4. 2H2O, 0.025 mg/l CuCO4. 5H2O, 0.025 mg/l CoCl2.6H2O, 0.83 mg/l KI, 27.8 mg/l FeSO4. 7H2O, 37.3 mg/l Disodium EDTA, pH 5.8). At the end of the incubation the mixture of inflorescence material and enzyme solution was passed through a size 60 sieve and then through a sieve with a pore size of 125 μm. Ovules greater than 125 μm in diameter were collected, rinsed twice in B5 liquid medium (2500 mg/l KNO3, 250 mg/l MgSO4.7H2O, 150 mg/l NaH2PO4.H2O, 150 mg/l CaCl2.2H2O, 134 mg/l (NH4)2 CaCl2.SO4, 3 mg/l H2BO3, 10 mg/l MnSO4.4H2O, 2 ZnSO4. 7H2O, 0.25 mg/l NaMoO4.2H2O, 0.025 mg/l CuCO4.5H2O, 0.025 mg/l CoCl2. 6 H2O, 0.75 mg/l KI, 40 mg/l EDTA sodium ferric salt, 20 g/l sucrose, 10 mg/l Thiamine hydrochloride, 1 mg/l Pyridoxine hydrochloride, 1 mg/l Nicotinic acid, 100 mg/l myo-inositol, pH 5.5)), rinsed once in deionized water and flash frozen in liquid nitrogen. The supernatant from the 125 μm sieving was passed through subsequent sieves of 50 μm and 32 μm. The tissue retained in the 32 μm sieve was collected and mRNA prepared for use as a control.
[0483]t) Wounding
[0484]Seeds of Arabidopsis thaliana (Wassilewskija) were sown in trays and left at 4° C. for three days to vernalize before being transferred to a growth chamber having 16 hr light/8 hr dark, 12,000-14,000 LUX, 70% humidity and 20° C. After 14 days, the leaves were wounded with forceps. Aerial tissues were harvested 1 hour and 6 hours after wounding. Aerial tissues from unwounded plants served as controls. Tissues were flash-frozen in liquid nitrogen and stored at -80° C.
[0485]Seeds of maize hybrid 35A (Pioneer) were sown in water-moistened sand in flats (10 rows, 5-6 seed/row) and covered with clear, plastic lids before being placed in a growth chamber having 16 hr light (25° C.)/8 hr dark (20° C.), 75% relative humidity and 13,000-14,000 LUX. Covered flats were watered every three days for 7 days. Seedlings were wounded (one leaf nicked by scissors) and placed in 1-liter beakers of water for treatment. Control plants were treated not wounded. After 1 hr and 6 hr aerial and root tissues were separated and flash frozen in liquid nitrogen prior to storage at -80° C.
[0486]u) Nitric Oxide Treatment
[0487]Seeds of Arabidopsis thaliana (Wassilewskija) were sown in trays and left at 4° C. for three days to vernalize before being transferred to a growth chamber having 16 hr light/8 hr dark, 12,000-14,000 LUX, 20° C. and 70% humidity. Fourteen day old plants were sprayed with 5 mM sodium nitroprusside in a 0.02% Silwett L-77 solution. Control plants were sprayed with a 0.02% Silwett L-77 solution. Aerial tissues were harvested 1 hour and 6 hours after spraying, flash-frozen in liquid nitrogen and stored at -80° C.
[0488]Seeds of maize hybrid 35A (Pioneer) were sown in water-moistened sand in flats (10 rows, 5-6 seed/row) and covered with clear, plastic lids before being placed in a growth chamber having 16 hr light (25° C.)/8 hr dark (20° C.), 75% relative humidity and 13,000-14,000 LUX. Covered flats were watered every three days for 7 days. Seedlings were carefully removed from the sand and placed in 1-liter beakers with 5 mM nitroprusside for treatment. Control plants were treated with water. After 1 hr, 6 hr and 12 hr, aerial and root tissues were separated and flash frozen in liquid nitrogen prior to storage at -80° C.
[0489]v) Root Hairless Mutants
[0490]Plants mutant at the rhl gene locus lack root hairs. This mutation is maintained as a heterozygote.
[0491]Seeds of Arabidopsis thaliana (Landsberg erecta) mutated at the rhl gene locus were sterilized using 30% bleach with 1 ul/ml 20% Triton-X 100 and then vernalized at 4° C. for 3 days before being plated onto GM agar plates. Plates were placed in growth chamber with 16 hr light/8 hr. dark, 23° C., 14,500-15,900 LUX, and 70% relative humidity for germination and growth.
[0492]After 7 days, seedlings were inspected for root hairs using a dissecting microscope. Mutants were harvested and the cotyledons removed so that only root tissue remained. Tissue was then flash frozen in liquid nitrogen and stored at -80 C.
[0493]Arabidopsis thaliana (Landsberg erecta) seedlings grown and prepared as above were used as controls.
[0494]Alternatively, seeds of Arabidopsis thaliana (Landsberg erecta), heterozygous for the rhl1 (root hairless) mutation, were surface-sterilized in 30% bleach containing 0.1% Triton X-100 and further rinsed in sterile water. They were then vernalized at 4° C. for 4 days before being plated onto MS agar plates. The plates were maintained in a growth chamber at 24° C., with 16 hr light/8 hr dark for germination and growth. After 10 days, seedling roots that expressed the phenotype (i.e. lacking root hairs) were cut below the hypocotyl junction, frozen in liquid nitrogen and stored at -80° C. Those seedlings with the normal root phenotype (heterozygous or wt) were collected as described for the mutant and used as controls.
[0495]w) Ap2
[0496]Seeds of Arabidopsis thaliana (ecotype Landesberg erecta) and floral mutant apetala2 (Jofuku et al., 1994, Plant Cell 6:1211-1225) were sown in pots and left at 4° C. for two to three days to vernalize. They were then transferred to a growth chamber. Plants were grown under long-day (16 hr light, 8 hr dark) conditions 7000-8000 LUX light intensity, 70% humidity and 22° C. temperature. Inflorescences containing immature floral buds (stages 1-7; Bowman, 1994) as well as the inflorescence meristem were harvested and flashfrozen. Polysomal polyA+ RNA was isolated from tissue according to Cox and Goldberg, 1988).
[0497]x) Salt
[0498]Arabidopsis thaliana ecotype Ws seeds were vernalized at 4° C. for 3 days before sowing in flats containing vermiculite soil. Flats were placed at 20° C. in a Conviron growth chamber having 16 hr light/8 hr dark. Whole plants (used as controls) received water. Other plants were treated with 100 mM NaCl. After 6 hr and 72 hr, aerial and root tissues were harvested and flash frozen in liquid nitrogen prior to storage at -80° C.
[0499]y) Petals
[0500]Arabidopsis thaliana ecotype Ws seeds were vernalized at 4° C. for 3 days before sowing in flats containing vermiculite soil. Flats were watered placed at 20° C. in a Conviron growth chamber having 16 hr light/8 hr dark. Whole plants (used as the control) and petals from inflorescences 23-25 days after germination were harvested, flash frozen in liquid nitrogen and stored at -80° C.
[0501]z) Pollen
[0502]Arabidopsis thaliana ecotype Ws seeds were vernalized at 4° C. for 3 days before sowing in flats containing vermiculite soil. Flats were watered and placed at 20° C. in a Conviron growth chamber having 16 hr light/8 hr dark. Whole plants (used as controls) and pollen from plants 38 dap was harvested, flash frozen in liquid nitrogen and stored at -80° C.
[0503]aa) Interploidy Crosses
[0504]Interploidy crosses involving a 6× parent are lethal. Crosses involving a 4× parent are complete and analyzed. The imbalance in the maternal/paternal ratio produced from the cross can lead to big seeds. Arabidopsis thaliana ecotype Ws seeds were vernalized at 4° C. for 3 days before sowing. Small siliques were harvested at 5 days after pollination, flash frozen in liquid nitrogen and stored at -80° C.
[0505]bb) Line Comparisons
[0506]Alkaloid 35S over-expressing lines were used to monitor the expression levels of terpenoid/alkaloid biosynthetic and P450 genes to identify the transcriptional regulatory points I the biosynthesis pathway and the related P450 genes. Arabidopsis thaliana ecotype Ws seeds were vernalized at 4° C. for 3 days before sowing in vermiculite soil (Zonolite) supplemented by Hoagland solution. Flats were placed in Conviron growth chambers under long day conditions (16 hr light, 23° C./8 hr dark, 20° C.). Basta spray and selection of the overexpressing lines was conducted about 2 weeks after germination. Approximately 2-3 weeks after bolting (approximately 5-6 weeks after germination), stem and siliques from the over-expressing lines and from wild-type plants were harvested, flash frozen in liquid nitrogen and stored at -80° C.
[0507]cc) DMT-II
[0508]Demeter (dmt) is a mutant of a methyl transferase gene and is similar to fie. Arabidopsis thaliana ecotype Ws seeds were vernalized at 4° C. for 3 days before sowing. Cauline leaves and closed flowers were isolated from 35S::DMT and dmt-/-plant lines, flash frozen in liquid nitrogen and stored at -80° C.
[0509]dd) CS6630 Roots and Shoots
[0510]Arabidopsis thaliana ecotype Ws seeds were vernalized at 4° C. for 3 days before sowing on MS media (1%) sucrose on bacto-agar. Roots and shoots were separated 14 days after germination, flash frozen in liquid nitrogen and stored at -80° C.
[0511]ee) CS237
[0512]CS237 is an ethylene triple response mutant that is insensitive to ethylene and which has an etr1-1 phenotype. Arabidopsis thaliana CS237 seeds were vernalized at 4° C. for 3 days before sowing. Aerial tissue was collected from mutants and wild-type Columbia ecotype plants, flash frozen in liquid nitrogen and stored at -80° C.
[0513]ff) Guard Cells
[0514]Arabidopsis thaliana ecotype Ws seeds were vernalized at 4° C. for 3 days before sowing. Leaves were harvested, homogenized and centrifuged to isolate the guard cell containing fraction. Homogenate from leaves served as the control. Samples were flash frozen in liquid nitrogen and stored at -80° C. Identical experiments using leaf tissue from canola were performed.
[0515]gg) 3642-1
[0516]3642-1 is a T-DNA mutant that affects leaf development. This mutant segregates 3:1, wild-type:mutant. Arabidopsis thaliana 3642-1 mutant seeds were vernalized at 4° C. for 3 days before sowing in flats of MetroMix 200. Flats were placed in the greenhouse, watered and grown to the 8 leaf, pre-flower stage. Stems and rosette leaves were harvested from the mutants and the wild-type segregants, flash frozen and stored at -80° C.
[0517]hh) Caf
[0518]Carple factory (Caf) is a double-stranded RNAse protein that is hypothesized to process small RNAs in Arabidopsis. The protein is closely related to a Drosophila protein named DICER that functions in the RNA degradation steps of RNA interference. Arabidopsis thaliana Caf mutant seeds were vernalized at 4° C. for 3 days before sowing in flats of MetroMix 200. Flats were placed in the greenhouse, watered and grown to the 8 leaf, pre-flower stage. Stems and rosette leaves were harvested from the mutants and the wild-type segregants, flash frozen and stored at -80° C.
[0519]ii) Drought Reproduction
[0520]Arabidopsis thaliana (ecotype Wassilewskija) seeds are kept at 4° C. in dark for three days and then sown in soil mix (Metromix 200) with a regular watering schedule (1.5-2 L per flat per week). Drought treatment by withholding water starts when plants are 30-days-old. The control samples are watered as before. Rosettes, flowers (with siliques less than 5 mm) and siliques (>5 mm) are harvested separately on day 5, 7 and 10 post-drought-treatment (PDT). By day 10 PDT, the majority of drought plants are wilted and unable to recover after re-watering and the experiment is terminated. The samples are harvested between 2-5 PM. Plants are grown in a walk-in growth chamber under these conditions: 16 h light/8 hr dark, 70% relative humidity, 20° C. light/18° C. dark for the first 10 days, and under 22° C. light/20° C. dark for the following days.
[0521](jj) Drought Stress
[0522]Seeds of Arabidopsis thaliana (ecotype Wassilewskija) are sown in pots and left at 4° C. for three days to vernalize before being transferred to a growth chamber having 16 hr light/8 hr dark, 150,000-160,000 LUX, 20° C. and 70% humidity. After 14 days, aerial tissues are cut and left to dry on 3MM Whatman paper in a petri-plate for 1 hour and 6 hours. Aerial tissues exposed for 1 hour and 6 hours to 3 MM Whatman paper wetted with 1× Hoagland's solution serve as controls. Tissues are harvested, flash-frozen in liquid nitrogen and stored at -80° C.
[0523]Alternatively, Arabidopsis thaliana (ecotype Wassilewskija) seed is vernalized at 4° C. for 3 days before sowing in Metromix soil type 350. Flats are placed in a growth chamber with 23° C., 16 hr light/8 hr. dark, 80% relative humidity, ˜13,000 LUX for germination and growth. Plants are watered with 1-1.5 L of water every four days. Watering is stopped 16 days after germination for the treated samples, but continues for the control samples. Rosette leaves and stems, flowers and siliques are harvested 2 d, 3 d, 4 d, 5 d, 6 d and 7 d after watering is stopped. Tissue is flash frozen in liquid nitrogen and kept at -80° C. until RNA is isolated. Flowers and siliques are also harvested on day 8 from plants that had undergone a 7 d drought treatment followed by 1 day of watering. Control plants (whole plants) are harvested after 5 weeks, flash frozen in liquid nitrogen and stored as above.
[0524]Seeds of maize hybrid 35A (Pioneer) are sown in water-moistened sand in flats (10 rows, 5-6 seed/row) and covered with clear, plastic lids before being placed in a growth chamber having 16 hr light (25° C.)/8 hr dark (20° C.), 75% relative humidity and 13,000-14,000 LUX. Covered flats are watered every three days for 7 days. Seedlings are carefully removed from the sand and placed in empty 1-liter beakers at room temperature for treatment. Control plants are placed in water. After 1 hr, 6 hr, 12 hr and 24 hr aerial and root tissues are separated and flash frozen in liquid nitrogen prior to storage at -80° C.
[0525](kk) Far-Red-Enriched
[0526]Seeds from wildtype Arabidopsis thaliana (ecotype Columbia) are vernalized in sterile water for 4 days at 4° C. prior to planting. Seeds are then sterilized and evenly planted on 0.5% sucrose MS media plates. Plates are sealed with Scotch micropore tape to allow for gas exchange and prevent contamination. Plates are grown in a growth room (16 h light/8 h dark, 22° C.; 6 bulbs total Gro-Lux); light measurements are as follows: Red=646.4 μW/cm2, Blue=387 μW/cm2, Far Red=158.7 μW/cm2. At 7 days after germination, the plates containing the seedlings are transferred to Far Red light only (Far Red=525 μW/cm2) for various durations of exposure time (1, 4, 8, and 24 hrs). After timed exposure, tissue is flash frozen with liquid nitrogen and stored at -80° C. Control seedlings are not transferred, but are collected at same time as the corresponding far-red exposed experimental samples.
[0527](ll) Far-Red-Induction--Adult
[0528]Wildtype Arabidopsis thaliana (ecotype Columbia) seeds are planted on soil and vernalized for 4 days at 4° C. Soil sown plants are grown in a growth room (16 h light/8 h dark, 22° C.; 4 bulbs total alternating Gro-Lux and cool whites); light measurements are as follows: Red=330.9 μW/cm2, Blue=267 μW/cm2, Far Red=56.1 μW/cm2. At 4 weeks after germination, the soil pots are transferred to shade environment (16 h light/8 h dark; Red=376 μW/cm2, Blue=266 μW/cm2, Far Red=552 μW/cm2) for various durations of exposure time (1, 4, 8, 16, 24, 48, and 72 hrs). After timed exposure, above ground tissue is flash frozen with liquid nitrogen and stored at -80° C. Control seedlings are not transferred, but are collected at same time as the corresponding shade-exposed experimental samples.
[0529](mm) Shoots
[0530]Sterilized wild-type Arabidopsis thaliana seeds (ecotype Wassilewskija) are sown on MS plates (0.5% sucrose, 1.5% agar) after 3 day-cold treatment. The plates are placed vertically in a Percival growth chamber (16:8 light cycles, 22° C.) so that roots grow vertically on the agar surface. The shoots or aerials, harvested after 7 d- and 14 d-growth in the chamber, are used as the experimental samples. The control sample is derived from tissues harvested from 3 week-old plants that are grown in soil in a Conviron chamber (16:8 light cycles, 22° C.), including rosettes, roots, stems, flowers, and siliques.
[0531](nn) Siliques
Wild type Arabidopsis thaliana (ecotype Wassilewskija) seeds are sown in moistened soil mix, metromix 200 with osmocote, and stratified at 4° C. for 3 days in dark. Flats are placed in a Conviron growth chamber maintained at 16 h light (22° C.), 8 h dark (20° C.) and 70% humidity. After 3 weeks, siliques (<5 mm long) are collected in liquid nitrogen. The control samples are 3-week old whole plants (including all tissue types) grown in the same Conviron growth chamber.
(oo) Cytokinin (BA)
[0532]Seeds of Arabidopsis thaliana (ecotype Wassilewskija) are sown in trays and left at 4° C. for 4 days to vernalize. They are then transferred to a growth chamber having 16 hr light/8 hr dark, 13,000 LUX, 70% humidity, 20° C. temperature and watered twice a week with 1 L of 1× Hoagland's solution. Approximately 1,000 14 day old plants are spayed with 200-250 mls of 100 μM BA in a 0.02% solution of the detergent Silwet L-77. Aerial tissues (everything above the soil line) are harvested within a 15 to 20 minute time period 1 hr and 6 hrs after treatment, flash-frozen in liquid nitrogen and stored at -80° C.
[0533]Seeds of maize hybrid 35A (Pioneer) are sown in water-moistened sand in flats (10 rows, 5-6 seed/row) and covered with clear, plastic lids before being placed in a growth chamber having 16 hr light (25° C.)/8 hr dark (20° C.), 75% relative humidity and 13,000-14,000 LUX. Covered flats were watered every three days for 7 days. Seedlings are carefully removed from the sand and placed in 1-liter beakers with 100 μM BA for treatment. Control plants are treated with water. After 6 hr, aerial and root tissues are separated and flash frozen in liquid nitrogen prior to storage at -80° C.
2. Microarray Hybridization Procedures
[0534]Microarray technology provides the ability to monitor mRNA transcript levels of thousands of genes in a single experiment. These experiments simultaneously hybridize two differentially labeled fluorescent cDNA pools to glass slides that have been previously spotted with cDNA clones of the same species. Each arrayed cDNA spot will have a corresponding ratio of fluorescence that represents the level of disparity between the respective mRNA species in the two sample pools. Thousands of polynucleotides can be spotted on one slide, and each experiment generates a global expression pattern.
Coating Slides
[0535]The microarray consists of a chemically coated microscope slide, referred herein as a "chip" with numerous polynucleotide samples arrayed at a high density. The poly-L-lysine coating allows for this spotting at high density by providing a hydrophobic surface, reducing the spreading of spots of DNA solution arrayed on the slides. Glass microscope slides (Gold Seal #3010 manufactured by Gold Seal Products, Portsmouth, N.H., USA) were coated with a 0.1% W/V solution of Poly-L-lysine (Sigma, St. Louis, Mo.) using the following protocol: [0536]1. Slides were placed in slide racks (Shandon Lipshaw #121). The racks were then put in chambers (Shandon Lipshaw #121). [0537]2. Cleaning solution was prepared: [0538]70 g NaOH was dissolved in 280 mL ddH2O. [0539]420 mL 95% ethanol was added. The total volume was 700 mL (=2×350 mL); it was stirred until completely mixed. If the solution remained cloudy, ddH2O was added until clear. [0540]3. The solution was poured into chambers with slides; the chambers were covered with glass lids. The solution was mixed on an orbital shaker for 2 hr. [0541]4. The racks were quickly transferred to fresh chambers filled with ddH2O. They were rinsed vigorously by plunging racks up and down. Rinses were repeated 4× with fresh ddH2O each time, to remove all traces of NaOH-ethanol. [0542]5. Polylysine solution was prepared: [0543]0 mL poly-L-lysine+70 mL tissue culture PBS in 560 mL water, using plastic graduated cylinder and beaker. [0544]6. Slides were transferred to polylysine solution and shaken for 1 hr. [0545]7. The rack was transferred to a fresh chambers filled with ddH2O. It was plunged up and down 5× to rinse. [0546]8. The slides were centrifuged on microtiter plate carriers (paper towels were placed below the rack to absorb liquid) for 5 min. @500 rpm. The slide racks were transferred to empty chambers with covers. [0547]9. Slide racks were dried in a 45 C oven for 10 min. [0548]10. The slides were stored in a closed plastic slide box. [0549]11. Normally, the surface of lysine coated slides was not very hydrophobic immediately after this process, but became increasingly hydrophobic with storage. A hydrophobic surface helped ensure that spots didn't run together while printing at high densities. After they aged for 10 days to a month the slides were ready to use. However, coated slides that have been sitting around for long periods of time were usually too old to be used. This was because they developed opaque patches, visible when held to the light, and these resulted in high background hybridization from the fluorescent probe. Alternatively, pre-coated glass slides were purchased from TeleChem International, Inc. (Sunnyvale, Calif., 94089; catalog number SMM-25, Superamine substrates).PCR Amplification of cDNA Clone Inserts
[0550]Polynucleotides were amplified from Arabidopsis cDNA clones using insert specific probes. The resulting 100 uL PCR reactions were purified with Qiaquick 96 PCR purification columns (Qiagen, Valencia, Calif., USA) and eluted in 30 uL of 5 mM Tris. 8.5 uL of the elution were mixed with 1.5 uL of 20×SSC to give a final spotting solution of DNA in 3×SSC. The concentrations of DNA generated from each clone varied between 10-100 ng/ul, but were usually about 50 ng/ul.
Arraying of PCR Products on Glass Slides
[0551]PCR products from cDNA clones were spotted onto the poly-L-Lysine coated glass slides using an arrangement of quill-tip pins (ChipMaker 3 spotting pins; Telechem, International, Inc., Sunnyvale, Calif., USA) and a robotic arrayer (PixSys 3500, Cartesian Technologies, Irvine, Calif., USA). Around 0.5 nl of a prepared PCR product was spotted at each location to produce spots with approximately 100 um diameters. Spot center-to-center spacing was from 180 um to 210 um depending on the array. Printing was conducted in a chamber with relative humidity set at 50%.
[0552]Slides containing maize sequences were purchased from Agilent Technology (Palo Alto, Calif. 94304).
Post-Processing of Slides
[0553]After arraying, slides were processed through a series of steps--rehydration, UV cross-linking, blocking and denaturation--required prior to hybridization. Slides were rehydrated by placing them over a beaker of warm water (DNA face down), for 2-3 sec, to distribute the DNA more evenly within the spots, and then snap dried on a hot plate (DNA side, face up). The DNA was then cross-linked to the slides by UV irradiation (60-65 mJ; 2400 Stratalinker, Stratagene, La Jolla, Calif., USA).
[0554]Following this a blocking step was performed to modify remaining free lysine groups, and hence minimize their ability to bind labeled probe DNA. To achieve this the arrays were placed in a slide rack. An empty slide chamber was left ready on an orbital shaker. The rack was bent slightly inwards in the middle, to ensure the slides would not run into each other while shaking. The blocking solution was prepared as follows: 3×350-ml glass chambers (with metal tops) were set to one side, and a large round Pyrex dish with dH2O was placed ready in the microwave. At this time, 15 ml sodium borate was prepared in a 50 ml conical tube.
[0555]6-g succinic anhydride was dissolved in approx. 325-350 mL 1-methyl-2-pyrrolidinone. Rapid addition of reagent was crucial.
[0556]a. Immediately after the last flake of the succinic anhydride dissolved, the 15-mL sodium borate was added.
[0557]b. Immediately after the sodium borate solution mixed in, the solution was poured into an empty slide chamber.
[0558]c. The slide rack was plunged rapidly and evenly in the solution. It was vigorously shaken up and down for a few seconds, making sure slides never left the solution.
[0559]d. It was mixed on an orbital shaker for 15-20 min. Meanwhile, the water in the Pyrex dish (enough to cover slide rack) was heated to boiling.
[0560]Following this, the slide rack was gently plunge in the 95 C water (just stopped boiling) for 2 min. Then the slide rack was plunged 5× in 95% ethanol. The slides and rack were centrifuged for 5 min. @500 rpm. The slides were loaded quickly and evenly onto the carriers to avoid streaking. The arrays were used immediately or store in slide box.
[0561]The Hybridization process began with the isolation of mRNA from the two tissues (see "Isolation of total RNA" and "Isolation of mRNA", below) in question followed by their conversion to single stranded cDNA (see "Generation of probes for hybridization", below). The cDNA from each tissue was independently labeled with a different fluorescent dye and then both samples were pooled together. This final differentially labeled cDNA pool was then placed on a processed microarray and allowed to hybridize (see "Hybridization and wash conditions", below).
Isolation of Total RNA
[0562]Approximately 1 g of plant tissue was ground in liquid nitrogen to a fine powder and transferred into a 50-ml centrifuge tube containing 10 ml of Trizol reagent. The tube was vigorously vortexed for 1 min and then incubated at room temperature for 10-20 min. on an orbital shaker at 220 rpm. Two ml of chloroform was added to the tube and the solution vortexed vigorously for at least 30-sec before again incubating at room temperature with shaking. The sample was then centrifuged at 12,000×g (10,000 rpm) for 15-20 min at 4° C. The aqueous layer was removed and mixed by inversion with 2.5 ml of 1.2 M NaCl/0.8 M Sodium Citrate and 2.5 ml of isopropyl alcohol added. After a 10 min. incubation at room temperature, the sample was centrifuged at 12,000×g (10,000 rpm) for 15 min at 4° C. The pellet was washed with 70% ethanol, re-centrifuged at 8,000 rpm for 5 min and then air dried at room temperature for 10 min. The resulting total RNA was dissolved in either TE (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) or DEPC (diethylpyrocarbonate) treated deionized water (RNAse-free water). For subsequent isolation of mRNA using the Qiagen kit, the total RNA pellet was dissolved in RNAse-free water.
