Patent application title: Cytokinin Oxidase-Like Sequences and Methods of Use
Inventors:
Norbert Brugiere (Johnston, IA, US)
Norbert Brugière (Johnston, IA, US)
Jeffrey E. Habben (Urbandale, IA, US)
Jeffrey E. Habben (Urbandale, IA, US)
Assignees:
PIONEER HI-BRED INTERNATIONAL, INC.
IPC8 Class: AC12N1582FI
USPC Class:
800287
Class name: Multicellular living organisms and unmodified parts thereof and related processes method of introducing a polynucleotide molecule into or rearrangement of genetic material within a plant or plant part the polynucleotide contains a tissue, organ, or cell specific promoter
Publication date: 2013-01-10
Patent application number: 20130014291
Abstract:
Methods and compositions for modulating plant development are provided.
Polynucleotide sequences and amino acid sequences encoding cytokinin
oxidase polypeptides are provided. The sequences can be used in a variety
of methods including modulating root development, modulating floral
development, modulating leaf and/or shoot development, modulating seed
size and/or weight, modulating tolerance under abiotic stress, and
modulating resistance to pathogens. Polynucleotides comprising CKX
promoters are also provided. The promoters can be used to regulate
expression of a sequence of interest. Transformed plants, plant cells,
tissues, and seed are also provided.Claims:
1. An isolated polypeptide comprising an amino acid sequence selected
from the group consisting of: (a) an amino acid sequence comprising SEQ
ID NO: 3, 6, 9, 12, 53, 59 or 62; (b) an amino acid sequence comprising
at least 85% sequence identity to the full length of SEQ ID NO: 3, 6, 9,
12, 53, 59 or 62, wherein said polypeptide has cytokinin oxidase
activity; (c) an amino acid sequence encoded by a nucleotide sequence
that hybridizes under stringent conditions to the complement of SEQ ID
NO: 2, 5, 8, 11, 52, 58 or 61, wherein said stringent conditions comprise
hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37.degree. C., and a
wash in 0.1.times.SSC at 60.degree. C. to 65.degree. C.; and, (d) an
amino acid sequence comprising a fragment of SEQ ID NO: 3, 6, 9, 12, 53,
59 or 62, wherein said polypeptide retains cytokinin oxidase activity.
2. An isolated polynucleotide comprising a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence comprising SEQ ID NO: 1, 2, 4, 5, 7, 8, 10, 11, 51, 52, 57, 58, 60 or 61; (b) a nucleotide sequence encoding an amino acid sequence comprising SEQ ID NO: 3, 6, 9, 12, 53, 59 or 62; (c) a nucleotide sequence comprising at least 85% sequence identity to SEQ ID NO: 1, 2, 4, 5, 7, 8, 10, 11, 51, 52, 57, 58, 60 or 61, or to the coding sequence thereof, wherein said polynucleotide encodes a polypeptide having cytokinin oxidase activity; (d) a nucleotide sequence comprising at least 50 consecutive nucleotides of SEQ ID NO: 1, 2, 4, 5, 7, 8, 10, 11, 51, 52, 57, 58, 60 or 61, or a complement thereof; and, (e) a nucleotide sequence that hybridizes under stringent conditions to the complement of the nucleotide sequence of a), wherein said stringent conditions comprise hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37.degree. C., and a wash in 0.1.times.SSC at 60.degree. C. to 65.degree. C.
3. An expression cassette comprising the polynucleotide of claim 2 operably linked to a promoter that drives expression in a plant.
4. A plant comprising the expression cassette of claim 3.
5. The plant of claim 4, wherein said plant has a modulated cytokinin level when compared to a control plant.
6. The expression cassette of claim 3, wherein said promoter is a tissue-preferred promoter, a constitutive promoter, or an inducible promoter.
7. The plant of claim 4, wherein said plant, when compared to a control plant, displays one or more traits selected from the group consisting of: modulated floral development, modulated root development, an altered shoot-to-root ratio, improved plant vigor, and increased plant biomass.
8. The plant of claim 4, wherein said plant has increased average seed size or increased total seed weight, or both, when compared to a control plant.
9. The plant of claim 4, wherein the stress tolerance of said plant is maintained or improved when compared to a control plant.
10. The plant of claim 9, wherein said plant is Zea mays and tip kernel abortion is reduced.
11. A transformed seed of the plant of claim 4.
12. A plant that is genetically modified to affect expression of a native genomic locus, said genomic locus encoding a polypeptide selected from the group consisting of: (a) an amino acid sequence comprising SEQ ID NO: 3, 6, 9, 12, 53, 59 or 62; and (b) an amino acid sequence comprising at least 85% sequence identity to the entire length of SEQ ID NO: 6, 9, 12, 53, 59 or 62, wherein said polypeptide has cytokinin oxidase activity; wherein said plant is genetically modified to reduce the level or activity of said polypeptide.
13. A method for modulating the level or activity of cytokinin oxidase in a plant or plant part, comprising introducing into said plant a polynucleotide comprising a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence comprising SEQ ID NO: 2, 5, 8, 11, 52, 58 or 61; (b) a nucleotide sequence encoding an amino acid sequence comprising SEQ ID NO: 3, 6, 9, 12, 53, 59 or 62; (c) a nucleotide sequence having at least 85% sequence identity to the full length of SEQ ID NO: 2, 5, 8, 11, 52, 58 or 61, wherein said polynucleotide encodes a polypeptide having cytokinin oxidase activity; and (d) a nucleotide sequence that hybridizes under stringent conditions to the complement of the polynucleotide of a), wherein said stringent conditions comprise hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37.degree. C., and a wash in 0.1.times.SSC at 60.degree. C. to 65.degree. C., and wherein said polynucleotide encodes a polypeptide having cytokinin oxidase activity.
14. The method of claim 13, wherein said polynucleotide is operably linked to a tissue-specific promoter, a constitutive promoter, or an inducible promoter.
15. The method of claim 13, wherein root development of the plant is modulated.
16. The method of claim 14, wherein the promoter drives root-preferred expression of the operably-linked polynucleotide.
17. The method of claim 16, wherein the promoter is the NAS2 promoter or the ROOTMET2 promoter.
18. The method of claim 17, wherein the promoter is operably linked to a polynucleotide encoding a polypeptide at least 85% identical to SEQ ID NO: 2.
19. A method for reducing the level or activity of cytokinin oxidase in a plant or plant part, comprising introducing into said plant a polynucleotide comprising a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence fragment comprising at least 50 consecutive nucleotides of SEQ ID NO: 1, 2, 4, 5, 7, 8, 10, 11, 51, 52, 57, 58, 61, 62 or a complement thereof; (b) a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of part (a), wherein said stringent conditions comprise hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37.degree. C., and a wash in 0.1.times.SSC at 60.degree. C. to 65.degree. C.; (c) a nucleotide sequence fragment comprising at least 50 consecutive nucleotides of SEQ ID NO: 17, 18, 13, 14, 15, 16, 69, 70 or 63; and (d) a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of part (c), wherein said stringent conditions comprise hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37.degree. C., and a wash in 0.1.times.SSC at 60.degree. C. to 65.degree. C.
20. The method of claim 19, wherein said plant has increased average seed size or increased total seed weight, or both, when compared to a control plant.
21. The method of claim 19, wherein said plant is maize, wheat, rice, barley, sorghum, or rye.
22. The method of claim 19, wherein the stress tolerance of said plant is maintained or improved when compared to a control plant.
23. The method of claim 22, wherein the plant is Zea mays and tip kernel abortion is reduced relative to a control.
24. The method of claim 19, wherein said polynucleotide is configured within a hairpin construct.
25. A method of suppressing cytokinin oxidase activity in a plant, comprising transformation of a plant host cell with the genetic construct of claim 24 and regenerating from said transformed cell a transgenic plant wherein expression of one or more endogenous cytokinin oxidase genes is reduced or eliminated.
26. A first polynucleotide comprising a nucleotide sequence comprising SEQ ID NO: 13, 14, 15, 16, 63, 69 or 70, wherein said polynucleotide drives expression of a second, operably-linked, polynucleotide.
27. A DNA construct comprising a promoter operably linked to a nucleotide sequence of interest, wherein said promoter comprises the polynucleotide of claim 26 or a functional fragment thereof.
Description:
[0001] This application is a continuation of U.S. patent application Ser.
No. 12/165,935, filed Jul. 1, 2008, which claims the benefit of U.S.
patent application Ser. No. 11/094,917, filed Mar. 31, 2005, and U.S.
Patent Application Ser. No. 60/559,252 filed Apr. 2, 2004, all of which
are hereby incorporated by reference.
FIELD OF THE INVENTION
[0002] The invention relates to the field of the genetic manipulation of plants, particularly the modulation of gene activity and development in plants.
BACKGROUND OF THE INVENTION
[0003] Cytokinins are a class of N6 substituted purine derivative plant hormones that regulate cell division, as well as a large number of developmental events, such as shoot development, root branching, control of apical dominance in the shoot, leaf development, chloroplast development, and leaf senescence (Mok, et al., (1994) Cytokinins. Chemistry, Action and Function CRC Press, Boca Raton, Fla., pp. 155-166). Active cytokinin pools are regulated by the rate of synthesis, storage, and/or degradation. In maize, cytokinins were found to play a role in establishing seed size, decreasing tip kernel abortion, and increasing seed set during unfavorable environmental conditions (Cheikh and Jones, (1994) Plant Physiol 106:45-51; Dietrich and Morris, (1995) Plant Physiol Biochem 33(5):327-336).
[0004] The irreversible degradation of cytokinins, catalyzed by cytokinin oxidase, is an important mechanism by which plants modulate their cytokinin levels (Houba-Herin, (1999) Plant Journal 17:615-626; Morris, et al., (1999) Biochemical and Biophysical Research Communications 255:328-333; Brugiere, et al., (2003) Plant Physiol 132:1228-1240). The catabolic enzyme cytokinin oxidase (CKX) plays a major role in controlling cytokinin levels in plant tissues, and CKX activity has been found in a great number of plant tissues. The CKX enzyme is a FAD-containing oxidoreductase that catalyzes the degradation of cytokinins bearing unsaturated isoprenoid side chains. The CKX enzymes irreversibly inactivate most cytokinins by cleaving the isoprenoid side chain from the adenine ring (Armstrong, et al., (1994) Cytokinins. Chemistry, Action and Function. CRC Press, Boca Raton, Fla., pp. 139-154).
[0005] It was earlier shown that ZmCkx1 gene expression is inducible in various organs by synthetic and natural cytokinins. ZmCkx1 is also induced by abscisic acid, which may control cytokinin oxidase expression in the kernel under abiotic stress. Under non-stress conditions, cytokinin oxidase in maize may play a role in controlling growth and development via regulation of cytokinin levels transiting in the xylem. Under environmental stress conditions, cytokinin oxidase gene induction by abscisic acid results in aberrant degradation of cytokinins, therefore impairing normal development (Brugiere, et al., 2003, supra).
[0006] In view of the influence of cytokinins on a wide variety of plant developmental processes, including root architecture, shoot and leaf development, and seed set, the ability to manipulate cytokinin levels in higher plant cells, and thereby affect plant growth and productivity, is of great commercial value.
BRIEF SUMMARY OF THE INVENTION
[0007] Compositions of the invention include cytokinin oxidase (CKX) polypeptides and polynucleotides that are involved in modulating plant development, morphology, and physiology. Compositions include isolated polypeptides comprising an amino acid sequence selected from the group consisting of: (a) the amino acid sequence comprising SEQ ID NO: 3, 6, 9, 12, 53, 59, 62 or 68; (b) the amino acid sequence comprising at least 70% sequence identity to SEQ ID NO: 3, 6, 9, 12, 53, 59, 62 or 68, wherein said polypeptide has cytokinin oxidase activity; (c) the amino acid sequence encoded by a nucleotide sequence that hybridizes under stringent conditions to the complement of SEQ ID NO: 2, 5, 8, 11, 52, 58, 61 or 67, wherein said stringent conditions comprise hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 0.1×SSC at 60° C. to 65° C.; and (d) the amino acid sequence comprising at least 30 consecutive amino acids of SEQ ID NO: 3, 6, 9, 12, 53, 59, 62 or 68, wherein said polypeptide retains cytokinin oxidase activity.
[0008] Compositions further include isolated polynucleotides comprising a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence comprising SEQ ID NO: 1, 2, 4, 5, 7, 8, 10, 11, 51, 52, 54, 55, 57, 58, 60, 61 or 67; (b) a nucleotide sequence encoding an amino acid sequence comprising SEQ ID NO: 3, 6, 9, 12, 53, 59, 62 or 68; (c) a nucleotide sequence comprising at least 60% sequence identity to SEQ ID NO: 1, 2, 4, 5, 7, 8, 10, 11, 51, 52, 54, 55, 57, 58, 60, 61 or 67, wherein said polynucleotide encodes a polypeptide having cytokinin oxidase activity; (d) a nucleotide sequence comprising at least 50 consecutive nucleotides of SEQ ID NO: 1, 2, 4, 5, 7, 8, 10, 11, 51, 52, 54, 55, 57, 58, 60, 61 or 67, or a complement thereof; and (e) a nucleotide sequence that hybridizes under stringent conditions to the complement of a nucleotide sequence of a), wherein said stringent conditions comprise hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 0.1×SSC at 60° C. to 65° C.
[0009] Compositions also include plants comprising a CKX polypeptide of the invention operably linked to a promoter that drives expression in the plant. The plants of the invention can have a modulated cytokinin level compared to a control plant. In some plants, the cytokinin level is modulated in a vegetative tissue, a reproductive tissue, or a vegetative tissue and a reproductive tissue. Plants of the invention may have at least one of the following phenotypes: modulated floral development, modulated flowering time, modulated root development, an altered shoot-to-root ratio, increased seed size and/or increased seed weight, increased plant yield and/or plant vigor, improved or maintained stress tolerance, or a decrease in shoot growth, when compared to a control plant.
[0010] Compositions further include plants that have been genetically modified at a genomic locus, wherein the genomic locus encodes a CKX polypeptide of the invention.
[0011] Methods for increasing the level or activity of a CKX polypeptide in a plant are provided, which may decrease the level of cytokinin in the plant. The method can comprise introducing into the plant a CKX polynucleotide of the invention. In certain methods, the activity of the CKX polypeptide is increased in a vegetative tissue, a reproductive tissue, or a vegetative tissue and a reproductive tissue. In certain embodiments, increasing the activity of the CKX polypeptide modulates root development, alters the shoot-to-root ratio and/or modulates floral development.
[0012] Methods for reducing or eliminating the level of a CKX polypeptide in a plant are also provided. The method can comprise introducing into said plant a CKX polynucleotide of the invention using techniques to result in downregulation. Reducing the level or activity of the CKX polypeptide can increase the level of a cytokinin in the plant. The level or activity of the polypeptide is reduced or eliminated in a vegetative tissue, a reproductive tissue or a vegetative tissue and a reproductive tissue. In certain methods, reducing the level and/or activity of the CKX polypeptide maintains or improves the stress tolerance of the plant, increases seed size and/or seed weight, increases the shoot growth of the plant, and/or delays leaf senescence.
[0013] Methods and compositions for regulating gene expression in a plant are also provided. Polynucleotides comprising promoter sequences are provided. Compositions include isolated polynucleotides comprising a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence comprising SEQ ID NO: 13, 14, 15, 16, 17, 18, 63, 69 or 70; b) a nucleotide sequence comprising at least 60% sequence identity to SEQ ID NO: 13, 14, 15, 16, 17, 18, 63, 69 or 70, wherein said polynucleotide retains the ability to regulate transcription; (c) a nucleotide sequence comprising at least 20 consecutive nucleotides of SEQ ID NO: 13, 14, 15, 16, 17, 18, 63, 69 or 70, wherein said polynucleotide retains the ability to regulate transcription; and, (d) a nucleotide sequence that hybridizes under stringent conditions to the complement of the nucleotide sequence of a), wherein said stringent conditions comprise hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 0.1×SSC at 60° C. to 65° C., wherein said sequence retains the ability to regulate transcription. Compositions further include plants and seed having a DNA construct comprising a nucleotide sequence of interest operably linked to a CKX promoter of the invention. In specific embodiments, the DNA construct is stably integrated into the genome of the plant. Other methods may comprise use of a fragment of the promoter in a hairpin construct designed to target the promoter and hence downregulate expression of an operably-linked polynucleotide.
[0014] Methods for regulating the expression of a nucleotide sequence of interest are also provided. The method comprises introducing into a plant a nucleotide sequence of interest operably linked to a CKX promoter of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] FIG. 1 provides a phylogenetic tree of maize and rice cytokinin oxidase protein sequences. The phylogenetic tree was calculated using the Unweighted Pair Group Method with Arithmetic Mean (UPGMA) method with Phylip (Phylogenetic Inference Package) Version 3.573c (Felsenstein, (1989) Cladistics 5:164-166) based on a ClustalW alignment using the Blosum matrix. The resulting radial tree was displayed using TreeView (Page, (1996) Comput Appl Biosci 12:357-358).
[0016] FIG. 2 provides a diagram of the structure of each of the ZmCkx genes.
[0017] FIG. 3 provides expression data for ZmCkx2a, ZmCkx2b, ZmCkx3, ZmCkx4, ZmCkx5, ZmCkx6, ZmCkx7, and ZmCkx8 in various maize tissues using Pioneer's Lynx database.
[0018] FIG. 4 (A-D) provides schematic representations of various Mu insertions in ZmCkx2a, ZmCkx2b, ZmCkx4, and ZmCkx7.
[0019] FIG. 5 shows increased in vitro root growth of Ubi:ZmCkx2a calli relative to control calli
[0020] FIG. 6 provides data as to number of shoots formed in transgenic Ubi:ZmCkx2a and control maize calli during the regeneration process.
[0021] FIG. 7 provides data as to phenotypic characteristics of transgenic Ubi:ZmCkx2a and control maize plants.
[0022] FIG. 8A shows the level of cytokinin oxidase activity in roots produced by calli expressing Ubi-ZmCkx2a compared to roots produced by control calli.
[0023] FIG. 8B shows the level of cytokinin oxidase activity in leaves of transgenic plants expressing Ubi-ZmCkx2a compared to transgenic controls.
[0024] FIG. 9 (A-M) provides the HmmerPfam (see, Bateman, et al., (2002) Nucleic Acids Research 30(1):276-280) FAD domain identification for ZmCkx2a, ZmCkx2b, ZmCkx3, ZmCkx4, ZmCkx5, ZmCkx6, ZmCkx7, and ZmCkx8. A Pfam consensus sequence is provided in SEQ ID NO: 56.
[0025] FIG. 10 provides InterPro data for ZmCkx2, ZmCkx3, ZmCkx4, ZmCkx5, ZmCkx6, ZmCkx7, and ZmCkx8.
[0026] FIG. 11 provides an amino acid alignment of AtCkx1 (SEQ ID NO: 35), AtCkx2 (SEQ ID NO: 36), AtCkx3 (SEQ ID NO: 37), AtCkx4 (SEQ ID NO: 38), AtCkx5 (SEQ ID NO: 39), AtCkx6 (SEQ ID NO: 40), AtCkx7 (SEQ ID NO: 41), DsCkx1 (SEQ ID NO: 42), HvCkx2 (SEQ ID NO: 43), HvCkx3 (SEQ ID NO: 44), OsCkx1 (SEQ ID NO: 45), OsCkx2 (SEQ ID NO: 46), OsCkx3 (SEQ ID NO: 47), OsCkx4 (SEQ ID NO: 48), OsCkx5 (SEQ ID NO: 49), OsCkx6 (SEQ ID NO: 73), OsCkx7 (SEQ ID NO: 74), OsCkx8 (SEQ ID NO: 75), OsCkx9 (SEQ ID NO: 76), OsCkx10 (SEQ ID NO: 77), OsCkx11 (SEQ ID NO: 78), ZmCkx1 (SEQ ID NO: 33), ZmCkx2a (SEQ ID NO: 3), ZmCkx2b (SEQ ID NO: 68), ZmCkx3 (SEQ ID NO: 6), ZmCkx4 (SEQ ID NO: 9) ZmCkx5 (SEQ ID NO: 12), ZmCkx6 (SEQ ID NO: 53), ZmCkx7 (SEQ ID NO: 59), and ZmCkx8 (SEQ ID NO: 62). The alignment was generated with AlignX from the VNTI suite using the blosum62mt2 matrix, a gap opening penalty of 10 and gap extension penalty of 0.05, a gap separation penalty range of 8 and a % identity for alignment delay of 40. Also see, Ashikari, et al., 2005 (Science 309:741-745) for rice cytokinin oxidase sequences.
[0027] FIG. 12A is a map of the PHP24865 plasmid showing the "head-to-tail" arrangement of the Ubi-ZmCkx1 PRO inverted repeat construct relative to the 35S promoter of the cauliflower mosaic virus (CaMV).
[0028] FIG. 12B is a map of the PHP24866 plasmid showing the "head-to-head" arrangement of the Ubi-ZmCkx1 PRO inverted repeat construct relative to the 35S promoter of the cauliflower mosaic virus (CaMV).
[0029] FIGS. 13-17 provide data on ZmCkx1 promoter hairpin expression as described in Example 10.
[0030] FIG. 18 shows details of the 3' UTR hairpin for ZmCkx2b.
[0031] FIG. 19 shows details of constructs PHP28930 and PHP28937.
[0032] FIG. 20 (A-B) shows root growth of PHP28930 and PHP28937 transgenic plants.
[0033] FIG. 21 shows data for height, leaf length, and leaf width for transgenic plants.
[0034] FIG. 22 provides details of optimized constructs.
[0035] FIG. 23 provides identity levels for ZmCkx1, ZmCkx2a, ZmCkx2b, ZmCkx3, ZmCkx4, ZmCkx5, ZmCkx6, ZmCkx7, ZmCkx8, OsCkx1, OsCkx2, OsCkx3, OsCkx4, OsCkx5, OsCkx6, OsCkx7, OsCkx8, OsCkx9, OsCkx10, and OsCkx11 polypeptides, calculated using the Multiple Sequences Pairwise Relationships Tool for global alignments using the Needleman-Wunsch Algorithm as implemented in the Needle program (EMBOSS tool suite), with a GAP creation penalty of 8 and a GAP extension penalty of 2.
[0036] FIG. 24 provides plant growth data (Z-scores) for Ckx2 RNAi events.
[0037] FIG. 25 (A-B) provides Northern data for PHP28930 and 28937.
[0038] FIG. 26 provides PHP28930 and PHP28937 ear phenotypes.
[0039] FIG. 27 provides data showing that improved yield under limited nitrogen conditions is associated with root-preferred overexpression of ZmCkx2.
DETAILED DESCRIPTION OF THE INVENTION
[0040] The present invention now will be described more fully hereinafter with reference to the accompanying drawings, in which some, but not all, embodiments of the invention are shown. Indeed, the invention may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements. Like numbers refer to like elements throughout.
[0041] Many modifications and other embodiments of the invention set forth herein will come to mind to one skilled in the art to which these inventions pertain, having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it is to be understood that the invention is not to be limited to the specific embodiments disclosed, and that modifications and other embodiments are intended to be included within the scope of the appended claims. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.
Compositions
[0042] Compositions of the invention include cytokinin oxidase (CKX) polypeptides and polynucleotides that are involved in modulating plant development, morphology, and physiology. Compositions of the invention further include CKX promoters that are capable of regulating transcription. In particular, the present invention provides for isolated polynucleotides comprising nucleotide sequences encoding the amino acid sequence shown in SEQ ID NO: 3, 6, 9, 12, 53, 59, 62 or 68. Further provided are polypeptides having an amino acid sequence encoded by a polynucleotide described herein, for example those set forth in SEQ ID NO: 1, 2, 4, 5, 7, 8, 10, 11, 51, 52, 54, 55, 57, 58, 60, 61 or 67. Additional compositions include the promoter sequences for ZmCkx2, ZmCkx3, ZmCkx4, ZmCkx5, ZmCkx8, ZmCkx6, and ZmCkx7, set forth in SEQ ID NO: 13, 14, 15, 16, 63, 69 and 70, respectively.
[0043] The cytokinin oxidase polypeptides of the invention share sequence identity with members of the cytokinin oxidase family of proteins. Changes in cytokinin oxidase activity alter the cytokinin concentration in tissues, and thus cytokinin oxidase enzymes are important in controlling local cytokinin-dependent processes. The cytokinin oxidase enzyme is a FAD-containing oxidoreductase that catalyzes the degradation of cytokinins bearing unsaturated isoprenoid side chains. The free bases, isopentenyl-adenine (iP) and zeatin (Z), and their respective ribosides, are exemplary substrates.
[0044] The CKX polypeptides of the invention contain a predicted FAD-binding domain (PFAM Accession Number PF01565), and are members of the recently identified PF09265 family of protein. Members of this family adopt an alpha+beta sandwich structure with an antiparallel beta-sheet, in a ferredoxin-like fold. They are predominantly found in plant cytokinin oxidase/dehydrogenases, where they are capable of binding both FAD and cytokinin substrates. The PF01565 and PF09265 domains of ZmCkx2a, ZmCkx2b, ZmCkx3, ZmCkx4, ZmCkx5, ZmCkx6, ZmCkx7, and ZmCkx8 are identified in FIG. 9. The CKX polypeptides of the invention also share homology with several polypeptides in the CKX family. FIG. 10 provides a graphic representation of the identified domains in ZmCkx2, ZmCkx3, ZmCkx4, ZmCkx5, ZmCkx6, ZmCkx7 and ZmCkx8. (Results for ZmCkx2a and ZmCkx2b were very similar due to their high level of identity.) This figure was prepared using InterPro, a program of the European Bioinformatics Institute, which integrates numerous protein signature databases to provide a unique, non-redundant characterization of a given protein family, domain or functional site. FIG. 23 provides a summary of identity of rice and maize cytokinin oxidase polypeptide sequences.
[0045] The invention encompasses isolated or substantially purified polynucleotide or protein compositions. An "isolated" or "purified" polynucleotide or protein, or biologically active portion thereof, is substantially or essentially free from components that normally accompany or interact with the polynucleotide or protein as found in its naturally occurring environment. Thus, an isolated or purified polynucleotide or protein is substantially free of other cellular material or culture medium when produced by recombinant techniques or substantially free of chemical precursors or other chemicals when chemically synthesized. Optimally, an "isolated" polynucleotide is free of sequences (optimally protein encoding sequences) that naturally flank the polynucleotide (i.e., sequences located at the 5' and 3' ends of the polynucleotide) in the genomic DNA of the organism from which the polynucleotide is derived. For example, in various embodiments, the isolated polynucleotide can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequence that naturally flank the polynucleotide in genomic DNA of the cell from which the polynucleotide is derived. A protein that is substantially free of cellular material includes preparations of protein having less than about 30%, 20%, 10%, 5% or 1% (by dry weight) of contaminating protein. When the protein of the invention or biologically active portion thereof is recombinantly produced, optimally culture medium represents less than about 30%, 20%, 10%, 5% or 1% (by dry weight) of chemical precursors or non-protein-of-interest chemicals.
[0046] Fragments and variants of the disclosed polynucleotides and proteins encoded thereby are also encompassed by the present invention. By "fragment" is intended a portion of the polynucleotide or a portion of the amino acid sequence and hence of the protein encoded thereby. Fragments of a polynucleotide may encode protein fragments that retain the biological activity of the native protein and hence exhibit cytokinin oxidase activity. Alternatively, fragments of a polynucleotide that are useful as hybridization probes generally do not encode protein fragments retaining biological activity. Thus, fragments of a nucleotide sequence may range from at least about 20 nucleotides, about 50 nucleotides, about 100 nucleotides and up to a full-length polynucleotide encoding a protein of the invention.
[0047] A fragment of a CKX polynucleotide that encodes a biologically active portion of a CKX protein of the invention will encode at least 15, 25, 30, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 525 or 537 contiguous amino acids, or up to the total number of amino acids present in a full-length CKX protein of the invention (for example, 519 amino acids, 538 amino acids, 521 amino acids and 542 amino acids for SEQ ID NO:3, 6, 9 and 12, respectively). Fragments of a CKX polynucleotide that are useful as hybridization probes or PCR primers generally need not encode a biologically active portion of a CKX protein.
[0048] Thus, a fragment of a CKX polynucleotide may encode a biologically active portion of a CKX protein, or it may be a fragment that can be used as a hybridization probe or PCR primer using methods disclosed below. A biologically active portion of a CKX protein can be prepared by isolating a portion of one of the CKX polynucleotides of the invention, expressing the encoded portion of the CKX protein (e.g., by recombinant expression in vitro), and assessing the activity of the encoded portion of the CKX protein. Polynucleotides that are fragments of a CKX nucleotide sequence comprise at least 16, 20, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600 or 1629 nucleotides, or up to the number of nucleotides present in a full-length CKX polynucleotide disclosed herein (for example, 3200 nucleotides, 1560 nucleotides, 3258 nucleotides, 2635 nucleotides, 1617 nucleotides, 6177 nucleotides, 1816 nucleotides, 1566 nucleotides, 5108 nucleotides or 1629 nucleotides for SEQ ID NO: 1, 2, 4, 5, 54, 7, 8, 55, 10 or 11, respectively).
[0049] "Variants" is intended to mean substantially similar sequences. For polynucleotides, a variant comprises a deletion and/or addition of one or more nucleotides at one or more sites within the native polynucleotide and/or a substitution of one or more nucleotides at one or more sites in the native polynucleotide. As used herein, a "native" polynucleotide or polypeptide comprises a naturally occurring nucleotide sequence or amino acid sequence, respectively. For polynucleotides, conservative variants include those sequences that, because of the degeneracy of the genetic code, encode the amino acid sequence of one of the cytokinin oxidase polypeptides of the invention. Naturally occurring allelic variants such as these can be identified with the use of well-known molecular biology techniques, as, for example, with polymerase chain reaction (PCR) and hybridization techniques as outlined below. Variant polynucleotides also include synthetically derived polynucleotides, such as those generated, for example, by using site-directed mutagenesis but which still encode a CKX protein of the invention. Generally, variants of a particular polynucleotide of the invention will have at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to that particular polynucleotide as determined by sequence alignment programs and parameters described elsewhere herein.
[0050] Variants of a particular polynucleotide of the invention (i.e., the reference polynucleotide) can also be evaluated by comparison of the percent sequence identity between the polypeptide encoded by a variant polynucleotide and the polypeptide encoded by the reference polynucleotide. Thus, for example, isolated polynucleotides that encode a polypeptide with a given percent sequence identity to the polypeptide of SEQ ID NO: 3, 6, 9, 12, 53, 59, 62 or 68 are disclosed. Percent sequence identity between any two polypeptides can be calculated using sequence alignment programs and parameters described elsewhere herein. Where any given pair of polynucleotides of the invention is evaluated by comparison of the percent sequence identity shared by the two polypeptides they encode, the percent sequence identity between the two encoded polypeptides is at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity.
[0051] "Variant" protein is intended to mean a protein derived from the native protein by deletion or addition of one or more amino acids at one or more sites in the native protein and/or substitution of one or more amino acids at one or more sites in the native protein. Variant proteins encompassed by the present invention are biologically active, that is they continue to possess the desired biological activity of the native protein, that is, cytokinin oxidase activity as described herein. Such variants may result from, for example, genetic polymorphism or from human manipulation. Biologically active variants of a native CKX protein of the invention will have at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the amino acid sequence for the native protein as determined by sequence alignment programs and parameters described elsewhere herein. A biologically active variant of a protein of the invention may differ from that protein by as few as 1-15 amino acid residues, as few as 1-10, such as 6-10, as few as 5, as few as 4, 3, 2 or even 1 amino acid residue. The upper limit of variation for an amino acid sequence of the invention which retains biological activity can be determined empirically, i.e., by testing variants in an assay for cytokinin oxidase activity as described elsewhere herein. A biologically active variant of a protein of the invention may differ from that protein by as much as 100, 200 or 300 amino acids. One of skill in the art would note that conservation of functional motifs, such as the FAD binding domain identified in FIG. 9 or cytokinin binding domains, is preferred.
[0052] The proteins of the invention may be altered in various ways including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known in the art. For example, amino acid sequence variants and fragments of the CKX proteins can be prepared by mutations in the DNA. Methods for mutagenesis and polynucleotide alterations are well known in the art. See, for example, Kunkel, (1985) Proc. Natl. Acad. Sci. USA 82:488-492; Kunkel, et al., (1987) Methods in Enzymol 154:367-382; U.S. Pat. No. 4,873,192; Walker and Gaastra, eds. (1983) Techniques in Molecular Biology (MacMillan Publishing Company, New York) and the references cited therein. Guidance as to appropriate amino acid substitutions that do not affect biological activity of the protein of interest may be found in the model of Dayhoff, et al., (1978) Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, D.C.), herein incorporated by reference. Conservative substitutions, such as exchanging one amino acid with another having similar properties, may be optimal.
[0053] Thus, the genes and polynucleotides of the invention include both the naturally occurring sequences as well as mutant forms. Likewise, the proteins of the invention encompass both naturally occurring proteins as well as variations and modified forms thereof. Such variants will continue to possess the desired cytokinin oxidase activity. The mutations that will be made in the DNA encoding the variant must not place the sequence out of reading frame and optimally will not create complementary regions that could produce secondary mRNA structure. See, EP Patent Application Publication Number 0075444.
[0054] The deletions, insertions and substitutions of the protein sequences encompassed herein are not expected to produce radical changes in the characteristics of the protein. However, when it is difficult to predict the exact effect of the substitution, deletion, or insertion in advance of doing so, one skilled in the art will appreciate that the effect will be evaluated by routine screening assays. That is, the activity can be evaluated by assaying for cytokinin oxidase activity.
[0055] Cytokinin oxidase activity can be assayed in a variety of ways. For example, a variety of cytokinin derivatives can be used as substrates to measure cytokinin oxidase activity. For instance, the polypeptide having CKX activity can be mixed with a cytokinin, for example, zeatin, and the net change of absorbance at 590 nm can be measured. See, U.S. Pat. No. 6,229,066. Alternatively, cytokinin oxidase activity can be measured by assaying for the conversion of [2-3H]iP to adenine. See, for example, Faiss, et al., (1997) Plant J. 12:401-415, herein incorporated by reference. For additional assays, see, Morris, et al., (1999) Biochem Biophys Res Comm 255:328-333, Bilyeu, et al., (2001) Plant Physiol 125:378-386, Jones, et al., (1990) Proceedings of the Plant Growth Regulation Society of America: (17th) pp 183-196, Dietrich, et al., (1995) Plant Physiol. Bioch. 268:327-336, Motyka, et al., (1996) Plant Physiol. 112:1035-1043, and Frebort, et al., (2002) Annu Biochem 306:1-7, each of which is herein incorporated by reference. In addition, a photospectrometric initial rate method which results in the formation of a formazan dye has been used to assay for cytokinin oxidase activity. See, for example, Frebort, et al., (2002) Annu Biochem 306:1-7. In addition, cytokinin oxidase activity can be measured by assaying for a decrease in cytokinin levels in vivo. Such a decrease in cytokinin levels can produce one or more symptoms of a cytokinin-deficiency syndrome. The various phenotypes associated with cytokinin-deficiency syndrome are known in the art. See, for example, Schmulling, et al., (2003) J. Plant Res 116:241-252, herein incorporated by reference.
[0056] Variant polynucleotides and proteins also encompass sequences and proteins derived from a mutagenic and recombinogenic procedure such as DNA shuffling. With such a procedure, one or more different CKX sequences can be manipulated to create a new CKX polypeptide possessing the desired properties. In this manner, libraries of recombinant polynucleotides are generated from a population of related sequence polynucleotides comprising sequence regions that have substantial sequence identity and can be homologously recombined in vitro or in vivo. For example, using this approach, sequence motifs encoding a domain of interest may be shuffled between the CKX gene of the invention and other known CKX genes to obtain a new gene coding for a protein with an improved property of interest, such as an increased Km in the case of an enzyme. Strategies for such DNA shuffling are known in the art. See, for example, Stemmer, (1994) Proc. Natl. Acad. Sci. USA 91:10747-10751; Stemmer, (1994) Nature 370:389-391; Crameri, et al., (1997) Nature Biotech. 15:436-438; Moore, et al., (1997) J. Mol. Biol. 272:336-347; Zhang, et al., (1997) Proc. Natl. Acad. Sci. USA 94:4504-4509; Crameri, et al., (1998) Nature 391:288-291; and U.S. Pat. Nos. 5,605,793 and 5,837,458.
[0057] The compositions of the invention also include isolated polynucleotides comprising the CKX promoter nucleotide sequences set forth in SEQ ID NOS: 13, 14, 15, 16, 63, 69 and 70. By "promoter" is intended a regulatory region of DNA usually comprising a TATA box capable of directing RNA polymerase II to initiate RNA synthesis at the appropriate transcription initiation site for a particular polynucleotide sequence. A promoter may additionally comprise other recognition sequences generally positioned upstream or 5' to the TATA box, referred to as upstream promoter elements, which influence the transcription initiation rate. The promoter sequences of the present invention regulate (i.e., repress or activate) transcription from the promoter region.
[0058] It is recognized that additional domains can be added to the promoter sequences of the invention and thereby modulate the level of expression, the developmental timing of expression, or tissue type in which expression occurs. See particularly, U.S. Pat. Nos. 5,466,785 and 5,635,618.
[0059] Fragments and variants of the disclosed CKX promoter polynucleotides are also encompassed by the present invention. Fragments of a promoter polynucleotide may retain biological activity and hence retain transcriptional regulatory activity. Alternatively, fragments of a polynucleotide that are useful as hybridization probes generally do not retain biological activity. Thus, fragments of a promoter nucleotide sequence may range from at least about 20 nucleotides, about 50 nucleotides, about 100 nucleotides, and up to the full-length polynucleotide of the invention.
[0060] Thus, a fragment of a CKX promoter polynucleotide may encode a biologically active portion of a CKX promoter, or it may be a fragment that can be used as a hybridization probe or PCR primer using methods disclosed below. A biologically active portion of a CKX promoter polynucleotide can be prepared by isolating a portion of one of the CKX promoter polynucleotides of the invention, and assessing the activity of the portion of the CKX promoter. Polynucleotides that are fragments of a CKX promoter polynucleotide comprise at least 16, 20, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900 or 2000 nucleotides, or up to the number of nucleotides present in a full-length CKX promoter polynucleotide disclosed herein (for example, 3003, 2001, 2448 or 2346 nucleotides for SEQ ID NO: 13, 14, 15 or 16, respectively).
[0061] For a promoter polynucleotide, a variant comprises a deletion and/or addition of one or more nucleotides at one or more sites within the native polynucleotide and/or a substitution of one or more nucleotides at one or more sites in the native polynucleotide. Generally, variants of a particular promoter polynucleotide of the invention will have at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to that particular polynucleotide as determined by sequence alignment programs and parameters described elsewhere herein.
[0062] Variant promoter polynucleotides also encompass sequences derived from a mutagenic and recombinogenic procedure such as DNA shuffling. With such a procedure, one or more different promoter sequences can be manipulated to create a new CKX promoter possessing the desired properties. Strategies for such DNA shuffling are described elsewhere herein.
[0063] Methods are available in the art for determining if a promoter sequence retains the ability to regulate transcription. Such activity can be measured by Northern blot analysis. See, for example, Sambrook, et al., (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.), herein incorporated by reference. Alternatively, biological activity of the promoter can be measured using assays specifically designed for measuring the activity and/or level of the polypeptide being expressed from the promoter. Such assays are known in the art.
[0064] The polynucleotides of the invention (i.e., the CKX sequences and the CKX promoter sequences) can be used to isolate corresponding sequences from other organisms, particularly other plants, and more particularly other monocots. In this manner, methods such as PCR, hybridization, and the like can be used to identify such sequences based on their sequence homology to the sequences set forth herein. Sequences isolated based on their sequence identity to the entire CKX sequences or the CKX promoter sequences set forth herein or to variants and fragments thereof are encompassed by the present invention. Such sequences include sequences that are orthologs of the disclosed sequences. "Orthologs" is intended to mean genes derived from a common ancestral gene and which are found in different species as a result of speciation. Genes found in different species are considered orthologs when their nucleotide sequences and/or their encoded protein sequences share at least 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity. Functions of orthologs are often highly conserved among species. Thus, isolated polynucleotides that encode a CKX protein and which hybridize under stringent conditions to the CKX sequences disclosed herein, or to variants or fragments or complements thereof, are encompassed by the present invention.
[0065] In a PCR approach, oligonucleotide primers can be designed for use in PCR reactions to amplify corresponding DNA sequences from cDNA or genomic DNA extracted from any plant of interest. Methods for designing PCR primers and PCR cloning are generally known in the art and are disclosed in Sambrook, et al., (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.). See also, Innis, et al., eds. (1990) PCR Protocols: A Guide to Methods and Applications (Academic Press, New York); Innis and Gelfand, eds. (1995) PCR Strategies (Academic Press, New York); and Innis and Gelfand, eds. (1999) PCR Methods Manual (Academic Press, New York). Known methods of PCR include, but are not limited to, methods using paired primers, nested primers, single specific primers, degenerate primers, gene-specific primers, vector-specific primers, partially-mismatched primers, and the like.
[0066] In hybridization techniques, all or part of a known polynucleotide is used as a probe that selectively hybridizes to other corresponding polynucleotides present in a population of cloned genomic DNA fragments or cDNA fragments (i.e., genomic or cDNA libraries) from a chosen organism. The hybridization probes may be genomic DNA fragments, cDNA fragments, RNA fragments, or other oligonucleotides, and may be labeled with a detectable group such as 32P, or any other detectable marker. Thus, for example, probes for hybridization can be made by labeling synthetic oligonucleotides based on the CKX polynucleotides or the CKX promoter sequences of the invention. Methods for preparation of probes for hybridization and for construction of cDNA and genomic libraries are generally known in the art and are disclosed in Sambrook, et al., (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.).
[0067] For example, an entire CKX polynucleotide or an entire CKX promoter sequence disclosed herein, or one or more portions thereof, may be used as a probe capable of specifically hybridizing to corresponding CKX polynucleotides and messenger RNAs. To achieve specific hybridization under a variety of conditions, such probes include sequences that are unique among CKX polynucleotide sequences and are optimally at least about 10 nucleotides in length, and most optimally at least about 20 nucleotides in length. Such probes may be used to amplify corresponding CKX polynucleotides from a chosen plant by PCR. This technique may be used to isolate additional coding sequences from a desired plant or as a diagnostic assay to determine the presence of coding sequences in a plant. Hybridization techniques include hybridization screening of plated DNA libraries (either plaques or colonies; see, for example, Sambrook, et al., (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.).
[0068] Hybridization of such sequences may be carried out under stringent conditions. By "stringent conditions" or "stringent hybridization conditions" is intended conditions under which a probe will hybridize to its target sequence to a detectably greater degree than to other sequences (e.g., at least 2-fold over background). Stringent conditions are sequence-dependent and will be different in different circumstances. By controlling the stringency of the hybridization and/or washing conditions, target sequences that are 100% complementary to the probe can be identified (homologous probing). Alternatively, stringency conditions can be adjusted to allow some mismatching in sequences so that lower degrees of similarity are detected (heterologous probing). Generally, a probe is less than about 1000 nucleotides in length, optimally less than 500 nucleotides in length.
[0069] Typically, stringent conditions will be those in which the salt concentration is less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. for short probes (e.g., 10 to 50 nucleotides) and at least about 60° C. for long probes (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. Exemplary low stringency conditions include hybridization with a buffer solution of 30 to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37° C., and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at 50 to 55° C. Exemplary moderate stringency conditions include hybridization in 40 to 45% formamide, 1.0 M NaCl, 1% SDS at 37° C., and a wash in 0.5× to 1×SSC at 55 to 60° C. Exemplary high stringency conditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 0.1×SSC at 60 to 65° C. Optionally, wash buffers may comprise about 0.1% to about 1% SDS. Duration of hybridization is generally less than about 24 hours, usually about 4 to about 12 hours. The duration of the wash time will be at least a length of time sufficient to reach equilibrium.
[0070] Specificity is typically the function of post-hybridization washes, the critical factors being the ionic strength and temperature of the final wash solution. For DNA-DNA hybrids, the Tm can be approximated from the equation of Meinkoth and Wahl, (1984) Anal. Biochem. 138:267-284: Tm=81.5° C.+16.6 (log M)+0.41 (% GC)-0.61 (% form)-500/L; where M is the molarity of monovalent cations, % GC is the percentage of guanosine and cytosine nucleotides in the DNA, % form is the percentage of formamide in the hybridization solution and L is the length of the hybrid in base pairs. The Tm is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridizes to a perfectly matched probe. Tm is reduced by about 1° C. for each 1% of mismatching; thus, Tm, hybridization, and/or wash conditions can be adjusted to hybridize to sequences of the desired identity. For example, if sequences with ≧90% identity are sought, the Tm can be decreased 10° C. Generally, stringent conditions are selected to be about 5° C. lower than the thermal melting point (Tm) for the specific sequence and its complement at a defined ionic strength and pH. However, severely stringent conditions can utilize a hybridization and/or wash at 1, 2, 3 or 4° C. lower than the thermal melting point (Tm); moderately stringent conditions can utilize a hybridization and/or wash at 6, 7, 8, 9 or 10° C. lower than the thermal melting point (Tm); low stringency conditions can utilize a hybridization and/or wash at 11, 12, 13, 14, 15 or 20° C. lower than the thermal melting point (Tm). Using the equation, hybridization and wash compositions, and desired Tm, those of ordinary skill will understand that variations in the stringency of hybridization and/or wash solutions are inherently described. If the desired degree of mismatching results in a Tm of less than 45° C. (aqueous solution) or 32° C. (formamide solution), it is optimal to increase the SSC concentration so that a higher temperature can be used. An extensive guide to the hybridization of nucleic acids is found in Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology--Hybridization with Nucleic Acid Probes, Part I, Chapter 2 (Elsevier, N.Y.); and Ausubel, et al., eds. (1995) Current Protocols in Molecular Biology, Chapter 2 (Greene Publishing and Wiley-Interscience, New York). See, Sambrook, et al., (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.).
[0071] The following terms are used to describe the sequence relationships between two or more polynucleotides or polypeptides: (a) "reference sequence", (b) "comparison window", (c) "sequence identity", and, (d) "percentage of sequence identity."
[0072] (a) As used herein, "reference sequence" is a defined sequence used as a basis for sequence comparison. A reference sequence may be a subset or the entirety of a specified sequence; for example, as a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence.
[0073] (b) As used herein, "comparison window" makes reference to a contiguous and specified segment of a polynucleotide sequence, wherein the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two polynucleotides. Generally, the comparison window is at least 20 contiguous nucleotides in length, and optionally can be 30, 40, 50, 100 or longer. Those of skill in the art understand that to avoid a high similarity to a reference sequence due to inclusion of gaps in the polynucleotide sequence a gap penalty is typically introduced and is subtracted from the number of matches.
[0074] Methods of alignment of sequences for comparison are well known in the art. Thus, the determination of percent sequence identity between any two sequences can be accomplished using a mathematical algorithm. Non-limiting examples of such mathematical algorithms are the algorithm of Myers and Miller (1988) CABIOS 4:11-17; the local alignment algorithm of Smith, et al., (1981) Adv. Appl. Math. 2:482; the global alignment algorithm of Needleman and Wunsch, (1970) J. Mol. Biol. 48:443-453; the search-for-local alignment method of Pearson and Lipman, (1988) Proc. Natl. Acad. Sci. 85:2444-2448; the algorithm of Karlin and Altschul, (1990) Proc. Natl. Acad. Sci. USA 872264, modified as in Karlin and Altschul, (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877.
[0075] Computer implementations of these mathematical algorithms can be utilized for comparison of sequences to determine sequence identity. Such implementations include, but are not limited to: CLUSTAL in the PC/Gene program (available from Intelligenetics, Mountain View, Calif.); the ALIGN program (Version 2.0) and GAP, BESTFIT, BLAST, FASTA and TFASTA in the GCG Wisconsin Genetics Software Package, Version 10 (available from Accelrys Inc., 9685 Scranton Road, San Diego, Calif., USA). Alignments using these programs can be performed using the default parameters. The CLUSTAL program is well described by Higgins, et al., (1988) Gene 73:237-244 (1988); Higgins, et al., (1989) CABIOS 5:151-153; Corpet, et al., (1988) Nucleic Acids Res. 16:10881-90; Huang, et al., (1992) CABIOS 8:155-65 and Pearson, et al., (1994) Meth. Mol. Biol. 24:307-331. The ALIGN program is based on the algorithm of Myers and Miller, (1988) supra. A PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used with the ALIGN program when comparing amino acid sequences. The BLAST programs of Altschul, et al., (1990) J. Mol. Biol. 215:403 are based on the algorithm of Karlin and Altschul, (1990) supra. BLAST nucleotide searches can be performed with the BLASTN program, score=100, wordlength=12, to obtain nucleotide sequences homologous to a nucleotide sequence encoding a protein of the invention. BLAST protein searches can be performed with the BLASTX program, score=50, wordlength=3, to obtain amino acid sequences homologous to a protein or polypeptide of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST (in BLAST 2.0) can be utilized as described in Altschul, et al., (1997) Nucleic Acids Res. 25:3389. Alternatively, PSI-BLAST (in BLAST 2.0) can be used to perform an iterated search that detects distant relationships between molecules. See, Altschul, et al., (1997) supra. When utilizing BLAST, Gapped BLAST, PSI-BLAST, the default parameters of the respective programs (e.g., BLASTN for nucleotide sequences, BLASTX for proteins) can be used. See www.ncbi.nlm.nih.gov. Alignment may also be performed manually by inspection.
[0076] Unless otherwise stated, sequence identity/similarity values provided herein refer to the value obtained using GAP Version 10 using the following parameters: % identity and % similarity for a nucleotide sequence using GAP Weight of 50 and Length Weight of 3, and the nwsgapdna.cmp scoring matrix; % identity and % similarity for an amino acid sequence using GAP Weight of 8 and Length Weight of 2, and the BLOSUM62 scoring matrix; or any equivalent program thereof. By "equivalent program" is intended any sequence comparison program that, for any two sequences in question, generates an alignment having identical nucleotide or amino acid residue matches and an identical percent sequence identity when compared to the corresponding alignment generated by GAP Version 10.
[0077] GAP uses the algorithm of Needleman and Wunsch, (1970) J. Mol. Biol. 48:443-453, to find the alignment of two complete sequences that maximizes the number of matches and minimizes the number of gaps. GAP considers all possible alignments and gap positions and creates the alignment with the largest number of matched bases and the fewest gaps. It allows for the provision of a gap creation penalty and a gap extension penalty in units of matched bases. GAP must make a profit of gap creation penalty number of matches for each gap it inserts. If a gap extension penalty greater than zero is chosen, GAP must, in addition, make a profit for each gap inserted of the length of the gap times the gap extension penalty. Default gap creation penalty values and gap extension penalty values in Version 10 of the GCG Wisconsin Genetics Software Package for protein sequences are 8 and 2, respectively. For nucleotide sequences the default gap creation penalty is 50 while the default gap extension penalty is 3. The gap creation and gap extension penalties can be expressed as an integer selected from the group of integers consisting of from 0 to 200. Thus, for example, the gap creation and gap extension penalties can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 or greater.
[0078] GAP presents one member of the family of best alignments. There may be many members of this family, but no other member has a better quality. GAP displays four figures of merit for alignments: Quality, Ratio, Identity and Similarity. The Quality is the metric maximized in order to align the sequences. Ratio is the quality divided by the number of bases in the shorter segment. Percent Identity is the percent of the symbols that actually match. Percent Similarity is the percent of the symbols that are similar. Symbols that are across from gaps are ignored. A similarity is scored when the scoring matrix value for a pair of symbols is greater than or equal to 0.50, the similarity threshold. The scoring matrix used in Version 10 of the GCG Wisconsin Genetics Software Package is BLOSUM62 (see, Henikoff and Henikoff, (1989) Proc. Natl. Acad. Sci. USA 89:10915).
[0079] (c) As used herein, "sequence identity" or "identity" in the context of two polynucleotides or polypeptide sequences makes reference to the residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window. When percentage of sequence identity is used in reference to proteins it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. When sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are said to have "sequence similarity" or "similarity". Means for making this adjustment are well known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated, e.g., as implemented in the program PC/GENE (Intelligenetics, Mountain View, Calif.).
[0080] (d) As used herein, "percentage of sequence identity" means the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) 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.
[0081] The invention further provides plants having altered levels and/or activities of the CKX polypeptides of the invention. In some embodiments, the plants of the invention have stably incorporated into their genomes the CKX sequences of the invention. In certain embodiments, plants that are genetically modified at a genomic locus encoding a CKX polypeptide of the invention are provided. By "native genomic locus" is intended a naturally occurring genomic sequence. In some embodiments, the genomic locus is set forth in SEQ ID NO: 1, 4, 7, 10, 51, 57 or 60. In still further embodiments, the genomic locus is modified to reduce or eliminate the activity of the CKX polypeptide. The term "genetically modified" as used herein refers to a plant or plant part that is modified in its genetic information by the introduction of one or more foreign polynucleotides, and that the insertion of the foreign polynucleotide leads to a phenotypic change in the plant. By "phenotypic change" is intended a measurable change in one or more cell functions. For example, plants having the genetic modification at the genomic locus encoding the CKX polypeptide can show reduced or eliminated expression or activity of the CKX polypeptide. Various methods to generate such a genetically modified genomic locus are described elsewhere herein, as are the variety of phenotypes that can result from the modulation of the level and/or activity of the CKX sequences of the invention.
[0082] As used herein, the term plant includes plant cells, plant protoplasts, plant cell tissue cultures from which a plant can be regenerated, plant calli, plant clumps, and plant cells that are intact in plants or parts of plants such as embryos, pollen, ovules, seeds, leaves, flowers, branches, fruit, kernels, ears, cobs, husks, stalks, roots, root tips, anthers, grain and the like. As used herein, by "grain" is intended the mature seed produced by commercial growers for purposes other than growing or reproducing the species. Progeny, variants, and mutants of the regenerated plants are also included within the scope of the invention, provided that these parts comprise the introduced nucleic acid sequences.
[0083] A "subject plant" or "subject plant cell" is one in which genetic alteration, such as transformation, has been affected as to a gene of interest or is a plant or plant cell which is descended from a plant or plant cell so altered and which comprises the alteration. A "control" or "control plant" or "control plant cell" provides a reference point for measuring changes in the subject plant or plant cell.
[0084] A control plant or control plant cell may comprise, for example: (a) a wild-type plant or plant cell, i.e., of the same genotype as the starting material for the genetic alteration which resulted in the subject plant or subject plant cell; (b) a plant or plant cell of the same genotype as the starting material but which has been transformed with a null construct (i.e., with a construct which has no known effect on the trait of interest, such as a construct comprising a marker gene); (c) a plant or plant cell which is a non-transformed segregant among progeny of a subject plant or subject plant cell; (d) a plant or plant cell genetically identical to the subject plant or subject plant cell but which is not exposed to conditions or stimuli that would induce expression of the gene of interest or (e) the subject plant or subject plant cell itself, under conditions in which the gene of interest is not expressed.
[0085] In the present case, for example, in various embodiments, changes in ctyokinin oxidase activity, cytokinin oxidase levels, cytokinin activity, cytokinin levels, cytokinin ratios, cytokinin distribution and/or changes in one or more traits such as flowering time, seed set, branching, senescence, stress tolerance or root mass, could be measured by comparing a subject plant or subject plant cell to a control plant or control plant cell.
Methods
I. Providing Sequences
[0086] The sequences of the present invention can be introduced/expressed in a host cell such as bacteria, yeast, insect, mammalian, or optimally plant cells. It is expected that those of skill in the art are knowledgeable in the numerous systems available for the introduction of a polypeptide or a nucleotide sequence of the present invention into a host cell. No attempt to describe in detail the various methods known for providing proteins in prokaryotes or eukaryotes will be made.
[0087] By "host cell" is meant a cell which comprises a heterologous nucleic acid sequence of the invention. Host cells may be prokaryotic cells such as E. coli, or eukaryotic cells such as yeast, insect, amphibian or mammalian cells. Host cells can also be monocotyledonous or dicotyledonous plant cells. In one embodiment, the monocotyledonous host cell is a maize host cell.
[0088] The use of the term "polynucleotide" is not intended to limit the present invention to polynucleotides comprising DNA. Those of ordinary skill in the art will recognize that polynucleotides can comprise ribonucleotides and combinations of ribonucleotides and deoxyribonucleotides. Such deoxyribonucleotides and ribonucleotides include both naturally occurring molecules and synthetic analogues. The polynucleotides of the invention also encompass all forms of sequences including, but not limited to, single-stranded forms, double-stranded forms, hairpins, stem-and-loop structures, and the like.
[0089] The CKX polynucleotide or CKX promoter sequences of the invention can be provided in expression cassettes for expression in the organism of interest. The cassette may include 5' and 3' regulatory sequences operably linked to a CKX polynucleotide of the invention. "Operably linked" is intended to mean a functional linkage between two or more elements. For example, an operable linkage between a polynucleotide of interest and a regulatory sequence (i.e., a promoter) is a functional link that allows for expression of the polynucleotide of interest. Operably linked elements may be contiguous or non-contiguous. When used to refer to the joining of two protein coding regions, by "operably linked" is intended that the coding regions are in the same reading frame. The cassette may additionally contain at least one additional gene to be cotransformed into the organism. Alternatively, any additional gene(s) can be provided on multiple expression cassettes. Such an expression cassette is provided with a plurality of restriction sites and/or recombination sites for insertion of the CKX polynucleotide to be under the transcriptional regulation of the regulatory regions. The expression cassette may additionally contain selectable marker genes.
[0090] The expression cassette may include in the 5'-3' direction of transcription, a transcriptional and translational initiation region (i.e., a promoter), a CKX polynucleotide of the invention, and a transcriptional and translational termination region (i.e., termination region) functional in the host cell (i.e., the plant). The regulatory regions (i.e., promoters, transcriptional regulatory regions, and translational termination regions) and/or the CKX polynucleotide of the invention may be native/analogous to the host cell or to each other. Alternatively, the regulatory regions and/or the CKX polynucleotide of the invention may be heterologous to the host cell or to each other. As used herein, "heterologous" in reference to a sequence is a sequence that originates from a foreign species, or, if from the same species, is substantially modified from its native form in composition and/or genomic locus by deliberate human intervention. For example, a promoter operably linked to a heterologous polynucleotide is from a species different from the species from which the polynucleotide was derived, or, if from the same/analogous species, one or both are substantially modified from their original form and/or genomic locus, or the promoter is not the native promoter for the operably linked polynucleotide. As used herein, a chimeric gene comprises a coding sequence operably linked to a transcription initiation region that is heterologous to the coding sequence.
[0091] While heterologous promoters can be used to express the sequences, the native promoter sequences (i.e., SEQ ID NOS: 13, 14, 15, 16, 63, 69 and 70) also may be used. Such constructs can change expression levels of CKX in the plant or plant cell. Thus, the phenotype of the plant or plant cell can be altered. Alternatively, in other methods, any CKX promoter sequence of the invention can be used to express a CKX sequence. In addition, other CKX promoters can be used, such as SEQ ID NOS: 17 and 18 herein; see also WO 2002/0708438; U.S. Pat. Nos. 6,921,815 and 7,371,925; and U.S. patent application Ser. No. 12/051,893 (SEQ ID NOS: 17 and 18 herein).
[0092] A termination region may be native with the transcriptional initiation region, may be native with the operably linked CKX polynucleotide of interest or with the CKX promoter sequences, may be native with the plant host, or may be derived from another source (i.e., foreign or heterologous) to the promoter, the CKX polynucleotide of interest, the plant host or any combination thereof. 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., (1991) Mol. Gen. Genet. 262:141-144; Proudfoot, (1991) Cell 64:671-674; Sanfacon, et al., (1991) 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; and Joshi, et al., (1987) Nucleic Acids Res. 15:9627-9639.
[0093] Where appropriate, the polynucleotides may be optimized for increased expression in the transformed plant. That is, the polynucleotides can be synthesized using plant-preferred codons for improved expression. See, for example, Campbell and Gowri, (1990) Plant Physiol. 92:1-11 for a discussion of host-preferred codon usage. Methods are available in the art for synthesizing plant-preferred genes. See, for example, U.S. Pat. Nos. 5,380,831 and 5,436,391 and Murray, et al., (1989) Nucleic Acids Res. 17:477-498, herein incorporated by reference.
[0094] Additional sequence modifications are known to enhance gene expression in a cellular host. These include elimination of sequences encoding spurious polyadenylation signals, exon-intron splice site signals, transposon-like repeats, and other such well-characterized sequences that may be deleterious to gene expression. The G-C content of the sequence may be adjusted to levels average for a given cellular host, as calculated by reference to known genes expressed in the host cell. When possible, the sequence is modified to avoid predicted hairpin secondary mRNA structures.
[0095] The expression cassettes may additionally contain 5' leader sequences. Such leader sequences can act to enhance translation. Translation leaders are known in the art and include: picornavirus leaders, for example, EMCV leader (Encephalomyocarditis 5' noncoding region) (Elroy-Stein, et al., (1989) Proc. Natl. Acad. Sci. USA 86:6126-6130); potyvirus leaders, for example, TEV leader (Tobacco Etch Virus) (Gallie, et al., (1995) Gene 165(2):233-238), MDMV leader (Maize Dwarf Mosaic Virus) (Johnson, et al., (1986) Virology 154:9-20), and human immunoglobulin heavy-chain binding protein (BiP) (Macejak, et al., (1991) Nature 353:90-94); untranslated leader from the coat protein mRNA of alfalfa mosaic virus (AMV RNA 4) (Jobling, et al., (1987) Nature 325:622-625); tobacco mosaic virus leader (TMV) (Gallie, et al., (1989) in Molecular Biology of RNA, ed. Cech (Liss, New York), pp. 237-256) and maize chlorotic mottle virus leader (MCMV) (Lommel, et al., (1991) Virology 81:382-385). See also, Della-Cioppa, et al., (1987) Plant Physiol. 84:965-968. Other methods known to enhance translation can also be utilized, for example, introns, and the like.
[0096] In preparing the expression cassette, the various DNA fragments may be manipulated, so as to provide for the DNA sequences in the proper orientation and, as appropriate, in the proper reading frame. Toward this end, adapters or linkers may be employed to join the DNA fragments or other manipulations may be involved to provide for convenient restriction sites, removal of superfluous DNA, removal of restriction sites, or the like. For this purpose, in vitro mutagenesis, primer repair, restriction, annealing, resubstitutions, e.g., transitions and transversions, may be involved.