Isolation of mRNA
[0563]mRNA was isolated using the Qiagen Oligotex mRNA Spin-Column protocol (Qiagen, Valencia, Calif.). Briefly, 500 μl OBB buffer (20 mM Tris-Cl, pH 7.5, 1 M NaCl, 2 mM EDTA, 0.2% SDS) was added to 500 μl of total RNA (0.5-0.75 mg) and mixed thoroughly. The sample was first incubated at 70° C. for 3 min, then at room temperature for 10 minutes and finally centrifuged for 2 min at 14,000-18,000×g. The pellet was resuspended in 400 μl OW2 buffer (10 mM Tris-Cl, pH 7.5, 150 mM NaCl, 1 mM EDTA) by vortexing, the resulting solution placed on a small spin column in a 1.5 ml RNase-free microcentrifuge tube and centrifuged for 1 min at 14,000-18,000×g. The spin column was transferred to a new 1.5 ml RNase-free microcentrifuge tube and washed with 400 μl of OW2 buffer. To release the isolated mRNA from the resin, the spin column was again transferred to a new RNase-free 1.5 ml microcentrifuge tube, 20-100 μl 70° C. OEB buffer (5 mM Tris-Cl, pH 7.5) added and the resin resuspended in the resulting solution via pipeting. The mRNA solution was collected after centrifuging for 1 min at 14,000-18,000×g.
[0564]Alternatively, mRNA was isolated using the Stratagene Poly(A) Quik mRNA Isolation Kit (Startagene, La Jolla, Calif.). Here, up to 0.5 mg of total RNA (maximum volume of 1 ml) was incubated at 65° C. for 5 minutes, snap cooled on ice and 0.1× volumes of 10× sample buffer (10 mM Tris-HCl (pH 7.5), 1 mM EDTA (pH 8.0) 5 M NaCl) added. The RNA sample was applied to a prepared push column and passed through the column at a rate of ˜1 drop every 2 sec. The solution collected was reapplied to the column and collected as above. 200 μl of high salt buffer (10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.5 NaCl) was applied to the column and passed through the column at a rate of ˜1 drop every 2 sec. This step was repeated and followed by three low salt buffer (10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.1 M NaCl) washes preformed in a similar manner. mRNA was eluted by applying to the column four separate 200 μl aliquots of elution buffer (10 mM Tris-HCl (pH 7.5), 1 mM EDTA) preheated to 65° C. Here, the elution buffer was passed through the column at a rate of 1 drop/sec. The resulting mRNA solution was precipitated by adding 0.1× volumes of 10× sample buffer, 2.5 volumes of ice-cold 100% ethanol, incubating overnight at -20° C. and centrifuging at 14,000-18,000×g for 20-30 min at 4° C. The pellet was washed with 70% ethanol and air dried for 10 min. at room temperature before resuspension in RNase-free deionized water.
Preparation of Yeast Controls
[0565]Plasmid DNA was isolated from the following yeast clones using Qiagen filtered maxiprep kits (Qiagen, Valencia, Calif.): YAL022c(Fun26), YAL031c(Fun21), YBR032w, YDL131w, YDL182w, YDL194w, YDL196w, YDR050c and YDR116c. Plasmid DNA was linearized with either BsrBI (YAL022c(Fun26), YAL031c(Fun21), YDL131w, YDL182w, YDL194w, YDL196w, YDR050c) or AflIII (YBR032w, YDR116c) and isolated.
In Vitro Transcription of Yeast Clones
[0566]The following solution was incubated at 37° C. for 2 hours: 17 μl of isolated yeast insert DNA (1 μg), 20 μl 5× buffer, 10 μl 100 mM DTT, 2.5 μl (100 U) RNasin, 20 μl 2.5 mM (ea.) rNTPs, 2.7 μl (40 U) SP6 polymerase and 27.8 μl RNase-free deionized water. 2 μl (2 U) Ampli DNase I was added and the incubation continued for another 15 min. 10 μl 5M NH4OAC and 100 μl phenol:chloroform:isoamyl alcohol (25:24:1) were added, the solution vortexed and then centrifuged to separate the phases. To precipitate the RNA, 250 μl ethanol was added and the solution incubated at -20° C. for at least one hour. The sample was then centrifuged for 20 min at 4° C. at 14,000-18,000×g, the pellet washed with 500 μl of 70% ethanol, air dried at room temperature for 10 min and resuspended in 100 μl of RNase-free deionized water. The precipitation procedure was then repeated.
[0567]Alternatively, after the two-hour incubation, the solution was extracted with phenol/chloroform once before adding 0.1 volume 3M sodium acetate and 2.5 volumes of 100% ethanol. The solution was centrifuged at 15,000 rpm, 4° C. for 20 minutes and the pellet resuspended in RNase-free deionized water. The DNase I treatment was carried out at 37° C. for 30 minutes using 2 U of Ampli DNase I in the following reaction condition: 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2. The DNase I reaction was then stopped with the addition of NH4OAC and phenol:chloroform:isoamyl alcohol (25:24:1), and RNA isolated as described above.
[0568]0.15-2.5 ng of the in vitro transcript RNA from each yeast clone were added to each plant mRNA sample prior to labeling to serve as positive (internal) probe controls.
Generation of Probes for Hybridization
[0569]Generation of Labeled Probes for Hybridization from First-Strand cDNA
[0570]Hybridization probes were generated from isolated mRNA using an Atlas® Glass Fluorescent Labeling Kit (Clontech Laboratories, Inc., Palo Alto, Calif., USA). This entails a two step labeling procedure that first incorporates primary aliphatic amino groups during cDNA synthesis and then couples fluorescent dye to the cDNA by reaction with the amino functional groups. Briefly, 5 μg of oligo(dT)18 primer d(TTTTTTTTTTTTTTTTTTV) was mixed with Poly A+ mRNA (1.5-2 μg mRNA isolated using the Qiagen Oligotex mRNA Spin-Column protocol or the Stratagene Poly(A) Quik mRNA Isolation protocol (Stratagene, La Jolla, Calif., USA)) in a total volume of 25 μl. The sample was incubated in a thermocycler at 70° C. for 5 min, cooled to 48° C. and 10 μl of 5× cDNA Synthesis Buffer (kit supplied), 5 μl 10× dNTP mix (dATP, dCTP, dGTP, dTTP and aminoallyl-dUTP; kit supplied), 7.5 μl deionized water and 2.5 μl MMLV Reverse Transcriptase (500 U) added. The reaction was then incubated at 48° C. for 30 minutes, followed by 1 hr incubation at 42° C. At the end of the incubation the reaction was heated to 70° C. for 10 min, cooled to 37° C. and 0.5 μl (5 U) RNase H added, before incubating for 15 min at 37° C. The solution was vortexed for 1 min after the addition of 0.5 μl 0.5 M EDTA and 5 μl of QuickClean Resin (kit supplied) then centrifuged at 14,000-18,000×g for 1 min. After removing the supernatant to a 0.45 μm spin filter (kit supplied), the sample was again centrifuged at 14,000-18,000×g for 1 min, and 5.5 μl 3 M sodium acetate and 137.5 μl of 100% ethanol added to the sample before incubating at -20° C. for at least 1 hr. The sample was then centrifuged at 14,000-18,000×g at 4° C. for 20 min, the resulting pellet washed with 500 μl 70% ethanol, air-dried at room temperature for 10 min and resuspended in 10 μl of 2× fluorescent labeling buffer (kit provided). 10 μl each of the fluorescent dyes Cy3 and Cy5 (Amersham Pharmacia (Piscataway, N.J., USA); prepared according to Atlas® kit directions of Clontech) were added and the sample incubated in the dark at room temperature for 30 min.
[0571]The fluorescently labeled first strand cDNA was precipitated by adding 2 μl 3M sodium acetate and 50 μl 100% ethanol, incubated at -20° C. for at least 2 hrs, centrifuged at 14,000-18,000×g for 20 min, washed with 70% ethanol, air-dried for 10 min and dissolved in 100 μl of water.
[0572]Alternatively, 3-4 μg mRNA, 2.5 (˜8.9 ng of in vitro translated mRNA) μl yeast control and 3 μg oligo dTV (TTTTTTTTTTTTTTTTTT(A/C/G) were mixed in a total volume of 24.7 μl. The sample was incubated in a thermocycler at 70° C. for 10 min. before chilling on ice. To this, 8 μl of 5× first strand buffer (SuperScript II RNase H--Reverse Transcriptase kit from Invitrogen (Carlsbad, Calif. 92008); cat no. 18064022), 0.8° C. of aa-dUTP/dNTP mix (50×; 25 mM dATP, 25 mM dGTP, 25 mM dCTP, 15 mM dTTP, 10 mM aminoallyl-dUTP), 4 μl of 0.1 M DTT and 2.5 μl (500 units) of Superscript R.T.II enzyme (Stratagene) were added. The sample was incubated at 42° C. for 2 hours before a mixture of 10° C. of 1M NaOH and 10° C. of 0.5 M EDTA were added. After a 15 minute incubation at 65° C., 25 μl of 1 M Tris pH 7.4 was added. This was mixed with 450 μl of water in a Microcon 30 column before centrifugation at 11,000×g for 12 min. The column was washed twice with 450 μl (centrifugation at 11,000 g, 12 min.) before eluting the sample by inverting the Microcon column and centrifuging at 11,000×g for 20 seconds. Sample was dehydrated by centrifugation under vacuum and stored at -20° C.
[0573]Each reaction pellet was dissolved in 9 μl of 0.1 M carbonate buffer (0.1M sodium carbonate and sodium bicarbonate, pH=8.5-9) and 4.5 μl of this placed in two microfuge tubes. 4.5 μl of each dye (in DMSO) were added and the mixture incubated in the dark for 1 hour. 4.5 μl of 4 M hydroxylamine was added and again incubated in the dark for 15 minutes.
[0574]Regardless of the method used for probe generation, the probe was purified using a Qiagen PCR cleanup kit (Qiagen, Valencia, Calif., USA), and eluted with 100 ul EB (kit provided). The sample was loaded on a Microcon YM-30 (Millipore, Bedford, Mass., USA) spin column and concentrated to 4-5 ul in volume. Probes for the maize microarrays were generated using the Fluorescent Linear Amplification Kit (cat. No. G2556A) from Agilent Technologies (Palo Alto, Calif.).
Hybridization and Wash Conditions
[0575]The following Hybridization and Washing Condition were developed:
Hybridization Conditions:
[0576]Labeled probe was heated at 95° C. for 3 min and chilled on ice. Then 25 quadratureL of the hybridization buffer which was warmed at 42 C was added to the probe, mixing by pipeting, to give a final concentration of:
50% formamide
[0577]4×SSC
[0578]0.03% SDS
5×Denhardt's solution0.1 μg/ml single-stranded salmon sperm DNA
[0579]The probe was kept at 42 C. Prior to the hybridization, the probe was heated for 1 more min., added to the array, and then covered with a glass cover slip. Slides were placed in hybridization chambers (Telechem, Sunnyvale, Calif.) and incubated at 42° C. overnight.
Washing Conditions:
[0580]A. Slides were washed in 1×SSC+0.03% SDS solution at room temperature for 5 minutes,B. Slides were washed in 0.2×SSC at room temperature for 5 minutes,C. Slides were washed in 0.05×SSC at room temperature for 5 minutes.
[0581]After A, B, and C, slides were spun at 800×g for 2 min. to dry. They were then scanned.
[0582]Maize microarrays were hybridized according to the instructions included Fluorescent Linear Amplification Kit (cat. No. G2556A) from Agilent Technologies (Palo Alto, Calif.).
Scanning of Slides
[0583]The chips were scanned using a ScanArray 3000 or 5000 (General Scanning, Watertown, Mass., USA). The chips were scanned at 543 and 633 nm, at 10 um resolution to measure the intensity of the two fluorescent dyes incorporated into the samples hybridized to the chips.
Data Extraction and Analysis
[0584]The images generated by scanning slides consisted of two 16-bit TIFF images representing the fluorescent emissions of the two samples at each arrayed spot. These images were then quantified and processed for expression analysis using the data extraction software Imagene® (Biodiscovery, Los Angeles, Calif., USA). Inagene output was subsequently analyzed using the analysis program Genespring® (Silicon Genetics, San Carlos, Calif., USA). In Genespring, the data was imported using median pixel intensity measurements derived from Inagene output. Background subtraction, ratio calculation and normalization were all conducted in Genespring. Normalization was achieved by breaking the data in to 32 groups, each of which represented one of the 32 pin printing regions on the microarray. Groups consist of 360 to 550 spots. Each group was independently normalized by setting the median of ratios to one and multiplying ratios by the appropriate factor.
[0585]Results
[0586]Table 4 presents the results of the differential expression experiments for the mRNAs, as reported by their corresponding cDNA ID number, that were differentially transcribed under a particular set of conditions as compared to a control sample. The cDNA ID numbers correspond to those utilized. Increases in mRNA abundance levels in experimental plants versus the controls are denoted with the plus sign (+). Likewise, reductions in mRNA abundance levels in the experimental plants are denoted with the minus (-) sign.
[0587]The Table is organized according to the clone number with each set of experimental conditions being denoted by the term "Expt Rep ID:" followed by a "short name". Table 5 links each "short name" with a short description of the experiment and the parameters.
[0588]The sequences showing differential expression in a particular experiment (denoted by either a "+" or "-" in the Table) thereby shows utility for a function in a plant, and these functions/utilities are described in detail below, where the title of each section (i.e. a "utility section") is correlated with the particular differential expression experiment in TABLE 5.
Organ-Affecting Genes, Gene Components, Products (Including Differentiation and Function)
Root Genes
[0589]The economic values of roots arise not only from harvested adventitious roots or tubers, but also from the ability of roots to funnel nutrients to support growth of all plants and increase their vegetative material, seeds, fruits, etc. Roots have four main functions. First, they anchor the plant in the soil. Second, they facilitate and regulate the molecular signals and molecular traffic between the plant, soil, and soil fauna. Third, the root provides a plant with nutrients gained from the soil or growth medium. Fourth, they condition local soil chemical and physical properties.
[0590]Root genes are active or potentially active to a greater extent in roots than in most other organs of the plant. These genes and gene products can regulate many plant traits from yield to stress tolerance. Root genes can be used to modulate root growth and development.
[0591]Differential Expression of the Sequences in Roots
[0592]The relative levels of mRNA product in the root versus the aerial portion of the plant was measured. Specifically, mRNA was isolated from roots and root tips of Arabidopsis plants and compared to mRNA isolated from the aerial portion of the plants utilizing microarray procedures.
Root Hair Genes, Gene Components and Products
[0593]Root hairs are specialized outgrowths of single epidermal cells termed trichoblasts. In many and perhaps all species of plants, the trichoblasts are regularly arranged around the perimeter of the root. In Arabidopsis, for example, trichoblasts tend to alternate with non-hair cells or atrichoblasts. This spatial patterning of the root epidermis is under genetic control, and a variety of mutants have been isolated in which this spacing is altered or in which root hairs are completely absent.
[0594]The root hair development genes of the instant invention are useful to modulate one or more processes of root hair structure and/or function including (1) development; (2) interaction with the soil and soil contents; (3) uptake and transport in the plant; and (4) interaction with microorganisms.
[0595]1.) Development
The surface cells of roots can develop into single epidermal cells termed trichoblasts or root hairs. Some of the root hairs will persist for the life of the plant; others will gradually die back; some may cease to function due to external influences. These genes and gene products can be used to modulate root hair density or root hair growth; including rate, timing, direction, and size, for example. These genes and gene products can also be used to modulate cell properties such as cell size, cell division, rate and direction and number, cell elongation, cell differentiation, lignified cell walls, epidermal cells (including trichoblasts) and root apical meristem cells (growth and initiation); and root hair architecture such as leaf cells under the trichome, cells forming the base of the trichome, trichome cells, and root hair responses.In addition these genes and gene products can be used to modulate one or more of the growth and development processes in response to internal plant programs or environmental stimuli in, for example, the seminal system, nodal system, hormone responses, Auxin, root cap abscission, root senescence, gravitropism, coordination of root growth and development with that of other organs (including leaves, flowers, seeds, fruits, and stems), and changes in soil environment (including water, minerals, Ph, and microfauna and flora).2.) Interaction with Soil and Soil ContentsRoot hairs are sites of intense chemical and biological activity and as a result can strongly modify the soil they contact. Roots hairs can be coated with surfactants and mucilage to facilitate these activities. Specifically, roots hairs are responsible for nutrient uptake by mobilizing and assimilating water, reluctant ions, organic and inorganic compounds and chemicals. In addition, they attract and interact with beneficial microfauna and flora. Root hairs also help to mitigate the effects of toxic ions, pathogens and stress. Thus, root hair genes and gene products can be used to modulate traits such as root hair surfactant and mucilage (including composition and secretion rate and time); nutrient uptake (including water, nitrate and other sources of nitrogen, phosphate, potassium, and micronutrients (e.g. iron, copper, etc.); microbe and nematode associations (such as bacteria including nitrogen-fixing bacteria, mycorrhizae, nodule-forming and other nematodes, and nitrogen fixation); oxygen transpiration; detoxification effects of iron, aluminum, cadmium, mercury, salt, and other soil constituents; pathogens (including chemical repellents) glucosinolates (GSL1), which release pathogen-controlling isothiocyanates; and changes in soil (such as Ph, mineral excess and depletion), and rhizosheath.
3.) Transport of Materials in Plants
[0596]Uptake of the nutrients by the root and root hairs contributes a source-sink effect in a plant. The greater source of nutrients, the more sinks, such as stems, leaves, flowers, seeds, fruits, etc. can draw sustenance to grow. Thus, root hair development genes and gene products can be used to modulate the vigor and yield of the overall plant as well as distinct cells, organs, or tissues of a plant. The genes and gene products, therefore, can modulate plant nutrition, growth rate (such as whole plant, including height, flowering time, etc., seedling, coleoptile elongation, young leaves, stems, flowers, seeds and fruit) and yield, including biomass (fresh and dry weight during any time in plant life, including maturation and senescence), number of flowers, number of seeds, seed yield, number, size, weight and harvest index (content and composition, e.g. amino acid, jasmonate, oil, protein and starch) and fruit yield (number, size, weight, harvest index, and post harvest quality).
Reproduction Genes, Gene Components and Products
[0597]Reproduction genes are defined as genes or components of genes capable of modulating any aspect of sexual reproduction from flowering time and inflorescence development to fertilization and finally seed and fruit development. These genes are of great economic interest as well as biological importance. The fruit and vegetable industry grosses over $1 billion USD a year. The seed market, valued at approximately $15 billion USD annually, is even more lucrative.
Inflorescence and Floral Development Genes, Gene Components and Products
[0598]During reproductive growth the plant enters a program of floral development that culminates in fertilization, followed by the production of seeds. Senescence may or may not follow. The flower formation is a precondition for the sexual propagation of plants and is therefore essential for the propagation of plants that cannot be propagated vegetatively as well as for the formation of seeds and fruits. The point of time at which the merely vegetative growth of plants changes into flower formation is of vital importance for example in agriculture, horticulture and plant breeding. Also the number of flowers is often of economic importance, for example in the case of various useful plants (tomato, cucumber, zucchini, cotton etc.) with which an increased number of flowers may lead to an increased yield, or in the case of growing ornamental plants and cut flowers.
[0599]Flowering plants exhibit one of two types of inflorescence architecture: indeterminate, in which the inflorescence grows indefinitely, or determinate, in which a terminal flower is produced. Adult organs of flowering plants develop from groups of stem cells called meristems. The identity of a meristem is inferred from structures it produces: vegetative meristems give rise to roots and leaves, inflorescence meristems give rise to flower meristems, and flower meristems give rise to floral organs such as sepals and petals. Not only are meristems capable of generating new meristems of different identity, but their own identity can change during development. For example, a vegetative shoot meristem can be transformed into an inflorescence meristem upon floral induction, and in some species, the inflorescence meristem itself will eventually become a flower meristem. Despite the importance of meristem transitions in plant development, little is known about the underlying mechanisms.
[0600]Following germination, the shoot meristem produces a series of leaf meristems on its flanks. However, once floral induction has occurred, the shoot meristem switches to the production of flower meristems. Flower meristems produce floral organ primordia, which develop individually into sepals, petals, stamens or carpels. Thus, flower formation can be thought of as a series of distinct developmental steps, i.e. floral induction, the formation of flower primordia and the production of flower organs. Mutations disrupting each of the steps have been isolated in a variety of species, suggesting that a genetic hierarchy directs the flowering process (see for review, Weigel and Meyerowitz, In Molecular Basis of Morphogenesis (ed. M. Bernfield). 51st Annual Symposium of the Society for Developmental Biology, pp. 93-107, New York, 1993).
[0601]Expression of many reproduction genes and gene products is orchestrated by internal programs or the surrounding environment of a plant. These genes can be used to modulate traits such as fruit and seed yield
Seed and Fruit Development Genes, Gene Components and Products
[0602]The ovule is the primary female sexual reproductive organ of flowering plants. At maturity it contains the egg cell and one large central cell containing two polar nuclei encased by two integuments that, after fertilization, develops into the embryo, endosperm, and seed coat of the mature seed, respectively. As the ovule develops into the seed, the ovary matures into the fruit or silique. As such, seed and fruit development requires the orchestrated transcription of numerous polynucleotides, some of which are ubiquitous, others that are embryo-specific and still others that are expressed only in the endosperm, seed coat, or fruit. Such genes are termed fruit development responsive genes and can be used to modulate seed and fruit growth and development such as seed size, seed yield, seed composition and seed dormancy.
[0603]Differential Expression of the Sequences in Siliques, Inflorescences and Flowers
The relative levels of mRNA product in the siliques relative to the plant as a whole was measured.
[0604]Differential Expression of the Sequences in Hybrid Seed Development
[0605]The levels of mRNA product in the seeds relative to those in a leaf and floral stems was measured.
Development Genes, Gene Components and Products
Imbibition and Germination Responsive Genes, Gene Components and Products
[0606]Seeds are a vital component of the world's diet. Cereal grains alone, which comprise ˜90% of all cultivated seeds, contribute up to half of the global per capita energy intake. The primary organ system for seed production in flowering plants is the ovule. At maturity, the ovule consists of a haploid female gametophyte or embryo sac surrounded by several layers of maternal tissue including the nucleus and the integuments. The embryo sac typically contains seven cells including the egg cell, two synergids, a large central cell containing two polar nuclei, and three antipodal cells. That pollination results in the fertilization of both egg and central cell. The fertilized egg develops into the embryo. The fertilized central cell develops into the endosperm. And the integuments mature into the seed coat. As the ovule develops into the seed, the ovary matures into the fruit or silique. Late in development, the developing seed ends a period of extensive biosynthetic and cellular activity and begins to desiccate to complete its development and enter a dormant, metabolically quiescent state. Seed dormancy is generally an undesirable characteristic in agricultural crops, where rapid germination and growth are required. However, some degree of dormancy is advantageous, at least during seed development. This is particularly true for cereal crops because it prevents germination of grains while still on the ear of the parent plant (preharvest sprouting), a phenomenon that results in major losses to the agricultural industry. Extensive domestication and breeding of crop species have ostensibly reduced the level of dormancy mechanisms present in the seeds of their wild ancestors, although under some adverse environmental conditions, dormancy may reappear. By contrast, weed seeds frequently mature with inherent dormancy mechanisms that allow some seeds to persist in the soil for many years before completing germination.
[0607]Germination commences with imbibition, the uptake of water by the dry seed, and the activation of the quiescent embryo and endosperm. The result is a burst of intense metabolic activity. At the cellular level, the genome is transformed from an inactive state to one of intense transcriptional activity. Stored lipids, carbohydrates and proteins are catabolized fueling seedling growth and development. DNA and organelles are repaired, replicated and begin functioning. Cell expansion and cell division are triggered. The shoot and root apical meristem are activated and begin growth and organogenesis. Schematic 4 summarizes some of the metabolic and cellular processes that occur during imbibition. Germination is complete when a part of the embryo, the radicle, extends to penetrate the structures that surround it. In Arabidopsis, seed germination takes place within twenty-four (24) hours after imbibition. As such, germination requires the rapid and orchestrated transcription of numerous polynucleotides. Germination is followed by expansion of the hypocotyl and opening of the cotyledons. Meristem development continues to promote root growth and shoot growth, which is followed by early leaf formation.
Imbibition and Germination Genes
[0608]Imbibition and germination includes those events that commence with the uptake of water by the quiescent dry seed and terminate with the expansion and elongation of the shoots and roots. The germination period exists from imbibition to when part of the embryo, usually the radicle, extends to penetrate the seed coat that surrounds it. Imbibition and germination genes are defined as genes, gene components and products capable of modulating one or more processes of imbibition and germination described above. They are useful to modulate many plant traits from early vigor to yield to stress tolerance.
[0609]Differential Expression of the Sequences in Germinating Seeds and Imbibed Embryos
[0610]The levels of mRNA product in the seeds versus the plant as a whole was measured.
Hormone Responsive Genes, Gene Components and Products
Abscissic Acid Responsive Genes, Gene Components and Products
[0611]Plant hormones are naturally occurring substances, effective in very small amounts, which act as signals to stimulate or inhibit growth or regulate developmental processes in plants. Abscisic acid (ABA) is a ubiquitous hormone in vascular plants that has been detected in every major organ or living tissue from the root to the apical bud. The major physiological responses affected by ABA are dormancy, stress stomatal closure, water uptake, abscission and senescence. In contrast to Auxins, cytokinins and gibberellins, which are principally growth promoters, ABA primarily acts as an inhibitor of growth and metabolic processes.
[0612]Changes in ABA concentration internally or in the surrounding environment in contact with a plant results in modulation of many genes and gene products. These genes and/or products are responsible for effects on traits such as plant vigor and seed yield.
While ABA responsive polynucleotides and gene products can act alone, combinations of these polynucleotides also affect growth and development. Useful combinations include different ABA responsive polynucleotides and/or gene products that have similar transcription profiles or similar biological activities, and members of the same or similar biochemical pathways. Whole pathways or segments of pathways are controlled by transcription factor proteins and proteins controlling the activity of signal transduction pathways. Therefore, manipulation of such protein levels is especially useful for altering phenotypes and biochemical activities of plants. In addition, the combination of an ABA responsive polynucleotide and/or gene product with another environmentally responsive polynucleotide is also useful because of the interactions that exist between hormone-regulated pathways, stress and defense induced pathways, nutritional pathways and development.
[0613]Differential Expression of the Sequences in ABA Treated Plants
[0614]The relative levels of mRNA product in plants treated with ABA versus controls treated with water were measured.
Brassinosteroid Responsive Genes, Gene Components and Products
[0615]Plant hormones are naturally occurring substances, effective in very small amounts, which act as signals to stimulate or inhibit growth or regulate developmental processes in plants. Brassinosteroids (BRs) are the most recently discovered, and least studied, class of plant hormones. The major physiological response affected by BRs is the longitudinal growth of young tissue via cell elongation and possibly cell division. Consequently, disruptions in BR metabolism, perception and activity frequently result in a dwarf phenotype. In addition, because BRs are derived from the sterol metabolic pathway, any perturbations to the sterol pathway can affect the BR pathway. In the same way, perturbations in the BR pathway can have effects on the later part of the sterol pathway and thus the sterol composition of membranes.
[0616]Changes in BR concentration in the surrounding environment or in contact with a plant result in modulation of many genes and gene products. These genes and/or products are responsible for effects on traits such as plant biomass and seed yield. These genes were discovered and characterized from a much larger set of genes by experiments designed to find genes whose mRNA abundance changed in response to application of BRs to plants.
[0617]While BR responsive polynucleotides and gene products can act alone, combinations of these polynucleotides also affect growth and development. Useful combinations include different BR responsive polynucleotides and/or gene products that have similar transcription profiles or similar biological activities, and members of the same or functionally related biochemical pathways. Whole pathways or segments of pathways are controlled by transcription factors and proteins controlling the activity of signal transduction pathways. Therefore, manipulation of such protein levels is especially useful for altering phenotypes and biochemical activities of plants. In addition, the combination of a BR responsive polynucleotide and/or gene product with another environmentally responsive polynucleotide is useful because of the interactions that exist between hormone-regulated pathways, stress pathways, nutritional pathways and development. Here, in addition to polynucleotides having similar transcription profiles and/or biological activities, useful combinations include polynucleotides that may have different transcription profiles but which participate in common or overlapping pathways.
[0618]Differential Expression of the Sequences in Epi-Brassinolide or Brassinozole Plants
[0619]The relative levels of mRNA product in plants treated with either epi-brassinolide or brassinozole were measured.
Metabolism Affecting Genes, Gene Components and Products
Nitrogen Responsive Genes, Gene Components and Products
[0620]Nitrogen is often the rate-limiting element in plant growth, and all field crops have a fundamental dependence on exogenous nitrogen sources. Nitrogenous fertilizer, which is usually supplied as ammonium nitrate, potassium nitrate, or urea, typically accounts for 40% of the costs associated with crops, such as corn and wheat in intensive agriculture. Increased efficiency of nitrogen use by plants should enable the production of higher yields with existing fertilizer inputs and/or enable existing yields of crops to be obtained with lower fertilizer input, or better yields on soils of poorer quality. Also, higher amounts of proteins in the crops could also be produced more cost-effectively. "Nitrogen responsive" genes and gene products can be used to alter or modulate plant growth and development.
[0621]Differential Expression of the Sequences in Whole Seedlings, Shoots and Roots
[0622]The relative levels of mRNA product in whole seedlings, shoots and roots treated with either high or low nitrogen media were compared to controls.
Viability Genes, Gene Components and Products
[0623]Plants contain many proteins and pathways that when blocked or induced lead to cell, organ or whole plant death. Gene variants that influence these pathways can have profound effects on plant survival, vigor and performance. The critical pathways include those concerned with metabolism and development or protection against stresses, diseases and pests. They also include those involved in apoptosis and necrosis. Viability genes can be modulated to affect cell or plant death.
Herbicides are, by definition, chemicals that cause death of tissues, organs and whole plants. The genes and pathways that are activated or inactivated by herbicides include those that cause cell death as well as those that function to provide protection.
[0624]Differential Expression of the Sequences in Herbicide Treated Plants and Herbicide Resistant Mutants
[0625]The relative levels of mRNA product in plants treated with herbicide and mutants resistant to herbicides were compared to control plants.
Stress Responsive Genes, Gene Components and Products
Wounding Responsive Genes, Gene Components and Products
[0626]Plants are continuously subjected to various forms of wounding from physical attacks including the damage created by pathogens and pests, wind, and contact with other objects. Therefore, survival and agricultural yields depend on constraining the damage created by the wounding process and inducing defense mechanisms against future damage.
[0627]Plants have evolved complex systems to minimize and/or repair local damage and to minimize subsequent attacks by pathogens or pests or their effects. These involve stimulation of cell division and cell elongation to repair tissues, induction of programmed cell death to isolate the damage caused mechanically and by invading pests and pathogens, and induction of long-range signaling systems to induce protecting molecules, in case of future attack. The genetic and biochemical systems associated with responses to wounding are connected with those associated with other stresses such as pathogen attack and drought.
[0628]Wounding responsive genes and gene products can be used to alter or modulate traits such as growth rate; whole plant height, width, or flowering time; organ development (such as coleoptile elongation, young leaves, roots, lateral roots, tuber formation, flowers, fruit, and seeds); biomass; fresh and dry weight during any time in plant life, such as at maturation; number of flowers; number of seeds; seed yield, number, size, weight, harvest index (such as content and composition, e.g., amino acid, nitrogen, oil, protein, and carbohydrate); fruit yield, number, size, weight, harvest index, post harvest quality, content and composition (e.g., amino acid, carotenoid, jasmonate, protein, and starch); seed and fruit development; germination of dormant and non-dormant seeds; seed viability, seed reserve mobilization, fruit ripening, initiation of the reproductive cycle from a vegetative state, flower development time, insect attraction for fertilization, time to fruit maturity, senescence; fruits, fruit drop; leaves; stress and disease responses; drought; heat and cold; wounding by any source, including wind, objects, pests and pathogens; uv and high light damage (insect, fungus, virus, worm, nematode damage).
Cold Responsive Genes, Gene Components and Products
[0629]The ability to endure low temperatures and freezing is a major determinant of the geographical distribution and productivity of agricultural crops. Even in areas considered suitable for the cultivation of a given species or cultivar, can give rise to yield decreases and crop failures as a result of aberrant, freezing temperatures. Even modest increases (1-2° C.) in the freezing tolerance of certain crop species would have a dramatic impact on agricultural productivity in some areas. The development of genotypes with increased freezing tolerance would provide a more reliable means to minimize crop losses and diminish the use of energy-costly practices to modify the microclimate.
[0630]Sudden cold temperatures result in modulation of many genes and gene products, including promoters. These genes and/or products are responsible for effects on traits such as plant vigor and seed yield.
[0631]Manipulation of one or more cold responsive gene activities is useful to modulate growth and development.
[0632]Differential Expression of the Sequences in Cold Treated Plants
[0633]The relative levels of mRNA product in cold treated plants were compared to control plants.
Heat Responsive Genes, Gene Components and Products
[0634]The ability to endure high temperatures is a major determinant of the geographical distribution and productivity of agricultural crops. Decreases in yield and crop failure frequently occur as a result of aberrant, hot conditions even in areas considered suitable for the cultivation of a given species or cultivar. Only modest increases in the heat tolerance of crop species would have a dramatic impact on agricultural productivity. The development of genotypes with increased heat tolerance would provide a more reliable means to minimize crop losses and diminish the use of energy-costly practices to modify the microclimate.
[0635]Changes in temperature in the surrounding environment or in a plant microclimate results in modulation of many genes and gene products.
[0636]Differential Expression of the Sequences in Heat Treated Plants
[0637]The relative levels of mRNA product in heat treated plants were compared to control plants.
Drought Responsive Genes, Gene Components and Products
[0638]The ability to endure drought conditions is a major determinant of the geographical distribution and productivity of agricultural crops. Decreases in yield and crop failure frequently occur as a result of aberrant, drought conditions even in areas considered suitable for the cultivation of a given species or cultivar. Only modest increases in the drought tolerance of crop species would have a dramatic impact on agricultural productivity. The development of genotypes with increased drought tolerance would provide a more reliable means to minimize crop losses and diminish the use of energy-costly practices to modify the microclimate.
[0639]Drought conditions in the surrounding environment or within a plant, results in modulation of many genes and gene products.
[0640]Differential Expression of the Sequences in Drought Treated Plants and Drought Mutants
[0641]The relative levels of mRNA product in drought treated plants and drought mutants were compared to control plants.
Methyl Jasmonate (Jasmonate) Responsive Genes, Gene Components and Products
[0642]Jasmonic acid and its derivatives, collectively referred to as jasmonates, are naturally occurring derivatives of plant lipids. These substances are synthesized from linolenic acid in a lipoxygenase-dependent biosynthetic pathway. Jasmonates are signaling molecules which have been shown to be growth regulators as well as regulators of defense and stress responses. As such, jasmonates represent a separate class of plant hormones. Jasmonate responsive genes can be used to modulate plant growth and development.
Differential Expression of the Sequences in Methyl Jasmonate Treated Plants
[0643]The relative levels of mRNA product in methyl jasmonate treated plants were compared to control plants.
Salicylic Acid Responsive Genes, Gene Components and Products
[0644]Plant defense responses can be divided into two groups: constitutive and induced. Salicylic acid (SA) is a signaling molecule necessary for activation of the plant induced defense system known as systemic acquired resistance or SAR. This response, which is triggered by prior exposure to avirulent pathogens, is long lasting and provides protection against a broad spectrum of pathogens. Another induced defense system is the hypersensitive response (HR). HR is far more rapid, occurs at the sites of pathogen (avirulent pathogens) entry and precedes SAR. SA is also the key signaling molecule for this defense pathway.
[0645]Differential Expression of the Sequences in Salicylic Acid Treated Plants
[0646]The relative levels of mRNA product in salicylic acid treated plants were compared to control plants.
Osmotic Stress Responsive Genes, Gene Components and Products
[0647]The ability to endure and recover from osmotic and salt related stress is a major determinant of the geographical distribution and productivity of agricultural crops. Osmotic stress is a major component of stress imposed by saline soil and water deficit. Decreases in yield and crop failure frequently occur as a result of aberrant or transient environmental stress conditions even in areas considered suitable for the cultivation of a given species or cultivar. Only modest increases in the osmotic and salt tolerance of a crop species would have a dramatic impact on agricultural productivity. The development of genotypes with increased osmotic tolerance would provide a more reliable means to minimize crop losses and diminish the use of energy-costly practices to modify the soil environment. Thus, osmotic stress responsive genes can be used to modulate plant growth and development.
[0648]Differential Expression of the Sequences in PEG Treated Plants
[0649]The relative levels of mRNA product in PEG treated plants were compared to control plants.
Shade Responsive Genes, Gene Components and Products
[0650]Plants sense the ratio of Red (R):Far Red (FR) light in their environment and respond differently to particular ratios. A low R:FR ratio, for example, enhances cell elongation and favors flowering over leaf production. The changes in R:FR ratios mimic and cause the shading response effects in plants. The response of a plant to shade in the canopy structures of agricultural crop fields influences crop yields significantly. Therefore manipulation of genes regulating the shade avoidance responses can improve crop yields. While phytochromes mediate the shade avoidance response, the down-stream factors participating in this pathway are largely unknown. One potential downstream participant, ATHB-2, is a member of the HD-Zip class of transcription factors and shows a strong and rapid response to changes in the R:FR ratio. ATHB-2 over-expressors have a thinner root mass, smaller and fewer leaves and longer hypocotyls and petioles. This elongation arises from longer epidermal and cortical cells, and a decrease in secondary vascular tissues, paralleling the changes observed in wild-type seedlings grown under conditions simulating canopy shade. On the other hand, plants with reduced ATHB-2 expression have a thick root mass and many larger leaves and shorter hypocotyls and petioles. Here, the changes in the hypocotyl result from shorter epidermal and cortical cells and increased proliferation of vascular tissue. Interestingly, application of Auxin is able to reverse the root phenotypic consequences of high ATHB-2 levels, restoring the wild-type phenotype. Consequently, given that ATHB-2 is tightly regulated by phytochrome, these data suggest that ATHB-2 may link the Auxin and phytochrome pathways in the shade avoidance response pathway.
[0651]Shade responsive genes can be used to modulate plant growth and development.
[0652]Differential Expression of the Sequences in Far-Red Light Treated Plants
[0653]The relative levels of mRNA product in far-red light treated plants were compared to control plants.
Viability Genes, Gene Components and Products
[0654]Plants contain many proteins and pathways that when blocked or induced lead to cell, organ or whole plant death. Gene variants that influence these pathways can have profound effects on plant survival, vigor and performance. The critical pathways include those concerned with metabolism and development or protection against stresses, diseases and pests. They also include those involved in apoptosis and necrosis. The applicants have elucidated many such genes and pathways by discovering genes that when inactivated lead to cell or plant death.
[0655]Herbicides are, by definition, chemicals that cause death of tissues, organs and whole plants. The genes and pathways that are activated or inactivated by herbicides include those that cause cell death as well as those that function to provide protection. The applicants have elucidated these genes.
[0656]The genes defined in this section have many uses including manipulating which cells, tissues and organs are selectively killed, which are protected, making plants resistant to herbicides, discovering new herbicides and making plants resistant to various stresses.
[0657]Viability genes were also identified from a much larger set of genes by experiments designed to find genes whose mRNA products changed in concentration in response to applications of different herbicides to plants. Viability genes are characteristically differentially transcribed in response to fluctuating herbicide levels or concentrations, whether internal or external to an organism or cell. The MA_diff Table reports the changes in transcript levels of various viability genes.
Early Seedling-Phase Specific Responsive Genes, Gene Components and Products
[0658]One of the more active stages of the plant life cycle is a few days after germination is complete, also referred to as the early seedling phase. During this period the plant begins development and growth of the first leaves, roots, and other organs not found in the embryo. Generally this stage begins when germination ends. The first sign that germination has been completed is usually that there is an increase in length and fresh weight of the radicle. Such genes and gene products can regulate a number of plant traits to modulate yield. For example, these genes are active or potentially active to a greater extent in developing and rapidly growing cells, tissues and organs, as exemplified by development and growth of a seedling 3 or 4 days after planting a seed.
[0659]Rapid, efficient establishment of a seedling is very important in commercial agriculture and horticulture. It is also vital that resources are approximately partitioned between shoot and root to facilitate adaptive growth. Phototropism and geotropism need to be established. All these require post-germination process to be sustained to ensure that vigorous seedlings are produced. Early seedling phase genes, gene components and products are useful to manipulate these and other processes.
Guard Cell Genes, Gene Components and Products
[0660]Scattered throughout the epidermis of the shoot are minute pores called stomata. Each stomal pore is surrounded by two guard cells. The guard cells control the size of the stomal pore, which is critical since the stomata control the exchange of carbon dioxide, oxygen, and water vapor between the interior of the plant and the outside atmosphere. Stomata open and close through turgor changes driven by ion fluxes, which occur mainly through the guard cell plasma membrane and tonoplast. Guard cells are known to respond to a number of external stimuli such as changes in light intensity, carbon dioxide and water vapor, for example. Guard cells can also sense and rapidly respond to internal stimuli including changes in ABA, auxin and calcium ion flux.
[0661]Thus, genes, gene products, and fragments thereof differentially transcribed and/or translated in guard cells can be useful to modulate ABA responses, drought tolerance, respiration, water potential, and water management as examples. All of which can in turn affect plant yield including seed yield, harvest index, fruit yield, etc.
To identify such guard cell genes, gene products, and fragments thereof, Applicants have performed a microarray experiment comparing the transcript levels of genes in guard cells versus leaves. Experimental data is shown below.
Nitric Oxide Responsive Genes, Gene Components and Products
[0662]The rate-limiting element in plant growth and yield is often its ability to tolerate suboptimal or stress conditions, including pathogen attack conditions, wounding and the presence of various other factors. To combat such conditions, plant cells deploy a battery of inducible defense responses, including synergistic interactions between nitric oxide (NO), reactive oxygen intermediates (ROS), and salicylic acid (SA). NO has been shown to play a critical role in the activation of innate immune and inflammatory responses in animals. At least part of this mammalian signaling pathway is present in plants, where NO is known to potentiate the hypersensitive response (HR). In addition, NO is a stimulator molecule in plant photomorphogensis.
[0663]Changes in nitric oxide concentration in the internal or surrounding environment, or in contact with a plant, results in modulation of many genes and gene products.
[0664]In addition, the combination of a nitric oxide responsive polynucleotide and/or gene product with other environmentally responsive polynucleotides is also useful because of the interactions that exist between hormone regulated pathways, stress pathways, pathogen stimulated pathways, nutritional pathways and development.
[0665]Nitric oxide responsive genes and gene products can function either to increase or dampen the above phenotypes or activities either in response to changes in nitric oxide concentration or in the absence of nitric oxide fluctuations. More specifically, these genes and gene products can modulate stress responses in an organism. In plants, these genes and gene products are useful for modulating yield under stress conditions. Measurements of yield include seed yield, seed size, fruit yield, fruit size, etc.
Shoot-Apical Meristem Genes, Gene Components and Products
[0666]New organs, stems, leaves, branches and inflorescences develop from the stem apical meristem (SAM). The growth structure and architecture of the plant therefore depends on the behavior of SAMs. Shoot apical meristems (SAMs) are comprised of a number of morphologically undifferentiated, dividing cells located at the tips of shoots. SAM genes elucidated here are capable of modifying the activity of SAMs and thereby many traits of economic interest from ornamental leaf shape to organ number to responses to plant density.
[0667]In addition, a key attribute of the SAM is its capacity for self-renewal. Thus, SAM genes of the instant invention are useful for modulating one or more processes of SAM structure and/or function including (I) cell size and division; (II) cell differentiation and organ primordia. The genes and gene components of this invention are useful for modulating any one or all of these cell division processes generally, as in timing and rate, for example. In addition, the polynucleotides and polypeptides of the invention can control the response of these processes to the internal plant programs associated with embryogenesis, and hormone responses, for example.
[0668]Because SAMs determine the architecture of the plant, modified plants will be useful in many agricultural, horticultural, forestry and other industrial sectors. Plants with a different shape, numbers of flowers and seed and fruits will have altered yields of plant parts. For example, plants with more branches can produce more flowers, seed or fruits. Trees without lateral branches will produce long lengths of clean timber. Plants with greater yields of specific plant parts will be useful sources of constituent chemicals.
GFP Experimental Procedures and Results
Procedures
[0669]The polynucleotide sequences of the present invention were tested for promoter activity using Green Fluorescent Protein (GFP) assays in the following manner.
[0670]Approximately 1-2 kb of genomic sequence occurring immediately upstream of the ATG translational start site of the gene of interest was isolated using appropriate primers tailed with BstXI restriction sites. Standard PCR reactions using these primers and genomic DNA were conducted. The resulting product was isolated, cleaved with BstXI and cloned into the BstXI site of an appropriate vector, such as pNewBin4-HAP1-GFP (see FIG. 1).
[0671]Transformation
[0672]The following procedure was used for transformation of plants
1. Stratification of WS-2 Seed.
[0673]Add 0.5 ml WS-2 (CS2360) seed to 50 ml of 0.2% Phytagar in a 50 ml Corning tube and vortex until seeds and Phytagar form a homogenous mixture. [0674]Cover tube with foil and stratify at 4° C. for 3 days.
2. Preparation of Seed Mixture.
[0674] [0675]Obtain stratified seed from cooler. [0676]Add seed mixture to a 1000 ml beaker. [0677]Add an additional 950 ml of 0.2% Phytagar and mix to homogenize.
3. Preparation of Soil Mixture.
[0677] [0678]Mix 24 L SunshineMix #5 soil with 16 L Therm-O-Rock vermiculite in cement mixer to make a 60:40 soil mixture. [0679]Amend soil mixture by adding 2 Tbsp Marathon and 3 Tbsp Osmocote and mix contents thoroughly. [0680]Add 1 Tbsp Peters fertilizer to 3 gallons of water and add to soil mixture and mix thoroughly. [0681]Fill 4-inch pots with soil mixture and round the surface to create a slight dome. [0682]Cover pots with 8-inch squares of nylon netting and fasten using rubber bands. [0683]Place 14 4-inch pots into each no-hole utility flat.
4. Planting.
[0683] [0684]Using a 60 ml syringe, aspirate 35 ml of the seed mixture. [0685]Exude 25 drops of the seed mixture onto each pot. [0686]Repeat until all pots have been seeded. [0687]Place flats on greenhouse bench, cover flat with clear propagation domes, place 55% shade cloth on top of flats and subirrigate by adding 1 inch of water to bottom of each flat.
5. Plant Maintenance.
[0687] [0688]3 to 4 days after planting, remove clear lids and shade cloth. [0689]Subirrigate flats with water as needed. [0690]After 7-10 days, thin pots to 20 plants per pot using forceps. [0691]After 2 weeks, subirrigate all plants with Peters fertilizer at a rate of 1 Tsp per gallon water. [0692]When bolts are about 5-10 cm long, clip them between the first node and the base of stem to induce secondary bolts. [0693]6 to 7 days after clipping, perform dipping infiltration.
6. Preparation of Agrobacterium.
[0693] [0694]Add 150 ml fresh YEB to 250 ml centrifuge bottles and cap each with a foam plug (Identi-Plug). [0695]Autoclave for 40 min at 121° C. [0696]After cooling to room temperature, uncap and add 0.1 ml each of carbenicillin, spectinomycin and rifampicin stock solutions to each culture vessel. [0697]Obtain Agrobacterium starter block (96-well block with Agrobacterium cultures grown to an OD600 of approximately 1.0) and inoculate one culture vessel per construct by transferring 1 ml from appropriate well in the starter block. [0698]Cap culture vessels and place on Lab-Line incubator shaker set at 27° C. and 250 RPM. [0699]Remove after Agrobacterium cultures reach an OD600 of approximately 1.0 (about 24 hours), cap culture vessels with plastic caps, place in Sorvall SLA 1500 rotor and centrifuge at 8000 RPM for 8 min at 4° C. [0700]Pour out supernatant and put bottles on ice until ready to use. [0701]Add 200 ml Infiltration Media (IM) to each bottle, resuspend Agrobacterium pellets and store on ice.
7. Dipping Infiltration.
[0701] [0702]Pour resuspended Agrobacterium into 16 oz polypropylene containers. [0703]Invert 4-inch pots and submerge the aerial portion of the plants into the Agrobacterium suspension and let stand for 5 min. [0704]Pour out Agrobacterium suspension into waste bucket while keeping polypropylene container in place and return the plants to the upright position. [0705]Place 10 covered pots per flat. [0706]Fill each flat with 1-inch of water and cover with shade cloth. [0707]Keep covered for 24 hr and then remove shade cloth and polypropylene containers. [0708]Resume normal plant maintenance. [0709]When plants have finished flowering cover each pot with a ciber plant sleeve. [0710]After plants are completely dry, collect seed and place into 2.0 ml micro tubes and store in 100-place cryogenic boxes.
Recipes:
0.2% Phytagar
[0711]2 g Phytagar
[0712]1 L nanopure water [0713]Shake until Phytagar suspended [0714]Autoclave 20 min
YEB (for 1 L)
[0715]5 g extract of meat
[0716]5 g Bacto peptone
[0717]1 g yeast extract
[0718]5 g sucrose
[0719]0.24 g magnesium sulfate [0720]While stirring, add ingredients, in order, to 900 ml nanopure water [0721]When dissolved, adjust pH to 7.2 [0722]Fill to 1 L with nanopure water [0723]Autoclave 35 min
Infiltration Medium (IM) (for 1 L)
[0724]2.2 g MS salts
[0725]50 g sucrose
[0726]5 ul BAP solution (stock is 2 mg/ml) [0727]While stirring, add ingredients in order listed to 900 ml nanopure water [0728]When dissolved, adjust pH to 5.8. [0729]Volume up to 1 L with nanopure water. [0730]Add 0.02% Silwet L-77 just prior to resuspending Agrobacterium
[0731]High Throughput Screening--T1 Generation
1. Soil Preparation. Wear gloves at all times. [0732]In a large container, mix 60% autoclaved SunshineMix #5 with 40% vermiculite. [0733]Add 2.5 Tbsp of Osmocote, and 2.5 Tbsp of 1% granular Marathon per 25 L of soil. [0734]Mix thoroughly.
2. Fill Com-Packs With Soil.
[0734] [0735]Loosely fill D601 Com-Packs level to the rim with the prepared soil. [0736]Place filled pot into utility flat with holes, within a no-hole utility flat. [0737]Repeat as necessary for planting. One flat set should contain 6 pots.
3. Saturate Soil.
[0737] [0738]Evenly water all pots until the soil is saturated and water is collecting in the bottom of the flats. [0739]After the soil is completely saturated, dump out the excess water.
4. Plant the Seed.
5. Stratify the Seeds.
[0739] [0740]After sowing the seed for all the flats, place them into a dark 4° C. cooler. [0741]Keep the flats in the cooler for 2 nights for WS seed. Other ecotypes may take longer. [0742]This cold treatment will help promote uniform germination of the seed.6. Remove Flats From Cooler and Cover With Shade Cloth. (Shade cloth is only needed in the greenhouse) [0743]After the appropriate time, remove the flats from the cooler and place onto growth racks or benches. [0744]Cover the entire set of flats with 55% shade cloth. The cloth is necessary to cut down the light intensity during the delicate germination period. [0745]The cloth and domes should remain on the flats until the cotyledons have fully expanded. This usually takes about 4-5 days under standard greenhouse conditions.
7. Remove 55% Shade Cloth and Propagation Domes.
[0745] [0746]After the cotyledons have fully expanded, remove both the 55% shade cloth and propagation domes.8. Spray Plants With Finale Mixture. Wear gloves and protective clothing at all times. [0747]Prepare working Finale mixture by mixing 3 ml concentrated Finale in 48 oz of water in the Poly-TEK sprayer. [0748]Completely and evenly spray plants with a fine mist of the Finale mixture. [0749]Repeat Finale spraying every 3-4 days until only transformants remain. (Approximately 3 applications are necessary.) [0750]When satisfied that only transformants remain, discontinue Finale spraying.
9. Weed Out Excess Transformants.
[0751]Weed out excess transformants such that a maximum number of five plants per pot exist evenly spaced throughout the pot.
[0752]GFP Assay
[0753]Tissues are dissected by eye or under magnification using INOX 5 grade forceps and placed on a slide with water and coversliped. An attempt is made to record images of observed expression patterns at earliest and latest stages of development of tissues listed below. Specific tissues will be preceded with High (H), Medium (M), Low (L) designations.