[0097] The expression cassette can also comprise a selectable marker gene for the selection of transformed cells. Selectable marker genes are utilized for the selection of transformed cells or tissues. Marker genes include genes encoding antibiotic resistance, such as those encoding neomycin phosphotransferase II (NEO) and hygromycin phosphotransferase (HPT), as well as genes conferring resistance to herbicidal compounds, such as glufosinate ammonium, bromoxynil, imidazolinones, and 2,4-dichlorophenoxyacetate (2,4-D). Additional selectable markers include phenotypic markers such as β-galactosidase and fluorescent proteins such as green fluorescent protein (GFP) (Su, et al., (2004) Biotechnol Bioeng 85:610-9 and Fetter, et al., (2004) Plant Cell 16:215-28), cyan florescent protein (CYP) (Bolte, et al., (2004) J. Cell Science 117:943-54 and Kato, et al., (2002) Plant Physiol 129:913-42), and yellow florescent protein (PhiYFP® from Evrogen, see, Bolte, et al., (2004) J. Cell Science 117:943-54). For additional selectable markers, see generally, Yarranton, (1992) Curr. Opin. Biotech. 3:506-511; Christopherson, et al., (1992) Proc. Natl. Acad. Sci. USA 89:6314-6318; Yao, et al., (1992) Cell 71:63-72; Reznikoff, (1992) Mol. Microbiol. 6:2419-2422; Barkley, et al., (1980) in The Operon, pp. 177-220; Hu, et al., (1987) Cell 48:555-566; Brown, et al., (1987) Cell 49:603-612; Figge, et al., (1988) Cell 52:713-722; Deuschle, et al., (1989) Proc. Natl. Acad. Aci. USA 86:5400-5404; Fuerst, et al., (1989) Proc. Natl. Acad. Sci. USA 86:2549-2553; Deuschle, et al., (1990) Science 248:480-483; Gossen (1993) Ph.D. Thesis, University of Heidelberg; Reines, et al., (1993) Proc. Natl. Acad. Sci. USA 90:1917-1921; Labow, et al., (1990) Mol. Cell. Biol. 10:3343-3356; Zambretti, et al., (1992) Proc. Natl. Acad. Sci. USA 89:3952-3956; Baim, et al., (1991) Proc. Natl. Acad. Sci. USA 88:5072-5076; Wyborski, et al., (1991) Nucleic Acids Res. 19:4647-4653; Hillenand-Wissman, (1989) Topics Mol. Struc. Biol. 10:143-162; Degenkolb, et al., (1991) Antimicrob. Agents Chemother. 35:1591-1595; Kleinschnidt, et al., (1988) Biochemistry 27:1094-1104; Bonin, (1993) Ph.D. Thesis, University of Heidelberg; Gossen, et al., (1992) Proc. Natl. Acad. Sci. USA 89:5547-5551; Oliva, et al., (1992) Antimicrob. Agents Chemother. 36:913-919; Hlavka, et al., (1985) Handbook of Experimental Pharmacology, Vol. 78 (Springer-Verlag, Berlin); Gill, et al., (1988) Nature 334:721-724. Such disclosures are herein incorporated by reference. The above list of selectable marker genes is not meant to be limiting. Any selectable marker gene can be used in the present invention.
[0098] A number of promoters can be used in the practice of the invention, including the native promoter of the polynucleotide sequence of interest. The promoters can be selected based on the desired outcome. The nucleic acids can be combined with constitutive, tissue-preferred, or other promoters for expression in plants.
[0099] Such constitutive promoters include, for example, the core promoter of the Rsyn7 promoter and other constitutive promoters disclosed in WO 99/43838 and U.S. Pat. No. 6,072,050; the core CaMV 35S promoter (Odell, et al., (1985) Nature 313:810-812); rice actin (McElroy, et al., (1990) Plant Cell 2:163-171); ubiquitin (Christensen, et al., (1989) Plant Mol. Biol. 12:619-632 and Christensen, et al., (1992) Plant Mol. Biol. 18:675-689); pEMU (Last, et al., (1991) Theor. Appl. Genet. 81:581-588); MAS (Velten, et al., (1984) EMBO J. 3:2723-2730); ALS promoter (U.S. Pat. No. 5,659,026), and the like. Other constitutive promoters include, for example, U.S. Pat. Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463; 5,608,142 and 6,177,611.
[0100] Tissue-preferred promoters can be utilized to target enhanced CKX expression within a particular plant tissue. Tissue-preferred promoters include those disclosed by Yamamoto, et al., (1997) Plant J. 12(2):255-265; Kawamata, et al., (1997) Plant Cell Physiol. 38(7):792-803; Hansen, et al., (1997) Mol. Gen Genet. 254(3):337-343; Russell, et al., (1997) Transgenic Res. 6(2):157-168; Rinehart, et al., (1996) Plant Physiol. 112(3):1331-1341; Van Camp, et al., (1996) Plant Physiol. 112(2):525-535; Canevascini, et al., (1996) Plant Physiol. 112(2):513-524; Yamamoto, et al., (1994) Plant Cell Physiol. 35(5):773-778; Lam, (1994) Results Probl. Cell Differ. 20:181-196; Orozco, et al., (1993) Plant Mol Biol. 23(6):1129-1138; Matsuoka, et al., (1993) Proc Natl. Acad. Sci. USA 90(20):9586-9590; and Guevara-Garcia, et al., (1993) Plant J. 4(3):495-505. Such promoters can be modified, if necessary, for weak expression. See, also, US Patent Application Publication Number 2003/0074698, herein incorporated by reference. Promoters active in maternal plant tissues, such as female florets, ovaries, aleurone, pedicel, and pedicel-forming region, either pre-pollination or upon pollination, may be of particular interest.
[0101] Leaf-preferred promoters are known in the art. See, for example, Yamamoto, et al., (1997) Plant J. 12(2):255-265; Kwon, et al., (1994) Plant Physiol. 105:357-67; Yamamoto, et al., (1994) Plant Cell Physiol. 35(5):773-778; Gotor, et al., (1993) Plant J. 3:509-18; Orozco, et al., (1993) Plant Mol. Biol. 23(6):1129-1138; Baszczynski, et al., (1988) Nucl. Acid Res. 16:4732; Mitra, et al., (1994) Plant Molecular Biology 26:35-93; Kayaya, et al., (1995) Molecular and General Genetics 248:668-674; and Matsuoka, et al., (1993) Proc. Natl. Acad. Sci. USA 90(20):9586-9590. Senescence regulated promoters are also of use, such as SAM22 (Crowell, et al., (1992) Plant Mol. Biol. 18:459-466).
[0102] Root-preferred or root-specific promoters are known and can be selected from the many available from the literature or isolated de novo from various compatible species. See, for example, Hire, et al., (1992) Plant Mol. Biol. 20(2):207-218 (soybean root-specific glutamine synthetase gene); Keller and Baumgartner, (1991) Plant Cell 3(10):1051-1061 (root-specific control element in the GRP 1.8 gene of French bean); Sanger, et al., (1990) Plant Mol. Biol. 14(3):433-443 (root-specific promoter of the mannopine synthase (MAS) gene of Agrobacterium tumefaciens); and Miao, et al., (1991) Plant Cell 3(1):11-22 (full-length cDNA clone encoding cytosolic glutamine synthetase (GS), which is expressed in roots and root nodules of soybean). See also, Bogusz, et al., (1990) Plant Cell 2(7):633-641, where two root-specific promoters isolated from hemoglobin genes from the nitrogen-fixing nonlegume Parasponia andersonii and the related non-nitrogen-fixing nonlegume Trema tomentosa are described. The promoters of these genes were linked to a β-glucuronidase reporter gene and introduced into both the nonlegume Nicotiana tabacum and the legume Lotus corniculatus, and in both instances root-specific promoter activity was preserved. Leach and Aoyagi, (1991) describe their analysis of the promoters of the highly expressed rolC and rolD root-inducing genes of Agrobacterium rhizogenes (see, Plant Science (Limerick) 79(1):69-76). They concluded that enhancer and tissue-preferred DNA determinants are dissociated in those promoters. Teeri, et al., (1989) used gene fusion to lacZ to show that the Agrobacterium T-DNA gene encoding octopine synthase is especially active in the epidermis of the root tip and that the TR2' gene is root specific in the intact plant and stimulated by wounding in leaf tissue, an especially desirable combination of characteristics for use with an insecticidal or larvicidal gene (see, EMBO J. 8(2):343-350). The TR1' gene, fused to nptII (neomycin phosphotransferase II) showed similar characteristics. Additional root-preferred promoters include the VfENOD-GRP3 gene promoter (Kuster, et al., (1995) Plant Mol. Biol. 29(4):759-772); rolB promoter (Capana, et al., (1994) Plant Mol. Biol. 25(4):681-691; and the CRWAQ81 root-preferred promoter with the ADH first intron (U.S. patent application Ser. No. 10/961,629, filed Oct. 8, 2004, herein incorporated by reference). See also, U.S. Pat. Nos. 5,837,876; 5,750,386; 5,633,363; 5,459,252; 5,401,836; 5,110,732 and 5,023,179. Promoters associated with the Ckx1 gene from maize may also be useful in modifying CKX activity in roots; see SEQ ID NOS: 17 and 18 herein and U.S. Pat. Nos. 6,921,815 and 7,371,925, and U.S. patent application Ser. No. 12/051,893. Other root-preferred promoters include Zm-NAS2 (U.S. patent application Ser. No. 12/030,455, filed Feb. 13, 2008), Zm-Cyclo1 promoter (U.S. Pat. No. 7,268,226), Zm-Metallothionein promoters (U.S. Pat. Nos. 6,774,282; 7,214,854 and 7,214,855 (also known as RootMET2)), Zm-MSY promoter (SEQ ID NO: 64; U.S. Patent Application Ser. No. 60/971,310 filed Sep. 11, 2007), or MsZRP promoter (SEQ ID NO: 65; see, U.S. Pat. No. 5,633,363); constructs may also include one or more of the CaMV35S enhancer, Odell, et al., (1988) Plant Mol. Biol. 10:263-272, the ADH1 INTRON1 (Callis, et al., (1987) Genes and Dev. 1:1183-1200), the UBI1ZM INTRON (PHI) as an enhancer, and PINII terminator.
[0103] "Seed-preferred" promoters include those promoters active during seed development, such as those expressed preferentially in female reproductive tissues, and those regulating seed storage proteins, as well as those promoters active during seed germination. See, Thompson, et al., (1989) BioEssays 10:108, herein incorporated by reference. Such seed-preferred promoters include, but are not limited to, maize zag2.1 promoter, (GenBank X80206); maize Zap promoter, also known as ZmMADS (US Patent Application Publication Number 2004/0025206); maize eep1 promoter (US Patent Publication Number 2004/0237147); maize lec1 promoter (U.S. patent application Ser. No. 09/718,754); maize F3.7 promoter (Baszczynski, et al., (1997) Maydica 42:189-201; maize tb1 promoter (Hubbarda, et al., (2002) Genetics 162:1927-1935); maize Zm40 promoter (U.S. Pat. No. 6,403,862 and WO 2001/21783); maize mLIP15 promoter, U.S. Pat. No. 6,479,734; maize ESR promoter, US Patent Application Publication Number 2004/0210960; maize PCNA 2 promoter (U.S. Patent application Ser. No. 10/388,359 and WO 2003/078591); Cim1 (cytokinin-induced message); cZ19B1 (maize 19 kDa zein); milps (myo-inositol-1-phosphate synthase) (see, WO 2000/11177 and U.S. Pat. No. 6,225,529, herein incorporated by reference); and a BETL (basal endosperm transfer layer) promoter, for example, see, U.S. Pat. No. 7,119,251. Several gamma-zein promoters are known to drive endosperm-specific expression. Globulin-1 (Glob-1) is a representative embryo-specific promoter. For dicots, seed-specific promoters include, but are not limited to, bean β-phaseolin, napin, β-conglycinin, soybean lectin, cruciferin, and the like. For monocots, seed-specific promoters include, but are not limited to, maize 15 kDa zein, 22 kDa zein, 27 kDa zein, gamma-zein, waxy, shrunken 1, shrunken 2, globulin 1, etc. See also, WO 2000/12733 and U.S. Pat. No. 6,528,704, where seed-preferred promoters from end1 and end2 genes are disclosed; herein incorporated by reference. Additional embryo specific promoters are disclosed in Sato, et al., (1996) Proc. Natl. Acad. Sci. 93:8117-8122; Nakase, et al., (1997) Plant J 12:235-46; and Postma-Haarsma, et al., (1999) Plant Mol. Biol. 39:257-71. Additional endosperm specific promoters are disclosed in Albani, et al., (1984) EMBO 3:1405-15; Albani, et al., (1999) Theor. Appl. Gen. 98:1253-62; Albani, et al., (1993) Plant J. 4:343-55; Mena, et al., (1998) The Plant Journal 116:53-62, and Wu, et al., (1998) Plant Cell Physiology 39:885-889.
[0104] Shoot-preferred promoters include shoot meristem-preferred promoters such as promoters disclosed in Weigal, et al., (1992) Cell 69:843-859; Accession Number AJ131822; Accession Number Z71981; Accession Number AF049870 and shoot-preferred promoters disclosed in McAvoy, et al., (2003) Acta Hort. (ISHS) 625:379-385.
[0105] Dividing cell or meristematic tissue-preferred promoters have been disclosed in Ito, et al., (1994) Plant Mol. Biol. 24:863-878; Reyad, et al., (1995) Mo. Gen. Genet. 248:703-711; Shaul, et al., (1996) Proc. Natl. Acad. Sci. 93:4868-4872; Ito, et al., (1997) Plant J. 11:983-992; and Trehin, et al., (1997) Plant Mol. Biol. 35:667-672.
[0106] Inflorescence-preferred promoters include the promoter of chalcone synthase (Van der Meer, et al., (1990) Plant Mol. Biol. 15:95-109), LAT52 (Twell, et al., (1989) Mol. Gen. Genet. 217:240-245), pollen specific genes (Albani et al (1990) Plant Mol. Biol. 15:605, Zm13 (Buerrero, et al., (1993) Mol. Gen. Genet. 224:161-168), maize pollen-specific gene (Hamilton, et al., (1992) Plant Mol. Biol. 18:211-218), sunflower pollen expressed gene (Baltz, et al., (1992) The Plant Journal 2:713-721), B. napus pollen specific genes (Arnoldo, et al., (1992) J. Cell. Biochem, Abstract Number Y101204).
[0107] Stress inducible promoters include salt/water stress-inducible promoters such as P5CS (Zang, et al., (1997) Plant Sciences 129:81-89); cold-inducible promoters, such as cor15a (Hajela, et al., (1990) Plant Physiol. 93:1246-1252), cor15b (Wlihelm, et al., (1993) Plant Mol Biol 23:1073-1077), wsc120 (Ouellet, et al., (1998) FEBS Lett. 423:324-328), ci7 (Kirch, et al., (1997) Plant Mol Biol. 33:897-909), ci21A (Schneider, et al., (1997) Plant Physiol. 113:335-45); drought-inducible promoters, such as, Trg-31 (Chaudhary, et al., (1996) Plant Mol. Biol. 30:1247-57), rd29 (Kasuga, et al., (1999) Nature Biotechnology 18:287-291); osmotic inducible promoters, such as, Rab17 (Vilardell, et al., (1991) Plant Mol. Biol. 17:985-93) and osmotin (Raghothama, et al., (1993) Plant Mol Biol 23:1117-28); and heat inducible promoters, such as heat shock proteins (Barros, et al., (1992) Plant Mol. 19:665-75; Marrs, et al., (1993) Dev. Genet. 14:27-41), and smHSP (Waters, et al., (1996) J. Experimental Botany 47:325-338). Other stress-inducible promoters include rip2 (U.S. Pat. No. 5,332,808 and US Patent Application Publication Number 2003/0217393) and rd29a (Yamaguchi-Shinozaki, et al., (1993) Mol. Gen. Genetics 236:331-334).
[0108] The methods of the invention comprise introducing a polypeptide or polynucleotide into a host cell (i.e., a plant). "Introducing" is intended to mean presenting to the plant the polynucleotide or polypeptide in such a manner that the sequence gains access to the interior of a cell. The methods of the invention do not depend on a particular method for introducing a sequence into the host cell, only that the polynucleotide or polypeptides gains access to the interior of at least one cell of the host. Methods for introducing polynucleotide or polypeptides into host cells (i.e., plants) are known in the art and include, but are not limited to, stable transformation methods, transient transformation methods, and virus-mediated methods.
[0109] "Stable transformation" is intended to mean that the nucleotide construct introduced into a host (i.e., a plant) integrates into the genome of the plant and is capable of being inherited by the progeny thereof. "Transient transformation" is intended to mean that a polynucleotide is introduced into the host (i.e., a plant) and expressed temporally or a polypeptide is introduced into a host (i.e., a plant).
[0110] Transformation protocols as well as protocols for introducing polypeptides or polynucleotide sequences into plants may vary depending on the type of plant or plant cell, i.e., monocot or dicot, targeted for transformation. Suitable methods of introducing polypeptides and polynucleotides into plant cells include microinjection (Crossway, et al., (1986) Biotechniques 4:320-334), electroporation (Riggs, et al., (1986) Proc. Natl. Acad. Sci. USA 83:5602-5606, Agrobacterium-mediated transformation (Townsend, et al., U.S. Pat. No. 5,563,055; Zhao, et al., U.S. Pat. No. 5,981,840), direct gene transfer (Paszkowski, et al., (1984) EMBO J. 3:2717-2722), and ballistic particle acceleration (see, for example, Sanford, et al., U.S. Pat. No. 4,945,050; Tomes, et al., U.S. Pat. No. 5,879,918; Tomes, et al., U.S. Pat. No. 5,886,244; Bidney, et al., U.S. Pat. No. 5,932,782; Tomes, et al., (1995) "Direct DNA Transfer into Intact Plant Cells via Microprojectile Bombardment," in Plant Cell, Tissue, and Organ Culture: Fundamental Methods, ed. Gamborg and Phillips (Springer-Verlag, Berlin); McCabe, et al., (1988) Biotechnology 6:923-926); and Lec1 transformation (WO 2000/28058). Also see, Weissinger, et al., (1988) Ann. Rev. Genet. 22:421-477; Sanford, et al., (1987) Particulate Science and Technology 5:27-37 (onion); Christou, et al., (1988) Plant Physiol. 87:671-674 (soybean); McCabe, et al., (1988) Bio/Technology 6:923-926 (soybean); Finer and McMullen, (1991) In Vitro Cell Dev. Biol. 27P:175-182 (soybean); Singh, et al., (1998) Theor. Appl. Genet. 96:319-324 (soybean); Datta, et al., (1990) Biotechnology 8:736-740 (rice); Klein, et al., (1988) Proc. Natl. Acad. Sci. USA 85:4305-4309 (maize); Klein, et al., (1988) Biotechnology 6:559-563 (maize); Tomes, U.S. Pat. No. 5,240,855; Buising, et al., U.S. Pat. Nos. 5,322,783 and 5,324,646; Tomes, et al., (1995) "Direct DNA Transfer into Intact Plant Cells via Microprojectile Bombardment," in Plant Cell, Tissue, and Organ Culture: Fundamental Methods, ed. Gamborg, (Springer-Verlag, Berlin) (maize); Klein, et al., (1988) Plant Physiol. 91:440-444 (maize); Fromm, et al., (1990) Biotechnology 8:833-839 (maize); Hooykaas-Van Slogteren, et al., (1984) Nature (London) 311:763-764; Bowen, et al., U.S. Pat. No. 5,736,369 (cereals); Bytebier, et al., (1987) Proc. Natl. Acad. Sci. USA 84:5345-5349 (Liliaceae); De Wet, et al., (1985) in The Experimental Manipulation of Ovule Tissues, ed. Chapman, et al., (Longman, N.Y.), pp. 197-209 (pollen); Kaeppler, et al., (1990) Plant Cell Reports 9:415-418 and Kaeppler, et al., (1992) Theor. Appl. Genet. 84:560-566 (whisker-mediated transformation); D'Halluin, et al., (1992) Plant Cell 4:1495-1505 (electroporation); Li, et al., (1993) Plant Cell Reports 12:250-255 and Christou and Ford, (1995) Annals of Botany 75:407-413 (rice); Osjoda, et al., (1996) Nature Biotechnology 14:745-750 (maize via Agrobacterium tumefaciens); all of which are herein incorporated by reference.
[0111] In specific embodiments, the CKX sequences of the invention can be provided to a plant using a variety of transient transformation methods. Such transient transformation methods include, but are not limited to, the introduction of the CKX protein or variants or fragments thereof directly into the plant, or the introduction of a CKX transcript into the plant. Such methods include, for example, microinjection or particle bombardment. See, for example, Crossway, et al., (1986) Mol Gen. Genet. 202:179-185; Nomura, et al., (1986) Plant Sci. 44:53-58; Hepler, et al., (1994) Proc. Natl. Acad. Sci. 91: 2176-2180 and Hush, et al., (1994) The Journal of Cell Science 107:775-784, all of which are herein incorporated by reference. Alternatively, the CKX polynucleotide can be transiently transformed into the plant using techniques known in the art. Such techniques include viral vector system and the precipitation of the polynucleotide in a manner that precludes subsequent release of the DNA. Thus, the transcription from the particle-bound DNA can occur, but the frequency with which it is released to become integrated into the genome is greatly reduced. Such methods include the use particles coated with polyethylimine (PEI; Sigma #P3143).
[0112] In certain embodiments, the polynucleotide of the invention may be introduced into plants by contacting plants with a virus or viral nucleic acids. Generally, such methods involve incorporating a nucleotide construct of the invention within a viral DNA or RNA molecule. It is recognized that the a CKX sequence of the invention may be initially synthesized as part of a viral polyprotein, which later may be processed by proteolysis in vivo or in vitro to produce the desired recombinant protein. Further, it is recognized that promoters of the invention also encompass promoters utilized for transcription by viral RNA polymerases. Methods for introducing polynucleotides into plants and expressing a protein encoded therein, involving viral DNA or RNA molecules, are known in the art. See, for example, U.S. Pat. Nos. 5,889,191, 5,889,190, 5,866,785, 5,589,367, 5,316,931 and Porta, et al., (1996) Molecular Biotechnology 5:209-221; herein incorporated by reference.
[0113] Methods are known in the art for the targeted insertion of a polynucleotide at a specific location in the plant genome. In one embodiment, the insertion of the polynucleotide at a desired genomic location is achieved using a site-specific recombination system. See, for example, WO 1999/25821, WO 1999/25854, WO 1999/25840, WO 1999/25855, and WO 1999/25853; also, U.S. Pat. Nos. 6,552,248, 6,624,297, 6,573,425, 6,455,315 and 6,458,594, all of which are herein incorporated by reference. Briefly, the polynucleotide of the invention can be contained in a transfer cassette flanked by two non-identical recombination sites. The transfer cassette is introduced into a plant having stably incorporated into its genome a target site which is flanked by two non-identical recombination sites that correspond to the sites of the transfer cassette. An appropriate recombinase is provided and the transfer cassette is integrated at the target site. The polynucleotide of interest is thereby integrated at a specific chromosomal position in the plant genome.
[0114] The cells that have been transformed may be grown into plants in accordance with conventional ways. See, for example, McCormick, et al., (1986) Plant Cell Reports 5:81-84. These plants may then be grown, and either pollinated with the same transformed strain or different strains, and the resulting progeny having appropriate expression of the desired phenotypic characteristic identified. Two or more generations may be grown to ensure that expression of the desired phenotypic characteristic is stably maintained and inherited, and then seeds harvested to ensure expression of the desired phenotypic characteristic has been achieved. In this manner, the present invention provides transformed seed (also referred to as "transgenic seed") having a polynucleotide of the invention, for example, an expression cassette of the invention, stably incorporated into its genome.
[0115] Pedigree breeding starts with the crossing of two genotypes, such as an elite line of interest and one other line having one or more desirable characteristics (e.g., having stably incorporated a polynucleotide of the invention, having a modulated activity and/or level of the polypeptide of the invention, etc.) which complements the elite line of interest. If the two original parents do not provide all the desired characteristics, other sources can be included in the breeding population. In the pedigree method, superior plants are selfed and selected in successive filial generations. In the succeeding filial generations the heterozygous condition gives way to homogeneous lines as a result of self-pollination and selection. Typically in the pedigree method of breeding, five or more successive filial generations of selfing and selection are practiced: F1→F2; F2→F3; F3→F4; F4→F5, etc. After a sufficient amount of inbreeding, successive filial generations will serve to increase seed of the developed inbred. Preferably, the inbred line comprises homozygous alleles at about 95% or more of its loci.
[0116] Backcrossing can be used to transfer one or more specifically desirable traits from one line, the donor parent, to an inbred called the recurrent parent, which has overall good agronomic characteristics yet lacks that desirable trait or traits. Backcrossing may be used in combination with pedigree breeding to modify an elite line of interest, and a hybrid is made using the modified elite line. However, the same procedure can be used to move the progeny toward the genotype of the recurrent parent but at the same time retain many components of the non-recurrent parent, by stopping the backcrossing at an early stage and proceeding with selfing and selection. For example, an F1, such as a commercial hybrid, is created. This commercial hybrid may be backcrossed to one of its parent lines to create a BC1 or BC2. Progeny are selfed and selected so that the newly developed inbred has many of the attributes of the recurrent parent and yet several of the desired attributes of the non-recurrent parent. This approach leverages the value and strengths of the recurrent parent for use in new hybrids and breeding.
[0117] Therefore, one embodiment of this invention is a method of making a backcross conversion of a maize inbred line of interest, comprising the steps of crossing a plant of the maize inbred line of interest with a donor plant comprising a mutant gene or transgene conferring a desired trait, selecting an F1 progeny plant comprising the mutant gene or transgene conferring the desired trait, and backcrossing the selected F1 progeny plant to a plant of the maize inbred line of interest. This method may further comprise the step of obtaining a molecular marker profile of the maize inbred line of interest and using the molecular marker profile to select for a progeny plant with the desired trait and the molecular marker profile of the inbred line of interest. In the same manner, this method may be used to produce F1 hybrid seed by adding a final step of crossing the desired trait-converted maize inbred line of interest with a different maize plant to make F1 hybrid maize seed comprising a mutant gene or transgene conferring the desired trait.
[0118] Recurrent selection is a method used in a plant breeding program to improve a population of plants. The method entails individual plants cross pollinating with each other to form progeny. The progeny are grown and the superior progeny selected by any number of selection methods, which include individual plant, half-sib progeny, full-sib progeny, selfed progeny and topcrossing. The selected progeny are cross-pollinated with each other to form progeny for another population. This population is planted and again superior plants are selected to cross pollinate with each other. Recurrent selection is a cyclical process and therefore can be repeated as many times as desired. The objective of recurrent selection is to improve the traits of a population. The improved population can then be used as a source of breeding material to obtain inbred lines to be used in hybrids or used as parents for a synthetic cultivar. A synthetic cultivar is the resultant progeny formed by the intercrossing of several selected inbreds.
[0119] Mass selection is a useful technique especially when used in conjunction with molecular marker enhanced selection. In mass selection seeds from individuals are selected based on phenotype and/or genotype. These selected seeds are then bulked and used to grow the next generation. Bulk selection requires growing a population of plants in a bulk plot, allowing the plants to self-pollinate, harvesting the seed in bulk and then using a sample of the seed harvested in bulk to plant the next generation. Instead of self pollination, directed pollination could be used as part of the breeding program.
[0120] Mutation breeding is one of many methods that could be used to introduce new traits into an elite line. Mutations that occur spontaneously or are artificially induced can be useful sources of variability for a plant breeder. The goal of artificial mutagenesis is to increase the rate of mutation for a desired characteristic. Mutation rates can be increased by many different means including temperature, long-term seed storage, tissue culture conditions, radiation (such as X-rays, Gamma rays (e.g., cobalt 60 or cesium 137), neutrons, (product of nuclear fission by uranium 235 in an atomic reactor), Beta radiation (emitted from radioisotopes such as phosphorus 32 or carbon 14), or ultraviolet radiation (preferably from 2500 to 2900 nm)), or chemical mutagens (such as base analogues (5-bromo-uracil), related compounds (8-ethoxy caffeine), antibiotics (streptonigrin), alkylating agents (sulfur mustards, nitrogen mustards, epoxides, ethylenamines, sulfates, sulfonates, sulfones, lactones), azide, hydroxylamine, nitrous acid, or acridines. Once a desired trait is observed through mutagenesis the trait may then be incorporated into existing germplasm by traditional breeding techniques, such as backcrossing. Details of mutation breeding can be found in Fehr's "Principles of Cultivar Development," 1993, Macmillan Publishing Company, the disclosure of which is incorporated herein by reference. In addition, mutations created in other lines may be used to produce a backcross conversion of an elite line comprising such mutations.
[0121] The present invention may be used for transformation of any plant species, including, but not limited to, monocots and dicots. Examples of plant species of interest include, but are not limited to, corn or maize (Zea mays), Brassica sp. (e.g., B. napus, B. rapa, B. juncea), particularly those Brassica species useful as sources of seed oil, alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), millet (e.g., pearl millet (Pennisetum glaucum), proso millet (Panicum miliaceum), foxtail millet (Setaria italica), finger millet (Eleusine coracana)), sunflower (Helianthus annuus), safflower (Carthamus tinctorius), wheat (Triticum aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanuts (Arachis hypogaea), cotton (Gossypium barbadense, Gossypium hirsutum), sweet potato (Ipomoea batatus), cassava (Manihot esculenta), coffee (Coffea spp.), coconut (Cocos nucifera), pineapple (Ananas comosus), citrus trees (Citrus spp.), cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musa spp.), avocado (Persea americana), fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica), olive (Olea europaea), papaya (Carica papaya), cashew (Anacardium occidentale), macadamia (Macadamia integrifolia), almond (Prunus amygdalus), sugar beets (Beta vulgaris), sugarcane (Saccharum spp.), oats, barley, vegetables, ornamentals, grasses and conifers.
[0122] Vegetables include tomatoes (Lycopersicon esculentum), lettuce (e.g., Lactuca sativa), green beans (Phaseolus vulgaris), lima beans (Phaseolus limensis), peas (Lathyrus spp.), and members of the genus Cucumis such as cucumber (C. sativus), cantaloupe (C. cantalupensis), and musk melon (C. melo). Ornamentals include azalea (Rhododendron spp.), hydrangea (Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosa spp.), tulips (Tulipa spp.), daffodils (Narcissus spp.), petunias (Petunia hybrida), carnation (Dianthus caryophyllus), poinsettia (Euphorbia pulcherrima), and chrysanthemum. Turfgrasses include, but are not limited to: annual bluegrass (Poa annua); annual ryegrass (Lolium multiflorum); Canada bluegrass (Poa compressa); Chewings fescue (Festuca rubra); colonial bentgrass (Agrostis tenuis); creeping bentgrass (Agrostis palustris); crested wheatgrass (Agropyron desertorum); fairway wheatgrass (Agropyron cristatum); hard fescue (Festuca longifolia); Kentucky bluegrass (Poa pratensis); orchardgrass (Dactylis glomerata); perennial ryegrass (Lolium perenne); red fescue (Festuca rubra); redtop (Agrostis alba); rough bluegrass (Poa trivialis); sheep fescue (Festuca ovina); smooth bromegrass (Bromus inermis); tall fescue (Festuca arundinacea); timothy (Phleum pratense); velvet bentgrass (Agrostis canina); weeping alkaligrass (Puccinellia distans); western wheatgrass (Agropyron smithii); Bermuda grass (Cynodon spp.); St. Augustine grass (Stenotaphrum secundatum); zoysia grass (Zoysia spp.); Bahia grass (Paspalum notatum); carpet grass (Axonopus affinis); centipede grass (Eremochloa ophiuroides); kikuyu grass (Pennisetum clandesinum); seashore paspalum (Paspalum vaginatum); blue grama (Bouteloua gracilis); buffalo grass (Buchloe dactyloids); sideoats grama (Bouteloua curtipendula).
[0123] In specific embodiments, plants of the present invention are crop plants (for example, corn (maize), alfalfa, sunflower, Brassica, soybean, cotton, safflower, peanut, sorghum, wheat, millet, tobacco, etc.).
[0124] Other plants of interest include grain plants that provide seeds of interest, oil-seed plants, and leguminous plants. Seeds of interest include grain seeds, such as corn, wheat, barley, rice, sorghum, rye, etc. Oil-seed plants include cotton, soybean, safflower, sunflower, Brassica, maize, alfalfa, palm, coconut, etc. Leguminous plants include beans and peas. Beans include guar, locust bean, fenugreek, soybean, garden beans, cowpea, mungbean, lima bean, fava bean, lentils, chickpea, etc.
[0125] Typically, an intermediate host cell will be used in the practice of this invention to increase the copy number of the cloning vector. With an increased copy number, the vector containing the nucleic acid of interest can be isolated in significant quantities for introduction into the desired plant cells. In one embodiment, plant promoters that do not cause expression of the polypeptide in bacteria are employed.
[0126] Prokaryotes most frequently are represented by various strains of E. coli; however, other microbial strains may also be used. Commonly used prokaryotic control sequences, which are defined herein to include promoters for transcription initiation, optionally with an operator, along with ribosome binding sequences, include such commonly used promoters as the beta lactamase (penicillinase) and lactose (lac) promoter systems (Chang, et al., (1977) Nature 198:1056), the tryptophan (trp) promoter system (Goeddel, et al., (1980) Nucleic Acids Res. 8:4057) and the lambda derived P L promoter and N-gene ribosome binding site (Shimatake, et al., (1981) Nature 292:128). The inclusion of selection markers in DNA vectors transfected in E coli. is also useful. Examples of such markers include genes specifying resistance to ampicillin, tetracycline, or chloramphenicol.
[0127] The vector is selected to allow introduction into the appropriate host cell. Bacterial vectors are typically of plasmid or phage origin. Appropriate bacterial cells are infected with phage vector particles or transfected with naked phage vector DNA. If a plasmid vector is used, the bacterial cells are transfected with the plasmid vector DNA. Expression systems for expressing a protein of the present invention are available using Bacillus sp. and Salmonella (Palva, et al., (1983) Gene 22:229-235); Mosbach, et al., (1983) Nature 302:543-545).
[0128] A variety of eukaryotic expression systems such as yeast, insect cell lines, plant and mammalian cells, are known to those of skill in the art. As explained briefly below, a polynucleotide of the present invention can be expressed in these eukaryotic systems. In some embodiments, transformed/transfected plant cells, as discussed infra, are employed as expression systems for production of the proteins of the instant invention.
[0129] Synthesis of heterologous polynucleotides in yeast is well known (Sherman, et al., (1982) Methods in Yeast Genetics, Cold Spring Harbor Laboratory). Two widely utilized yeasts for production of eukaryotic proteins are Saccharomyces cerevisiae and Pichia pastoris. Vectors, strains, and protocols for expression in Saccharomyces and Pichia are known in the art and available from commercial suppliers (e.g., Invitrogen). Suitable vectors usually have expression control sequences, such as promoters, including 3-phosphoglycerate kinase or alcohol oxidase, and an origin of replication, termination sequences and the like as desired.
[0130] A protein of the present invention, once expressed, can be isolated from yeast by lysing the cells and applying standard protein isolation techniques to the lysate. The monitoring of the purification process can be accomplished by using Western blot techniques or radioimmunoassay or other standard immunoassay techniques.
[0131] The sequences of the present invention can also be ligated to various expression vectors for use in transfecting cell cultures of, for instance, mammalian, insect, or plant origin. Illustrative cell cultures useful for the production of the peptides are mammalian cells. A number of suitable host cell lines capable of expressing intact proteins have been developed in the art, and include the HEK293, BHK21, and CHO cell lines. Expression vectors for these cells can include expression control sequences, such as an origin of replication, a promoter (e.g., the CMV promoter, a HSV tk promoter or pgk (phosphoglycerate kinase) promoter), an enhancer (Queen, et al., (1986) Immunol. Rev. 89:49), and necessary processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites (e.g., an SV40 large T Ag poly A addition site), and transcriptional terminator sequences. Other animal cells useful for production of proteins of the present invention are available, for instance, from the American Type Culture Collection.
[0132] Appropriate vectors for expressing proteins of the present invention in insect cells are usually derived from the SF9 baculovirus. Suitable insect cell lines include mosquito larvae, silkworm, armyworm, moth and Drosophila cell lines such as a Schneider cell line (See, Schneider, (1987) J. Embryol. Exp. Morphol. 27:353-365).
[0133] As with yeast, when higher animal or plant host cells are employed, polyadenylation or transcription terminator sequences are typically incorporated into the vector. An example of a terminator sequence is the polyadenylation sequence from the bovine growth hormone gene. Sequences for accurate splicing of the transcript may also be included. An example of a splicing sequence is the VP1 intron from SV40 (Sprague, et al., (1983) J. Virol. 45:773-781). Additionally, gene sequences to control replication in the host cell may be incorporated into the vector such as those found in bovine papilloma virus type-vectors (Saveria-Campo, (1985) DNA Cloning Vol. II a Practical Approach, D. M. Glover, Ed., IRL Press, Arlington, Va., pp. 213-238).
[0134] Animal and lower eukaryotic (e.g., yeast) host cells are competent or rendered competent for transfection by various means. There are several well-known methods of introducing DNA into animal cells. These include: calcium phosphate precipitation, fusion of the recipient cells with bacterial protoplasts containing the DNA, treatment of the recipient cells with liposomes containing the DNA, DEAE dextrin, electroporation, biolistics and micro-injection of the DNA directly into the cells. The transfected cells are cultured by means well known in the art (Kuchler, (1997) Biochemical Methods in Cell Culture and Virology, Dowden, Hutchinson and Ross, Inc.).
[0135] In certain embodiments, the polynucleotides of the present invention can be stacked with any combination of other polynucleotide sequences of interest in order to create a plant with a desired phenotype with respect to one or more traits. The combinations generated may include multiple copies of any one or more of the polynucleotides of interest.
[0136] These stacked combinations can be created by any method including, but not limited to, cross breeding plants by any conventional or TopCross methodology, or genetic transformation. If the traits are stacked by genetically transforming the plants, the polynucleotide sequences of interest can be combined at any time and in any order. For example, a transgenic plant comprising one or more desired traits can be used as the target to introduce further traits by subsequent transformation. The traits can be introduced simultaneously in a co-transformation protocol with the polynucleotides of interest provided by any combination of transformation cassettes. For example, if two sequences will be introduced, the two sequences can be contained in separate transformation cassettes (trans) or contained on the same transformation cassette (cis). Expression of the sequences can be driven by the same promoter or by different promoters. In certain cases, it may be desirable to introduce a transformation cassette that will suppress the expression of a polynucleotide of interest. This may be combined with any combination of other suppression cassettes or overexpression cassettes to generate the desired combination of traits in the plant.
II. Modulating the Concentration and/or Activity of a CKX Polypeptide
[0137] A method for modulating the concentration and/or activity of a polypeptide of the present invention in a plant is provided. In general, concentration and/or activity is increased or decreased by at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% relative to a native control plant, plant part, or cell which did not have the sequence of the invention introduced. Modulation in the present invention may occur during and/or subsequent to growth of the plant to the desired stage of development. In specific embodiments, the polypeptides of the present invention are modulated in monocots, particularly maize.
[0138] A variety of methods can be employed to assay for a modulation in the concentration and/or activity of a CKX polypeptide. For instance, the expression level of the CKX polypeptide may be measured directly, for example, by assaying for the level of the CKX polypeptide in the plant (i.e., Western or Northern blot), or indirectly, for example, by assaying the cytokinin oxidase activity of the CKX polypeptide in the plant. Methods for measuring the cytokinin oxidase activity are described elsewhere herein. In specific embodiments, modulation of CKX polypeptide concentration and/or activity comprises the modulation (i.e., an increase or a decrease) in the level of cytokinin in the plant. Methods to measure the level and/or activity of cytokinin are known in the art and are discussed elsewhere herein. In still other embodiments, the level and/or activity of the CKX polypeptide is modulated in vegetative tissue, in reproductive tissue, or in both vegetative and reproductive tissue.
[0139] In one embodiment, the activity and/or concentration of the CKX polypeptide is modulated by introducing the polypeptide or the polynucleotide of the invention into the plant. Subsequently, a plant having the introduced sequence of the invention is selected using methods known to those of skill in the art such as, but not limited to, Southern blot analysis, DNA sequencing, PCR analysis, or phenotypic analysis. A plant or plant part altered or modified by the foregoing embodiments is grown under plant forming conditions for a time sufficient to modulate the concentration and/or activity of the CKX polypeptide in the plant. Plant forming conditions are well known in the art and discussed briefly elsewhere herein.
[0140] It is also recognized that the level and/or activity of the polypeptide may be modulated by employing a polynucleotide that is not capable of directing, in a transformed plant, the expression of a protein or an RNA. For example, the polynucleotides of the invention may be used to design polynucleotide constructs that can be employed in methods for altering or mutating a genomic nucleotide sequence in an organism. Such polynucleotide constructs include, but are not limited to, RNA:DNA vectors, RNA:DNA mutational vectors, RNA:DNA repair vectors, mixed-duplex oligonucleotides, self-complementary RNA:DNA oligonucleotides, and recombinogenic oligonucleobases. Such nucleotide constructs and methods of use are known in the art. See, U.S. Pat. Nos. 5,565,350; 5,731,181; 5,756,325; 5,760,012; 5,795,972 and 5,871,984, all of which are herein incorporated by reference. See also, WO 1998/49350, WO 1999/07865, WO 1999/25821 and Beetham, et al., (1999) Proc. Natl. Acad. Sci. USA 96:8774-8778; herein incorporated by reference. It is therefore recognized that methods of the present invention do not depend on the incorporation of the entire polynucleotide into the genome, only that the plant or cell thereof is altered as a result of the introduction of the polynucleotide into a cell. In one embodiment of the invention, the genome may be altered following the introduction of the polynucleotide into a cell. For example, the polynucleotide, or any part thereof, may be incorporated into the genome of the plant. Alterations to the genome of the present invention include, but are not limited to, additions, deletions, and substitutions of nucleotides into the genome. While the methods of the present invention do not depend on additions, deletions, and substitutions of any particular number of nucleotides, it is recognized that such additions, deletions, or substitutions comprise at least one nucleotide.
[0141] Genetic constructs providing reduced expression of cytokinin oxidase genes may be used in combination with constructs providing further modulation of effective levels of cytokinin in a plant, including increased biosynthesis of cytokinins, as described in co-pending U.S. patent application Ser. No. 09/545,334 filed Apr. 16, 1999 and US Patent Application Publication Number 2004/0237147, published Nov. 24, 2004, herein incorporated by reference.
A Increasing the Activity and/or Level of a CKX Polypeptide
[0142] Methods are provided to increase the activity and/or level of a CKX polypeptide of the invention in a plant. Such increase in the level and/or activity of a CKX polypeptide of the invention can be achieved by providing to the plant a CKX polypeptide. The CKX polypeptide can be provided by introducing the amino acid sequence encoding the CKX polypeptide into the plant, introducing into the plant a nucleotide sequence encoding a CKX polypeptide, or by modifying a genomic locus encoding the CKX polypeptide of the invention.
[0143] As discussed elsewhere herein, many methods are known in the art for providing a polypeptide to a plant including, but not limited to, direct introduction of the polypeptide into the plant, and introducing into the plant (transiently or stably) a polynucleotide construct encoding a polypeptide having cytokinin oxidase activity. It is also recognized that the methods of the invention may employ a polynucleotide that is not capable of directing, in the transformed plant, the expression of a protein or an RNA. Thus, the level and/or activity of a CKX polypeptide may be increased by altering the gene encoding the CKX polypeptide or by altering or affecting its promoter. See, e.g., Kmiec, U.S. Pat. No. 5,565,350; Zarling, et al., PCT/US93/03868. Therefore mutagenized plants that carry mutations in CKX genes, where the mutations increase expression of the CKX gene or increase the cytokinin oxidase activity of the encoded CKX polypeptide, are provided.
B. Reducing the Activity and/or Level of a CKX Polypeptide
[0144] Methods are provided to reduce or eliminate the activity of a CKX polypeptide of the invention by transforming a plant cell with an expression cassette that expresses a polynucleotide that inhibits the expression of the CKX polypeptide. The polynucleotide may inhibit the expression of the CKX polypeptide directly, by preventing translation of the CKX messenger RNA, or indirectly, by encoding a polypeptide that inhibits the transcription or translation of a CKX gene encoding a CKX polypeptide. Methods for inhibiting or eliminating the expression of a gene in a plant are well known in the art, and any such method may be used in the present invention to inhibit the expression of a CKX polypeptide.
[0145] In accordance with the present invention, the expression of a CKX polypeptide is inhibited if the protein level of the CKX polypeptide is less than the protein level of the same CKX polypeptide in a plant or plant part that has not been genetically modified or mutagenized to inhibit the expression of that CKX polypeptide. In particular embodiments of the invention, the protein level of the CKX polypeptide in a modified plant or plant part according to the invention is less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5% or less than 2% of the protein level of the same CKX polypeptide in a plant or plant part that is not a mutant or that has not been genetically modified to inhibit the expression of that CKX polypeptide. The expression level of the CKX polypeptide may be measured directly, for example, by assaying for the level of CKX polypeptide expressed in the plant cell or plant, or indirectly, for example, by measuring the cytokinin oxidase activity of the CKX polypeptide in the plant cell or plant, or by measuring the cytokinin level or activity in the plant or plant cell. Methods for performing such assays are described elsewhere herein.
[0146] In certain embodiments of the invention, the activity of the CKX polypeptides is reduced or eliminated by transforming a plant cell with an expression cassette comprising a polynucleotide encoding a polypeptide that inhibits the activity of a CKX polypeptide. The cytokinin oxidase activity of a CKX polypeptide is inhibited according to the present invention if the cytokinin oxidase activity of the CKX polypeptide is less than the cytokinin oxidase activity of the same CKX polypeptide in a plant that has not been modified to inhibit the cytokinin oxidase activity of that CKX polypeptide. In particular embodiments of the invention, the cytokinin oxidase activity of the CKX polypeptide in a modified plant according to the invention is less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10% or less than 5% of the cytokinin oxidase activity of the same CKX polypeptide in a plant that that has not been modified to inhibit the expression of that CKX polypeptide. The cytokinin oxidase activity of a CKX polypeptide is "eliminated" according to the invention when it is not detectable by the assay methods described elsewhere herein. Methods of determining the cytokinin oxidase activity of a CKX polypeptide are described elsewhere herein.
[0147] In other embodiments, the activity of a CKX polypeptide may be reduced or eliminated by disrupting the gene encoding the CKX polypeptide. The invention encompasses mutagenized plants that carry mutations in CKX genes, where the mutations reduce expression of the CKX gene or inhibit the cytokinin oxidase activity of the encoded CKX polypeptide.
[0148] Thus, many methods may be used to reduce or eliminate the activity of a CKX polypeptide. In addition, more than one method may be used to reduce the activity of a single CKX polypeptide. Non-limiting examples of methods of reducing or eliminating the expression of CKX polypeptides are given below.
1. Polynucleotide-Based Methods:
[0149] In some embodiments of the present invention, a plant is transformed with an expression cassette that is capable of expressing a polynucleotide that inhibits the expression of a CKX polypeptide of the invention. The term "expression" as used herein refers to the biosynthesis of a gene product, including the transcription and/or translation of said gene product. For example, for the purposes of the present invention, an expression cassette capable of expressing a polynucleotide that inhibits the expression of at least one CKX polypeptide is an expression cassette capable of producing an RNA molecule that inhibits the transcription and/or translation of at least one CKX polypeptide of the invention. The "expression" or "production" of a protein or polypeptide from a DNA molecule refers to the transcription and translation of the coding sequence to produce the protein or polypeptide, while the "expression" or "production" of a protein or polypeptide from an RNA molecule refers to the translation of the RNA coding sequence to produce the protein or polypeptide.
[0150] Examples of polynucleotides that inhibit the expression of a CKX polypeptide are given below.
i. Sense Suppression/Cosuppression
[0151] In some embodiments of the invention, inhibition of the expression of a CKX polypeptide may be obtained by sense suppression or cosuppression. For cosuppression, an expression cassette is designed to express an RNA molecule corresponding to all or part of a messenger RNA encoding a CKX polypeptide in the "sense" orientation. Overexpression of this RNA molecule can result in reduced expression of the native gene. Accordingly, multiple plant lines transformed with the cosuppression expression cassette are screened to identify those that show the greatest inhibition of CKX polypeptide expression.
[0152] The polynucleotide used for cosuppression may correspond to all or part of the sequence encoding the CKX polypeptide, all or part of the 5' and/or 3' untranslated region of a CKX polypeptide transcript, or all or part of both the coding sequence and the untranslated regions of a transcript encoding a CKX polypeptide. In some embodiments where the polynucleotide comprises all or part of the coding region for the CKX polypeptide, the expression cassette is designed to eliminate the start codon of the polynucleotide so that no protein product will be translated.
[0153] Cosuppression may be used to inhibit the expression of plant genes to produce plants having undetectable protein levels for the proteins encoded by these genes. See, for example, Broin, et al., (2002) Plant Cell 14:1417-1432. Cosuppression may also be used to inhibit the expression of multiple proteins in the same plant. See, for example, U.S. Pat. No. 5,942,657. Methods for using cosuppression to inhibit the expression of endogenous genes in plants are described in Flavell, et al., (1994) Proc. Natl. Acad. Sci. USA 91:3490-3496; Jorgensen, et al., (1996) Plant Mol. Biol. 31:957-973; Johansen and Carrington, (2001) Plant Physiol. 126:930-938; Broin, et al., (2002) Plant Cell 14:1417-1432; Stoutjesdijk, et al., (2002) Plant Physiol. 129:1723-1731; Yu, et al., (2003) Phytochemistry 63:753-763 and U.S. Pat. Nos. 5,034,323, 5,283,184 and 5,942,657, each of which is herein incorporated by reference. The efficiency of cosuppression may be increased by including a poly-dT region in the expression cassette at a position 3' to the sense sequence and 5' of the polyadenylation signal. See, US Patent Application Publication Number 2002/0048814, herein incorporated by reference. Typically, such a nucleotide sequence has substantial sequence identity to the sequence of the transcript of the endogenous gene, optimally greater than about 65% sequence identity, more optimally greater than about 85% sequence identity, most optimally greater than about 95% sequence identity. See, U.S. Pat. Nos. 5,283,184 and 5,034,323, herein incorporated by reference.
ii. Antisense Suppression
[0154] In some embodiments of the invention, inhibition of the expression of the CKX polypeptide may be obtained by antisense suppression. For antisense suppression, the expression cassette is designed to express an RNA molecule complementary to all or part of a messenger RNA encoding the CKX polypeptide. Overexpression of the antisense RNA molecule can result in reduced expression of the native gene. Accordingly, multiple plant lines transformed with the antisense suppression expression cassette are screened to identify those that show the greatest inhibition of CKX polypeptide expression.
[0155] The polynucleotide for use in antisense suppression may correspond to all or part of the complement of the sequence encoding the CKX polypeptide, all or part of the complement of the 5' and/or 3' untranslated region of the CKX transcript or all or part of the complement of both the coding sequence and the untranslated regions of a transcript encoding the CKX polypeptide. In addition, the antisense polynucleotide may be fully complementary (i.e., 100% identical to the complement of the target sequence) or partially complementary (i.e., less than 100% identical to the complement of the target sequence) to the target sequence. Antisense suppression may be used to inhibit the expression of multiple proteins in the same plant. See, for example, U.S. Pat. No. 5,942,657. Furthermore, portions of the antisense nucleotides may be used to disrupt the expression of the target gene. Generally, sequences of at least 50 nucleotides, 100 nucleotides, 200 nucleotides, 300, 400, 450, 500, 550 or greater may be used. Methods for using antisense suppression to inhibit the expression of endogenous genes in plants are described, for example, in Liu, et al., (2002) Plant Physiol. 129:1732-1743 and U.S. Pat. Nos. 5,759,829 and 5,942,657, each of which is herein incorporated by reference. Efficiency of antisense suppression may be increased by including a poly-dT region in the expression cassette at a position 3' to the antisense sequence and 5' of the polyadenylation signal. See, US Patent Application Publication Number 2002/0048814, herein incorporated by reference.
iii. Double-Stranded RNA Interference
[0156] In some embodiments of the invention, inhibition of the expression of a CKX polypeptide may be obtained by double-stranded RNA (dsRNA) interference. For dsRNA interference, a sense RNA molecule like that described above for cosuppression and an antisense RNA molecule that is fully or partially complementary to the sense RNA molecule are expressed in the same cell, resulting in inhibition of the expression of the corresponding endogenous messenger RNA.
[0157] Expression of the sense and antisense molecules can be accomplished by designing the expression cassette to comprise both a sense sequence and an antisense sequence. Alternatively, separate expression cassettes may be used for the sense and antisense sequences. Multiple plant lines transformed with the dsRNA interference expression cassette or expression cassettes are then screened to identify plant lines that show the greatest inhibition of CKX polypeptide expression. Methods for using dsRNA interference to inhibit the expression of endogenous plant genes are described in Waterhouse, et al., (1998) Proc. Natl. Acad. Sci. USA 95:13959-13964, Liu, et al., (2002) Plant Physiol. 129:1732-1743 and WO 1999/49029, WO 1999/53050, WO 1999/61631 and WO 2000/49035, each of which is herein incorporated by reference.
iv. Hairpin RNA Interference and Intron-Containing Hairpin RNA Interference
[0158] In some embodiments of the invention, inhibition of the expression of one or more CKX polypeptides may be obtained by hairpin RNA (hpRNA) interference or intron-containing hairpin RNA (ihpRNA) interference. These methods are highly efficient at inhibiting the expression of endogenous genes. See, Waterhouse and Helliwell, (2003) Nat. Rev. Genet. 4:29-38 and the references cited therein.
[0159] For hpRNA interference, the expression cassette is designed to express an RNA molecule that hybridizes with itself to form a hairpin structure that comprises a single-stranded loop region and a base-paired stem. The base-paired stem region comprises a sense sequence corresponding to all or part of the endogenous messenger RNA encoding the gene whose expression is to be inhibited, and an antisense sequence that is fully or partially complementary to the sense sequence. Thus, the base-paired stem region of the molecule generally determines the specificity of the RNA interference. Alternatively, the base-paired stem region may comprise complementary sequences corresponding to a selected promoter region, resulting in silencing of a coding sequence operably linked to said selected promoter. See, for example, Mette, et al., (2000) EMBO J. 19(19):5194-5201. hpRNA molecules are highly efficient at inhibiting the expression of endogenous genes, and the RNA interference they induce is inherited by subsequent generations of plants. See, for example, Chuang and Meyerowitz, (2000) Proc. Natl. Acad. Sci. USA 97:4985-4990; Stoutjesdijk, et al., (2002) Plant Physiol. 129:1723-1731 and Waterhouse and Helliwell, (2003) Nat. Rev. Genet. 4:29-38. Methods for using hpRNA interference to inhibit or silence the expression of genes are described, for example, in Chuang and Meyerowitz, (2000) Proc. Natl. Acad. Sci. USA 97:4985-4990; Stoutjesdijk, et al., (2002) Plant Physiol. 129:1723-1731; Waterhouse and Helliwell, (2003) Nat. Rev. Genet. 4:29-38; Pandolfini, et al., BMC Biotechnology 3:7 and US Patent Application Publication Number 2003/0175965, each of which is herein incorporated by reference. A transient assay for the efficiency of hpRNA constructs to silence gene expression in vivo has been described by Panstruga, et al., (2003) Mol. Biol. Rep. 30:135-140, herein incorporated by reference.
[0160] For ihpRNA, the interfering molecules have the same general structure as for hpRNA, but the RNA molecule additionally comprises an intron that is capable of being spliced in the cell in which the ihpRNA is expressed. The use of an intron minimizes the size of the loop in the hairpin RNA molecule following splicing, and this increases the efficiency of interference. See, for example, Smith, et al., (2000) Nature 407:319-320. In fact, Smith, et al., show 100% suppression of endogenous gene expression using ihpRNA-mediated interference. Methods for using ihpRNA interference to inhibit the expression of endogenous plant genes are described, for example, in Smith, et al., (2000) Nature 407:319-320; Wesley, et al., (2001) Plant J. 27:581-590; Wang and Waterhouse, (2001) Curr. Opin. Plant Biol. 5:146-150; Waterhouse and Helliwell, (2003) Nat. Rev. Genet. 4:29-38; Helliwell and Waterhouse, (2003) Methods 30:289-295 and US Patent Application Publication Number 2003/0180945, each of which is herein incorporated by reference. See also, Cigan, et al., (2005) Plant Journal 43:929-940, demonstrating downregulation using a hairpin construct which targets the DNA regulating expression of a gene of interest.
[0161] The expression cassette for hpRNA interference may also be designed such that the sense sequence and the antisense sequence do not correspond to an endogenous RNA. In this embodiment, the sense and antisense sequence flank a loop sequence that comprises a nucleotide sequence corresponding to all or part of the endogenous messenger RNA of the target gene. Thus, it is the loop region that determines the specificity of the RNA interference. See, for example, WO 2002/00904, herein incorporated by reference.
v. Amplicon-Mediated Interference
[0162] Amplicon expression cassettes comprise a plant virus-derived sequence that contains all or part of the target gene but generally not all of the genes of the native virus. The viral sequences present in the transcription product of the expression cassette allow the transcription product to direct its own replication. The transcripts produced by the amplicon may be either sense or antisense relative to the target sequence (i.e., the messenger RNA for the CKX polypeptide). Methods of using amplicons to inhibit the expression of endogenous plant genes are described, for example, in Angell and Baulcombe, (1997) EMBO J. 16:3675-3684, Angell and Baulcombe, (1999) Plant J. 20:357-362 and U.S. Pat. No. 6,646,805, each of which is herein incorporated by reference.
vi. Ribozymes
[0163] In some embodiments, the polynucleotide expressed by the expression cassette of the invention is catalytic RNA or has ribozyme activity specific for the messenger RNA of the CKX polypeptide. Thus, the polynucleotide causes the degradation of the endogenous messenger RNA, resulting in reduced expression of the CKX polypeptide. This method is described, for example, in U.S. Pat. No. 4,987,071, herein incorporated by reference.
vii Small Interfering RNA or Micro RNA
[0164] In some embodiments of the invention, inhibition of the expression of a CKX polypeptide may be obtained by RNA interference by expression of a gene encoding a micro RNA (miRNA). miRNAs are regulatory agents consisting of about 22 ribonucleotides. miRNA are highly efficient at inhibiting the expression of endogenous genes. See, for example, Javier, et al., (2003) Nature 425:257-263, herein incorporated by reference.
[0165] For miRNA interference, the expression cassette is designed to express an RNA molecule that is modeled on an endogenous miRNA gene. The miRNA gene encodes an RNA that forms a hairpin structure containing a 22-nucleotide sequence that is complementary to another endogenous gene (target sequence). For suppression of CKX expression, the 22-nucleotide sequence is selected from a CKX transcript sequence and contains 22 nucleotides of said CKX sequence in sense orientation and 21 nucleotides of a corresponding antisense sequence that is complementary to the sense sequence. miRNA molecules are highly efficient at inhibiting the expression of endogenous genes, and the RNA interference they induce is inherited by subsequent generations of plants.
2. Polypeptide-Based Inhibition of Gene Expression
[0166] In one embodiment, the polynucleotide encodes a zinc finger protein that binds to a gene encoding a CKX polypeptide, resulting in reduced expression of the gene. In particular embodiments, the zinc finger protein binds to a regulatory region of a CKX gene. In other embodiments, the zinc finger protein binds to a messenger RNA encoding a CKX polypeptide and prevents its translation. Methods of selecting sites for targeting by zinc finger proteins have been described, for example, in U.S. Pat. No. 6,453,242, and methods for using zinc finger proteins to inhibit the expression of genes in plants are described, for example, in US Patent Application Publication Number 2003/0037355; each of which is herein incorporated by reference.
3. Polypeptide-Based Inhibition of Protein Activity
[0167] In some embodiments of the invention, the polynucleotide encodes an antibody that binds to at least one CKX polypeptide, and reduces the cytokinin oxidase activity of the CKX polypeptide. In another embodiment, the binding of the antibody results in increased turnover of the antibody-CKX complex by cellular quality control mechanisms. The expression of antibodies in plant cells and the inhibition of molecular pathways by expression and binding of antibodies to proteins in plant cells are well known in the art. See, for example, Conrad and Sonnewald, (2003) Nature Biotech. 21:35-36, incorporated herein by reference.
4. Gene Disruption
[0168] In some embodiments of the present invention, the activity of a CKX polypeptide is reduced or eliminated by disrupting the gene encoding the CKX polypeptide. The gene encoding the CKX polypeptide may be disrupted by any method known in the art. For example, in one embodiment, the gene is disrupted by transposon tagging. In another embodiment, the gene is disrupted by mutagenizing plants using random or targeted mutagenesis, and selecting for plants that have reduced cytokinin oxidase activity.
i. Transposon Tagging
[0169] In one embodiment of the invention, transposon tagging is used to reduce or eliminate the CKX activity of one or more CKX polypeptides. Transposon tagging comprises inserting a transposon within an endogenous CKX gene to reduce or eliminate expression of the CKX polypeptide. "CKX gene" is intended to mean the gene that encodes a CKX polypeptide according to the invention.
[0170] In this embodiment, the expression of one or more CKX polypeptide is reduced or eliminated by inserting a transposon within a regulatory region or coding region of the gene encoding the CKX polypeptide. A transposon that is within an exon, intron, 5' or 3' untranslated sequence, a promoter, or any other regulatory sequence of a CKX gene may be used to reduce or eliminate the expression and/or activity of the encoded CKX polypeptide.
[0171] Methods for the transposon tagging of specific genes in plants are well known in the art. See, for example, Maes, et al., (1999) Trends Plant Sci. 4:90-96; Dharmapuri and Sonti, (1999) FEMS Microbiol. Lett. 179:53-59; Meissner, et al., (2000) Plant J. 22:265-274; Phogat, et al., (2000) J. Biosci. 25:57-63; Walbot, (2000) Curr. Opin. Plant Biol. 2:103-107; Gai, et al., (2000) Nucleic Acids Res. 28:94-96; Fitzmaurice, et al., (1999) Genetics 153:1919-1928). In addition, the TUSC process for selecting Mu insertions in selected genes has been described in Bensen, et al., (1995) Plant Cell 7:75-84; Mena, et al., (1996) Science 274:1537-1540 and U.S. Pat. No. 5,962,764; each of which is herein incorporated by reference.
ii. Mutant Plants with Reduced Activity
[0172] Additional methods for decreasing or eliminating the expression of endogenous genes in plants are also known in the art and can be similarly applied to the instant invention. These methods include other forms of mutagenesis, such as ethyl methanesulfonate-induced mutagenesis, deletion mutagenesis and fast neutron deletion mutagenesis used in a reverse genetics sense (with PCR) to identify plant lines in which the endogenous gene has been deleted. For examples of these methods see, Ohshima, et al., (1998) Virology 243:472-481; Okubara, et al., (1994) Genetics 137:867-874 and Quesada, et al., (2000) Genetics 154:421-436; each of which is herein incorporated by reference. In addition, a fast and automatable method for screening for chemically induced mutations, TILLING (Targeting Induced Local Lesions In Genomes), using denaturing HPLC or selective endonuclease digestion of selected PCR products is also applicable to the instant invention. See, McCallum, et al., (2000) Nat. Biotechnol. 18:455-457, herein incorporated by reference.
[0173] Mutations that impact gene expression or that interfere with the function (cytokinin oxidase activity) of the encoded protein are well known in the art. Insertional mutations in gene exons usually result in null-mutants. Mutations in conserved residues are particularly effective in inhibiting the cytokinin oxidase activity of the encoded protein. Conserved residues of plant CKX polypeptides suitable for mutagenesis with the goal to eliminate cytokinin oxidase activity have been described. See, for example, FIGS. 4, 9 and 10 and Example 3. Such mutants can be isolated according to well-known procedures, and mutations in different CKX loci can be stacked, for example by genetic crossing. See, for example, Gruis, et al., (2002) Plant Cell 14:2863-2882.
[0174] In another embodiment of this invention, dominant mutants can be used to trigger RNA silencing due to gene inversion and recombination of a duplicated gene locus. See, for example, Kusaba, et al., (2003) Plant Cell 15:1455-1467.
[0175] The invention encompasses additional methods for reducing or eliminating the activity of one or more CKX polypeptides. Examples of other methods for altering or mutating a genomic nucleotide sequence in a plant are known in the art and include, but are not limited to, the use of RNA:DNA vectors, RNA:DNA mutational vectors, RNA:DNA repair vectors, mixed-duplex oligonucleotides, self-complementary RNA:DNA oligonucleotides and recombinogenic oligonucleobases. Such vectors and methods of use are known in the art. See, for example, U.S. Pat. Nos. 5,565,350; 5,731,181; 5,756,325; 5,760,012; 5,795,972 and 5,871,984; each of which are herein incorporated by reference. See also, WO 1998/49350, WO 1999/07865, WO 1999/25821 and Beetham, et al., (1999) Proc. Natl. Acad. Sci. USA 96:8774-8778; each of which is herein incorporated by reference.
iii. Modulating Cytokinin Level/Activity
[0176] As used herein a "cytokinin" refers to a class of plant-specific hormones that play a central role during the cell cycle and influence numerous developmental programs. Cytokinins comprise an N6-substituted purine derivative. Representative cytokinins include isopentenyladenine (N6-(Δ2-isopentenyl)adenine (hereinafter, iP), zeatin (6-(4-hydroxy-3methylbut-trans-2-enylamino) purine) (hereinafter, Z) and dihydrozeatin (DZ). The free bases and their ribosides (iPR, ZR and DZR) are believed to be the active compounds. Additional cytokinins are known. See, for example, U.S. Pat. No. 5,211,738.