TABLE-US-00001 Flower pedicel receptacle nectary sepal petal filament anther pollen carpel style papillae vascular epidermis stomata trichome Silique stigma style carpel septum placentae transmitting tissue vascular epidermis stomata abcission zone ovule Ovule Pre-fertilization: inner integument outer integument embryo sac funiculus chalaza micropyle gametophyte Embryo Post-fertilization: zygote inner integument outer integument seed coat primordia chalaza micropyle early endosperm mature endosperm embryo suspensor preglobular globular heart torpedo late mature provascular hypophysis radicle cotyledons hypocotyl Stem epidermis cortex vascular xylem phloem pith stomata trichome Leaf petiole mesophyll vascular epidermis trichome primordia stomata stipule margin
[0754]T1 Mature These are the T1 plants resulting from independent transformation events. These are screened between stage 6.50-6.90 (means the plant is flowering and that 50-90% of the flowers that the plant will make have developed) which is 4-6 weeks of age. At this stage the mature plant possesses flowers, siliques at all stages of development, and fully expanded leaves. We do not generally differentiate between 6.50 and 6.90 in the report but rather just indicate 6.50. The plants are initially imaged under UV with a Leica Confocal microscope. This allows examination of the plants on a global level. If expression is present, they are imaged using scanning laser confocal microcopy.
[0755]T2 Seedling: Progeny are collected from the T1 plants giving the same expression pattern and the progeny (T2) are sterilized and plated on agar-solidified medium containing M&S salts. In the event that there was no expression in the T1 plants, T2 seeds are planted from all lines. The seedlings are grown in Percival incubators under continuous light at 22° C. for 10-12 days. Cotyledons, roots, hypocotyls, petioles, leaves, and the shoot meristem region of individual seedlings were screened until two seedlings were observed to have the same pattern. Generally found the same expression pattern was found in the first two seedlings. However, up to 6 seedlings were screened before "no expression pattern" was recorded. All constructs are screened as T2 seedlings even if they did not have an expression pattern in the T1 generation.
[0756]T2 Mature: The T2 mature plants were screened in a similar manner to the T1 plants. The T2 seeds were planted in the greenhouse, exposed to selection and at least one plant screened to confirm the T1 expression pattern. In instances where there were any subtle changes in expression, multiple plants were examined and the changes noted in the tables.
[0757]T3 Seedling: This was done similar to the T2 seedlings except that only the plants for which we are trying to confirm the pattern are planted.
Image Data:
[0758]Images are collected by scanning laser confocal microscopy. Scanned images are taken as 2-D optical sections or 3-D images generated by stacking the 2-D optical sections collected in series. All scanned images are saved as TIFF files by imaging software, edited in Adobe Photoshop, and labeled in Powerpoint specifying organ and specific expressing tissues.
Instrumentation:
Microscope
Inverted Leica DM IRB
[0759]Fluorescence filter blocks:Blue excitation BP 450-490; long pass emission LP 515.Green excitation BP 515-560; long pass emission LP 590
Objectives
HC PL FLUOTAR 5×/0.5
[0760]HCPL APO 10×/0.4 IMM water/glycerol/oilHCPL APO 20×/0.7 IMM water/glycerol/oilHCXL APO 63×/1.2 IMM water/glycerol/oil
Leica TCS SP2 Confocal Scanner
[0761]Spectral range of detector optics 400-850 nm.Variable computer controlled pinhole diameter.Optical zoom 1-32×.Four simultaneous detectors:Three channels for collection of fluorescence or reflected light.One channel for transmitted light detector.Laser sources:Blue Ar 458/5 mW, 476 nm/5 mW, 488 nm/20 mW, 514 nm/20 mW.Green HeNe 543 nm/1.2 mWRed HeNe 633 nm/10 mW
Results
[0762]Table 2 presents the results of the GFP assays as reported by their corresponding cDNA ID number, construct number and line number. Unlike the microarray results, which measure the difference in expression of the endogenous cDNA under various conditions, the GFP data gives the location of expression that is visible under the imaging parameters.
[0763]The invention being thus described, it will be apparent to one of ordinary skill in the art that various modifications of the materials and methods for practicing the invention can be made. Such modifications are to be considered within the scope of the invention as defined by the following claims.
[0764]Each of the references from the patent and periodical literature cited herein is hereby expressly incorporated in its entirety by such citation.
Sequence CWU
1
6811952DNAUnknownPromoter and/or promoter control element identified
from Arabidopsis thaliana or Oryza sativa. 1ctgcattcac acatattttg
ggctctcacg tgtttgtgaa tttaatatat ttgactacac 60gatctttcaa cgtatgaaaa
agttttatac tactattttc gtttgagtgg gaaataaaca 120aatgatagct acagttatct
atatggtata attttacact tttataacta ataatgatga 180gtgatgacaa tcgagtgtcg
gatataacag gccaacaagt ggaatggact tatgtaactt 240tttaatcacg ggattaaatc
acgtaaccca atgtcctaat tggtatttaa ttttgattat 300ctcgatgcta catattgtca
taggactcat atctttgatc acgtgccgct accaatccag 360acattttagt atacaaaaaa
aaagaagata caaacttaag atatggaata tatatcagaa 420ctatcagttt tagactttaa
taattcgaat tgaataacta cgatcaatat ataaattggc 480aaatagattg gtcaattgta
gtgcaagaaa tttgtgaact ttattacagt acgaagagag 540taagagaagc aagatccggt
ttttaggcaa caagtaacat ttttgagttc agagagtttg 600cttcttactt taagttacgt
cactacaaaa gccaagttcc tacttcttag gtctaaagtc 660aattttcgaa tattcagaaa
aattgtactc tactagatcg aatagttttc accggtgaaa 720cgatatataa atgaagacta
caatattttt taattttttt aagcgtatga gttctagacc 780tttggcacgt aaatttctcc
ggtacctggg accaatcgtt gataatatca cgtttaagat 840ttaatcatcc atcccaagta
gagttgaact agtaaccttg agcacttttt ctcgagacaa 900ctaaaccatc atccacttag
tgcaataaag cgtcattctt ttttttcttt tcaaaaattc 960gtatttaatt ttaatttatt
aaaaatattt cttttgtttt aaattgggac agaattatca 1020tttaacatat ttaaaattta
tatttttaat taaaaatagg gtaaaatata tttttcaaac 1080aaaaattcaa aaatagggca
attttcaaaa tcatccattc ttaaatctaa agtcggctac 1140agtcttttcg ttgttttgtt
gctaatttca atttatatac atgcaaatta caaaatataa 1200tagtttttgg gggataatta
tcttcttgcg cctttttatt aaattaatat gctcatatag 1260cagttcttac aattaatata
actagggttt taaatttcaa tatcgagttg acaaaatgaa 1320ttgtttacaa gtttttttct
tttcaatatg cattgttcat cacgtattcg tagtgatgca 1380aaaacaaact ataaattata
attgcactag tgagattagc aagaagtgtt ataaattaga 1440ataaacggaa ctatcaaact
gtgttatgta caccatttat ttttgttaaa gaatatgtgt 1500agtagttaga aaactgatca
aattaaactg aaaattcaca ttacggagat caagttacat 1560tgtctattga tgaaaaaaac
aaaataaatc caaatggcac taaaagttgt agaaattgaa 1620agaagaaaat agatttttgt
ctaggaataa aagtcaaaat gggaaagaca aaaaaaagag 1680aggcaaataa gcagtgatgg
agctaaagca acgctttact cttttaatta tgaattattt 1740gatttgacct ccactcgcct
ggcttttttt ggttgttctt tatagaaaag taaaataaca 1800caattagcac ataacatgag
ttatcgagaa accaattctc tttgtggtgt tttagttaat 1860ttctataact tatgaaacca
ttttctcagt ttatcatgat aattgatcct ctatttaaaa 1920ccctaaagtt tatattttgt
ttgttcaaac ac 195221033DNAUnknownPromoter
and/or promoter control element identified from Arabidopsis thaliana
or Oryza sativa. 2agcagaacaa ctatatttat tgtgtcacat aaatctgaga tcatttataa
ccaccaaaga 60acctatacac agtaaatgac aaatgtatct ccctctatct ctattgccca
tatgtagatg 120ctaaagtaag atttctcttt tttttaatgt actttttttt gtataaagta
tattccataa 180gaaaaaggaa aagcttgttt atggatcaat tgaccccaaa aaaagttttt
agatcaaagc 240ccaatataaa aaaaaaacac agtagtgaca caaaggaact taaataaacc
atgaattgat 300ctataaacag tagagatcga taaggcgaac attttccatg tgaagtgtct
tctttcatct 360ataatatttt tgacatccaa taatttcctc tataatatca ttcacataat
tgatagaaac 420attatgttag aattgtccac atcatttgag ctgtaatata ttctgtttta
acaaattata 480tggtagttgc ttaatcttat gtccatcttc ttctatgcat cgttttcgcg
cctagttgtc 540cagtccattt caactaccta cctctaattc ttatcttaaa acaacatttt
ttaatttaag 600tattatgctc aaagactaac tagatagaaa accgttatta aacattaaac
gaattaaaag 660tcttacatgg aaaatgtagg tttataaacc acgagttatg attgacaata
aaaaaaatgc 720aaatcatcaa tcaaaagaga cttgagtgcg actctatatc aaccattgca
attaaaatta 780tctatcacaa aaattttaga cagattaagt taatttagtc taaattcact
aatttatttt 840ctataattag taattaacta tatttattta tttacacatt ttctgataat
ttagaaattt 900gcatgaataa caaatataag attttggaaa ttagtagcaa atttaattaa
taattatttt 960tgcctaaatg aaccaaacta taaaacctcc acatacacca gtcatcaaat
ttacagagac 1020aacaaactaa agt
10333999DNAUnknownPromoter and/or promoter control element
identified from Arabidopsis thaliana or Oryza sativa. 3atcttgtgat
acacaattta ttactatttg gtacattttg aagtatttgt ttttgcatga 60tatatgacgt
taatttgaac tgatattagt caatttatgg gtacaaaagt tgaaagttta 120gagcactatg
ttggatttat taaaaatgat atcatacaat ggttcaatat atatatattt 180ttttccacgt
ttttaataac atttttgtaa acaagtcttc tactattgtc tttattgtta 240atgagtttct
agtacctaat taggaatttt gaggatatac gatacattaa tgagttacat 300tatcccgaaa
acaaaatctt gaaaacgaac aaagataatt tggacattac tcgttatgta 360tacgtatgga
attggataga gccgttgaac catcaagtgg gtcttcaagt caacgaactg 420aatttgattt
tacactcatg tacatcggcc acaattttat tcacacacta ctaacacctc 480tggtgtccac
ttttttcttt ctctagattg atgtgttaag atttttgttg caattcattt 540attcaggtat
ttttatatat atatatatat aaattagaat aaactaattt aaagaaagat 600atagcaatta
tgtttcacat tttaacattc tcaatcattt ataaaactaa tgtggtgatg 660aatggtatat
atatatatat atatatatat atatatatat attttgttgt gaactaatgg 720taaatattta
aaataagaca tacgtacata aatccacgga ctcttaaagt catgatgcgg 780ttaataaatg
ttcacataac ggtaaccaag tggctcaaaa tcatgaaaca acgtcacata 840atttatctta
taatgtggat aattagtacc gcattatttg taagaaaatt aaattaatta 900tagattcaca
gctaagaaaa tacgaaaaga cagctcaaca cttttccact tctattcccc 960actgtctata
aactctgata aataatctct gatctctcc
99941000DNAUnknownPromoter and/or promoter control element
identified from Arabidopsis thaliana or Oryza sativa. 4ttcatcttta
tatttaagag tttaaaaact gcaacttttg tttttctttc actaagtctt 60atggccacag
ttaattaaaa gcagatgaaa ggtggtccaa tggaaaagga gaatgtgatt 120gggctagttg
ggagagttct gatgtctagt gttgggtaca cgtgtccgtc agttacacat 180agcattaaat
cagacggcat gtcattattc aaatctagtt cacatagtac gactaatagc 240tgataaatta
atgattatac agcatatgaa ttatgaattc aaaaaaaaaa aaaaattgaa 300aatgttaagg
agatgctata ttttacaaaa ttcatcgcaa tgctttctac taatttgcta 360agtggtcttc
tccagttagt cttgtcgatt ccaagcgata ttattaaatc ttgaagcatc 420gctcaaagca
ttatagctta agataaccaa attgttatta aaaacaccta gtgaaatttt 480taaattaaaa
caattttgat atctttgtaa tatctaatac tactctttct gtgtctaaaa 540ggattaattt
tcaaaaattt cacacatatt aaaaaaaaaa aaaaattact agctaaacaa 600ttttcaataa
tcataaaaca atagtaactt aataattttt ttttattttc aaaatagtcc 660ttcaagttta
caattcattt tagtattata atcaacaaaa tttgtattaa aaagttggaa 720aattaatctt
tgtggaacaa aaaaatctag aaatcatttt ttagaattag agagaggttt 780gataaaaaaa
aataaaaaaa aatagagaga ggtagtacat actaaacgat gtgatactac 840tattgacaaa
atcttaattc tcagtttagt agaataaact agaaggaatg aatgaagtaa 900atgcgaatcc
aactactaac aaaccctact tagtcatcat attttcccat atgaaatccc 960tatataaacc
catcatcatc tcccactttt ttcatatcca
10005999DNAUnknownPromoter and/or promoter control element
identified from Arabidopsis thaliana or Oryza sativa. 5tatatagttt
ttatgcattc tcctcttgtg taatacataa accaaatatg agataggtta 60atctgtattt
cagataatat taaattccaa acaatatttt tacttgttat aagaaggcaa 120ttaatatctc
tctgttaatg gcaagtggta ccaagtagta ttaaactatt aatgcaatgg 180aagagtactg
ttggaaatta taatcctcta tcacacattc aaacagatct cctgaaatct 240tctcttccaa
acttgtactt ctctgatcca aatgtaggct ccaaaatata gacatttacc 300atttactaag
tccacaactc ctttcttgtc tccttcaaaa atgactcttg tgtaaccatc 360atatgactcc
gacagttcgg cattgccatg atgagagctt aaaaattcac cttcctgagc 420atttcaagtc
ttcactccct tagcttgacc tgaaccaaga taaaatgcct ttgtcgtccc 480gtaatatcca
tcctgctttg gacggcatca tagttacatt cgatccatcc tatttacaat 540gttattttag
tattaaaaac atgacaataa atttgttgtt aaacatattc aaatacaata 600tgattggatt
tataagtaat tgtaatatga aatgtcctta gtaatatgtt aaaaaataca 660tagatacaca
cacgtactaa aagaggcaac gcgggagatg tcattagagg aagaactagg 720aagcagagcg
ttcatgcaaa atgctaccaa aaacgttaat gcaatatctc aactaatcag 780cacagtccat
ttcatactga gaatgtaaaa accaatcagc atcgtccatt ttttcatcta 840attatttgtt
aactcttaat tggccacaac ttccaaccac atgacgctct ttctattccc 900tttatatatt
cccatctcaa atgttcttgg agacacaaaa tatcataaac atataaacat 960aaacgccaat
cgcagctttt gtacttttgg cggtttaca
9996999DNAUnknownPromoter and/or promoter control element identified
from Arabidopsis thaliana or Oryza sativa. 6tttattttat tttttgaatg
aaaatgtctt ctttattcgt aattttaaac tcactggtgg 60tggatatatt gttatgtccc
caattcgtct ggcaactctc gtatattagt gagaaaaatt 120tgtccattat ttactgcact
attaccctgt gttaattttt tgtattgaaa ttgtttttta 180gtaattcacg tcatatagcg
aatgattctt taattttaaa aattcagtct taagtttaca 240aattaaataa cgctactgta
accaactctg tacgaccaac atgttcgagt ttttgtatat 300acggccatat atgtacatat
tttactataa agcgaaaaaa tccataaatt atttaattaa 360tatataaagg tgccattcta
tttccaatgt gcttaggaaa atgcagaacc tcgtgctata 420tctctgtcgc cacgtgcaaa
tataacaata tgaaatagaa ctagcaaatc ttgaaatcta 480actcttaaga ctaattcaag
cacatacgta gagaaagttg accaacggtt atcagcattt 540taacatggac cttatcaaca
ttttaacaaa gtccacaaac aaccagtctt acaatcgcat 600tggtacaaga taatcgaatt
catcttccat ataacaaaac ctaaaccttg gtgtgaaaag 660gagaagatat gtatgttaaa
ggccgcctat gcctctggtt tggggtatat gattctaaga 720ttagggtttg aatattttcg
ttagcctgcc atgagatata tttatgtgat aattagagcc 780tcttatgcat taatgcataa
ccgactagat catgtggtat tcagctaatc agtacacaca 840agacaaagta gtaaatgagt
ttgatgaaga ctgtggtctg ataattccta tcaacgttaa 900atctgtcggg gccaggcagc
cagcaacatt ttgcctaaca acgctctgaa ttcaattgaa 960cctaggctat ataatagcag
gctaacttaa ctaagagtt 99971000DNAUnknownPromoter
and/or promoter control element identified from Arabidopsis thaliana
or Oryza sativa. 7tggattacaa atcattaagc taatatcttc gatgaattaa gaagataagt
ggataacaag 60tacctaaccg caatagtcca taaattaaaa cattaatgta tttgtcgttg
aaaatttggc 120cgacttttat ttgttattct agtttccaca tcaaaaatgt ttgtacttcg
tagcaatcca 180tccacctaaa ccccaaatct taatttatat ttgttgcgtt taaatttggg
tgagatttga 240ttctaagtag ttgagataaa ttgatattct attcattagt aaaatgatag
agaaattggt 300ttataataat tttaccctag aacatgacat gatattggta accattaatc
aaagaaagag 360caaagcattt aatttaccct actctccaac cactccagcc tttattagtt
gcagttggga 420atcatttctt tatgattctt atgtcattgt ctcctaaatc aatgaagtgc
cttgaccttg 480ttactaattc gaacatagca aagccaacta catagatcct ttacaaagtt
ctaaaaacag 540gttgtttagg cgtctagaca aacaaaacca ttttgtacga ttcaacaaat
tggtccatag 600aatgttattg atctttcttg tttaggcatt cgataaatcg gctaatacat
tatttttttg 660ttttgctttt tccttattaa aaatatgcaa agtattatga tgtttaacct
gaactgaatt 720ttacatttaa ctggatatag gaaaatattg ggttgaattt aataattaag
caattgtcac 780gtaaatcaaa ttgggcttaa tatatattgt tgatttcagc aaagacaaag
ttgggccgtt 840tcaatagtct tcacgcgatg taagcgttca ctaaccaact agagaagaca
atcaaatgaa 900tacgttccac gtgacgctta cgaacttgtc agtcactttg gtaatatgac
agacagtaac 960cagtaaacta ctaatctctt tcgctaacga acacacaaaa
100081000DNAUnknownPromoter and/or promoter control element
identified from Arabidopsis thaliana or Oryza sativa. 8aaacatgttt
tatgtaacta ctttgcttat gtgattgcct gaggatacta ttattctctg 60tctttattct
cttcacacca catttaaata gtttaagagc atagaaatta attattttca 120aaaaggtgat
tatatgcatg caaaatagca caccatttat gtttatattt tcaaattatt 180taatacattt
caatatttca taagtgtgat tttttttttt tttgtcaatt tcataagtgt 240gatttgtcat
ttgtattaaa caattgtatc gcgcagtaca aataaacagt gggagaggtg 300aaaatgcagt
tataaaactg tccaataatt tactaacaca tttaaatatc taaaaagagt 360gtttcaaaaa
aaattctttt gaaataagaa aagtgataga tatttttacg ctttcgtctg 420aaaataaaac
aataatagtt tattagaaaa atgttatcac cgaaaattat tctagtgcca 480ctcgctcgga
tcgaaattcg aaagttatat tctttctctt tacctaatat aaaaatcaca 540agaaaaatca
atccgaatat atctatcaac atagtatatg cccttacata ttgtttctga 600cttttctcta
tccgaatttc tcgcttcatg gttttttttt aacatattct catttaattt 660tcattactat
tatataacta aaagatggaa ataaaataaa gtgtctttga gaatcgaacg 720tccatatcag
taagatagtt tgtgtgaagg taaaatctaa aagatttaag ttccaaaaac 780agaaaataat
atattacgct aaaaaagaag aaaataatta aatacaaaac agaaaaaaat 840aatatacgac
agacacgtgt cacgaagata ccctacgcta tagacacagc tctgttttct 900cttttctatg
cctcaaggct ctcttaactt cactgtctcc tcttcggata atcctatcct 960tctcttccta
taaatacctc tccactcttc ctcttcctcc
100091000DNAUnknownPromoter and/or promoter control element
identified from Arabidopsis thaliana or Oryza sativa. 9taaagatcag
aagaggaagg tttcgccgcg gcggttgcat cttcaccgtc gatttcatcg 60ttacagcgac
gccggtaatt cctaggttgc ttagttccca ttctctctct aaaattaggg 120ctcgaaatga
attgttgaac aagatagaga tctttttctg atccccgtcg aacatttatt 180caaggccaaa
aaaagcacac gggaatttag agtaccaata catatcaaaa cctaatgggc 240tttgaatggt
tgcatgtgtg tgtttatttc tgatatgcaa agcgatcgat agtcttttcc 300atacaagtgt
aaactgtaaa caacgtaatt aagcataaca atacaactct ttcttctctt 360tttttttgta
aacacaaaat aaaattacat caattcatgc ttttcctagt tcatctgaca 420ttttccaaaa
ttcatgttcc attgagtccc taatacttgt tcatattcat attagggtac 480atgaataaaa
gttatcattc ttgaaactac taaattttca tagtttattt ttcttctttt 540cgtttcactt
tcgaacaaaa cactacgcgt ggcatttgca atgaattcca cattatatgg 600aataacacca
tgatgaacat tctacatata taattattat gtttaagcac ttagacagca 660taaattcttt
ctaattatat aaatctaacc ttgttacatt gtacatctat aaattacttg 720aagaaataac
gagttctatt tctttttaaa aattaaaaat actataccat atctcagtga 780ttaagttgaa
ccaaaaggta cggaggagaa acaagcattt gattcttcct tattttattt 840tattcatctc
tcactaatga tggtggagaa aaaaagaaaa tacctaacaa acaaatatat 900attgtcatac
aaaaatattt ctatattttt agttaattag tttatattcc tcacttttca 960gggcttatat
aagaaagtga gcaaacacaa atcaaaatgc
100010999DNAUnknownPromoter and/or promoter control element
identified from Arabidopsis thaliana or Oryza sativa. 10aaacttccaa
atttctaaac ggatgcaata agaacttaca tattctcttt cattagtcat 60ttattggcca
gatttattaa aaaaagtttt actcaatgac caaggattag agttaaagat 120aatatagatt
attacatata ttattcgaaa aaatatacgc atgtccgact ttttaaacct 180caaaaatatc
aaaaccagaa aagatgatac cacacaaaaa aacaataaaa taataagtgg 240aagagatatc
atcggacaac agtacaagta cagcaccagc tctgccaaaa gccaaaacca 300tttgtcaatt
acagaaagat actattgttt gcaattacta aattacccct cggactttac 360aaaagcatct
ctaacttatc cacgtgtcag tcatctattg attgtttcaa taccaccttg 420tattaacgcc
ccacgattcg tggttgggta cacctgatag tccgaggata tttaaatctc 480acgcgctcgt
gtctataatt cgactgtact cgcttttctt gtcgtgattt tagcaattta 540cgaagtcaaa
tgtttgactc aatcagactt gcgcataaga gagcgagtat aaatgtttac 600tatactcacg
caagtggggc tttattgaaa ctactctttt gtaataaaac cagcagtggt 660tttgttctga
atccgctctc ttgccatata taccacaaac agaaaccaca gaagatatct 720tttgagaagg
aaaaaaaaaa agaagcttct cctcttcctc tgccttcttc tttccattta 780ttgcaaaccc
tgatcaagta agtcaaatct tcacgaacac atatgtatat aaattcaatc 840caagaaacta