[0177] "Modulating the level and/or activity of cytokinin" includes any decrease or increase in cytokinin level and/or activity in the plant. For example, modulating the level and/or activity can comprise either an increase or a decrease in overall cytokinin content of about 0.1%, 0.5%, 1%, 3% 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or greater when compared to a control plant or plant part. Alternatively, the modulated level and/or activity of the cytokinin can include about a 0.5 fold, 1 fold, 2 fold, 4 fold, 8 fold, 16 fold or 32 fold increase or decrease in cytokinin level/activity in the plant or a plant part when compared to a control plant or plant part.
[0178] It is further recognized that the modulation of the cytokinin level/activity need not be an overall increase/decrease in cytokinin level and/or activity, but also includes a change in tissue distribution of the cytokinin. For example, CKX polypeptides may influence the amount of cytokinin imported into specific tissues or exported from a cytokinin producing tissue. For example, import of cytokinin in sink tissues may involve an apoplastic transport step, where CKX polypeptides control the level of physiologically active cytokinins. See, for example, Jones, et al., (1997) Plant Growth Regul 23:123-134, Turner, et al., (1985) Plant Physiol 79:321-322, and Mok, et al., (2001) Annu Rev Plant Physiol Plant Mol Biol 52:89-118, each of which are herein incorporated by reference.
[0179] Moreover, the modulation of the cytokinin level/activity need not be an overall increase/decrease in cytokinins, but also includes a change in the ratio of various cytokinin derivatives. For example, the ratio of various cytokinin derivatives such as isopentenyladenine-type, zeatin-type, or dihydrozeatin-type cytokinins, and the like, could be altered and thereby modulate the level/activity of the cytokinin of the plant or plant part when compared to a control plant.
[0180] Methods for assaying for a modulation in cytokinin level and/or activity are known in the art. For example, representative methods for cytokinin extraction, immunopurification, HPLC separation, and quantification by ELISA methods can be found in Faiss, et al., (1997) Plant J. 12:401-415. See also, Werner, et al., (2001) PNAS 98:10487-10492) and Dewitte, et al., (1999) Plant Physiol. 119:111-121. Each of these references is herein incorporated by reference.
[0181] In specific methods, the level and/or activity of a cytokinin in a plant is decreased by increasing the level or activity of the CKX polypeptide in the plant. Methods for increasing the level and/or activity of CKX polypeptides in a plant are discussed elsewhere herein. Briefly, such methods comprise providing a CKX polypeptide of the invention to a plant and thereby increasing the level and/or activity of the CKX polypeptide. In other embodiments, a CKX nucleotide sequence encoding a CKX polypeptide can be provided by introducing into the plant a polynucleotide comprising a CKX nucleotide sequence of the invention, expressing the CKX sequence, increasing the activity of the CKX polypeptide and thereby decreasing the level and/or activity of a cytokinin in the plant or plant part. In certain embodiments, the CKX nucleotide construct introduced into the plant is stably incorporated into the genome of the plant.
[0182] In other methods, the level and/or activity of a cytokinin in a plant is increased by decreasing the level and/or activity of the CKX polypeptide in the plant. Such methods are disclosed in detail elsewhere herein. In one such method, a CKX nucleotide sequence is introduced into the plant and expression of said CKX nucleotide sequence decreases the activity of the CKX polypeptide, and thereby increasing the level and/or activity of a cytokinin in the plant or plant part. In certain embodiments, the CKX nucleotide construct introduced into the plant is stably incorporated into the genome of the plant.
[0183] As discussed above, one of skill will recognize the appropriate promoter to use to modulate the level/activity of a cytokinin in the plant. Exemplary promoters for this embodiment have been disclosed elsewhere herein.
[0184] Accordingly, the present invention further provides plants having a modulated level/activity of a cytokinin when compared to the cytokinin level/activity of a control plant. In one embodiment, the plant of the invention has an increased level/activity of the CKX polypeptide of the invention and thus has a decreased level/activity of cytokinin. In other embodiments, the plant of the invention has a reduced or eliminated level of the CKX polypeptide of the invention and thus has an increased level/activity of a cytokinin. In certain embodiments, such plants have stably incorporated into their genome a nucleic acid molecule comprising a CKX nucleotide sequence of the invention operably linked to a promoter that drives expression in the plant cell.
iv. Modulating Root Development
[0185] Methods for modulating root development in a plant are provided. By "modulating root development" is intended any alteration in the development of the plant root when compared to a control plant. Such alterations in root development include, but are not limited to, alterations in the growth rate of the primary root, the fresh root weight, the extent of lateral and adventitious root formation, the vasculature system, meristem development, or radial expansion.
[0186] Methods for modulating root development in a plant are provided. The methods comprise modulating the level and/or activity of the CKX polypeptide in the plant. In one method, a CKX sequence of the invention is provided to the plant. In another method, the CKX nucleotide sequence is provided by introducing into the plant a polynucleotide comprising a CKX nucleotide sequence of the invention, expressing the CKX sequence, and thereby modifying root development. In still other methods, the CKX nucleotide construct introduced into the plant is stably incorporated into the genome of the plant.
[0187] In other methods, root development is modulated by increasing the level or activity of the CKX polypeptide in the plant. An increase in CKX activity can result in one or more alterations to root development, including, but not limited to, larger root meristems, increased root growth, enhanced radial expansion, an enhanced vasculature system, increased root branching, more adventitious roots, and/or an increase in fresh root weight when compared to a control plant.
[0188] As used herein, "root growth" encompasses all aspects of growth of the different parts that make up the root system at different stages of its development in both monocotyledonous and dicotyledonous plants. It is to be understood that enhanced root growth can result from enhanced growth of one or more of its parts including the primary root, lateral roots, adventitious roots, etc.
[0189] Methods of measuring such developmental alterations in the root system are known in the art. See, for example, US Patent Application Publication Number 2003/0074698 and Werner, et al., (2001) PNAS 18:10487-10492, both of which are herein incorporated by reference.
[0190] As discussed above, one of skill will recognize the appropriate promoter to use to modulate root development in the plant. Exemplary promoters for this embodiment include constitutive promoters and root-preferred promoters. Exemplary root-preferred promoters have been disclosed elsewhere herein.
[0191] Stimulating root growth and increasing root mass by increasing the activity and/or level of the CKX polypeptide also finds use in improving the standability of a plant. The term "resistance to lodging" or "standability" refers to the ability of a plant to fix itself to the soil. For plants with an erect or semi-erect growth habit, this term also refers to the ability to maintain an upright position under adverse conditions, such as adverse environments. This trait relates to the size, depth and morphology of the root system. In addition, stimulating root growth and increasing root mass by increasing the level and/or activity of the CKX polypeptide also finds use in promoting in vitro propagation of explants.
[0192] Furthermore, higher root biomass production due to an increased level and/or activity of CKX has a direct effect on the yield and an indirect effect on production of compounds produced by root cells or transgenic root cells or cell cultures of said transgenic root cells. One example of an interesting compound produced in root cultures is shikonin, the yield of which can be advantageously enhanced by said methods.
[0193] Higher root biomass production resulting from an increased level and/or activity of CKX may also impact the plant's assimilation of water and/or nutrients, favorably impacting yield of vegetative and/or reproductive tissues, including seed. Further, improved root structure may result in increased tolerance to drought, or improved nitrogen use efficiency, or improved disease resistance, or improved insect resistance, particularly when combined with an insecticidal trait. Such characteristics may be apparent at various points throughout the plant life cycle, affecting, for example, flowering, early seed development and/or senescence. Modified plants may be more productive with current fertilizer application rates, or may maintain their productivity even under significantly reduced fertilizer input or on less fertile soils. Increased nitrogen use efficiency can result from enhanced uptake and assimilation of nitrogen fertilizer and/or the subsequent remobilization and reutilization of accumulated nitrogen reserves, enhancing yield. Improving nitrogen use efficiency in maize would increase corn harvestable yield per unit of input nitrogen, both in developing nations where access to nitrogen fertilizer is limited and in developed nations where the level of nitrogen use is high. Nitrogen utilization improvement also allows decreases in on-farm input costs, reduces dependence on non-renewable energy sources required for synthetic nitrogen fertilizer production and decreases the environmental impact of nitrogen fertilizer manufacturing and its agricultural use.
[0194] Evaluation for improved nitrogen use efficiency may include testing in field plots where yield is limited by reducing fertilizer application by 30% or more. Improvement in nitrogen utilization resulting from expression of transgenic events is measured by assessing yield, yield components, or other agronomic traits of transgenic plants compared to non-transgenic plants in these reduced-nitrogen-fertility plots. Similar comparisons are made in plots supplemented with recommended nitrogen fertility rates. Effective transgenic events may achieve similar yields in the nitrogen-limited and normal-nitrogen environments.
[0195] Accordingly, the present invention further provides plants having modulated root development when compared to the root development of a control plant. In some embodiments, the plant of the invention has an increased level/activity of the CKX polypeptide of the invention and has enhanced root growth and/or root biomass. In certain embodiments, such plants have stably incorporated into their genome a nucleic acid molecule comprising a CKX nucleotide sequence of the invention operably linked to a promoter that drives expression in the plant cell. The CKX sequence may be preferentially expressed in cells of root tissues.
v. Modulating Shoot and Leaf Development
[0196] Methods are also provided for modulating shoot and leaf development in a plant. By "modulating shoot and/or leaf development" is intended any alteration in the development of the plant shoot and/or leaf. Such alterations in shoot and/or leaf development include, but are not limited to, alterations in shoot meristem development, in leaf number, leaf size, leaf and stem vasculature, internode length, and leaf senescence. As used herein, "leaf development" and "shoot development" encompasses all aspects of growth of the different parts that make up the leaf system and the shoot system, respectively, at different stages of their development, both in monocotyledonous and dicotyledonous plants. Methods for measuring such developmental alterations in the shoot and leaf system are known in the art. See, for example, Werner, et al., (2001) PNAS 98:10487-10492 and US Patent Application Publication Number 2003/0074698, each of which is herein incorporated by reference.
[0197] The method for modulating shoot and/or leaf development in a plant comprises modulating the activity and/or level of a CKX polypeptide of the invention. In one embodiment, a CKX polypeptide sequence of the invention is provided. In other embodiments, the CKX nucleotide sequence can be provided by introducing into the plant a polynucleotide comprising a CKX nucleotide sequence of the invention, expressing the CKX sequence, and thereby modifying shoot and/or leaf development. In certain embodiments, the CKX nucleotide construct introduced into the plant is stably incorporated into the genome of the plant.
[0198] In specific embodiments, shoot or leaf development is modulated by increasing the level and/or activity of the CKX polypeptide in the plant. An increase in CKX activity can result in one or more alterations in shoot and/or leaf development, including, but not limited to, smaller apical meristems, reduced leaf number, reduced leaf surface, reduced vasculature, shorter internodes and stunted growth, and retarded leaf senescence, when compared to a control plant. Thus, the methods of the invention may find use in producing dwarf plants.
[0199] In certain embodiments, the level and/or activity of the CKX polypeptide in the plant is decreased to result in higher cytokinin levels. As discussed elsewhere herein, targeted reduction in CKX polypeptide level and/or activity may result in one or more of modulated floral development, modulated flowering time, increased seed size and/or increased seed weight, increased plant yield and/or plant vigor, improved or maintained stress tolerance, altered root/shoot ratio, or an increase in shoot growth, when compared to a control plant.
[0200] As discussed above, one of skill will recognize the appropriate promoter to use to modulate shoot and leaf development of the plant. Exemplary promoters for this embodiment include constitutive promoters, shoot-preferred promoters, shoot meristem-preferred promoters, and leaf-preferred promoters. Exemplary promoters have been disclosed elsewhere herein.
[0201] Accordingly, the present invention further provides plants having modulated shoot and/or leaf development when compared to a control plant. In some embodiments, the plant of the invention has an increased level/activity of the CKX polypeptide of the invention. In other embodiments, the plant of the invention has a decreased level/activity of the CKX polypeptide of the invention.
vi. Modulating Reproductive Tissue Development
[0202] Methods for modulating reproductive tissue development are provided. In one embodiment, methods are provided to modulate floral development in a plant. By "modulating floral development" is intended any alteration in a structure of a plant's reproductive tissue as compared to a control plant in which the activity or level of the CKX polypeptide has not been modulated. "Modulating floral development" further includes any alteration in the timing of the development of a plant's reproductive tissue (i.e., a delayed or a accelerated timing of floral development) when compared to a control plant in which the activity or level of the CKX polypeptide has not been modulated. Macroscopic alterations may include changes in size, shape, number, or location of reproductive organs, the developmental time period over which these structures form, and/or the ability to maintain or proceed through the flowering process in times of environmental stress. Microscopic alterations may include changes to the types or shapes of cells that make up the reproductive organs.
[0203] The method for modulating floral development in a plant comprises modulating CKX activity in a plant. In one method, a CKX sequence of the invention is provided. A CKX nucleotide sequence can be provided by introducing into the plant a polynucleotide comprising a CKX nucleotide sequence of the invention, expressing the CKX sequence and thereby modifying floral development. In certain embodiments, the CKX nucleotide construct introduced into the plant is stably incorporated into the genome of the plant.
[0204] In specific methods, floral development is modulated by increasing the level or activity of the CKX polypeptide in the plant. An increase in CKX activity can result in one or more alterations in floral development, including, but not limited to, retarded flowering, reduced number of flowers, partial male sterility, and reduced seed set, when compared to a control plant. Inducing delayed flowering or inhibiting flowering can be used to enhance yield in forage crops such as alfalfa. Methods for measuring such developmental alterations in floral development are known in the art. See, for example, Mouradov, et al., (2002) The Plant Cell S111-S130, herein incorporated by reference.
[0205] As discussed above, one of skill will recognize the appropriate promoter to use to modulate floral development of the plant. Exemplary promoters for this embodiment include constitutive promoters, inducible promoters, shoot-preferred promoters and inflorescence-preferred promoters.
[0206] In other methods, floral development is modulated by decreasing the level and/or activity of the CKX sequence of the invention. Such methods can comprise introducing a CKX nucleotide sequence into the plant and decreasing the activity of the CKX polypeptide. In other methods, the CKX nucleotide construct introduced into the plant is stably incorporated into the genome of the plant. Decreasing expression of the CKX sequence of the invention can modulate floral development during periods of stress. Such methods are described elsewhere herein.
[0207] Accordingly, the present invention further provides plants having modulated floral development when compared to the floral development of a control plant. Compositions include plants having an increased level/activity of the CKX polypeptide of the invention and having an altered floral development. Compositions also include plants having a decreased level/activity of the CKX polypeptide of the invention wherein the plant maintains or proceeds through the flowering process in times of stress.
[0208] Methods are also provided for the use of the CKX sequences of the invention to increase seed size and/or weight. The method comprises decreasing the activity of the CKX sequences in a plant or plant part, such as the seed, by means of downregulation techniques described elsewhere herein. An increase in seed size and/or weight comprises an increased size or weight of the seed and/or an increase in the size or weight of one or more seed parts including, for example, the embryo, endosperm, seed coat, aleurone and/or cotyledon.
[0209] As discussed above, one of skill will recognize an appropriate promoter to use to increase seed size and/or seed weight. Exemplary promoters of this embodiment include constitutive promoters, inducible promoters, seed-preferred promoters, embryo-preferred promoters, endosperm-preferred promoters and promoters active in female reproductive tissues immediately pre- and post-pollination.
[0210] It is further recognized that increasing seed size and/or weight can also be accompanied by an increase in the speed of growth of seedlings or an increase in early vigor. As used herein, the term "early vigor" refers to the ability of a plant to grow rapidly during early development, and relates to the successful establishment, after germination, of a well-developed root system and a well-developed photosynthetic apparatus. In addition, an increase in seed size and/or weight can result in an increase in plant yield when compared to a control.
[0211] Accordingly, the present invention further provides plants having an increased seed weight and/or seed size when compared to a control plant. In other embodiments, plants having an increased vigor and plant yield are also provided. In some embodiments, the plant of the invention has a decreased level/activity of the CKX polypeptide of the invention and has an increased seed weight and/or seed size. In certain embodiments, such plants have stably incorporated into their genome a nucleic acid molecule comprising a CKX nucleotide sequence of the invention operably linked to a promoter that drives expression in the plant cell.
vii. Modulating the Stress Tolerance of a Plant
[0212] Methods are provided for the use of the CKX sequences of the invention to modify the tolerance of a plant to abiotic stress. Increases in the growth of seedlings or early vigor are often associated with increase in stress tolerance. For example, faster development of seedlings, including the root system of seedlings upon germination, is critical for survival, particularly under adverse conditions such as drought or low temperatures. Promoters that can be used in this method are described elsewhere herein and include constitutive, root-preferred, or stress-induced promoters. Accordingly, in one method of the invention, a plant's tolerance to stress is increased or maintained when compared to a control plant by decreasing the level of CKX activity in one or more parts of the plant. In other methods, a CKX nucleotide sequence is provided by introducing into the plant a polynucleotide comprising a CKX nucleotide sequence of the invention, expressing the CKX sequence and thereby increasing the plant's tolerance to stress. In certain embodiments, the CKX nucleotide construct introduced into the plant is stably incorporated into the genome of the plant.
[0213] Methods are also provided to increase or maintain seed set during abiotic stress episodes. During periods of stress (i.e., drought, salt, heavy metals, temperature extremes, etc.) embryo development is often aborted. In maize, halted embryo development results in aborted kernels on the ear. Preventing this kernel loss will maintain yield. Accordingly, methods are provided to increase the stress resistance in a plant (for example, by targeted downregulation of cytokinin oxidase in an early developing embryo or endosperm). Decreasing expression of the CKX sequence of the invention in appropriate tissues can also modulate floral development during periods of stress and thus methods are provided to maintain or improve the flowering process in plants under stress. The method comprises decreasing the level and/or activity of the CKX sequence of the invention by means of downregulation techniques described elsewhere herein.
[0214] Significant yield instability can occur as a result of unfavorable environments, especially during the lag phase of seed development. During this period, seeds undergo dramatic changes in ultra structure, biochemistry, and sensitivity to environmental perturbation, yet demonstrate little change in dry mass accumulation. Two important events that occur during the lag phase are initiation and division of endosperm cells and amyloplasts (which are the sites for starch deposition). It has been demonstrated that during the lag phase (in maize, from pollination to about 10 to 12 days after pollination (DAP)), a dramatic increase in cytokinin concentration immediately precedes maximum rates of endosperm cell division and amyloplast formation, indicating that this hormone plays a central role in these processes and in what is called the `sink strength` of the developing seed. Cytokinins have been demonstrated to play an important role in establishing seed size, decreasing tip kernel abortion, and increasing seed set during unfavorable environmental conditions. See, for example, Brugiere, et al., (2003) Plant Physiology 132:1228-1240; Setter, et al., (2001) Crop Sci. 41:1530-1540.
[0215] Methods are therefore provided to decrease activity and/or level of CKX polypeptides in the developing female inflorescence, thereby elevating cytokinin levels and allowing developing seed to achieve their full genetic potential for size, minimizing tip kernel abortion, and buffering seed set during unfavorable environments. The methods further allow the plant to maintain and/or improve the flowering process during unfavorable environments.
[0216] In this embodiment, a variety of promoters could be used to direct the expression of a sequence capable of decreasing the level and/or activity of the CKX polypeptide. In one method, a stress insensitive/lag phase/developing kernel-preferred promoter is used. By "insensitive to stress" is intended that the expression level of a sequence operably linked to the promoter is not altered or only minimally altered under stress conditions. Such promoters are known in the art and include Zag2.1 (Schmidt, et al., (1993) Plant Cell 5:729-737, Genbank Accession Number X80206). Also useful are ZmCkx1-2 promoter (U.S. Pat. Nos. 6,921,815 and 7,371,925 and U.S. patent application Ser. No. 12/051,893), ZmCkx2 promoter (SEQ ID NO: 13), ZmCkx3 promoter (SEQ ID NO: 14), ZmCkx4 promoter (SEQ ID NO: 15), ZmCkx5 promoter (SEQ ID NO: 16), ZmCkx6 promoter (SEQ ID NO: 69), ZmCkx7 promoter (SEQ ID NO: 70), ZmCkx8 promoter (SEQ ID NO: 63) any other CKX promoter and mzE40 (Zm40) (U.S. Pat. No. 6,403,862 and WO 2001/2178). Alternatively, a stress-responsive promoter may be used, such as rd29a (Yamaguchi-Shinozaki, et al., (1993) Mol. Gen. Genetics 236:331-334). Also of interest are promoters directing expression preferentially within seed tissues such as the endosperm or the basal endosperm transfer layer, as listed elsewhere herein. Methods to assay for an increase in seed set during abiotic stress are known in the art. For example, plants having the reduced CKX activity can be monitored under various stress conditions and compared to control plants. For instance, the plant having the reduced CKX activity can be subjected to various degrees of stress during flowering and seed set. Under identical conditions, the genetically modified plant having the reduced CKX activity will have a higher number of developing kernels than will a wild type (non-transformed) plant.
[0217] Accordingly, the present invention further provides plants having increased yield or maintained yield and/or an increased or maintained flowering process during periods of abiotic stress (e.g., drought, salt, heavy metals, temperature extremes, etc.). In some embodiments, the plants having an increased or maintained yield during abiotic stress have a decreased level/activity of the CKX polypeptide of the invention. In other embodiments, the plant comprises a CKX nucleotide sequence of the invention operably linked to a promoter that drives expression in the plant cell. In certain embodiments, such plants have stably incorporated into their genome a nucleic acid molecule comprising a CKX nucleotide sequence of the invention operably linked to a promoter that drives expression in the plant cell.
viii. Modulating Pathogen Resistance
[0218] Methods for modulating pathogen resistance in a plant are provided. Plant pathogens can produce cytokinins (Mills, et al., (1978) Physiol Plant Pathol 13:73-80 and Angra, et al., (1990) Mycopathologia 109:177-182). Accordingly, increasing CKX activity in a plant or plant part can increase the plant's resistance to the pathogen. See, for example, Bilyeu, et al., (2001) Plant Physiol. 125:378-386. Thus, compositions and methods for inducing resistance in a plant to plant pests are provided. In specific embodiments, the CKX polypeptide is provided to the developing seed and thereby increases the pathogen resistance of the seed. Accordingly, the compositions and methods are also useful in protecting plants against fungal pathogens, viruses, nematodes, insects and the like.
[0219] By "disease resistance" is intended that the plants avoid the disease symptoms that are the outcome of plant-pathogen interactions. That is, pathogens are prevented from causing plant diseases and the associated disease symptoms or alternatively, the disease symptoms caused by the pathogen are minimized or lessened. By "antipathogenic compositions" is intended that the compositions of the invention have antipathogenic activity and thus are capable of suppressing, controlling and/or killing the invading pathogenic organism. An antipathogenic composition of the invention will reduce the disease symptoms resulting from pathogen challenge by at least about 2% to about 6%, at least about 5% to about 50%, at least about 10% to about 60%, at least about 30% to about 70%, at least about 40% to about 80% or at least about 50% to about 90% or greater. Hence, the methods of the invention can be utilized to protect plants from disease, particularly those diseases that are caused by plant pathogens.
[0220] The method for increasing pathogen resistance in a plant comprises increasing the level or activity of the CKX polypeptides of the invention. In specific methods, a CKX sequence of the invention is provided. A CKX nucleotide sequence can be provided by introducing into the plant a polynucleotide comprising a CKX nucleotide sequence of the invention, expressing the CKX sequence, and thereby increasing pathogen resistance in the plant. In certain embodiments, the CKX nucleotide construct introduced into the plant is stably incorporated into the genome of the plant.
[0221] As discussed above, one of skill will recognize the appropriate promoter to use to increase pathogen resistance in the plant. Exemplary promoters for this embodiment include constitutive promoters, tissue-preferred promoters, pathogen-inducible promoters and seed-preferred promoters.
[0222] Assays that measure antipathogenic activity are commonly known in the art, as are methods to quantitate disease resistance in plants following pathogen infection. See, for example, U.S. Pat. No. 5,614,395, herein incorporated by reference. Such techniques include measuring over time the average lesion diameter, the pathogen biomass, and the overall percentage of decayed plant tissues. For example, a plant either expressing an antipathogenic polypeptide or having an antipathogenic composition applied to its surface shows a decrease in tissue necrosis (i.e., lesion diameter) or a decrease in plant death following pathogen challenge when compared to a control plant that was not exposed to the antipathogenic polypeptide or composition. Alternatively, antipathogenic activity can be measured by a decrease in pathogen biomass. For example, a plant expressing an antipathogenic polypeptide or exposed to an antipathogenic composition is challenged with a pathogen of interest. Over time, tissue samples from the pathogen-inoculated tissues are obtained and RNA is extracted. The percent of a specific pathogen RNA transcript relative to the level of a plant specific transcript allows the level of pathogen biomass to be determined. See, for example, Thomma, et al., (1998) Plant Biology 95:15107-15111, herein incorporated by reference.
[0223] Pathogens of the invention include, but are not limited to, viruses or viroids, bacteria, insects, nematodes, fungi, and the like. Viruses include any plant virus, for example, tobacco or cucumber mosaic virus, ringspot virus, necrosis virus, or maize dwarf mosaic virus.
ix. Method of Use for CKX Promoter Polynucleotides
[0224] The polynucleotides comprising the CKX promoters disclosed in the present invention, as well as variants and fragments thereof, are useful in the genetic manipulation of any host cell, preferably plant cell, when assembled with a DNA construct such that the promoter sequence is operably linked to a nucleotide sequence comprising a polynucleotide of interest. In this manner, the CKX promoter polynucleotides of the invention are provided in expression cassettes along with a polynucleotide sequence of interest for expression in the host cell of interest. As discussed in Example 2 below, the CKX promoter sequences of the invention drive native expression in a variety of tissues and thus the promoter sequences can find use in regulating temporal and/or spatial expression of polynucleotides of interest.
[0225] Synthetic hybrid promoter regions are known in the art. Such regions comprise upstream promoter elements of one polynucleotide operably linked to the promoter element of another polynucleotide. In an embodiment of the invention, heterologous sequence expression is controlled by a synthetic hybrid promoter comprising a CKX promoter sequence of the invention, or a variant or fragment thereof, operably linked to upstream promoter element(s) from a heterologous promoter. Upstream promoter elements that are involved in the plant defense system have been identified and may be used to generate a synthetic promoter. See, for example, Rushton, et al., (1998) Curr. Opin. Plant Biol. 1:311-315. Alternatively, a synthetic CKX promoter sequence may comprise duplications of the upstream promoter elements found within the CKX promoter sequences.
[0226] It is recognized that a promoter sequence of the invention may be used with its native CKX coding sequence. A DNA construct comprising a CKX promoter operably linked with its native CKX gene may be used to transform any plant of interest to bring about a desired phenotypic change, such as modulating cytokinin levels, modulating root, shoot, leaf, floral, and embryo development, stress tolerance and any other phenotype described elsewhere herein.
[0227] The promoter nucleotide sequences and methods disclosed herein are useful in regulating expression of any nucleotide sequence in a host plant in order to vary the phenotype of a plant. Various changes in phenotype are of interest including modifying the fatty acid composition in a plant, altering the amino acid content of a plant, altering a plant's pathogen defense mechanism, and the like. These results can be achieved by providing expression of heterologous products or increased expression of endogenous products in plants. Alternatively, the results can be achieved by providing for a reduction of expression of one or more endogenous products, particularly enzymes or cofactors in the plant. These changes result in a change in phenotype of the transformed plant.
[0228] Genes of interest are reflective of the commercial markets and interests of those involved in the development of the crop. Crops and markets of interest change, and as developing nations open up world markets, new crops and technologies will emerge also. In addition, as our understanding of agronomic traits and characteristics such as yield and heterosis increases, the choice of genes for transformation will change accordingly. General categories of genes of interest include, for example, those genes involved in information, such as zinc fingers, those involved in communication, such as kinases, and those involved in housekeeping, such as heat shock proteins. More specific categories of transgenes, for example, include genes encoding important traits for agronomics, insect resistance, disease resistance, herbicide resistance, sterility, grain characteristics and commercial products. Genes of interest include, generally, those involved in oil, starch, carbohydrate or nutrient metabolism, particularly nitrogen assimilation, as well as those affecting kernel size, sucrose loading, and the like.
[0229] In one embodiment, sequences of interest improve plant growth and/or crop yields. In more specific embodiments, expression of the nucleotide sequence of interest improves the plant's response to stress induced under high density growth conditions. For example, sequences of interest include agronomically important genes that result in improved primary or lateral root systems. Such genes include, but are not limited to, nutrient/water transporters and growth inducers. Examples of such genes include, but are not limited to, maize plasma membrane H+-ATPase (MHA2) (Frias, et al., (1996) Plant Cell 8:1533-44); AKT1, a component of the potassium uptake apparatus in Arabidopisis, (Spalding, et al., (1999) J Gen Physiol 113:909-18); RML genes which activate cell division cycle in the root apical cells (Cheng, et al., (1995) Plant Physiol 108:881); maize glutamine synthetase genes (Sukanya, et al., (1994) Plant Mol Biol 26:1935-46) and hemoglobin (Duff, et al., (1997) J. Biol. Chem. 27:16749-16752, Arredondo-Peter, et al., (1997) Plant Physiol. 115:1259-1266; Arredondo-Peter, et al., (1997) Plant Physiol 114:493-500 and references cited therein) and isopentenyl transferase or ipt (Strabala, et al., (1989) Mol. Gen. Genet. 216:388-394, (Agrobacterium); U.S. Patent Application Ser. Nos. 60/610,656 filed Sep. 17, 2004 and 60/637,230 filed Dec. 17, 2004 (maize); Takei, et al., (2001) J. Biol. Chem. 276:26405-26410 (Arabidopsis); Zubko, et al., (2002) Plant J. 29(6):797-808 (petunia); Sakano, et al., (2004) Phytochem 65:2439-2446 (hop); and GenBank Accession Number XM--477138 (rice, 2004)). The sequence of interest may also be useful in expressing antisense nucleotide sequences of genes that negatively affect root development.
[0230] Additional, agronomically important traits such as oil, starch, and protein content can be genetically altered in addition to using traditional breeding methods. Modifications include increasing content of oleic acid, saturated and unsaturated oils, increasing levels of lysine and sulfur, providing essential amino acids, and also modification of starch. Hordothionin protein modifications are described in U.S. Pat. Nos. 5,703,049, 5,885,801, 5,885,802 and 5,990,389, herein incorporated by reference. Another example is lysine and/or sulfur rich seed protein encoded by the soybean 2S albumin described in U.S. Pat. No. 5,850,016, and the chymotrypsin inhibitor from barley, described in Williamson, et al., (1987) Eur. J. Biochem. 165:99-106, the disclosures of which are herein incorporated by reference.
[0231] Derivatives of the coding sequences can be made by site-directed mutagenesis to increase the level of preselected amino acids in the encoded polypeptide. For example, the gene encoding the barley high lysine polypeptide (BHL) is derived from barley chymotrypsin inhibitor, U.S. patent application Ser. No. 08/740,682, filed Nov. 1, 1996, and WO 1998/20133, the disclosures of which are herein incorporated by reference. Other proteins include methionine-rich plant proteins such as from sunflower seed (Lilley, et al., (1989) Proceedings of the World Congress on Vegetable Protein Utilization in Human Foods and Animal Feedstuffs, ed. Applewhite (American Oil Chemists Society, Champaign, Ill.), pp. 497-502; herein incorporated by reference); corn (Pedersen, et al., (1986) J. Biol. Chem. 261:6279; Kirihara, et al., (1988) Gene 71:359; both of which are herein incorporated by reference); and rice (Musumura, et al., (1989) Plant Mol. Biol. 12:123, herein incorporated by reference). Other agronomically important genes encode latex, Floury 2, growth factors, seed storage factors and transcription factors.
[0232] Insect resistance genes may encode resistance to pests that cause significant yield penalty such as rootworm, cutworm, European Corn Borer, and the like. Such genes include, for example, Bacillus thuringiensis toxin genes (see, for example, U.S. Pat. Nos. 5,366,892 5,747,450; 5,736,514; 5,723,756; 5,593,881; 5,188,960; 5,689,052; 5,880,275; 7,105,332; WO 1991/14778; WO 1999/31248; WO 2001/12731; WO 1999/24581; WO 1997/40162 and U.S. patent application Ser. Nos. 10/032,717; 10/414,637; 10/746,914 and 11/224,624 and Geiser, et al., (1986) Gene 48:109). Other insect resistance genes may encode an insect-specific hormone or pheromone such as an ecdysteroid and juvenile hormone, a variant thereof, a mimetic based thereon or an antagonist or agonist thereof; an insect-specific peptide which, upon expression, disrupts the physiology of the affected pest (for example, see, Regan, (1994) J. Biol. Chem. 269:9; Pratt, et al., (1989) Biochem. Biophys. Res. Comm. 163:1243; Chattopadhyay, et al., (2004) Critical Reviews in Microbiology 30(1):33-54 2004; Zjawiony, (2004) J Nat Prod 67(2):300-310; Carlini and Grossi-de-Sa, (2002) Toxicon, 40(11):1515-1539; Ussuf, et al., (2001) Curr Sci. 80(7):847-853 and Vasconcelos and Oliveira, (2004) Toxicon 44(4):385-403 and U.S. Pat. No. 5,266,317); an enzyme responsible for a hyperaccumulation of a monoterpene, a sesquiterpene, a steroid, hydroxycinnamic acid, a phenylpropanoid derivative or another non-protein molecule with insecticidal activity; or an enzyme involved in the modification, including the post-translational modification, of a biologically active molecule, such as a glycolytic enzyme, a proteolytic enzyme, a lipolytic enzyme, a nuclease, a cyclase, a transaminase, an esterase, a hydrolase, a phosphatase, a kinase, a phosphorylase, a polymerase, an elastase, a chitinase and a glucanase, whether natural or synthetic (for example, see, WO 1993/02197; Kramer, et al., (1993) Insect Biochem. Molec. Biol. 23:691, Kawalleck, et al., (1993) Plant Molec. Biol. 21:673, U.S. Pat. Nos. 6,563,020, 7,145,060 and 7,087,810.
[0233] Genes encoding disease resistance traits include detoxification genes, such as against fumonosin (U.S. Pat. No. 5,792,931); avirulence (avr) and disease resistance (R) genes (Jones, et al., (1994) Science 266:789; Martin, et al., (1993) Science 262:1432; and Mindrinos, et al., (1994) Cell 78:1089); and the like.
[0234] Herbicide resistance traits may include genes coding for resistance to herbicides that act to inhibit the action of acetolactate synthase (ALS), in particular the sulfonylurea-type herbicides (e.g., the acetolactate synthase (ALS) gene containing mutations leading to such resistance, in particular the S4 and/or Hra mutations), genes coding for resistance to herbicides that act to inhibit action of glutamine synthase, such as phosphinothricin or basta (e.g., the bar gene), genes encoding proteins which break down glyphosate, or other such genes known in the art. The bar gene encodes resistance to the herbicide basta, the nptII gene encodes resistance to the antibiotics kanamycin and geneticin, and the ALS-gene mutants encode resistance to the herbicide chlorsulfuron.
[0235] Sterility genes can also be encoded in an expression cassette and provide an alternative to physical detasseling. Examples of genes used in such ways include male tissue-preferred genes and genes with male sterility phenotypes such as QM, described in U.S. Pat. No. 5,583,210. Other genes include kinases and those encoding compounds toxic to either male or female gametophytic development.
[0236] The quality of grain is reflected in traits such as levels and types of oils, saturated and unsaturated, quality and quantity of essential amino acids, and levels of cellulose. In corn, modified hordothionin proteins are described in U.S. Pat. Nos. 5,703,049, 5,885,801, 5,885,802 and 5,990,389.
[0237] Commercial traits can also be encoded on a gene or genes that could increase for example, starch for ethanol production, or provide expression of proteins. Another important commercial use of transformed plants is the production of polymers and bioplastics such as described in U.S. Pat. No. 5,602,321. Genes such as β-Ketothiolase, PHBase (polyhydroxyburyrate synthase), and acetoacetyl-CoA reductase (see, Schubert, et al., (1988) J. Bacteriol. 170:5837-5847) facilitate expression of polyhyroxyalkanoates (PHAs).
[0238] Exogenous products include plant enzymes and products as well as those from other sources including procaryotes and other eukaryotes. Such products include enzymes, cofactors, hormones, and the like. The level of proteins, particularly modified proteins having improved amino acid distribution to improve the nutrient value of the plant, can be increased. This is achieved by the expression of such proteins having enhanced amino acid content.
[0239] All publications and patents herein referred to are hereby incorporated by reference to the same extent as if each was individually so incorporated.
[0240] The following examples are intended to illustrate but not to limit the invention.
EXPERIMENTAL
Example 1
Analysis of CKX Sequences
[0241] The CKX polypeptides of the invention share sequence similarity with a number of CKX polypeptides. FIG. 23 provides identity values for ZmCkx1-8 and OsCkx 1-11 polypeptides compared to each other.
[0242] The amino acid alignment of the CKX polypeptides of the invention with other known CKX polypeptides is provided in FIG. 11. Specifically, the alignment provides the sequence relationship of AtCkx1 (SEQ ID NO: 35), AtCkx2 (SEQ ID NO: 36), AtCkx3 (SEQ ID NO: 37), AtCkx4 (SEQ ID NO: 38), AtCkx5 (SEQ ID NO: 39), AtCkx6 (SEQ ID NO: 40), AtCkx7 (SEQ ID NO: 41), DsCkx1 (SEQ ID NO: 42), HvCkx2 (SEQ ID NO: 43), HvCkx3 (SEQ ID NO: 44), OsCkx1 (SEQ ID NO: 45), OsCkx2 (SEQ ID NO: 46), OsCkx3 (SEQ ID NO: 47), OsCkx4 (SEQ ID NO: 48), OsCkx5 (SEQ ID NO: 49), OsCkx6 (SEQ ID NO: 73), OsCkx7 (SEQ ID NO: 74), OsCkx8 (SEQ ID NO: 75), OsCkx9 (SEQ ID NO: 76), OsCkx10 (SEQ ID NO: 77), OsCkx11 (SEQ ID NO: 78), ZmCkx1 (SEQ ID NO: 33), ZmCkx2 or 2a (SEQ ID NO: 3), ZmCkx2b (SEQ ID NO: 68) ZmCkx3 (SEQ ID NO: 6), ZmCkx4 (SEQ ID NO: 9) ZmCkx5 (SEQ ID NO: 12), ZmCkx6 (SEQ ID NO: 53), ZmCkx7 (SEQ ID NO: 59) and ZmCkx8 (SEQ ID NO: 62).
[0243] The CKX polypeptides of the invention contain a predicted FAD-binding domain (PFAM Accession Number PF01565). The PFAM consensus sequence is provided in SEQ ID NO: 56.
[0244] Analysis of the subcellular location of the CKX polypeptides of the invention was also performed using ProtComp (Softberry, Inc.; version 5 or 6.1) trained onto plants. The program is based on complex neural-network recognizers, which identify probability of subcellular localization in nuclear, plasma membrane, extracellular, cytoplasmic, mitochondrial, chloroplast, endoplasmic reticulum, peroxisomal, lysosomal or Golgi compartments. The results of these analyses are set forth below.
A. Analysis of ZmCkx2:
[0245] The results for ZmCkx2a follow and predict that the ZmCkx2a polypeptide is extracellularly localized.
TABLE-US-00001 ProtComp Version 5. Identifying sub-cellular location (Plants) Seq name: ZmCkx2a 519 Significant similarity in Location DB - Location: Extracellular (Secreted) Database sequence: AC = Q9T0N8 Location: Extracellular (Secreted) DE Cytokinin oxidase 1 precursor (EC Score = 11145, Sequence length = 534, Alignment length = 392 Predicted by Neural Nets - Plasma membrane with score 0.9 ******** Transmembrane segments are found: .-325: 337+. Integral Prediction of protein location: Membrane bound Extracellular (Secreted) with score 4.3 Location weights: LocDB PotLocDB Neural Nets Integral Nuclear 0.0 0.0 0.74 0.74 Plasma membrane 0.0 0.0 0.92 0.92 Extracellular 11145.0 9230.0 0.81 4.31 Cytoplasmic 0.0 0.0 0.64 0.64 Mitochondrial 0.0 0.0 0.76 0.76 Chloroplast 0.0 0.0 0.73 0.73 Endoplasm. retic. 0.0 0.0 0.77 0.77 Peroxisomal 0.0 0.0 0.76 0.76 SPScan in SeqWeb 1. 1 mkppslvhcfkllvllalarltmh{circumflex over ( )}vp 26 Score: 7.7 Probability: 7.225E-01 SP Length: 24 McGeoch scan succeeded: Charged-region statistics: Length: 11 Charge: 2 Hydrophobic-region statistics: Length: 8 Offset: 12 Total hydropathy: 62.3 Maximum 8-residue hydropathy: 62.3, starting at 13
[0246] The results for ZmCkx2b follow and predict that the ZmCkx2b polypeptide is extracellularly localized.
TABLE-US-00002 ProtComp Version 6.1. Identifying sub-cellular location (Plants) Seq name: ZmCkx2b 525 Significant similarity in Location DB - Location: Extracellular (Secreted) Database sequence: AC = Q9T0N8 Location: Extracellular (Secreted) DE Cytokinin dehydrogenase 1 precurso Score = 11240, Sequence length = 534, Alignment length = 384 Predicted by Neural Nets - Endoplasmic reticulum with score 1.1 Integral Prediction of protein location: Extracellular (Secreted) with score 5.3 Neural Pen- Inte- Location weights: LocDB / PotLocDB / Nets / tamers / gral Nuclear 0.0 / 0.0 / 0.74 / 0.00 / 0.74 Plasma membrane 0.0 / 0.0 / 0.72 / 0.63 / 1.35 Extracellular 11240.0 / 15415.0 / 0.77 / 0.31 / 5.29 Cytoplasmic 0.0 / 0.0 / 0.78 / 0.05 / 0.84 Mitochondrial 0.0 / 0.0 / 0.77 / 0.22 / 1.00 Chloroplast 0.0 / 0.0 / 0.73 / 0.00 / 0.73 Endoplasm. retic. 0.0 / 0.0 / 1.13 / 0.00 / 1.13 Peroxisomal 0.0 / 0.0 / 0.75 / 0.00 / 0.75 SPScan in SeqWeb 1.1 mkppsslvhyfkllvllalarltmh{circumflex over ( )}vp 27 Score: 7.7 Probability: 8.044E-01 SP length: 25 McGeoch scan succeeded: Charged-region statistics: Length: 2 Charge: 1 Hydrophobic-region statistics: Length: 15 Offset: 3 Total hydropathy: 83.7 Maximum 8-residue hydropathy: 52.5, starting at 11
B. Analysis of ZmCkx3:
[0247] The results for ZmCkx3 follow and predict that the ZmCkx3 polypeptide is extracellularly localized.
TABLE-US-00003 ProtComp Version 5. Identifying sub-cellular location (Plants) Seq name: ZmCkx3 538 Significant similarity in Location DB - Location: Extracellular (Secreted) Database sequence: AC = Q9T0N8 Location: Extracellular (Secreted) DE Cytokinin oxidase Score = 13520, Sequence length = 534, Alignment length = 500 Predicted by Neural Nets - Plasma membrane with score 1.3 Integral Prediction of protein location: Extracellular (Secreted) with score 5.3 Neural Inte- Location weights: LocDB / PotLocDB / Nets / gral Nuclear 0.0 / 0.0 / 1.18 / 1.18 Plasma membrane 0.0 / 0.0 / 1.32 / 1.32 Extracellular 13520.0 / 11200.0 / 1.07 / 5.32 Cytoplasmic 0.0 / 0.0 / 0.72 / 0.72 Mitochondrial 0.0 / 0.0 / 0.98 / 0.98 Chloroplast 0.0 / 0.0 / 0.71 / 0.71 Endoplasm. retic. 0.0 / 0.0 / 0.56 / 0.56 Peroxisomal 0.0 / 0.0 / 0.42 / 0.42 ZmCkx3 SPScan in SeqWeb 1 marrtrfvaiaalltsflnvaag{circumflex over ( )}hs 25 Score: 8.7 Probability: 4.497E-01 SP length: 23
C. Analysis of ZmCkx4:
[0248] The results for ZmCkx4 follow and predict that the ZmCkx4 polypeptide is extracellularly localized.
TABLE-US-00004 ProtComp Version 5. Identifying sub-cellular location (Plants) Seq name: ZmCkx4 521 Significant similarity in Location DB - Location: Extracellular (Secreted) Database sequence: AC = Q9LTS3 Location: Extracellular (Secreted) DE Cytokinin oxidase Score = 10155, Sequence length = 523, Alignment length = 360 Predicted by Neural Nets - Plasma membrane with score 1.3 Integral Prediction of protein location: Extracellular (Secreted) with score 4.4 Neural Inte- Location weights: LocDB / PotLocDB / Nets / gral Nuclear 0.0 / 0.0 / 1.18 / 1.18 Plasma membrane 0.0 / 0.0 / 1.32 / 1.32 Extracellular 10155.0 / 9925.0 / 1.07 / 4.43 Cytoplasmic 0.0 / 0.0 / 0.72 / 0.72 Mitochondrial 0.0 / 0.0 / 0.95 / 0.95 Chloroplast 0.0 / 0.0 / 0.71 / 0.71 Endoplasm. retic. 0.0 / 0.0 / 0.56 / 0.56 Peroxisomal 0.0 / 0.0 / 0.42 / 0.42 ZmCkx4 SPScan in SeqWeb 1 mlaymdrataaaepedagrepatmaggcaaaatdfgglgsampaavvrpasa{circumflex over ( )}dd 54 Score: 6.7 Probability: 9.945E-01 SP length: 52 McGeoch scan succeeded: Charged-region statistics: Length: 7 Charge: 0 Hydrophobic-region statistics: Length: 8 Offset: 8 Total hydropathy: 33.9 Maximum 8-residue hydropathy: 33.9, starting at 9
D. Analysis of ZmCkx5:
[0249] The results for ZmCkx5 follow and predict that the ZmCkx5 polypeptide is extracellularly localized.
TABLE-US-00005 ProtComp Version 5. Identifying sub-cellular location (Plants) Seq name: ZmCkx5 542 Significant similarity in Location DB - Location: Extracellular (Secreted) Database sequence: AC = Q9LTS3 Location: Extracellular (Secreted) DE Cytokinin oxidase Score = 9405, Sequence length = 523, Alignment length = 390 Predicted by Neural Nets - Plasma membrane with score 1.3 Integral Prediction of protein location: Extracellular (Secreted) with score 4.3 Neural Inte- Location weights: LocDB / PotLocDB / Nets / gral Nuclear 0.0 / 0.0 / 1.18 / 1.18 Plasma membrane 0.0 / 0.0 / 1.32 / 1.32 Extracellular 9405.0 / 10020.0 / 1.08 / 4.28 Cytoplasmic 0.0 / 0.0 / 0.72 / 0.72 Mitochondrial 0.0 / 0.0 / 0.98 / 0.98 Chloroplast 0.0 / 0.0 / 0.71 / 0.71 Endoplasm. retic. 0.0 / 0.0 / 0.56 / 0.56 Peroxisomal 0.0 / 0.0 / 0.42 / 0.42 ZmCkx5 SPScan in SeqWeb 1 MEVAMVVSARASLLILVLSLCSP{circumflex over ( )}YK 25 Score: 7.4 Probability: 9.387E-01 SP length: 23 McGeoch scan succeeded: Charged-region statistics: Length: 10 Charge: 0 Hydrophobic-region statistics: Length: 10 Offset: 11 Total hydropathy: 72.2 Maximum 8-residue hydropathy: 62.3, starting at 14
E. Analysis of ZmCkx6:
[0250] The results for ZmCkx6 follow and predict that the ZmCkx6 polypeptide is extracellularly localized.
TABLE-US-00006 Seq name: ZmCkx6 540 Significant similarity in Location DB - Location: Extracellular (Secreted) Database sequence: AC = Q9T0N8 Location: Extracellular (Secreted) DE Cytokinin dehydrogenase 1 precurso Score = 12595, Sequence length = 534, Alignment length = 439 Predicted by Neural Nets - Chloroplast with score 1.6 ******** Signal 1-47 is found ******** Transmembrane segments are found: .-348:360+. Integral Prediction of protein location: Membrane bound Extracellular (Secreted) with score 6.3 Location weights: LocDB / PotLocDB / Neural Nets / Pentamers / Integral Nuclear 0.0 / 0.0 / 0.73 / 0.05 / 0.78 Plasma membrane 0.0 / 0.0 / 1.31 / 0.73 / 2.04 Extracellular 12595.0 / 17440.0 / 1.11 / 0.49 / 6.34 Cytoplasmic 0.0 / 0.0 / 0.66 / 0.06 / 0.72 Mitochondrial 0.0 / 0.0 / 0.72 / 0.04 / 0.76 Chloroplast 0.0 / 0.0 / 1.58 / 0.00 / 1.63 Endoplasm.retic. 0.0 / 0.0 / 1.04 / 0.05 / 1.04 Peroxisomal 0.0 / 0.0 / 0.71 / 0.00 / 0.71 ZmCkx6 SPScan in SeqWeb 1 mtrclmftllflvsslistvg{circumflex over ( )}lp 23 Score: 8.8 Probability: 4.377E-01 SP length: 21 McGeoch scan succeeded: Charged-region statistics: Length: 3 Charge: 1 Hydrophobic-region statistics: Length: 10 Offset: 4 Total hydropathy: 73.1 Maximum 8-residue hydropathy: 57.9, starting at 7
E. Analysis of ZmCkx7:
[0251] The results for ZmCkx7 follow and predict that the ZmCkx7 polypeptide is extracellularly localized.
TABLE-US-00007 Seq name: ZmCkx7 582 Significant similarity in Location DB - Location: Extracellular (Secreted) Database sequence: AC = Q9T0N8 Location: Extracellular (Secreted) DE Cytokinin dehydrogenase 1 precurso Score = 13695, Sequence length = 534, Alignment length = 481 Predicted by Neural Nets - Mitochondrial with score 1.7 Integral Prediction of protein location: Extracellular (Secreted) with score 6.2 Location weights: LocDB / PotLocDB / Neural Nets / Pentamers / Integral Nuclear 0.0 / 0.0 / 0.71 / 0.19 / 0.90 Plasma membrane 0.0 / 0.0 / 0.72 / 0.37 / 1.09 Extracellular 13695.0 / 12375.0 / 1.27 / 0.54 / 6.23 Cytoplasmic 0.0 / 0.0 / 0.70 / 0.35 / 1.05 Mitochondrial 0.0 / 0.0 / 1.74 / 0.00 / 1.74 Chloroplast 0.0 / 0.0 / 1.40 / 0.00 / 1.40 Endoplasm. retic. 0.0 / 0.0 / 0.72 / 0.00 / 0.72 Peroxisomal 0.0 / 0.0 / 0.48 / 0.00 / 0.48 ZmCkx7 SPScan in SeqWeb 1 marattstvaalcfllscvsa{circumflex over ( )}tp 23 Score: 11.3 Probability: 7.910E-03 SP length: 21 McGeoch scan succeeded: Charged-region statistics: Length: 3 Charge: 1 Hydrophobic-region statistics: Length: 13 Offset: 4 Total hydropathy: 81.4 Maximum 8-residue hydropathy: 60.1, starting at 10
E. Analysis of ZmCkx8:
[0252] The results for ZmCkx8 follow and predict that the ZmCkx8 polypeptide is extracellularly localized.
TABLE-US-00008 Seq name: ZmCkx8 528 Significant similarity in Location DB - Location: Extracellular (Secreted) Database sequence: AC = Q9T0N8 Location: Extracellular (Secreted) DE Cytokinin dehydrogenase 1 precurso Score = 9365, Sequence length = 534, Alignment length = 350 Predicted by Neural Nets - Mitochondrial with score 1.3 ******** Signal 1-17 is found ******** Transmembrane segments are found: .o133:146-. Integral Prediction of protein location: Membrane bound Extracellular (Secreted) with score 4.8 Location weights: LocDB / PotLocDB / Neural Nets / Pentamers / Integral Nuclear 0.0 / 0.0 / 0.73 / 0.04 / 0.78 Plasma membrane 0.0 / 0.0 / 1.26 / 0.30 / 1.56 Extracellular 9365.0 / 10875.0 / 1.11 / 0.37 / 4.78 Cytoplasmic 0.0 / 0.0 / 0.73 / 0.00 / 0.73 Mitochondrial 0.0 / 0.0 / 1.31 / 0.13 / 1.45 Chloroplast 0.0 / 0.0 / 0.72 / 0.00 / 0.72 Endoplasm. retic. 0.0 / 0.0 / 1.01 / 0.00 / 1.01 Peroxisomal 0.0 / 0.0 / 1.13 / 0.00 / 1.13 ZmCkx8 SPScan in SeqWeb 1 megkvlctyagivalllcssvnfiqspsdvfgpvalleptasa{circumflex over ( )}ar 45 Score: 6.7 Probability: 9.988E-01 SP length: 43 McGeoch scan succeeded: Charged-region statistics: Length: 4 Charge: 0 Hydrophobic-region statistics: Length: 14 Offset: 5 Total hydropathy: 96.9 Maximum 8-residue hydropathy: 59.7, starting at 12
Example 2
Expression Profiles of Cytokinin Oxidase Genes
[0253] Several cytokinin oxidase ESTs were identified and genomic sequences isolated from corresponding BAC clones. Expression profiles of the CKX sequences were studied using Northern blots and RT-PCR for ZmCkx2, ZmCkx3, ZmCkx4, ZmCkx5 and ZmCkx6. In addition, expression of ZmCkx2-8 was evaluated using a proprietary Lynx database (Lynx Therapeutics, Hayward Calif., USA; see, for example, Brenner, et al., (2000) Nature Biotechnology 18:630-634).
A. Analysis of ZmCkx2
[0254] Northern analysis of ZmCkx2 was performed using ExpressHyb® Hybridization Solution from BD Biosciences Clontech (Palo Alto, Calif.) with a final wash in 0.1×SSC, 0.1% SDS at 65° C. for 20 minutes.
[0255] ZmCkx2 exists as a duplicated gene in maize, identified on Chromosome 3 (ZmCkx2a, SEQ ID NO: 1-3; NCBI CAE55200) and on Chromosome 8 (ZmCkx2b, SEQ ID NO: 67-68; NCBI CAE55201) (Massoneau, et al., 2004). The ZmCkx2b polypeptide (GenBank entry AJ606944) is 94% identical to the ZmCkx2a polypeptide of SEQ ID NO: 3. Because of the high degree of identity between the two ZmCkx2 sequences, analysis of expression will likely reflect activity of both ZmCkx2a and ZmCkx2b.
[0256] A tight relationship exists between Lynx and Northern data for ZmCkx2. This provided confidence when the Lynx database was mined for ZmCkx2 expression in various plant parts. For example, both Northern and Lynx analyses showed that ZmCkx2 had a 2-fold increase in expression in leaf discs incubated with 10 μM benzyladenine (a synthetic cytokinin). Lynx data in FIG. 3 show that expression is highest in leaves, stalk, whorl, roots and seedlings. Similarly, Northern data indicated strongest signals from ear leaf and midrib tissues; intermediate levels in tassel, husk leaves, young leaves, stalk, and pulvini; and lower levels in cob and ovary tissue. Little to no ZmCkx2 activity was detected by Northern analysis of roots or silks.
[0257] In addition, analyses of the Lynx data revealed that expression of ZmCkx2 increases during root aging and is induced 4-fold in seedlings submitted to a freezing stress. In the stalk, expression is 3-fold higher in the pith than in the rind.
[0258] RT-PCR was performed to determine the expression profile of ZmCkx2 in various maize tissues. RT-PCR was performed on maize mature and seedling tissue employing the following PCR parameters: 94° C. for 45 sec, 60° C. for 1 min, 72° C. for 3 min, for 30 cycles. ZmCkx2 expression was strongest in mature stalk tissue and in seedling leaf and mesocotyl. Weaker expression was noted in midribs and young and mature leaves of mature plants, as well as seedling roots. Similar RT-PCR studies were also performed during various stages of maize kernel development, including 0, 5, 10, 15, 20, 25 and 30 days after pollination. An expression peak was detected at 5 DAP.
[0259] A proprietary Agilent database (Agilent Technologies, Palo Alto, Calif.) was also analyzed to identify trends in ZmCkx2 expression. Tissues that showed the most dramatic differences in ZmCkx2 expression are from stalk. These samples were collected from the internodal zone of the 3rd or 4th internode below the ear before and after flowering. It was found that ZmCkx2 expression goes up more than 10-fold in the stalk after flowering (Table 1).
TABLE-US-00009 TABLE 1 Experiment Fold Id Experiment Name Ratio Change P-value 2340 Pt#1 preflowering at 59K vs 0.08 -12.8 1.73E-27 Pt#2 postflowering at 59K 2364 Pt#2 preflowering at 27K vs 0.09 -11.3 7.77E-26 Pt#2 postflowering at 59K 2349 Pt#1 preflowering at 27K vs 0.09 -11.1 5.73E-23 Pt#3 postflowering at 59K 2336 Pt#3 preflowering at 59K vs 0.09 -10.7 7.05E-25 Pt#1 postflowering at 59K 2345 Pt#3 preflowering at 27K vs 0.1 -10.3 3.67E-24 Pt33 postflowering at 27K 2332 Pt#2 postflowering at 27K vs 6.82 6.82 7.37E-17 Pt#1 preflowering at 59K 2359 Pt#1 postflowering at 59K vs 9.16 9.16 6.71E-22 Pt#2 preflowering at 27K 2368 Pt#2 postflowering at 27K vs 11.17 11.17 1.07E-25 Pt#3 preflowering at 59K 2366 Pt#1 postflowering at 27K vs 11.78 11.78 2.04E-26 Pt#3 preflowering at 59K 2333 Pt#3 postflowering at 27K vs 17.7 17.7 1.72E-31 Pt#1 preflowering at 27K
[0260] Table 1 shows fold changes identified in stalk samples collected from the internodal zone of the 3rd or 4th internode below ear, before and after flowering. This increase in ZmCkx2 expression could be associated with the flowering process. An increase of cytokinin flux from roots to shoots is often regarded as a flowering signal and is consistent with previous findings that increased cytokinin levels induce ZmCkx1 and ZmCkx2 expression. ZmCkx2 expression was also found to increase an average of 10-fold during ear development. Thus, manipulation of ZmCkx2 expression may be useful in modulation of flowering time.
B. Analysis of ZmCkx3
[0261] Expression of ZmCkx3 could not be detected using Northern blots. Mining of the Agilent and Lynx database confirmed that the gene is expressed at extremely low levels. The EST for ZmCkx3 came from a tassel library and it is believed that this gene could be tightly expressed in a particular cell type at a particular stage of tassel development. It remains possible that ZmCkx3 expresses during anther development at very low levels. The only tags from Lynx are from roots at an average of 4-5 ppm (See, FIG. 3).
C. Analysis of ZmCkx4
[0262] Analysis of the Lynx database for ZmCkx4 showed low constitutive expression of the gene in most organs, with higher levels observed in ear, silk and vascular bundles as well as intermediate levels in leaf and pedicels (FIG. 3). Interestingly, in 15-20 mm ears, ZmCkx4 is expressed at higher levels at the base of the ear than at the ear tip. This stage of ear growth coincides with the appearance of silk structure on the ear, which, taken together with strong expression in the silk, suggests a role for this gene in silk development.
D. Analysis of ZmCkx5
[0263] Analysis of the Lynx database for ZmCkx5 showed highest levels of expression to be in root and vascular bundles. (See, FIG. 3)
E. Analysis of ZmCkx6
[0264] Analysis of the Lynx database showed that ZmCkx6 is expressed at low levels in most maize tissues with stronger expression in anthers and pedicels. (See, FIG. 3)
F. Analysis of ZmCkx7
[0265] Analysis of the Lynx database showed that ZmCkx7 is also expressed at low levels in most tissues but with stronger levels in endosperm and pedicel. (See, FIG. 3)
G. Analysis of ZmCkx8
[0266] Analysis of the Lynx database showed that ZmCkx8 is expressed at low levels with stronger levels in anther, endosperm, and meristems. (See FIG. 3)
Example 3
Identification of ZmCkx2a, ZmCkx2b, ZmCkx4, and ZmCkx7 TUSC Events
[0267] In order to better define the roles of ZmCkx genes in plant development, knockout mutants were obtained for ZmCkx2a, ZmCkx2b, ZmCkx4, and ZmCkx7 using methods previously described (see, U.S. Pat. Nos. 5,962,764 and 6,300,542; Trait Utility System for Corn (TUSC)).
A. ZmCkx2a TUSC Summary
[0268] Two genomic sequences for cytokinin oxidase orthologues were provided for knockout screening. ZmCkx2a is a ˜3200 bp genomic sequence with five exons and four introns. Using this annotation, six PCR primers were designed across various intervals of the ZmCkx2a gene and then tested in control reactions against wild type maize (B73) gDNA. Primers were identified as 71936 (SEQ ID NO: 19), 71937 (SEQ ID NO: 20), 71938 (SEQ ID NO: 21), 71939 (SEQ ID NO: 22), 71940 (SEQ ID NO: 23), 71941 (SEQ ID NO: 24) and 9242 MuTIR (SEQ ID NO: 25). Verification and clean results were obtained for 71936+71937, 71940+71937, 71940+71941, 71940+71939, 71938+71941 and 71938+71939. No amplification results were observed for 71936+71941 and 71936+71939.
[0269] The 71936+71937 and 71938+71939 amplification products were cut out of the agarose gel, purified and used as probes for hybridization. These two intervals effectively segment the ZmCkx2a gene into 5' and 3' halves for insertion screening. Primer sequences are shown below along with the expected and observed amplicon sizes for each primer combination.
TABLE-US-00010 TABLE 2 Primer Pair cDNA (bp) observed (bp) 71936 + 71937 798 ~800 71936 + 71941 1350 No product 71936 + 71939 1841 No product 71940 + 71937 245 ~250 71940 + 71941 797 ~800 71940 + 71939 1288 ~1300 71938 + 71941 310 ~300 71938 + 71939 801 ~800
[0270] The pooled TUSC population was screened with gene primers 71936, 71937, 71938, and 71939 each in combination with the Mutator TIR primer 9242. Results of the pool hybridizations were fair with some PCR-positive pools detected by hybridization. Overall, hybridization signals were cross-confirmed between the primers.
[0271] Pools were selected for fragment sizing analysis based on hybridization signal intensity and reproducibility of the pool dot blots. In this phase of the screen, sizes of target::Mu PCR products are determined by reamplification, electrophoresis, and Southern analysis. Fourteen positive pools for primer 71936, fifty-one positive pools for primer 71937, forty-four positive pools for primer 71939, and thirty-seven positive pools for 71938 were screened through fragment-sizing. A number of pools were identified with strong EtBr and Southern bands.
[0272] Eight pools were selected for individual analysis based on the putative Mutator insertion location within ZmCKX2a, determined from the size data, and the overall quality of the hybridization signals throughout the screening process. The pools are shown in the table below, along with their size data. Each plate listed consists of individuals from two pools: those assayed in the sizing analysis (highlighted in bold type), as well as individuals from its companion pools. Individuals in the companion pools are often, but not necessarily, related to those in the targeted pools.
TABLE-US-00011 TABLE 3 Plate Pools Size (Bp) for Pool PV03 60 119 and 120 350 PV03 70 139 and 140 425 PV03 71 141 and 142 600 PV03 94 187 and 188 750 PV03 118 235 and 236 750 PV03 119 237 and 238 225 PV03 159 317 and 318 350 BT94 166 841 and 842 425 71937
[0273] Individual DNAs were arrayed, and a dot blot screen conducted with 71936 and 71937. Note that these selections are focused on the best candidates from the 5' half of the gene, targeting primarily the first large exon. In individual screens, PCR-positive individuals were identified for all of the targeted pools. To ensure germinal transmission of target::Mu alleles, F2 transmission testing was performed on thirty individual families harboring putative ZmCKX2a::Mu alleles. F2 genomic DNA was isolated from dry kernels (5K/individual) and amplified with the appropriate primers. Template controls on these preps were also performed using the gene-specific pair 71936+71937.
[0274] FIG. 4 provides a schematic of various Mu insertions in ZmCkx2 and ZmCkx4. Results indicate the genetic transmission of five ZmCkx2::Mu alleles.
[0275] 1) Insertion A: This insertion is inherited uniquely by this F2 family in Pool 139. The insertion is cross-confirmed from both flanks of the insertion, producing strong EtBr and hybridization signals in F2 tests. The allele amplifies a ˜625 fragment with 71936+9242, cross-confirmed with a ˜375 bp fragment using 71937+9242. This provides evidence for a knockout allele in the first exon of ZmCkx2, near nt 800 of the genomic reference sequence.
[0276] 2) Insertion B: Several related sibling families inherit the same insertion allele, suggesting a pre-meiotic origin for this allele; a parental insertion would have been evident in many more positive families. Five strong positive individuals were subjected to F2 tests; all were positive for the insertion allele. This insertion is cross-confirmed by amplification from both flanks. The 71936+9242 combination produces a small product of ˜150 bp, and the 3' flank primer pair 71937+9242 produces a fragment of ˜800 bp. The insertion site is thus predicted to be near the beginning of Exon I, and may be in the untranslated region. A Mu-suppressible phenotype may be one outcome of an insertion in this position.
[0277] 3) Insertion C: This is a uniquely inherited Mu insertion in the 5' end of ZmCkx2. The allele is of a distinct pedigree from that of Allele 2, yet it produces very similar PCR product sizes as those listed above from 5' and 3' flanks.
[0278] 4) Insertion D: This is another uniquely inherited and cross-confirmed insertion in the 5' end of the ZmCKX2a gene. This insertion produces fragments of ˜775 bp and ˜225 bp with 5' (71936) and 3' (71937) primer combinations, respectively. Based on the genomic annotation, this insertion occurs in Intron I of the gene, and thus may not provide a strong knockout allele. DNA sequence confirmation will be necessary to substantiate the expectations for this allele.
[0279] 5) Insertion E: This is a uniquely inherited insertion, again cross-confirmed by amplification from both flanks of the insertion site. The allele produces strong EtBr and hybridization fragments of ˜525 bp with the 71936+9242 combination, and ˜475 bp with the 71937+9242 combination. This insertion position appears to squarely interrupt Exon I of the gene, and is perhaps the best candidate for a good null in the ZmCkx2a gene.
B. ZmCKX4 TUSC Summary
[0280] As for ZmCkx2, a complete genomic sequence for ZmCkx4 was provided to facilitate knockout screening. Alignments of the two genes were used, and known intron sequences identified to enable the design of primers specific for insertions in ZmCkx4. Following these analyses, six PCR primers were designed across various intervals of ZmCkx4 and tested in control pairs against wild-type (wt) maize (B73) gDNA. Primers were identified as 71942 (SEQ ID NO: 26), 71943 (SEQ ID NO: 27), 71944 (SEQ ID NO: 28), 71945 (SEQ ID NO: 29), 71946 (SEQ ID NO: 30), 71947 (SEQ ID NO: 31), and 9249 MuTIR (SEQ ID NO: 32). Verification and clean results were obtained solely for the 71944+71947 primer combination. Further screening targeted Exon IV.
[0281] For Exon IV screening, the 71944+71947 amplification product was cut out of the agarose gel, purified, and used as probe for hybridization. Primer sequences are shown below along with the expected and observed amplicon sizes for each primer combination.
TABLE-US-00012 TABLE 4 Primer Pair cDNA (bp) observed by 71942 + 71943 1575 No product 71942 + 71947 2072 No product 71942 + 71945 3075 No product 71946 + 71943 763 No product 71946 + 71947 1260 No product 71946 + 71945 2263 No product 71944 + 71947 448 450 71944 + 71945 1451 No product
[0282] The pooled TUSC population was screened with gene primers 71944 and 71947, each in combination with the Mutator TIR primer 9242. Results of the pool hybridizations were fair with some PCR-positive pools detected by hybridization: some signals were reproducible, and were cross-confirmed between the primers.
[0283] Pools were selected for fragment sizing analysis based on hybridization signal intensity and reproducibility of the pool dot blots. In this phase of the screen, sizes of target::Mu PCR products are determined by reamplification, electrophoresis, and Southern analysis. Forty-five positive pools for primer 71944 and seven positive pools for primer 71947 were screened through fragment-sizing. A number of pools were identified with strong EtBr and Southern bands.
[0284] Six pools were selected for individual analysis based on the putative Mutator insertion location within ZmCkx4, determined from the size-data, and the overall quality of the hybridization signals throughout the screening process. The pools are shown in the table below, along with their size data. Insertions detected outside the bounds of the primer interval are useful to expand the search for insertions beyond exon IV. Each plate listed consists of individuals from two pools: those assayed in the sizing analysis (highlighted in bold type), as well as individuals from its companion pools. Individuals in the companion pools are often, but not necessarily, related to those in the targeted pools.
TABLE-US-00013 TABLE 5 Plate Pools Size (bp) for Pool PV03 47 93 and 94 1800 PV03 119 237 and 238 1550 PV03 170 339 and 340 400; 225 PV03 253 505 and 506 175 BT94 19 547 and 548 1675, 775, 350 BT94 96 705 and 706 1175, 350 71944 71947
[0285] Individual DNAs were arrayed, and a dot blot screen conducted with 71944 and 71947. PCR-positive individuals were identified for all of the targeted pools. To ensure germinal transmission of target::Mu alleles, F2 transmission testing was performed on thirty individual families harboring putative ZmCkx4::Mu alleles. F2 genomic DNA was isolated from dry kernels (5K/individual) and amplified with the appropriate primers. Template controls on these preps were also performed using the gene-specific pair 71944+71947.
[0286] FIG. 4 provides a schematic of various Mu insertions in ZmCkx4. Results indicate the genetic transmission of three ZmCKX4::Mu alleles.
[0287] 1) Insertion A: This unique insertion allele is detected solely with primer 71947+9242, and produces a large fragment of >1600 bp. This is a positive signal and likely represents an insertion into Exon I of the ZmCkx4 gene. Further characterization of this allele will include DNA sequencing and the design and testing of alternative 5' primers.
[0288] 2) Insertion B: A uniquely inherited insertion, this is cross-confirmed by amplification with both F and R primers from Exon IV. As such, this represents an excellent candidate for a knockout. The allele produces a strong product of ˜200 bp with 71944+9242; cross-confirmed by the ˜400 bp product with 71947+9242. These primers may be useful for genotyping assays during propagation.
[0289] 3) Insertion C: This is another uniquely inherited insertion into Exon IV. This insertion is near that of Allele 2. The insertion produces a small ˜175 bp product with the 71944+9242 combination and is cross-confirmed by a ˜425 bp product with the right flank combination 71947+9242.
[0290] All three of these alleles are excellent candidates for ZmCkx4 knockouts.
C. ZmCKX2b TUSC Summary
[0291] Mu-insertion mutants have been isolated using gene-specific primers for ZmCkx2b and techniques similar to those described above. Insertions are diagrammed in FIG. 4.
D. ZmCkx7 TUSC Summary
[0292] Mu-insertion mutants have been isolated using gene-specific primers for ZmCkx7 and techniques similar to those described above. Insertions are diagrammed in FIG. 4.
Example 4
Altered Expression of ZmCkx2 Modulates Plant Development
[0293] A DNA construct comprising ZmCkx2a operably linked to the ubiquitin promoter was introduced into maize plants as described in Zhao, et al., U.S. Pat. No. 5,981,840 and PCT Publication Number WO98/32326, herein incorporated by reference, and herein at Example 7.
[0294] Maize plants comprising a plasmid containing the ZmCkx2a sequence operably linked to a ubiquitin promoter were obtained (PHP21533). As a control, a non-cytokinin-related construct was also introduced into maize plants using the transformation method outlined above. Northern analysis indicated elevated levels of ZmCkx2a expression in transgenic events. The phenotypes of these transgenic maize plants having an elevated level of the ZmCkx2a polypeptide were further studied.
[0295] Callus cultures of the transgenic maize tissue produced significantly more roots (see, FIG. 5) and only one-sixth as many shoots as control plants during the regeneration process. (See FIG. 6) In addition, transgenic roots cultured in vitro and leaves of T0 plants in the greenhouse showed a 2-fold increase in cytokinin oxidase activity. (See, FIG. 8)
[0296] Plants growing in the greenhouse and expressing the ZmCkx2a sequence at high levels showed a phenotype typical of plants with lower cytokinin levels, including developmental problems as shorter plants with thinner leaves and a green/gray color. These differences were evident through the vegetative growth period. Out of 23 plants expressing the Ubi:ZmCkx2a sequence, 6 transgenic plants appeared to be of normal size, 8 transgenic plants displayed a medium size, 6 transgenic plants were small but viable, and 3 transgenic plants were very small. FIG. 7 provides data as to plant height, leaf length, and leaf width of transgenic plants compared to controls, showing a strong difference in plant height and leaf width but very similar leaf length relative to control plants.
[0297] Certain Ubi:ZmCkx2a plants produced tassels lacking spikelets but generated silks capable of setting seed.
Example 5
Assaying for Cytokinin Oxidase Activity
[0298] The level of cytokinin oxidase activity in the maize plants generated in Example 4 was measured. The assay to determine the level of cytokinin oxidase activity was carried out as described in Brugiere, et al., (2003) Plant Physiol. 132:1228-1240, herein incorporated by reference.
[0299] As demonstrated in FIG. 8A, cytokinin oxidase activity in transgenic root tissue is significantly higher than cytokinin oxidase activity in control root tissue. In addition, as demonstrated in FIG. 8B, cytokinin oxidase activity in leaves is higher in plants expressing ZmCkx2 than in the control plants.
Example 6
Maintaining or Increasing Seed Set During Stress
[0300] Immature maize embryos from greenhouse donor plants are bombarded with a plasmid designed to achieve post-transcriptional gene silencing (PTGS) with an appropriate promoter. For example, the plasmid may comprise the ZmCkx2 promoter (SEQ ID NO: 13) operably linked to a sequence encoding a hairpin structure corresponding to at least a portion of the coding sequence of the ZmCkx2 polynucleotide (SEQ ID NO: 2 or SEQ ID NO: 67). The plasmid may also contain the selectable marker gene PAT (Wohlleben, et al., (1988) Gene 70:25-37), which confers resistance to the herbicide Bialaphos.
[0301] Transformation is performed as follows. Media recipes follow below.
[0302] The ears are husked and surface sterilized in 30% Clorox® bleach plus 0.5% Micro detergent for 20 minutes, and rinsed two times with sterile water. The immature embryos are excised and placed embryo axis side down (scutellum side up), 25 embryos per plate, on 560Y medium for 4 hours and then aligned within the 2.5 cm target zone in preparation for bombardment.
[0303] A plasmid vector is made comprising the ZmCkx2 promoter sequence operably linked to a sequence encoding a hairpin structure corresponding to the CDS of the ZmCkx2 polynucleotide. This plasmid DNA plus plasmid DNA containing a PAT selectable marker is precipitated onto 1.1 μm (average diameter) tungsten pellets using a CaCl2 precipitation procedure as follows: 100 μl prepared tungsten particles in water; 10 μl (1 μg) DNA in Tris EDTA buffer (1 μg total DNA); 100 μl 2.5 M CaCl2; and, 10 μl 0.1 M spermidine.
[0304] Each reagent is added sequentially to the tungsten particle suspension, while maintained on the multitube vortexer. The final mixture is sonicated briefly and allowed to incubate under constant vortexing for 10 minutes. After the precipitation period, the tubes are centrifuged briefly, liquid removed, washed with 500 ml 100% ethanol, and centrifuged for 30 seconds. Again the liquid is removed, and 105 μl 100% ethanol is added to the final tungsten particle pellet. For particle gun bombardment, the tungsten/DNA particles are briefly sonicated and 10 μl spotted onto the center of each macrocarrier and allowed to dry about 2 minutes before bombardment.
[0305] The sample plates are bombarded at level #4 in particle gun #HE34-1 or #HE34-2. All samples receive a single shot at 650 PSI, with a total of ten aliquots taken from each tube of prepared particles/DNA.
[0306] Following bombardment, the embryos are kept on 560Y medium for 2 days, then transferred to 560R selection medium containing 3 mg/liter Bialaphos, and subcultured every 2 weeks. After approximately 10 weeks of selection, selection-resistant callus clones are transferred to 288J medium to initiate plant regeneration. Following somatic embryo maturation (2-4 weeks), well-developed somatic embryos are transferred to medium for germination and transferred to the lighted culture room. Approximately 7-10 days later, developing plantlets are transferred to 272V hormone-free medium in tubes for 7-10 days until plantlets are well established. Plants are then transferred to inserts in flats (equivalent to 2.5'' pot) containing potting soil and grown for 1 week in a growth chamber, subsequently grown an additional 1-2 weeks in the greenhouse, then transferred to classic 600 pots (1.6 gallon) and grown to maturity. Plants are monitored and scored under various stress conditions and compared to control plants. The maintenance of or an increase in seed set during an abiotic stress episode is monitored.
[0307] Bombardment medium (560Y) comprises 4.0 g/l N6 basal salts (SIGMA C-1416), 1.0 ml/l Eriksson's Vitamin Mix (1000×SIGMA-1511), 0.5 mg/l thiamine HCl, 120.0 g/l sucrose, 1.0 mg/l 2,4-D, and 2.88 g/l L-proline (brought to volume with D-I H2O following adjustment to pH 5.8 with KOH); 2.0 g/l Gelrite® (added after bringing to volume with D-I H2O); and 8.5 mg/l silver nitrate (added after sterilizing the medium and cooling to room temperature). Selection medium (560R) comprises 4.0 g/l N6 basal salts (SIGMA C-1416), 1.0 ml/I Eriksson's Vitamin Mix (1000×SIGMA-1511), 0.5 mg/l thiamine HCl, 30.0 g/l sucrose, and 2.0 mg/l 2,4-D (brought to volume with D-I H2O following adjustment to pH 5.8 with KOH); 3.0 g/l Gelrite® (added after bringing to volume with D-I H2O); and 0.85 mg/l silver nitrate and 3.0 mg/l bialaphos (both added after sterilizing the medium and cooling to room temperature).
[0308] Plant regeneration medium (288J) comprises 4.3 g/l MS salts (GIBCO 11117-074), 5.0 ml/I MS vitamins stock solution (0.100 g nicotinic acid, 0.02 g/l thiamine HCL, 0.10 g/l pyridoxine HCL, and 0.40 g/l glycine brought to volume with polished D-I H2O) (Murashige and Skoog, (1962) Physiol. Plant. 15:473), 100 mg/l myo-inositol, 0.5 mg/l zeatin, 60 g/l sucrose, and 1.0 ml/l of 0.1 mM abscisic acid (brought to volume with polished D-I H2O after adjusting to pH 5.6); 3.0 g/l Gelrite® (added after bringing to volume with D-I H2O); and 1.0 mg/l indoleacetic acid and 3.0 mg/l bialaphos (added after sterilizing the medium and cooling to 60° C.). Hormone-free medium (272V) comprises 4.3 g/l MS salts (GIBCO 11117-074), 5.0 ml/l MS vitamins stock solution (0.100 g/l nicotinic acid, 0.02 g/l thiamine HCL, 0.10 g/l pyridoxine HCL, and 0.40 g/l glycine brought to volume with polished D-I H2O), 0.1 g/1 myo-inositol, and 40.0 g/l sucrose (brought to volume with polished D-I H2O after adjusting pH to 5.6); and 6 g/l Bacto®-agar (added after bringing to volume with polished D-I H2O), sterilized and cooled to 60° C.
Example 7
Modulating Root Development
[0309] For Agrobacterium-mediated transformation of maize with the ZmCkx4 sequence operably linked to the CRWAQ81 root-preferred promoter::ADH intron, the method of Zhao is employed (U.S. Pat. No. 5,981,840, and PCT Patent Publication Number WO98/32326, the contents of which are hereby incorporated by reference). Briefly, immature embryos are isolated from maize and the embryos contacted with a suspension of Agrobacterium, where the bacteria are capable of transferring the zmCkx4 to at least one cell of at least one of the immature embryos (step 1: the infection step). In this step the immature embryos are immersed in an Agrobacterium suspension for the initiation of inoculation. The embryos are co-cultured for a time with the Agrobacterium (step 2: the co-cultivation step). The immature embryos are cultured on solid medium following the infection step. Following this co-cultivation period an optional "resting" step is contemplated. In this resting step, the embryos are incubated in the presence of at least one antibiotic known to inhibit the growth of Agrobacterium without the addition of a selective agent for plant transformants (step 3: resting step). The immature embryos are cultured on solid medium with antibiotic, but without a selecting agent, for elimination of Agrobacterium and for a resting phase for the infected cells. Next, inoculated embryos are cultured on medium containing a selective agent and growing transformed callus is recovered (step 4: the selection step). The immature embryos are cultured on solid medium with a selective agent resulting in the selective growth of transformed cells. The callus is then regenerated into plants (step 5: the regeneration step), and calli grown on selective medium are cultured on solid medium to regenerate the plants.
[0310] Plants are monitored and scored for a modulation in root development. The modulation in root development includes monitoring for enhanced root growth of one or more root parts including the primary root, lateral roots, adventitious roots, etc. Methods of measuring such developmental alterations in the root system are known in the art. See, for example, US Patent Application Publication Number 2003/0074698 and Werner, et al., (2001) PNAS 18:10487-10492, both of which are herein incorporated by reference.
Example 8
Soybean Embryo Transformation
[0311] Soybean embryos are bombarded with a plasmid containing the ZmCkx3 sequence operably linked to a root-preferred promoter. To induce somatic embryos, cotyledons, 3-5 mm in length dissected from surface-sterilized, immature seeds of the soybean cultivar A2872, are cultured in the light or dark at 26° C. on an appropriate agar medium for six to ten weeks. Somatic embryos producing secondary embryos are then excised and placed into a suitable liquid medium. After repeated selection for clusters of somatic embryos that multiplied as early, globular-staged embryos, the suspensions are maintained as described below.
[0312] Soybean embryogenic suspension cultures can maintained in 35 ml liquid media on a rotary shaker, 150 rpm, at 26° C. with florescent lights on a 16:8 hour day/night schedule. Cultures are subcultured every two weeks by inoculating approximately 35 mg of tissue into 35 ml of liquid medium.
[0313] Soybean embryogenic suspension cultures may then be transformed by the method of particle gun bombardment (Klein, et al., (1987) Nature (London) 327:70-73, U.S. Pat. No. 4,945,050). A Du Pont Biolistic PDS1000/HE instrument (helium retrofit) can be used for these transformations.
[0314] A selectable marker gene that can be used to facilitate soybean transformation is a transgene composed of the 35S promoter from Cauliflower Mosaic Virus (Odell, et al., (1985) Nature 313:810-812), the hygromycin phosphotransferase gene from plasmid pJR225 (from E. coli; Gritz, et al., (1983) Gene 25:179-188), and the 3' region of the nopaline synthase gene from the T-DNA of the Ti plasmid of Agrobacterium tumefaciens. The expression cassette comprising the ZmCkx2 sequence operably linked to the root-preferred promoter can be isolated as a restriction fragment. This fragment can then be inserted into a unique restriction site of the vector carrying the marker gene.
[0315] To 50 μl of a 60 mg/ml 1 μm gold particle suspension is added (in order): 5 μl DNA (1 μg/μl), 20 μl spermidine (0.1 M), and 50 μl CaCl2 (2.5 M). The particle preparation is then agitated for three minutes, spun in a microfuge for 10 seconds and the supernatant removed. The DNA-coated particles are then washed once in 400 μl 70% ethanol and resuspended in 40 μl of anhydrous ethanol. The DNA/particle suspension can be sonicated three times for one second each. Five microliters of the DNA-coated gold particles are then loaded on each macro carrier disk.
[0316] Approximately 300-400 mg of a two-week-old suspension culture is placed in an empty 60×15 mm petri dish and the residual liquid removed from the tissue with a pipette. For each transformation experiment, approximately 5-10 plates of tissue are normally bombarded. Membrane rupture pressure is set at 1100 psi, and the chamber is evacuated to a vacuum of 28 inches mercury. The tissue is placed approximately 3.5 inches away from the retaining screen and bombarded three times. Following bombardment, the tissue can be divided in half and placed back into liquid and cultured as described above.
[0317] Five to seven days post bombardment, the liquid media may be exchanged with fresh media, and eleven to twelve days post-bombardment with fresh media containing 50 mg/ml hygromycin. This selective media can be refreshed weekly. Seven to eight weeks post-bombardment, green, transformed tissue may be observed growing from untransformed, necrotic embryogenic clusters. Isolated green tissue is removed and inoculated into individual flasks to generate new, clonally propagated, transformed embryogenic suspension cultures. Each new line may be treated as an independent transformation event. These suspensions can then be subcultured and maintained as clusters of immature embryos or regenerated into whole plants by maturation and germination of individual somatic embryos.
Example 9
Variants of CKX Sequences
[0318] A. Variant Nucleotide Sequences of CKX (SEQ ID NO: 2, 5, 8, 11, 52, 58, 61 or 67) that do not Alter the Encoded Amino Acid Sequence
[0319] The CKX nucleotide sequences set forth in SEQ ID NO: 2, 5, 8, 11, 52, 58, 61 and 67 are used to generate variant nucleotide sequences having the nucleotide sequence of the open reading frame with about 70%, 75%, 80%, 85%, 90% and 95% nucleotide sequence identity when compared to the starting unaltered ORF nucleotide sequence of the corresponding SEQ ID NO. These functional variants are generated using a standard codon table. While the nucleotide sequence of the variants are altered, the amino acid sequence encoded by the open reading frames do not change.
B. Variant Amino Acid Sequences of CKX Polypeptides
[0320] Variant amino acid sequences of the CKX polypeptides are generated. In this example, one amino acid is altered. Specifically, the open reading frames set forth in SEQ ID NOS: 3, 6, 9, 12, 53, 59, 62 and 68 are reviewed to determine the appropriate amino acid alteration. The selection of the amino acid to change is made by consulting the protein alignment (with the other orthologs and other gene family members from various species). See, FIG. 11. An amino acid is selected that is deemed not to be under high selection pressure (not highly conserved) and which is rather easily substituted by an amino acid with similar chemical characteristics (i.e., similar functional side-chain). Using the protein alignment set forth in FIG. 11, an appropriate amino acid can be changed. Once the targeted amino acid is identified, the procedure outlined in Example 9A is followed. Variants having about 70%, 75%, 80%, 85%, 90% and 95% sequence identity to each of SEQ ID NO: 3, 6, 9, 12, 53, 59, 62 or 68 are generated using this method.
C. Additional Variant Amino Acid Sequences of CKX Polypeptides
[0321] In this example, artificial protein sequences are created having 80%, 85%, 90% and 95% identity relative to the reference protein sequence. This latter effort requires identifying conserved and variable regions from the alignments and analyses set forth in FIGS. 9, 10, and 11 and then the judicious application of an amino acid substitution table. These parts will be discussed in more detail below.
[0322] Largely, the determination of which amino acid sequences are altered is made based on the conserved regions among CKX protein or among the other CKX polypeptides. See, FIGS. 9, 10 and 11. Based on the sequence alignment, the various regions of the CKX polypeptide that can likely be altered are represented in lower case letters, while the conserved regions are represented by capital letters. It is recognized that conservative substitutions can be made in the conserved regions below without altering function. In addition, one of skill will understand that functional variants of the CKX sequence of the invention can have minor non-conserved amino acid alterations in the conserved domain.
[0323] Artificial protein sequences are then created that are different from the original in the intervals of 80-85%, 85-90%, 90-95% and 95-100% identity. Midpoints of these intervals are targeted, with liberal latitude of plus or minus 1%, for example. The amino acids substitutions will be effected by a custom Perl script. The substitution table is provided below in Table 6.
TABLE-US-00014 TABLE 6 Substitution Table Strongly Similar and Optimal Rank of Order to Amino Acid Substitution Change Comment I L, V 1 50:50 substitution L I, V 2 50:50 substitution V I, L 3 50:50 substitution A G 4 G A 5 D E 6 E D 7 W Y 8 Y W 9 S T 10 T S 11 K R 12 R K 13 N Q 14 Q N 15 F Y 16 M L 17 First methionine cannot change H Na No good substitutes C Na No good substitutes P Na No good substitutes
[0324] First, any conserved amino acids in the protein that should not be changed is identified and "marked off" for insulation from the substitution. The start methionine will of course be added to this list automatically. Next, the changes are made.
[0325] H, C and P are not changed in any circumstance. The changes will occur with isoleucine first, sweeping N-terminal to C-terminal. Then leucine, and so on down the list until the desired target it reached. Interim number substitutions can be made so as not to cause reversal of changes. The list is ordered 1-17, so start with as many isoleucine changes as needed before leucine, and so on down to methionine. Clearly many amino acids will in this manner not need to be changed. L, I and V will involve a 50:50 substitution of the two alternate optimal substitutions.
[0326] The variant amino acid sequences are written as output. Perl script is used to calculate the percent identities. Using this procedure, variants of the CKX polypeptides are generating having about 80%, 85%, 90% and 95% amino acid identity to the starting unaltered ORF nucleotide sequence of SEQ ID NO: 3, 6, 9, 12, 53, 59, 62 or 68.
Example 10
Downregulation of Cytokinin Catabolism
[0327] The promoters of the present invention can be used in constructs designed to downregulate cytokinin oxidase activity to prevent the adverse effects of cytokinin oxidase expression on plant performance under normal or stress conditions. For example, certain embodiments comprise a construct comprising a segment of an endogenous cytokinin oxidase promoter such that, upon expression, self-hybridization of the RNA results in formation of hairpin RNA (hpRNA), resulting in transcriptional gene silencing of the native cytokinin oxidase gene. Thus, the embodiment comprises a nucleotide sequence which, when expressed in a cell, forms a hairpin RNA molecule (hpRNA), which suppresses (i.e., reduces or eliminates) expression of the endogenous cytokinin oxidase gene from its endogenous promoter. The ability of hpRNAs to suppress expression of a gene has been described (see, e.g., Matzke, et al., (2001) Curr. Opin. Genet. Devel. 11:221-227; Scheid, et al., (2002) Proc. Natl. Acad. Sci. USA 99:13659-13662; Waterhouse and Helliwell, (2003) Nature Reviews Genetics 4:29-38; Aufsaftz, et al., (2002) Proc. Nat'l. Acad. Sci. 99(4):16499-16506; Sijen, et al., (2001) Curr. Biol. 11:436-440).
[0328] The promoter which is operably linked to the nucleotide sequence encoding the hpRNA can be any promoter that is active in plant cells, particularly a promoter that is active (or can be activated) in reproductive tissues of a plant. As such, the promoter can be, for example, a constitutively active promoter, an inducible promoter, a tissue-specific promoter, a tissue-preferred promoter, a developmental-stage-specific promoter or a developmental-stage-preferred promoter.
[0329] A hairpin may target a single promoter or may target two or more promoters by means of a single transcribed RNA. The hairpin-encoding region may be located in any appropriate position within the construct, such as within an intron of an encoded gene or within 5' or 3' non-coding regions, or may be the sole expressed element of the construct.
[0330] Methods for preparing said constructs and transforming plants may be as previously described (for example, see, Cigan, et al., (2001) Sex Plant Reprod. 14:135-142).
[0331] Said construct for downregulating cytokinin oxidase expression may be used in combination with other constructs or methods, such as those which result in increased cytokinin biosynthesis activity.
[0332] This example demonstrates the effectiveness of this approach at down-regulating cytokinin-induced expression of ZmCkx1 in leaves. The inverted repeat constructs were prepared using a strategy designed by Cigan, et al., (2005, The Plant Journal 43:929-940).
[0333] An approximately 500 bp fragment, nucleotides 942-1470 of the ZmCkx1 promoter (Accession Number CQ895592; U.S. Pat. No. 6,921,815) was PCR amplified and cloned in inverse orientations separated by a portion of the Nos gene (nucleotide 259-568, accession number V00087). The ZmCkx1 PRO-Nos-ZmCkx1 PRO fragment was placed under transcriptional control of the Ubiquitin promoter (Ubiquitin-1) (Christensen, et al., (1992) Plant Molecular Biology 18:675) in a plasmid containing the 35S::PAT selectable marker (Unger, et al., (2001) Transgenic Research 10:409) to yield PHP24865 (FIG. 12A). A second version of the construct, PHP24866 (FIG. 12B), was obtained by inversion of the HindIII fragment by digestion and re-ligation, and screening for the inversion by restriction digests. Plasmids were introduced in Agrobacterium strain LBA4404 and used for transformation as described earlier (Zhao, et al., (1998) Maize Genetics Cooperation Newsletter 72:34-37) and at Example 7 herein.
RNA Isolation, RT-PCR and Northern Blot
[0334] Total RNA extractions were performed as previously described (Brugiere, et al., (2003) Plant Physiol. 132:1228-1240). Hybridizations were performed overnight at 65° C. using the procedure previously described (Brugiere, et al., (1999) Plant Cell 11:1995-2012; Abarca, (2001) Physiologia Plantarum 113:409-415). Successive washes were performed as follows: twice at 25° C. for 10 min each with 2×SSC; 0.1% (w/v) SDS (1×SSC is 150 mM NaCl and 15 mM sodium citrate), and twice for 20 min at 65° C. with 0.1×SSC; 0.1% (w/v) SDS. Blots were hybridized with quadrature-32P-dCTP labeled probes corresponding to the fragment of Nos gene present in the inverted repeat or ZmCkx1. Relative mRNA abundance was quantified using a phosphor imager (Typhoon®, Molecular Dynamics, Sunnyvale, Calif.) with imaging software (ImageQuant®, Molecular Dynamics). RNA was quantified using a spectrophotometer. Semi-quantitative RT-PCR was carried out from 5 μg of total RNA using Superscript III kit from Invitrogen (Carlsbad, Calif.) according to the manufacturer's instructions. PCR was carried out using the following primers, 5'-GGTGCACGGCGAGGAGGT-3' (SEQ ID NO: 71) and 5'TCGCCGCCGACATGCCGTCGTCCC-3' (SEQ ID NO: 72) using the following conditions: 94° C. 2 min, 25 cycles of 94° C. for 1 min, 62° C. for 1 min and 72° C. for 1.5 min, followed by 7 min at 72° C. After electrophoresis, the DNA amplification products were quantified by density using LabWorks analysis software (UVP, Inc., Upland, Calif.).
Cytokinin Treatment
[0335] Leaf discs (5 mm in diameter) were collected from fully expanded leaves of 8-week-old transgenic and non-transgenic plants and incubated in petri dishes containing water or water supplemented with 10 μM benzyladenine (BA). Approximately 100 discs per sample collected from individual TO plants were used for each treatment, and discs were incubated at 25° C. for 24 h.
Expression of the Inverted Repeat Construct in T0 Plants
[0336] FIGS. 16 and 17 show the pattern of expression of the ZmCkx1 PRO-Nos-ZmCkx1 PRO inverted repeat in transgenic T0 PHP24865 and PHP24866 plants corresponding to the constructs described in FIGS. 12A and 12B, respectively. Total RNA was extracted from leaf of T0 plants harvested at the V8 stage in the greenhouse. After electrophoresis and transfer to nylon membrane, blots were probed with a DNA probe corresponding to the Nos sequence present in the hairpin. As previously seen when constitutively expressing this kind of inverted repeat in corn (Cigan, et al., (2005) supra), two major transcripts were identified in plants transformed with the constructs, most likely being the result of the absence of terminator at the 3'-end of the inverted repeat construct.
Induction of ZmCkx1 Expression by BA in Transgenic Ubi-ZmCkx1 PRO Hairpin Compared to Transgenic Control Plants
[0337] Because native ZmCkx1 expression is high only in developing kernels (Brugiere, et al., (2003) supra) and destructive sampling of TO plants is undesirable, the effect of the inverted repeat constructs on ZmCkx1 cytokinin-induced expression in leaves was studied. Using the technique described in Brugiere, et al., (2003, supra), leaf discs of selected PHP24865 and PHP24866 transgenic TO plants, as well as leaf discs of transgenic control plants, were treated with 10 μM of the cytokinin benzyladenine (BA) for 24 h, and induction of ZmCkx1 was compared. The Northern blot of FIG. 15 shows a strong down-regulation of BA-induced ZmCkx1 expression in leaf discs of PHP24865 and PHP24866 transgenics compared to transgenic controls.
[0338] In order to quantify the degree of down-regulation of BA-induced ZmCkx1 expression in transgenic plants compared to controls, the radioactive signal of FIG. 15 was quantified with a phosphor imager and compared to the signal obtained after hybridization of the same blot with a probe corresponding to the ubiquitously expressed 18S RNA. The ratio of ZmCkx1 vs. 18S RNA expression was calculated, and results are presented in FIG. 16. Results show that ZmCkx1 was down-regulated by between 18% and 61% depending on the construct and the event considered. On the average, down-regulation was 50% in PHP24865 and 44% in PHP24866 (54% if not considering Event 14). As seen in FIG. 17, similar results were obtained using a semi-quantitative RT-PCR procedure with PHP24865 samples (PHP24866 samples were not tested). Results show down-regulation ranging from 58 to 69% with an average of 65% compared to transgenic control.
[0339] The promoter inverted repeat strategy was previously found to be effective for transcriptional gene silencing (Cigan, et al., (2005) supra). Here, we show that this approach is efficacious to down-regulate BA-induced ZmCkx1 expression in leaves by a measurable and reproducible extent. Expression levels were reduced by 50-60% as measured by Northern blot or semi-quantitative RT-PCR. Optimization of the construct, for example by using different sections of the promoter in hairpin configurations, and/or by using alternative promoters, may result in a stronger down-regulation effect and/or in a more tissue-preferred downregulation.
Example 11
Yield Improvement Through ZmCkx2b 3'UTR-RNAi
[0340] ZmCkx2 exists as a duplicated gene in maize, identified on Chromosome 3 (ZmCkx2a, SEQ ID NO: 1-3; NCBI CAE55200) and on Chromosome 8 (ZmCkx2b, SEQ ID NO: 67-68; NCBI CAE55201) (Massoneau, et al., 2004). The ZmCkx2b polypeptide (GenBank entry AJ606943) is 94% identical to the ZmCkx2a polypeptide of SEQ ID NO: 3.
[0341] The ZmCkx2b(TR1) genetic element (SEQ ID NO: 66) corresponds to the 3'-UTR of ZmCkx2b. The ZmCKx2b(TR1) element can be used in a hairpin construct to down-regulate the expression of ZmCkx2, improving seed yield, for example, in maize. The ZmCkx2b(TR1) sequence (SEQ ID NO: 66; see also, NM--001111693) is over 99% identical to the corresponding 3' region of ZmCkx2b (SEQ ID NO: 67). While not being bound by any particular mode of action, Applicants propose that targeted downregulation results from the activity of small interfering RNAs produced from the double-stranded RNA of a hairpin construct with significant complentarity to the target sequence, as has been previously described (McManus and Sharp, (2002) Nature Reviews Genetics 3:737-747; Johnston and Hobert, (2003) Nature 426:845-849; Brugiere, et al., (1999) supra).
[0342] Data were gathered from maize plants transformed with a construct comprising the ubiquitin promoter operably linked to a 447-base-pair portion of the ZmCkx2b genomic locus (a 3' UTR segment, designated p0081.chcag31r in plasmid PHP27911; see, FIG. 18) in direct and reverse orientations, separated by a 539-base-pair Adh1 intron sequence, to produce a hairpin configuration when transcribed. The construct also comprises the UBI1Zm intron (PHI) as an enhancer and PINII as terminator. This hairpin, or a similar one, could also be expressed under the control of a drought-inducible promoter such as Rab17 (Vilardell, et al., (1991) Plant Molecular Biology 17(5):985-993.) Similar constructs could be created using fragments of the ZmCkx2a and/or b coding sequence in inverted repeats separated by a fragment of the Adh1 intron driven by UBI1Zm PRO. Each such exemplary construct is designed to suppress expression of the endogenous gene(s) using a hairpin strategy.
[0343] The plants were of the fast-cycling type (Gaspe/Flint) described in US Patent Application Publication Number 2003/0221212. Ten plants transformed with PHP27911 were scored for (1) rate of growth to half of maximum volume ("rt halfmaxvol"); (2) rate of growth to maximum volume ("rt maxvolume"); and (3) estimated seed yield ("yield estimate") using a high-throughput system (Functional Analysis System for Traits or FASTcorn). These scores were then compared to the scores obtained for 13,968 other FASTcorn transgenic plants tested with the same system. Each of the FASTcorn plants in the pool represents an independent transformation event for an assortment of proprietary constructs tested for improved agronomic traits.
[0344] The total pool of 13,968 FASTcorn transgenic plants for which a Zscore was available for the rate to half maximum volume (Zscore rt half max vol) was first filtered to retain only those events with a Zscore>1. A Zscore of 1 indicates a value that is two standard deviations away from the mean and is therefore substantially different. Seven events out of ten events transformed with the PHP27911 construct met this criterion (FIG. 24).
[0345] A second filter was then applied to retain only those events which had a Zscore>1 for the trait rt maxvolume. Seven out of ten events corresponding to the PHP27911 construct, met this criterion alone.
[0346] Six events out of the ten generated were retained when both filters were applied (FIG. 24). Thus, a significant improvement in growth rate was observed in more than half of the PHP27911 events tested.
[0347] The total volume growth rate was determined for the ten PHP27911 events. For the six identified in FIG. 24 as meeting both criteria, a clear growth rate advantage was demonstrated relative to all FASTcorn transgenics.
[0348] FASTcorn events remaining in the pool following application of the two filters as described above (1557 events total) were further filtered to identify those with a Zscore>1 for the "yield estimate" trait. Yield estimate is calculated based on seed count and single kernel mass. Four of the six PHP27911 events retained based on the previous filters were again retained after filtering for improved yield estimate (FIG. 24). (For one of the six events, no Zscore for yield estimate was available.) These data strongly suggest that the improved growth rate observed in the PHP27911 events generally translates to improved yield estimate.
Example 12
Root-Preferred Overexpression of Zmckx2a for Increased Root Biomass and Improved Nitrogen Use Efficiency
[0349] As described in Example 4 and shown in FIGS. 5, 6 and 7, constitutive overexpression of a genomic ZmCkx2a sequence resulted in increased root growth but had negative effects on overall plant growth. In contrast, over-expression of ZmCkx2a genomic or cDNA sequences in maize using a root-specific or root-preferred promoter improves root biomass and yield of transgenic plants growing in drought-stressed or low-nitrogen conditions compared to control plants. The improved root biomass may improve the plant's ability to mine for water and/or essential nutrients, such as nitrogen, in the soil. Improved root growth may also improve resistance to insects and other biotic or abiotic stresses. Delayed leaf senescence may also result.
[0350] Preferred promoters include Zm-NAS2 promoter (U.S. patent application Ser. No. 12/030,455); Zm-Cyclo1 promoter (U.S. Pat. No. 7,268,226); Zm-Metallothionein promoters (U.S. Pat. Nos. 6,774,282, 7,214,854 and 7,214,855 (also known as RootMET2)); ZM-MSY promoter (SEQ ID NO: 64; U.S. Patent Application Ser. No. 60/971,310 filed Sep. 11, 2007) or ZRP promoter (SEQ ID NO: 65; see, U.S. Pat. No. 5,633,363); constructs may also include one or more of the CaMV35S enhancer, Odell, et al., (1988) Plant Mol. Biol. 10:263-272, the ADH1 INTRON1 (Callis, et al., (1987) Genes and Dev. 1:1183-1200), the UBI1ZM INTRON (PHI) as an enhancer, and PINII as terminator.