ggagaaatct atgaaagagg acaaatctaa gtcaagtttg aatcaggaag 900attatctaga
tttgatcatt ttgacattta cgatgtgctt acttattctt gataaacttt 960gatgcagttg
gttttggtgt tagtcttttg gggagagag
99911915DNAUnknownPromoter and/or promoter control element
identified from Arabidopsis thaliana or Oryza sativa. 11acatcaattt
gcctgcttgt agggtgattc gtcaaatcta ttatcaggtt ttaaatatac 60tcgaattgac
ttccaaattc ttagtctcta gtgtaatgat tttgagaatc acttaactcc 120aaaaatataa
tccacgatcc cgtgttaatt attgaagaat caatcgtttt taatttctca 180ccaatagatg
ttgctcttat tacttaaaac aaattgttta gacaaatgta gcaagtgtga 240tacttagtgg
gatcttaaag acgatttctc ctataacaga ggacaaacag gtcggtcaat 300tacaatgtca
tccctcttta ccctgtcttt ttttttcttc ttaaaaccta accatttgat 360tgtttctaaa
ggtatttcaa gaatatatga tcaatctaga tgaatactat accgacgatg 420actacacaca
caaggaaata tatatatcag ctttcttttc acctaaaagt ggtcccggtt 480tagaatctaa
ttcctttatc tctcattttc ttctgcttca cattcccgct agtcaaatgt 540taataagtgc
acacaacgtt ttctcgaagc attagaatgt cctcctctta attaatctcc 600ttctgattag
attctcaata gagtttaaat ttgttaatgg agagatatat tgggaccctc 660aaggcttcta
attataccac gtttggcata attctctatc gtttggggcc acatctttca 720cacttcatta
ccttatcacc aaaacataaa atcaatcaac ttttttttgc cttattgatt 780gtgttggatc
cctccaaaat taaaacttgt gttccccaca aaagcttacc caatttcact 840tcaatcttaa
caaataggac caccactacc acgtacggtt tgcatcatac aaaccacaaa 900ctccttcttc
attac
915121000DNAUnknownPromoter and/or promoter control element
identified from Arabidopsis thaliana or Oryza sativa. 12tggagcttta
ttgaaatgca agaaagtaaa caaaggaaga tctttagatt gtcaccaaga 60gtggtctgaa
actctcataa cactcaatcc tcctcctcct catcaccacc actacaaaat 120attatattct
ctatctctca atctatgagg agatgtattc tatcaagcat ttgaaatgat 180aagaaactgg
cgatcatcct ctacgtcacc atcactccaa aattatcctc tttctaggtt 240taagttttgt
aatgatcgcc tttatttgtt gagatctcta acttctcgca tttccaaaat 300gttaagtcca
ataactgcat tggttaagtt ggggcgttac tagtcggctt aaatccaaat 360atggatttga
ttccatatgt atgtgacagt ttcttaacgt tcatattaca atgaatgatg 420gatccttgac
tagacaaaga gaaaatggat tgtcacttcg taggaaaaat agaaattctc 480cacgaaggct
ggtctccttt atttaacgac aaattcactc atagtctcat tcacaatttg 540aacttgtcta
acacaatgtg ttatatactc gcgaaaagaa gcataatagg ctcttaaggg 600taatccacga
aaccaaaaca catataaaac attaatattt ttctctaaat ttattcatat 660caataataaa
gtttacaaaa aatataaaac aataatccat acttagccca tagcttcgtg 720tggaagaaga
cttgattttt gactagtcaa cgaaaatgag taaatgacgt attcagctat 780agtaaaaggg
atcataagcg gaaattacaa agaagctttg agggtaaaat agtcaaaaag 840cataatcaga
aataacttag gcccaaagca aaaaggaaag gactctggat ccagccgcaa 900atcagaatct
ggtaagttcg aacgccacgt catcacctaa atatctgaaa tatctaatta 960agacttgtct
atatataaag gcttctcctt tcacaatccc
1000131000DNAUnknownPromoter and/or promoter control element
identified from Arabidopsis thaliana or Oryza sativa. 13aaagattgag
ttgagagaga tggtggagac gcagaacaga caaagggagt ttaccatata 60gtgctctaaa
gggcaatgag attgcagtga tgtggctatc cggggaatca tcgcaggtta 120ttccttccca
tgagcaacaa tcaatggatg ggttccaatt cagaggagaa acagaagaag 180aaacgtttcc
agagaaccac agtagggatt ctcgatcttg cgagttgcag agagcctctg 240aaactgcaat
agaaaggaca ctgatgaaaa gaacacactg aaggagtatg ccaatcatgt 300gaaaactcag
agcttgtatt ggtcttgtgg ttgatgaagt tctcacaaaa cctttggctt 360tgaatctccc
ctcattagtc atggtgagaa caagaacaag acgagaaaca gacaaagaag 420atgaaaaaac
ttgttggcca gtgttgacta agggggaata gcccagacat aacaaaatta 480gacttgtcgt
acatctttaa tatttttttt atctgtttct ttgtcctgac gctttcatta 540ttcctgtgat
caattttctc ataccattgg tccatcgtta atcctttctc aatttcattt 600tctacgtaac
atgagaggag accaagtcct atgagaacag ttgacgtaac agtggttgtt 660aagttaagtt
aaaaagagga agctagtgag agtgaccgtt aggtagagaa gtgagatctt 720taaccactct
tctttctctc tctctctgct tttttcgtcg tctttcacat ctactgttcg 780caaactctct
tatgcttcca ataatggtga taccaattga gacttgcagg agaatctcct 840cttctccaca
ctctatcaac tggtcagcca tggaatggtc gtttcagttt caatattcct 900ggattctttt
taaggattcc tgtttctctt ctgttcctgg tatattctta acgacgaaat 960tagtatcgga
tcctggtaat acattttgaa gcttttaagt
1000141000DNAUnknownPromoter and/or promoter control element
identified from Arabidopsis thaliana or Oryza sativa. 14tatgaagaaa
ttataataga ctctcataaa aatagtgtta caacttacat tctcttatat 60agaaattagg
ataaacagaa atgtaaataa tatatttcga aataatgtta aatttcctaa 120attctaatat
taatatttat aaatggtcat ttaacttttt cgtaccggtt cgatgggaca 180tgtgttatat
tcagttaagg ttaccaccat gcgccaactt ggcctctacc aagtcaacat 240ggatatggac
cttatggtta catgccgcct ccgcctccac cgctaccggg atatggatac 300agaggtccgc
cacctcagca accgacgagg aatgaaacaa ggcaataata tattgatgct 360attgtggatt
tagttactga taattagtgc cttagtgaca gttcaaaaat gttgttcatc 420aataatctac
aatttaaggt ttgtgttgtg gaatgtttca tgattttatg aagtcttgct 480tatcaaaaag
tatgatgatt aagaatttga cttcatggca tattcatttg agttagcaaa 540acttttttgt
gttgcacctt caaatttata aatttatgat ttttaaccat cgaaattata 600tatttgaaaa
gactatctct acaagccaaa cccactgggc caccaatatg ggtttatctg 660cgaaatctgt
gaaccttaga aaatcaaagc ccatatccac tttgctggaa ctttgctgga 720atgtaggtta
gacaaaacct taagacgcag ctacaagtct cttatgtggc agatgtcaaa 780attaatgagc
acgtataatt tacccaagag gagcaaaata agattagcag cttaaattaa 840ttgtgttgga
ttaaatgaaa cttgcactat gaatggcaaa aaagaggtta caatctagca 900accacctcat
aaaccctcat taatgagata ctgactcgtg aaccaatcaa atctcaagtt 960tcgtagttta
aataagtagt aaacacctcc tgatcaaagc
100015999DNAUnknownPromoter and/or promoter control element
identified from Arabidopsis thaliana or Oryza sativa. 15ttcctcgacc
atgccgttgc cggaaccggc tagcgcggcc ggccggcggc ggcggggagg 60ccgcagtggg
acgacgggtg aaggatcctc cagctgcgga aggaggtggt cctcgaggcc 120gaaggggaga
ggctacggag atggagggaa gccgaagaga agggaggctg ctgctgctgc 180tgcatttggg
agacgagaac tcgactcgag ccatggcggc agattggtgt ttcacggcgg 240aatgctaact
agatccagca tctccatagc aaaggtagaa tggtagattg aggtgagttt 300tttttcccct
cttctgcagt tttgatgtat tattactgcc ctcatctgat ctgggtaaca 360tatttctgag
ctcagtagaa ctgttaaaaa aaggcagaaa tgcacaaact cttctcacaa 420aacaacatac
aaatgcttat attttggagc ggaggcaata catggtatat tttttaaagt 480gaaaaaaaca
atcagacaca tggtattgag tgatagcaaa gctgggtgac cacagaaaat 540acctcctgct
ttaaatactt tatacctggg ctgtcaatcc tcggagttcc tcccaatgta 600atgtctgagg
aagaagtatt gcagctaaat tttaagggtt tcttgtacga aacagggaca 660atcagagatt
aagaaactct atgtggaaaa ggccatgcgc attttgttat gtgattcaac 720aaataagatg
aggaggcaaa gtcatggttc tgttctaatt aacaaatcta ctatgggggc 780cgttgctccc
tattgtccac gctccttttc ttcatttctc tcctgcagga tatcttgtct 840tttgattctt
cattttaggt cttataaata tcacgtggtt caggcctcca atgtcaaatt 900atcattacgt
ggaactctct tagatgcttg agaaaagtta gctcttacct gtccatagaa 960gctccaagga
agcgagaata gtagatactt tggttggcc
999161000DNAUnknownPromoter and/or promoter control element
identified from Arabidopsis thaliana or Oryza sativa. 16atttggttga
taacgttttc actcgactaa ttatatactt cagaaggata gtaatagaat 60accaaaataa
ttaaatgatt ggttagtgcc ttagtggaga ctttttaacc gattctaata 120gactaatgat
gtagctaagc atttatttgg gatcatcact gtttgaaaac gtgaaatgtg 180ataaaagtta
tgaaacgatt aaaatataaa ataaccgtac aaaacattat gtaccgtttt 240tttctctgtt
cttttggcga tttggtttag ttcgttacac tctaaatgtt attgcagata 300tatatataat
gatgcatttg catctgagga acatataatt ccggttaaca cttccaaatc 360ttatatccgt
ctaggtaggg attttataaa tcatttgtgt catcatgcgt tatgcttgtc 420ggctttgacc
ataacgcaga gatatagaac tagcttttac ttaactttta gatttattat 480ttgatctaga
gttaagtgga gatatatagt gtttttgtta gattattggt ggatgtgaga 540gtttgtcttt
agtttcaagt tgagaatata aggcaagagg agactctgag gcaatcagag 600gttttgattg
gcaaaatatc caaaaggccc aaaccaagtc gaagcccatc tcgtacaaaa 660aaagaaagag
atctgtaaga aaaaatattc tttgatattc ttacaaaaat aagtgtaaaa 720cttttattag
tcaaaatctt caatctttaa aaactctcat cactcctacg aaagcgcgtg 780agagttatga
gacattcctt aatagcatta ctcacaagtc acaagttcaa aacgtctgac 840tgaaacagaa
acaagccttt gttgaagtct tgaagaagag acattagtac tcgtcgtata 900gccataaaag
gtaatatacg aaatttcttc gctaatctct tcaccttcct ctacgcgttt 960cactttcact
ttataaatcc aaatctccct tcgaaaacat
1000171001DNAUnknownPromoter and/or promoter control element
identified from Arabidopsis thaliana or Oryza sativa. 17gttttgaaga
acaatctgga tcgaaatcta acataaggtc atcgtattca agttacgcag 60tcaaggactt
gacatcatcc tactctggtc tgaggttacc acttccaaag atgggatttt 120tcgactcggt
atgcttccta agaaattcgt tttattgaac ctagcaaata tcttgtaatg 180taagattcct
gagatgatga agaaaaaaca aacttttgtt acagcaggag aacggagaga 240aagaaaacag
agaaccaaat gctcttgaag caaacagaag aagaagacac aaatccaaac 300ttgagacttc
ttctacacca gaaaaccgca gcattctggg acaacgcaaa acacgaaagt 360gaaacgggca
atgatatata tgtcttgggt gcgttacaag gcatcgtttg catgttgagt 420tggataagtc
aactgtcttc ttttcttttg gttgtagtag ctgccttttt tttcctttgt 480tgctttaaga
aatagcccga aaaaaagaat gttctacatt tcggagcaga aaactaaccg 540aatgagtttt
tggtcggatc atcggatcga tcagatatat tttgagttac gaactgttat 600aaaaaaagcc
ataattttgt gttgagtttg caaaatacct tataacttgt tatttgagat 660tgcacctcca
tatatattaa ttcgtaagag tatttattaa gtaagcttta gtataaatcc 720ttttttcctt
taaagtaagt taatgttcta ctaaataata gtaaagttga agaaccgctc 780cgttttacac
catgcacgtg ttatctaaca aagaaaatat ggtacaccta atggctaatg 840caaaggacaa
cacaatgaaa ctaacttgac tctgtgttat acaaacccat agacatctgc 900atacatccta
gtatttgtat aaattggact caaattcctg aggacaatca tagcaaacaa 960tcacatcatc
gcaatataca taaacaaaag aggaagaaaa a
1001181000DNAUnknownPromoter and/or promoter control element
identified from Arabidopsis thaliana or Oryza sativa. 18taacaatcct
tgggaacatt gcatccatag atatccggtt aagatcgatc tttgaactca 60taaaaactag
tagattggtt ggttggtttc catgtaccag aaggcttacc ctattagttg 120aaagttgaaa
ctttgttccc tactcaattc ctagttgtgt aaatgtatgt atatgtaatg 180tgtataaaac
gtagtactta aatgactagg agtggttctt gagaccgatg agagatggga 240gcagaactaa
agatgatgac ataattaaga acgaatttga aaggctctta ggtttgaatc 300ctattcgaga
atgtttttgt caaagatagt ggcgattttg aaccaaagaa aacatttaaa 360aaatcagtat
ccggttacgt tcatgcaaat agaaagtggt ctaggatctg attgtaattt 420tagacttaaa
gagtctctta agattcaatc ctggctgtgt acaaaactac aaataatata 480ttttagacta
tttggcctta actaaacttc cactcattat ttactgaggt tagagaatag 540acttgcgaat
aaacacattc ccgagaaata ctcatgatcc cataattagt cagagggtat 600gccaatcaga
tctaagaaca cacattccct caaattttaa tgcacatgta atcatagttt 660agcacaattc
aaaaataatg tagtattaaa gacagaaatt tgtagacttt tttttggcgt 720taaaagaaga
ctaagtttat acgtacattt tattttaagt ggaaaaccga aattttccat 780cgaaatatat
gaatttagta tatatatttc tgcaatgtac tattttgcta ttttggcaac 840tttcagtgga
ctactacttt attacaatgt gtatggatgc atgagtttga gtatacacat 900gtctaaatgc
atgctttgta aaacgtaacg gaccacaaaa gaggatccat acaaatacat 960ctcatagctt
cctccattat tttccgacac aaacagagca
1000191000DNAUnknownPromoter and/or promoter control element
identified from Arabidopsis thaliana or Oryza sativa. 19tcatctgcta
ggcgattagg tttcatacac acatgagtaa actgcactat ctagttcata 60tacactccat
cttattgatg atatttcaat tttaaatagt aactcatata cttttcagta 120tttaatttat
tatttcctta aaccaaattt caatcttaca atttcgaatt tgcaatacaa 180tttaaatatc
tattttatga taataaaaat aaaatttaat ttgattgtat aaaattcaaa 240tacaattcga
ttttgcaata gaaaacaatt taattctata cactccatct actattaatt 300ttccattata
gttataaatt agtatatgta aatttgttta ttttttttag gttttttctc 360ttctaagaga
aaaaaaaaaa gttaaaatct tttccgatac atgtcaaaat ataagatcga 420tagatttgcc
atgtgttacg atcgtatgag ttattaactt tgaaaatcat actttatata 480atacaaaaca
tgtaaataca tgtttataca tatatttaca actaaaaaca tgtgtaaaat 540ctaatggatt
tttaaataca tgcttttagc tcgaaaaaaa tttgatacgg agaaaaaaat 600ttgacgggaa
ataacatacg taaatatctg atcaaattat ctatagtacg attttgacgg 660gaaaaaaaat
tattttaaag gaagagctta actttgaatc tcactaaacc agatcataca 720taatcaatcc
tttcttttat cttttttttt cttttcatta cgtgtaatcg tgttgtgtct 780aatatatcag
tttgatttgt aataatttga ataaaaaagg gagtgttgtt atctttaagt 840ttgcccaaaa
tctatagtca tgttcgatgt aaacgtatct taaacaaaat tattaaatgt 900taaagatagt
aacatacaat tattaatgaa taaatgttta actaattaaa tatcatttag 960tgattgtcct
ataaaatctc ttgttttctt gtttcatatc
1000201000DNAUnknownPromoter and/or promoter control element
identified from Arabidopsis thaliana or Oryza sativa. 20ttatgtgccc
tgatgtccta tgcagatggt gcaactactg cttttggtga gaagcttcgc 60gaacaagttg
aggaaaggct agaattttat gacaaaggtg ttgccccacg caagaacgtg 120gatgtaatga
aggaggtgat agagaatcta aagcaaggta tttcttgtag ctgttttttt 180ttggttgtaa
tcagagtcct ctttatgatg gcaaactcag tgttttttta tctgttcctc 240ctttagaaga
ggaagggaag gagccagttg atgcctcggt gaagaaaagc aagaagaaga 300aggcaaaggg
tgaagaagaa gaagaggtgg tggcaatgga ggaggacaag tcagagaaaa 360agaagaagaa
agagaagagg aagatggaga ctgcagagga gaacgagaaa tcagagaaga 420agaagacaaa
gaagagtaaa gctggaggag aagaggagac tgatgatggt cacagcacca 480agaagaagaa
gaagaagtct aagagcgctg aatagaaagg gatgcaacat taacaaaccc 540tgtattgtat
tttttttttg agctaaatta atgtcgtctg tttttcgtag tgaacatcgg 600agaatttttg
ttttggtctg gaaacgattc aaggtttggc aatatcttaa gtttgtttag 660gttttcacta
ttttgacgtt tgcaaccgtg aaggaggctc ctccatttta taaaatacaa 720ttaccaattc
cagtgctttg caaatgtttc aataatagct aaactaacta ccaaattgga 780aaactagctt
aacaagtttg tgaaaatgaa tttggagcca tatgatttat tattttaccc 840aaatggagta
atagaagaag agcagctcgc gtttgaatgg tcagttaaca ttaacaaaag 900gtaaaattga
atagatgtta aaacttgtgt aagtaaacaa tagagctacc tccttttgag 960aaggatagat
aaactcgtga ccaaccacat tcccagtccc
100021935DNAUnknownPromoter and/or promoter control element
identified from Arabidopsis thaliana or Oryza sativa. 21aaacgcctct
tcggtccacg ctgtcgtttt attgaaggaa ttatatttta ttttaattgg 60gcctgcaggc
taaactataa gtccgtctga tatgggtcgg gttgggctta tgagttatgg 120gtctggtagg
ggtcaattag cttaatttcg atatgtgccc tactctcgac ctaacgtttt 180gaacacgtaa
gagagagttt ctaatattga gttgtctaat taactcgata ggcttataca 240aagtgtttcc
gcattttacc ttcttaataa ctcatcattc actaactaag aaaagtttta 300ctcagaccat
atcttccgct tcttgattat tgtcaatttg ttgtcactca atttatctct 360tgcaaaattt
agttgaaatc atttggtttc atctttggct cttgaatagt tgcatgtgtg 420tatttagtaa
gttcttttca attaagaagg aagaataaaa caaattgtgg ccagaaacaa 480ttatgttgag
ttttatctca tacgttggct cattcatccc catctctctg cttttgaatc 540attctactcc
tcccattttt tgatcgtcct ttttcctgct tctgaacatg gatcattgtg 600catgttcgga
tgttcctcga tcgtgctgaa actcaaagtc tgaatcgatt accatagact 660ctcaacccat
ctttgatata taaaaaagag ccttaaccca tctcttctac tctccctctc 720tagaaacaaa
cacatcacgt gatgatctgt ttccccccat acttacggga tgatcagaat 780gtggcatgag
gaaaaagcca agaaataagt tgataaattt aaggtttaat ttaacaaaaa 840tgagagatta
atcttttcat tttagggtcg cacgcggtgt tttgtgcaac cgcagaaact 900tcctataaat
accgatacaa tgtgcatgct ttcta
935221000DNAUnknownPromoter and/or promoter control element
identified from Arabidopsis thaliana or Oryza sativa. 22aatcacagtc
ctttatgata aaacgaactc ataattattc caccgacaac atgcgtttta 60aattattttt
tcttaaatta tattatatta tattgatatc aacctagcta aaataattcg 120gatggcgaaa
tcggacaatt tttaatagaa aaaatgggta tgaagatagt ctatgattcc 180gttcttagcg
actagaggga cctgctcaaa tctcccgggt gatacgcgat gtcaagctca 240atagaacccc
acaaccgacg agaccgagaa atccttgatt tgggctagaa gattttgaaa 300tgaatttaat
atattctaag taacttgctt aaattttttt tcaaactcta aagacataac 360taacataaag
taaaaaaaaa aagttaatac atgggaagaa aaaaattaaa ctaatgatta 420gctctctaac
gtgtttaatc tcgtatcaag ttttttttta aaaattatat tgctattaaa 480acattgtact
attgtttcta ttttgtttag ctattattct tgtgaaatga aaagttgtgt 540ttattcaatt
actaaatggc aatatttatc ttggaaaact atacctctaa ttggattagg 600ccctagacat
cctctttagc ttattgacgt taaaattatt cccaaaacta ttaaagttta 660gtagtttgaa
agatgcatca agacctactc agataggtaa aagtagaaaa ctacagttag 720tgtgattata
ttttaaaata tataaaacaa tcttattaaa ctaaatattc aagatatata 780ctcaaatgga
agataaaaac atttagtctg ttaccactac cagcctagct agtcactaat 840agtcactttg
gaactgagta gatatttgca tcttgagtta ccatggactc aaaagtccaa 900aaagagaccc
cgagtgaaaa tgctaccaac ttaataacaa agaagcattt acagcggtca 960aaaagtatct
ataaatgttt acacaacagt agtcataagc
1000231000DNAUnknownPromoter and/or promoter control element
identified from Arabidopsis thaliana or Oryza sativa. 23aattgagaaa
ggtgcctcaa tttcagtaga acctgacgca aaatttcgcg atcatgcatg 60actcaaattg
gtttattcac ttaaataaaa aagttgtttc cctatctagt tgaagttctc 120aattcaaacg
caacttctta ctttttcttt ttatttatac tggaatgaat ttttcgtcaa 180tgctagacct
caatatttgg tgattaagtc caaaaaatta tagcaatatt cattagttaa 240atcataataa
tatttgttat ttctgctaaa tatattagtt ttaaattggt aaatatatca 300gtcatcatac
tttatatatg tgcacaagaa aaagaggaaa aaaaactaac ttttaataaa 360ttgaacgcta
tcctctatat ctcgtcctgg tccaaatgta aacttcaata tccttttgat 420tttattgctg
attgctttaa aaaatttcac aaacactttt atcattcttt tattccacca 480aaatctacag
acataatact ttgtaatttt atgtaaaaat cttcaaaatt tgggaaaaga 540aaaatcattt
aaaatcaatt tgcattaact ggatttattt ccaaaggtgt ggtattgtgt 600ttatatatgt
ggagttgttg gctagtaata taataaggaa aagagtgaaa catatgtagt 660ataacgtatt
tctagttttt ttctctgtat taatgaatca ctaattaagt agtatgcatt 720aattgaatta
tcagaagctg gtcacaaaag tctaccaaaa aaaacaaaaa aattggtcag 780aagaaaatga
aaataatgag aataaaaaag ggaaaaaaaa taagaagcta gcaaacaaag 840caattaacat
ttcaaggcag ttaattcatc atgcaaggtg cttatgtgtg acaacgtcat 900gcgttacttt
ttgcgtctac actcatctct ctaacgcaat ccactaattc tggtaatgga 960ttctgctatt
tagaccagcc agtttcttcg tctctcaatc
1000241000DNAUnknownPromoter and/or promoter control element
identified from Arabidopsis thaliana or Oryza sativa. 24cattgtatct
gagatgtgac tgtgaagaac aaagattcat gacatggtat tgttaagccg 60cccattggat
gatcataacc aaactcttcc tcagatttac tcaacagttg ttgaaacaaa 120ggctggttta
agtatgaaac cggcaccaca tatctcttct tcttctgatc attctctcct 180acatagaccg
ccatgaatcc tcttggtgtc gacgatgatt cccttcgaat aatttgctta 240gcacccaaga
aactcctcaa aaaagccata ttttccctta tgttttcctg aagcttaaat 300gtttcttagt
cttggagaaa gctttgagat tttaaaattg gatcttcttt agtttgtgaa 360tctaaagggg
tttagttact tgttatataa acgaacgtat gaaagaaatg attaagtatt 420tttgaggttt
ttctttttaa ttacagagca catggctttg ggttgtagat actaaaccaa 480gaacaaatca
ataaatggtg tctgagaagt tagtgtctaa tgatgtccta catgataact 540tcattggggc
ttatttgtct caaagacatc acatgccaaa tctctctata gattatgtag 600ggacatgaag
ttgtgtacct aatgaaccac aagtctctat cactgattaa gtcatacctt 660cttctcaatg
atattcaaaa gacaggacca catgatttga ttatatactg acaaagtcac 720aaaagccttc
aaaaaaattc tgtggcaaga aaggaaaatt tgactagtta tagtgtctat 780ctaacaaaca
agtggtcata ttgatttctg tcttcacatc agaaatcatg aagattgatc 840actatagggc
ccttacttat catgccgtgg tccggcaaag ccatgtgctt gcttgttggt 900gtaaaaattt
atgagctgaa acttttgaaa ccaataaagg gttatctaca agtaatgttc 960ttatctatat
atactcatca ctgactcctt tctgctctgc
1000251041DNAUnknownPromoter and/or promoter control element
identified from Arabidopsis thaliana or Oryza sativa. 25acgtttaaag
ttgagacata aaacagtgat ttcaaatttg tattagggtg gtcttattgt 60gtgtctagct
actagctaga gaatactaga agaagaatac gtagcaagat acgcacaaca 120tttggtcctc
tctttttttt actttctttt aacacattgt cctcttatga tttgcttatt 180gatttcagta
tctttttgta tcaataattc cctccaaatg attaaaccct aaaaaaatgt 240gattcattca
ccacccgaag attagcatca tcaagtaaca cacaataact accaataacc 300tagttttcat
ttttctatac taaaatccta aacatcccat aaaaatacaa acaactctga 360accaataatt
tcctctaatc cacgtgcacc ccatcgtctc ctgacgtaag atttgtctat 420aacttatcaa
atcccaaatt cagctttgtt ttcattatat agtacgtact cttataaaaa 480agagaagagt
acacatcttt aatactttaa cttaaaagaa gaaagtaata ctaatataag 540aggagtctga
gtcagcgaca agtgttcgcg gagaaacgga aacgctctct ttctctctct 600tcccccaacg
ccaatacctt tggaatccct ccctaactct gtcctgtcct ttcgtcctca 660ctttctctct
ttttacattt tctacacacc aataaaattg aaaccagcaa cttataaatc 720aactcaagtt
tgaattaatg atcgaaaaac tagtttattt gtgtcaatat gacccattct 780ttattcacat
aagtatttta acttttcaaa atgttatctc aatctccttt gagtttctgt 840cttccccata
ataaatttca aataattaat acacatggtt ttttaattag aaataatgga 900aaagaaagga
caaaggaata aaaaagaaac acaagttggc acactctctt tattattcac 960tcccctctat
aaatctcata ctatcttctc tcatcttctt aaatattgga tatatttctt 1020tttcaaattt
cggaaaagaa a
104126975DNAUnknownPromoter and/or promoter control element
identified from Arabidopsis thaliana or Oryza sativa. 26gataaactga
taatggaaaa gaacaaagaa accagttttt aactatttgc atatgtaatt 60tatttgttgc
aaattatatt tagttaaaat gtttcctcta tttatatata tatcagtcaa 120gcactatgta
taagaaatgt caatttataa atttttacat gtcctttaac agaaagaaaa 180tgaattttta
catgtcattc atagagagtc actcgtttat ttcttatata gagaataaca 240cactcacatg
catatgcatg caatatgata cattttatga caaagataat caacggaaac 300ggtcaagaca
taatttgata aacaacttgc acgatgcaca gatctgatca aatatataac 360tctttaacat
atccaaaata ttcaaaaaga aaaactcgat ccaaactagc aacatcacgc 420tcacgcgtag
gctaaaaatt tattaatctc caaaagtctt tcttatgaac actgcaaaca 480caacaacttg
aaaagtcata taggtttaga tgatgacgcg tattggctat cgcttaccgg 540agtggctcat
aaatacaata aacaatacgt aaaagtcaaa gtcaaatata tttagtcaac 600tataaccatt
aatcgggcaa aacctttagc tgtcaaaaca acgtgaaaac gatatttgta 660tatatcatca
agaatcagta gataagagaa tgatttaatc ccctgactat tacaattttg 720gtgtaataaa
cagtctctat tggtttttat tctttgtttt aatttctcat gacctataga 780gagaattagg
tagtttcgaa aattggctaa tcaacttttg aaaactactg tctactttgc 840ttaaattctc
tacacttagt ttcggataag ataattgtcg gactaatagt taatcccttg 900acaatctttg
atattataaa aggtttagtt aatctcttct ctatataaat attcatacac 960cagctttcaa
aaata
97527999DNAUnknownPromoter and/or promoter control element
identified from Arabidopsis thaliana or Oryza sativa. 27cagagcagtg
catatttttt tttttttttt tttggtgtta gtgcatatct atatatatag 60tactattata
atatatttca atatatatat tttaagaaaa tatctgattc ttaagtttgg 120acttatttgt
caacaatagc cagtaaaaaa caaaagcgaa gtttcactaa cttaaaaaat 180aaccacattt
gtatatttcg aatacatact ataaattaat aaatttatca aaacaactat 240agaaactgtt
atttccaatc aatttcttta tcaagattat atctgaaata tatttattaa 300aattaatagt
tatttacaag aactattttt atgaaagtgt aagaactctc tgaaaacttg 360ataagtcaat
attttttcta acatcgtaaa cataaactag attcaaattc gaatctagtt 420attcaaaaac
ttataaaaac ataaaaatga aatactgtta cttcaacaaa aaaacattat 480tattattttg
tttaaatatc taaatttatt catcaacagc aaaatattta aaagagtggg 540aaacaaataa
aaattaaact ctgttttggt atgataaaat tatttactaa actaaactca 600atttttttta
gtatcacggt tataactata acaataatcg aactttgtta ttttcttggt 660actggtttta
gtagtataga tagatatttt agtcataact cataagatac atgtacaaat 720atttgctata
tatgatcagt gataactgaa tttcgtgctg aaaattgcca tagtttgctt 780attttactct
tgaaacaata acgatatggt cgttacttaa aacaacattt taaaaacgaa 840gaaaattaaa
cagagtttgt taaaataaat taaataccat aaatttctct ttgactcttc 900ctatatagta
aaatctctca tccccttctc tctctctctc atagcatgtt ggtctttagg 960ttcctatata
aacaacgcca cacacaccca tttagtccc
99928999DNAUnknownPromoter and/or promoter control element
identified from Arabidopsis thaliana or Oryza sativa. 28tagttccatt
acaatttcca aatgatttgt tacaaagcta caagattatt cgaaatagga 60tttcatccat
aagagagaat ggtgtggtcg acgctacaat gttgatttat tggttgtggt 120ttgcatcttg
gggatgtcaa atcctaagtt tcaagttctt gtaaaaacgt tttcaggttt 180ctttaatata
ttttaatatt aatgtaaaaa gaaaagatat agcttttgta caaaaaaatt 240tgtttaatca
ctatgtagga ggatgcgatc aaattcatgg aatgatgtat tattagcttt 300tctatcctca
ctctaaaaac aatactatag tgagttaaat aatttgatca tttcaatgta 360gattaaaatt
ttattaaaag aagaaaaatt taaaagccta taacaaaata aaaaaggagg 420ctcgaggtat
gatgggtgta gcagaagagc tggcaacagc tatcgactga gtgattacga 480actcagtact
cagtgttctc agctcacaca ctcttttttt gttctctttc ttttggacag 540ctttcatttt
ctcttttctt ttttctattt tgtttcaaaa ttccatccat attaaaatag 600gcctgatcat
gagaataaag gaaatactaa tgatgagttt ctcaataatg caataagatg 660caattattat
gagctattta ctattgaaaa tgagcaaata aatgtcaaaa cacaatctgg 720ttaagttaga
gcaactccat tgtataggat tcatgtagtt tctaagaaaa caaaatgtat 780taatatttta
cttttacatc caaaaaacca acttatatga gtaatagaaa cgatcctaat 840attaggaatt
ttagagattt tctctcatct gtttcttaac ttttcaatat ttttattttt 900taaaattgta
tgagtttcta ctaagaaact actgctggag ttggtcttag cttcccaatg 960cttctccacc
tatatatatg catatctcct tcttaaaac
99929999DNAUnknownPromoter and/or promoter control element
identified from Arabidopsis thaliana or Oryza sativa. 29tgttaaggga
aggtttgcac ctaagaattt tgaaggaatt ttgcggcgat atatcagtaa 60gtaactttct
tcttagtctc aaaatttaag ttgccataaa agtatatcag tttggagttg 120ttaacctctt
gttttattat ttctcagctg actacgtcat ttgccttggt tgcaagagcc 180cagacaccat
tctctccaag gagaaccgtc tcttctttct gagatgtgaa aaggtataag 240ttaatctaat
tagtcctgat cttgatatgc attcctttgt ttctgtttta cagttttact 300ttctgcgcaa
caaagtaata aagtattttg tgtgtttgaa tttgctaatg tgattaacga 360gtgggctaca
tggtttttgc agtgtggatc tcaacgatct gtggctccga tcaaaacagg 420gtttgttgct
cgtgttagtc gcaggaagac ttgagaaatt agaaggtgaa gtgaccttgg 480tatggagttt
ggagctattc tactgcttct gtatgagttt atgagttgaa gaaatacttg 540tcttgttttt
tttattttgt tttggaatat gattatgact tgacttttaa aatgggatag 600gatcaaaacc
ttttactctg tcaggttcat gtggtcacct tgaaggttga tttagtaaat 660ccatggactt
cttttttgtg ttaagattat tcttagttca aaattaatag actaatgata 720ttaacgtcca
caggcattgc gttcaacatc tcaaattaaa gcgtggaagg ctcagaaagt 780ccaatataca
ctatgtttat ctacagttac aatcatacta caaaaaacaa ataatgtata 840cggtttggtc
taatatagcc gcatacgatt tagtatttac caacaaaaaa ttggtctcaa 900accaaaccga
acaattggta attaacaatt gttcttttgg tcttgaaccg aaccaaaccg 960aactgaacta
tattaaccga ccgacttcgt cctttcctc
99930999DNAUnknownPromoter and/or promoter control element
identified from Arabidopsis thaliana or Oryza sativa. 30tagtacttga
aacacttggt tggtttcatg tatttggcct atatataaac aaacatcgta 60attatatacg
gatttttttc ggaattttac gccatatctg taagtatata taacatgcat 120gtcgttttca
aattcatatg atgaacgatc cacgtaagtg ctactactcc tacaatattg 180catgagagag
atatgtattt ataaatttta ttttgaagaa gaaataagag ggaaggttac 240ttgggtggat
cgatgtgaaa acaaaagaag aaaaagcgaa acccactaag ccattacatg 300atatcgacct
tcttatcttt ttcctcttta ttttattttt ctcaggactt ttttctactt 360aatgaaacct
ccaaactatc taactaatac actcccatgt agaataaaga aaattatata 420agatattgtt
gatattttgt aactagaaaa tatatttgct ctgtaatttt tcgtaagtta 480aatcaacatt
tttcagtaga aacaaatatt actgcaaaaa gtaggatcat tatttttgtc 540caaaatctca
gttagctata gggttgtagt aaaaacaaaa cacattcttg atttgcccca 600aaaaataaag
agagagaaga atattgttca aaagtggtct cttctctctc taattatgtt 660ttcactaaac
ccaattagat tcaaacagtc tacaaagtcc aaaagataaa catgggacaa 720caattcgatg
caaaaaatcc tcttttcatg ctcttttttt attctctagt cttttaaatt 780actaataaaa
actcacaaat ccaccaaacc cattctctac aactcacctt catctagatt 840tacccactcc
caccgagaaa cacaagaaaa aaaatataca tatataaata tacaagacaa 900cacatgatgc
tgatgcaata tacacaacaa agtattaaat cttagatatt gtgggtctcc 960ctttcttcta
ttcattttct tattcattaa aaaaaaaaa
99931999DNAUnknownPromoter and/or promoter control element
identified from Arabidopsis thaliana or Oryza sativa. 31tacttgcctc
atgtgtttgg atacgagatt actgaacgtt gtggtgtatt ttatagtcat 60gggtttgtta
attgttatca tgcttgccta cttaactagc gtaattatgt ttttttgtac 120tacctcggaa
gtagctattt tgtcgcttat tgacaacgag atactttaag atgttccaca 180tccacgtcgt
aatcggttga tcgaatggtg cctaatagat caaagttatc ctcaacaaat 240atcgatgtgt
agtatatacg tgaatatata gtagtctctt gcatgcatat catatacaac 300ttaaatactc
tttttgtttc aaaataaata atgttttagg aaaaagatta ttgtgtcaaa 360ttaagtgttg
gtctattcat ccaaacaaga aagaaaaaaa atacgaattt gttttatata 420tcattgacga
acaatgttta gctaataata aataattatt tatttataaa aattaaaagt 480tagatagttt
cttaatttag gtgcatataa gttctttaac aaaaaaaaca tttaggtgca 540taagtcttaa
atatcaaata ttttggaaca gtaattttat gtataacttt tttcgtacct 600atcttcacac
cgcataaatt gccaaagtca accttttgat atttcattcc tcacaaaacc 660atattaattt
atacacctca atattgttta atagtattat catgttggct ttcgctgaat 720ttatcaaagt
gcaacatgtt ttatcttaca aaaaaataaa aagaaattca cgttgtgtga 780tcttgagagt
tgacttttaa atatatcaca acttatataa atacgcagca acattccaat 840ctctcaagaa
aatctacagt tcctccaaat aataataccc tccctctaag gtttaaaact 900atacctcatt
aacacattaa gaagctagtc attacttcat ttctatattt taaataatgt 960ttattgataa
caattgcagg caactaattt tcagcaatc
99932999DNAUnknownPromoter and/or promoter control element
identified from Arabidopsis thaliana or Oryza sativa. 32agcttatttt
gttctattct atcgtatttg attcttcttt cgtttttttt ttgtttgact 60taagaaaccg
attgtttata gtagtaaaca tttgttttta atgttgctcg attccagtgc 120acatgtccag
gctagacact tgtcgttata aaggttgctt tggttcaata ttgatccact 180agagatgtta
caactattgt tgacatctga gattgtgtga taagaaaata tgaaactgga 240tttagtgaaa
gttacaatat ataatcatac atcatagata ggaaataagg aaatgtcaga 300tatacttgaa
gaatacatca aatagacaag gtcctttttc ttattgtcga ctattataga 360gccgtacaga
accttttcac gtctttagta attagtacat tctccatttc ggctctctct 420tatttttttt
ccatctcttt tacttctcca aataataaca ataaaagctt cgattttgtg 480tgtgtttgta
tttacatctt gacatcgata ttcttttcat caatttttta ccaaaaatgt 540aataaaaaca
aaaaaaaacc aacgctgaac acagacatgg tttctccatc cgtttatatt 600catcgtttgt
atgtttactt aacaacttat ttcaaaatag tacatatcat ggttgtgttt 660ttaaaaaaag
tatacagaac agaaaagcac atggtagaca aaataatgaa gccaaaatta 720atacaaagaa
gaagttcaac ttgtatttat taacacattt tctttccttg tcaaagacat 780gcaaattggt
tttgttttct tattcccatt ttttttttat aataaaaaga agaagagtaa 840aacaaaaaaa
ctatcatttc ttcttatcgc aaaactctta tctaagcaag aaaccgacaa 900aacctatatc
tacatatatt ctcatcaaca tctcttgaga catattcatt ttggttaaag 960caaaagattt
taagagagaa agggggagaa gtgagagag
99933999DNAUnknownPromoter and/or promoter control element
identified from Arabidopsis thaliana or Oryza sativa. 33tacttgaggg
aaacatcata tttttaaacc ttgtctcagt aagctaacac acaccccttg 60tgattactta
tccatgttta tccacaagaa tgcagttgga ttgagatatt ttcttctttg 120ttgaaatcag
gcctcaaggt gttcatgtgg tctgcaaaaa aattcccaaa aataaagata 180gtgacatctg
aaatcgataa tggattagac gaagagtttc gtgttattcc ttggtatggg 240cgggtttggg
gacagatatt ttggcacaga cgaggactag gccactgtgg tcctgcagca 300ttaggtgtcc
cttccatgtc ctgcattaca ttttattgat ggattcatca ccctatctac 360tacaacggct
acacaaacta tgaagagttt tgtttactaa taaatgccca agtgaggggt 420cgatcgaacc
cgggacacgt ttttcagttt accatataga attatccttg gaacccttga 480tactccataa
aacatcacca cctctgttgt catctcatga atccaggttc aaacctagtc 540tctctctccc
tagtgggagg tatatggcca ctgggccaat gatgacaaaa tgcaaaaaaa 600ataaaataca
tttgggttca ttatctaaaa tatctcttgt gtttgtaagt tttggttgca 660cactcgtgtg
gttgaagtgt gtgtgagagg tactatacaa tacactctgc ttttgttttg 720tacctatctc
tttctcttct ccacatatcc aagactttgg ggataaagct gagatcattg 780gttgccattt
ggttgtgtag aagcaatcac ccatttgctt tatccgaggt tgataaattt 840cctcgggttc
tccttctgac acgtatgaca aattctaata gtatattcct cgtagatatt 900acctatatat
tctcaatagt tgcaggtact taaggctttg tcttggcatc ctcgtcctct 960tcagcaaaac
tcgtctctct tgcactccaa aaagcaacc
99934999DNAUnknownPromoter and/or promoter control element
identified from Arabidopsis thaliana or Oryza sativa. 34cccatcacat
gtaacatcat tgggctatcc aaaagtctaa ccaataatgt caatctataa 60accacattaa
gtagttcatt ttttttgtag tcgtgtttag cttgttaaac ctcataaaat 120atgttttcac
ttacgttaac aaaacaaata tcttcacgaa aaaaaataaa ataaaatatc 180tttttgatac
cgaaaaaata aaataaaata attttccctt tcgatcataa aatgcgtaga 240taagagaaac
tgtgtttgag gctccatttc atgttcacct accagtctac cacgtcattt 300ctcaaagacg
caaattttct aattagggat gtgctctttt tacatataga tcaatatcct 360aaaaaaattt
aagatattca tattttcgta catatatatc gagtttcccg aaaaatccat 420aaaatgggta
taatgatagt cctttttcac ctttaataat aatttctgaa caaaattata 480tcataataaa
cttgtgattt tatacaaaat ttatttgtat atataatttt actaaccaac 540gtgaacgata
aaaataatat tctcataaaa tgttgattaa aaattactta aaataaataa 600ttatttagga
ttatgtatta gtagtactcg aaccattttt ttagttatct gcatgaagac 660cctaattttt
cacatatatc gaaactaaaa ctttggatat acactgtaat ttgaaaacgc 720ttggaacgga
taatgtagtt acctcacaag attttgtaca tccctgacat tttatattca 780ttaaagtgtg
tttttttctt cagaaaagaa aacacttttt ttttttgtgc ttttagttta 840aattaacaaa
aaaatggaca ccatgagatt ccactaactc atgtgtatat aacattaggg 900aagcagtcaa
ttcatttcag catccacaca cactttgaat gctcaatcaa agcttcttca 960tagttaaact
tccacacaac gtcaaaactc gagaagaag
99935999DNAUnknownPromoter and/or promoter control element
identified from Arabidopsis thaliana or Oryza sativa. 35ataggaatct
gcttcggtag aagattcgag agaggagagg aagcatcggt ggttttggag 60ttccttattc
ttctcttctt tccaaagttt tgtcattcgc caagattcct taaaaacttg 120ttcacacatc
ataattatgc accaataggt tataaatcat aatccaacaa gttagtcatt 180ggctttaatt
ttaaaaaatc ccataagagt aaaatctttt agaaagttaa tcaacccaca 240catgggctag
aaaaccaaaa accccacgaa cattgagatt acaagaaaca tttttaagtc 300ctaaatgagc
ccaagagcat tgcttaatga agaagaactg atattaatta actaatatta 360ggacacataa
aaaaatacga aaacaccaat cttcatgcca caaaatcaaa caaaaacgaa 420aaaatcaatt
ttcatgaaat ggataaagag agagcgtaat tatcaggaat ttgattgagt 480acggttgtta
tgatgatcat tcacaattat ctttgatctt gagatttagc aatagttaat 540tttcggatgt
ttttttgtta cttgctgctc acttcttgta tgcagattaa tttataagag 600agaccagtta
caactctttc ttatttgaat aagattttat aagatgtagt gtggccatgt 660gggtttattg
catgcagctc tctgcgttgg tcccaagtcc acgacaatag agagtttctg 720cacttcacgg
tatcgtcgtc gtcacaagtt ctttacctta tcattggcac aagttagcca 780ccgtctttgc
gcaagttagc atgttgtgct acatacgtgt catgaactga ttggtcaaat 840ttggatatat
tttattcccg tcggttatgt ttggataaaa atataaaacg gaaatttctg 900tttcagcctt
ccttggtccc aaagaaaaat acgcacacct actcccttca ttctctatcc 960tctccactca
taatatatac atctaaatgc aatctctcc
99936998DNAUnknownPromoter and/or promoter control element
identified from Arabidopsis thaliana or Oryza sativa. 36aaattgggga
gtggggagat gtttggttat attcccttct catcgatggt ctagatgtgc 60gaggtgactc
tcatggaggt aaagaacaat ggtgattttg tgaagaaccc aacgtaatgg 120taattcctaa
aaaggttaga agttttttca gcttgttgta ttgctaaaat ggggttgatg 180tactcaacga
catccaagtg tacttgagtg agcttttttg gggttgagta cctcgaccca 240ttattcaaac
taatgtaaat ggtgaatgca gcagtgactt tgttgccttt tgcaagaact 300aaagaagaca
gaaacaggtt gtaaaagaga gccaagtgtg tgtttatggt agaaagagca 360aagtgaacga
aaggtgtacc tttttgactt gttgtcactg gttttctccc acttcatccg 420tttcatgctg
catcagaaaa caacataagg aatgaatgac gtaacgcgaa gcattaggag 480ttgcttgtaa
attaatacat tgccattact aacgtaattc agtagattct aactacaaat 540gaagtcaatg
tatctatttg tctactttag ccaatgtatg ataagaccaa atagtcttct 600cttttttcag
aaactctcta ggattaaaaa gtttgtgggt gaaagaaata ttatcgtgtg 660gatgataaga
ataattgatc ttgtgttagt aaattaggaa tagatataca agtaggtttc 720tctctaaata
aaaaataaaa gagtttaaat tgcatgcgta taaaagaaaa aagtaagaag 780aaaatatgtt
ccggttaatg gttgggtgca tccgaatcga accggcgcaa accaaaaaat 840ctaaaggaga
tttgaggtga taaaaggaaa tcagacattg aaccaaaaaa acaaaagcga 900gacggtggaa
agaaaaaact ggaaaagaca gttttagccc ctcctaaaag caaagaaaaa 960aaagataata
aatagcttcg tcgtcgtgat cgacctct
99837999DNAUnknownPromoter and/or promoter control element
identified from Arabidopsis thaliana or Oryza sativa. 37tgtccttaag
actcttatag taaagctgga attatatggt tcaaggaatc gtctagtcta 60tatacactgg
tttgaacaat tgtgatatat aatatagtta ggggtatatt atatttaatc 120tgttagataa
cggttggggt acttgaagat ctgtaggagt tgacagcacg tagaggcaga 180ggtaagaaca
cttctgcatg tagtgtgtct acataataaa atatagagtg tattttttac 240acacaccaaa
aagagagatt ataattaatg tattatgtca aagcatatat gaaggtcagc 300ttagctagag
acacgtcttt tgtttatctc tcgactaaac aacatggcgt tttaataaaa 360tcaaaactta
aaaggtccaa ttcagaacgg ccccatagta tatagtctac gttgaataaa 420taaacctcaa
gatagcgtca aactctttag tctttaccca aaaatatttt tttttaaata 480acgtcaaaac
tctaagtctt gacctcaaca ccaatatata tttgccttct ccaatatctg 540atttttttaa
ttgtttatcc gagtcttctt ggtcttttcg aatgtttgcc cgaaccagac 600cttcccacgt
tcggtggttg gtggccgcct cggcctttgg ttgatttctg tccacatttt 660ggtccttttc
attcatgtac catgttctag ggtcatttga cttgttgacc ataaatctac 720taaaacaggc
ctaataccga tgggccgtag cccgttaata aacaagacaa tttatatttg 780tttcacttag
cttgggagcc acggatctct agaaacatcc agagaaatat caatctcccc 840acttctccag
aacattcact cactgacaat atcccacctt caacacttaa ctcctgtata 900tagtcctccc
ctgtctccag tttcgtcgca cacagttctc agataaatac taaactcact 960gttaaaactt
tctcaacaaa gcttcctgtt tctctacaa
99938999DNAUnknownPromoter and/or promoter control element
identified from Arabidopsis thaliana or Oryza sativa. 38ttacgcggcg
ctacactctt atcaaagttt gaagattttt caagagacac aacagattca 60agattttctg
gtggctaaac ttacaatgac agtacatgga ggatctccgc gaatggactt 120ctgcaatgta
ctagcgtaga acaaacactt tttgttaaag tcatcaacca acatagcata 180gagttgttta
tctgaacaga acactgaaag tcttggtttt gtttgtgttc cagtaaactg 240tttcaaaatg
aaagaaaata cttattaaca agttcggcaa aaaaaattca aacttttgtg 300cattattata
tgaaagcact tctagaaagc taccttcttc ctgctcctcc tgttcctagt 360tttcggactc
tccactcgag tgttccctct cgcttcaatc acaaacggct ttactacaga 420catagctgat
aaaagggtcg aaaaatcatg aaccaagtaa gcgaaacaga ggataataaa 480catggaagaa
gaacagagta agacgaatta taccactcac ttgttattcg aattggaaac 540tggggataag
gtttcaaacg agttccgaga atgtcagaga ctctaaactg aacagtagaa 600agagaagtca
aagcagccat gccaagtatc attcgtaaag catcgaaagt cagaacatta 660ccctcagcgg
aatttaatca aacaccttct gtgcaggaat aatctctggg ggttttatca 720acactccaaa
aaaactggaa ctttgtaaat aaaattataa atgttcgtac ctttatgcaa 780aatttctcac
agcgtaatta tctatttcct ttttgtcctt tatgaaagag gataaggttt 840ttaaataata
aatactaaat tgtttttaaa agaaactaaa aataaatgga aagtcttaag 900cgtcgtcaat
ggttctagag tcttctgcaa ctttcttttc atgaaactac tgtaatcttc 960tgctaacata
tataatctca aacactatct tctccaatt
99939999DNAUnknownPromoter and/or promoter control element
identified from Arabidopsis thaliana or Oryza sativa. 39tgaattgagt
aaaatgtgtt ttcaaacagt taggtggtag aaggtaaagg taataacatc 60atgatcttac
taaaagaatt gttgcatact aactatcaat attctcaaca acataatata 120atgttttttt
aggtaatttt ccattttaat tttttgtgat taaacaatta aacaactcga 180atgatgatga
taaaaaaaaa aaattaacaa ctcgaataag ttaaagtagc aatacacatg 240tcgttcaatt
caaccaataa agtaagactt atatttttaa gaagttgact aatagcttaa 300taagttggaa
aacttgtgta gtttcttaat tcccacgtgc agtaagaaat aaaaatgaaa 360aaaattatta
tatccttccc actctgcgac ttttctttta ttttatcaaa tattaaaaag 420attcatatca
cagtttacac attgaaatca taaacgataa ttatgtattt tgtaataaaa 480agttagttct
gaagctcata ctttggatag tcgctagtcg ctaatatgct ccttgtaata 540attaaagtca
ctacgacgca cgtcaaagcc gatatttagg gcttaattga tgcgtgtttt 600tcttttcata
taatagtaat ataaattagt actaataaag tatgatggat ggttgagaca 660gaaaagaaaa
aagatgactg tatggtcatc attacaaaga agaatgtatt cttcatgttc 720ttaagaataa
taaaatgtca cttgtaaatc aagttggtaa gcattttgag aactttgttc 780gatgcaacgt
atgatgattt atgtagacaa aagataaaac cgtatcttca actattgcca 840agaaaagata
aaacctaatc tagtcagtct ctcaacataa atacaaccca atagccaaac 900tgtgtccaat
tcggagagaa actaaactaa aacaaaacac aaaagcccaa cataagccca 960ataaaaccca
ttttataaac agaacattac taacactca
99940990DNAUnknownPromoter and/or promoter control element