[0351] Maize was transformed as described in Zhao, et al., (1998) Maize Genetics Cooperation Newsletter 72:34-37, and at Example 7 herein, but with a construct comprising either the ZmCyclo1 promoter (plasmid PHP28930) or the ZmROOTMET2 promoter (plasmid PHP28937) operably linked to the ZmCkx2a genomic sequence (SEQ ID NO: 1) and the PinII terminator (FIG. 19). Plants were regenerated and pollinated, and next-generation plants were observed in the field. Both constructs resulted in increased branching on brace roots (FIG. 20A) and increased root mass overall (FIG. 20B), with no (PHP28937) or minimal (PHP28930) reduction in above-ground biomass (FIG. 21). Northern data indicated ROOTMET2-driven expression (FIG. 25A) was more tightly targeted to root tissue than was ZmCyclo1-driven expression (FIG. 25B). Ears (FIG. 26) harvested from the transgenic plants confirmed that more favorable ear phenotypes and yield result from more highly specific root overexpression of ZmCkx2a. Optimization of the constructs (FIG. 22) will further improve the positive effect on roots while avoiding negative impact on above-ground growth.
[0352] Also, maize was transformed as described in Zhao, et al., (1998) Maize Genetics Cooperation Newsletter 72:34-37, but with a construct comprising either the NAS2 promoter (PHP22524) or the ZRP promoter and Adh1 intron (PHP22532) operably linked to ZmCkx2a cDNA (SEQ ID NO: 2). This root-specific overexpression of ZmCkx2 resulted in yield improvement under conditions of limited nitrogen. FIG. 27 shows the increase in hybrid grain yield for two events of PHP22514 (ZM-NAS2 PRO::ZM-CKX2) in limited nitrogen environments at Iowa and California test locations. Yield data from 6 replicates of each event per location are compared to that of the bulked transgenic nulls for the construct (NULL). Asterisks (*) mark those that are significantly different from the NULL at P<0.1. The yield data from hybrid 3245 (WT) are also included and shown to be not different from that of the NULL.
[0353] All publications and patent applications mentioned in the specification are indicative of the level of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.
[0354] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.
Sequence CWU
1
11213200DNAZea
maysexon(267)...(849)exon(957)...(1084)exon(1175)...(1435)exon(1509)...(1-
771)exon(1871)...(2195)polyA_signal(2859)...(2864)misc_feature(0)...(0)Gen-
omic sequence for ZmCkx2 1cctatataga gaggccccct ccctccccct gcatggacag
ccaccgcctt cttcaaccct 60ccttccgtct tcctcctcta gtcttacctc gttgcacctc
aagaaacttg gcgcgcaacc 120aggaaacccc ctcttctctc tctctctctc tctctctctc
tgccttctga ttccaagctc 180cccaactgcc cagcaccaac ctgccgaact cccctccttt
ttgttggttt gtcgaattat 240aaattgagcc cggccggctg actaccatga agccgccatc
actggtgcac tgcttcaagc 300tgctggtcct gctggcgctc gccaggctga ccatgcacgt
ccccgacgag gacatgctat 360cgcccctcgg cgcgctgcgc ctcgacggtc atttcagctt
ccatgacgtc tccgccatgg 420cgcgggactt cggcaaccag tgcagcttcc tgccggccgc
cgtgctccac ccaggctcgg 480tctccgatat cgccgccacc gtgaggcacg tcttctccct
gggcgagggc tcgccgctca 540ccgtcgcggc gcgcgggcat ggacactccc tcatgggtca
gtcccaggcc gcccagggga 600tcgtggtcag gatggagtcg ctccggggcg ctaggctcca
ggtccacgac ggctttgtcg 660atgcccccgg aggagagctc tggatcaatg tcctgcgtga
gacgctgaag cacggcctgg 720cacccaagtc gtggacggac tatctccatc tcacggtcgg
tggcaccttg tctaatgcgg 780gggtcagcgg ccaggcgttc cgccacggac cgcaggtcag
caatgtcaat caactggaga 840ttgtgacagg tctcaaacga actcacaaag cattcaatca
actagcttgg agcatacata 900acgaaacata aaaaaaaaca gtcgctgatc gtaataatcg
taaaaaccaa atgcaggaag 960gggagacgtc gttacctgct cacccgagga taactctgat
ctcttctatg ctgctctcgg 1020cggtcttggt cagttcggga tcataaccag agcaaggatt
gcacttgagc ctgctccaga 1080gatggtaagt catcagacaa gcgattcagt taaatgaaat
ctccagacag catgcagtca 1140tttagtaaat ggatgtatat atatacaatg acaggtgagg
tggataagag ttctttactc 1200ggattttgaa agcttcaccg aagaccagga gatgttgatc
atggcagaga actcctttga 1260ctacattgaa ggttttgtca tcataaacag gacaggcatc
ctcaacaact ggagggcgtc 1320cttcaagcca caggacccag tccaagcaag ccatttccag
tcagatggaa gagtgctata 1380ctgcctcgaa ctaaccaaga acttcaatag tggcgacact
gataccatgg aacaggtgag 1440cctgttattt cactttgcac caagatatta gactccaatg
ataataactg taaattttat 1500gtttacagga agttgctgta ctgctatctc ggcttagatt
catacagtct actctattcc 1560acaccgatgt cacgtacctg gagtttttgg acagggtgca
cacctctgag ctgaagctga 1620gggcacaaag cctctgggaa gttccacacc cttggttgaa
tcttctgata ccgaggagct 1680caatccgcag atttgctacg gaagtctttg gcaggatcct
gaaagatagc aacaatggtc 1740ctatattgct ttatccagtg aacaaatcaa agtaacttcc
ttcacttgca aaaattactg 1800tcacaaataa taagttaatc tagttgcgca cggttaaggt
agctcaattc gtctgttcgt 1860tctgatgcag gtgggacaac aaaacgtcag tggtcatacc
agatgaggaa attttctacc 1920tagtgggatt cctttcttca gcaccgtctc tctcaggtca
cggcagcatt gcacatgcga 1980tgagcctgaa cagccaaata gtagagttct gtgaagaggc
tgatattggg atgaaacagt 2040atctagcaca ctacaccaca caggagcagt ggaaaaccca
ctttggagca aggtgggaga 2100catttgaacg gaggaaacac agatatgatc ccctagccat
cctagcacca ggacagagaa 2160tattcccaaa ggcgtcactc ccattgtctt tgtgacggtt
cctgctattt aaaggcttct 2220gtagagcata cattgtacaa aagtgtaggt aaaagtatcc
cctgtaaaga caatatctac 2280ggaaggtagc tagcctgaag aacacagcat agcgactttt
tcagtggcca aagatacctc 2340aaagcagtac ttcaatgtgg agcaacgtca cctgaaccct
gaaggtggtg agtgcaactt 2400tggaggcaat cactggtagt ggagcctgga gggttgtagc
ggtccaagga acctgtctgt 2460tgttacagcg ttgagatgag ctgtgctgat caactgatca
ctaaccagtg tcccgaggaa 2520atcatgttgg tctgtatgta ttttccgtta acaacagtgc
agaagtttgc atgagggtag 2580tgcattgatt agcaaatagc actgcctgtt atttcacttg
taactggcat ctcatctcaa 2640ggagagcctg cgtaactgta gcaggttata ttgttttcca
tgagtcagaa actcagaata 2700ttaatgctcg tgcaaaaaca gtgtagtcgc ttatcaatca
tggtgttcag aaacagaaaa 2760actcttggaa ttctctcaac ttgttcatta aatgttagca
acctatagtg tgagcttgga 2820taacaaataa gaaatcaaga gcgcaaatat tgaaactgaa
taaatgttaa atgataattt 2880tctaaagtcc aatcaagcag aattataagt ttgcaagata
acttgataac tgacatctca 2940gttttttgta tcaagcaaat gtgctgtcaa aaacaaaagc
aaatacaccc ttttcagtta 3000gtgggccata ctgtggtctt aatcagacct ttgtttccgc
aataaggtgt tcatggaact 3060gcatgtgcga tacagcttct gctcatgata caaccaacat
aagatctcaa tagagaagta 3120ttcatatccc gtaacagcgc aatgttcaaa gatatttgcc
tggttcaaat acgggcatgc 3180agttcttcac gatcgtggta
320021560DNAZea
maysCDS(1)...(1560)misc_feature(0)...(0)ZmCkx2a cDNA 2atg aag ccg cca tca
ctg gtg cac tgc ttc aag ctg ctg gtc ctg ctg 48Met Lys Pro Pro Ser
Leu Val His Cys Phe Lys Leu Leu Val Leu Leu1 5
10 15gcg ctc gcc agg ctg acc atg cac gtc ccc gac
gag gac atg cta tcg 96Ala Leu Ala Arg Leu Thr Met His Val Pro Asp
Glu Asp Met Leu Ser 20 25
30ccc ctc ggc gcg ctg cgc ctc gac ggt cat ttc agc ttc cat gac gtc
144Pro Leu Gly Ala Leu Arg Leu Asp Gly His Phe Ser Phe His Asp Val
35 40 45tcc gcc atg gcg cgg gac ttc ggc
aac cag tgc agc ttc ctg ccg gcc 192Ser Ala Met Ala Arg Asp Phe Gly
Asn Gln Cys Ser Phe Leu Pro Ala 50 55
60gcc gtg ctc cac cca ggc tcg gtc tcc gat atc gcc gcc acc gtg agg
240Ala Val Leu His Pro Gly Ser Val Ser Asp Ile Ala Ala Thr Val Arg65
70 75 80cac gtc ttc tcc ctg
ggc gag ggc tcg ccg ctc acc gtc gcg gcg cgc 288His Val Phe Ser Leu
Gly Glu Gly Ser Pro Leu Thr Val Ala Ala Arg 85
90 95ggg cat gga cac tcc ctc atg ggt cag tcc cag
gcc gcc cag ggg atc 336Gly His Gly His Ser Leu Met Gly Gln Ser Gln
Ala Ala Gln Gly Ile 100 105
110gtg gtc agg atg gag tcg ctc cgg ggc gct agg ctc cag gtc cac gac
384Val Val Arg Met Glu Ser Leu Arg Gly Ala Arg Leu Gln Val His Asp
115 120 125ggc ttt gtc gat gcc ccc gga
gga gag ctc tgg atc aat gtc ctg cgt 432Gly Phe Val Asp Ala Pro Gly
Gly Glu Leu Trp Ile Asn Val Leu Arg 130 135
140gag acg ctg aag cac ggc ctg gca ccc aag tcg tgg acg gac tat ctc
480Glu Thr Leu Lys His Gly Leu Ala Pro Lys Ser Trp Thr Asp Tyr Leu145
150 155 160cat ctc acg gtc
ggt ggc acc ttg tct aat gcg ggg gtc agc ggc cag 528His Leu Thr Val
Gly Gly Thr Leu Ser Asn Ala Gly Val Ser Gly Gln 165
170 175gcg ttc cgc cac gga ccg cag gtc agc aat
gtc aat caa ctg gag att 576Ala Phe Arg His Gly Pro Gln Val Ser Asn
Val Asn Gln Leu Glu Ile 180 185
190gtg aca gga agg gga gac gtc gtt acc tgc tca ccc gag gat aac tct
624Val Thr Gly Arg Gly Asp Val Val Thr Cys Ser Pro Glu Asp Asn Ser
195 200 205gat ctc ttc tat gct gct ctc
ggc ggt ctt ggt cag ttc ggg atc ata 672Asp Leu Phe Tyr Ala Ala Leu
Gly Gly Leu Gly Gln Phe Gly Ile Ile 210 215
220acc aga gca agg att gca ctt gag cct gct cca gag atg gtg agg tgg
720Thr Arg Ala Arg Ile Ala Leu Glu Pro Ala Pro Glu Met Val Arg Trp225
230 235 240ata aga gtt ctt
tac tcg gat ttt gaa agc ttc acc gaa gac cag gag 768Ile Arg Val Leu
Tyr Ser Asp Phe Glu Ser Phe Thr Glu Asp Gln Glu 245
250 255atg ttg atc atg gca gag aac tcc ttt gac
tac att gaa ggt ttt gtc 816Met Leu Ile Met Ala Glu Asn Ser Phe Asp
Tyr Ile Glu Gly Phe Val 260 265
270atc ata aac agg aca ggc atc ctc aac aac tgg agg gcg tcc ttc aag
864Ile Ile Asn Arg Thr Gly Ile Leu Asn Asn Trp Arg Ala Ser Phe Lys
275 280 285cca cag gac cca gtc caa gca
agc cat ttc cag tca gat gga aga gtg 912Pro Gln Asp Pro Val Gln Ala
Ser His Phe Gln Ser Asp Gly Arg Val 290 295
300cta tac tgc ctc gaa cta acc aag aac ttc aat agt ggc gac act gat
960Leu Tyr Cys Leu Glu Leu Thr Lys Asn Phe Asn Ser Gly Asp Thr Asp305
310 315 320acc atg gaa cag
gaa gtt gct gta ctg cta tct cgg ctt aga ttc ata 1008Thr Met Glu Gln
Glu Val Ala Val Leu Leu Ser Arg Leu Arg Phe Ile 325
330 335cag tct act cta ttc cac acc gat gtc acg
tac ctg gag ttt ttg gac 1056Gln Ser Thr Leu Phe His Thr Asp Val Thr
Tyr Leu Glu Phe Leu Asp 340 345
350agg gtg cac acc tct gag ctg aag ctg agg gca caa agc ctc tgg gaa
1104Arg Val His Thr Ser Glu Leu Lys Leu Arg Ala Gln Ser Leu Trp Glu
355 360 365gtt cca cac cct tgg ttg aat
ctt ctg ata ccg agg agc tca atc cgc 1152Val Pro His Pro Trp Leu Asn
Leu Leu Ile Pro Arg Ser Ser Ile Arg 370 375
380aga ttt gct acg gaa gtc ttt ggc agg atc ctg aaa gat agc aac aat
1200Arg Phe Ala Thr Glu Val Phe Gly Arg Ile Leu Lys Asp Ser Asn Asn385
390 395 400ggt cct ata ttg
ctt tat cca gtg aac aaa tca aag tgg gac aac aaa 1248Gly Pro Ile Leu
Leu Tyr Pro Val Asn Lys Ser Lys Trp Asp Asn Lys 405
410 415acg tca gtg gtc ata cca gat gag gaa att
ttc tac cta gtg gga ttc 1296Thr Ser Val Val Ile Pro Asp Glu Glu Ile
Phe Tyr Leu Val Gly Phe 420 425
430ctt tct tca gca ccg tct ctc tca ggt cac ggc agc att gca cat gcg
1344Leu Ser Ser Ala Pro Ser Leu Ser Gly His Gly Ser Ile Ala His Ala
435 440 445atg agc ctg aac agc caa ata
gta gag ttc tgt gaa gag gct gat att 1392Met Ser Leu Asn Ser Gln Ile
Val Glu Phe Cys Glu Glu Ala Asp Ile 450 455
460ggg atg aaa cag tat cta gca cac tac acc aca cag gag cag tgg aaa
1440Gly Met Lys Gln Tyr Leu Ala His Tyr Thr Thr Gln Glu Gln Trp Lys465
470 475 480acc cac ttt gga
gca agg tgg gag aca ttt gaa cgg agg aaa cac aga 1488Thr His Phe Gly
Ala Arg Trp Glu Thr Phe Glu Arg Arg Lys His Arg 485
490 495tat gat ccc cta gcc atc cta gca cca gga
cag aga ata ttc cca aag 1536Tyr Asp Pro Leu Ala Ile Leu Ala Pro Gly
Gln Arg Ile Phe Pro Lys 500 505
510gcg tca ctc cca ttg tct ttg tga
1560Ala Ser Leu Pro Leu Ser Leu 5153519PRTZea mays 3Met Lys Pro
Pro Ser Leu Val His Cys Phe Lys Leu Leu Val Leu Leu1 5
10 15 Ala Leu Ala Arg Leu Thr Met His
Val Pro Asp Glu Asp Met Leu Ser 20 25
30 Pro Leu Gly Ala Leu Arg Leu Asp Gly His Phe Ser Phe
His Asp Val 35 40 45
Ser Ala Met Ala Arg Asp Phe Gly Asn Gln Cys Ser Phe Leu Pro Ala 50
55 60 Ala Val Leu His Pro
Gly Ser Val Ser Asp Ile Ala Ala Thr Val Arg65 70
75 80 His Val Phe Ser Leu Gly Glu Gly Ser Pro
Leu Thr Val Ala Ala Arg 85 90
95 Gly His Gly His Ser Leu Met Gly Gln Ser Gln Ala Ala Gln Gly
Ile 100 105 110 Val
Val Arg Met Glu Ser Leu Arg Gly Ala Arg Leu Gln Val His Asp 115
120 125 Gly Phe Val Asp Ala Pro
Gly Gly Glu Leu Trp Ile Asn Val Leu Arg 130 135
140 Glu Thr Leu Lys His Gly Leu Ala Pro Lys Ser
Trp Thr Asp Tyr Leu145 150 155
160 His Leu Thr Val Gly Gly Thr Leu Ser Asn Ala Gly Val Ser Gly Gln
165 170 175 Ala Phe Arg
His Gly Pro Gln Val Ser Asn Val Asn Gln Leu Glu Ile 180
185 190 Val Thr Gly Arg Gly Asp Val Val
Thr Cys Ser Pro Glu Asp Asn Ser 195 200
205 Asp Leu Phe Tyr Ala Ala Leu Gly Gly Leu Gly Gln Phe
Gly Ile Ile 210 215 220
Thr Arg Ala Arg Ile Ala Leu Glu Pro Ala Pro Glu Met Val Arg Trp225
230 235 240 Ile Arg Val Leu Tyr
Ser Asp Phe Glu Ser Phe Thr Glu Asp Gln Glu 245
250 255 Met Leu Ile Met Ala Glu Asn Ser Phe Asp
Tyr Ile Glu Gly Phe Val 260 265
270 Ile Ile Asn Arg Thr Gly Ile Leu Asn Asn Trp Arg Ala Ser Phe
Lys 275 280 285 Pro
Gln Asp Pro Val Gln Ala Ser His Phe Gln Ser Asp Gly Arg Val 290
295 300 Leu Tyr Cys Leu Glu Leu
Thr Lys Asn Phe Asn Ser Gly Asp Thr Asp305 310
315 320 Thr Met Glu Gln Glu Val Ala Val Leu Leu Ser
Arg Leu Arg Phe Ile 325 330
335 Gln Ser Thr Leu Phe His Thr Asp Val Thr Tyr Leu Glu Phe Leu Asp
340 345 350 Arg Val His
Thr Ser Glu Leu Lys Leu Arg Ala Gln Ser Leu Trp Glu 355
360 365 Val Pro His Pro Trp Leu Asn Leu
Leu Ile Pro Arg Ser Ser Ile Arg 370 375
380 Arg Phe Ala Thr Glu Val Phe Gly Arg Ile Leu Lys Asp
Ser Asn Asn385 390 395
400 Gly Pro Ile Leu Leu Tyr Pro Val Asn Lys Ser Lys Trp Asp Asn Lys
405 410 415 Thr Ser Val Val
Ile Pro Asp Glu Glu Ile Phe Tyr Leu Val Gly Phe 420
425 430 Leu Ser Ser Ala Pro Ser Leu Ser Gly
His Gly Ser Ile Ala His Ala 435 440
445 Met Ser Leu Asn Ser Gln Ile Val Glu Phe Cys Glu Glu Ala
Asp Ile 450 455 460
Gly Met Lys Gln Tyr Leu Ala His Tyr Thr Thr Gln Glu Gln Trp Lys465
470 475 480 Thr His Phe Gly Ala
Arg Trp Glu Thr Phe Glu Arg Arg Lys His Arg 485
490 495 Tyr Asp Pro Leu Ala Ile Leu Ala Pro Gly
Gln Arg Ile Phe Pro Lys 500 505
510 Ala Ser Leu Pro Leu Ser Leu 515
43258DNAZea maysmisc_feature(0)...(0)Genomic sequence for ZmCkx3
4aaaaaatgtt ntncagatat atgtataaaa atgtgtacct agtacctacg catgtcttag
60ttcaacatac ttgatagctg tagttttctg aaacctgttc aaattaacct ttttcctacc
120tgatggtgaa tagagagaaa agctttacct ttgtctgaat aagaaaacta acagaaagct
180tacattttgg ccactctacc tgcccgagta ttttctaagc aagcaaaggc gcatgaaaat
240tttctcggaa tccatgacct tttacgcgca tgaaaatttt gaccaatgat cattttgata
300ctctccacaa gtcaacatct caaaaccaca agatggggcc catcaacata agttcacgag
360tgtgccttca ggtacattgt tctttttttt tgttttgcta aagtcaatca gctgcaaaat
420attcagaaca atttcaataa cccgaaaggc tgttgtgcct ccatttgtca acgtttgcga
480ggccaaatgg tacccccgct ataaatacca tggaagttct tggcctctag gacacacaag
540cgatctctcc tcctatagtt tctataaccc cacaaagcgt ccaggtcccg tagtcacctc
600cgattgcatt gcgttgccgc aagacaagca tggcaagaag gactcgtttc gtggccatcg
660ccgccctcct cacaagcttc ctcaacgtcg cagccgggca ttcccggcca ctgtccggtg
720ccggcctccc gggcgatctt ttcgggctgg gcatcgcgtc gaggatccgc acggacagca
780actcgacggc gaaggcggcg acggacttcg gccagatggt gagggccgcg ccggaggccg
840tgttccaccc cgccacgccg gccgacatcg ccgcgctcgt ccggttctcc gccacgtcgg
900cggcgccgtt ccccgttgcg ccgcgcgggc agggccactc ctggcgcggc caggcgctcg
960ccccgggcgg cgtcgtcgtg gacatgggct cgctggggcg cggcccccgc atcaacgtgt
1020ccgccgtggc cggcgcggag ccgttcgtcg acgccggcgg ggagcagctg tgggtcgacg
1080tcctccgcgc cacgctgcga cacggcctgg cgccccgcgt gtggaccgac tacctccggc
1140tcaccgtcgg cggcacgctc tccaacgcgg gaatcggcgg gcaggcgttc cgacacggtc
1200cgcagatcgc caacgtgcat gaactcgacg tcgtcacagg tatcgaccga tcgatggtta
1260cactcccagt gacaattaca taagcagcta atcacacacg aatgctaata atagtttata
1320catgcgatga aaaatgtagg cacaggtgag atggtgacat gctccatgga cgtgaactcg
1380gacctgttca tggcggctct aggcgggtta ggccagttcg gggtcataac cagagcacgg
1440atccggcttg agccggcgcc caagagggtg cgctgggttc gacttgccta caccgacgtc
1500gctactttca ccaaggatca ggagtttctc atatcaaacc gggctagcca agtcgggttc
1560gactacgtcg aaggccaggt ccagctcagc cggtccttgg tcgaaggccc caaatcaaca
1620cccttcttct ccggcgccga tgttgctagg cttgctggac tcgcgtccag gaccggacct
1680gctgcaatct actacatcga aggcgccatg tactacacca aggacaccgc catatctgtg
1740gacaaggtac agatcagctt gaacacacac acaaaaaaac gaactttatt attgctttca
1800atgctttgga cgaaaggaaa ttcattcgtt gttgctatat gaaacgttgc agaaaatgaa
1860ggcactcctg gatcagctga gcttcgagcc agggtttgcg ttcaccaagg acgtgacgtt
1920cgtgcagttc ctcgatcggg tgcgcgagga ggagagggtg ctccggtcag ccggcgcgtg
1980ggaggtgccg cacccatggc tgaacctctt cgtcccacgg tcgcgcatcc tcgacttcga
2040cgacggagtg ttcaaggctc tgctcaagga ctccaaccca gctgggatca tcctcatgta
2100ccccatgaac aaggataggt gggacgaccg gatgacagcg atgaccccag ccacggacga
2160cgacgacatg ttctatgccg ttagtttcct ttggtcagca ctgtccgcag acgacgtgcc
2220ccagctcgag agatggaaca aggcagtgct ggacttctgt gatcggtcag gaatagaatg
2280caagcagtac ctgccacact acacatctca agacgggtgg cgacggcatt tcggggcgaa
2340atggagcagg atcgctgagc tgaaggccag atatgaccct cgggcattgt tgtcgccggg
2400ccagaggatt tttccggtgc cagtagaggc atctggcatt gcttctgcct gattgcccgg
2460tctcgtagtc tcgaagcaaa cataaatgat tttcttgtgt agattgtaga atgtacatga
2520taggtctttt tcattgtaag aaagataggt cttattttgt acataatttt tcttttgggt
2580tgctagcacg gacggagggg ccattgtggc tagagaaagg taataatagt cacaaattat
2640ataattcaca tctcaccttt taagtattaa gaagtcattt aaaaagaagg atagacaaat
2700gcaattgcct tagtctctaa gatttatata gcaaaattat cagtataact ttgtattgtg
2760tattatttac cccgcatatg tttcttaaca gtttattctt attttagaca atattctatt
2820ttatacaatt tttttacagt agaatcccac ggtacactcg aaactaggat ggggctccaa
2880atcggagcag gttttaatat aagagatggg aaagaagaac cagattaata ctaccctctc
2940ctatcaaata aatttgcatt ccattaaaaa aattgaaaaa ctgaaaaaaa atcagtacaa
3000gtagagccaa gatttgaatt tggcaaatac ttataaaaca ttttgaggca ttgatatgtt
3060agctagaggc cgagagccaa gtatgtcgga tggtaaatcg agaagcgggc agtttgctaa
3120aatatctttg ttttattttt gtcatttaaa ctagggatga caatggggat ttttccgtcg
3180gggaatggct ccccatcccc gtcctcgtgg ggtggaaaat tcctcgtccc cgtccccgcg
3240aacacccacg ggaagctt
325852635DNAZea maysmisc_feature(0)...(0)ZmCkx3 cDNA 5aaaaaatgtt
ntncagatat atgtataaaa atgtgtacct agtacctacg catgtcttag 60ttcaacatac
ttgatagctg tagttttctg aaacctgttc aaattaacct ttttcctacc 120tgatggtgaa
tagagagaaa agctttacct ttgtctgaat aagaaaacta acagaaagct 180tacattttgg
ccactctacc tgcccgagta ttttctaagc aagcaaaggc gcatgaaaat 240tttctcggaa
tccatgacct tttacgcgca tgaaaatttt gaccaatgat cattttgata 300ctctccacaa
gtcaacatct caaaaccaca agatggggcc catcaacata agttcacgag 360tgtgccttca
ggtacattgt tctttttttt tgttttgcta aagtcaatca gctgcaaaat 420attcagaaca
atttcaataa cccgaaaggc tgttgtgcct ccatttgtca acgtttgcga 480ggccaaatgg
tacccccgct ataaatacca tggaagttct tggcctctag gacacacaag 540cgatctctcc
tcctatagtt tctataaccc cacaaagcgt ccaggtcccg tagtcacctc 600cgattgcatt
gcgttgccgc aagacaagc atg gca aga agg act cgt ttc gtg 653
Met Ala Arg Arg Thr Arg Phe Val
1 5gcc atc gcc gcc ctc ctc aca agc ttc ctc aac gtc
gca gcc ggg cat 701Ala Ile Ala Ala Leu Leu Thr Ser Phe Leu Asn Val
Ala Ala Gly His 10 15 20tcc cgg cca
ctg tcc ggt gcc ggc ctc ccg ggc gat ctt ttc ggg ctg 749Ser Arg Pro
Leu Ser Gly Ala Gly Leu Pro Gly Asp Leu Phe Gly Leu25 30
35 40ggc atc gcg tcg agg atc cgc acg
gac agc aac tcg acg gcg aag gcg 797Gly Ile Ala Ser Arg Ile Arg Thr
Asp Ser Asn Ser Thr Ala Lys Ala 45 50
55gcg acg gac ttc ggc cag atg gtg agg gcc gcg ccg gag gcc
gtg ttc 845Ala Thr Asp Phe Gly Gln Met Val Arg Ala Ala Pro Glu Ala
Val Phe 60 65 70cac ccc gcc
acg ccg gcc gac atc gcc gcg ctc gtc cgg ttc tcc gcc 893His Pro Ala
Thr Pro Ala Asp Ile Ala Ala Leu Val Arg Phe Ser Ala 75
80 85acg tcg gcg gcg ccg ttc ccc gtt gcg ccg cgc
ggg cag ggc cac tcc 941Thr Ser Ala Ala Pro Phe Pro Val Ala Pro Arg
Gly Gln Gly His Ser 90 95 100tgg cgc
ggc cag gcg ctc gcc ccg ggc ggc gtc gtc gtg gac atg ggc 989Trp Arg
Gly Gln Ala Leu Ala Pro Gly Gly Val Val Val Asp Met Gly105
110 115 120tcg ctg ggg cgc ggc ccc cgc
atc aac gtg tcc gcc gtg gcc ggc gcg 1037Ser Leu Gly Arg Gly Pro Arg
Ile Asn Val Ser Ala Val Ala Gly Ala 125 130
135gag ccg ttc gtc gac gcc ggc ggg gag cag ctg tgg gtc
gac gtc ctc 1085Glu Pro Phe Val Asp Ala Gly Gly Glu Gln Leu Trp Val
Asp Val Leu 140 145 150cgc gcc
acg ctg cga cac ggc ctg gcg ccc cgc gtg tgg acc gac tac 1133Arg Ala
Thr Leu Arg His Gly Leu Ala Pro Arg Val Trp Thr Asp Tyr 155
160 165ctc cgg ctc acc gtc ggc ggc acg ctc tcc
aac gcg gga atc ggc ggg 1181Leu Arg Leu Thr Val Gly Gly Thr Leu Ser
Asn Ala Gly Ile Gly Gly 170 175 180cag
gcg ttc cga cac ggt ccg cag atc gcc aac gtg cat gaa ctc gac 1229Gln
Ala Phe Arg His Gly Pro Gln Ile Ala Asn Val His Glu Leu Asp185
190 195 200gtc gtc aca ggc aca ggt
gag atg gtg aca tgc tcc atg gac gtg aac 1277Val Val Thr Gly Thr Gly
Glu Met Val Thr Cys Ser Met Asp Val Asn 205
210 215tcg gac ctg ttc atg gcg gct cta ggc ggg tta ggc
cag ttc ggg gtc 1325Ser Asp Leu Phe Met Ala Ala Leu Gly Gly Leu Gly
Gln Phe Gly Val 220 225 230ata
acc aga gca cgg atc cgg ctt gag ccg gcg ccc aag agg gtg cgc 1373Ile
Thr Arg Ala Arg Ile Arg Leu Glu Pro Ala Pro Lys Arg Val Arg 235
240 245tgg gtt cga ctt gcc tac acc gac gtc
gct act ttc acc aag gat cag 1421Trp Val Arg Leu Ala Tyr Thr Asp Val
Ala Thr Phe Thr Lys Asp Gln 250 255
260gag ttt ctc ata tca aac cgg gct agc caa gtc ggg ttc gac tac gtc
1469Glu Phe Leu Ile Ser Asn Arg Ala Ser Gln Val Gly Phe Asp Tyr Val265
270 275 280gaa ggc cag gtc
cag ctc agc cgg tcc ttg gtc gaa ggc ccc aaa tca 1517Glu Gly Gln Val
Gln Leu Ser Arg Ser Leu Val Glu Gly Pro Lys Ser 285
290 295aca ccc ttc ttc tcc ggc gcc gat gtt gct
agg ctt gct gga ctc gcg 1565Thr Pro Phe Phe Ser Gly Ala Asp Val Ala
Arg Leu Ala Gly Leu Ala 300 305
310tcc agg acc gga cct gct gca atc tac tac atc gaa ggc gcc atg tac
1613Ser Arg Thr Gly Pro Ala Ala Ile Tyr Tyr Ile Glu Gly Ala Met Tyr
315 320 325tac acc aag gac acc gcc ata
tct gtg gac aag aaa atg aag gca ctc 1661Tyr Thr Lys Asp Thr Ala Ile
Ser Val Asp Lys Lys Met Lys Ala Leu 330 335
340ctg gat cag ctg agc ttc gag cca ggg ttt gcg ttc acc aag gac gtg
1709Leu Asp Gln Leu Ser Phe Glu Pro Gly Phe Ala Phe Thr Lys Asp Val345
350 355 360acg ttc gtg cag
ttc ctc gat cgg gtg cgc gag gag gag agg gtg ctc 1757Thr Phe Val Gln
Phe Leu Asp Arg Val Arg Glu Glu Glu Arg Val Leu 365
370 375cgg tca gcc ggc gcg tgg gag gtg ccg cac
cca tgg ctg aac ctc ttc 1805Arg Ser Ala Gly Ala Trp Glu Val Pro His
Pro Trp Leu Asn Leu Phe 380 385
390gtc cca cgg tcg cgc atc ctc gac ttc gac gac gga gtg ttc aag gct
1853Val Pro Arg Ser Arg Ile Leu Asp Phe Asp Asp Gly Val Phe Lys Ala
395 400 405ctg ctc aag gac tcc aac cca
gct ggg atc atc ctc atg tac ccc atg 1901Leu Leu Lys Asp Ser Asn Pro
Ala Gly Ile Ile Leu Met Tyr Pro Met 410 415
420aac aag gat agg tgg gac gac cgg atg aca gcg atg acc cca gcc acg
1949Asn Lys Asp Arg Trp Asp Asp Arg Met Thr Ala Met Thr Pro Ala Thr425
430 435 440gac gac gac gac
atg ttc tat gcc gtt agt ttc ctt tgg tca gca ctg 1997Asp Asp Asp Asp
Met Phe Tyr Ala Val Ser Phe Leu Trp Ser Ala Leu 445
450 455tcc gca gac gac gtg ccc cag ctc gag aga
tgg aac aag gca gtg ctg 2045Ser Ala Asp Asp Val Pro Gln Leu Glu Arg
Trp Asn Lys Ala Val Leu 460 465
470gac ttc tgt gat cgg tca gga ata gaa tgc aag cag tac ctg cca cac
2093Asp Phe Cys Asp Arg Ser Gly Ile Glu Cys Lys Gln Tyr Leu Pro His
475 480 485tac aca tct caa gac ggg tgg
cga cgg cat ttc ggg gcg aaa tgg agc 2141Tyr Thr Ser Gln Asp Gly Trp
Arg Arg His Phe Gly Ala Lys Trp Ser 490 495
500agg atc gct gag ctg aag gcc aga tat gac cct cgg gca ttg ttg tcg
2189Arg Ile Ala Glu Leu Lys Ala Arg Tyr Asp Pro Arg Ala Leu Leu Ser505
510 515 520ccg ggc cag agg
att ttt ccg gtg cca gta gag gca tct ggc att gct 2237Pro Gly Gln Arg
Ile Phe Pro Val Pro Val Glu Ala Ser Gly Ile Ala 525
530 535tct gcc tga ttgcccggtc tcgtagtctc
gaagcaaaca taaatgattt 2286Ser Ala tcttgtgtag attgtagaat
gtacatgata ggtctttttc attgtaagaa agataggtct 2346tattttgtac ataatttttc
ttttgggttg ctagcacgga cggaggggcc attgtggcta 2406gagaaaggta ataatagtca
caaattatat aattcacatc tcacctttta agtattaaga 2466agtcatttaa aaagaaggat
agacaaatgc aattgcctta gtctctaaga tttatatagc 2526aaaattatca gtataacttt
gtattgtgta ttatttaccc cgcatatgtt tcttaacagt 2586ttattcttat tttagacaat
attctatttt atacaatttt tttacagta 26356538PRTZea mays 6Met
Ala Arg Arg Thr Arg Phe Val Ala Ile Ala Ala Leu Leu Thr Ser1
5 10 15 Phe Leu Asn Val Ala Ala
Gly His Ser Arg Pro Leu Ser Gly Ala Gly 20 25
30 Leu Pro Gly Asp Leu Phe Gly Leu Gly Ile Ala
Ser Arg Ile Arg Thr 35 40 45
Asp Ser Asn Ser Thr Ala Lys Ala Ala Thr Asp Phe Gly Gln Met Val
50 55 60 Arg Ala Ala
Pro Glu Ala Val Phe His Pro Ala Thr Pro Ala Asp Ile65 70
75 80 Ala Ala Leu Val Arg Phe Ser Ala
Thr Ser Ala Ala Pro Phe Pro Val 85 90
95 Ala Pro Arg Gly Gln Gly His Ser Trp Arg Gly Gln Ala
Leu Ala Pro 100 105 110
Gly Gly Val Val Val Asp Met Gly Ser Leu Gly Arg Gly Pro Arg Ile
115 120 125 Asn Val Ser Ala
Val Ala Gly Ala Glu Pro Phe Val Asp Ala Gly Gly 130
135 140 Glu Gln Leu Trp Val Asp Val Leu
Arg Ala Thr Leu Arg His Gly Leu145 150
155 160 Ala Pro Arg Val Trp Thr Asp Tyr Leu Arg Leu Thr
Val Gly Gly Thr 165 170
175 Leu Ser Asn Ala Gly Ile Gly Gly Gln Ala Phe Arg His Gly Pro Gln
180 185 190 Ile Ala Asn
Val His Glu Leu Asp Val Val Thr Gly Thr Gly Glu Met 195
200 205 Val Thr Cys Ser Met Asp Val Asn
Ser Asp Leu Phe Met Ala Ala Leu 210 215
220 Gly Gly Leu Gly Gln Phe Gly Val Ile Thr Arg Ala Arg
Ile Arg Leu225 230 235
240 Glu Pro Ala Pro Lys Arg Val Arg Trp Val Arg Leu Ala Tyr Thr Asp
245 250 255 Val Ala Thr Phe
Thr Lys Asp Gln Glu Phe Leu Ile Ser Asn Arg Ala 260
265 270 Ser Gln Val Gly Phe Asp Tyr Val Glu
Gly Gln Val Gln Leu Ser Arg 275 280
285 Ser Leu Val Glu Gly Pro Lys Ser Thr Pro Phe Phe Ser Gly
Ala Asp 290 295 300
Val Ala Arg Leu Ala Gly Leu Ala Ser Arg Thr Gly Pro Ala Ala Ile305
310 315 320 Tyr Tyr Ile Glu Gly
Ala Met Tyr Tyr Thr Lys Asp Thr Ala Ile Ser 325
330 335 Val Asp Lys Lys Met Lys Ala Leu Leu Asp
Gln Leu Ser Phe Glu Pro 340 345
350 Gly Phe Ala Phe Thr Lys Asp Val Thr Phe Val Gln Phe Leu Asp
Arg 355 360 365 Val
Arg Glu Glu Glu Arg Val Leu Arg Ser Ala Gly Ala Trp Glu Val 370
375 380 Pro His Pro Trp Leu Asn
Leu Phe Val Pro Arg Ser Arg Ile Leu Asp385 390
395 400 Phe Asp Asp Gly Val Phe Lys Ala Leu Leu Lys
Asp Ser Asn Pro Ala 405 410
415 Gly Ile Ile Leu Met Tyr Pro Met Asn Lys Asp Arg Trp Asp Asp Arg
420 425 430 Met Thr Ala
Met Thr Pro Ala Thr Asp Asp Asp Asp Met Phe Tyr Ala 435
440 445 Val Ser Phe Leu Trp Ser Ala Leu
Ser Ala Asp Asp Val Pro Gln Leu 450 455
460 Glu Arg Trp Asn Lys Ala Val Leu Asp Phe Cys Asp Arg
Ser Gly Ile465 470 475
480 Glu Cys Lys Gln Tyr Leu Pro His Tyr Thr Ser Gln Asp Gly Trp Arg
485 490 495 Arg His Phe Gly
Ala Lys Trp Ser Arg Ile Ala Glu Leu Lys Ala Arg 500
505 510 Tyr Asp Pro Arg Ala Leu Leu Ser Pro
Gly Gln Arg Ile Phe Pro Val 515 520
525 Pro Val Glu Ala Ser Gly Ile Ala Ser Ala 530
535 76177DNAZea maysmisc_feature(0)...(0)Genomic
sequence for ZmCkx4 7ctttatgttg tagccaagga aagtatactg ttaagatcag
aatgaacctt ataggagttg 60tatgggcata aagccagcaa gtatagccaa aggtacacaa
ggctaatata gtcaagttgt 120tgatgtgtga gacgttcaag gaagtgaact attggaggag
tcgactaaaa gtacgattaa 180taaggtagac atgatggtaa aatctttgat ctagaattta
agtggtatgg atgcgagggt 240gagaatggca agcacaactt caaatatagg gtgatgctta
tgcttggctg agccatttca 300ttcatgagca taggaacatg agacatggtg ggatatggat
acttgcacaa aaaaaggaat 360taagtttatg atattcacct cccagtcagt ttgcatggta
aaaaaattcc tatcaatttg 420gttctcaact agggcctaaa attctcaaaa tatctgttgg
ggaccattat cgtcgacgat 480cctcagaatc tgttattacc aaattaaaag gtgtgtttca
ggtactgtgc aaagcagcag 540cgaagctatc cttcgtcaaa agtggctcaa tgaaccaggt
ggagaagcta tggagcttcg 600tctgcgtaga gcgtgccgga ggaggaagct ttggctctga
atgcatcgac ttacgaagca 660tgggagaaga agactcagaa ggcttgtcca gcgtgggaat
aaaaaggaga aaatacaatt 720ttgcccttgt gggatttgta aatcatgtgc aaggctcatg
gatatgtttg taattttata 780tgatatgttt gtaaatcatg gatatgtttt gtaaatcagg
tggactagag gagagggagg 840gtggacatag tgacttgcat cttgatcatg gtagagtggt
catggtagag ggaaaggggt 900aggtcaattc tggagtgcgg ccacggtggc ttgagtgtcg
gccacggtag gggaaagggg 960tagcccaatt ctagggccgg catcggagaa ggccgacatg
tgcacgtcag gaggtagtgt 1020tagaggtttg aacggaaaaa attgaacatg ttagtatgat
gagttgtgta attgctggga 1080attgtggata atttccactt aactacggcc ctgtttattt
acccctagat tataaaatcc 1140aacttaaaaa agttgagatg taaacaaaca acacatatta
ttaggtggat tatgttatct 1200agaaatctgg atgataataa tttataagtc ggttaatagg
tgtttacata atcgataagc 1260tggattatat aatcctggaa cacggctttc gcgagagcgt
attaaaacag gattccgtga 1320agcacactat ctgaggagct ccaccaaaag ctgaatctag
cccgcactct tttttggagg 1380attcaaattt ggtgtcactg gagcattcgg cattttgttt
catggcgtga agctattttt 1440actaattaca gaagctgttt caaatagacc tttaaatgat
ggctgagtat aaaaggaggc 1500aattttttta tctcgccgat ggagccaggt cgcgtcgcgc
cgcggccgtg ctgcgctctc 1560gacgcgatct agcggcgatg tgcacagtac agttttgcca
tgccattggt taagcctgca 1620tacaacacac cagcgtactg ccctgcacaa gatctcctcg
gctcggcctc tcctgatgga 1680acgttcagct tgaacagcgg agcgtggggg catcccgggg
atgggcgccg cggccgagaa 1740attttgcaac ctggcaaatc tgccctgtcg catactacca
tccacctcca ggcgccaaga 1800acgcctccga gtttcaggct tgcagctcag ctctgtgttg
aattggaacg ggcggagttt 1860ctgggttcca gacttccagt acaaggcgat caattggtag
ggcgaattac ttgcaggccc 1920agatgcatgg cccatctatc tggttctcta tcggttgctt
ttacttgcac aatagtggca 1980gacaaactac aagtcagatc cgatcctatc catccatcca
tctcgcagcg cgatgcaaat 2040atgcaatcgt ctgtggaact cgaaaaaaaa cagaggtccg
gcctcgcacg aggttaaggg 2100aaaaaaaacg aagcgtttgg aactttggtt ggcattcgca
gcatgctgtg ctgccaccgt 2160atgtttttat ttttgctttg tttgtcttct ttgagaaacg
tgagggagcc gcgtgtccgc 2220tcgttataaa acccccccgg cgacccaaac taccacgagc
tcaagcctca agcctcaagc 2280ctcaagcaag cagagcgccg tgacatcacg aaacaaacat
atagagctag ctgctctgcc 2340tctgcttcac caatcacctg cttggccgcg cggaggggag
ggtttccccc tttgacacag 2400ctgagctccc ctccatcagc agccagctcc tcgtcgcaaa
gcaagaagat gatgctcgcg 2460tacatggacc gcgcgacggc ggccgccgag ccagaggacg
ccggccgcga gcccgccacc 2520atggcgggcg ggtgcgcggc ggcggcgacg gatttcggcg
ggctggggag cgccatgccc 2580gcggccgtgg tccgcccggc gagcgcggac gacgtggcca
gcgccatccg cgcggcggcg 2640ctgacgccgc acctcaccgt ggccgcccgc gggaacgggc
actcggtggc cggccaggcc 2700atggccgagg gcgggctggt cctcgacatg cgctcgctcg
cggcgccgtc ccggcgcgcg 2760cagatgcagc tcgtcgtgca gtgccccgac ggcggcggcg
gccgccgctg cttcgccgac 2820gtccccggcg gcgcgctctg ggaggaggtg ctccactggg
ccgtcgacaa ccacgggctc 2880gccccggcgt cctggacgga ctacctccgc ctcaccgtgg
gcggcacgct ctccaatggc 2940ggcgtcagcg gccagtcctt ccgctacggg ccccaggtgt
ccaacgtggc cgagctcgag 3000gtggtcaccg gcgacggcga gcgccgcgtc tgctcgccct
cctcccaccc ggacctcttc 3060ttcgccgtgc tcggcgggct cggccagttt ggcgtcatca
cgcgcgcccg catcccgctc 3120cacagggcgc ccaaggcggt gagcgcgcgg acatcggggg
cgaaagctaa agcttgcttt 3180ttgcttgggc actactaact gactgacgtt gccattcagg
tgcggtggac gcgcgtggtg 3240tacgcgagca tcgcggacta cacggcggac gcggagtggc
tggtgacgcg gccccccgac 3300gcggcgttcg actacgtgga gggcttcgcg ttcgtgaaca
gcgacgaccc cgtgaacggc 3360tggccgtccg tgcccatccc cggcggcgcc cgcttcgacc
cgtccctcct ccccgccggc 3420gccggccccg tcctctactg cctggaggtg gccctgtacc
agtacgcgca ccggcccgac 3480gacgacgacg aggaggacca ggtaggtagc agtaattgcc
aacctctccc cccgcttggc 3540gcattcccgt acttgacccc ctcgcccgct ctggcgtgta
cttttccgcg ggcagggcat 3600gtctgactcg cctcgtcgtg tatctcccgc tggattcggt
gacgggggtg ctgcgtcctg 3660ccaaaccaaa ccaccctaga ctagacagac ccccaggggc
aggggtcgcg ccattggccg 3720cacgcgggga ccggcgccag tgagtgcgcc gcgccgcacg
gccgcgcccc gatctcgctc 3780gctcgctcgc tggtgatcga atcggcgcgt acaatgcggc
atggccccga gccccacacc 3840cgcagtggcc gtgacgcgat tgcgctgcct ccggtccggc
ccatgaccca gcggatcgcg 3900tcgcgtcttt tggcaacgcc cgcgtcatca tatcgcgctc
tttgtcgtcc ccacggagca 3960cagcgcagcg cagcgcagcg cagccaacct tttctccgcc
acgcacgctt cggcggcatt 4020cattatttgg attttgttcc taccggtcga tccgcgtccg
tccgtgcact gcaggcggct 4080accgtcatgc tgaccaaccc attgccattg gttttgtttc
ttctctctct ctccctctcg 4140ttggttatgg ttcgtgcgtg cctgcaggcg gcggtgaccg
tgagccggat gatggcgccg 4200ctcaagcacg tgcggggcct ggagttcgcg gcggacgtcg
ggtacgtgga cttcctgtcc 4260cgcgtgaacc gggtggagga ggaggcccgg cgcaacggca
gctgggacgc gccgcacccg 4320tggctcaacc tcttcgtctc cgcgcgcgac atcgccgact
tcgaccgcgc cgtcatcaag 4380ggcatgctcg ccgacggcat cgacgggccc atgctcgtct
accctatgct caagagcaag 4440tgagttgccc tccgctccgc tccttcgcac tgcgtgcagt
agtacagtac aggagtggct 4500gagtggtggt actgccattc agtgtgcagt tgccgtttgc
ggcccgccaa gctagctagg 4560ggccgggacg catgtgagcc gccctgcctt ctctctgctc
gtcgtgtcac tgacgcctgg 4620tcctccggga cagttgctga gccggcccgt acgtacctgt
aagacgacgg tcccgagcct 4680ccaccgccgc ttctgttttg gatttagccg tgtcacacag
atcttacgga ggaggaggag 4740tactatgatt gacaaattat tgcttcgccc gacccgaggc
tagcgcacag tccatgtcat 4800gtgggcctgg ctgtgtggtt tccgtcctga tgctgatgcc
tgaagggaac tgcgtgcgtg 4860tgcgtgcgtg caggtgggac cccaacacgt cggtggcgct
gccggagggc gaggtcttct 4920acctggtggc gctgctgcgg ttctgccgga gcggcgggcc
ggcggtggac gagctggtgg 4980cgcagaacgg cgccatcctc cgcgcctgcc gcgccaacgg
ctacgactac aaggcctact 5040tcccgagcta ccgcggcgag gccgactggg cgcgccactt
cggcgccgcc aggtggaggc 5100gcttcgtgga ccgcaaggcc cggtacgacc cgctggcgat
cctcgcgccg ggccagaaga 5160tcttccctcg ggtcccggcg tccgtcgccg tgtagagcaa
ggggggagga ccagccagct 5220gccagccaag acaggaggag gaggagggga ggctgatgga
tcgccgctgc tgttgccggt 5280aatgatggcg attacgctgc tgatcctggt gatgatgatg
gacgatcgag gaagccgcag 5340ggccgggcaa tgatggcgat agggccaccg ttaggtgtgc
atccgggggc acaaattaaa 5400gggattgctg tgtggagatc tgcacgagtt tttgctccat
gcatgcttgc cgttcgtgtc 5460cgcgtgtccc tctccccctt gttattattc cttcgcccgc
cgaggccgag cgagcgggtg 5520gtggcgacgc tggatttgtc tgctctgctc tgctccgccg
ccgtggccac cccggtggcg 5580tgcgcccgca agctgttcct tccgcgcgct tctgttccgt
tcggttcccc cgtggtagct 5640tccccccctc gccgtcctgg tccccccgcc cgcccggcac
cccacgtggc acaccagccc 5700gatccaaacg ccgcgaccgc gacgcgcggg gccgttggtt
cgcgttcccg ttccgtagca 5760gcttgcccgc agcacacgac gaccgcgaac aaagcgcggc
caaaaccgac gggtctcgcc 5820gccgccgccg cggacgcgcc cacgggacag gaggaatatc
actctggggc catccgcgcg 5880ggaccataga actggtcggg tcgatgtcga tcgatatcgg
cactctgtgc tggctggcga 5940cgcggaccga gcggcaggga cgtgacggtt gctgccgccc
gagcgcgacg gcgaccgtcc 6000ttcgtctctg gggcggggcg gcgttttcgt ttggaaaatt
tgtggacttc tacttgtata 6060taaaaaaaca cgatcggcgc acgtatacaa ccagtcttcc
tttccctgtc gtgcccagtc 6120gcattccgtg atgcgagccg gatcgcgacg gaagcggctc
aacgagcgtc gtccctg 617781816DNAZea maysmisc_feature(0)...(0)cDNA for
ZmCkx4 8atg atg ctc gcg tac atg gac cgc gcg acg gcg gcc gcc gag cca gag
48Met Met Leu Ala Tyr Met Asp Arg Ala Thr Ala Ala Ala Glu Pro Glu1
5 10 15gac gcc ggc cgc gag
ccc gcc acc atg gcg ggc ggg tgc gcg gcg gcg 96Asp Ala Gly Arg Glu
Pro Ala Thr Met Ala Gly Gly Cys Ala Ala Ala 20
25 30gcg acg gat ttc ggc ggg ctg ggg agc gcc atg ccc
gcg gcc gtg gtc 144Ala Thr Asp Phe Gly Gly Leu Gly Ser Ala Met Pro
Ala Ala Val Val 35 40 45cgc ccg
gcg agc gcg gac gac gtg gcc agc gcc atc cgc gcg gcg gcg 192Arg Pro
Ala Ser Ala Asp Asp Val Ala Ser Ala Ile Arg Ala Ala Ala 50
55 60ctg acg ccg cac ctc acc gtg gcc gcc cgc ggg
aac ggg cac tcg gtg 240Leu Thr Pro His Leu Thr Val Ala Ala Arg Gly
Asn Gly His Ser Val65 70 75
80gcc ggc cag gcc atg gcc gag ggc ggg ctg gtc ctc gac atg cgc tcg
288Ala Gly Gln Ala Met Ala Glu Gly Gly Leu Val Leu Asp Met Arg Ser
85 90 95ctc gcg gcg ccg tcc
cgg cgc gcg cag atg cag ctc gtc gtg cag tgc 336Leu Ala Ala Pro Ser
Arg Arg Ala Gln Met Gln Leu Val Val Gln Cys 100
105 110ccc gac ggc ggc ggc ggc cgc cgc tgc ttc gcc gac
gtc ccc ggc ggc 384Pro Asp Gly Gly Gly Gly Arg Arg Cys Phe Ala Asp
Val Pro Gly Gly 115 120 125gcg ctc
tgg gag gag gtg ctc cac tgg gcc gtc gac aac cac ggg ctc 432Ala Leu
Trp Glu Glu Val Leu His Trp Ala Val Asp Asn His Gly Leu 130
135 140gcc ccg gcg tcc tgg acg gac tac ctc cgc ctc
acc gtg ggc ggc acg 480Ala Pro Ala Ser Trp Thr Asp Tyr Leu Arg Leu
Thr Val Gly Gly Thr145 150 155
160ctc tcc aat ggc ggc gtc agc ggc cag tcc ttc cgc tac ggg ccc cag
528Leu Ser Asn Gly Gly Val Ser Gly Gln Ser Phe Arg Tyr Gly Pro Gln
165 170 175gtg tcc aac gtg gcc
gag ctc gag gtg gtc acc ggc gac ggc gag cgc 576Val Ser Asn Val Ala
Glu Leu Glu Val Val Thr Gly Asp Gly Glu Arg 180
185 190cgc gtc tgc tcg ccc tcc tcc cac ccg gac ctc ttc
ttc gcc gtg ctc 624Arg Val Cys Ser Pro Ser Ser His Pro Asp Leu Phe
Phe Ala Val Leu 195 200 205ggc ggg
ctc ggc cag ttt ggc gtc atc acg cgc gcc cgc atc ccg ctc 672Gly Gly
Leu Gly Gln Phe Gly Val Ile Thr Arg Ala Arg Ile Pro Leu 210
215 220cac agg gcg ccc aag gcg gtg cgg tgg acg cgc
gtg gtg tac gcg agc 720His Arg Ala Pro Lys Ala Val Arg Trp Thr Arg
Val Val Tyr Ala Ser225 230 235
240atc gcg gac tac acg gcg gac gcg gag tgg ctg gtg acg cgg ccc ccc
768Ile Ala Asp Tyr Thr Ala Asp Ala Glu Trp Leu Val Thr Arg Pro Pro
245 250 255gac gcg gcg ttc gac
tac gtg gag ggc ttc gcg ttc gtg aac agc gac 816Asp Ala Ala Phe Asp
Tyr Val Glu Gly Phe Ala Phe Val Asn Ser Asp 260
265 270gac ccc gtg aac ggc tgg ccg tcc gtg ccc atc ccc
ggc ggc gcc cgc 864Asp Pro Val Asn Gly Trp Pro Ser Val Pro Ile Pro
Gly Gly Ala Arg 275 280 285ttc gac
ccg tcc ctc ctc ccc gcc ggc gcc ggc ccc gtc ctc tac tgc 912Phe Asp
Pro Ser Leu Leu Pro Ala Gly Ala Gly Pro Val Leu Tyr Cys 290
295 300ctg gag gtg gcc ctg tac cag tac gcg cac cgg
ccc gac gac gac gac 960Leu Glu Val Ala Leu Tyr Gln Tyr Ala His Arg
Pro Asp Asp Asp Asp305 310 315
320gag gag gac cag gcg gcg gtg acc gtg agc cgg atg atg gcg ccg ctc
1008Glu Glu Asp Gln Ala Ala Val Thr Val Ser Arg Met Met Ala Pro Leu
325 330 335aag cac gtg cgg ggc
ctg gag ttc gcg gcg gac gtc ggg tac gtg gac 1056Lys His Val Arg Gly
Leu Glu Phe Ala Ala Asp Val Gly Tyr Val Asp 340
345 350ttc ctg tcc cgc gtg aac cgg gtg gag gag gag gcc
cgg cgc aac ggc 1104Phe Leu Ser Arg Val Asn Arg Val Glu Glu Glu Ala
Arg Arg Asn Gly 355 360 365agc tgg
gac gcg ccg cac ccg tgg ctc aac ctc ttc gtc tcc gcg cgc 1152Ser Trp
Asp Ala Pro His Pro Trp Leu Asn Leu Phe Val Ser Ala Arg 370
375 380gac atc gcc gac ttc gac cgc gcc gtc atc aag
ggc atg ctc gcc gac 1200Asp Ile Ala Asp Phe Asp Arg Ala Val Ile Lys
Gly Met Leu Ala Asp385 390 395
400ggc atc gac ggg ccc atg ctc gtc tac cct atg ctc aag agc aag tgg
1248Gly Ile Asp Gly Pro Met Leu Val Tyr Pro Met Leu Lys Ser Lys Trp
405 410 415gac ccc aac acg tcg
gtg gcg ctg ccg gag ggc gag gtc ttc tac ctg 1296Asp Pro Asn Thr Ser
Val Ala Leu Pro Glu Gly Glu Val Phe Tyr Leu 420
425 430gtg gcg ctg ctg cgg ttc tgc cgg agc ggc ggg ccg
gcg gtg gac gag 1344Val Ala Leu Leu Arg Phe Cys Arg Ser Gly Gly Pro
Ala Val Asp Glu 435 440 445ctg gtg
gcg cag aac ggc gcc atc ctc cgc gcc tgc cgc gcc aac ggc 1392Leu Val
Ala Gln Asn Gly Ala Ile Leu Arg Ala Cys Arg Ala Asn Gly 450
455 460tac gac tac aag gcc tac ttc ccg agc tac cgc
ggc gag gcc gac tgg 1440Tyr Asp Tyr Lys Ala Tyr Phe Pro Ser Tyr Arg
Gly Glu Ala Asp Trp465 470 475
480gcg cgc cac ttc ggc gcc gcc agg tgg agg cgc ttc gtg gac cgc aag
1488Ala Arg His Phe Gly Ala Ala Arg Trp Arg Arg Phe Val Asp Arg Lys
485 490 495gcc cgg tac gac ccg
ctg gcg atc ctc gcg ccg ggc cag aag atc ttc 1536Ala Arg Tyr Asp Pro
Leu Ala Ile Leu Ala Pro Gly Gln Lys Ile Phe 500
505 510cct cgg gtc ccg gcg tcc gtc gcc gtg tag
agcaaggggg gaggaccagc 1586Pro Arg Val Pro Ala Ser Val Ala Val
515 520cagctgccag ccaagacagg aggaggagga ggaggggagg
ctgatggatc gccgctgctg 1646ttgccggtaa tgatggcgat tacgctgctg atcctggtga
tgatgatgga cgatcgagga 1706agccgcaggg ccgggcaatg atggcgatag ggccaccgtt
aggtgtgcat ccgggggcgc 1766aaattaaagg gattgctgtg tggagatctg cacgagtttt
tgctccatgc 18169521PRTZea mays 9Met Met Leu Ala Tyr Met Asp
Arg Ala Thr Ala Ala Ala Glu Pro Glu1 5 10
15 Asp Ala Gly Arg Glu Pro Ala Thr Met Ala Gly Gly
Cys Ala Ala Ala 20 25 30
Ala Thr Asp Phe Gly Gly Leu Gly Ser Ala Met Pro Ala Ala Val Val
35 40 45 Arg Pro Ala Ser
Ala Asp Asp Val Ala Ser Ala Ile Arg Ala Ala Ala 50 55
60 Leu Thr Pro His Leu Thr Val Ala Ala
Arg Gly Asn Gly His Ser Val65 70 75
80 Ala Gly Gln Ala Met Ala Glu Gly Gly Leu Val Leu Asp Met
Arg Ser 85 90 95
Leu Ala Ala Pro Ser Arg Arg Ala Gln Met Gln Leu Val Val Gln Cys
100 105 110 Pro Asp Gly Gly Gly
Gly Arg Arg Cys Phe Ala Asp Val Pro Gly Gly 115
120 125 Ala Leu Trp Glu Glu Val Leu His Trp
Ala Val Asp Asn His Gly Leu 130 135
140 Ala Pro Ala Ser Trp Thr Asp Tyr Leu Arg Leu Thr Val
Gly Gly Thr145 150 155
160 Leu Ser Asn Gly Gly Val Ser Gly Gln Ser Phe Arg Tyr Gly Pro Gln
165 170 175 Val Ser Asn Val
Ala Glu Leu Glu Val Val Thr Gly Asp Gly Glu Arg 180
185 190 Arg Val Cys Ser Pro Ser Ser His Pro
Asp Leu Phe Phe Ala Val Leu 195 200
205 Gly Gly Leu Gly Gln Phe Gly Val Ile Thr Arg Ala Arg Ile
Pro Leu 210 215 220
His Arg Ala Pro Lys Ala Val Arg Trp Thr Arg Val Val Tyr Ala Ser225
230 235 240 Ile Ala Asp Tyr Thr
Ala Asp Ala Glu Trp Leu Val Thr Arg Pro Pro 245
250 255 Asp Ala Ala Phe Asp Tyr Val Glu Gly Phe
Ala Phe Val Asn Ser Asp 260 265
270 Asp Pro Val Asn Gly Trp Pro Ser Val Pro Ile Pro Gly Gly Ala
Arg 275 280 285 Phe
Asp Pro Ser Leu Leu Pro Ala Gly Ala Gly Pro Val Leu Tyr Cys 290
295 300 Leu Glu Val Ala Leu Tyr
Gln Tyr Ala His Arg Pro Asp Asp Asp Asp305 310
315 320 Glu Glu Asp Gln Ala Ala Val Thr Val Ser Arg
Met Met Ala Pro Leu 325 330
335 Lys His Val Arg Gly Leu Glu Phe Ala Ala Asp Val Gly Tyr Val Asp
340 345 350 Phe Leu Ser
Arg Val Asn Arg Val Glu Glu Glu Ala Arg Arg Asn Gly 355
360 365 Ser Trp Asp Ala Pro His Pro Trp
Leu Asn Leu Phe Val Ser Ala Arg 370 375
380 Asp Ile Ala Asp Phe Asp Arg Ala Val Ile Lys Gly Met
Leu Ala Asp385 390 395
400 Gly Ile Asp Gly Pro Met Leu Val Tyr Pro Met Leu Lys Ser Lys Trp
405 410 415 Asp Pro Asn Thr
Ser Val Ala Leu Pro Glu Gly Glu Val Phe Tyr Leu 420
425 430 Val Ala Leu Leu Arg Phe Cys Arg Ser
Gly Gly Pro Ala Val Asp Glu 435 440
445 Leu Val Ala Gln Asn Gly Ala Ile Leu Arg Ala Cys Arg Ala
Asn Gly 450 455 460
Tyr Asp Tyr Lys Ala Tyr Phe Pro Ser Tyr Arg Gly Glu Ala Asp Trp465
470 475 480 Ala Arg His Phe Gly
Ala Ala Arg Trp Arg Arg Phe Val Asp Arg Lys 485
490 495 Ala Arg Tyr Asp Pro Leu Ala Ile Leu Ala
Pro Gly Gln Lys Ile Phe 500 505
510 Pro Arg Val Pro Ala Ser Val Ala Val 515
520 105108DNAZea maysmisc_feature(0)...(0)Genomic sequence for
ZmCkx5 10atcccgaata aacaaatgaa gtaggtcctc agtcaccctt gccctgttag
ctgcaagaga 60gctcatggtt tccagccaca caatcagtcc atggctcctt cttcttggcc
taagtggtgg 120ccaatcattg tgggtgatcg agtcttgggc cctctgaaca gtattacaca
acagtaatcc 180tgcaaaagat ttggtatatc tagattctag agtgagcgcc gtgttgtgcc
cagctaggaa 240tgggttgtca agtgcaacag gaggaggacc caggatggtc aggtgtaata
ggctctcatt 300aaaagactgt tcagatggat tagagcaacg acggggaagc cgggaaaaaa
tggttggttc 360tgctttcctc tcgctccccg gccgggttca tatatgaatc tgagaacgat
attttttgct 420tcatttttca tttgctatat atttaaactg tttttttgtg tgtgtgtgtg
tgttcattga 480gctcaatact tgaggcttga tagggagagg agtgaggcag ctgatcacat
ggacctccat 540ctgaggacag ttcctcttcc gaaacagaaa ggagagtgca gggaccagcg
tggcctgtac 600agtattgtgt ttgccctttt cctttggcag ggacagagag cttcaggctt
gtcctcttta 660tgtatgctgc tcgcctgctt cagagtcaga gcttcccctt ctcacttctc
agagagagag 720agagagaaga gagagagagg agagccctcc acagctcccc tgtcctgccc
tcaggcattc 780tttgtcacag ggggcgaggg ctgaagatca tcacatggtg gccttttttg
ggtctgtggc 840ctttggtctt ttagtgcttc ttccttttac ctcctcatga catgaacccc
ctttttaaac 900ctccctcaaa atcaaatcac cctccttctc ctttaagagc cctcaacccc
ttcccctcat 960tttccttcat ccctcagcct ttgcacaaag ggcaagaata acgcagtatg
atcatctgat 1020catactcccg ccgccatcac aatcccacac gaacgtgaga caaaggtaac
agacgcaaga 1080agctagcagc tgcaggagat tgctcagccc atctccatgg aggttgccat
ggtcgtgagc 1140gcaagagcca gcctgctgat cctcgtcctc tccctctgct ctccgtacaa
attcatacag 1200agccccatgg acctgggccc cctgaacctg ctccccacca ccagcaccgc
ggccgcgtcc 1260agcgacttcg gcaggatact cttccgcgcc ccggccgcgg tgctgaggcc
ccagtcgccg 1320agggacatct ccatgctgct cagcttcctc tccggctcgc cctcgctgag
cagggtcacg 1380gtggcggcca ggggggcagg ccactccatc cacgggcagg cgcaggcccc
ggacggcatt 1440gtggtggaga cgcgctcctt gcccggcgag atggagttcc accacgtccg
cgggggaggc 1500gaagggcgtg cctcctacgc cgacgtgggc ggcggggttc tgtggatcga
gctcctggag 1560cggagcctga agcttgggct ggctcccagg tcctggaccg actacctcta
cctcactgtc 1620ggcgggacgc tgtccaatgc cggcatcagc gggcagacgt tcaagcacgg
gccacagatc 1680agcaacgtcc tccagctgga ggtagtcaca ggtgagacac acgcacgcat
gcatgcgtgc 1740atgcatggta catagatgaa acacaaagat cagatttttt tttctgcctc
tctttcttga 1800ccaacaaaca acctctctct ctctctctct ctctctctct ctctctataa
caaacaggac 1860gaggggagat tgtggaatgc tcacccagca aggaggccga cctgttcaat
gccgtcctgg 1920gaggcctagg ccagttcggc atcataacca gggccaggat cctgctgcag
gaggctccgg 1980agaaggtgac gtgggtgagg gccttctacg acgacttggg cgccttcacc
agggaccagg 2040agctgctggt gtcgattccg gattcggtgg actacgtgga agggttcatg
gtcctgaacg 2100agcggtccct ccacagctcc tccatcgcct tccccgcgag cgtggacttc
agcccggatt 2160tcggcaccag gagcagccct aggatctact actgcgtcga gttcgcggtc
caccaccacc 2220acggttacca gcagcagtct caggcggccg tggaggccat ctcgaggcgg
atgagccaca 2280tggcgtccca gctgtacagc gtggaggtgt cctacttgga cttcctgaac
cgggtcagga 2340tggaggaggt gagcctgcgg agcgccggga tgtgggagga ggtgcaccac
ccgtggctca 2400acatgttcgt gcccaaggcc ggggtcgctg gcttcaggga tctgctcatg
gacaacgtct 2460cgccggatag cttccagggc ctcatcctca tctacccact cctcagagac
aagtaagtac 2520cactcttcta ataataataa taataatgat gatgacacaa aagctatata
gtagtacatc 2580catgaagata cgcctacact ttcggctttt ctcaaagcaa agcttatctg
ctttattaat 2640cccggcctct tccggccgtc ctcacttcat attagcttac aagaagtcca
agttgggcaa 2700gcaagcattt ctttgcatta tccacagcaa gttgcctcct tgcgctgcct
acagtggtaa 2760cgacatgggt agggtttggt tttggagtaa tagcgggata tgaagccttt
ccgctcttaa 2820tttgttgttt ttagattgat tttatatagg acatagtgac acttaaaaaa
tatatgttca 2880aatattgaac cattttggtt tcagaaaatc tcttggaacg agccgttcta
gccgttctct 2940ctctagaacg gagtcgctcc atcctaacta gcttcacaat caaacattac
cttggatgac 3000gacgaagcgc cgagaaaagt ggtcactcat cgtgcgtgca ttagggatga
tagatccctt 3060ttgcttaatt agcatgttgg tttgtttttt ccctatgtcc agcaaaggcg
ctcgtgggac 3120ccacgccgtc gtatagaggc gaatatgggt tgttctatcg tgtgtttgta
ctagtggtcc 3180ctcggatagc atcacatctg cgtcatcatc agcattgtat gaatcagcta
aaactgtaga 3240tgagctcaat cagtcagtag catcaacctt gagagtggcg acagggaaat
tataaaacat 3300agtagtagct agctgatctg ctttggaatt agtcttcgtt tttcttcttt
tttcacctaa 3360gggctagttt aggaacacaa ttttctcaaa aaaaaaattg aactaattac
tcttaagaaa 3420atggaaattc ctagaaaaaa aatgaggttg ccaaactagg cttaaaagat
ttttttaaca 3480ggtgcaaaca acgtcgtaaa cttgtaatcg atagcacaag ttattcagtt
acaagcgtct 3540tgttatgtgt tcataccctc cgaataaatt tagacaagtt tacatggatg
caaaaggggt 3600ttaaatatgt gtatgtgcta gatcgcatcc cagtgtcaac tctgaactga
ttgagcttta 3660acaaaagatg aagtcgtcac ccaaagttcc acgttcaccg cactggacta
gtgtatatat 3720aatctacagg cccaacagag acctctgact tgcctcagga aaaaccacta
aaccaaaagt 3780ctctctctct cgagagagag tcagaatacg ctgaactgca gaatgtcagc
tacaccaccc 3840atacacaaat tcctgctacc gcttatttga aattcacagc gtctcgtcag
caagctctaa 3900aaaactgttt gttgctgtct tcatcctttc ttttttttta tttgatttga
ttgacgacag 3960gtgggacacc aacacgtcgg tcgtgatccc ggactccggg cccaccgcgg
acgacccggt 4020gatgtacgtg gtcggcatcc tcaggtccgc gaaccctggt ccagaagaag
acggtgacgg 4080ctgctcccac cgctgcctgc acgagctcct ccgcagccac cgccggatcg
ccgacgccgc 4140ggaggcgcgc ctcggcgcca agcagtacct gcctcaccac ccgaccccgg
cccgctggca 4200gcagcacctg ggccggcgct gggagcgctt cgcggaccgc aaggcccggt
tcgacccgct 4260gcgcatcctg gggcccggcc agggcatatt ccctcggacg gcccaggatg
ctgccgccgc 4320tgctgcgtac gggagctagc attttatata tatacggcat gaaacagtat
agattattaa 4380ttatcagctg actagctagg atgcgatctt ctttcttctt cttcttctct
cttcagtttc 4440ctctgcattt tgagggcatg tggggccggt tgttgggtta ttctgtgagg
ctctggcctc 4500cggggcactt tgagaggacc tgatggtggt ggtggtggtg gtgattggtg
attggtgaat 4560gtcactggga attttggaac ttttgtacag gttgatggag gaagcacagg
ctttaagttt 4620ataagggaat agacatagcg cttctattcg gtctctctat tccttgttcg
atatggccat 4680tgctagtgtc actattgtcc tcggtaattc cgtttctagg ctgataaatt
ccttccactt 4740tgtcagagcc atcttcttta ggagaagtgg tgacaacgtg ccagagccct
ttgggttagc 4800tcgagatcag aatgtagtcc atggatgagg ggccatgggt gctagattta
cagtgaattt 4860gatccgtccc caatgttccg ttagctaaaa tgcatgcggt gaagtcgtac
aagcatgcat 4920gatgcatgcc ctttttttct ggagtacccg tcattttgcc ttctgaaact
ccggaagccc 4980ttgcattgac cgtttgacac aattatgaag tgaagatgat aagtcgtgaa
tggattcatg 5040acagatcaac acgtggcacc tccgtacata caaacacgcg cccgaagttc
cctctaggaa 5100acttcaat
5108111629DNAZea maysmisc_feature(0)...(0)cDNA for ZmCkx5
11atg gag gtt gcc atg gtc gtg agc gca aga gcc agc ctg ctg atc ctc
48Met Glu Val Ala Met Val Val Ser Ala Arg Ala Ser Leu Leu Ile Leu1
5 10 15gtc ctc tcc ctc tgc tct
ccg tac aaa ttc ata cag agc ccc atg gac 96Val Leu Ser Leu Cys Ser
Pro Tyr Lys Phe Ile Gln Ser Pro Met Asp 20 25
30ctg ggc ccc ctg aac ctg ctc ccc acc acc agc acc gcg
gcc gcg tcc 144Leu Gly Pro Leu Asn Leu Leu Pro Thr Thr Ser Thr Ala
Ala Ala Ser 35 40 45agc gac ttc
ggc agg ata ctc ttc cgc gcc ccg gcc gcg gtg ctg agg 192Ser Asp Phe
Gly Arg Ile Leu Phe Arg Ala Pro Ala Ala Val Leu Arg 50
55 60ccc cag tcg ccg agg gac atc tcc atg ctg ctc agc
ttc ctc tcc ggc 240Pro Gln Ser Pro Arg Asp Ile Ser Met Leu Leu Ser
Phe Leu Ser Gly65 70 75
80tcg ccc tcg ctg agc agg gtc acg gtg gcg gcc agg ggg gca ggc cac
288Ser Pro Ser Leu Ser Arg Val Thr Val Ala Ala Arg Gly Ala Gly His
85 90 95tcc atc cac ggg cag gcg
cag gcc ccg gac ggc att gtg gtg gag acg 336Ser Ile His Gly Gln Ala
Gln Ala Pro Asp Gly Ile Val Val Glu Thr 100
105 110cgc tcc ttg ccc ggc gag atg gag ttc cac cac gtc
cgc ggg gga ggc 384Arg Ser Leu Pro Gly Glu Met Glu Phe His His Val
Arg Gly Gly Gly 115 120 125gaa ggg
cgt gcc tcc tac gcc gac gtg ggc ggc ggg gtt ctg tgg atc 432Glu Gly
Arg Ala Ser Tyr Ala Asp Val Gly Gly Gly Val Leu Trp Ile 130
135 140gag ctc ctg gag cgg agc ctg aag ctt ggg ctg
gct ccc agg tcc tgg 480Glu Leu Leu Glu Arg Ser Leu Lys Leu Gly Leu
Ala Pro Arg Ser Trp145 150 155
160acc gac tac ctc tac ctc act gtc ggc ggg acg ctg tcc aat gcc ggc
528Thr Asp Tyr Leu Tyr Leu Thr Val Gly Gly Thr Leu Ser Asn Ala Gly
165 170 175atc agc ggg cag acg
ttc aag cac ggg cca cag atc agc aac gtc ctc 576Ile Ser Gly Gln Thr
Phe Lys His Gly Pro Gln Ile Ser Asn Val Leu 180
185 190cag ctg gag gta gtc aca gga cga ggg gag att gtg
gaa tgc tca ccc 624Gln Leu Glu Val Val Thr Gly Arg Gly Glu Ile Val
Glu Cys Ser Pro 195 200 205agc aag
gag gcc gac ctg ttc aat gcc gtc ctg gga ggc cta ggc cag 672Ser Lys
Glu Ala Asp Leu Phe Asn Ala Val Leu Gly Gly Leu Gly Gln 210
215 220ttc ggc atc ata acc agg gcc agg atc ctg ctg
cag gag gct ccg gag 720Phe Gly Ile Ile Thr Arg Ala Arg Ile Leu Leu
Gln Glu Ala Pro Glu225 230 235
240aag gtg acg tgg gtg agg gcc ttc tac gac gac ttg ggc gcc ttc acc
768Lys Val Thr Trp Val Arg Ala Phe Tyr Asp Asp Leu Gly Ala Phe Thr
245 250 255agg gac cag gag ctg
ctg gtg tcg att ccg gat tcg gtg gac tac gtg 816Arg Asp Gln Glu Leu
Leu Val Ser Ile Pro Asp Ser Val Asp Tyr Val 260
265 270gaa ggg ttc atg gtc ctg aac gag cgg tcc ctc cac
agc tcc tcc atc 864Glu Gly Phe Met Val Leu Asn Glu Arg Ser Leu His
Ser Ser Ser Ile 275 280 285gcc ttc
ccc gcg agc gtg gac ttc agc ccg gat ttc ggc acc agg agc 912Ala Phe
Pro Ala Ser Val Asp Phe Ser Pro Asp Phe Gly Thr Arg Ser 290
295 300agc cct agg atc tac tac tgc gtc gag ttc gcg
gtc cac cac cac cac 960Ser Pro Arg Ile Tyr Tyr Cys Val Glu Phe Ala
Val His His His His305 310 315
320ggt tac cag cag cag tct cag gcg gcc gtg gag gcc atc tcg agg cgg
1008Gly Tyr Gln Gln Gln Ser Gln Ala Ala Val Glu Ala Ile Ser Arg Arg
325 330 335atg agc cac atg gcg
tcc cag ctg tac agc gtg gag gtg tcc tac ttg 1056Met Ser His Met Ala
Ser Gln Leu Tyr Ser Val Glu Val Ser Tyr Leu 340
345 350gac ttc ctg aac cgg gtc agg atg gag gag gtg agc
ctg cgg agc gcc 1104Asp Phe Leu Asn Arg Val Arg Met Glu Glu Val Ser
Leu Arg Ser Ala 355 360 365ggg atg
tgg gag gag gtg cac cac ccg tgg ctc aac atg ttc gtg ccc 1152Gly Met
Trp Glu Glu Val His His Pro Trp Leu Asn Met Phe Val Pro 370
375 380aag gcc ggg gtc gct ggc ttc agg gat ctg ctc
atg gac aac gtc tcg 1200Lys Ala Gly Val Ala Gly Phe Arg Asp Leu Leu
Met Asp Asn Val Ser385 390 395
400ccg gat agc ttc cag ggc ctc atc ctc atc tac cca ctc ctc aga gac
1248Pro Asp Ser Phe Gln Gly Leu Ile Leu Ile Tyr Pro Leu Leu Arg Asp
405 410 415aag tgg gac acc aac
acg tcg gtc gtg atc ccg gac tcc ggg ccc acc 1296Lys Trp Asp Thr Asn
Thr Ser Val Val Ile Pro Asp Ser Gly Pro Thr 420
425 430gcg gac gac ccg gtg atg tac gtg gtc ggc atc ctc
agg tcc gcg aac 1344Ala Asp Asp Pro Val Met Tyr Val Val Gly Ile Leu
Arg Ser Ala Asn 435 440 445cct ggt
cca gaa gaa gac ggt gac ggc tgc tcc cac cgc tgc ctg cac 1392Pro Gly
Pro Glu Glu Asp Gly Asp Gly Cys Ser His Arg Cys Leu His 450
455 460gag ctc ctc cgc agc cac cgc cgg atc gcc gac
gcc gcg gag gcg cgc 1440Glu Leu Leu Arg Ser His Arg Arg Ile Ala Asp
Ala Ala Glu Ala Arg465 470 475
480ctc ggc gcc aag cag tac ctg cct cac cac ccg acc ccg gcc cgc tgg
1488Leu Gly Ala Lys Gln Tyr Leu Pro His His Pro Thr Pro Ala Arg Trp
485 490 495cag cag cac ctg ggc
cgg cgc tgg gag cgc ttc gcg gac cgc aag gcc 1536Gln Gln His Leu Gly
Arg Arg Trp Glu Arg Phe Ala Asp Arg Lys Ala 500
505 510cgg ttc gac ccg ctg cgc atc ctg ggg ccc ggc cag
ggc ata ttc cct 1584Arg Phe Asp Pro Leu Arg Ile Leu Gly Pro Gly Gln
Gly Ile Phe Pro 515 520 525cgg acg
gcc cag gat gct gcc gcc gct gct gcg tac ggg agc tag 1629Arg Thr
Ala Gln Asp Ala Ala Ala Ala Ala Ala Tyr Gly Ser 530
535 54012542PRTZea mays 12Met Glu Val Ala Met Val Val Ser
Ala Arg Ala Ser Leu Leu Ile Leu1 5 10
15 Val Leu Ser Leu Cys Ser Pro Tyr Lys Phe Ile Gln Ser
Pro Met Asp 20 25 30
Leu Gly Pro Leu Asn Leu Leu Pro Thr Thr Ser Thr Ala Ala Ala Ser
35 40 45 Ser Asp Phe Gly
Arg Ile Leu Phe Arg Ala Pro Ala Ala Val Leu Arg 50 55
60 Pro Gln Ser Pro Arg Asp Ile Ser Met
Leu Leu Ser Phe Leu Ser Gly65 70 75
80 Ser Pro Ser Leu Ser Arg Val Thr Val Ala Ala Arg Gly Ala
Gly His 85 90 95
Ser Ile His Gly Gln Ala Gln Ala Pro Asp Gly Ile Val Val Glu Thr
100 105 110 Arg Ser Leu Pro Gly
Glu Met Glu Phe His His Val Arg Gly Gly Gly 115
120 125 Glu Gly Arg Ala Ser Tyr Ala Asp Val
Gly Gly Gly Val Leu Trp Ile 130 135
140 Glu Leu Leu Glu Arg Ser Leu Lys Leu Gly Leu Ala Pro
Arg Ser Trp145 150 155
160 Thr Asp Tyr Leu Tyr Leu Thr Val Gly Gly Thr Leu Ser Asn Ala Gly
165 170 175 Ile Ser Gly Gln
Thr Phe Lys His Gly Pro Gln Ile Ser Asn Val Leu 180
185 190 Gln Leu Glu Val Val Thr Gly Arg Gly
Glu Ile Val Glu Cys Ser Pro 195 200
205 Ser Lys Glu Ala Asp Leu Phe Asn Ala Val Leu Gly Gly Leu
Gly Gln 210 215 220
Phe Gly Ile Ile Thr Arg Ala Arg Ile Leu Leu Gln Glu Ala Pro Glu225
230 235 240 Lys Val Thr Trp Val
Arg Ala Phe Tyr Asp Asp Leu Gly Ala Phe Thr 245
250 255 Arg Asp Gln Glu Leu Leu Val Ser Ile Pro
Asp Ser Val Asp Tyr Val 260 265
270 Glu Gly Phe Met Val Leu Asn Glu Arg Ser Leu His Ser Ser Ser
Ile 275 280 285 Ala
Phe Pro Ala Ser Val Asp Phe Ser Pro Asp Phe Gly Thr Arg Ser 290
295 300 Ser Pro Arg Ile Tyr Tyr
Cys Val Glu Phe Ala Val His His His His305 310
315 320 Gly Tyr Gln Gln Gln Ser Gln Ala Ala Val Glu
Ala Ile Ser Arg Arg 325 330
335 Met Ser His Met Ala Ser Gln Leu Tyr Ser Val Glu Val Ser Tyr Leu
340 345 350 Asp Phe Leu
Asn Arg Val Arg Met Glu Glu Val Ser Leu Arg Ser Ala 355
360 365 Gly Met Trp Glu Glu Val His His
Pro Trp Leu Asn Met Phe Val Pro 370 375
380 Lys Ala Gly Val Ala Gly Phe Arg Asp Leu Leu Met Asp
Asn Val Ser385 390 395
400 Pro Asp Ser Phe Gln Gly Leu Ile Leu Ile Tyr Pro Leu Leu Arg Asp
405 410 415 Lys Trp Asp Thr
Asn Thr Ser Val Val Ile Pro Asp Ser Gly Pro Thr 420
425 430 Ala Asp Asp Pro Val Met Tyr Val Val
Gly Ile Leu Arg Ser Ala Asn 435 440
445 Pro Gly Pro Glu Glu Asp Gly Asp Gly Cys Ser His Arg Cys
Leu His 450 455 460
Glu Leu Leu Arg Ser His Arg Arg Ile Ala Asp Ala Ala Glu Ala Arg465
470 475 480 Leu Gly Ala Lys Gln
Tyr Leu Pro His His Pro Thr Pro Ala Arg Trp 485
490 495 Gln Gln His Leu Gly Arg Arg Trp Glu Arg
Phe Ala Asp Arg Lys Ala 500 505
510 Arg Phe Asp Pro Leu Arg Ile Leu Gly Pro Gly Gln Gly Ile Phe
Pro 515 520 525 Arg
Thr Ala Gln Asp Ala Ala Ala Ala Ala Ala Tyr Gly Ser 530
535 540 133003DNAZea
maysmisc_feature(0)...(0)ZmCkx2 promoter 13ctgccatcct catgcagatg
agacggagag aagatgagaa aagtacaaga tcccagaagc 60aagcagcagg atggggccat
cccccccccc ccccactggg ccccacgggc cgaaagccac 120cggcgaaaat gtccagaagg
ccacgtgggg catgggtccc cggagtccac ttccgcgcga 180tctcgaggcc gggccgcacc
ggcaatcgct ctccggccac ctccctgctt cctcaggtcc 240ggtctcccat agtccaatgc
atgcatgcac gagcatcccc tcagaacgct ggcagtgagt 300gtcttgctcg cacatcagct
tggccagtca gtgcgagaac acagcagcaa caacaacaac 360aacacctgtg cacaatggcg
tctatcattg gtaccatctc aatcggctga cttgtctata 420actactgtta acggaggtcc
cttgtgcatc atgcagtttt agaagagcac ctcgatcgca 480agcgcctcat tattatcatc
attctcttaa actggtcaga aaactgacca tcagctaaag 540tgatactgac atactgtatc
tttgtagata attaaatgga gaaaaatctc cttctgttcc 600gtctggccgt taaatgccga
atccatgcat atataaatct gtacgtaggc tcaaagcaca 660gtgtgcattt tggctttcca
gctagcatac atacatgtga ctgctgacga tgaattgtgt 720ggaccacatt ggcacaacgg
tgcattgcaa cggacgggcg ccgtcaaggt caaacgcata 780aaaggctgtc atttggcaac
acaatgaatc agtggcgcca cgccatccgt ccacgatcca 840ccgttcttgg tgtagtggtt
ggtcccagcg cttgaaggcc aggccgaggc cgtgttctgg 900aaggtggcct gtggtgagca
ctaaacatgt gtgtgctttt gcctttccaa gccagagggc 960cggtctctta atatacataa
catacacacc actttttcat tttgttcatt attacggtct 1020aatgcaaaca aagccatttg
cagaatgtgc tacatagcag gtatgtttct ctttttttcc 1080ctgtaaaatt tgtagactta
tcacaagaat aagtttaacc attactagaa tagttcctca 1140catgtttgtt taccatcggg
gcgggaacag cttgcattgc aaaagctgcg caagtattag 1200ggccctctag atttttttaa
tagtagtagt atatataata tataggtgtt actatttgag 1260ttgttaggcc atctgcggca
gattttctat gacatccctt atttcaaact ttattttgca 1320aacagttgtc atatacccta
ttttaggcga atcactgaag acaggtaagt tttggcacgg 1380atgaggtgga gagtggacaa
gaatctccgt tgtggagtct gcctaccagt accaggcaaa 1440gtaatgcatg cgcgcggaca
ggatggacgg tcgaagtggc ctccctgcct ccaccccgac 1500gacgacgcat gggctccgtc
cccttcgctt gcttcctgct ccagctagct ccatcgccta 1560gtgctccgct ccgccgcaca
ggaacggaac ggaacggacc gaaccacttg gtcgcatccc 1620gatgcgttgc cgtctgccgg
tgtccatcgt gtcggtttca cctctgcact agcataaatt 1680ccttgacacc aacagcgagc
gacatcatcg gctcagccct acaagtcacg agtgttctga 1740ctgaccagct agcaatagca
atctgctgct ctgcttgact tgctcggacg atccgccgct 1800gcttgcgttc ggctccagta
ggctatcctc cgcgacgtcg tcgatctgga ctccatggcg 1860tccacacaga atcgacacga
gcttggtgtg ccgcgtacgc atgtgtgcgt atgtatgcct 1920cgtcttccac atgcaaacat
acgcagagga aggggaaagg cggcagcaaa cgcgacggtc 1980caagtcgtac cacagaagtg
gtcgcgcatg tgtgcccaag ttgccatcac ccggatgcta 2040ttagatttcc agaaactaac
ttgtgaggac ccctggtgtc tgctagctgc tctccaactc 2100caacctgtca atcaattccc
agacggacaa gctgagctca cagctcaagc tcaacaacga 2160tggccggccg ggtcaccatg
gaactgatcc tctacagtac aggcatggga aaatggagga 2220ggagagcagg gcagtgaggc
cacagaatca gaggctgatt agtgttggtg agctccaatc 2280caacagcata tgaccagcga
gcagaacata gggatgtcct gtgggcttgc ccagggacag 2340acgcatgcaa gccatgtgac
tgtccggaga gagagccggt gatactggaa cagaggatcc 2400gatcctgccc cccttctttt
gcctctccct ctctcacaca cacagtctca cctatatgtg 2460gctatgtcgt ctccattagg
ctgttaacta gccaacacat gttcccccgt tgcttaagac 2520agcagctaca aagcgagaac
atcatgctct aaaaagaaac ttccgcaatg caccactagc 2580acatgtctgc gcctcaattc
gcaaccggca agcaagcaag ccggcaagca gacagtcgcc 2640atacggtttt taccaaacag
ctagcgccca cagctgacta gctgaccacc gcaccaccca 2700cactcctcct cgcgagtcgc
gaggcaagcc gcaagctcct atatagagag gccccctccc 2760tccccctgca tggacagcca
ccgccttctt caaccctcct tccgtcttcc tcctctagtc 2820ttacctcgtt gcacctcaag
aaacttggcg cgcaaccagg aaaccccctc ttctctctct 2880ctctctctct ctctctctgc
cttctgattc caagctcccc aactgcccag caccaacctg 2940ccgaactccc ctcctttttg
ttggtttgtc gaattataaa ttgagcccgg ccggctgact 3000acc
3003142001DNAZea
maysN_region(0)...(0)ZmCkx3 promoter 14ccggggtgtg acaggagcat tgaagcatgc
atgctctgct cagcatataa ttaaagaaag 60aagcatcaaa atgcactgga gcagttgacc
aaaacttgca gctacgtcaa aatatatacg 120agggctggca tcaaggtgtg ctcagcccga
gccccgtcag gtaacttggt cttttgtttt 180ctggccttgc ggcttcatta aaggccgccg
gccgcgagcg aggcaaaaca gtgaagggga 240ggggaggtgc ccgccactaa cctctcggtc
ggatatatta gtattcaagc agttgacaaa 300tctgtgcgga tttgatttgg tctgaggaaa
atatatatat atatatatat agcccctcgt 360cgttcatgca ccctctcgca gcctgcaacc
ttacaatatt gttcttgcat ccggttttat 420ttatattttt attttttaaa aaaaaaatcc
atagtcctgc cgtcttgaag gatatgtttt 480tctttaccca tgcacggcgg agtttaaatt
tgcgctgacc cgactgctcg tgaacagaga 540caagtatgac agatatcgtt gagttccaaa
ttttaaaaaa aaaatcaata aaaaatttaa 600aacagaatgt tgacgaggaa aaaaaatatg
aaggtgcttg cacacctgtc actccatgcc 660ggacatcaac aaattaattg ttcaagtggt
gggagtcagc tgcttccagt ttaccttcct 720gcgccagcgg ttggtagaca ggattgttgc
cacgtggacg aaatctcctg ccgccagctg 780gttgatcacg gcaggcagtc acatgcttct
tgccaagatt accgcgggtt gtaatcatct 840gaaatatatt aacctgagca cgtgatagag
taaaaaaatt ggtcgactaa gggggtgttt 900ggtttctagg gactaatgtt tagtccctac
attttattcc attttagttc taaaattacc 960aaatatagaa actaaaactt tattttagtt
tctatattag caatttatag actaaaaaag 1020aataaaatga agggactaaa tattaatccc
tagaaaccaa acacccccta actttaggta 1080agttgtggca tgcattctct ggaacggcag
ttctagagag cacttgagat gtcaacaggt 1140gaagaattga agattggcca acacaggcgt
tcaaggagat tcaaccaccc atccacatac 1200cgcgcaaaca cttggggggc attcttgctg
ctgccacatt tggaagaagc gcagcaatgt 1260ggtgttcaga agaagcacag ctattttagc
tcttgataac tatctttttt tttgcataga 1320ttaatttatt tcttcgatat atactagctt
gtaaaaaaat gttttncaga tatatgtata 1380aaaatgtgta cctagtacct acgcatgtct
tagttcaaca tacttgatag ctgtagtttt 1440ctgaaaacct gttcaaatta acctttttcc
taccctgatg gtgaatagag agaaaagctt 1500tacctttgtc tgaataagaa aactaacaga
aagcttacat tttggccact ctacctgccc 1560gagtattttc taagcaagca aaggcgcatg
aaaattttct cggaatccat gaccttttac 1620gcgcantgnw aaayawwgwm mattgmtcmg
accaatgatc attttgatac tctccacaag 1680tcaacatctc aaaaaaacca caagatgggg
cccatcaaca taagttcacg agtgtgcctt 1740caggtacatt gttctttttt tttgttttgc
taaagtcaat cagctgcaaa atattcagaa 1800caatttcaat aacccgaaag gctgttgtgc
ctccatttgt caacgtttgc gaggccaaat 1860ggtacccccg ctataaatac catggaagtt
cttggcctct aggacacaca agcgatctct 1920cctcctatag tttctataac cccacaaagc
gtccaggtcc cgtagtcacc tccgattgca 1980ttgcgttgcc gcaagacaag c
2001152448DNAZea
maysmisc_feature(0)...(0)ZmCkx4 promoter 15ctttatgttg tagccaagga
aagtatactg ttaagatcag aatgaacctt ataggagttg 60tatgggcata aagccagcaa
gtatagccaa aggtacacaa ggctaatata gtcaagttgt 120tgatgtgtga gacgttcaag
gaagtgaact attggaggag tcgactaaaa gtacgattaa 180taaggtagac atgatggtaa
aatctttgat ctagaattta agtggtatgg atgcgagggt 240gagaatggca agcacaactt
caaatatagg gtgatgctta tgcttggctg agccatttca 300ttcatgagca taggaacatg
agacatggtg ggatatggat acttgcacaa aaaaaggaat 360taagtttatg atattcacct
cccagtcagt ttgcatggta aaaaaattcc tatcaatttg 420gttctcaact agggcctaaa
attctcaaaa tatctgttgg ggaccattat cgtcgacgat 480cctcagaatc tgttattacc
aaattaaaag gtgtgtttca ggtactgtgc aaagcagcag 540cgaagctatc cttcgtcaaa
agtggctcaa tgaaccaggt ggagaagcta tggagcttcg 600tctgcgtaga gcgtgccgga
ggaggaagct ttggctctga atgcatcgac ttacgaagca 660tgggagaaga agactcagaa
ggcttgtcca gcgtgggaat aaaaaggaga aaatacaatt 720ttgcccttgt gggatttgta
aatcatgtgc aaggctcatg gatatgtttg taattttata 780tgatatgttt gtaaatcatg
gatatgtttt gtaaatcagg tggactagag gagagggagg 840gtggacatag tgacttgcat
cttgatcatg gtagagtggt catggtagag ggaaaggggt 900aggtcaattc tggagtgcgg
ccacggtggc ttgagtgtcg gccacggtag gggaaagggg 960tagcccaatt ctagggccgg
catcggagaa ggccgacatg tgcacgtcag gaggtagtgt 1020tagaggtttg aacggaaaaa
attgaacatg ttagtatgat gagttgtgta attgctggga 1080attgtggata atttccactt
aactacggcc ctgtttattt acccctagat tataaaatcc 1140aacttaaaaa agttgagatg
taaacaaaca acacatatta ttaggtggat tatgttatct 1200agaaatctgg atgataataa
tttataagtc ggttaatagg tgtttacata atcgataagc 1260tggattatat aatcctggaa
cacggctttc gcgagagcgt attaaaacag gattccgtga 1320agcacactat ctgaggagct
ccaccaaaag ctgaatctag cccgcactct tttttggagg 1380attcaaattt ggtgtcactg
gagcattcgg cattttgttt catggcgtga agctattttt 1440actaattaca gaagctgttt
caaatagacc tttaaatgat ggctgagtat aaaaggaggc 1500aattttttta tctcgccgat
ggagccaggt cgcgtcgcgc cgcggccgtg ctgcgctctc 1560gacgcgatct agcggcgatg
tgcacagtac agttttgcca tgccattggt taagcctgca 1620tacaacacac cagcgtactg
ccctgcacaa gatctcctcg gctcggcctc tcctgatgga 1680acgttcagct tgaacagcgg
agcgtggggg catcccgggg atgggcgccg cggccgagaa 1740attttgcaac ctggcaaatc
tgccctgtcg catactacca tccacctcca ggcgccaaga 1800acgcctccga gtttcaggct
tgcagctcag ctctgtgttg aattggaacg ggcggagttt 1860ctgggttcca gacttccagt
acaaggcgat caattggtag ggcgaattac ttgcaggccc 1920agatgcatgg cccatctatc
tggttctcta tcggttgctt ttacttgcac aatagtggca 1980gacaaactac aagtcagatc
cgatcctatc catccatcca tctcgcagcg cgatgcaaat 2040atgcaatcgt ctgtggaact
cgaaaaaaaa cagaggtccg gcctcgcacg aggttaaggg 2100aaaaaaaacg aagcgtttgg
aactttggtt ggcattcgca gcatgctgtg ctgccaccgt 2160atgtttttat ttttgctttg
tttgtcttct ttgagaaacg tgagggagcc gcgtgtccgc 2220tcgttataaa acccccccgg
cgacccaaac taccacgagc tcaagcctca agcctcaagc 2280ctcaagcaag cagagcgccg
tgacatcacg aaacaaacat atagagctag ctgctctgcc 2340tctgcttcac caatcacctg
cttggccgcg cggaggggag ggtttccccc tttgacacag 2400ctgagctccc ctccatcagc
agccagctcc tcgtcgcaaa gcaagaag 2448162346DNAZea
maysmisc_feature(0)...(0)ZmCkx5 promoter 16tacagatttg cgttcatcaa
tggcagcgcg ggatctcatg aggtcactgg gttcttgcaa 60gtggggagag aaagggagat
ctacgaaaga cttgttagtg ggccaccttt tccctctttc 120cccacaagga cgagatcgtg
gattagagta ggaaagtgat tccgcattgg tctcaaatct 180tggcgaaaga ttgcattgtg
tactctccac cactcgaccg gcaacgaggc attttgttat 240tgcacgatgc atcctttgca
catgagctag gcttgtgcct ttgagtattc agttagcatt 300gcaaccccat ttcaattcac
atgcttgtct ttccaaggaa ctttctaagc cacctaacag 360acattagggt ttatatcaga
atcgagctca tggcgtactt tatgctgcac gaacaatggg 420ttgggggcgt cgtttcttgc
atgagagcat gcgcatcctg gtaaggattt cgccaaaaga 480actttagtcc tctaccgact
ttgtgtttgc gtgatctcgt gatttgaagc ctgtggtggt 540gtgctgaggc agcatattgg
aaggtatctc tgtgttgata tggcatccgt ccgtggacaa 600atcgatacca catactgttc
ttggattcta ttcttgggat tgctaaatga tctagataga 660ttatattctc ttgttgcagc
ccctattgct tcaatacgaa gaaaacccaa cgtttagaac 720ttaataaaac catttgtgag
cttagctgct taggcaattc atttttatgc atgacaaata 780tataataata ttagctatac
tattattgat gcaacctgtg ggagcgtata aaatggtact 840tccccaattc taaattataa
gacgttttga ctatatattc tacatacata tgtttaattt 900tatatttaga taatcgctat
gccttaatat atagtaaaaa gtagtatatc tagaaaagat 960aaaacatctt ataatttaaa
aatgggtaga gtattatatt agatatgaac agtgcttaga 1020tgccaccaaa attttgccat
gccatcctaa ggccagcaaa agtttgtgtc ttcttttgtt 1080ttccaaacca ctagatgcca
atatactatt tatcatcgat cgagatgtag gtcttagtta 1140attgtgtcgg gtgcccttga
gaaagaaaag aaaaaggtgg gattttgttt tcgcttagac 1200gatgattgga tctcttggtc
tctgaattcc atcccgaata aacaaatgaa gtaggtcctc 1260agtcaccctt gccctgttag
ctgcaagaga gctcatggtt tccagccaca caatcagtcc 1320atggctcctt cttcttggcc
taagtggtgg ccaatcattg tgggtgatcg agtcttgggc 1380cctctgaaca gtattacaca
acagtaatcc tgcaaaagat ttggtatatc tagattctag 1440agtgagcgcc gtgttgtgcc
cagctaggaa tgggttgtca agtgcaacag gaggaggacc 1500caggatggtc aggtgtaata
ggctctcatt aaaagactgt tcagatggat tagagcaacg 1560acggggaagc cgggaaaaaa
tggttggttc tgctttcctc tcgctccccg gccgggttca 1620tatatgaatc tgagaacgat
attttttgct tcatttttca tttgctatat atttaaactg 1680tttttttgtg tgtgtgtgtg
tgttcattga gctcaatact tgaggcttga tagggagagg 1740agtgaggcag ctgatcacat
ggacctccat ctgaggacag ttcctcttcc gaaacagaaa 1800ggagagtgca gggaccagcg
tggcctgtac agtattgtgt ttgccctttt cctttggcag 1860ggacagagag cttcaggctt
gtcctcttta tgtatgctgc tcgcctgctt cagagtcaga 1920gcttcccctt ctcacttctc
agagagagag agagagaaga gagagagagg agagccctcc 1980acagctcccc tgtcctgccc
tcaggcattc tttgtcacag ggggcgaggg ctgaagatca 2040tcacatggtg gccttttttg
ggtctgtggc ctttggtctt ttagtgcttc ttccttttac 2100ctcctcatga catgaacccc
ctttttaaac ctccctcaaa atcaaatcac cctccttctc 2160ctttaagagc cctcaacccc
ttcccctcat tttccttcat ccctcagcct ttgcacaaag 2220ggcaagaata acgcagtatg
atcatctgat catactcccg ccgccatcac aatcccacac 2280gaacgtgaga caaaggtaac
agacgcaaga agctagcagc tgcaggagat tgctcagccc 2340atctcc
2346171470DNAZea
maysmisc_feature(0)...(0)ZmCkx1-2 promoter 17gagctcgccc ttgcatgctt
gagtcatatc ttggaaaaaa aaactgtaac ttaaagtatg 60atctatatat ggattatttg
gatgggatgt cattttcgta tcaccaacca aaattacagt 120ttggtcgtgc gtagaaattc
tacctactag ctgaaacaac ggctgctatg tataactact 180ggtactggaa agaatattag
tcattgactc aaaattagaa tgcatgtgta agtcatgcgt 240gctaatttgt tctatcagca
ttcggcgaat tccgaagtcc gtacgtgttg ttcgtggagg 300agaggaaaac atcagaaatg
acaaaactag acggcgtgtg cttctacact gaattcatca 360acatttgttt tacttttact
agagaatggc atcagatgga aaaccgctga aaaaacaaga 420aaacaattgg accccaaata
tgtacagacg ctagctatag ccagccacac tgaagttgac 480atgcggcaac tagctaacca
ccttctctga aacactaaca tttgtacctt ggtcgtgtaa 540gtgtagttag taacgtatgt
tgacgcgact taccgaacaa aaatataatt gtcccaatca 600agctagggac gattgtttgt
ttccaaaatg ttgccatttg cttaatcaat cctatattga 660ttcatggctg ttaaggtgag
ataaagcgac aagaaatctc tctctatata tatatataag 720atcccgaagg ctagcgacat
ttttgatagc aaaatatgag aagttggcag gttctggtag 780caaatcaaat aatatggcca
gaataatcgt ggctagcttg attaaacctt cagcttggtg 840tattttggaa gtcgaccaac
cagctgggcc ggggctcgtc gtagtaccaa aattacagcc 900tgcttccttc gtcgtcctgt
acgtaatgca gtacagctgt ctgtctagta gagacgattt 960tgagcaggca cacacattaa
gtgataacat aaaagacggc ttcattttat ttcataacca 1020aacgatatgg tcaacacaca
cctatagcta ccaaatttgt acaactattt agtgcgaaaa 1080ctatttcatt ctcaagaatt
gatcgcttat atttattatt acaggttttt aaatgtataa 1140atacgctata ttgcatggca
aaagggggta ataattaggc aggactatat atataatagt 1200tttttttcct ttaaattctt
gggaggatgg taaagttggt aactaggcac cttgtgcgca 1260tatttttctg tggtcaaaca
gaataaaact agacgggatg cagaattttt ttttccttgg 1320aaagcagctc atctctgtgt
tcgagtacgt aattgaagaa gtatgtgatc gcactacacc 1380tacacgtatg tgccgccgta
tccgtcctat atatatacgg ggtgcaatca cctagttacc 1440aaacactcac acataagggc
ggatccatgg 1470183317DNAZea
maysmisc_feature(0)...(0)ZmCkx1-2 promoter extended 18tcttgttccc
aagtgttttg taagcaaggc aagagacacc aagtgtgtgg tggtccttgc 60ggggtctaag
tgacccattt gattaaggag aaggctcact cggtctaagt gaccgtttga 120gagagtgaaa
gggttgaaag agacccggtc tttgtgacca cctcaatggg gactaggttc 180tttagaaccg
aacctcggta aaagaaatca tcgtgttcat ccgctttatt tcttggttga 240tttgtttttc
ccctctctcc cgaactcgga tttaattcta acgctaaccc cggcttgtag 300tttatgtttt
aagttgtaaa tttcagatta cgcctattca ccccctaggc aactttcagt 360tccaccactc
ctccccacct tgtacttacg aaagattgtg ttagtagatc tcgagattta 420caaacttacc
ataagcaact aaaaataata taaacctaaa aatatgaaaa cccggaaaga 480gcctaaaact
tgaacccgga aaaaaattcc tgcaataatc ataatcaact aagttatgct 540cacacattag
gtctgcgttg ttgaattaac tttcaagacc agaaaacagg cacgcgtgca 600tcgtacaagt
ggaacttcca aacctttaag taaatagaga tagacacgcc cacatgtgga 660acttcttggt
acgtaatgcg tgacaagtta gaagtgaaca aggcaaccac gtcgctactt 720gtgatgaata
ctgggctgtg tgtgggtttt gtcgtcgcag gagtacatgt gttctctgaa 780ttctaaatcg
atcatgatgt ccgtcttttt tttattttag ccctttttta gttttttttc 840ataaatatgc
cctttctagt tttaactcaa aaaatggacc ctctggtcga caccattact 900attggcggca
acctaacacg tctaggcgcc tagacggcta gaccttgttc gtggcattga 960cgtggtgcac
ccgtggctag tgaggtggca gaggtaggcg ctaagaacta tagtgccgaa 1020ggtaggcggc
atatatcttg gcacaggccc cctttaatac gggcccacgt gtaattttat 1080tttccttttc
ctcactctcc accctcgtcg ttcgcctggc tcaacatagc cgcccctctc 1140cctctccgcg
tctcgccatc gtcgcccctc tcctcgcagt gacttgccat ggtcgcggct 1200caccgtggcc
acgccatgcc agcgtcagcc cgctccctca tttatcgatg cgggctcggt 1260ggtcggcgac
gagcgcaagg ctgaggagcg tgggcgggcg acggtggtga gcgacgtagg 1320acgccgacga
cggtgacggg cgacttaggg caccgacgac gtatggcgtg ggcgggcgcc 1380agcggcctca
ccgacggagg atggcgcggg caacgctgga gagtggccga gcagcgaccg 1440ggtcgaatgg
aagagagaga aagaggaaaa gaacgtgaac cgattggtat atagagggtc 1500ggcgctaaga
tctatggcgt cgatccatac catgcctcag ggtccttcct ggccacgtca 1560tcgatacata
tgcgtcaatt gcattggcac caatacgtgt taggttggca ccaatagtga 1620gagcgtcaac
ctaagggtcc atattctgaa ttcaaacaaa aaataaccta tttgtgaaaa 1680tgaaacacta
aaaaggctaa aatagaaaaa aaatcggtcg cgatgcagac gcatcgtcgg 1740tttcaccgcc
gtcacgcgcg cgtctgtatg cgctgccagg agtcactgca agcggcaagc 1800agccaaaaaa
ataaaaattg gctgcatccg atctcgagac tccgacgaga ggaggctgcg 1860catgcttgag
tcatatcttg gaaaaaaaaa ctgtaactta aagtatgatc tatatatgga 1920ttatttggat
gggatgtcat tttcgtatca ccaaccaaaa ttacagtttg gtcgtgcgta 1980gaaattctac
ctactagctg aaacaacggc tgctatgtat aactactggt actggaaaga 2040atattagtca
ttgactcaaa attagaatgc atgtgtaagt catgcgtgct aatttgttct 2100atcagcattc
ggcgaattcc gaagtccgta cgtgttgttc gtggaggaga ggaaaacatc 2160agaaatgaca
aaactagacg gcgtgtgctt ctacactgaa ttcatcaaca tttgttttac 2220ttttactaga
gaatggcatc agatggaaaa ccgctgaaaa aacaagaaaa caattggacc 2280ccaaatatgt
acagacgcta gctatagcca gccacactga agttgacatg cggcaactag 2340ctaaccacct
tctctgaaac actaacattt gtaccttggt cgtgtaagtg tagttagtaa 2400cgtatgttga
cgcgacttac cgaacaaaaa tataattgtc ccaatcaagc tagggacgat 2460tgtttgtttc
caaaatgttg ccatttgctt aatcaatcct atattgattc atggctgtta 2520aggtgagata
aagcgacaag aaatctctct ctatatatat atataagatc ccgaaggcta 2580gcgacatttt
tgatagcaaa atatgagaag ttggcaggtt ctggtagcaa atcaaataat 2640atggccagaa
taatcgtggc tagcttgatt aaaccttcag cttggtgtat tttggaagtc 2700gaccaaccag
ctgggccggg gctcgtcgta gtaccaaaat tacagcctgc ttccttcgtc 2760gtcctgtacg
taatgcagta cagctgtctg tctagtagag acgattttga gcaggcacac 2820acattaagtg
ataacataaa agacggcttc attttatttc ataaccaaac gatatggtca 2880acacacacct
atagctacca aatttgtaca actatttagt gcgaaaacta tttcattctc 2940aagaattgat
cgcttatatt tattattaca ggtttttaaa tgtataaata cgctatattg 3000catggcaaaa
gggggtaata attaggcagg actatatata taatagtttt ttttccttta 3060aattcttggg
aggatggtaa agttggtaac taggcacctt gtgcgcatat ttttctgtgg 3120tcaaacagaa
taaaactaga cgggatgcag aatttttttt tccttggaaa gcagctcatc 3180tctgtgttcg
agtacgtaat tgaagaagta tgtgatcgca ctacacctac acgtatgtgc 3240cgccgtatcc
gtcctatata tatacggggt gcaatcacct agttaccaaa cactcacaca 3300taagggcgga
tccatgg
33171925DNAArtificial Sequenceprimer 19ctgactacca tgaagccgcc atcat
252029DNAArtificial Sequenceprimer
20ttgctctggt tatgatcccg aactgacca
292124DNAArtificial Sequenceprimer 21atcctcaaca actggagggc gtcc
242227DNAArtificial Sequenceprimer
22tcccaccttg ctccaaagtg ggttttc
272327DNAArtificial SequencePrimer 23cgcaggtcag caatgtcaat caactgg
272427DNAArtificial SequencePrimer
24agaggtgtgc accctgtcca aaaactc
272532DNAArtificial SequencePrimer 25agagaagcca acgccawcgc ctcyatttcg tc
322624DNAArtificial SequencePrimer
26ggagggtttc cccctttgac acag
242727DNAArtificial SequencePrimer 27gggacgacaa agagcgcgat atgatga
272828DNAArtificial SequencePrimer
28acgcacgctt cggcggcatt cattattt
282928DNAArtificial SequencePrimer 29ggcaagcatg catggagcaa aaactcgt
283029DNAArtificial SequencePrimer
30gcactactaa ctgactgacg ttgccattc
293128DNAArtificial SequencePrimer 31gcaactcact tgctcttgag catagggt
283232DNAArtificial SequencePrimer
32agagaagcca acgccawcgc ctcyatttcg tc
3233534PRTZea mays 33Met Ala Val Val Tyr Tyr Leu Leu Leu Ala Gly Leu Ile
Ala Cys Ser1 5 10 15
His Ala Leu Ala Ala Gly Thr Pro Ala Leu Gly Asp Asp Arg Gly Arg
20 25 30 Pro Trp Pro Ala Ser
Leu Ala Ala Leu Ala Leu Asp Gly Lys Leu Arg 35 40
45 Thr Asp Ser Asn Ala Thr Ala Ala Ala Ser
Thr Asp Phe Gly Asn Ile 50 55 60
Thr Ser Ala Leu Pro Ala Ala Val Leu Tyr Pro Ser Ser Thr Gly
Asp65 70 75 80 Leu
Val Ala Leu Leu Ser Ala Ala Asn Ser Thr Pro Gly Trp Pro Tyr
85 90 95 Thr Ile Ala Phe Arg Gly
Arg Gly His Ser Leu Met Gly Gln Ala Phe 100
105 110 Ala Pro Gly Gly Val Val Val Asn Met Ala
Ser Leu Gly Asp Ala Ala 115 120
125 Ala Pro Pro Arg Ile Asn Val Ser Ala Asp Gly Arg Tyr Val
Asp Ala 130 135 140
Gly Gly Glu Gln Val Trp Ile Asp Val Leu Arg Ala Ser Leu Ala Arg145
150 155 160 Gly Val Ala Pro Arg
Ser Trp Asn Asp Tyr Leu Tyr Leu Thr Val Gly 165
170 175 Gly Thr Leu Ser Asn Ala Gly Ile Ser Gly
Gln Ala Phe Arg His Gly 180 185
190 Pro Gln Ile Ser Asn Val Leu Glu Met Asp Val Ile Thr Gly His
Gly 195 200 205 Glu
Met Val Thr Cys Ser Lys Gln Leu Asn Ala Asp Leu Phe Asp Ala 210
215 220 Val Leu Gly Gly Leu Gly
Gln Phe Gly Val Ile Thr Arg Ala Arg Ile225 230
235 240 Ala Val Glu Pro Ala Pro Ala Arg Ala Arg Trp
Val Arg Phe Val Tyr 245 250
255 Thr Asp Phe Ala Ala Phe Ser Ala Asp Gln Glu Arg Leu Thr Ala Pro
260 265 270 Arg Pro Gly
Gly Gly Gly Ala Ser Phe Gly Pro Met Ser Tyr Val Glu 275
280 285 Gly Ser Val Phe Val Asn Gln Ser
Leu Ala Thr Asp Leu Ala Asn Thr 290 295
300 Gly Phe Phe Thr Asp Ala Asp Val Ala Arg Ile Val Ala
Leu Ala Gly305 310 315
320 Glu Arg Asn Ala Thr Thr Val Tyr Ser Ile Glu Ala Thr Leu Asn Tyr
325 330 335 Asp Asn Ala Thr
Ala Ala Ala Ala Ala Val Asp Gln Glu Leu Ala Ser 340
345 350 Val Leu Gly Thr Leu Ser Tyr Val Glu
Gly Phe Ala Phe Gln Arg Asp 355 360
365 Val Ala Tyr Ala Ala Phe Leu Asp Arg Val His Gly Glu Glu
Val Ala 370 375 380
Leu Asn Lys Leu Gly Leu Trp Arg Val Pro His Pro Trp Leu Asn Met385
390 395 400 Phe Val Pro Arg Ser
Arg Ile Ala Asp Phe Asp Arg Gly Val Phe Lys 405
410 415 Gly Ile Leu Gln Gly Thr Asp Ile Val Gly
Pro Leu Ile Val Tyr Pro 420 425
430 Leu Asn Lys Ser Met Trp Asp Asp Gly Met Ser Ala Ala Thr Pro
Ser 435 440 445 Glu
Asp Val Phe Tyr Ala Val Ser Leu Leu Phe Ser Ser Val Ala Pro 450
455 460 Asn Asp Leu Ala Arg Leu
Gln Glu Gln Asn Arg Arg Ile Leu Arg Phe465 470
475 480 Cys Asp Leu Ala Gly Ile Gln Tyr Lys Thr Tyr
Leu Ala Arg His Thr 485 490
495 Asp Arg Ser Asp Trp Val Arg His Phe Gly Ala Ala Lys Trp Asn Arg
500 505 510 Phe Val Glu
Met Lys Asn Lys Tyr Asp Pro Lys Arg Leu Leu Ser Pro 515
520 525 Gly Gln Asp Ile Phe Asn 530
34579PRTArtificial SequenceConsensus Sequence 34Val Xaa Xaa
Phe Leu Leu Leu Val Leu Leu Leu Leu Ser Ser Leu Ala1 5
10 15 Leu Ala Ala Gly Xaa Pro Xaa Asp
Leu Xaa Xaa Leu Gly Xaa Xaa Xaa 20 25
30 Xaa Gly Xaa Leu Xaa Gly Xaa Xaa Xaa Xaa Xaa Arg Leu
Xaa Thr Asp 35 40 45
Ala Xaa Ser Thr Ala Ala Ala Ala Thr Asp Phe Gly Asn Ile Xaa Ser 50
55 60 Ala Leu Pro Ala Ala
Val Leu His Pro Ala Ser Xaa Gly Asp Ile Ala65 70
75 80 Ala Leu Val Arg Ala Ala Xaa Xaa Ser Xaa
Ser Ala Ser Pro Leu Thr 85 90
95 Val Ala Ala Arg Gly Xaa Gly His Ser Ile Xaa Gly Gln Ala Gln
Ala 100 105 110 Pro
Gly Gly Val Val Val Asp Met Xaa Ser Leu Gly Gly Ala Xaa Xaa 115
120 125 Xaa Xaa Xaa Xaa Xaa Xaa
Ile Xaa Xaa Xaa Xaa Val Ala Gly Gly Gly 130 135
140 Arg Xaa Xaa Phe Val Asp Val Gly Gly Gly Xaa
Leu Trp Ile Asp Val145 150 155
160 Leu Arg Xaa Thr Leu Lys His Gly Xaa Leu Ala Pro Arg Ser Trp Thr
165 170 175 Asp Tyr Leu
Tyr Leu Thr Val Gly Gly Thr Leu Ser Asn Ala Gly Ile 180
185 190 Ser Gly Gln Ala Phe Arg His Gly
Pro Gln Ile Ser Asn Val Xaa Glu 195 200
205 Leu Asp Val Val Thr Gly Arg Gly Glu Met Val Thr Cys
Ser Pro Ser 210 215 220
Xaa Asn Ala Asp Leu Phe Phe Ala Val Leu Gly Gly Leu Gly Gln Phe225
230 235 240 Gly Ile Ile Thr Arg
Ala Arg Ile Ala Leu Glu Pro Ala Pro Lys Arg 245
250 255 Val Arg Trp Val Arg Val Leu Tyr Ser Asp
Phe Ala Ala Phe Thr Ala 260 265
270 Asp Gln Glu Xaa Leu Ile Ser Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ala
Xaa 275 280 285 Xaa
Xaa Ser Phe Asp Tyr Val Glu Gly Phe Val Val Leu Asn Xaa Ser 290
295 300 Leu Ile Xaa Asn Xaa Xaa
Xaa Ser Ser Phe Phe Ser Pro Ala Asp Val305 310
315 320 Xaa Arg Leu Xaa Ser Leu Ala Ser Xaa Ser Xaa
Xaa Xaa Val Leu Tyr 325 330
335 Cys Leu Glu Val Thr Leu Asn Tyr Xaa Xaa Gly Thr Ala Xaa Ser Xaa
340 345 350 Xaa Xaa Xaa
Xaa Xaa Val Asp Gln Glu Val Ala Ala Leu Leu Gly Xaa 355
360 365 Leu Ser Phe Val Xaa Gly Xaa Leu
Phe Thr Xaa Asp Val Thr Tyr Val 370 375
380 Asp Phe Leu Asp Arg Val His Xaa Glu Glu Leu Xaa Leu
Arg Ala Xaa385 390 395
400 Gly Leu Trp Glu Val Pro Xaa His Pro Trp Leu Asn Leu Phe Val Pro
405 410 415 Arg Ser Arg Ile
Ala Asp Phe Asp Arg Gly Val Phe Lys Gly Ile Leu 420
425 430 Xaa Asp Xaa Xaa Xaa Xaa Xaa Xaa Gly
Pro Ile Leu Ile Tyr Pro Met 435 440
445 Asn Lys Ser Lys Trp Asp Xaa Arg Thr Ser Val Val Thr Pro
Asp Xaa 450 455 460
Xaa Xaa Xaa Xaa Xaa Glu Glu Val Phe Tyr Leu Val Ala Phe Leu Arg465
470 475 480 Ser Ala Leu Ser Gly
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 485
490 495 Ser Leu Xaa Xaa Leu Leu Arg Gln Asn Arg
Arg Ile Leu Asp Phe Cys 500 505
510 Asp Ala Ala Gly Ile Gly Xaa Lys Gln Tyr Leu Pro His His Thr
Thr 515 520 525 Xaa
Ala Asp Trp Xaa Arg His Phe Gly Ala Ala Arg Trp Glu Arg Phe 530
535 540 Ala Asp Arg Lys Ala Arg
Tyr Asp Pro Leu Ala Ile Leu Ala Pro Gly545 550
555 560 Gln Arg Ile Phe Pro Arg Xaa Xaa Xaa Xaa Ala
Ala Ile Ala Xaa Ala 565 570
575 Xaa Xaa Xaa35575PRTArabidopsis thaliana 35Met Gly Leu Thr Ser
Ser Leu Arg Phe His Arg Gln Asn Asn Lys Thr1 5
10 15 Phe Leu Gly Ile Phe Met Ile Leu Val Leu
Ser Cys Ile Pro Gly Arg 20 25
30 Thr Asn Leu Cys Ser Asn His Ser Val Ser Thr Pro Lys Glu Leu
Pro 35 40 45 Ser
Ser Asn Pro Ser Asp Ile Arg Ser Ser Leu Val Ser Leu Asp Leu 50
55 60 Glu Gly Tyr Ile Ser Phe
Asp Asp Val His Asn Val Ala Lys Asp Phe65 70
75 80 Gly Asn Arg Tyr Gln Leu Pro Pro Leu Ala Ile
Leu His Pro Arg Ser 85 90
95 Val Phe Asp Ile Ser Ser Met Met Lys His Ile Val His Leu Gly Ser
100 105 110 Thr Ser Asn
Leu Thr Val Ala Ala Arg Gly His Gly His Ser Leu Gln 115
120 125 Gly Gln Ala Leu Ala His Gln Gly
Val Val Ile Lys Met Glu Ser Leu 130 135
140 Arg Ser Pro Asp Ile Arg Ile Tyr Lys Gly Lys Gln Pro
Tyr Val Asp145 150 155
160 Val Ser Gly Gly Glu Ile Trp Ile Asn Ile Leu Arg Glu Thr Leu Lys
165 170 175 Tyr Gly Leu Ser
Pro Lys Ser Trp Thr Asp Tyr Leu His Leu Thr Val 180
185 190 Gly Gly Thr Leu Ser Asn Ala Gly Ile
Ser Gly Gln Ala Phe Lys His 195 200
205 Gly Pro Gln Ile Asn Asn Val Tyr Gln Leu Glu Ile Val Thr
Gly Lys 210 215 220
Gly Glu Val Val Thr Cys Ser Glu Lys Arg Asn Ser Glu Leu Phe Phe225
230 235 240 Ser Val Leu Gly Gly
Leu Gly Gln Phe Gly Ile Ile Thr Arg Ala Arg 245
250 255 Ile Ser Leu Glu Pro Ala Pro His Met Val
Lys Trp Ile Arg Val Leu 260 265
270 Tyr Ser Asp Phe Ser Ala Phe Ser Arg Asp Gln Glu Tyr Leu Ile
Ser 275 280 285 Lys
Glu Lys Thr Phe Asp Tyr Val Glu Gly Phe Val Ile Ile Asn Arg 290
295 300 Thr Asp Leu Leu Asn Asn
Trp Arg Ser Ser Phe Ser Pro Asn Asp Ser305 310
315 320 Thr Gln Ala Ser Arg Phe Lys Ser Asp Gly Lys
Thr Leu Tyr Cys Leu 325 330
335 Glu Val Val Lys Tyr Phe Asn Pro Glu Glu Ala Ser Ser Met Asp Gln
340 345 350 Glu Thr Gly
Lys Leu Leu Ser Glu Leu Asn Tyr Ile Pro Ser Thr Leu 355
360 365 Phe Ser Ser Glu Val Pro Tyr Ile
Glu Phe Leu Asp Arg Val His Ile 370 375
380 Ala Glu Arg Lys Leu Arg Ala Lys Gly Leu Trp Glu Val
Pro His Pro385 390 395
400 Trp Leu Asn Leu Leu Ile Pro Lys Ser Ser Ile Tyr Gln Phe Ala Thr
405 410 415 Glu Val Phe Asn
Asn Ile Leu Thr Ser Asn Asn Asn Gly Pro Ile Leu 420
425 430 Ile Tyr Pro Val Asn Gln Ser Lys Trp
Lys Lys His Thr Ser Leu Ile 435 440
445 Thr Pro Asn Glu Asp Ile Phe Tyr Leu Val Ala Phe Leu Pro
Ser Ala 450 455 460
Val Pro Asn Ser Ser Gly Lys Asn Asp Leu Glu Tyr Leu Leu Lys Gln465
470 475 480 Asn Gln Arg Val Met
Asn Phe Cys Ala Ala Ala Asn Leu Asn Val Lys 485
490 495 Gln Tyr Leu Pro His Tyr Glu Thr Gln Lys
Glu Trp Lys Ser His Phe 500 505
510 Gly Lys Arg Trp Glu Thr Phe Ala Gln Arg Lys Gln Ala Tyr Asp
Pro 515 520 525 Leu
Ala Ile Leu Ala Pro Gly Gln Arg Ile Phe Gln Lys Thr Thr Gly 530
535 540 Lys Leu Ser Pro Ile Gln
Leu Ala Lys Ser Lys Ala Thr Gly Ser Pro545 550
555 560 Gln Arg Tyr His Tyr Ala Ser Ile Leu Pro Lys
Pro Arg Thr Val 565 570
575 36501PRTArabidopsis thaliana 36Met Ala Asn Leu Arg Leu Met Ile Thr
Leu Ile Thr Val Leu Met Ile1 5 10
15 Thr Lys Ser Ser Asn Gly Ile Lys Ile Asp Leu Pro Lys Ser
Leu Asn 20 25 30
Leu Thr Leu Ser Thr Asp Pro Ser Ile Ile Ser Ala Ala Ser His Asp 35
40 45 Phe Gly Asn Ile Thr
Thr Val Thr Pro Gly Gly Val Ile Cys Pro Ser 50 55
60 Ser Thr Ala Asp Ile Ser Arg Leu Leu Gln
Tyr Ala Ala Asn Gly Lys65 70 75
80 Ser Thr Phe Gln Val Ala Ala Arg Gly Gln Gly His Ser Leu Asn
Gly 85 90 95 Gln
Ala Ser Val Ser Gly Gly Val Ile Val Asn Met Thr Cys Ile Thr
100 105 110 Asp Val Val Val Ser
Lys Asp Lys Lys Tyr Ala Asp Val Ala Ala Gly 115
120 125 Thr Leu Trp Val Asp Val Leu Lys Lys
Thr Ala Glu Lys Gly Val Ser 130 135
140 Pro Val Ser Trp Thr Asp Tyr Leu His Ile Thr Val Arg
Gly Thr Leu145 150 155
160 Ser Asn Gly Gly Ile Gly Gly Gln Val Phe Arg Asn Gly Pro Leu Val
165 170 175 Ser Asn Val Leu
Glu Leu Asp Val Ile Thr Gly Lys Gly Glu Met Leu 180
185 190 Thr Cys Ser Arg Gln Leu Asn Pro Glu
Leu Phe Tyr Gly Val Leu Gly 195 200
205 Gly Leu Gly Gln Phe Gly Ile Ile Thr Arg Ala Arg Ile Val
Leu Asp 210 215 220
His Ala Pro Lys Arg Ala Lys Trp Phe Arg Met Leu Tyr Ser Asp Phe225
230 235 240 Thr Thr Phe Thr Lys
Asp Gln Glu Arg Leu Ile Ser Met Ala Asn Asp 245
250 255 Ile Gly Val Asp Tyr Leu Glu Gly Gln Ile
Phe Leu Ser Asn Gly Val 260 265
270 Val Asp Thr Ser Phe Phe Pro Pro Ser Asp Gln Ser Lys Val Ala
Asp 275 280 285 Leu
Val Lys Gln His Gly Ile Ile Tyr Val Leu Glu Val Ala Lys Tyr 290
295 300 Tyr Asp Asp Pro Asn Leu
Pro Ile Ile Ser Lys Val Ile Asp Thr Leu305 310
315 320 Thr Lys Thr Leu Ser Tyr Leu Pro Gly Phe Ile
Ser Met His Asp Val 325 330
335 Ala Tyr Phe Asp Phe Leu Asn Arg Val His Val Glu Glu Asn Lys Leu
340 345 350 Arg Ser Leu
Gly Leu Trp Glu Leu Pro His Pro Trp Leu Asn Leu Tyr 355
360 365 Val Pro Lys Ser Arg Ile Leu Asp
Phe His Asn Gly Val Val Lys Asp 370 375
380 Ile Leu Leu Lys Gln Lys Ser Ala Ser Gly Leu Ala Leu
Leu Tyr Pro385 390 395
400 Thr Asn Arg Asn Lys Trp Asp Asn Arg Met Ser Ala Met Ile Pro Glu
405 410 415 Ile Asp Glu Asp
Val Ile Tyr Ile Ile Gly Leu Leu Gln Ser Ala Thr 420
425 430 Pro Lys Asp Leu Pro Glu Val Glu Ser
Val Asn Glu Lys Ile Ile Arg 435 440
445 Phe Cys Lys Asp Ser Gly Ile Lys Ile Lys Gln Tyr Leu Met
His Tyr 450 455 460
Thr Ser Lys Glu Asp Trp Ile Glu His Phe Gly Ser Lys Trp Asp Asp465
470 475 480 Phe Ser Lys Arg Lys
Asp Leu Phe Asp Pro Lys Lys Leu Leu Ser Pro 485
490 495 Gly Gln Asp Ile Phe 500
37523PRTArabidopsis thaliana 37Met Ala Ser Tyr Asn Leu Arg Ser Gln Val
Arg Leu Ile Ala Ile Thr1 5 10
15 Ile Val Ile Ile Ile Thr Leu Ser Thr Pro Ile Thr Thr Asn Thr
Ser 20 25 30 Pro
Gln Pro Trp Asn Ile Leu Ser His Asn Glu Phe Ala Gly Lys Leu 35
40 45 Thr Ser Ser Ser Ser Ser
Val Glu Ser Ala Ala Thr Asp Phe Gly His 50 55
60 Val Thr Lys Ile Phe Pro Ser Ala Val Leu Ile
Pro Ser Ser Val Glu65 70 75
80 Asp Ile Thr Asp Leu Ile Lys Leu Ser Phe Asp Ser Gln Leu Ser Phe
85 90 95 Pro Leu Ala
Ala Arg Gly His Gly His Ser His Arg Gly Gln Ala Ser 100
105 110 Ala Lys Asp Gly Val Val Val Asn
Met Arg Ser Met Val Asn Arg Asp 115 120
125 Arg Gly Ile Lys Val Ser Arg Thr Cys Leu Tyr Val Asp
Val Asp Ala 130 135 140
Ala Trp Leu Trp Ile Glu Val Leu Asn Lys Thr Leu Glu Leu Gly Leu145
150 155 160 Thr Pro Val Ser Trp
Thr Asp Tyr Leu Tyr Leu Thr Val Gly Gly Thr 165
170 175 Leu Ser Asn Gly Gly Ile Ser Gly Gln Thr
Phe Arg Tyr Gly Pro Gln 180 185
190 Ile Thr Asn Val Leu Glu Met Asp Val Ile Thr Gly Lys Gly Glu
Ile 195 200 205 Ala
Thr Cys Ser Lys Asp Met Asn Ser Asp Leu Phe Phe Ala Val Leu 210
215 220 Gly Gly Leu Gly Gln Phe
Gly Ile Ile Thr Arg Ala Arg Ile Lys Leu225 230
235 240 Glu Val Ala Pro Lys Arg Ala Lys Trp Leu Arg
Phe Leu Tyr Ile Asp 245 250
255 Phe Ser Glu Phe Thr Arg Asp Gln Glu Arg Val Ile Ser Lys Thr Asp
260 265 270 Gly Val Asp
Phe Leu Glu Gly Ser Ile Met Val Asp His Gly Pro Pro 275
280 285 Asp Asn Trp Arg Ser Thr Tyr Tyr
Pro Pro Ser Asp His Leu Arg Ile 290 295
300 Ala Ser Met Val Lys Arg His Arg Val Ile Tyr Cys Leu
Glu Val Val305 310 315
320 Lys Tyr Tyr Asp Glu Thr Ser Gln Tyr Thr Val Asn Glu Glu Met Glu
325 330 335 Glu Leu Ser Asp
Ser Leu Asn His Val Arg Gly Phe Met Tyr Glu Lys 340
345 350 Asp Val Thr Tyr Met Asp Phe Leu Asn
Arg Val Arg Thr Gly Glu Leu 355 360
365 Asn Leu Lys Ser Lys Gly Gln Trp Asp Val Pro His Pro Trp
Leu Asn 370 375 380
Leu Phe Val Pro Lys Thr Gln Ile Ser Lys Phe Asp Asp Gly Val Phe385
390 395 400 Lys Gly Ile Ile Leu
Arg Asn Asn Ile Thr Ser Gly Pro Val Leu Val 405
410 415 Tyr Pro Met Asn Arg Asn Lys Trp Asn Asp
Arg Met Ser Ala Ala Ile 420 425
430 Pro Glu Glu Asp Val Phe Tyr Ala Val Gly Phe Leu Arg Ser Ala
Gly 435 440 445 Phe
Asp Asn Trp Glu Ala Phe Asp Gln Glu Asn Met Glu Ile Leu Lys 450
455 460 Phe Cys Glu Asp Ala Asn
Met Gly Val Ile Gln Tyr Leu Pro Tyr His465 470
475 480 Ser Ser Gln Glu Gly Trp Val Arg His Phe Gly
Pro Arg Trp Asn Ile 485 490
495 Phe Val Glu Arg Lys Tyr Lys Tyr Asp Pro Lys Met Ile Leu Ser Pro
500 505 510 Gly Gln Asn
Ile Phe Gln Lys Ile Asn Ser Ser 515 520
38524PRTArabidopsis thaliana 38Met Thr Asn Thr Leu Cys Leu Ser Leu Ile
Thr Leu Ile Thr Phe Phe1 5 10
15 Ile Ser Leu Thr Pro Thr Leu Ile Lys Ser Asp Glu Gly Ile Asp
Val 20 25 30 Phe
Leu Pro Ile Ser Leu Asn Leu Thr Val Leu Thr Asp Pro Phe Ser 35
40 45 Ile Ser Ala Ala Ser His
Asp Phe Gly Asn Ile Thr Asp Glu Asn Pro 50 55
60 Gly Ala Val Leu Cys Pro Ser Ser Thr Thr Glu
Val Ala Arg Leu Leu65 70 75
80 Arg Phe Ala Asn Gly Gly Phe Ser Tyr Asn Lys Gly Ser Thr Ser Pro
85 90 95 Ala Ser Thr