identified from Arabidopsis thaliana or Oryza sativa. 40ttattgttga
aacggatggt atccagattc atagagttat agttgttgac ctcgtaagga 60tgaattcatt
atcttcttct tcttttgcag catggaggtg atcgatggta tgactttgat 120gatagccatg
tccaccaaat cagccaagaa aagatcaaga cctcggctgc ttacgttctg 180ttctataaac
gccttgtaga ctaaagaaac tgaagcggaa aagacaagaa agtggtattt 240gcatttttgc
cgggtttggc ttatttaaaa acatcattgg cttgattcta attcactaca 300agatcaagat
gaaagcagct ctgcgttgag gctaatttac agaagagaga gagagagttg 360ggaagaagag
caaaagaccg agaggacatg ttgcggggaa tttattttat tcttacaaaa 420attggtatct
gattatttta ttaaccatat tcaattagag aatagaagaa tagagaaaag 480cccttttgtg
ggatatggtt ctaaattgtt gtttagttct tgtgtgtcag ttttggctct 540cgtcgaccaa
agaagattaa agaaacctct accttatttt aactcaattc ttttgttttt 600gcaatgtcct
ttgctttcca aaattgttag tcttactttt cactactttg atagacattg 660cctttgcgtt
tccctgatta ataagccaga gtacttaaat caaaattgac tgttttgtgc 720atcctgcatc
acgtttccaa tcagaaccat agtgttgtcg ttgtgtcatt atccgaattt 780aagtggagac
attggtaagt tatttataaa ctaattacaa tctatttttc taattatttc 840aaataacata
tttaagctct gtagcttcca ctagacggtg aagatttgaa gtgagagctc 900tctttgcatt
gctcacccac caatggatct acctaccctt cttcttcttc tcctcctttt 960aaaccctaaa
agtttctctt tccttcaaca
99041999DNAUnknownPromoter and/or promoter control element
identified from Arabidopsis thaliana or Oryza sativa. 41tggacaatta
ctcttgtgtg tatccttgga gttgctgttt catatgtaag tggacaatta 60ctcttgtgtg
tagccttgga gtttttttat ttacgttatt ttggtcagcc tttaattatt 120ttgcaaaaaa
tgtatctgtt tttgccacat gcccacataa tacatttcgc aaatttgata 180cattatgctt
tggcccttgt atattcggta aaaaaaaaag ctcaggctac tctcaaaacc 240ggctctgagt
attcgtaggc cacaatcgaa gaaaaaaagt gccgatttac atatttttca 300tacaaaaaat
taaaactgtt atgtattatt caaaagctat ttacatatgt tttactaaca 360cgttttcaat
attttcttaa tccttttcaa aatttaacta agtataatac tttttttgtg 420tgttatttcg
ttgttttggt taaagaaaaa cgaaaaaaag agagagttat tcatccttgc 480agataaggct
agggttggtt gaataaagat gtgcatatct tataccacta gaccaaagaa 540acagtcacaa
gtaaaaggcc gaatcctttt tataaaatat aaacagacga aagctaatgc 600ttcatgggct
tggcccaagt gcaggctctc gctagtcgct acgctacaac tatcccatat 660ttaattagtg
aagagtattt tattattttg gtcaacgggc tatctttgtt gacaaaacta 720tcccattggt
aaagaaatag caaaataggc gtttcattct ctatatttaa acttgatttt 780atgaagagtt
gaatagctga accaggaaga tatttaagaa gcccgtactt cacgctttaa 840ctgtcaatcg
atagatcata ataaatgact atctatggat aggaactata actgaattca 900gaaagaatct
actactacta taaatactaa aagagtatta atacaacgga aaaaacaaaa 960caaaaaaaag
ggggaacaag ggagtttcat gttaaaaag
99942996DNAUnknownPromoter and/or promoter control element
identified from Arabidopsis thaliana or Oryza sativa. 42ttggattttt
tttttgttga gtcagcagac catctaatct ctctttttcc accacagcct 60gctttctatg
aagcatttgg gcttacggtt gtggaatcaa tgacttgtgc actcccaacg 120tttgctacct
gtcatggtgg acccgcagag attatcgaaa acggagtttc tgggttccac 180attgacccat
atcatccaga ccaggttgca gctaccttgg tcagcttctt tgagacctgt 240aacaccaatc
caaatcattg ggttaaaatc tctgaaggag ggctcaagcg aatctatgaa 300aggttggccc
attctccttg acaggcttaa caatacaact tgtatcgctt caacaagatg 360atggcttaat
aaggattttt gcatgtatag gtacacatgg aagaagtact cagagagact 420gcttaccctg
gctggagtct atgcattctg gaaacatgtg tctaagctcg aaaggagaga 480aacacgacgt
tacctagaga tgttttactc attgaaattt cgtgatttgg ttagtgtaac 540ccactgttat
tcttttgatg tctacatcta ctttacttac attattcttt tcttcggttt 600gcaggccaat
tcaatcccgc tggcaacaga tgagaactga tcatgacagg gtaggatttt 660atttcctgca
ctttctttag atcttttgtt tgtgttatct tgaataaaaa ttgttgggtt 720ttgtttcctt
cagtggtttg attttggact tatttgtgtt aatgttgttt tggctgttct 780cttaatatca
ataacaaata aatttactgg ttggtatcta agatctaaca atagttacta 840tttttagagg
taaagacacc aaccttgtta tattggtcag agagctaaaa ccttgacttg 900ttgggaaaac
aaaactctaa tgacagaaaa tctgacatga tgccttataa ttagcctcat 960gttctacata
aatcctaaca atagcacttt gtttct
99643999DNAUnknownPromoter and/or promoter control element
identified from Arabidopsis thaliana or Oryza sativa. 43tgcaaaattg
aaaaattgaa gggtgagaca aatttaaaga taatatctat taaatcctct 60aattttaaaa
atttagcaaa aattgtattt tcttatggat cagttagttc acacgtatct 120tagttagtat
caaatcatat ctaatgatta gtgataaaac tagttagata tctatatgtg 180tctttaccat
ttaacttgaa tccttcttct ttttttacgt aaacaacttg aatccttcgt 240taatatataa
atttaaagca ttttttcttt aattctattg atcggtatat atttactata 300agttttagct
catatgcaat ttcaaatgat atgcttttaa attttgtcta ggtgtgatag 360ttgtatcttt
aacataaatc ttatagcaaa actatacttg atattctaaa tttatctatt 420tgctcttgtg
aacctcatat tagtctagag aaactttgaa atcctttcaa ttagttgtat 480gtccaataca
tttttactaa catttattag tctttttaat taagattatt gttagaaaaa 540aaaagatttt
ttaaaaataa ataatatgtt ttagatacaa tgtgagttag gcttcttata 600ttttaaaaaa
taaatttatt tcatacttaa aaatagtttg gaatttcaat ttatttggct 660gaataccata
aaatatgtca atttgaacct tatacccatt gactatttgg tgttagaaac 720cctttaacaa
aaaaaaacta tttggtgtta gatatcaaaa taaaaaaaaa ttaaccattg 780gtttcttata
ttgaattgga tattgttaca tgtattaaag tttttttggt ttaattttga 840aacgttgata
gaaactatta agtttaagtt tggtagtata tttatttgtg gaaaatttaa 900ttgccattaa
atataacgtc aacttttttt gttttttttt gagaagttac gttgtgattt 960tgatttccta
tataaaagtt agattacgtc attttttaa
99944999DNAUnknownPromoter and/or promoter control element
identified from Arabidopsis thaliana or Oryza sativa. 44tatttttata
aattatctta gtaaaagtat gtattttcta atagatctgt tagttcatac 60atatcttaat
tagtgttaaa ttagatctaa tgattagtga taaagttttt agatatcgat 120ataggtgtct
ttaccattta acttgaatcc tttgttaatg taaaatttta aaatattttg 180ctttgattct
acttattggt atataatttt aacatatcaa tccaatgcca ctcttaaatt 240atcatgtact
tttcgatata tgttatgact cacttgttat gaaacgatgg attttcacca 300attttggtta
tttattaact agaagtttta gctctagtgc aattttaaat aatatgcttt 360taaaattggt
ctagttataa tagttgtatc tataacataa aacttataac aaaactatac 420ttgatattca
aaaattattg attttctctt gtgaacttca tattagccta gagaaacttt 480gaaaaccttt
caataaattg tatgtcgaat aaagttttac aaacatttat tagccatttc 540gattaagact
attgtgagca aaagtttttt ttattataaa ataaataatt tgtttaagat 600aaattgtgaa
ttaggcttct tatattttaa aaattatata aatttatact gaaaaattgt 660tagaattttc
aaattttaaa tttatttggc ttaagaacat aaatatgtca atttgaacct 720tatacccact
aaatattcca tgttagatat ctaaataaaa gaaaattaac tattgatttc 780ttatattgaa
ttggatattg ttacttgtat ttatgttttt tgtttcattt ttaaacgttg 840ataaaatcat
taaactaaag ttttgtagta tatttatttg tcgaaaattt attcccatta 900aatataacgt
taaatttatt tgtctttatt aaaaaagtta ctttgtgatt ttgatttcct 960atataaaatt
tagataactt caattttcaa ataaaaaat
99945999DNAUnknownPromoter and/or promoter control element
identified from Arabidopsis thaliana or Oryza sativa. 45ttagctgaac
caggaaattg atctcttata ccagtttccg ggtttagatt ggtttgatgg 60cgatttgatt
aaacccccga aattttatgt cgtagttgtg catagtatta ttattctttg 120cggacaatag
acgtatcggg accaagttct gtagcaaaat tgtataagct taagtttgat 180gaaatttaaa
ggtaatcact aaaacccaaa tgggacaata aaccggtgaa gatttagagt 240ttttaatttt
gactcatgaa tctggagaaa gagccctcgt taaaaggagt gaatcaatcc 300ataggggaaa
aagttttgtc tttttaaaaa ctaaagaacc aaaccttaat agaagcagct 360caatgtgtga
caactttcca ctggcactaa gataaagtga ctagcgatga gtgcaattat 420tgaaatagta
gatggtaaat attacataca agagtaaaaa tatctttatg tcaatgctta 480attcagtgtt
tctggttaac aagagaaact tctctaactt tcgtaattgg gtcttataaa 540attttatgca
attatgattt taccctttta ctacttttca ttagctttca cgaatctatt 600ttgacaagag
aaatcattag aggtaaacat gctttttggt caagggcctt aacagttcca 660ccaatcaagc
tcaaaagttg tacttaaccg acatcttctg tgaaaacata taattacatg 720tacaaatcaa
aactacctta tgaaataaat agaaatattg cagttcattt ctaatttaac 780ctcttcaact
tttaaaacta tttacatttc tttatgtcat ttctagtcat tttgatgcaa 840attgtaccat
ttatggatta tcttcacaaa tttttaagtt ggtgaaaact ttttggtggg 900tagttaaaac
ttgaaataga aatttacttt accaaaataa actaatgaaa agtaatcact 960ccactcccta
taataagatt tccaacgttc ccactaagc
99946999DNAUnknownPromoter and/or promoter control element
identified from Arabidopsis thaliana or Oryza sativa. 46cctactttag
gcttaaacaa gaagaaaata tgactgctaa gtcatatttt tcaactctca 60tgagcaaccg
taaagttgca ccgcaatatc caacaaatga cattcgtgtt atctacaatc 120taatgttgaa
aatttggctc atctaataaa ggagacaaaa gttatatctc tttcacacac 180acgttaatgg
aagtgtaaag gcggtgagag tgtgggagag acttggggaa caagaagaag 240gacgcggtca
aaaagtgacg gtgggctacg gcttttcttg gtagcagttg gaaattccat 300taatgactta
aaaagtgtaa atcttatctt ctttttattt tgtgatttga tatgcacatt 360catttcatga
aaatatttgt atagtttgat gatcatacga caaacttata gggttcacaa 420agtagatgca
atagttgcat acctctgttt aaatgttctt gttaatatta tacttgatga 480tgaaactcgt
gaatgttatt caaaatgtcc atgtaataca agatcatgca ctataataag 540taatctatca
atttcagcac aacaattttg acaaaaagta aaaataaaat aaaataaact 600gatatcatat
ttccgaatta tatgtaaacg ttttctgttt ctcaatggtc tctttcactc 660ttgtgttttc
taatatttca tttaaaccta tttctaaact aagcacatct ttgttgattg 720attgcatttc
aaccaaaatc gataaccgaa tcattgtttt tttatgtttt atttcagctt 780accacacacg
tttagaattt taaaaataaa acaaaaaaaa gttaactcgt tacaaatgaa 840aatgatattt
ttaattggac tcgatggaaa ggaccaattt attcaacact attgtttagt 900ccgaacactt
gccgcgtaag ttttccaact ccccccattg acctttcgca ctttcacaaa 960ctccgtatat
atataatgga tacactctct ctttgatct
99947999DNAUnknownPromoter and/or promoter control element
identified from Arabidopsis thaliana or Oryza sativa. 47tgtgtgtcct
aaatagtttc tttttaaaat ttgtaaatac caagacgcgt atttaagagt 60attttgaaaa
gatatttgat tataaaaaga aagaaaaaga gaaggctgag gattaactgc 120aacgtctacc
gttggaaaag aaaaacgatc agaaaacaca gaaattaata aaaagagaga 180aaaaaaaata
gagtatgaga gatgcacatg ggtgcctgca aaaaaaaggt agaagaaatt 240tgtctgaaag
tgtcacaggc acactctctc gaaccacatt taacaacact ccaaacactc 300ttcttctact
ttgtaccctt cagtacatta ctctttccaa agtccgtgat ttacgctctt 360cgatgacacc
tctcaacaga gagagactac atgtgtacat tttcttctac cattaaattt 420tgaagatttt
cgatgattca atttagtata tatatggaag ataaaatttt cattgtcttt 480ctacatgata
gtaacggttt tagaagggtg gttatcactt atagtatttg agttaagaaa 540tataaaaata
tacgtgactg tttttccttg taaactattt ttaggccctt atttttattc 600aagtagtcac
atacgtgttt gaagtgtatt taactaagaa aaagaaagta ggaaatgaaa 660aggatatagt
atttatggtg taatcttggt aaggaccagg agatcagaag gggccacaat 720gtcacaaaga
ggaccaacaa tgaaattaaa tcctcagctg gcctttaaca ttttggctcc 780caccatctcc
ttccacacat atgcacatgt cttcatgtct ctctctctct atacgttacc 840tacacaaata
tgtacagaca aatagcccat tacaaaatct ttatttataa atatatactc 900ctcaactccc
tcaatatcca cccatctcct tctccataac tctctctctc tctccctaaa 960cacaaccaaa
gacttttatc tctcaggaac cccaaaaac
99948999DNAUnknownPromoter and/or promoter control element
identified from Arabidopsis thaliana or Oryza sativa. 48tcctcctact
gtctgctacg tcaacaagtg gattgcaatc agacggtgat tgtgtctctt 60ttcattctct
ctcttttact aatttctctg ataattaaac tgagaatgta tattaagaaa 120aaaaaacaaa
aacaagagag gaattttcat acacactaac ttaagactct ttgtaagttt 180tcccaaatat
ggattttcta gtataaatat gagttcatta gtttcaccaa gcctacaagc 240atctctccat
ctcaaatcat attcacctaa aaatcaggtc ccctctcttt atatctctaa 300cattcttata
tcagatcata ttttttggat ttcttgttaa gtaacaccaa tcttttaaaa 360gtgttttcag
gttaatataa aagaataatg atgttttcgg tgacggttgc gatccttgtt 420tgtcttattg
gctacattta ccgatcattt aagcctccac caccgcgaat ctgcggccat 480cctaacggtc
ctccggttac ttctccgaga atcaagctca gtgatggaag atatcttgct 540tatagagaat
ctggggttga tagagacaat gctaactaca agatcattgt cgttcatggc 600ttcaacagct
ccaaagacac tgaatttccc atccctaagg ttcactctta ttctcaatat 660taactctcgt
acatgtcaca tgcccatttt caccatttta gatatacagt tttgatactt 720tactttgcat
ttattttgct atatgtaatt gaggatattg ttttaatttc tttgggtttt 780ttttttggct
aaatgagaat tcagtgtctt tggttcttaa aaaaaaagta tttgttaatg 840gtaaacgcta
aacgctattt gagtttatgt tttttcaaga actgaaaacg ttttattgaa 900aatatacact
ttttttgcta tttatagaaa ggcatatcac atctagacgc aaacgcaaaa 960ttgacttttg
aagcaaccac aatcttaaat gcaatgaaa
99949999DNAUnknownPromoter and/or promoter control element
identified from Arabidopsis thaliana or Oryza sativa. 49aactaattag
gtcgttaatt gtccaagggt ttttcatagt tgatatagtt ctgttcaaat 60atagccatcc
ttaatcgatt catgggatcg taaattacta cttcgagtgt tgtaaaaaaa 120aatgaaactt
ctacattaca aactcgaatt taatgcatct ggagtgatac tataaaagta 180gggatgctct
caggtcgcat ttgagagaca cagaaatgat tttaatggaa ttaatatatt 240ttcagttttt
cacaaaaaaa aattgtgttt ataacaactg cagattcaat gctgatttta 300tgagtctcac
ctatagaatt tatatttcta tattcataga ggcagtatag gtgttgaccc 360aacatcgaaa
gaacacttcg taaaaaattc tttggaacaa ggctgaaaat ttactcccaa 420atttagctat
ccgatgaaga taaatcattt accgtttatt aaagaattat cgagatttta 480gtccaaacca
aaagagatta tgagcctaag attttgaatt tgtattggta aaagaaattg 540aacgaaaatt
tcagaaaaaa atattaataa attgaacgat agagttcact tactacatag 600tcaactagtg
cctagctata atagtttcaa aagacaaaaa aaaacaaaat cggttaacta 660cttccgtgac
ataattctca ttttgatttt tgaatccagt ctaatttgaa aagtatattc 720aaaatcttta
aatccattaa tgataacttt tataatacgt tgacacacgc aattgtatat 780acaatattct
tgaattttaa atgtaaattc tagaatatat tgcgatcacc acactaatca 840aaatctttgg
gacaacttga acccacattt gacttttctt ggtcaaatat tttggcatca 900tgcatgatct
tctctataaa aaccaaaagg cctcaacgac attcataaac tcagtcatta 960tatttatttt
tgttgtattt caacgttcaa tctctgaaa
999501823DNAUnknownPromoter and/or promoter control element
identified from Arabidopsis thaliana or Oryza sativa. 50gtctcttaaa
aaggatgaac aaacacgaaa ctggtggatt atacaaatgt cgccttatac 60atatatcggt
tattggccaa aagagctatt ttaccttatg gataatggtg ctactatggt 120tggagttgga
ggtgtagttc aggcttcacc ttctggttta agccctccaa tgggtaatgg 180taaatttccg
gcaaaaggtc ctttgagatc agccatgttt tccaatgttg atgtcttata 240ttccaagtat
gagaaaggta aaataaatgc gtttcctata gtggagttgc tagatagtag 300tagatgttat
gggctacgaa ttggtaagag agttcgattt tggactagtc cactcggata 360ctttttcaat
tatggtggtc ctggaggaat ctcttgtgga gtttgatatt tgcgagtata 420atctttgaac
ttgtgtagat tgtacccaaa accgaaaaca tatcctatat aaatttcatt 480atgagagtaa
aattgtttgt tttatgtatc atttctcaac tgtgattgag ttgactattg 540aaaacatatc
ttagataagt ttcgttatga gagttaatga tgattgatga catacacact 600cctttatgat
ggtgattcaa cgttttggag aaaatttatt tataatctct cataaattct 660ccgttattag
ttgaataaaa tcttaaatgt ctcctttaac catagcaaac caacttaaaa 720atttagattt
taaagttaag atggatattg tgattcaacg attaattatc gtaatgcata 780ttgattatgt
aaaataaaat ctaactaccg gaatttattc aataactcca ttgtgtgact 840gcatttaaat
atatgtttta tgtcccatta attaggctgt aatttcgatt tatcaattta 900tatactagta
ttaatttaat tccatagatt tatcaaagcc aactcatgac ggctagggtt 960ttccgtcacc
ttttcgatca tcaagagagt ttttttataa aaaaatttat acaattatac 1020aatttcttaa
ccaaacaaca cataattata agctatttaa catttcaaat tgaaaaaaaa 1080aatgtatgag
aattttgtgg atccattttt gtaattcttt gttgggtaaa ttcacaacca 1140aaaaaataga
aaggcccaaa acgcgtaagg gcaaattagt aaaagtagaa ccacaaagag 1200aaagcgaaaa
ccctagacac ctcgtagcta taagtaccct cgagtcgacc aggattaggg 1260tgcgctctca
tatttctcac attttcgtag ccgcaagact cctttcagat tcttacttgc 1320aggttagata
ttttctctct ttagtgtctc cgatcttcat cttcttatga ttattgtagc 1380tgtttagggt
ttagattctt agttttagct ctatattgac tgtgattatc gcttattctt 1440tgctgttgtt
atactgcttt tgattctcta gctttagatc cgtttactcg tcgatcaata 1500ttgttcctat
tgagtctgat gtataatcct ctgattaatt gatagcgttt agttttgata 1560tcgtcttcgc
atgtttttta tcatgtcgat ctgtatctgc tctggttata gttgattctg 1620atgtatttgg
ttggtgatgt tccttagatt tgatatacct gttgtctcgt ggtttgatat 1680gatagctcaa
ctggtgatat gtggttttgt ttcagtggat ctgtgtttga ttatattgtt 1740gacgttttgg
ttgttgtata gttgatggtt gatgtatttt tgttgattct gatgtttcga 1800tttttgtttt
tgttttgaca gct
1823511539DNAUnknownPromoter and/or promoter control element
identified from Arabidopsis thaliana or Oryza sativa. 51aagcttatgt
caaaaatatt taattaaaat atatgtaatt tatatgttga ttgagttatg 60agtatcaagt
aaaaacccta atccgttatt aaaatatcaa tgattataac gtatttataa 120acgaaaaaaa
aaagaacatc tagaattttc gatatttgat cctcaagtta aacttggaaa 180aatttggatg
tatgaaatat tttgtcgtcc acttatacaa taaagtatga aacatggatg 240catgaaggct
agacatccaa tgtctaaaaa tactatatat aatgcttttg gtagggtctt 300ttctttatca
tgtctcactt ctgtttctat ccctcatttt aaatagccaa tataatttca 360ctctttacta
taaaattatt atataaacat cattttgatt gaactaccta aaaggaagaa 420acgtatagga
atttttggag cctcaagatt gtaataatgt ctcatagttt gacttgcaaa 480agctaaatta
aacgcctaaa tcattaccat taaataaatg aacttttgta cgcaattgat 540tcagacacaa
ggaccgacca attcgaaaac aatgaatgga tatgattcat ccttatgaaa 600gcttgacaac
aaactcggtt ttggctggtt aacctagact cggtttattt aaaccagaca 660ataatttctt
tcgtcgtcgt tttatttgaa taggtgcgtc aaaaataaaa gctgaaattc 720ttggttgcaa
aagcccaaca ggcctgtgga gatagctttt tagattgatt aaatgggccg 780aattgggctg
acacatgacg agaatgtggc tatagaaatt gttagtgaga gggtccgggt 840ccaaaaatgt
tgcagaagtg atatagtatt tatttaatta aaaacatatt attcgacgta 900tttttaacgc
tcactggatt tataagtaga gattttttgt gtctcacaaa aacaaaaaaa 960tcatcgtgaa
acgttcgaag gccattttct ttggacgacc atcggcgtta aggagagagc 1020ttagatctcg
tgccgtcgtg cgacgttgtt ttccggtacg tttattcctg ttgattcctt 1080ctctgtctct
ctcgattcac tgctacttct gtttggattc ctttcgcgcg atctctggat 1140ccgtgcgtta
ttcattggct cgtcgttttc agatctgttg cgtttcttct gttttctgtt 1200atgagtggat
gcgttttctt gtgattcgct tgtttgtaat gctggatctg tatctgcgtc 1260gtgggaattc
aaagtgatag tagttgatat tttttccaga tcaggcatgt tctcgtataa 1320tcaggtctaa
tggttgatga ttctgcggaa ttatagatct aagatcttga ttgatttaga 1380tttgaggata
tgaatgagat tcgtaggtcc acaaaggtct tgttatctct gctgctagat 1440agatgattat
ccaattgcgt ttcgtagtta tttttatgga ttcaaggaat tgcgtgtaat 1500tgagagtttt
actctgtttt gtgaacaggc ttgatcaaa
1539521954DNAUnknownPromoter and/or promoter control element
identified from Arabidopsis thaliana or Oryza sativa. 52gtgggtaaaa
gtatccttct ttgtgcattt ggtattttta agcatgtaat aagaaaaacc 60aaaatagacg
gctggtattt aataaaagga gactaatgta tgtatagtat atgatttgtg 120tggaatataa
taaagttgta aaatatagat gtgaagcgag tatctatctt ttgactttca 180aaggtgatcg
atcgtgttct ttgtgatagt tttggtcgtc ggtctacaag tcaacaacca 240ccttgaagtt
ttcgcgtctc ggtttcctct tcgcatctgg tatccaatag catacatata 300ccagtgcgga
aaatggcgaa gactagtggg cttgaaccat aaggtttggc cccaatacgg 360attccaaaca
acaagcctag cgcagtcttt tgggatgcat aagactaaac tgtcgcagtg 420atagacgtaa
gatatatcga cttgattgga atcgtctaag ctaataagtt taccttgacc 480gtttatagtt
gcgtcaacgt ccttatggag attgatgccc atcaaataaa cctgaaaatc 540catcaccatg
accaccataa actcccttgc tgccgctgct ttggcttgag caaggtgttt 600ccttgtaaag
ctccgatctt tggataaagt gttccacttt ttgcaagtag ctctgacccc 660tctcagagat
gtcaccggaa tcttagacag aacctcctct gccaaatcac ttggaagatc 720ggacaatgtc
atcatttttg caggtaattt ctccttcgtt gctgctttgg cttgagcacg 780gtgcttcttt
gtaaagctcc gatctttgga taagagcgga tcggaatcct ctaggaggtg 840ccagtccctt
gacctattaa tttatagaag gttttagtgt attttgttcc aatttcttct 900ctaacttaac
aaataacaac tgcctcatag tcatgggctt caaattttat cgcttggtgt 960atttcgttat
ttgcaaggcc ttggcccatt ttgagcccaa taactaaatc tagccttttc 1020agaccggaca
tgaacttcgc atattggcgt aactgtgcag ttttaccttt ttcggatcag 1080acaagatcag
atttagacca cccaacaata gtcagtcata tttgacaacc taagctagcc 1140gacactacta
aaaagcaaac aaaagaagaa ttctatgttg tcattttacc ggtggcaagt 1200ggacccttct
ataaaagagt aaagagacag cctgtgtgtg tataatctct aattatgttc 1260accgacacaa
tcacacaaac ccttctctaa tcacacaact tcttcatgat ttacgacatt 1320aattatcatt
aactctttaa attcacttta catgctcaaa aatatctaat ttgcagcatt 1380aatttgagta