Phe Lys Val Ala Ala Arg Gly Gln Gly His Ser Leu Arg 100
105 110 Gly Gln Ala Ser Ala Pro Gly Gly
Val Val Val Asn Met Thr Cys Leu 115 120
125 Ala Met Ala Ala Lys Pro Ala Ala Val Val Ile Ser Ala
Asp Gly Thr 130 135 140
Tyr Ala Asp Val Ala Ala Gly Thr Met Trp Val Asp Val Leu Lys Ala145
150 155 160 Ala Val Asp Arg Gly
Val Ser Pro Val Thr Trp Thr Asp Tyr Leu Tyr 165
170 175 Leu Ser Val Gly Gly Thr Leu Ser Asn Ala
Gly Ile Gly Gly Gln Thr 180 185
190 Phe Arg His Gly Pro Gln Ile Ser Asn Val His Glu Leu Asp Val
Ile 195 200 205 Thr
Gly Lys Gly Glu Met Met Thr Cys Ser Pro Lys Leu Asn Pro Glu 210
215 220 Leu Phe Tyr Gly Val Leu
Gly Gly Leu Gly Gln Phe Gly Ile Ile Thr225 230
235 240 Arg Ala Arg Ile Ala Leu Asp His Ala Pro Thr
Arg Val Lys Trp Ser 245 250
255 Arg Ile Leu Tyr Ser Asp Phe Ser Ala Phe Lys Arg Asp Gln Glu Arg
260 265 270 Leu Ile Ser
Met Thr Asn Asp Leu Gly Val Asp Phe Leu Glu Gly Gln 275
280 285 Leu Met Met Ser Asn Gly Phe Val
Asp Thr Ser Phe Phe Pro Leu Ser 290 295
300 Asp Gln Thr Arg Val Ala Ser Leu Val Asn Asp His Arg
Ile Ile Tyr305 310 315
320 Val Leu Glu Val Ala Lys Tyr Tyr Asp Arg Thr Thr Leu Pro Ile Ile
325 330 335 Asp Gln Val Ile
Asp Thr Leu Ser Arg Thr Leu Gly Phe Ala Pro Gly 340
345 350 Phe Met Phe Val Gln Asp Val Pro Tyr
Phe Asp Phe Leu Asn Arg Val 355 360
365 Arg Asn Glu Glu Asp Lys Leu Arg Ser Leu Gly Leu Trp Glu
Val Pro 370 375 380
His Pro Trp Leu Asn Ile Phe Val Pro Gly Ser Arg Ile Gln Asp Phe385
390 395 400 His Asp Gly Val Ile
Asn Gly Leu Leu Leu Asn Gln Thr Ser Thr Ser 405
410 415 Gly Val Thr Leu Phe Tyr Pro Thr Asn Arg
Asn Lys Trp Asn Asn Arg 420 425
430 Met Ser Thr Met Thr Pro Asp Glu Asp Val Phe Tyr Val Ile Gly
Leu 435 440 445 Leu
Gln Ser Ala Gly Gly Ser Gln Asn Trp Gln Glu Leu Glu Asn Leu 450
455 460 Asn Asp Lys Val Ile Gln
Phe Cys Glu Asn Ser Gly Ile Lys Ile Lys465 470
475 480 Glu Tyr Leu Met His Tyr Thr Arg Lys Glu Asp
Trp Val Lys His Phe 485 490
495 Gly Pro Lys Trp Asp Asp Phe Leu Arg Lys Lys Ile Met Phe Asp Pro
500 505 510 Lys Arg Leu
Leu Ser Pro Gly Gln Asp Ile Phe Asn 515 520
39524PRTArabidopsis thaliana 39Met Ile Ala Tyr Ile Glu Pro Tyr
Phe Leu Glu Asn Asp Ala Glu Ala1 5 10
15 Ala Ser Ala Ala Thr Ala Ala Gly Lys Ser Thr Asp Gly
Val Ser Glu 20 25 30
Ser Leu Asn Ile Gln Gly Glu Ile Leu Cys Gly Gly Ala Ala Ala Asp
35 40 45 Ile Ala Gly Arg
Asp Phe Gly Gly Met Asn Cys Val Lys Pro Leu Ala 50 55
60 Val Val Arg Pro Val Gly Pro Glu Asp
Ile Ala Gly Ala Val Lys Ala65 70 75
80 Ala Leu Arg Ser Asp Lys Leu Thr Val Ala Ala Arg Gly Asn
Gly His 85 90 95
Ser Ile Asn Gly Gln Ala Met Ala Glu Gly Gly Leu Val Val Asp Met
100 105 110 Ser Thr Thr Ala Glu
Asn His Phe Glu Val Gly Tyr Leu Ser Gly Gly 115
120 125 Asp Ala Thr Ala Phe Val Asp Val Ser
Gly Gly Ala Leu Trp Glu Asp 130 135
140 Val Leu Lys Arg Cys Val Ser Glu Tyr Gly Leu Ala Pro
Arg Ser Trp145 150 155
160 Thr Asp Tyr Leu Gly Leu Thr Val Gly Gly Thr Leu Ser Asn Ala Gly
165 170 175 Val Ser Gly Gln
Ala Phe Arg Tyr Gly Pro Gln Thr Ser Asn Val Thr 180
185 190 Glu Leu Asp Val Val Thr Gly Asn Gly
Asp Val Val Thr Cys Ser Glu 195 200
205 Ile Glu Asn Ser Glu Leu Phe Phe Ser Val Leu Gly Gly Leu
Gly Gln 210 215 220
Phe Gly Ile Ile Thr Arg Ala Arg Val Leu Leu Gln Pro Ala Pro Asp225
230 235 240 Met Val Arg Trp Ile
Arg Val Val Tyr Thr Glu Phe Asp Glu Phe Thr 245
250 255 Gln Asp Ala Glu Trp Leu Val Ser Gln Lys
Asn Glu Ser Ser Phe Asp 260 265
270 Tyr Val Glu Gly Phe Val Phe Val Asn Gly Ala Asp Pro Val Asn
Gly 275 280 285 Trp
Pro Thr Val Pro Leu His Pro Asp His Glu Phe Asp Pro Thr Arg 290
295 300 Leu Pro Gln Ser Cys Gly
Ser Val Leu Tyr Cys Leu Glu Leu Gly Leu305 310
315 320 His Tyr Arg Asp Ser Asp Ser Asn Ser Thr Ile
Asp Lys Arg Val Glu 325 330
335 Arg Leu Ile Gly Arg Leu Arg Phe Asn Glu Gly Leu Arg Phe Glu Val
340 345 350 Asp Leu Pro
Tyr Val Asp Phe Leu Leu Arg Val Lys Arg Ser Glu Glu 355
360 365 Ile Ala Lys Glu Asn Gly Thr Trp
Glu Thr Pro His Pro Trp Leu Asn 370 375
380 Leu Phe Val Ser Lys Arg Asp Ile Gly Asp Phe Asn Arg
Thr Val Phe385 390 395
400 Lys Glu Leu Val Lys Asn Gly Val Asn Gly Pro Met Leu Val Tyr Pro
405 410 415 Leu Leu Arg Ser
Arg Trp Asp Asp Arg Thr Ser Val Val Ile Pro Glu 420
425 430 Glu Gly Glu Ile Phe Tyr Ile Val Ala
Leu Leu Arg Phe Val Pro Pro 435 440
445 Cys Ala Lys Val Ser Ser Val Glu Lys Met Val Ala Gln Asn
Gln Glu 450 455 460
Ile Val His Trp Cys Val Lys Asn Gly Ile Asp Tyr Lys Leu Tyr Leu465
470 475 480 Pro His Tyr Lys Ser
Gln Glu Glu Trp Ile Arg His Phe Gly Asn Arg 485
490 495 Trp Ser Arg Phe Val Asp Arg Lys Ala Met
Phe Asp Pro Met Ala Ile 500 505
510 Leu Ser Pro Gly Gln Lys Ile Phe Asn Arg Ser Leu 515
520 40540PRTArabidopsis thaliana 40Met Asn
Arg Glu Met Thr Ser Ser Phe Leu Leu Leu Thr Phe Ala Ile1 5
10 15 Cys Lys Leu Ile Ile Ala Val
Gly Leu Asn Val Gly Pro Ser Glu Leu 20 25
30 Leu Arg Ile Gly Ala Ile Asp Val Asp Gly His Phe
Thr Val His Pro 35 40 45
Ser Asp Leu Ala Ser Val Ser Ser Asp Phe Gly Met Leu Lys Ser Pro
50 55 60 Glu Glu Pro
Leu Ala Val Leu His Pro Ser Ser Ala Glu Asp Val Ala65 70
75 80 Arg Leu Val Arg Thr Ala Tyr Gly
Ser Ala Thr Ala Phe Pro Val Ser 85 90
95 Ala Arg Gly His Gly His Ser Ile Asn Gly Gln Ala Ala
Ala Gly Arg 100 105 110
Asn Gly Val Val Val Glu Met Asn His Gly Val Thr Gly Thr Pro Lys
115 120 125 Pro Leu Val Arg
Pro Asp Glu Met Tyr Val Asp Val Trp Gly Gly Glu 130
135 140 Leu Trp Val Asp Val Leu Lys Lys
Thr Leu Glu His Gly Leu Ala Pro145 150
155 160 Lys Ser Trp Thr Asp Tyr Leu Tyr Leu Thr Val Gly
Gly Thr Leu Ser 165 170
175 Asn Ala Gly Ile Ser Gly Gln Ala Leu His His Gly Pro Gln Ile Ser
180 185 190 Asn Val Leu
Glu Leu Asp Val Val Thr Gly Lys Gly Glu Val Met Arg 195
200 205 Cys Ser Glu Glu Glu Asn Thr Arg
Leu Phe His Gly Val Leu Gly Gly 210 215
220 Leu Gly Gln Phe Gly Ile Ile Thr Arg Ala Arg Ile Ser
Leu Glu Pro225 230 235
240 Ala Pro Gln Arg Val Arg Trp Ile Arg Val Leu Tyr Ser Ser Phe Lys
245 250 255 Val Phe Thr Glu
Asp Gln Glu Tyr Leu Ile Ser Met His Gly Gln Leu 260
265 270 Lys Phe Asp Tyr Val Glu Gly Phe Val
Ile Val Asp Glu Gly Leu Val 275 280
285 Asn Asn Trp Arg Ser Ser Phe Phe Ser Pro Arg Asn Pro Val
Lys Ile 290 295 300
Ser Ser Val Ser Ser Asn Gly Ser Val Leu Tyr Cys Leu Glu Ile Thr305
310 315 320 Lys Asn Tyr His Asp
Ser Asp Ser Glu Ile Val Asp Gln Glu Val Glu 325
330 335 Ile Leu Met Lys Lys Leu Asn Phe Ile Pro
Thr Ser Val Phe Thr Thr 340 345
350 Asp Leu Gln Tyr Val Asp Phe Leu Asp Arg Val His Lys Ala Glu
Leu 355 360 365 Lys
Leu Arg Ser Lys Asn Leu Trp Glu Val Pro His Pro Trp Leu Asn 370
375 380 Leu Phe Val Pro Lys Ser
Arg Ile Ser Asp Phe Asp Lys Gly Val Phe385 390
395 400 Lys Gly Ile Leu Gly Asn Lys Thr Ser Gly Pro
Ile Leu Ile Tyr Pro 405 410
415 Met Asn Lys Asp Lys Trp Asp Glu Arg Ser Ser Ala Val Thr Pro Asp
420 425 430 Glu Glu Val
Phe Tyr Leu Val Ala Leu Leu Arg Ser Ala Leu Thr Asp 435
440 445 Gly Glu Glu Thr Gln Lys Leu Glu
Tyr Leu Lys Asp Gln Asn Arg Arg 450 455
460 Ile Leu Glu Phe Cys Glu Gln Ala Lys Ile Asn Val Lys
Gln Tyr Leu465 470 475
480 Pro His His Ala Thr Gln Glu Glu Trp Val Ala His Phe Gly Asp Lys
485 490 495 Trp Asp Arg Phe
Arg Ser Leu Lys Ala Glu Phe Asp Pro Arg His Ile 500
505 510 Leu Ala Thr Gly Gln Arg Ile Phe Gln
Asn Pro Ser Leu Ser Leu Phe 515 520
525 Pro Pro Ser Ser Ser Ser Ser Ser Ala Ala Ser Trp 530
535 540 41504PRTArabidopsis thaliana 41Met
Leu Ile Val Arg Ser Phe Thr Ile Leu Leu Leu Ser Cys Ile Ala1
5 10 15 Phe Lys Leu Ala Cys Cys
Phe Ser Ser Ser Ile Ser Ser Leu Lys Ala 20 25
30 Leu Pro Leu Val Gly His Leu Glu Phe Glu His
Val His His Ala Ser 35 40 45
Lys Asp Phe Gly Asn Arg Tyr Gln Leu Ile Pro Leu Ala Val Leu His
50 55 60 Pro Lys Ser
Val Ser Asp Ile Ala Ser Thr Ile Arg His Ile Trp Met65 70
75 80 Met Gly Thr His Ser Gln Leu Thr
Val Ala Ala Arg Gly Arg Gly His 85 90
95 Ser Leu Gln Gly Gln Ala Gln Thr Arg His Gly Ile Val
Ile His Met 100 105 110
Glu Ser Leu His Pro Gln Lys Leu Gln Val Tyr Ser Val Asp Ser Pro
115 120 125 Ala Pro Tyr Val
Asp Val Ser Gly Gly Glu Leu Trp Ile Asn Ile Leu 130
135 140 His Glu Thr Leu Lys Tyr Gly Leu
Ala Pro Lys Ser Trp Thr Asp Tyr145 150
155 160 Leu His Leu Thr Val Gly Gly Thr Leu Ser Asn Ala
Gly Ile Ser Gly 165 170
175 Gln Ala Phe Arg His Gly Pro Gln Ile Ser Asn Val His Gln Leu Glu
180 185 190 Ile Val Thr
Gly Lys Gly Glu Ile Leu Asn Cys Thr Lys Arg Gln Asn 195
200 205 Ser Asp Leu Phe Asn Gly Val Leu
Gly Gly Leu Gly Gln Phe Gly Ile 210 215
220 Ile Thr Arg Ala Arg Ile Ala Leu Glu Pro Ala Pro Thr
Met Asp Gln225 230 235
240 Glu Gln Leu Ile Ser Ala Gln Gly His Lys Phe Asp Tyr Ile Glu Gly
245 250 255 Phe Val Ile Ile
Asn Arg Thr Gly Leu Leu Asn Ser Trp Arg Leu Ser 260
265 270 Phe Thr Ala Glu Glu Pro Leu Glu Ala
Ser Gln Phe Lys Phe Asp Gly 275 280
285 Arg Thr Leu Tyr Cys Leu Glu Leu Ala Lys Tyr Leu Lys Gln
Asp Asn 290 295 300
Lys Asp Val Ile Asn Gln Glu Val Lys Glu Thr Leu Ser Glu Leu Ser305
310 315 320 Tyr Val Thr Ser Thr
Leu Phe Thr Thr Glu Val Ala Tyr Glu Ala Phe 325
330 335 Leu Asp Arg Val His Val Ser Glu Val Lys
Leu Arg Ser Lys Gly Gln 340 345
350 Trp Glu Val Pro His Pro Trp Leu Asn Leu Leu Val Pro Arg Ser
Lys 355 360 365 Ile
Asn Glu Phe Ala Arg Gly Val Phe Gly Asn Ile Leu Thr Asp Thr 370
375 380 Ser Asn Gly Pro Val Ile
Val Tyr Pro Val Asn Lys Ser Lys Trp Asp385 390
395 400 Asn Gln Thr Ser Ala Val Thr Pro Glu Glu Glu
Val Phe Tyr Leu Val 405 410
415 Ala Ile Leu Thr Ser Ala Ser Pro Gly Ser Ala Gly Lys Asp Gly Val
420 425 430 Glu Glu Ile
Leu Arg Arg Asn Arg Arg Ile Leu Glu Phe Ser Glu Glu 435
440 445 Ala Gly Ile Gly Leu Lys Gln Tyr
Leu Pro His Tyr Thr Thr Arg Glu 450 455
460 Glu Trp Arg Ser His Phe Gly Asp Lys Trp Gly Glu Phe
Val Arg Arg465 470 475
480 Lys Ser Arg Tyr Asp Pro Leu Ala Ile Leu Ala Pro Gly His Arg Ile
485 490 495 Phe Gln Lys Ala
Val Ser Tyr Ser 500 42536PRTDendrobium 42Met
Asn Leu His Ala Met Pro Pro Phe Leu Asn Pro Thr Ser Leu Leu1
5 10 15 Leu Thr Thr Thr Leu Met
Ser Ile Leu Ile Gln Ser Pro Asn Ser Leu 20 25
30 Pro Thr Asn Leu Leu Thr His Pro Thr Ser Thr
His Leu Arg Phe Asp 35 40 45
Ser Leu Ser Leu Ser Ala Ala Ser Ser Asp Phe Gly Asp Ile Ile His
50 55 60 Ser Leu Pro
Ser Ala Val Phe Leu Pro Ser Ser Pro Ser Asp Ile Ala65 70
75 80 Thr Leu Leu Arg Leu Ser His Phe
Ser Pro His Ser Phe Thr Val Ser 85 90
95 Ala Arg Gly Leu Gly His Ser Thr Arg Gly Gln Ala Gln
Ala Phe Gly 100 105 110
Gly Ile Val Ile Asn Met Pro Ser Leu Asp Gly Gly Ile Thr Val Ser
115 120 125 Ile Asp Gly Met
Phe Val Asp Ala Gly Ala Glu Gln Met Trp Ile Asp 130
135 140 Val Leu Arg Glu Thr Leu Arg His
Gly Leu Thr Pro Lys Ser Trp Thr145 150
155 160 Asp Tyr Leu Tyr Leu Thr Leu Gly Gly Thr Leu Ser
Asn Gly Gly Ile 165 170
175 Ser Gly Gln Ala Phe Leu His Gly Pro Gln Ile Ser Asn Val His Glu
180 185 190 Leu Asp Ile
Val Thr Gly Lys Gly Glu Met Val Thr Cys Ser Glu Ser 195
200 205 Asn Asn Pro Asp Leu Phe Phe Ser
Val Leu Gly Gly Leu Gly Gln Phe 210 215
220 Gly Ile Ile Thr Arg Ala Arg Ile Ala Leu Glu Lys Ala
Pro Gln Ser225 230 235
240 Val Arg Trp Met Arg Leu Met Tyr Thr Asp Phe Glu Leu Phe Thr Lys
245 250 255 Asp Gln Glu Leu
Leu Ile Ser Ile Lys Ala Glu Gly Glu Gly Trp Lys 260
265 270 Leu Asn Tyr Val Glu Gly Ser Leu Leu
Met Glu His Ser Leu Lys Ser 275 280
285 Asn Trp Arg Ser Pro Phe Phe Ser Glu Lys Asp Leu Lys Lys
Ile Lys 290 295 300
Lys Leu Ala Ser Gly Asn Glu Gly Val Ile Tyr Cys Leu Glu Ala Ser305
310 315 320 Phe Tyr Tyr Asp Tyr
Gly His Glu Met Asn Phe Ser Arg Ala Asp Lys 325
330 335 Ala Gln Met Asp Gln Asp Ile Glu Glu Leu
Leu Arg Lys Leu Ser Phe 340 345
350 Val Ser Gly Phe Ala Phe Arg Asn Asp Val Ser Tyr Met Gly Phe
Leu 355 360 365 Asn
Arg Val His Asp Gly Glu Leu Lys Leu Arg Ala Met Gly Leu Trp 370
375 380 Asp Val Pro His Pro Trp
Leu Asn Leu Phe Val Ser Lys Ser Asn Ile385 390
395 400 Met Asp Phe His Ile Gly Val Phe Lys Gly Ile
Met Lys Asn Ser Lys 405 410
415 Ser Met Gly Pro Ile Leu Val Tyr Pro Thr Lys Arg Ser Lys Trp Asp
420 425 430 Lys Arg Met
Ser Thr Ser Ile Pro Asp Glu Glu Val Phe Tyr Ser Ile 435
440 445 Gly Ile Leu Leu Ser Ser Glu Met
Asn Asp Leu Glu His Leu Glu Ser 450 455
460 His Asn Ala Glu Ile Leu Lys Phe Cys Asp Gln Gln Gly
Met Asn Tyr465 470 475
480 Lys Gln Tyr Leu Pro His Tyr Thr Ser Ile Glu Asp Trp Lys Lys His
485 490 495 Phe Gly Lys Lys
Trp Glu Arg Phe Val Glu Met Lys Ser Arg Tyr Asp 500
505 510 Pro Lys Ala Ile Leu Ser Pro Gly Gln
Lys Ile Phe Thr His Leu Val 515 520
525 Asp Glu Leu Cys Leu Ser Asp His 530
535 43526PRTHordeum vulgare 43Met Arg Gln Leu Leu Leu Gln Tyr Leu Lys
Leu Phe Leu Leu Leu Gly1 5 10
15 Leu Gly Ala Val Thr Ala Glu His Val Leu Lys His Asp Val Leu
Ala 20 25 30 Ser
Leu Gly Thr Leu Pro Leu Asp Gly His Phe Ser Phe His Asp Leu 35
40 45 Ser Ala Ala Ala Met Asp
Phe Gly Asn Leu Ser Ser Phe Pro Pro Val 50 55
60 Ala Val Leu His Pro Gly Ser Val Ala Asp Ile
Ala Thr Thr Val Arg65 70 75
80 His Val Phe Leu Met Gly Glu His Ser Ala Leu Thr Val Ala Ala Arg
85 90 95 Gly His Gly
His Ser Leu Tyr Gly Gln Ser Gln Ala Ala Gly Gly Ile 100
105 110 Val Ile Arg Met Glu Ser Leu Arg
Ser Val Lys Met Gln Val His Pro 115 120
125 Gly Ala Ser Pro Tyr Val Asp Ala Ser Gly Gly Glu Leu
Trp Ile Asn 130 135 140
Val Leu Asn Lys Thr Leu Lys Tyr Gly Leu Ala Pro Lys Ser Trp Thr145
150 155 160 Asp Tyr Leu His Leu
Thr Val Gly Gly Thr Leu Ser Asn Ala Gly Val 165
170 175 Ser Gly Gln Thr Phe Arg His Gly Pro Gln
Ile Ser Asn Val Asn Glu 180 185
190 Leu Glu Ile Val Thr Gly Arg Gly Asp Ile Val Thr Cys Ser Pro
Glu 195 200 205 Gln
Asn Ser Asp Leu Phe Arg Ala Ala Leu Gly Gly Leu Gly Gln Phe 210
215 220 Gly Ile Ile Thr Arg Ala
Arg Ile Ala Leu Glu Pro Ala Pro Gln Met225 230
235 240 Val Arg Trp Ile Arg Val Leu Tyr Leu Asp Phe
Met Ser Phe Thr Glu 245 250
255 Asp Gln Glu Met Leu Ile Ser Ala Glu Lys Thr Phe Asp Tyr Ile Glu
260 265 270 Gly Phe Val
Ile Ile Asn Arg Thr Gly Ile Leu Asn Asn Trp Arg Ser 275
280 285 Ser Phe Asn Pro Gln Asp Pro Glu
Arg Ala Ser Arg Phe Glu Thr Asp 290 295
300 Arg Lys Val Leu Phe Cys Leu Glu Met Thr Lys Asn Phe
Asn Pro Glu305 310 315
320 Glu Ala Asp Ile Met Glu Gln Glu Val His Ala Leu Leu Ser Gln Leu
325 330 335 Arg Tyr Thr Pro
Ala Ser Leu Phe His Thr Asp Val Thr Tyr Ile Glu 340
345 350 Phe Leu Asp Arg Val His Ser Ser Glu
Met Lys Leu Arg Ala Lys Gly 355 360
365 Leu Trp Glu Val Pro His Pro Trp Leu Asn Leu Ile Ile Pro
Arg Ser 370 375 380
Thr Ile His Thr Phe Ala Glu Gln Val Phe Gly Lys Ile Leu Glu Asp385
390 395 400 Asn Asn Asn Gly Pro
Ile Leu Leu Tyr Pro Val Lys Lys Ser Arg Trp 405
410 415 Asp Asn Arg Thr Ser Val Val Ile Pro Asp
Glu Glu Val Phe Tyr Leu 420 425
430 Val Gly Phe Leu Ser Ser Ala Ile Gly Pro His Ser Ile Glu His
Thr 435 440 445 Leu
Asn Leu Asn Asn Gln Ile Ile Glu Phe Ser Asn Lys Ala Ser Ile 450
455 460 Gly Val Lys Gln Tyr Leu
Pro Asn Tyr Thr Thr Glu Pro Glu Trp Lys465 470
475 480 Ala His Tyr Gly Ala Arg Trp Asp Ala Phe Gln
Gln Arg Lys Asn Thr 485 490
495 Tyr Asp Pro Leu Ala Ile Leu Ala Pro Gly Gln Lys Ile Phe Gln Lys
500 505 510 Lys Pro Ala
Ser Leu Pro Leu Ser Ser Leu Gln Tyr Leu Leu 515
520 525 44520PRTHordeum vulgare 44Met Lys Gln Leu Leu
Leu Gln Tyr Leu Lys Leu Phe Leu Leu Leu Gly1 5
10 15 Leu Ser Arg Val Thr Thr Glu His Val Pro
Lys Tyr Asp Val Leu Ala 20 25
30 Ser Leu Gly Thr Leu Pro Leu Asp Gly His Phe Ser Phe His Asp
Leu 35 40 45 Pro
Ala Ala Ala Arg Asp Phe Gly Asn Leu Ser Ser Phe Pro Pro Val 50
55 60 Ala Val Leu His Pro Gly
Ser Val Ala Asp Ile Ala Arg Thr Val Arg65 70
75 80 His Val Phe Leu Met Gly Glu His Ser Thr Leu
Thr Val Ala Ala Arg 85 90
95 Gly His Gly His Ser Leu Tyr Gly Gln Ser Gln Ala Ala Gly Gly Ile
100 105 110 Val Ile Arg
Met Glu Ser Leu Gln Ser Val Lys Met Gln Val His Pro 115
120 125 Gly Ala Ser Pro Tyr Val Asp Ala
Ser Gly Gly Glu Leu Trp Ile Asn 130 135
140 Val Leu Asn Lys Thr Leu Lys Tyr Gly Leu Ala Pro Lys
Ser Trp Thr145 150 155
160 Asp Tyr Leu His Leu Thr Val Gly Gly Thr Leu Ser Asn Ala Gly Val
165 170 175 Ser Gly Gln Thr
Phe Arg His Gly Pro Gln Ile Ser Asn Val Asn Glu 180
185 190 Leu Glu Ile Val Thr Gly Arg Gly Asp
Ile Ile Thr Cys Ser Pro Glu 195 200
205 Gln Asn Ser Asp Leu Phe His Ala Ala Leu Gly Gly Leu Gly
Gln Phe 210 215 220
Gly Ile Ile Thr Arg Ala Arg Ile Ala Leu Glu Pro Ala Pro Gln Met225
230 235 240 Val Arg Trp Ile Arg
Val Leu Tyr Leu Asp Phe Met Ser Leu Thr Glu 245
250 255 Asp Gln Glu Met Leu Ile Ser Ala Glu Lys
Thr Phe Asp Tyr Ile Glu 260 265
270 Gly Phe Val Ser Ile Asn Arg Thr Gly Ile Leu Asn Asn Trp Arg
Ser 275 280 285 Ser
Phe Asn Pro Gln Asp Pro Glu Arg Ala Ser Gln Phe Glu Thr Asp 290
295 300 Arg Lys Val Leu Phe Cys
Leu Glu Met Thr Lys Asn Phe Asn Pro Glu305 310
315 320 Glu Ala Gly Ile Met Glu Gln Ile His Ala Leu
Leu Ser Gln Leu Arg 325 330
335 Tyr Thr Pro Pro Ser Leu Phe His Thr Asp Val Thr Tyr Met Glu Phe
340 345 350 Leu Asp Arg
Val His Ser Ser Glu Ile Lys Leu Arg Ala Lys Gly Leu 355
360 365 Trp Glu Val Pro His Pro Trp Leu
Asn Leu Ile Ile Pro Arg Ser Thr 370 375
380 Val His Thr Phe Ala Lys Gln Val Phe Gly Lys Ile Leu
Glu Asp Asn385 390 395
400 Asn Asn Gly Pro Ile Leu Leu Tyr Pro Val Asn Lys Ser Arg Trp Asp
405 410 415 Asn Arg Thr Ser
Val Val Leu Pro Asp Glu Glu Val Ser Tyr Leu Val 420
425 430 Gly Phe Leu Pro Ser Ala Met Gly Pro
His Ser Ile Lys Arg Thr Leu 435 440
445 Asn Leu Asn Asn Gln Ile Ile Glu Phe Ser Asn Lys Ala Ser
Ile Gly 450 455 460
Val Lys Gln Tyr Leu Pro His Tyr Ser Thr Glu Pro Glu Trp Lys Ala465
470 475 480 His Tyr Gly Ala Arg
Trp Asp Ala Phe Gln Gln Arg Lys Asn Thr Tyr 485
490 495 Asp Pro Leu Ala Ile Leu Ala Pro Gly Gln
Arg Ile Phe Gln Lys Thr 500 505
510 Pro Ala Ser Leu Pro Leu Ser Ser 515
520 45532PRTOryza sativa 45Met Ala Ala Ile Tyr Leu Leu Ile Ala Ala Leu
Ile Ala Ser Ser His1 5 10
15 Ala Leu Ala Ala His Gly Ala Gly Gly Gly Val Pro Leu Ala Ala Ala
20 25 30 Ala Pro Leu
Pro Phe Pro Gly Asp Leu Ala Ala Ser Gly Lys Leu Arg 35
40 45 Thr Asp Pro Asn Ala Thr Val Pro
Ala Ser Met Asp Phe Gly Asn Ile 50 55
60 Thr Ala Ala Leu Pro Ala Ala Val Leu Phe Pro Gly Ser
Pro Gly Asp65 70 75 80
Val Ala Glu Leu Leu Arg Ala Ala Tyr Ala Ala Pro Gly Arg Pro Phe
85 90 95 Thr Val Ser Phe Arg
Gly Arg Gly His Ser Thr Met Gly Gln Ala Leu 100
105 110 Ala Ala Gly Gly Val Val Val His Met Gln
Ser Met Gly Gly Gly Gly 115 120
125 Ala Pro Arg Ile Asn Val Ser Ala Asp Gly Ala Tyr Val Asp
Ala Gly 130 135 140
Gly Glu Gln Leu Trp Val Asp Val Leu Arg Ala Ala Leu Ala Arg Gly145
150 155 160 Val Ala Pro Arg Ser
Trp Thr Asp Tyr Leu His Leu Thr Val Gly Gly 165
170 175 Thr Leu Ser Asn Ala Gly Val Ser Gly Gln
Thr Tyr Arg His Gly Pro 180 185
190 Gln Ile Ser Asn Val Leu Glu Leu Asp Val Ile Thr Gly His Gly
Glu 195 200 205 Thr
Val Thr Cys Ser Lys Ala Val Asn Ser Asp Leu Phe Asp Ala Val 210
215 220 Leu Gly Gly Leu Gly Gln
Phe Gly Val Ile Thr Arg Ala Arg Val Ala225 230
235 240 Val Glu Pro Ala Pro Ala Arg Ala Arg Trp Val
Arg Leu Val Tyr Ala 245 250
255 Asp Phe Ala Ala Phe Ser Ala Asp Gln Glu Arg Leu Val Ala Ala Arg
260 265 270 Pro Asp Gly
Ser His Gly Pro Trp Ser Tyr Val Glu Gly Ala Val Tyr 275
280 285 Leu Ala Gly Arg Gly Leu Ala Val
Ala Leu Lys Ser Ser Gly Gly Phe 290 295
300 Phe Ser Asp Ala Asp Ala Ala Arg Val Val Ala Leu Ala
Ala Ala Arg305 310 315
320 Asn Ala Thr Ala Val Tyr Ser Ile Glu Ala Thr Leu Asn Tyr Ala Ala
325 330 335 Asn Ala Thr Pro
Ser Ser Val Asp Ala Ala Val Ala Ala Ala Leu Gly 340
345 350 Asp Leu His Phe Glu Glu Gly Phe Ser
Phe Ser Arg Asp Val Thr Tyr 355 360
365 Glu Glu Phe Leu Asp Arg Val Tyr Gly Glu Glu Glu Ala Leu
Glu Lys 370 375 380
Ala Gly Leu Trp Arg Val Pro His Pro Trp Leu Asn Leu Phe Val Pro385
390 395 400 Gly Ser Arg Ile Ala
Asp Phe Asp Arg Gly Val Phe Lys Gly Ile Leu 405
410 415 Gln Thr Ala Thr Asp Ile Ala Gly Pro Leu
Ile Ile Tyr Pro Val Asn 420 425
430 Lys Ser Lys Trp Asp Ala Ala Met Ser Ala Val Thr Pro Glu Gly
Glu 435 440 445 Glu
Glu Val Phe Tyr Val Val Ser Leu Leu Phe Ser Ala Val Ala Asn 450
455 460 Asp Val Ala Ala Leu Glu
Ala Gln Asn Arg Arg Ile Leu Arg Phe Cys465 470
475 480 Asp Leu Ala Gly Ile Gly Tyr Lys Ala Tyr Leu
Ala His Tyr Asp Ser 485 490
495 Arg Gly Asp Trp Val Arg His Phe Gly Ala Lys Trp Asp Arg Phe Val
500 505 510 Gln Arg Lys
Asp Lys Tyr Asp Pro Lys Lys Leu Leu Ser Pro Gly Gln 515
520 525 Asp Ile Phe Asn 530
46558PRTOryza sativa 46Met Ala Val Leu Leu Met Leu Asn Cys Phe Val Lys
Ala Thr Ala Pro1 5 10 15
Pro Pro Trp Pro Pro Ser Ala Ser Ser Ala Ser Phe Leu Asp Asp Leu
20 25 30 Gly Asp Leu Gly
Ile Ala Pro Leu Ile Arg Ala Asp Glu Ala Gly Thr 35
40 45 Ala Arg Ala Ser Ala Asp Phe Gly Asn
Leu Ser Val Ala Gly Val Gly 50 55 60
Ala Pro Arg Leu Ala Ala Ala Ala Ala Val Leu Tyr Pro Ser
Arg Pro65 70 75 80
Ala Asp Ile Ala Ala Leu Leu Arg Ala Ser Cys Ala Arg Pro Ala Pro
85 90 95 Phe Ala Val Ser Ala
Arg Gly Cys Gly His Ser Val His Gly Gln Ala 100
105 110 Ser Ala Pro Asp Gly Val Val Val Asp Met
Ala Ser Leu Gly Arg Leu 115 120
125 Gln Gly Gly Gly Ala Arg Arg Leu Ala Val Ser Val Glu Gly
Arg Tyr 130 135 140
Val Asp Ala Gly Gly Glu Gln Leu Trp Val Asp Val Leu Arg Ala Ser145
150 155 160 Met Ala His Gly Leu
Thr Pro Val Ser Trp Thr Asp Tyr Leu His Leu 165
170 175 Thr Val Gly Gly Thr Leu Ser Asn Ala Gly
Ile Ser Gly Gln Ala Phe 180 185
190 Arg His Gly Pro Gln Ile Ser Asn Val Leu Glu Leu Asp Val Ile
Thr 195 200 205 Gly
Val Gly Glu Met Val Thr Cys Ser Lys Glu Lys Ala Pro Asp Leu 210
215 220 Phe Asp Ala Val Leu Gly
Gly Leu Gly Gln Phe Gly Val Ile Thr Arg225 230
235 240 Ala Arg Ile Pro Leu Ala Pro Ala Pro Ala Arg
Ala Arg Trp Val Arg 245 250
255 Phe Val Tyr Thr Thr Ala Ala Ala Met Thr Ala Asp Gln Glu Arg Leu
260 265 270 Ile Ala Val
Asp Arg Ala Gly Gly Ala Gly Ala Val Gly Gly Leu Met 275
280 285 Asp Tyr Val Glu Gly Ser Val His
Leu Asn Gln Gly Leu Val Glu Thr 290 295
300 Trp Arg Thr Gln Pro Gln Pro Pro Ser Pro Ser Ser Ser
Ser Ser Ser305 310 315
320 Ser Phe Phe Ser Asp Ala Asp Glu Ala Arg Val Ala Ala Leu Ala Lys
325 330 335 Glu Ala Gly Gly
Val Leu Tyr Phe Leu Glu Gly Ala Ile Tyr Phe Gly 340
345 350 Gly Ala Ala Gly Pro Ser Ala Ala Asp
Val Asp Lys Arg Met Asp Val 355 360
365 Leu Arg Arg Glu Leu Arg His Glu Arg Gly Phe Val Phe Ala
Gln Asp 370 375 380
Val Ala Tyr Ala Gly Phe Leu Asp Arg Val His Asp Gly Glu Leu Lys385
390 395 400 Leu Arg Ala Ala Gly
Leu Trp Asp Val Pro His Pro Trp Leu Asn Leu 405
410 415 Phe Leu Pro Arg Ser Gly Val Leu Ala Phe
Ala Asp Gly Val Phe His 420 425
430 Gly Ile Leu Ser Arg Thr Pro Ala Met Gly Pro Val Leu Ile Tyr
Pro 435 440 445 Met
Asn Arg Asn Lys Trp Asp Ser Asn Met Ser Ala Val Ile Thr Asp 450
455 460 Asp Asp Gly Asp Glu Val
Phe Tyr Thr Val Gly Ile Leu Arg Ser Ala465 470
475 480 Ala Ala Ala Gly Asp Val Gly Arg Leu Glu Glu
Gln Asn Asp Glu Ile 485 490
495 Leu Gly Phe Cys Glu Val Ala Gly Ile Ala Tyr Lys Gln Tyr Leu Pro
500 505 510 Tyr Tyr Gly
Ser Gln Ala Glu Trp Gln Lys Arg His Phe Gly Ala Asn 515
520 525 Leu Trp Pro Arg Phe Val Gln Arg
Lys Ser Lys Tyr Asp Pro Lys Ala 530 535
540 Ile Leu Ser Arg Gly Gln Gly Ile Phe Thr Ser Pro Leu
Ala545 550 555 47527PRTOryza
sativa 47Met Glu Val Ala Met Val Cys Thr Arg Val Asn Leu Leu Ile Leu Ile1
5 10 15 Leu Ser Leu
Cys Ser Pro Tyr Lys Phe Ile Gln Ser Pro Met Asp Phe 20
25 30 Gly Pro Leu Asn Leu Leu Pro Thr
Thr Thr Thr Ala Ser Ser Asp Phe 35 40
45 Gly Arg Ile Leu Phe His Ser Pro Ser Ala Val Leu Lys
Pro Gln Ala 50 55 60
Pro Arg Asp Ile Ser Leu Leu Leu Ser Phe Leu Ser Ala Ser Pro Leu65
70 75 80 Gly Lys Val Thr Val
Ala Ala Arg Gly Ala Gly His Ser Ile His Gly 85
90 95 Gln Ala Gln Ala Leu Asp Gly Ile Val Val
Glu Met Ser Ser Leu Pro 100 105
110 Ser Glu Ile Glu Phe Tyr Arg Arg Gly Glu Gly Asp Val Ser Tyr
Ala 115 120 125 Asp
Val Gly Gly Gly Ile Met Trp Ile Glu Leu Leu Glu Gln Ser Leu 130
135 140 Lys Leu Gly Leu Ala Pro
Arg Ser Trp Thr Asp Tyr Leu Tyr Leu Thr145 150
155 160 Ile Gly Gly Thr Leu Ser Asn Ala Gly Ile Ser
Gly Gln Thr Phe Lys 165 170
175 His Gly Pro Gln Ile Ser Asn Val Leu Gln Leu Glu Val Val Thr Gly
180 185 190 Arg Gly Glu
Ile Val Thr Cys Ser Pro Thr Lys Asp Ala Glu Leu Phe 195
200 205 Asn Ala Val Leu Gly Gly Leu Gly
Gln Phe Gly Ile Ile Thr Arg Ala 210 215
220 Arg Ile Leu Leu Gln Glu Ala Pro Gln Lys Val Lys Trp
Val Arg Ala225 230 235
240 Phe Tyr Asp Asp Phe Ala Thr Phe Thr Lys Asp Gln Glu Leu Leu Val
245 250 255 Ser Met Pro Val
Leu Val Asp Tyr Val Glu Gly Phe Ile Val Leu Asn 260
265 270 Glu Gln Ser Leu His Ser Ser Ser Ile
Ala Phe Pro Thr Asn Val Asp 275 280
285 Phe Asn Pro Asp Phe Gly Thr Lys Asn Asn Pro Lys Ile Tyr
Tyr Cys 290 295 300
Ile Glu Phe Ala Val His Asp Tyr Gln Asn Lys Asn Ile Asn Val Glu305
310 315 320 Gln Val Val Glu Val
Ile Ser Arg Gln Met Ser His Ile Ala Ser His 325
330 335 Leu Tyr Ser Val Glu Val Ser Tyr Phe Asp
Phe Leu Asn Arg Val Arg 340 345
350 Met Glu Glu Met Ser Leu Arg Asn Ser Gly Leu Trp Glu Val His
His 355 360 365 Pro
Trp Leu Asn Met Phe Val Pro Ser Ala Gly Ile Ser Asp Phe Arg 370
375 380 Asp Leu Leu Met Asp Ser
Ile Ser Pro Asp Asn Phe Glu Gly Leu Ile385 390
395 400 Leu Ile Tyr Pro Leu Leu Arg His Lys Trp Asp
Thr Asn Thr Ser Val 405 410
415 Val Leu Pro Asp Ser Gly Ser Thr Asp Gln Val Met Tyr Ala Val Gly
420 425 430 Ile Leu Arg
Ser Ala Asn Pro Asp Asp Gly Cys Ser His His Cys Leu 435
440 445 Gln Glu Leu Leu Leu Arg His Arg
Arg Leu Ala Gly Ala Ala Ala Ser 450 455
460 Gly Leu Gly Ala Lys Gln Tyr Leu Ala His His Pro Thr
Pro Ala Gly465 470 475
480 Trp Arg Arg His Phe Gly Arg Arg Trp Glu Arg Phe Ala Asp Arg Lys
485 490 495 Ala Arg Phe Asp
Pro Arg Cys Ile Leu Gly Pro Gly Gln Gly Ile Phe 500
505 510 Pro Arg Asp Ser Ser Ser Ser Asn Gly
Ala Phe Ala Ser Tyr Ser 515 520
525 48525PRTOryza sativa 48Met Lys Pro Ser Ile Val His Cys Leu
Lys Leu Leu Met Leu Leu Ala1 5 10
15 Leu Gly Gly Val Thr Met His Val Pro Asp Glu Asp Asp Val
Val Ala 20 25 30
Ser Leu Gly Ala Leu Arg Leu Asp Gly His Phe Ser Phe Asp Asp Ala 35
40 45 His Ala Ala Ala Arg
Asp Phe Gly Asn Arg Cys Ser Leu Leu Pro Ala 50 55
60 Ala Val Leu His Pro Gly Ser Val Ser Asp
Val Ala Ala Thr Val Arg65 70 75
80 Arg Val Phe Gln Leu Gly Arg Ser Ser Pro Leu Thr Val Ala Ala
Arg 85 90 95 Gly
His Gly His Ser Leu Leu Gly Gln Ser Gln Ala Ala Gly Gly Ile
100 105 110 Val Val Lys Met Glu
Ser Leu Ala Ala Ala Ala Ala Arg Ala Val Arg 115
120 125 Val His Gly Gly Ala Ser Pro His Val
Asp Ala Pro Gly Gly Glu Leu 130 135
140 Trp Ile Asn Val Leu His Glu Thr Leu Lys His Gly Leu
Ala Pro Arg145 150 155
160 Ser Trp Thr Asp Tyr Leu His Leu Thr Val Gly Gly Thr Leu Ser Asn
165 170 175 Ala Gly Val Ser
Gly Gln Ala Phe Arg His Gly Pro Gln Val Ser Asn 180
185 190 Val Asn Gln Leu Glu Ile Val Thr Gly
Arg Gly Glu Val Val Thr Cys 195 200
205 Ser His Glu Val Asn Ser Asp Leu Phe Tyr Ala Ala Leu Gly
Gly Leu 210 215 220
Gly Gln Phe Gly Ile Ile Thr Arg Ala Arg Ile Ala Leu Glu Pro Ala225
230 235 240 Pro Lys Met Val Arg
Trp Ile Arg Val Leu Tyr Ser Asp Phe Glu Thr 245
250 255 Phe Thr Glu Asp Gln Glu Lys Leu Ile Ala
Ser Glu Lys Thr Phe Asp 260 265
270 Tyr Ile Glu Gly Phe Val Ile Ile Asn Arg Thr Gly Ile Leu Asn
Asn 275 280 285 Trp
Arg Thr Ser Phe Lys Pro Gln Asp Pro Val Gln Ala Ser Gln Phe 290
295 300 Gln Ser Asp Gly Arg Val
Leu Tyr Cys Leu Glu Leu Thr Met Asn Phe305 310
315 320 Asn His Asp Glu Ala Asp Ile Met Glu Gln Glu
Val Gly Ala Leu Leu 325 330
335 Ser Arg Leu Arg Tyr Ile Ser Ser Thr Leu Phe Tyr Thr Asp Val Thr
340 345 350 Tyr Leu Glu
Phe Leu Asp Arg Val His Thr Ser Glu Leu Lys Leu Arg 355
360 365 Ala Gln Gly Leu Trp Glu Val Pro
His Pro Trp Leu Asn Leu Leu Ile 370 375
380 Pro Arg Ser Thr Val His Lys Phe Ala Lys Glu Val Phe
Gly Lys Ile385 390 395
400 Leu Lys Asp Ser Asn Asn Gly Pro Ile Leu Leu Tyr Pro Val Asn Arg
405 410 415 Thr Lys Trp Asp
Asn Arg Thr Ser Val Val Ile Pro Asp Glu Glu Ile 420
425 430 Phe Tyr Leu Val Gly Phe Leu Ser Ser
Ala Pro Ser Ser Ser Gly His 435 440
445 Gly Ser Val Glu His Ala Met Asn Leu Asn Asn Lys Ile Val
Asp Phe 450 455 460
Cys Glu Lys Asn Gly Val Gly Met Lys Gln Tyr Leu Ala Pro Tyr Thr465
470 475 480 Thr Gln Lys Gln Trp
Lys Ala His Phe Gly Ala Arg Trp Glu Thr Phe 485
490 495 Glu Arg Arg Lys His Thr Tyr Asp Pro Leu
Ala Ile Leu Ala Pro Gly 500 505
510 Gln Arg Ile Phe Pro Lys Ala Ser Leu Pro Met Ser Leu
515 520 525 49532PRTOryza sativa 49Met
Ala Trp Cys Leu Val Phe Met Val Phe Leu Ile Tyr Cys Leu Ile1
5 10 15 Ser Thr Val Gly Leu Pro
Val Ala Pro Ala Asp Glu Ala Ala Met Gln 20 25
30 Leu Gly Gly Val Gly Gly Gly Arg Leu Ser Val
Glu Pro Ser Asp Val 35 40 45
Met Glu Ala Ser Leu Asp Phe Gly Arg Leu Thr Ser Ala Glu Pro Leu
50 55 60 Ala Val Phe
His Pro Arg Gly Ala Gly Asp Val Ala Ala Leu Val Lys65 70
75 80 Ala Ala Tyr Gly Ser Ala Ser Gly
Ile Arg Val Ser Ala Arg Gly His 85 90
95 Gly His Ser Ile Ser Gly Gln Ala Gln Ala Ala Gly Gly
Val Val Val 100 105 110
Asp Met Ser His Gly Trp Arg Ala Glu Ala Ala Glu Arg Thr Leu Pro
115 120 125 Val Tyr Ser Pro
Ala Leu Gly Gly His Tyr Ile Asp Val Trp Gly Gly 130
135 140 Glu Leu Trp Ile Asp Val Leu Asn
Trp Thr Leu Ala His Gly Gly Leu145 150
155 160 Ala Pro Arg Ser Trp Thr Asp Tyr Leu Tyr Leu Ser
Val Gly Gly Thr 165 170
175 Leu Ser Asn Ala Gly Ile Ser Gly Gln Ala Phe His His Gly Pro Gln
180 185 190 Ile Ser Asn
Val Tyr Glu Leu Asp Val Val Thr Lys Gly Glu Val Val 195
200 205 Thr Cys Ser Glu Ser Asn Asn Pro
Asp Leu Phe Phe Gly Ala Leu Gly 210 215
220 Gly Leu Gly Gln Leu Gly Ile Ile Thr Arg Ala Arg Ile
Ala Leu Glu225 230 235
240 Pro Ala Pro His Arg Val Arg Trp Ile Arg Ala Leu Tyr Ser Asn Phe
245 250 255 Thr Glu Phe Thr
Ala Asp Gln Glu Arg Leu Ile Ser Leu Gln His Gly 260
265 270 Gly Arg Arg Phe Asp Tyr Val Glu Gly
Phe Val Val Ala Ala Glu Gly 275 280
285 Leu Ile Asn Asn Trp Arg Ser Ser Phe Phe Ser Pro Gln Asn
Pro Val 290 295 300
Lys Leu Ser Ser Leu Lys His His Ser Gly Val Leu Tyr Cys Leu Glu305
310 315 320 Val Thr Lys Asn Tyr
Asp Asp Ser Thr Ala Val Thr Val Asp Gln Asp 325
330 335 Val Glu Ala Leu Leu Gly Glu Leu Asn Phe
Ile Pro Gly Thr Val Phe 340 345
350 Thr Thr Asp Leu Pro Tyr Val Asp Phe Leu Asp Arg Val His Lys
Ala 355 360 365 Glu
Leu Lys Leu Arg Gly Lys Gly Met Trp Glu Val Pro His Pro Trp 370
375 380 Leu Asn Leu Phe Val Pro
Ala Ser Arg Ile Ala Asp Phe Asp Arg Gly385 390
395 400 Val Phe Arg Gly Val Leu Gly Ser Arg Thr Ala
Gly Gly Pro Ile Leu 405 410
415 Ile Tyr Pro Met Asn Arg His Trp Asp Pro Arg Ser Ser Val Val Thr
420 425 430 Pro Glu Glu
Asp Val Phe Tyr Leu Val Ala Phe Leu Arg Ser Ala Val 435
440 445 Pro Gly Ser Thr Asp Pro Ala Gln
Ser Leu Glu Ala Leu Glu Arg Gln 450 455
460 Asn Arg Glu Ile Leu Glu Phe Cys Asp Glu Ala Gly Ile
Gly Ala Lys465 470 475
480 Gln Tyr Leu Pro Asn His Lys Ala Gln Arg Glu Trp Glu Ala His Phe
485 490 495 Gly Ala Arg Trp
Ala Arg Phe Ala Arg Leu Lys Ala Glu Phe Asp Pro 500
505 510 Arg Ala Met Leu Ala Thr Gly Gln Gly
Ile Phe Asp Ser Pro Pro Leu 515 520
525 Leu Ala Glu Ser 530 50650PRTArtificial
SequenceConsensus sequence 50Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa1 5 10
15 Leu Leu Xaa Xaa Xaa Leu Leu Xaa Leu Leu Xaa Xaa Leu Xaa Xaa Xaa
20 25 30 Xaa Ala Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ser Xaa Leu Xaa Xaa 35
40 45 Leu Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 50 55
60 Xaa Xaa Xaa Leu Xaa Xaa Xaa Xaa Xaa Xaa Val Ala Ala
Ala Ser Xaa65 70 75 80
Asp Phe Gly Asn Ile Xaa Xaa Xaa Xaa Pro Xaa Xaa Xaa Xaa Xaa Xaa
85 90 95 Xaa Xaa Ala Ala Val
Leu His Pro Xaa Ser Xaa Xaa Asp Ile Ala Xaa 100
105 110 Leu Leu Arg Xaa Ala Xaa Xaa Ser Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa 115 120
125 Xaa Xaa Xaa Ser Xaa Leu Thr Val Ala Ala Arg Gly Xaa Gly
His Ser 130 135 140
Leu Xaa Gly Gln Ala Xaa Ala Xaa Gly Xaa Gly Val Val Val Xaa Met145
150 155 160 Xaa Ser Leu Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Val Xaa Xaa Val 165
170 175 Xaa Xaa Xaa Xaa Xaa Gly Xaa Xaa Xaa Xaa
Xaa Tyr Val Asp Val Xaa 180 185
190 Gly Gly Xaa Leu Trp Ile Asp Val Leu Arg Xaa Thr Leu Lys His
Gly 195 200 205 Xaa
Leu Ala Pro Arg Ser Trp Thr Asp Tyr Leu His Leu Thr Val Gly 210
215 220 Gly Thr Leu Ser Asn Ala
Gly Ile Ser Gly Gln Ala Phe Arg His Gly225 230
235 240 Pro Gln Ile Ser Asn Val Xaa Glu Leu Asp Val
Val Thr Gly Lys Gly 245 250
255 Glu Ile Val Thr Cys Ser Xaa Xaa Xaa Asn Ser Asp Leu Phe Phe Ala
260 265 270 Val Leu Gly
Gly Leu Gly Gln Phe Gly Ile Ile Thr Arg Ala Arg Ile 275
280 285 Ala Leu Glu Pro Ala Pro Xaa Arg
Val Arg Trp Ile Arg Val Leu Tyr 290 295
300 Ser Asp Phe Xaa Xaa Phe Thr Xaa Asp Gln Glu Xaa Leu
Ile Ser Xaa305 310 315
320 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Phe Asp Tyr Val
325 330 335 Glu Gly Phe Val
Ile Ile Asn Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 340
345 350 Xaa Xaa Xaa Xaa Xaa Xaa Ile Leu Asn
Xaa Trp Arg Ser Ser Phe Phe 355 360
365 Xaa Pro Xaa Asp Xaa Xaa Arg Ile Ser Xaa Leu Xaa Xaa Xaa
Xaa Xaa 370 375 380
Xaa Val Leu Tyr Cys Leu Glu Val Ala Lys Tyr Tyr Xaa Xaa Xaa Xaa385
390 395 400 Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Val Asp Gln Glu Val 405
410 415 Glu Xaa Leu Leu Xaa Xaa Leu Xaa Phe Ile
Xaa Gly Xaa Leu Phe Xaa 420 425
430 Xaa Asp Val Thr Tyr Val Asp Phe Leu Asp Arg Val His Xaa Xaa
Glu 435 440 445 Leu
Lys Leu Arg Ala Xaa Gly Leu Trp Xaa Glu Val Pro His Pro Trp 450
455 460 Leu Asn Leu Phe Val Pro
Arg Ser Xaa Ile Xaa Asp Phe Xaa Xaa Gly465 470
475 480 Val Phe Lys Gly Ile Leu Xaa Xaa Xaa Xaa Xaa
Xaa Gly Gly Pro Ile 485 490
495 Leu Ile Tyr Pro Val Asn Arg Ser Lys Trp Asp Xaa Arg Thr Ser Val
500 505 510 Val Ile Pro
Xaa Xaa Xaa Xaa Xaa Xaa Asp Glu Glu Val Phe Tyr Leu 515
520 525 Val Gly Leu Leu Xaa Ser Ala Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 530 535
540 Xaa Xaa Xaa Xaa Xaa Val Glu Xaa Leu Leu Xaa Xaa Asn
Xaa Arg Ile545 550 555
560 Leu Xaa Phe Cys Glu Xaa Ala Gly Ile Gly Val Lys Gln Tyr Leu Pro
565 570 575 His Tyr Thr Thr
Xaa Xaa Glu Trp Xaa Arg Xaa His Phe Gly Xaa Ala 580
585 590 Arg Trp Asp Arg Phe Xaa Xaa Arg Lys
Ala Arg Tyr Asp Pro Lys Ala 595 600
605 Ile Leu Ala Pro Gly Gln Arg Ile Phe Gln Lys Xaa Xaa Xaa
Xaa Xaa 610 615 620
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa625
630 635 640 Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa 645 650 517366DNAZea
maysmisc_feature(1)...(7366)ZmCkx6 genomic sequence 51aaaaactgtg
cagctagcta agagaagctg aaaaacagtt ttttttttta aaaaaaatct 60gtctactctt
agagcatctc caacaacgtg acctataaaa ttgccctata atttgaaaat 120aagtatattt
tatagaattt agggcaccaa caaaacacct cgctccaaca gtaaagtccc 180aaatctagat
tatagggcag accactacag tgtagtatat ttgagtcact tgagagggtg 240ctctatagtt
ttttgacaaa aaattatgaa atatggcact gttggagtag tttttcctgt 300gtagagccct
atatttcaat tttaggcact agtttaaggc attgttggag atgctcttat 360tttttaacga
aaagctgaaa aactggcctt cgattgataa aaaaacattc agattaataa 420tgttgtgagt
ggtacctatg ccttctctta tttttttctt aatgatttat gagaaactat 480aaattcttat
attaacatat agagaaaaag gctctttgtt ttgcgaccga gcgagggagt 540atacacggat
acaccggtac ctccgctccg cacgtacctg gaggctggag cagacgtttg 600actgggacgc
gccgagtgtc cggccaatga gagcgacgca cgtagcgcgg gggcgccgct 660gcggcggcac
atcatcacgt gcatgcggcc acgcgcgcgg gcgacagaca acgcgcgagc 720gacaggtcga
cccccgtggc cgaaccgaat cgcgtagggg atctcgacct atggcagcaa 780atttaacgcc
gcgttccggt ggcggtcccg ctccagcgat ggccgcgtac cgtacctacg 840gcgaccagac
cacgggataa tgcgtgcgat tgttcttttg ggtgggggag aatgctcgat 900cgatcgcaaa
tgccggtgct ccccggccgt tcgtcgtcgg ccggtcgatc acaggtacat 960actggcagta
aaaacagacg tgcaggttcc cgacctgtca tcgtattata ttcggcgtta 1020ctgacaccat
ggcaatggca tgcatggtac gaagccaagt aaggagcaga cgtgttcgta 1080cgcctgtcgt
cgtcttcgcg cgcgcgccca cgagcagcat gtctcacgcg cccagcaaat 1140tcgcgcgcgc
ggatgcagcc cgatcggtta tattcgatcg gttataatgc atcatcgtca 1200acggcgtcaa
aacaacgcga gagaggacac ctacattttt cccctccgga aattaatctt 1260aaaatttgcg
cctcttatgc tattaatata cgtattaaaa tttgtataat ttaaaactca 1320aaaaacattg
ccaaatgcat tgacgcgatt aaaaagttaa aaaaacaaaa aggataagaa 1380taagtgtagc
tacttttgaa ctttaaaacg tggtaaggct acagtgcagc tacctttgtc 1440tagttactgc
ctcgtgcgtg gaagattaga attccaccta gagtacgttt ttttccttct 1500ttttgttagt
tattactaac aataaagttc taactagaga caatttggct aattaaaaga 1560aggaaagcag
aggatgcaag ctgcctgttc tgtacagagc ctgaatatgc acgtcatctc 1620tgaagttact
aaccgtaatt taggagagaa aatatagcag agacaggaaa atcgttcggt 1680gtatctggaa
actcacgaat gagttatgtt ttcagagaaa cttgctcgag aagcatggag 1740ctgttactac
acacgcgata agcggacttt cacagaaatg gaaaacttta cgcccgccag 1800aaacgaaaga
gcaattggag atcagatcac cgtggagaaa aataatagcg tgtttggttt 1860gtaggttggg
ctgcttctgg agccatccag acctgtgtcc gagcctacat cagcgtttgg 1920tttgaatcgc
agaatgatgt cgtccgccac tgtattgttc taataataaa ctagcatgcg 1980ggttcaactc
actccacaag gaactgccgg acggctccat ccggagccaa gccacgacgg 2040atgagcgaaa
ccgccggacc aaacgcgctg taaaagaatg cagataggtt aggttttggg 2100agttgtgtga
tcttcagctt tctgccgata ggctgtctgt aagaggtctt tcagttttgt 2160ttggttctgt
ttctggttgg aaccagttcc cttggcctca ggcttcagca caagtctagg 2220tgtgatttaa
actgcactgt attgaatact ttagtctttt gacaatactg tagttaaaag 2280gccggggggt
tttgccttgg aactctaaaa aaatatacag tattaaccat ggactctgaa 2340ctctgtctgc
gtccacaggc aagtcatctt tcttccttgc actggttatc ttattgaaac 2400agaacggaaa
tcttttttgg aacaagagaa tttcgtcaca tcttgcctgc agtaaagttt 2460cccatctaga
tgcatactcc ctccgtccaa gtttaactgg cgttttagct tttctcagac 2520ataaatacca
gccaagagaa tagacgcatg tacccctgtg atctagcgtg aagtattaat 2580tgcaattact
gctgaggaca cgaaacggtt cacaacctcc agccctccac ggtggatgag 2640aggagaccaa
gagtccgttg gtgtgggaac gaagcgaacg ggtgtgtgaa acgaggagat 2700aactataatg
gcatcgaggt gtagaccacg aacgacacat aattctggac aaataaaatg 2760agctaaaacg
ccattaaact tggacggagg gagtatacta tcaacatttc gatcaaaagt 2820tactatacaa
aatttgcact gtccgaaaag cgatccttat caggaaggcg caggattcgt 2880cccagctaag
cgcaccggcc acaagtattc caccaccccc ggtcaatagc taaagaaatt 2940gggcggcaag
tgaaagtctc cgggatggga atgtgcatga gtcatgacgc gcctccgccc 3000tccggcctcc
gcagttgttt attcgcagcg cgcgggtggc ggcccgcccg tccgtgttct 3060ctgctccctg
tgttcggcac atcgtcaccc ccaccgtttc ctgtgcctct ctctcctatc 3120ttcctcggtc
tcctcccgta atcctttgcc tgataccccg ctctaccagg ccgccaccac 3180ctccctccag
gctccagcag cctataaata cgcccgcgtc gcccaccacc gcacaccact 3240tgaatactcc
atctcaactt cccttcctct cccgtgctgc gctgagctat atagctgctc 3300ctcgacctcc
aagaagcacg cgggcggagc ccggagcgag tgattagtga aaggcatagc 3360ataaggccgc
ccggccggga agtggtggca atgacgcggt gcctcatgtt cacgctgctg 3420ttcctcgtct
cctccctcat ctccaccgtg gggctccccg tcgagccgcc cgcggagctc 3480ctgcagctgg
gcggcgggga cgtcggcggc gggcgcctga gcgtcgacgc gtccgacatc 3540gcggaggcgt
cgcgcgactt cgggggcgtc gcccgcgccg agcccatggc ggtgttccat 3600ccgcgcgcgg
ccggcgacgt ggcgggcctg gtcggcgccg cgttccggtc ggcgcgcggc 3660ttccgcgtct
cggcgcgggg ccacggccac tccatcagcg gccaggcgca ggcggccggc 3720ggcgtggtcg
tggacatgag ccgcggccgc ggccccggcg ccgccgtggc gcgggcattg 3780cccgtgcact
cggcggcgct gggcgggcac tacgtggacg tctggggcgg cgagctgtgg 3840gtggacgtgc
tcaactggac gctgtcccac ggcgggctgg cgccgcggtc gtggacggac 3900tacctgtacc
tgtccgtggg cggcaccctc tccaacgccg gcatcagcgg gcaggcgttc 3960caccacgggc
cacagatcag caatgtctac gagctcgacg tcgtcacagg tagcgtagca 4020gctagctagg
cgatcgagcc ggccgacgag tcgtagatgc aaggcgctgt ctctggcggg 4080ccgatcgacg
aatgaaccga ctgactgaca cacgtgcgct gtgcgttggc agggaaggga 4140gaggtggtga
cctgctcgga gacggagaac ccggacctgt tcttcggcgt cctgggcggg 4200ctgggccagt
tcggcatcat cacgcgggcg cgcatcgccc tggagcgtgc tcccaagagg 4260gtaagtaaac
agcttaggca gcccacaact gcgttctcct cgctcccttc tgctctctgt 4320cgtgtctgga
cctcccttca ggcgcgcgcc ctgatggatc accacctgca ccgattagat 4380agcctttaat
ttccctcccg tgagtcgtgt ctcgatcgtg tggcaccggg aaaggaacac 4440agggcggggc
gggcagtgca tctcctcccg tggcgtgtgt caccggcctc gctttccaac 4500taatgaccga
cccagcacac cggccggcca tgcaagtggc gagcgagcgc gctgctgatc 4560cgttgaggaa
agcgcccctc tgccgtccgc taataaaacg gctgccacat gtgtagcgtc 4620cgaaaaaaga
tccacgcgta gtacgtgtac gtcttgataa tcgccactgt agtatccgtg 4680ggtctatcta
taagcagggc ccccagccgc ccgggccagc catggccagt ccgacacata 4740cgtacgcgtc
atccggtcag gtgcatggtc cacggtgccc ctgctcaacg attagccgcc 4800gccgccgtgt
cgtcgtcatc gagtccgcct ttttcttttt gtttagctcg tgggatcttt 4860cgcaagattt
tgttcctctg ctaattaatc ggcctgtaat cttagtagca gctaagaatt 4920gacgggcgga
atgcatgggt ggtggtggtg gtggtggttg cttgcaggtt cggtggatcc 4980gggcgctcta
ctccaacttc agcgagttca cggcggacca ggagcgcctc atctccctcg 5040gcagcggcgg
cggacgccgg ttcgactacg tggagggctt cgtcgtcgcc gccgagggcc 5100tcatcaacaa
ctggaggtcc tccttcttct cgccgcagaa ccccgtgaag ctcacctcgc 5160tcaagcacca
ttccagcgtc ctctactgcc tcgaggtcac caagaactac gacgacgaaa 5220ccgcagggtc
ggtcgaccag gtaggtactc ggactcggag ccttgctttt cagttactag 5280ccgtatgata
acagtagcag gcagtgtact gtgtgtgcta atatttttct tcttctctcc 5340gttcaggacg
tggatacgct gctgggcgag ctgaacttcc tccctggcac ggtgttcact 5400acggacctgc
cgtacgtgga cttcctggac cgcgtgcaca aggcggagct gaagctgcgc 5460gccaagggga
tgtgggaggt gccgcacccg tggctcaacc tcttcgtgcc ggcgtcccgc 5520atcgccgact
tcgaccgcgg cgtcttccgt ggcgtgctgg ggggccgcac cgccggcgcc 5580ggcggccccg
tcctcatcta ccccatgaac aagcacaagt aagtcgtcaa atcacaaagg 5640cacacacgga
ccacaagcca aaggccgcgc gcgctcatcg atctgccgcc acgcacgcgg 5700aggcgcggcg
cgtcgccctc gccgtccccg cccccgcccc gcgcttccaa ccaaccaacc 5760ttccattcca
ttccatccac atgcggcgct cgggacacgg ggacaggcgg acagcaccct 5820ccctcacatg
cgccgcccac acgcggacgg caccgcacgc gcagcgcagg tgccggctcg 5880ctcatgtgtc
atgtgttgcg attgcgatgg acttgtgccg cccattattg gcgccacgca 5940cgcaaatcac
cagccagccc atccgtagca gcggattatt atttgcccaa ctgcgcgagc 6000cgaatctgga
gccccgatag atagaatggc cgtggacaaa ccgcgcactc gcgcgacagg 6060gcctactcta
ttctgttgca atggacgcca cgcaactgcg gccggtgacg ccgacctgcc 6120atttataaac
cggcgccgcg acgagagctt tacagggcgc aacgagctcg gagcggtcag 6180aatcggaatt
ccttagctga gacctgctcg ccttttagta ctttgtttgc attggggtca 6240gtgggccggg
tagacgccta gacggcacca atttgtttgt tgctttcagc ttcagcgcat 6300acacaaccaa
cctaaaaaaa agacgtatca gaatcaccag acgatcctgc taaaaaccgc 6360tgctttttta
tttatttttt cccgtactct attctgtact agtatcgtgc agtatcccaa 6420acccttttcg
cgtcgattag cgactgcagc tctgagatct ggactgggcc gtgtggctga 6480cgggctgctt
cgtgcgcgca ggtgggaccc gaggagctcg gcggtgaccc cggacgagga 6540ggtgttctac
ctggtggcgt tcctgcggtc ggcgctgccg ggcgcgccgg agagcctgga 6600ggcgctggcg
cggcagaacc agcggatcct cgacttctgc gcgggcgcgg gcatcggcgc 6660caagcagtac
ctgccgggcc acaaggcgcg gcacgagtgg gcggagcact tcggcgccgc 6720gaggtgggac
cggttcgcga ggctcaaggc cgagttcgac ccgcgggcca tcctggcggc 6780ggggcagggc
atcttcaggc cgcccggctc gccggcgctc gcggccgact cgtaacgtaa 6840tccagctgct
tatactaatt attaggcgcg tttagtgtaa ggtagaggta ctagctacag 6900cagtaaccat
acaggattgt ttagttagct ccggttggtt catgtacaaa tgtggggttg 6960ttaatcgcgt
gctctgccat ggctgctgtg atcggttctg tacaggggat gaggggagcc 7020aaatatgaac
gtggcaaaat cgatacttct tataaagaaa aaatatctat ggtaaatact 7080ggtgcctgcc
tctgtttccc gtcaaactac agtgcagtgt attgttttct ccgtggtcca 7140ctggatataa
ggcattgcct gcaccttcat cctaaattat aattcatttg actttttctg 7200tcacgtttga
ccgatcttcc tatttattta ttttttaaaa taaatgaaaa ctcaaatata 7260aagtatatta
tgtgctaaac aatattacga taaaaaaata acaataatta tgatattttt 7320taattcgaac
gggttttcca agcacaaaaa cgcatatata tgtatg 7366521623DNAZea
maysCDS(1)...(1623) 52atg acg cgg tgc ctc atg ttc acg ctg ctg ttc ctc gtc
tcc tcc ctc 48Met Thr Arg Cys Leu Met Phe Thr Leu Leu Phe Leu Val
Ser Ser Leu1 5 10 15atc
tcc acc gtg ggg ctc ccc gtc gag ccg ccc gcg gag ctc ctg cag 96Ile
Ser Thr Val Gly Leu Pro Val Glu Pro Pro Ala Glu Leu Leu Gln 20
25 30ctg ggc ggc ggg gac gtc ggc ggc
ggg cgc ctg agc gtc gac gcg tcc 144Leu Gly Gly Gly Asp Val Gly Gly
Gly Arg Leu Ser Val Asp Ala Ser 35 40
45gac atc gcg gag gcg tcg cgc gac ttc ggg ggc gtc gcc cgc gcc gag
192Asp Ile Ala Glu Ala Ser Arg Asp Phe Gly Gly Val Ala Arg Ala Glu
50 55 60ccc atg gcg gtg ttc cat ccg cgc
gcg gcc ggc gac gtg gcg ggc ctg 240Pro Met Ala Val Phe His Pro Arg
Ala Ala Gly Asp Val Ala Gly Leu65 70 75
80gtc ggc gcc gcg ttc cgg tcg gcg cgc ggc ttc cgc gtc
tcg gcg cgg 288Val Gly Ala Ala Phe Arg Ser Ala Arg Gly Phe Arg Val
Ser Ala Arg 85 90 95ggc
cac ggc cac tcc atc agc ggc cag gcg cag gcg gcc ggc ggc gtg 336Gly
His Gly His Ser Ile Ser Gly Gln Ala Gln Ala Ala Gly Gly Val
100 105 110gtc gtg gac atg agc cgc ggc
cgc ggc ccc ggc gcc gcc gtg gcg cgg 384Val Val Asp Met Ser Arg Gly
Arg Gly Pro Gly Ala Ala Val Ala Arg 115 120