ccgataacta ttattataat cgtcgtgatt cgcaatcttc ttcattagat 1440gctgtcaagt
tgtactcgca cgcggtggtc cagtgaagca aatccaacgg tttaaaacct 1500tcttacattt
ctagatctaa tctgaaccgt cagatatcta gatctcattg tctgaacaca 1560gttagatgaa
actgggaatg aatctggacg aaattacgat cttacaccaa ccccctcgac 1620gagctcgtat
atataaagct tatacgctcc tccttcacct tcgtactact actaccacca 1680catttcttta
gctcaacctt cattactaat ctccttttaa ggtatgttca cttttcttcg 1740attcatactt
tctcaagatt cctgcatttc tgtagaattt gaaccaagtg tcgatttttg 1800tttgagagaa
gtgttgattt atagatctgg ttattgaatc tagattccaa tttttaattg 1860attcgagttt
gttatgtgtg tttatactac ttctcattga tcttgtttga tttctctgct 1920ctgtattagg
tttctttcgt gaatcagatc ggaa
195453999DNAUnknownPromoter and/or promoter control element
identified from Arabidopsis thaliana or Oryza sativa. 53tagaaacctc
aacttgaata taatagtttg tttgtttgct tgaagttaat ctctctcttt 60tttatcagct
aaagctgcat ttataaaaat tctagtttaa cttttaccat ttgctataat 120ttagagattt
taacaagaaa tctggcctag tccgcaaaac ttatagaata aatcaaacat 180tcttcaatat
tttacacatc cacaataccc aatccaagaa atggattgca gttgcaccag 240agattacatg
tctcgtttta gtttgctagt cactcaaact cacaaagcat aaattgtaat 300agaaaataga
actttattta aatctgaaga aagatatata taaaaaaaaa aaaaaaagaa 360gaagcaggct
ccagttttga tgggagaaga aaagagagct ggcaacagct attcactgat 420agaccgatca
ctctcttctg tcccgcactc ttttcttctt ttgtttcttt cttttcgaca 480gctttcattt
ttcctccatt tttaaatttg aattatttta cagtcataaa agtacttcaa 540acgtatatgt
aaataacgag caacaaaaca attaactaca acaaaactag ttctagctaa 600gagaattagt
tagaaatttt tattataata gttagtatat gtttattcat aacacaatta 660attaacacac
aaaatacatg taatttcctc tataccctct tcacatataa ttagagtagt 720gctttaattt
aagattaatt atcgatttac atcattaatg atcatctagt cttacacaga 780gagtttcagt
atctgcatga gattatataa aggaatgtat tcatgttttt acttcttttt 840attcatggtt
aaggatgata cattataatt ataaatccat aatctatgaa ctcaactatt 900ctttataaaa
aaggaattaa attctgaaaa taaacaactg tagttggctt cccaaggctg 960ctgcttcacc
tataaatacc ctatcctctt tgaaaactc
99954999DNAUnknownPromoter and/or promoter control element
identified from Arabidopsis thaliana or Oryza sativa. 54ataggcccta
cttctaatta aagcccattt acttctctcc ttgtcttctt attcctcttt 60tctccccatc
acgtgacgac gatgctataa acgccgtcgg attatataac tggtgccgtt 120gacaagacgg
cgacagaaga aagaaagaag aaaccacagg ctctagggaa cgtaacgtta 180tgtcctgtct
atagcattta taacggtcag atcaacgccg tttagataaa gatctgtcaa 240tgttaaagaa
gagatgcatc tctacaccgt taaatttaaa acgccgtgaa cctcttatct 300attgattttt
gtttgatgaa gccaaaacaa atcgtgtcag aagacttatc agagaagaag 360aaaacgacga
cgttcccgtt tctccatgtc taataagtgt agtagtggcg gctactaaaa 420actctaaagt
ttgactccag taaaactgcc tttctagtgt aattccagtg attttagagt 480ttgaatagtg
tgtgaccaaa tttgaaagta caatctcagc aatattattg atcactcgtt 540ataaaagaat
cgaatgtaaa aatagccaat gagagactga gacgtatgtg tttgaccata 600agtcgtatag
tttgtatcta tctacctgca agatcagcag atggttctct gatcaattgt 660accttaatta
tcttttattt tcgtaaaatt tctctattca caaatgataa atctacttaa 720gacagtaacc
ataacaagat ttacaagata atttgaaaaa tgaacacata aaagtatttt 780ggcgcattat
ttttaataat aacaatattt atgtaaagtc acataaaagt atatattcgc 840tcacaaagtc
ttacggtatt tagaacagta gtaccacatc gattctcttc atcttcttct 900tcataatatg
ccattgttca tgtctctgtg tcctatcgca taacactcac gctatcttat 960tattttctct
cgctctttct cactgagagg acactaaaa
99955999DNAUnknownPromoter and/or promoter control element
identified from Arabidopsis thaliana or Oryza sativa. 55aagcggcaat
ttagtaagaa gtactcaaag tatcatttac caaaagtata tggttttggg 60aagagttgtt
agggatgtat tctttctaaa cagatgatat gacgatgttc ttgaaaacta 120atgttaaaga
cggaatctct ggcatcttca ctcgggagat atattaaacc gttgattgta 180gttagccatg
tacttagctt agtgcacaaa taatctgctg caagaaatct ttttctatta 240taatatctct
catttaaaca ttagaacata ttgtttaact tgttcttcta gaaataaaac 300tgctaatttc
ttatggtaaa ctattttcct ttagattgca caatcgaact cgaaaatcta 360gtggagacta
tgtgactatg tttatatata tgaaacctaa atcaaattat cccaataatt 420gggagacaca
aaagaaaaat tacgaaagaa aacaggaaat caaatcaaaa gataaagaga 480aggtaaaaaa
aggcaagaag cactaatgtt taatatttat agttttctcc attaaagaaa 540aagcgatgat
gtgtgttctc atcttttgtg aaagtatata tattgctttt gcttttctca 600aaagcaaaag
actcatccaa caagaacaaa aaaaaaaact aaagctcaat ccaaaagacg 660aagaatgcat
tggatactac aacttctttt tcacttttct ttcaaattta caattatgat 720tttcacaata
cagtttattc aaaaataaat aaaaaaacga ggcatgaaaa taatgattat 780cctcttcact
tattaagcca ctcactataa gcagagcaac tccagaacat agtgagcccc 840caaaacatta
aagcatgatg atgtctaatg atgatgatct tcttcgttcc atttctctaa 900atttttggga
tttctgcgaa gacccttctt ctctttctct tctctgaact tcaagattcg 960tgtcggacaa
atttttgttt ttatttttct gatgttaca
99956999DNAUnknownPromoter and/or promoter control element
identified from Arabidopsis thaliana or Oryza sativa. 56tagtttttga
tttaatctac gtttttctta atcataaatg ggtaattatt agtttttgca 60aaatcaaaat
ccaaaaattg ttctaaacac tgcaaccatt taaggcctat atcactcaga 120aaatttctgg
tgggagaact aatcgtttgt cctttctaaa tctcacatat tagaatttag 180aattagtgtg
ctacataaaa atattagttc agctcggaac aactattttt tggtaaaaca 240gagaacttaa
acaaatgcat tattttatca acatgcattt tgaattgaat ataaaatttc 300ataattgtaa
agacataaat tacataaaat tttacatgaa aaaatagata tagaaagaaa 360atgaaactaa
ctgatgatat gctctctaaa ttttttaatc tcataacaag aattcaaatt 420aattagttca
tatttttggt taatataaca tttacctgtc taagttggaa ctttcatttt 480ttttctgttt
tgtttagtca gtattcttaa tgtgaaacgg aaagttgaat ttattcaaac 540ttaaattcaa
tagcattaat taaaggcgaa agctattatc tctacatgtg gttcaaacta 600gacatccaat
ttaattagct tattgacgtt gaaatgtttt ccaaaactac tatagtttgg 660caatttgaaa
gatgcatcag aactactcag acaggtaaaa gtagaacctc tagctgtgtg 720aattgtatgt
tagtccataa agaacatctt gtaaacttca tacttaagat atatattaca 780atatatactt
gaatggtaga taaaaacgat tagtctgatt gctagcatac tcacaactat 840ttggaaatga
gtaagatatt ggcatctaga gttactacta tggagacaaa agtcgaataa 900aagagacctc
acgtgaaaat gttacgagct agtaactaaa gcatttacac taacggtaaa 960aaaagtatct
ataaatgttt acacaaggta gtagtcatt
99957999DNAUnknownPromoter and/or promoter control element
identified from Arabidopsis thaliana or Oryza sativa. 57agtttaatta
tttgttatct atccaatcaa ttttttttct aaactgtttg gaccaatgta 60cgtacgtacc
atcctttttg attttttttg taaactaaat tttcgaatta gcaggttctt 120aataattgaa
cgaagaaaat aaagaataga ggtagacacc tgtagtattt tcttggtcag 180accaataatt
tataattcaa cgtcaaagaa gaagaaaaat ataaaccatt atttcattat 240gacttacgta
taccaaaata cacaaattaa atgtataatt gtgaggcatt ttatatgcgg 300gaaaaaataa
aataaaaaga atattaatat ttcttttgaa aattgtaaag cattttgacc 360cacttgtgat
atatatatat atatagatat atatagagag agagattaaa acattgatgg 420ctagctatag
agtctatggc agggtcatga tcacctgtct tctgatctct gaagagatac 480caatctgatt
ttttctcttc ctaggtttaa ttttatttta ccattttata attctttatt 540tttgcctgta
gtacaattta cagacccata ctaaaagaaa aattaaattt tgtcaaagta 600caaaacaaag
agagaggtga agccacacaa tctcttttct tctctctctc tctgttatat 660ctcttctgtt
taattctttt attcttcttc gtctatcttc tcctataatc tcttctctct 720ccctcttcac
ctaaagaata agaagaaaaa taattcacat ctttatgcaa actactttct 780tgtagggttt
taggagctat ctctattgtc ttggttctga tacaaagttt tgtaattttc 840atggtatgag
aagatttgcc tttctatttt gtttattggt tctttttaac tttttcttgg 900agatgggttc
ttgtagatct taatgaaact tctgtttttg tcccaaaaag agttttcttt 960tttcttctct
tctttttggg ttttcaattc ttgagagac
99958999DNAUnknownPromoter and/or promoter control element
identified from Arabidopsis thaliana or Oryza sativa. 58ttttgtttct
aatagtttga tgtttatatc aacattatta tttactttca tttgttaccg 60atagaaagag
gagaaaattg ttgacaaaaa caaagaaaaa agtaaaatta atattattaa 120attaataaaa
ataacaaact gtaaaagcta tttttaaaaa tttttcttgt aaaacatcta 180aaaattattc
ttgtagaaac agaggaatat cattgaagat aatagtgtga aattatatat 240atatatagaa
atatataaag taggattttt ttctgtatac aaatatacgt ttccaatttt 300atcaaaaact
gtaaagattt ttttctttgt cagtacctgc taaacttgtt aattttttta 360ttaaaaaaaa
atcaaattac aattcttcta taatcatttt aaattccatt tctttatacc 420acaaaagatt
atattgcctt tatcgtcttt ggtatgtatg cgtgaatata tttatttatt 480ttcttttctt
tcattttctt tttaaagaac tttataaatg aaataaggaa caaacaatat 540acacatgtac
taacgtatat aaataatatc atcaatatct atccaaaact tggatttcat 600ggttgacgtg
gcccaaccaa aatctcaagt tctctgcgga tgacgaacca tctcaccatc 660tctttttttc
tctctctttt ttttttttaa tatcatcagc acggttacat aaaattcgtg 720atccatgaag
ttggctttct tgtcgtttta cttcatcacc ccattttttt aaagtctcca 780tctttatact
tcttcaactc tccaccacca ccattgtcac caccacattt aaacacacac 840tttcacttgt
agtgggattc gaaagtgcgt tttattcatt tgttttactg tttttgataa 900cctcaaaatt
tgcctaaatt ttattctcta taaatcctta tatgttttac ttacattcct 960aaagttttca
actttcttga gcttcaaaaa gtacctcca
99959999DNAUnknownPromoter and/or promoter control element
identified from Arabidopsis thaliana or Oryza sativa. 59ataaaattat
ctataaatca ttaaatcttt gatgagaaat atccaatcta ctaatgtata 60tcgatgattt
aaatgaaatt acttatttga acacaaaaat aaatgaattt actaataaat 120aaatagcgta
gttggagcaa gtggctaaaa aaattacaaa tctagtttcc attctcagcg 180tcggctgctt
ggaacgtcac cgttttctgg aaaacgcaat cttctccctt ccgtgacgtc 240tcaccggaat
tttctcgctt ttgtctactc tcctccatct ccgaggttct ccaagctcag 300ctcctcttcc
catcattcat ccgaccgcct tatccggtca gatcctttac gtatttctat 360tttcctgatc
gtcgattttt gagaaatgta aaaacagatc gtataaggcc tcgaagtttt 420taatttgaaa
gtggtatcga aattttttgg tctttgatta ggttagggca ccgtagctct 480gggtattgaa
tttgtagggt tttcctctgg ttattggtct ttggagcttg gtaatttctg 540ctgaattgat
tgatcccttt tccatctttt gaagtaaagt ctcgagcttt cgtgtctcga 600tgtagatgaa
ttctattttg aatatgagat ttgataagac gtcaattgct gataatttgg 660agtctttgtg
tctgaatttg ttcatatgaa gttttctgag ggatgtgaat tttattgtct 720gctaattttg
aaacgttcct tttggaattt ggtttgtgag gagtcctaga tctttttctg 780tgaagtttct
tgcttgtaag ttttctggat cacttgattg agtctagaat ctagatagat 840tacatgtacg
gtttgattcc tttggctgat tttccaaagt tttgttcaaa tttcaggaga 900actacaaaga
ggaaaccaag attgttttgt tttgttagac tctacccctt ttccgattca 960catggtaagg
acattgaggt agagaataat actaaaaag
99960998DNAUnknownPromoter and/or promoter control element
identified from Arabidopsis thaliana or Oryza sativa. 60gtcgattggt
tgtaaattag ttttatcgta gaagtaccaa atcaagtgat tcaatggtta 60aattaaggta
ttaagttaca tttgatattt aaaagtatcc agaccttcat tatagctcat 120aagggttaaa
attttgtcgt tcttttgtat attcatggca agctctaatt catgactaag 180tcacattttt
caaatatgtt tttagttttt acttatgttg gtaattagtg gatttatagt 240taagttaaaa
agttggcgag ttctagcttt gaaactcatt tagaaatata tatatatata 300tatatattca
attttagtaa attgttaatc tattctaatg gtgtaactgt aacaaatgag 360aatgaaaaaa
atatactatt gtgaataaaa ccccacacaa cacattacta taataagtta 420aacttctttt
tttataggcg cctggaaaaa aaagaaaagc aacaagaggg stgtgaggac 480gcatcaccng
gtttcgtagc acacatgtgc atttgtctct ttgctttttc ggtttttttc 540ttgccaatca
atttattttg ttcctcagaa aaaagaaaat ctaaaaccaa aatatatatt 600ataacctcat
ttaataaaca acaaaaatgt ttgttgaaaa aaaaaaagtt tttatttatc 660ttgaccttat
ttctttgaag aaaataaagc ttggttatta aagaagtcca agttagttgc 720caccatcagt
ggcataacgg taaattaaag ccaacttcct ctaactaaag ttttctataa 780attcaaccac
tcacctccca ctctaaaacc caacaacata atttcacata tctctctttc 840tttctcttga
aggaaagacg aagatctcca agtcccaagt acgtaactac tttctccatc 900tacattcaat
tgtttctcct taatttctct agtacatatt tacttgtgct ataagtaatt 960gattttatat
cacccatgtg caggttgtta acacaaga
99861999DNAUnknownPromoter and/or promoter control element
identified from Arabidopsis thaliana or Oryza sativa. 61tattatatat
acgattaaat aaataaaaaa attgtaatgt gaaaatatca tagtcgagag 60gggaactgac
aagtgtacat atgtatctag ctgtggattc caccaaaatt ctggcagggc 120catgatctaa
aaactgagac tgcgcgtgtt gttttgcagt gatttgtatt tcatatttgc 180accatcctac
acagtccact tggtatcgta accaaacata aggagaacct aattacatta 240ttgttttaat
ttcgtcaaac tggtttttac cttttagtta catagttgat tcttcatttg 300ttttagtagt
tatggagcac aataatgtgc aacaaagaaa gatcatagtg gattaatatg 360ttgagaggtc
agaaattctt ggttaacaaa aaaaaaaaag ttacaaggac tgagattttg 420ggtgggagaa
agccatagct tttaaaacat gattgaactt aaaagtgatg ttatggtttg 480aggggaaaaa
ggttgatgtc aactaagata gttgaagtaa tgtcttaaac taaagtaaac 540caccggtcca
aacgtggtcc ggaagcatct ctggtatgat ttatcctaaa aatcaaaata 600gtagaaacat
actttaaata tatacattga tcggacgaaa attgtaaact agtatagttt 660caaaaactag
ttgaacaggt tatgtacctt aaacatttat ttcaaactta aacactaaag 720aacatatatg
aatagaagtt tatataaatt actatatatc taccataaat ctcttataat 780tatgatgtca
cgatgaggaa gtgttgaaac gttaaaatgc caaaatataa gcatgcgacg 840gaattttggc
agaagattgt agagttgtaa tctgtcgcaa tcattactca tgctagcatt 900tttcattttc
ccttcatttg tggataacgc acgatataac attctacaca ccaacaagat 960tctataaaaa
cgcaaaggtt gtctccatag aatatcgtc
99962999DNAUnknownPromoter and/or promoter control element
identified from Arabidopsis thaliana or Oryza sativa. 62atctctgatt
ttttttatca ggaacaagta aataaatagc tttgagtttt tgtttttttt 60ctacattctt
cgcccaaaag atgtaagaaa ataaaggatt tgaaaccttg ttctgttgtt 120actcctttaa
attcttaaaa actataaatc attatatctt tgatctgttt cacaaactaa 180tcatattcgt
tgcaaagtga gaattcgtcc cactttactc tttacaccga tactagtatt 240atagatgtac
agcatagtat tccatatcta gttatttagt caaaactcta tatattaaga 300ggtaggttaa
ttaattaagg agtaattgaa gattatagaa agaataaaaa ataccattta 360atggacagaa
ccaaagataa ctaactatca tactataatg ttgaatttct tccacgatcc 420aatgcatgga
taacaacatc aatcaaatca tacattcatg ctatataaca tagttttcag 480ttacaaactc
tcttttttat ttatttcagt tgttcctttt catgaccata ttaacatcaa 540ataatgcatt
tttttcaacg tctcttgact tacacccact aatattgaca aattgaacat 600ctatacgact
atacacacat aagttaaaaa tgcatgcaag tgctaaggga atttataaca 660tctaaggtta
ataagactaa gaaagtataa aataagaata cgtattatga atttatgata 720tactttacta
atctttttga aaaatacttt aatttaatct actatagggg gtaaaaagta 780aaaaagaaat
aaagatacgt ttatccgcat atagtacctg gaaataacag aaaataaaaa 840cacaggtaag
tactttgcct gagctagtat atgaacacta aagagataca cacacacaaa 900aagagagcag
aaacaaaaca cacacactta aagctttcgt ctttacctct tcccttctct 960ctctctatct
aaaaagagtt ccgagaagaa gatcatcat
99963999DNAUnknownPromoter and/or promoter control element
identified from Arabidopsis thaliana or Oryza sativa. 63cgctttatta
taggtttaac aattgatttt tcattatttt gttttcaatc tccaaatcat 60ttctcaataa
ctctcaaaca ttgtttaaag ctttttttct taattaacat tataacaaaa 120aaataaatag
agaaatttac tttgattcaa acaccagtca ttgtagatta gccaagagtt 180ttcagtaaca
aaatttacct tataaacctt ttgaatggct atttctgaaa tggaatagaa 240atctttagtc
gtggaagtat ctctatccat aagaaaactc gttttacaaa gtaattttaa 300atcaatacaa
aaagtgaaaa aatccactgg tggaccccat tcattccaga attgccgatt 360acgagctatc
ttgtcccttc ttcaccattc gctcactctc tctctctctc tctcgtcttc 420ttcttcccac
cactctctct gtttctccac aacttctctt ctcaaagtta aaattacccc 480taaaccaaaa
aaaaaaaaaa cgctcttcac tatttattta ctaaactctc ctttgtttgt 540tactaagctc
tcactaaaac cctaatcttt ctcctcttat atatctcgtg actcttcttt 600ctcctccaat
ctctctctcc ctcttcacaa accaattagc ttctttctgt aaaacctcac 660tcgttggcca
attcttttgg ttttcataca cataaatctc agattccaaa tgggttttct 720tagctctttc
tttcaaatga tgaacatttg ttagcagaat cttcctcttt ccctaaagtt 780ttgatctttt
tttccccctt caattttgta ttttctcacc aaataaaaaa aggtttcttc 840agtgggtttt
aagggtttat tattatctta aaattaaaca caattcttta atcaaaaggc 900aaaaatctta
atttcatcac tctcttctca ctcacaaaag ttcttacaat cttcaaagtt 960ttggtcttgt
ttcttttccg atttcaccgg acaaaaaaa
99964999DNAUnknownPromoter and/or promoter control element
identified from Arabidopsis thaliana or Oryza sativa. 64gggattatat
atgatagacg attgtatttg cgggacattg agatgtttcc gaaaatagtc 60atcaaatatc
aaaccagaat ttgatgtgaa aacactaatt aaaacatata attgacaact 120agactatatc
atttgttaag ttgagcgttg aaagaaaatg aaagagtgta gactgtagta 180tgtatgagtt
tcccaaagga tggtgcttga atattattgg gaagagactt tggttggttc 240ggttgaatga
agatttttac ctgccatgtt gatagagaaa ggcaaataaa tgtaggggtc 300gatgtctaac
gtaaagactg gatcaaccaa gagtcctcct cctcgtcttc accaaaaaaa 360aagagtcctc
ctcgtggaaa cttatttctt ctccagccaa gatctcatct catctcttca 420ctctatgaaa
tataaaggaa tcttatggtt tttctaaaaa ctatagtacg tctatatacc 480aaaggaaaca
atataaaatc agttaatctg ataaattttg agtaaataat aaagttaact 540ttgtacttac
ctatatcaaa ctaattcaca aaataaagta ataataacaa agaattttta 600gtagatccac
aatatacaca cacactatga gaaatcataa tagagaattt taatgatttt 660gtctaactca
tagcaacaag tcgctttggc cgagtggtta aggcgtgtgc ctgctaagta 720catgggctct
gcccgcgaga gttcgaatct ctcaggcgac gtttcttttg ttttcggcca 780taaaggaaaa
agcccaatta acacgtctcg cttataagcc cataaagcaa acaatgggct 840gtctctgtct
cactcacaca cgcgttttcc tactttttga ctatttttat aaccggcggg 900tctgacttaa
ttagggtttt ctttaataat cagacactct ctcactcgtt tcgtcaacat 960tgaacacaga
caaaaccgcg tcacaaaaca aaactcgct
99965882DNAUnknownPromoter and/or promoter control element
identified from Arabidopsis thaliana or Oryza sativa. 65tggatctgct
agatatatga gaacggaaag aaccagaagc tattagaggc gggaggagat 60atgtggggat
gatttcagtg caattccacg acgcaccatt tccactttcg taacacctaa 120acgaccgctt
cggccgtata aaatcgcaaa tgtttggtct cagtgtattt ttccaatttc 180caaatacatc
aattcaaatt atataatatc tagtggcaat tataagtata tcatatattt 240tcaaaattaa
ttaaaaagat tactaaatta tgtttgacta caactattat aatagttaaa 300aacataaaca
aaaacaaaga aactatttta ataaaaaaat caagtaaaca ttaaaacata 360agcaaaaaat
aatgttaaag aaattattaa ttattaattt actaataatt aatacctcta 420taaattaatt
gttagaggtt taacgtaatt tataaggaaa actaaagaag actttaaccc 480ataaagaaaa
aaacaaagac tgaattgaag gcccatattt agaagaagag aaagaagacc 540caaatatgat
ataaaatcca gcccatttat atatttttat tttgtttctg gaaggaaaat 600aagaaaatgg
caaaaacgaa ataatctgaa aaagtaaggt cttttaccaa aaaggatatt 660ttttttataa
acagagcata aagttttcac ttttcttctg ctcctttctc gtctctgtct 720tcttcgtcct
cattcgtttt aaagcatcaa aatttcatca acccaaaata gattaaaaaa 780atctgtagct
ttcgcatgta aatctctctt tgaaggttcc taactcgtta atcgtaactc 840acagtgactc
gttcgagtca aagtctctgt ctttagctca aa
88266999DNAUnknownPromoter and/or promoter control element
identified from Arabidopsis thaliana or Oryza sativa. 66ttgagcctta
ttgttgttat tgacttttag ccaatagaaa gagatggaaa ttcaataatt 60atccacaaaa
ttccaaatca ttggtgtaca aaaagatcta aggctgttat attttcaaaa 120aagaaagaaa
agaaatgcaa caaatatgga ttaaactgtg gtttgtaaat tgagctttgc 180atgaaaactt
tatcactatg atttcactac tccatattta ttgactaaag tggcactaat 240gaatttctta
atcatgaaat cttgtatcaa aaagtactaa aataaacatg acattggcaa 300ttaggaaaat
tctaaattag aaattagtaa aaatgaaagg tgaaagggaa agatgatgat 360atgaattggt
tggtgaccag gagaaatgta tcccgatttt tgcagacact ttcagtgtcc 420ccattcatat
aattatggcc cacctcgtta agatttttca ttcaccacca taacaagatc 480taagcttaga
tttcatgtaa ttaaacatat aatatacttg ccaatactat ctaataaagt 540atacttaagc
aaaaattatt actctagtgt aaggcgatga aatataagtt tagttgaaaa 600tttatgtcga
tataacaaag tataatgaat taagaccttg gttttcgatt aacaaactaa 660ttaaacacta
gttttgccta ataaaaccgg gaatcgtatt caaaaccgaa cgacaaaaca 720agggacaagt
tgagagacaa aaccaaatca gcatctttct tccagaaatg tcatgaccac 780atgacgtcat
cttgaccctt cttcattgtg atatctgtgg ataaagcgca cgtgtttaat 840tcacgaacct
tcgtagtaac gaaaaatcca caactttcat attttttaat tacccactaa 900actaaaacaa
atttggaaaa acatgaaaaa ctttttcttt ttttccaggt tcgtgaacct 960cgtaccctct
atataaacct cttaaccacc ttccacata
9996719DNAArtificial Sequenceoligo(dT)18 primer 67tttttttttt ttttttttv
196819DNAArtificial
Sequenceoligo dTV primer 68tttttttttt ttttttttn
19
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