125gca ttg ccc gtg cac tcg gcg gcg ctg ggc ggg cac tac gtg gac
gtc 432Ala Leu Pro Val His Ser Ala Ala Leu Gly Gly His Tyr Val Asp
Val 130 135 140tgg ggc ggc gag ctg tgg
gtg gac gtg ctc aac tgg acg ctg tcc cac 480Trp Gly Gly Glu Leu Trp
Val Asp Val Leu Asn Trp Thr Leu Ser His145 150
155 160ggc ggg ctg gcg ccg cgg tcg tgg acg gac tac
ctg tac ctg tcc gtg 528Gly Gly Leu Ala Pro Arg Ser Trp Thr Asp Tyr
Leu Tyr Leu Ser Val 165 170
175ggc ggc acc ctc tcc aac gcc ggc atc agc ggg cag gcg ttc cac cac
576Gly Gly Thr Leu Ser Asn Ala Gly Ile Ser Gly Gln Ala Phe His His
180 185 190ggg cca cag atc agc aat
gtc tac gag ctc gac gtc gtc aca ggg aag 624Gly Pro Gln Ile Ser Asn
Val Tyr Glu Leu Asp Val Val Thr Gly Lys 195 200
205gga gag gtg gtg acc tgc tcg gag acg gag aac ccg gac ctg
ttc ttc 672Gly Glu Val Val Thr Cys Ser Glu Thr Glu Asn Pro Asp Leu
Phe Phe 210 215 220ggc gtc ctg ggc ggg
ctg ggc cag ttc ggc atc atc acg cgg gcg cgc 720Gly Val Leu Gly Gly
Leu Gly Gln Phe Gly Ile Ile Thr Arg Ala Arg225 230
235 240atc gcc ctg gag cgt gct ccc aag gtt cgg
tgg atc cgg gcg ctc tac 768Ile Ala Leu Glu Arg Ala Pro Lys Val Arg
Trp Ile Arg Ala Leu Tyr 245 250
255tcc aac ttc agc gag ttc acg gcg gac cag gag cgc ctc atc tcc ctc
816Ser Asn Phe Ser Glu Phe Thr Ala Asp Gln Glu Arg Leu Ile Ser Leu
260 265 270ggc agc ggc ggc gga cgc
cgg ttc gac tac gtg gag ggc ttc gtc gtc 864Gly Ser Gly Gly Gly Arg
Arg Phe Asp Tyr Val Glu Gly Phe Val Val 275 280
285gcc gcc gag ggc ctc atc aac aac tgg agg tcc tcc ttc ttc
tcg ccg 912Ala Ala Glu Gly Leu Ile Asn Asn Trp Arg Ser Ser Phe Phe
Ser Pro 290 295 300cag aac ccc gtg aag
ctc acc tcg ctc aag cac cat tcc agc gtc ctc 960Gln Asn Pro Val Lys
Leu Thr Ser Leu Lys His His Ser Ser Val Leu305 310
315 320tac tgc ctc gag gtc acc aag aac tac gac
gac gaa acc gca ggg tcg 1008Tyr Cys Leu Glu Val Thr Lys Asn Tyr Asp
Asp Glu Thr Ala Gly Ser 325 330
335gtc gac cag gac gtg gat acg ctg ctg ggc gag ctg aac ttc ctc cct
1056Val Asp Gln Asp Val Asp Thr Leu Leu Gly Glu Leu Asn Phe Leu Pro
340 345 350ggc acg gtg ttc act acg
gac ctg ccg tac gtg gac ttc ctg gac cgc 1104Gly Thr Val Phe Thr Thr
Asp Leu Pro Tyr Val Asp Phe Leu Asp Arg 355 360
365gtg cac aag gcg gag ctg aag ctg cgc gcc aag ggg atg tgg
gag gtg 1152Val His Lys Ala Glu Leu Lys Leu Arg Ala Lys Gly Met Trp
Glu Val 370 375 380ccg cac ccg tgg ctc
aac ctc ttc gtg ccg gcg tcc cgc atc gcc gac 1200Pro His Pro Trp Leu
Asn Leu Phe Val Pro Ala Ser Arg Ile Ala Asp385 390
395 400ttc gac cgc ggc gtc ttc cgt ggc gtg ctg
ggg ggc cgc acc gcc ggc 1248Phe Asp Arg Gly Val Phe Arg Gly Val Leu
Gly Gly Arg Thr Ala Gly 405 410
415gcc ggc ggc ccc gtc ctc atc tac ccc atg aac aag cac aag tgg gac
1296Ala Gly Gly Pro Val Leu Ile Tyr Pro Met Asn Lys His Lys Trp Asp
420 425 430ccg agg agc tcg gcg gtg
acc ccg gac gag gag gtg ttc tac ctg gtg 1344Pro Arg Ser Ser Ala Val
Thr Pro Asp Glu Glu Val Phe Tyr Leu Val 435 440
445gcg ttc ctg cgg tcg gcg ctg ccg ggc gcg ccg gag agc ctg
gag gcg 1392Ala Phe Leu Arg Ser Ala Leu Pro Gly Ala Pro Glu Ser Leu
Glu Ala 450 455 460ctg gcg cgg cag aac
cag cgg atc ctc gac ttc tgc gcg ggc gcg ggc 1440Leu Ala Arg Gln Asn
Gln Arg Ile Leu Asp Phe Cys Ala Gly Ala Gly465 470
475 480atc ggc gcc aag cag tac ctg ccg ggc cac
aag gcg cgg cac gag tgg 1488Ile Gly Ala Lys Gln Tyr Leu Pro Gly His
Lys Ala Arg His Glu Trp 485 490
495gcg gag cac ttc ggc gcc gcg agg tgg gac cgg ttc gcg agg ctc aag
1536Ala Glu His Phe Gly Ala Ala Arg Trp Asp Arg Phe Ala Arg Leu Lys
500 505 510gcc gag ttc gac ccg cgg
gcc atc ctg gcg gcg ggg cag ggc atc ttc 1584Ala Glu Phe Asp Pro Arg
Ala Ile Leu Ala Ala Gly Gln Gly Ile Phe 515 520
525agg ccg ccc ggc tcg ccg gcg ctc gcg gcc gac tcg taa
1623Arg Pro Pro Gly Ser Pro Ala Leu Ala Ala Asp Ser 530
535 54053540PRTZea mays 53Met Thr Arg Cys
Leu Met Phe Thr Leu Leu Phe Leu Val Ser Ser Leu1 5
10 15 Ile Ser Thr Val Gly Leu Pro Val Glu
Pro Pro Ala Glu Leu Leu Gln 20 25
30 Leu Gly Gly Gly Asp Val Gly Gly Gly Arg Leu Ser Val Asp
Ala Ser 35 40 45
Asp Ile Ala Glu Ala Ser Arg Asp Phe Gly Gly Val Ala Arg Ala Glu 50
55 60 Pro Met Ala Val Phe
His Pro Arg Ala Ala Gly Asp Val Ala Gly Leu65 70
75 80 Val Gly Ala Ala Phe Arg Ser Ala Arg Gly
Phe Arg Val Ser Ala Arg 85 90
95 Gly His Gly His Ser Ile Ser Gly Gln Ala Gln Ala Ala Gly Gly
Val 100 105 110 Val
Val Asp Met Ser Arg Gly Arg Gly Pro Gly Ala Ala Val Ala Arg 115
120 125 Ala Leu Pro Val His Ser
Ala Ala Leu Gly Gly His Tyr Val Asp Val 130 135
140 Trp Gly Gly Glu Leu Trp Val Asp Val Leu Asn
Trp Thr Leu Ser His145 150 155
160 Gly Gly Leu Ala Pro Arg Ser Trp Thr Asp Tyr Leu Tyr Leu Ser Val
165 170 175 Gly Gly Thr
Leu Ser Asn Ala Gly Ile Ser Gly Gln Ala Phe His His 180
185 190 Gly Pro Gln Ile Ser Asn Val Tyr
Glu Leu Asp Val Val Thr Gly Lys 195 200
205 Gly Glu Val Val Thr Cys Ser Glu Thr Glu Asn Pro Asp
Leu Phe Phe 210 215 220
Gly Val Leu Gly Gly Leu Gly Gln Phe Gly Ile Ile Thr Arg Ala Arg225
230 235 240 Ile Ala Leu Glu Arg
Ala Pro Lys Val Arg Trp Ile Arg Ala Leu Tyr 245
250 255 Ser Asn Phe Ser Glu Phe Thr Ala Asp Gln
Glu Arg Leu Ile Ser Leu 260 265
270 Gly Ser Gly Gly Gly Arg Arg Phe Asp Tyr Val Glu Gly Phe Val
Val 275 280 285 Ala
Ala Glu Gly Leu Ile Asn Asn Trp Arg Ser Ser Phe Phe Ser Pro 290
295 300 Gln Asn Pro Val Lys Leu
Thr Ser Leu Lys His His Ser Ser Val Leu305 310
315 320 Tyr Cys Leu Glu Val Thr Lys Asn Tyr Asp Asp
Glu Thr Ala Gly Ser 325 330
335 Val Asp Gln Asp Val Asp Thr Leu Leu Gly Glu Leu Asn Phe Leu Pro
340 345 350 Gly Thr Val
Phe Thr Thr Asp Leu Pro Tyr Val Asp Phe Leu Asp Arg 355
360 365 Val His Lys Ala Glu Leu Lys Leu
Arg Ala Lys Gly Met Trp Glu Val 370 375
380 Pro His Pro Trp Leu Asn Leu Phe Val Pro Ala Ser Arg
Ile Ala Asp385 390 395
400 Phe Asp Arg Gly Val Phe Arg Gly Val Leu Gly Gly Arg Thr Ala Gly
405 410 415 Ala Gly Gly Pro
Val Leu Ile Tyr Pro Met Asn Lys His Lys Trp Asp 420
425 430 Pro Arg Ser Ser Ala Val Thr Pro Asp
Glu Glu Val Phe Tyr Leu Val 435 440
445 Ala Phe Leu Arg Ser Ala Leu Pro Gly Ala Pro Glu Ser Leu
Glu Ala 450 455 460
Leu Ala Arg Gln Asn Gln Arg Ile Leu Asp Phe Cys Ala Gly Ala Gly465
470 475 480 Ile Gly Ala Lys Gln
Tyr Leu Pro Gly His Lys Ala Arg His Glu Trp 485
490 495 Ala Glu His Phe Gly Ala Ala Arg Trp Asp
Arg Phe Ala Arg Leu Lys 500 505
510 Ala Glu Phe Asp Pro Arg Ala Ile Leu Ala Ala Gly Gln Gly Ile
Phe 515 520 525 Arg
Pro Pro Gly Ser Pro Ala Leu Ala Ala Asp Ser 530 535
540 541617DNAZea maysmisc_feature(1)...(1617)ZmCkx3 cds
54atggcaagaa ggactcgttt cgtggccatc gccgccctcc tcacaagctt cctcaacgtc
60gcagccgggc attcccggcc actgtccggt gccggcctcc cgggcgatct tttcgggctg
120ggcatcgcgt cgaggatccg cacggacagc aactcgacgg cgaaggcggc gacggacttc
180ggccagatgg tgagggccgc gccggaggcc gtgttccacc ccgccacgcc ggccgacatc
240gccgcgctcg tccggttctc cgccacgtcg gcggcgccgt tccccgttgc gccgcgcggg
300cagggccact cctggcgcgg ccaggcgctc gccccgggcg gcgtcgtcgt ggacatgggc
360tcgctggggc gcggcccccg catcaacgtg tccgccgtgg ccggcgcgga gccgttcgtc
420gacgccggcg gggagcagct gtgggtcgac gtcctccgcg ccacgctgcg acacggcctg
480gcgccccgcg tgtggaccga ctacctccgg ctcaccgtcg gcggcacgct ctccaacgcg
540ggaatcggcg ggcaggcgtt ccgacacggt ccgcagatcg ccaacgtgca tgaactcgac
600gtcgtcacag gcacaggtga gatggtgaca tgctccatgg acgtgaactc ggacctgttc
660atggcggctc taggcgggtt aggccagttc ggggtcataa ccagagcacg gatccggctt
720gagccggcgc ccaagagggt gcgctgggtt cgacttgcct acaccgacgt cgctactttc
780accaaggatc aggagtttct catatcaaac cgggctagcc aagtcgggtt cgactacgtc
840gaaggccagg tccagctcag ccggtccttg gtcgaaggcc ccaaatcaac acccttcttc
900tccggcgccg atgttgctag gcttgctgga ctcgcgtcca ggaccggacc tgctgcaatc
960tactacatcg aaggcgccat gtactacacc aaggacaccg ccatatctgt ggacaagaaa
1020atgaaggcac tcctggatca gctgagcttc gagccagggt ttgcgttcac caaggacgtg
1080acgttcgtgc agttcctcga tcgggtgcgc gaggaggaga gggtgctccg gtcagccggc
1140gcgtgggagg tgccgcaccc atggctgaac ctcttcgtcc cacggtcgcg catcctcgac
1200ttcgacgacg gagtgttcaa ggctctgctc aaggactcca acccagctgg gatcatcctc
1260atgtacccca tgaacaagga taggtgggac gaccggatga cagcgatgac cccagccacg
1320gacgacgacg acatgttcta tgccgttagt ttcctttggt cagcactgtc cgcagacgac
1380gtgccccagc tcgagagatg gaacaaggca gtgctggact tctgtgatcg gtcaggaata
1440gaatgcaagc agtacctgcc acactacaca tctcaagacg ggtggcgacg gcatttcggg
1500gcgaaatgga gcaggatcgc tgagctgaag gccagatatg accctcgggc attgttgtcg
1560ccgggccaga ggatttttcc ggtgccagta gaggcatctg gcattgcttc tgcctga
1617551566DNAZea maysmisc_feature(1)...(1566)ZmCkx4 cds 55atgatgctcg
cgtacatgga ccgcgcgacg gcggccgccg agccagagga cgccggccgc 60gagcccgcca
ccatggcggg cgggtgcgcg gcggcggcga cggatttcgg cgggctgggg 120agcgccatgc
ccgcggccgt ggtccgcccg gcgagcgcgg acgacgtggc cagcgccatc 180cgcgcggcgg
cgctgacgcc gcacctcacc gtggccgccc gcgggaacgg gcactcggtg 240gccggccagg
ccatggccga gggcgggctg gtcctcgaca tgcgctcgct cgcggcgccg 300tcccggcgcg
cgcagatgca gctcgtcgtg cagtgccccg acggcggcgg cggccgccgc 360tgcttcgccg
acgtccccgg cggcgcgctc tgggaggagg tgctccactg ggccgtcgac 420aaccacgggc
tcgccccggc gtcctggacg gactacctcc gcctcaccgt gggcggcacg 480ctctccaatg
gcggcgtcag cggccagtcc ttccgctacg ggccccaggt gtccaacgtg 540gccgagctcg
aggtggtcac cggcgacggc gagcgccgcg tctgctcgcc ctcctcccac 600ccggacctct
tcttcgccgt gctcggcggg ctcggccagt ttggcgtcat cacgcgcgcc 660cgcatcccgc
tccacagggc gcccaaggcg gtgcggtgga cgcgcgtggt gtacgcgagc 720atcgcggact
acacggcgga cgcggagtgg ctggtgacgc ggccccccga cgcggcgttc 780gactacgtgg
agggcttcgc gttcgtgaac agcgacgacc ccgtgaacgg ctggccgtcc 840gtgcccatcc
ccggcggcgc ccgcttcgac ccgtccctcc tccccgccgg cgccggcccc 900gtcctctact
gcctggaggt ggccctgtac cagtacgcgc accggcccga cgacgacgac 960gaggaggacc
aggcggcggt gaccgtgagc cggatgatgg cgccgctcaa gcacgtgcgg 1020ggcctggagt
tcgcggcgga cgtcgggtac gtggacttcc tgtcccgcgt gaaccgggtg 1080gaggaggagg
cccggcgcaa cggcagctgg gacgcgccgc acccgtggct caacctcttc 1140gtctccgcgc
gcgacatcgc cgacttcgac cgcgccgtca tcaagggcat gctcgccgac 1200ggcatcgacg
ggcccatgct cgtctaccct atgctcaaga gcaagtggga ccccaacacg 1260tcggtggcgc
tgccggaggg cgaggtcttc tacctggtgg cgctgctgcg gttctgccgg 1320agcggcgggc
cggcggtgga cgagctggtg gcgcagaacg gcgccatcct ccgcgcctgc 1380cgcgccaacg
gctacgacta caaggcctac ttcccgagct accgcggcga ggccgactgg 1440gcgcgccact
tcggcgccgc caggtggagg cgcttcgtgg accgcaaggc ccggtacgac 1500ccgctggcga
tcctcgcgcc gggccagaag atcttccctc gggtcccggc gtccgtcgcc 1560gtgtag
156656173PRTArtificial SequenceConsensus sequence of FAD domains of
ZmCkx2, 3, 4, and 5 56Pro Ala Ala Val Leu Xaa Pro Ala Ser Pro Xaa
Asp Ile Ala Ala Leu1 5 10
15 Val Arg Xaa Xaa Phe Ser Ala Xaa Ser Xaa Ser Pro Leu Thr Val Ala
20 25 30 Ala Arg Gly
Xaa Gly His Ser Xaa Xaa Gly Gln Ala Gln Ala Pro Gly 35
40 45 Gly Ile Val Val Asp Met Arg Ser
Leu Xaa Arg Gly Xaa Arg Xaa Xaa 50 55
60 Xaa Xaa Xaa Leu Xaa Val His Xaa Xaa Xaa Gly Gly Glu
Gly Arg Xaa65 70 75 80
Xaa Phe Xaa Asp Xaa Xaa Gly Gly Xaa Leu Trp Ile Glu Val Leu Arg
85 90 95 Xaa Thr Leu Xaa Lys
His Gly Leu Ala Pro Arg Ser Trp Thr Asp Tyr 100
105 110 Leu Arg Leu Thr Val Gly Gly Thr Leu Ser
Asn Ala Gly Xaa Ser Gly 115 120
125 Gln Ala Phe Arg His Gly Pro Gln Xaa Ser Asn Val Xaa Xaa
Leu Glu 130 135 140
Val Val Thr Gly Arg Gly Glu Xaa Val Thr Cys Ser Pro Ser Xaa Asn145
150 155 160 Ser Asp Leu Phe Xaa
Ala Xaa Leu Gly Gly Leu Gly Gln 165 170
575737DNAZea
maysexon(1514)...(2183)exon(2317)...(2768)exon(4501)...(5127)misc_feature-
(0)...(0)genomic sequence for ZmCkx7 57gctcggattg tcgcacgcga gggtagtgta
cgctacctgc cgcggtgact tcggaatggt 60cgaaatactg aagactgtcg aaacggtctt
tttttgacac gttgcgtctg agttttgttt 120ttgtgggggc ttttgttggt atattacatg
gctccgcctt gttaaaaacc tcacccccgg 180ggggaaaaga gtgcgggccg gaataacatt
gtttgctgga ttacaagggc gcatgggccc 240tgatgattaa aaaaattgcg gtggttgtca
atgttccagg agtgctctaa gtcttcacca 300gatgtcgtta ctagcctata cgcgctgggg
gatgccttcg acatgacgat gaatgggcct 360tctcatttcg gctctagatt gccccgtgat
tctgtctggg tggttcggac gagtacgagg 420tctcctttgt cgaactctgt tgggacgacc
gcgtggttgc gctaggcctt agtttgagcc 480tgatatttgt tgagggcttg tagggcgagg
acgcggtctc cgtcgatgag gtctttggac 540attggttcgt cgacatcggg gaccgctgat
gtgcttgttc ggggtgagcc atgctttatc 600tcctgcggta tcatgacctc ggatccatac
aatagacgaa aaggtgtgaa tccggttgcc 660cgacattctg tcgtgtttag ggcccagacc
acttcaggta gttggttggc ccatttgccc 720ttcttgtcat cgagatgtcg tttcttgatg
gcagtgaaga tcttgccgtt ggcgcgctcc 780accaatccaa cgcgcgcaac agcaatttac
gtggacttgc aggcttggag caaggaacaa 840caccaaaaca aaaaagaaac atgcaacaag
taatattgaa atttactttg aaacaggtat 900gcatgtttat ttaatatatt ttgtacttga
tgtttggact atttcatatt aacttgcata 960ctaaattatt tatgaaaaat ttcttatggc
atacctcatg aataaatcct agctacgcca 1020ctattaccag tggtttttgg tttttatgat
ttttttaaat cttttgaatt tagaacgaat 1080tttttaaaaa acggtgattt atcgaaaccg
tatcccgact ggtttaccgt cagtttttac 1140cggttttgta aaccatgcct acgctacaca
tatatacata cggtattacg gtgtatgtac 1200gtcgtatata tatcttagct tatatatctt
attgcatggt tctgtacgtg tccgacgagt 1260gacgacggct atcttagctt atactctctc
cctctgtttt tttagttgtt gctggatagt 1320ttaattttac actatccagc gacaactaaa
acgaaacgaa gggagtatat atcttactct 1380caatcgttcg taacaataat aatggtaata
ataacagcag tttaatctat atataggcca 1440ccacggctct ccactgctgc gtgcgtgcgt
gcgtacatcg tcaaaaacct ccatcaagca 1500actgatcatg acgatggcta gagctacgac
ctccacggtc gcagcactct gcttcctcct 1560cagctgcgtc tccgcgaccc cttccacgct
cgccgcgtcc tccgccatca tccacgacat 1620catccgcggc ctcgcggaca ccacggcggc
gcgcgtccgc acggacgccg aggccacggc 1680gcgcgcgtcc accgacttcg gcaccaacgc
gaccgcggac gacgcgaccc ggccggcggc 1740cgtgttctac ccgtcgtgcg ccgccgacat
cgccgcgctg ctgcgggcgt ccagcgcgag 1800cgcctcgccg ttcccggtct ccgcgagggg
ccgcgggcac tcgacccggg gccaggccac 1860ggcccccggc ggcgtcgtcg tcgacatggc
gtcgctagca gtagctgcag ggcgcgacga 1920gaccgccacc accaacgcct cctccacctc
cgcctccgca aggctcgccg tgtcggtgga 1980cgggcgctac atcgacgccg gcggcgagca
gctgtgggtg gacgtgctgc acgccgccct 2040ggcgcacggc ctcacgcctc gctcctggac
ggactacctc cgcctcaccg tcggcggcac 2100gctctccaac gcaggcatca gcggccaggc
cttccgccac ggcccgcaga tatccaacgt 2160cctagagctc gacgtcgtca cgggtacgtg
tacgtggctg tgcttatgat aagatcgatc 2220aataacatgt ggagtatacg tcctatggaa
tggacatgga cgtggaaacc gggtgatata 2280tatagctagt tttggaactc gcgtgaaccc
tcacaggaac aggtgacatg gtgacgtgct 2340ccaaggagaa ggacgccgac ctcttcgacg
ccgtgctggg agggctgggg cagttcggca 2400tcataacgcg cgcgcggatc ccgctggcgc
cggcgccggc gagggcgcgc tggctgcggc 2460tcctctacac cggcgccgcc gacctcacgg
ccgaccagga gcggctcatc gccgacgacg 2520agcgccgcgg cggcgcgctg gccgggctca
tggactacgt cgagggctcc gtcgtcaccg 2580acctccagca gggcctcatc ggcagctggc
gctcgcagcc gccgccgtcc tcctcgtcct 2640tctactcggc taccgacgcc gcgcgcatcg
cggcgctagc cgaggaggcc ggcggcgtcc 2700tctacttcct cgagggcgcg gtgtactacg
gcggcgccag cgacacgacc gccgcagacg 2760ttgacaaggt aacacgatcg acgtacgacc
caccgccggt ttctacgtgc atacgccagt 2820gccaccgaag ccacgtgatc tgtggatggt
tgatctggat tgtcgtcttc agcctttggc 2880ttgtgatgca tgcatgtctg gtggggcagc
atcggcagtc gtgcagcgtc aacatcgtac 2940ttgcatggcc tgccgttgtc gggccctcgt
cgtgcatatc attccaaagc tttttgggca 3000tccccccccc cccctgctta cctatagttt
tcaattttat aagacctgca cgcatccatt 3060tgaagagtca aaacctcgtg attttgaagc
gacagttgta gcctgtacaa cagttttgtt 3120gataccatat ttttttatgg aactacgcca
tccaaacagc tccgtaaact tttgctggat 3180aataattaag tcgtagatgg ctagcgaatt
aaaaccttat atattgcatc tattatatat 3240aagaaaaaaa aacctagcta gtttactata
ttaaggtgca taaaatacta ctatcatatt 3300tcgtgaactc aaaacaaaga aataagaaat
actctcgacg acaagttgat acaaatccta 3360tgtaatttac attgctagta ccttaattca
tcaactatgg tatatctgca tgcagttggc 3420taatgttttt ggtaatacta ttccagttgg
atgacaggaa caagtcatga ccactatgca 3480tgcatggagc agctaacaaa agccagctcg
atatagaact gattcaagta gtcatatgat 3540aaggctagcg ctaaaaaagt aggaatatag
ttacaaatgt ggcaagtatt ggtacttggc 3600caatggccac atagaaacga gtcattcacc
tatccaaata tttcttcaca atttcttagg 3660gcgctaaatt aattttagtt cgaaaataaa
taaaaataga gttcgatatt aatcagatct 3720gatcttctgt aaaaatttat aacccattgt
catcccctac cctactagtt attagaggat 3780actcttataa tagataatgc aagagatcta
agcactgcac catccaagca aagctactgt 3840agctcgacat gtgagtacac ttcactgtga
ccaaccagat actgcacagc tcgcattcac 3900aagcgctata gctagaagga cgaccgcatc
agctctgcaa acgactcgga tctgttcgct 3960gttcgcacca aggacagcaa cagtgctagt
gtctctttct tttctgtttt ttttatcgga 4020gaggcaacac catatatttc cagacaaatc
ttgagctata tatagagact ttcgtacata 4080tgtgttttag acgaccgcat cctagcaact
ttttttactt gagcagactt tcgtacattc 4140tatattcaag gtcacaagaa gtcaagctcg
atctcacatc aaattatgtt gtagtggtat 4200ttttttagtc cccaaaatga aatggttttt
ggacatcccc aacgaacgac gatcggttat 4260atatgtgtca gttgggacag tcacgtttct
cgatgaaagt gaccgtgcaa gcaaggtgca 4320agttgacgtg tagtcgtgta gtcgcgcgcg
catatcccta catatatgga cacatcacac 4380acatgcaaag aaacactagt gaccaaatca
tatacctttt gcacacaacg tgacactact 4440tgtagagtat acgtagttag ttcctgcatg
catggattaa cacaaaatgc gtacatgcag 4500cgcgtggacg tgatgctgcg tgagctgcgg
tacgcgcggg ggttcgcgta cgtgcaggac 4560gtgtcgtacg agcagttcct ggaccgcgtg
agcgccggcg agcgcaggct ccgcggcgag 4620ggcctctggg acgtgccgca cccgtggctc
aacctcttcc tcccgcgctc ccgcatcctc 4680gacttcgccg cgggcgtctt ccacggcgtg
ctgctcccca cgcgcacggc tggcggcggc 4740ggcggcgggc ccgtgctggt ctaccccatg
aaccggggca agtgggacgg cgcgacgtcg 4800gcggtgctcc cctacgacga tggcgacggc
gacggcgacg aggtgttcta cacggtgggg 4860atcctgcggt cggccgtggc ggacggcgac
ctgcgccgca tggaggagca gaacgccgag 4920gtggcgcgct tctgcgaggc cgccggcatc
ccctgcacgc agtacctgcc ctcctacgcc 4980acgcaggcgg actgggcggc gcgccacttc
ggccccgccg gcagcggcag gtgggacacc 5040ttcctccgcc gcaagaggaa atacgacccc
atggcgatct tgtcgcgcgg ccagaggatt 5100ttctcgtccc cgctacttgc ctcatgatcc
gccggttccg gtctctcgat cgtcgtgtgt 5160tgctgttgct ggcatgggct agctgcatga
aataatagtg caagcaagca aaggcaaagc 5220aagcttcaag gcatgactgt tttgctttag
cgttttactg atcagtagtc aagtgacaca 5280gttactacgt actacgatct gtctctgact
ctcttccaga gggtcccaaa tatatgatgc 5340tttttattaa ctttatttga ccgttattaa
aaatatagtg atattgttta ctttaaacga 5400gttattttat tgttacagaa ttttatcatt
tacataaaaa tataaattta aataaatgat 5460caaatacaat aatatctaaa aagtaaaaaa
aaaccatcgt ctatgtcaaa agaaacatca 5520tcaaaacgag ggaggaattt tactagtaat
agacatgcat gcacgtgatc gagcaataat 5580gcaacactac gataactata tatgcaagcg
cgcgtgatac tgataagagt gtaccgtacg 5640attgaagaac attgtacatg tactactgca
cgtactcagc tgatgcctga cgatttgtaa 5700tcttcacctt tgtgtttacg tacatagcgg
tttgaac 5737581749DNAZea maysCDS(1)...(1749)
58atg gct aga gct acg acc tcc acg gtc gca gca ctc tgc ttc ctc ctc
48Met Ala Arg Ala Thr Thr Ser Thr Val Ala Ala Leu Cys Phe Leu Leu1
5 10 15agc tgc gtc tcc gcg acc
cct tcc acg ctc gcc gcg tcc tcc gcc atc 96Ser Cys Val Ser Ala Thr
Pro Ser Thr Leu Ala Ala Ser Ser Ala Ile 20 25
30atc cac gac atc atc cgc ggc ctc gcg gac acc acg gcg
gcg cgc gtc 144Ile His Asp Ile Ile Arg Gly Leu Ala Asp Thr Thr Ala
Ala Arg Val 35 40 45cgc acg gac
gcc gag gcc acg gcg cgc gcg tcc acc gac ttc ggc acc 192Arg Thr Asp
Ala Glu Ala Thr Ala Arg Ala Ser Thr Asp Phe Gly Thr 50
55 60aac gcg acc gcg gac gac gcg acc cgg ccg gcg gcc
gtg ttc tac ccg 240Asn Ala Thr Ala Asp Asp Ala Thr Arg Pro Ala Ala
Val Phe Tyr Pro65 70 75
80tcg tgc gcc gcc gac atc gcc gcg ctg ctg cgg gcg tcc agc gcg agc
288Ser Cys Ala Ala Asp Ile Ala Ala Leu Leu Arg Ala Ser Ser Ala Ser
85 90 95gcc tcg ccg ttc ccg gtc
tcc gcg agg ggc cgc ggg cac tcg acc cgg 336Ala Ser Pro Phe Pro Val
Ser Ala Arg Gly Arg Gly His Ser Thr Arg 100
105 110ggc cag gcc acg gcc ccc ggc ggc gtc gtc gtc gac
atg gcg tcg cta 384Gly Gln Ala Thr Ala Pro Gly Gly Val Val Val Asp
Met Ala Ser Leu 115 120 125gca gta
gct gca ggg cgc gac gag acc gcc acc acc aac gcc tcc tcc 432Ala Val
Ala Ala Gly Arg Asp Glu Thr Ala Thr Thr Asn Ala Ser Ser 130
135 140acc tcc gcc tcc gca agg ctc gcc gtg tcg gtg
gac ggg cgc tac atc 480Thr Ser Ala Ser Ala Arg Leu Ala Val Ser Val
Asp Gly Arg Tyr Ile145 150 155
160gac gcc ggc ggc gag cag ctg tgg gtg gac gtg ctg cac gcc gcc ctg
528Asp Ala Gly Gly Glu Gln Leu Trp Val Asp Val Leu His Ala Ala Leu
165 170 175gcg cac ggc ctc acg
cct cgc tcc tgg acg gac tac ctc cgc ctc acc 576Ala His Gly Leu Thr
Pro Arg Ser Trp Thr Asp Tyr Leu Arg Leu Thr 180
185 190gtc ggc ggc acg ctc tcc aac gca ggc atc agc ggc
cag gcc ttc cgc 624Val Gly Gly Thr Leu Ser Asn Ala Gly Ile Ser Gly
Gln Ala Phe Arg 195 200 205cac ggc
ccg cag ata tcc aac gtc cta gag ctc gac gtc gtc acg gga 672His Gly
Pro Gln Ile Ser Asn Val Leu Glu Leu Asp Val Val Thr Gly 210
215 220aca ggt gac atg gtg acg tgc tcc aag gag aag
gac gcc gac ctc ttc 720Thr Gly Asp Met Val Thr Cys Ser Lys Glu Lys
Asp Ala Asp Leu Phe225 230 235
240gac gcc gtg ctg gga ggg ctg ggg cag ttc ggc atc ata acg cgc gcg
768Asp Ala Val Leu Gly Gly Leu Gly Gln Phe Gly Ile Ile Thr Arg Ala
245 250 255cgg atc ccg ctg gcg
ccg gcg ccg gcg agg gcg cgc tgg ctg cgg ctc 816Arg Ile Pro Leu Ala
Pro Ala Pro Ala Arg Ala Arg Trp Leu Arg Leu 260
265 270ctc tac acc ggc gcc gcc gac ctc acg gcc gac cag
gag cgg ctc atc 864Leu Tyr Thr Gly Ala Ala Asp Leu Thr Ala Asp Gln
Glu Arg Leu Ile 275 280 285gcc gac
gac gag cgc cgc ggc ggc gcg ctg gcc ggg ctc atg gac tac 912Ala Asp
Asp Glu Arg Arg Gly Gly Ala Leu Ala Gly Leu Met Asp Tyr 290
295 300gtc gag ggc tcc gtc gtc acc gac ctc cag cag
ggc ctc atc ggc agc 960Val Glu Gly Ser Val Val Thr Asp Leu Gln Gln
Gly Leu Ile Gly Ser305 310 315
320tgg cgc tcg cag ccg ccg ccg tcc tcc tcg tcc ttc tac tcg gct acc
1008Trp Arg Ser Gln Pro Pro Pro Ser Ser Ser Ser Phe Tyr Ser Ala Thr
325 330 335gac gcc gcg cgc atc
gcg gcg cta gcc gag gag gcc ggc ggc gtc ctc 1056Asp Ala Ala Arg Ile
Ala Ala Leu Ala Glu Glu Ala Gly Gly Val Leu 340
345 350tac ttc ctc gag ggc gcg gtg tac tac ggc ggc gcc
agc gac acg acc 1104Tyr Phe Leu Glu Gly Ala Val Tyr Tyr Gly Gly Ala
Ser Asp Thr Thr 355 360 365gcc gca
gac gtt gac aag cgc gtg gac gtg atg ctg cgt gag ctg cgg 1152Ala Ala
Asp Val Asp Lys Arg Val Asp Val Met Leu Arg Glu Leu Arg 370
375 380tac gcg cgg ggg ttc gcg tac gtg cag gac gtg
tcg tac gag cag ttc 1200Tyr Ala Arg Gly Phe Ala Tyr Val Gln Asp Val
Ser Tyr Glu Gln Phe385 390 395
400ctg gac cgc gtg agc gcc ggc gag cgc agg ctc cgc ggc gag ggc ctc
1248Leu Asp Arg Val Ser Ala Gly Glu Arg Arg Leu Arg Gly Glu Gly Leu
405 410 415tgg gac gtg ccg cac
ccg tgg ctc aac ctc ttc ctc ccg cgc tcc cgc 1296Trp Asp Val Pro His
Pro Trp Leu Asn Leu Phe Leu Pro Arg Ser Arg 420
425 430atc ctc gac ttc gcc gcg ggc gtc ttc cac ggc gtg
ctg ctc ccc acg 1344Ile Leu Asp Phe Ala Ala Gly Val Phe His Gly Val
Leu Leu Pro Thr 435 440 445cgc acg
gct ggc ggc ggc ggc ggc ggg ccc gtg ctg gtc tac ccc atg 1392Arg Thr
Ala Gly Gly Gly Gly Gly Gly Pro Val Leu Val Tyr Pro Met 450
455 460aac cgg ggc aag tgg gac ggc gcg acg tcg gcg
gtg ctc ccc tac gac 1440Asn Arg Gly Lys Trp Asp Gly Ala Thr Ser Ala
Val Leu Pro Tyr Asp465 470 475
480gat ggc gac ggc gac ggc gac gag gtg ttc tac acg gtg ggg atc ctg
1488Asp Gly Asp Gly Asp Gly Asp Glu Val Phe Tyr Thr Val Gly Ile Leu
485 490 495cgg tcg gcc gtg gcg
gac ggc gac ctg cgc cgc atg gag gag cag aac 1536Arg Ser Ala Val Ala
Asp Gly Asp Leu Arg Arg Met Glu Glu Gln Asn 500
505 510gcc gag gtg gcg cgc ttc tgc gag gcc gcc ggc atc
ccc tgc acg cag 1584Ala Glu Val Ala Arg Phe Cys Glu Ala Ala Gly Ile
Pro Cys Thr Gln 515 520 525tac ctg
ccc tcc tac gcc acg cag gcg gac tgg gcg gcg cgc cac ttc 1632Tyr Leu
Pro Ser Tyr Ala Thr Gln Ala Asp Trp Ala Ala Arg His Phe 530
535 540ggc ccc gcc ggc agc ggc agg tgg gac acc ttc
ctc cgc cgc aag agg 1680Gly Pro Ala Gly Ser Gly Arg Trp Asp Thr Phe
Leu Arg Arg Lys Arg545 550 555
560aaa tac gac ccc atg gcg atc ttg tcg cgc ggc cag agg att ttc tcg
1728Lys Tyr Asp Pro Met Ala Ile Leu Ser Arg Gly Gln Arg Ile Phe Ser
565 570 575tcc ccg cta ctt gcc
tca tga 1749Ser Pro Leu Leu Ala
Ser 58059582PRTZea mays 59Met Ala Arg Ala Thr Thr Ser Thr
Val Ala Ala Leu Cys Phe Leu Leu1 5 10
15 Ser Cys Val Ser Ala Thr Pro Ser Thr Leu Ala Ala Ser
Ser Ala Ile 20 25 30
Ile His Asp Ile Ile Arg Gly Leu Ala Asp Thr Thr Ala Ala Arg Val
35 40 45 Arg Thr Asp Ala
Glu Ala Thr Ala Arg Ala Ser Thr Asp Phe Gly Thr 50 55
60 Asn Ala Thr Ala Asp Asp Ala Thr Arg
Pro Ala Ala Val Phe Tyr Pro65 70 75
80 Ser Cys Ala Ala Asp Ile Ala Ala Leu Leu Arg Ala Ser Ser
Ala Ser 85 90 95
Ala Ser Pro Phe Pro Val Ser Ala Arg Gly Arg Gly His Ser Thr Arg
100 105 110 Gly Gln Ala Thr Ala
Pro Gly Gly Val Val Val Asp Met Ala Ser Leu 115
120 125 Ala Val Ala Ala Gly Arg Asp Glu Thr
Ala Thr Thr Asn Ala Ser Ser 130 135
140 Thr Ser Ala Ser Ala Arg Leu Ala Val Ser Val Asp Gly
Arg Tyr Ile145 150 155
160 Asp Ala Gly Gly Glu Gln Leu Trp Val Asp Val Leu His Ala Ala Leu
165 170 175 Ala His Gly Leu
Thr Pro Arg Ser Trp Thr Asp Tyr Leu Arg Leu Thr 180
185 190 Val Gly Gly Thr Leu Ser Asn Ala Gly
Ile Ser Gly Gln Ala Phe Arg 195 200
205 His Gly Pro Gln Ile Ser Asn Val Leu Glu Leu Asp Val Val
Thr Gly 210 215 220
Thr Gly Asp Met Val Thr Cys Ser Lys Glu Lys Asp Ala Asp Leu Phe225
230 235 240 Asp Ala Val Leu Gly
Gly Leu Gly Gln Phe Gly Ile Ile Thr Arg Ala 245
250 255 Arg Ile Pro Leu Ala Pro Ala Pro Ala Arg
Ala Arg Trp Leu Arg Leu 260 265
270 Leu Tyr Thr Gly Ala Ala Asp Leu Thr Ala Asp Gln Glu Arg Leu
Ile 275 280 285 Ala
Asp Asp Glu Arg Arg Gly Gly Ala Leu Ala Gly Leu Met Asp Tyr 290
295 300 Val Glu Gly Ser Val Val
Thr Asp Leu Gln Gln Gly Leu Ile Gly Ser305 310
315 320 Trp Arg Ser Gln Pro Pro Pro Ser Ser Ser Ser
Phe Tyr Ser Ala Thr 325 330
335 Asp Ala Ala Arg Ile Ala Ala Leu Ala Glu Glu Ala Gly Gly Val Leu
340 345 350 Tyr Phe Leu
Glu Gly Ala Val Tyr Tyr Gly Gly Ala Ser Asp Thr Thr 355
360 365 Ala Ala Asp Val Asp Lys Arg Val
Asp Val Met Leu Arg Glu Leu Arg 370 375
380 Tyr Ala Arg Gly Phe Ala Tyr Val Gln Asp Val Ser Tyr
Glu Gln Phe385 390 395
400 Leu Asp Arg Val Ser Ala Gly Glu Arg Arg Leu Arg Gly Glu Gly Leu
405 410 415 Trp Asp Val Pro
His Pro Trp Leu Asn Leu Phe Leu Pro Arg Ser Arg 420
425 430 Ile Leu Asp Phe Ala Ala Gly Val Phe
His Gly Val Leu Leu Pro Thr 435 440
445 Arg Thr Ala Gly Gly Gly Gly Gly Gly Pro Val Leu Val Tyr
Pro Met 450 455 460
Asn Arg Gly Lys Trp Asp Gly Ala Thr Ser Ala Val Leu Pro Tyr Asp465
470 475 480 Asp Gly Asp Gly Asp
Gly Asp Glu Val Phe Tyr Thr Val Gly Ile Leu 485
490 495 Arg Ser Ala Val Ala Asp Gly Asp Leu Arg
Arg Met Glu Glu Gln Asn 500 505
510 Ala Glu Val Ala Arg Phe Cys Glu Ala Ala Gly Ile Pro Cys Thr
Gln 515 520 525 Tyr
Leu Pro Ser Tyr Ala Thr Gln Ala Asp Trp Ala Ala Arg His Phe 530
535 540 Gly Pro Ala Gly Ser Gly
Arg Trp Asp Thr Phe Leu Arg Arg Lys Arg545 550
555 560 Lys Tyr Asp Pro Met Ala Ile Leu Ser Arg Gly
Gln Arg Ile Phe Ser 565 570
575 Ser Pro Leu Leu Ala Ser 580 608113DNAZea
maysexon(2080)...(2668)exon(2891)...(3018)exon(3144)...(3416)exon(3574)..-
.(3836)exon(6286)...(6619)misc_feature(1)...(8113)ZmCkx8 genomic
60gaattctatc ttttcttgag ttattttatg atacaactgt tgctttgtct ggaatttatt
60atcctacttc aacattgatg cttcatcata tacttaaaat tgctagacat ctaaatgctt
120ttgaaaatga tgctttgctt agagatgcta ttgttcctat gaaaacaaaa tatttgaaat
180attggaggaa gatacctgtt ttatattgct ttgcttttgt attggatcct agagcaaaaa
240tgagggggtt taataagctt cttatgaggt tgtctggact taatggaact gattattcaa
300ggtatcctac atacattcgg tctaaactaa ctaagatttt tcagatatat gaattgaaat
360ttggtgaagt gtgcttgagt gcacaacaac ataagagtgc tggtacggca ggtaaggcta
420cagaggcatg ggatgacata tatggggatg atatccttat gccttcccaa tctactagag
480ctactcctac agctgtatca tctactgctg ctgctatatc tgagttgtca tcatatcttg
540atagtgatac tgtcacccag tttgactctg atttcattct tctaaactgg tggcagcgac
600acaagttgac atatcctgtg ctttctatac ttgctaaaga tgttataatt gtgcctgctt
660ccactgtatc atcagagtcc actttcagtt tagctggcag ggtgcttgaa gaccgacggc
720ggcgcctaac tcctgatatg gttgaagttt tgtcttgcat aaaggactgg gagcttgctg
780acttgcatag tcagcacacg gtggagaaag ataccaaaga acttgaagtt gtttttgaag
840caatgtacct agaagaaact ggtggaggca aagaaagaag aggtggagga tctggtggag
900cgggtagatc ttgagcagct gaattattgc tattactata ctctgttctt ctgttgtaac
960ttgtgatgaa ctattaaact ctggacttaa attgaaccta tataggagct ggctctactc
1020tttttcttcc tagggttttc tcacgaggtg tgagttttta cctaggaagg tttttaatga
1080ggcagcattg cactaaggct ccattagtat attgtttgca taaacttttg tgaactgtga
1140ttttgtttct gagatgtttt gtgaactgtg tgaattgact gaaatctgat ataggaactg
1200tgtgaaatct gatataggaa ctgtgtgaat tgactgaaat ttgatatatg aactgtgtga
1260aatttgattc agctgtttat tgtgaaatta ctgtgcttcg ggtcagcccg gccctatggg
1320ctgaccgggc cagaggcacg gcacgacaca acccgtttaa gccactttcg tgccgtgctt
1380gtgccaacag tttagcccgc gggccagcac ggcacggcac ggaagtagga tcgtgccgtg
1440cccggcacgc acagtaacgt gctgtgcttg gccgtgcccg tgccgtgccg gcccgacaca
1500cacgaatgga catgtatagt cctgactgtc ccgtaaccaa acggacccca acatactcga
1560tgttgtttag accgaccgac tgatcgtgcc acattgcact gcgcgtgaag aggtcgatac
1620cgatcgttta gaccaccatg tcagctgatg gtactgtccc acgttggcat tggagcagct
1680tacctatcat acatatcatc tattttttat ttaaaaattt actataaata gtgtagtata
1740caatataaaa tagtatcata tgctcaatat gcttgagaca gctttaatag gatcaaacta
1800agattctcgg gcccggcatc ggtagcgacg acaccggcta tatataatgc actcagtgag
1860cttcctggtg gctcttgctg cttcttcctt gctgttccat ccgtccacag ttcttgtggg
1920aacccaagat cgatcttgac ggggacgtga gcacggcacg tcgcgacctt attcttccgt
1980cttggccccg tgcaccggca agcggcaacc aaatgcgcat gcccctgtga aagctaatag
2040tagctacatc acacagcaag acactatagc cagctagcca tggagggcaa ggtgctgtgc
2100acgtacgccg ggatcgtggc cctactgctc tgctcgtcgg tgaatttcat acagagcccc
2160tccgacgtgt tcggccccgt ggcgctgctg gagccgacag catccgcggc acgcgacttc
2220ggcggcgtgg tctcggaggc ggccatcgcg gtcatgcagc ccgggtcccc cgccgacatc
2280gcgcggctcc tgggcgcgct gtcgtcgacg gggccggggc cggggccgaa ggcggccgtg
2340gcggcgcgcg gcgcggggca ctcgctccac gggcaggccc aggcgcgcgg cggcattgtg
2400gtggagacgc gcgccctgcc gcgcctcgtg gaggtggtgc gacgcgggga cggggacggc
2460ggcggcgcgg cgtacgcgga cgtgggcggc ggcgcgctgt gggtggaggt gctggaggag
2520tgcctgaggg ccgggctggc gccgcggtcg tggacggact acctgtacct gaccgtgggc
2580gggacgctgt cgaacggcgg catcagcggg caggcgttca agcacggccc gcagatcagc
2640aacgtgctgc agctggaggt ggtcacaggt acgttacgcg cgccgtacac gcatcatgca
2700cttccgcacg gcctcgctgc tcgcgtgcac catctgccgc gcgcctaatt gatctctctt
2760ttccgtgtct ctctctctcc ctcccttttc ttcggtgaaa gaaagaaaga aagaaagaaa
2820agatacgggt gatcggtggc ctttgttcct ttgccggttg tttatgtgtt gtttgtcccg
2880tcggttgcag gcacagggga ggtggtgaca tgctcgccca cccagagccc ggagcttttc
2940ttcgccgtac ttggtgggct tggccagttc ggtatcataa cccgcgcaag gattccgctg
3000caagttgctc cgcccaaggt acacagacac aattgcgcac gccctcgatc tgcttcctct
3060ctagctggta gcagaaatag aaagaaatca ggaaagctgg taactaaaag cagctttggc
3120cgataatttt ctatgattat taggtgagat gggtgagggc cttctacgac agcttcgaga
3180cgttcaccaa ggaccaggag ctgctggtct caatgccaga gctggtggac tacgtggagg
3240ggttcatggt cctgaacgag cagtccctcc gcagctcctc cgtggccttc cccgcccagg
3300tcaacttcag accggacttc ggctccgacg acggcaccaa caagaaggtc tgctactact
3360actgcatcga gttcgcggtg catgacttcc aacggcagga ctccgctgct gaccatgtca
3420gtctctacta gctcatcgtc tctaccactc tatcaggagc gcgtgacagt atccccgtca
3480cgttagcatc gtcaacagta gcctcgagaa ggaaagcact cactgaacga ccggccgggc
3540ttgtggcctg cctccgaatt ttgctctgtg caggttgtgg acctggtgtc ggggaagctg
3600agctatctga ggccccacgc gtacagcgtg gaggtggcct actgggattt cctcaacagg
3660gtgcggatgg aggaggagag cctcaggagg cggggcctct gggacgtgcc gcacccctgg
3720ctcaacctct tcgtgcccag gcatggcgtc gcgcggttca tggacctgct catggccacc
3780atcgcgcagg gggacttcga ggggcccgtc ctcgtctacc ccctcctcac tcacaggtac
3840ggtgcgaccc acgtccactc atattttttt tttattttat acgtatgaaa gacggcatgc
3900ttggtccatt ggttgcatgg atgcatgaac tatagtggct taggatggat tagccagtca
3960taaaagatgt tcatcatact gtactatata ttagtaagag tatatattta ttcataaaag
4020tagttcttca aactgtaaaa agggttcagt ggtttattta ttttttgaaa aaaaaaagaa
4080gtgatggcca cggccctcgt ggtcgtgggt gggtagataa aggagtccgt ccctttcaca
4140ccacactggt attaatctgc atgattttcc tgcgcaaaaa aaaaacgttt ggatggtgtg
4200gccggcgtga tcgccttgtc atcacccgca gggaatctca aagcattcca aaaacatgag
4260acaaaagctc gtgcctcctt ttttttcttt catctgcgtg atctatgaac acggccaggc
4320agattgcaca tcgtcaggtg cagcctagct cgaggtacct ggcccgttgt ttttcctccc
4380agaaatgggt tgccaaacac caagcgcaac atgttggtcc aattgtgaca atgctatggc
4440atatcgccgg ctctccctct cagtcgtcgt cacaggggcg tcgtagcggt ggtcacctca
4500taggccgtgt cctttcccag caggactgca cctttgcgtc gcttgtcaac gatcacactc
4560acacgtcttt atgcacagtg aaaactagac attttatttt tttcactttc agaaaagaaa
4620agaaactata cgtagccatt tcttctcgca aactttgcac tgcagtgtac gctgctgcta
4680tagtagtgca caattaggat cggtaatgca tcacgattct aatgtttctt tacaattttg
4740tttgagacat taataaattt tagtccaaaa ataaatagaa aataaactcg attctagtct
4800aatccgattt tttatattgt aaaatttaga gactattatt gatccgatgc acaagccaag
4860cccagccgtg atggccttgc atattttctg aaaggcgtcg tctctccata gctaccctac
4920cttaggaata tcgtgaatct ctgtgtagtc gcctgggcgc ctggcctttt gcaagttgca
4980gcgcttgctt gcacgtgacg gtataaatgg tcatataaaa tactatttgt aatgtagatt
5040acactgtttg cgaagtgaaa tttgaaacaa ctggtaatag agtgtgatag tgattcttgt
5100gaagataagc tactgagcta tgtgcagtcg cctggccttg ctaccactag ccatggcacg
5160gctgcagcaa gtcgcgaggg caatcagtgc agtgcagtct cctcaaatgg cagtgccagt
5220atggtggcag tggcactggc accctagcgt gtccacaccc ttcctggctg gctgactcgt
5280cactttgata ggagaaagta ccgtggcaac atgatggtga ttattccccg tgtgcagtcg
5340cctggtcggt tgcaaatcgc ttgacactga cttttgaaat gtgcgtaacc gtgtcgtgta
5400attctatatc ttatcaaaaa aaaatcatgc aaaaggtatg ttaaaaaatt ttaccacgag
5460aaagtagtgt ggcaacatga tgttgattac tcactccatc ttagaaaagc tgtgatttta
5520gttttctgcc ggatcaatca atctgtctca actctaacac aatatatatg tactgaaatt
5580cgaatttttt actaaataat ataattacat ttatcatgat ttttttaata gtgtaactgt
5640ttagagtcat agaaattaat tatattttct acaaactgag acgaacttat taagaaagtt
5700tgaccaatct aaatctgaaa ttccattatt ttttggggac tgagaggttg tactacatgt
5760ttcatgacat ttttttttgt aatgtacagt gccagtttcc ttcagttttg tttgcatgtg
5820ggtgtgttaa gcaactcttg aacaaaagct gcagctcgtg aggttcagaa cccgggatta
5880gcaggcctgg cccaggcgtt caggggtttt gtcacgttcg caggctctgc cgctatgctg
5940gcaggcagca gcacaggatg cttgcacagc ctaccttttc tagtggtcaa attgcctttg
6000agcctttccc agacagcgaa tctacatcct gccacaccac atggcagggc ctacgcccta
6060caccctgacc ctgtcacggc ctcctcctcc tcctccatgc cattttagga aggcgtcgca
6120gggcacgcac cggcaccacc accgcccgaa agcctatagt cgggcataca ctgtcggatc
6180atggcatcct ttataataac tctggttctc gtcatacgcc agctcgtgct gctaccgcac
6240ccgtcatcct gctcatccac gcttggtgcg ctgcaatatt ttcaggtggg acggcaacat
6300gtcggcggtg gttccggcgg cgccagacgg tgtgatgtac gtcttcagcg tgctacggtc
6360gacggacccg gcgcggtgcg gccgcgcctg catggagagg atcctggagc agcaccgccg
6420tgtcgccgac gaggcctgcc ggcgcctcgg cgccaagcag tacctggcca ggcagccgtc
6480gctggcgcac tggcgcgacc acttcggggc cagctgggac cgcttcgtgg ccaggaaggc
6540ccgcttcgac cccatgaacg tgctcgggcc gggccaggga attttcccct ggacggactc
6600ctcctcgagc ccgatgtgac ggcggctgtc gtgggctttg gtccacccac gtacggagga
6660agtagctagg ctgatgagga gctgaagccg atgatgctag ttttgatgag attgtttatt
6720attgttcatg tggaattcct atttaggttc ctaggtacta gtactatgtt ggcaagtggc
6780tcaaggtaaa atgttcattc ttttttttcc ctacttctta tgtgctgtgt ttagctgatt
6840agtggcctcc taaaattagt tgtttgccga gatgggcaaa taattcatcc tctcgtgttg
6900ttccgtttcg atcattgaaa tgtctatcgt ttctctgcct aggttctttt tgtatttgat
6960ctattatcat attcttgtct tctcatatta tttattttaa gctcaagatt tcgcagttta
7020ttttataaat gatcatacag tgtttgcctt gttacattat ttatactcac aaaacgtaaa
7080aatctagggc catgtgccgt cacccccgtc acacttggca ctggacagcc ctggccgtga
7140agataataag tgtgagatta attttaagcg gctctttata tcattcataa ttgttagaaa
7200tagatatctt acggcagagc actctccaac atctttttta aattggctat ccaagaatat
7260cgtcatctat ttcaaaattc tcgctagcca cggatagaaa gttaaaataa ttctttagag
7320tgtgtataag atatataaat gtcggaggat gaaaaaaatt taaaactatt tagagggtga
7380gtttataaaa aactctaact acaaaatgat aatgatagaa ccaaaggtta aaaacaacaa
7440gtctttggaa gaaataaaat ggctatctgt gtccgttctc atgagacacg ctaaaaaatc
7500tataaaattt tcatagaggt aaaaaagatc tggcaaccaa ccaattctat atacattaat
7560caccatttct cataataaca tctaattttt aaaaaaaata tccccttgtt aagaataacc
7620taacataagc ccataaacat gtatctaaat tacccacctc acttaatata atcataatct
7680aaagtgacgc acaaaattta cattttaatt gcctaactat gattataata tataatctaa
7740gcaacacatt aagtttacat ctaaattgca agctcttata tttttcaaaa ttaaataaaa
7800tatacaaatc aacaaaattt tgttatgaaa aatgtaaatt accataaatt atatttatgc
7860atggttctaa gttgcccact tctatcgctg atggcctata aaaacatgtg ttttgcatgt
7920tcaacattcc taaaaagtgc cccttgaaag gctcatatga attcatctgc aattatattg
7980atatccatca caaaaaattg taactacttt atataaatct atttctataa aattatgagg
8040acattttaca attaataata taattataaa cacactgttt ttttaaatac tgctacacat
8100tacttctagt taa
8113611587DNAZea maysCDS(1)...(1587)ZmCkx8 cds 61atg gag ggc aag gtg ctg
tgc acg tac gcc ggg atc gtg gcc cta ctg 48Met Glu Gly Lys Val Leu
Cys Thr Tyr Ala Gly Ile Val Ala Leu Leu1 5
10 15ctc tgc tcg tcg gtg aat ttc ata cag agc ccc tcc
gac gtg ttc ggc 96Leu Cys Ser Ser Val Asn Phe Ile Gln Ser Pro Ser
Asp Val Phe Gly 20 25 30ccc
gtg gcg ctg ctg gag ccg aca gca tcc gcg gca cgc gac ttc ggc 144Pro
Val Ala Leu Leu Glu Pro Thr Ala Ser Ala Ala Arg Asp Phe Gly 35
40 45ggc gtg gtc tcg gag gcg gcc atc gcg
gtc atg cag ccc ggg tcc ccc 192Gly Val Val Ser Glu Ala Ala Ile Ala
Val Met Gln Pro Gly Ser Pro 50 55
60gcc gac atc gcg cgg ctc ctg ggc gcg ctg tcg tcg acg ggg ccg ggg
240Ala Asp Ile Ala Arg Leu Leu Gly Ala Leu Ser Ser Thr Gly Pro Gly65
70 75 80ccg ggg ccg aag gcg
gcc gtg gcg gcg cgc ggc gcg ggg cac tcg ctc 288Pro Gly Pro Lys Ala
Ala Val Ala Ala Arg Gly Ala Gly His Ser Leu 85
90 95cac ggg cag gcc cag gcg cgc ggc ggc att gtg
gtg gag acg cgc gcc 336His Gly Gln Ala Gln Ala Arg Gly Gly Ile Val
Val Glu Thr Arg Ala 100 105
110ctg ccg cgc ctc gtg gag gtg gtg cga cgc ggg gac ggg gac ggc ggc
384Leu Pro Arg Leu Val Glu Val Val Arg Arg Gly Asp Gly Asp Gly Gly
115 120 125ggc gcg gcg tac gcg gac gtg
ggc ggc ggc gcg ctg tgg gtg gag gtg 432Gly Ala Ala Tyr Ala Asp Val
Gly Gly Gly Ala Leu Trp Val Glu Val 130 135
140ctg gag gag tgc ctg agg gcc ggg ctg gcg ccg cgg tcg tgg acg gac
480Leu Glu Glu Cys Leu Arg Ala Gly Leu Ala Pro Arg Ser Trp Thr Asp145
150 155 160tac ctg tac ctg
acc gtg ggc ggg acg ctg tcg aac ggc ggc atc agc 528Tyr Leu Tyr Leu
Thr Val Gly Gly Thr Leu Ser Asn Gly Gly Ile Ser 165
170 175ggg cag gcg ttc aag cac ggc ccg cag atc
agc aac gtg ctg cag ctg 576Gly Gln Ala Phe Lys His Gly Pro Gln Ile
Ser Asn Val Leu Gln Leu 180 185
190gag gtg gtc aca ggc aca ggg gag gtg gtg aca tgc tcg ccc acc cag
624Glu Val Val Thr Gly Thr Gly Glu Val Val Thr Cys Ser Pro Thr Gln
195 200 205agc ccg gag ctt ttc ttc gcc
gta ctt ggt ggg ctt ggc cag ttc ggt 672Ser Pro Glu Leu Phe Phe Ala
Val Leu Gly Gly Leu Gly Gln Phe Gly 210 215
220atc ata acc cgc gca agg att ccg ctg caa gtt gct ccg ccc aag gtg
720Ile Ile Thr Arg Ala Arg Ile Pro Leu Gln Val Ala Pro Pro Lys Val225
230 235 240aga tgg gtg agg
gcc ttc tac gac agc ttc gag acg ttc acc aag gac 768Arg Trp Val Arg
Ala Phe Tyr Asp Ser Phe Glu Thr Phe Thr Lys Asp 245
250 255cag gag ctg ctg gtc tca atg cca gag ctg
gtg gac tac gtg gag ggg 816Gln Glu Leu Leu Val Ser Met Pro Glu Leu
Val Asp Tyr Val Glu Gly 260 265
270ttc atg gtc ctg aac gag cag tcc ctc cgc agc tcc tcc gtg gcc ttc
864Phe Met Val Leu Asn Glu Gln Ser Leu Arg Ser Ser Ser Val Ala Phe
275 280 285ccc gcc cag gtc aac ttc aga
ccg gac ttc ggc tcc gac gac ggc acc 912Pro Ala Gln Val Asn Phe Arg
Pro Asp Phe Gly Ser Asp Asp Gly Thr 290 295
300aac aag aag gtc tgc tac tac tac tgc atc gag ttc gcg gtg cat gac
960Asn Lys Lys Val Cys Tyr Tyr Tyr Cys Ile Glu Phe Ala Val His Asp305
310 315 320ttc caa cgg cag
gac tcc gct gct gac cat gtt gtg gac ctg gtg tcg 1008Phe Gln Arg Gln
Asp Ser Ala Ala Asp His Val Val Asp Leu Val Ser 325
330 335ggg aag ctg agc tat ctg agg ccc cac gcg
tac agc gtg gag gtg gcc 1056Gly Lys Leu Ser Tyr Leu Arg Pro His Ala
Tyr Ser Val Glu Val Ala 340 345
350tac tgg gat ttc ctc aac agg gtg cgg atg gag gag gag agc ctc agg
1104Tyr Trp Asp Phe Leu Asn Arg Val Arg Met Glu Glu Glu Ser Leu Arg
355 360 365agg cgg ggc ctc tgg gac gtg
ccg cac ccc tgg ctc aac ctc ttc gtg 1152Arg Arg Gly Leu Trp Asp Val
Pro His Pro Trp Leu Asn Leu Phe Val 370 375
380ccc agg cat ggc gtc gcg cgg ttc atg gac ctg ctc atg gcc acc atc
1200Pro Arg His Gly Val Ala Arg Phe Met Asp Leu Leu Met Ala Thr Ile385
390 395 400gcg cag ggg gac
ttc gag ggg ccc gtc ctc gtc tac ccc ctc ctc act 1248Ala Gln Gly Asp
Phe Glu Gly Pro Val Leu Val Tyr Pro Leu Leu Thr 405
410 415cac agg tgg gac ggc aac atg tcg gcg gtg
gtt ccg gcg gcg cca gac 1296His Arg Trp Asp Gly Asn Met Ser Ala Val
Val Pro Ala Ala Pro Asp 420 425
430ggt gtg atg tac gtc ttc agc gtg cta cgg tcg acg gac ccg gcg cgg
1344Gly Val Met Tyr Val Phe Ser Val Leu Arg Ser Thr Asp Pro Ala Arg
435 440 445tgc ggc cgc gcc tgc atg gag
agg atc ctg gag cag cac cgc cgt gtc 1392Cys Gly Arg Ala Cys Met Glu
Arg Ile Leu Glu Gln His Arg Arg Val 450 455
460gcc gac gag gcc tgc cgg cgc ctc ggc gcc aag cag tac ctg gcc agg
1440Ala Asp Glu Ala Cys Arg Arg Leu Gly Ala Lys Gln Tyr Leu Ala Arg465
470 475 480cag ccg tcg ctg
gcg cac tgg cgc gac cac ttc ggg gcc agc tgg gac 1488Gln Pro Ser Leu
Ala His Trp Arg Asp His Phe Gly Ala Ser Trp Asp 485
490 495cgc ttc gtg gcc agg aag gcc cgc ttc gac
ccc atg aac gtg ctc ggg 1536Arg Phe Val Ala Arg Lys Ala Arg Phe Asp
Pro Met Asn Val Leu Gly 500 505
510ccg ggc cag gga att ttc ccc tgg acg gac tcc tcc tcg agc ccg atg
1584Pro Gly Gln Gly Ile Phe Pro Trp Thr Asp Ser Ser Ser Ser Pro Met
515 520 525tga
158762528PRTZea mays 62Met Glu Gly
Lys Val Leu Cys Thr Tyr Ala Gly Ile Val Ala Leu Leu1 5
10 15 Leu Cys Ser Ser Val Asn Phe Ile
Gln Ser Pro Ser Asp Val Phe Gly 20 25
30 Pro Val Ala Leu Leu Glu Pro Thr Ala Ser Ala Ala Arg
Asp Phe Gly 35 40 45
Gly Val Val Ser Glu Ala Ala Ile Ala Val Met Gln Pro Gly Ser Pro 50
55 60 Ala Asp Ile Ala Arg
Leu Leu Gly Ala Leu Ser Ser Thr Gly Pro Gly65 70
75 80 Pro Gly Pro Lys Ala Ala Val Ala Ala Arg
Gly Ala Gly His Ser Leu 85 90
95 His Gly Gln Ala Gln Ala Arg Gly Gly Ile Val Val Glu Thr Arg
Ala 100 105 110 Leu
Pro Arg Leu Val Glu Val Val Arg Arg Gly Asp Gly Asp Gly Gly 115
120 125 Gly Ala Ala Tyr Ala Asp
Val Gly Gly Gly Ala Leu Trp Val Glu Val 130 135
140 Leu Glu Glu Cys Leu Arg Ala Gly Leu Ala Pro
Arg Ser Trp Thr Asp145 150 155
160 Tyr Leu Tyr Leu Thr Val Gly Gly Thr Leu Ser Asn Gly Gly Ile Ser
165 170 175 Gly Gln Ala
Phe Lys His Gly Pro Gln Ile Ser Asn Val Leu Gln Leu 180
185 190 Glu Val Val Thr Gly Thr Gly Glu
Val Val Thr Cys Ser Pro Thr Gln 195 200
205 Ser Pro Glu Leu Phe Phe Ala Val Leu Gly Gly Leu Gly
Gln Phe Gly 210 215 220
Ile Ile Thr Arg Ala Arg Ile Pro Leu Gln Val Ala Pro Pro Lys Val225
230 235 240 Arg Trp Val Arg Ala
Phe Tyr Asp Ser Phe Glu Thr Phe Thr Lys Asp 245
250 255 Gln Glu Leu Leu Val Ser Met Pro Glu Leu
Val Asp Tyr Val Glu Gly 260 265
270 Phe Met Val Leu Asn Glu Gln Ser Leu Arg Ser Ser Ser Val Ala
Phe 275 280 285 Pro
Ala Gln Val Asn Phe Arg Pro Asp Phe Gly Ser Asp Asp Gly Thr 290
295 300 Asn Lys Lys Val Cys Tyr
Tyr Tyr Cys Ile Glu Phe Ala Val His Asp305 310
315 320 Phe Gln Arg Gln Asp Ser Ala Ala Asp His Val
Val Asp Leu Val Ser 325 330
335 Gly Lys Leu Ser Tyr Leu Arg Pro His Ala Tyr Ser Val Glu Val Ala
340 345 350 Tyr Trp Asp
Phe Leu Asn Arg Val Arg Met Glu Glu Glu Ser Leu Arg 355
360 365 Arg Arg Gly Leu Trp Asp Val Pro
His Pro Trp Leu Asn Leu Phe Val 370 375
380 Pro Arg His Gly Val Ala Arg Phe Met Asp Leu Leu Met
Ala Thr Ile385 390 395
400 Ala Gln Gly Asp Phe Glu Gly Pro Val Leu Val Tyr Pro Leu Leu Thr
405 410 415 His Arg Trp Asp
Gly Asn Met Ser Ala Val Val Pro Ala Ala Pro Asp 420
425 430 Gly Val Met Tyr Val Phe Ser Val Leu
Arg Ser Thr Asp Pro Ala Arg 435 440
445 Cys Gly Arg Ala Cys Met Glu Arg Ile Leu Glu Gln His Arg
Arg Val 450 455 460
Ala Asp Glu Ala Cys Arg Arg Leu Gly Ala Lys Gln Tyr Leu Ala Arg465
470 475 480 Gln Pro Ser Leu Ala
His Trp Arg Asp His Phe Gly Ala Ser Trp Asp 485
490 495 Arg Phe Val Ala Arg Lys Ala Arg Phe Asp
Pro Met Asn Val Leu Gly 500 505
510 Pro Gly Gln Gly Ile Phe Pro Trp Thr Asp Ser Ser Ser Ser Pro
Met 515 520 525
632080DNAZea mayspromoter(1)...(2080)ZmCkx8 promoter region 63gaattctatc
ttttcttgag ttattttatg atacaactgt tgctttgtct ggaatttatt 60atcctacttc
aacattgatg cttcatcata tacttaaaat tgctagacat ctaaatgctt 120ttgaaaatga
tgctttgctt agagatgcta ttgttcctat gaaaacaaaa tatttgaaat 180attggaggaa
gatacctgtt ttatattgct ttgcttttgt attggatcct agagcaaaaa 240tgagggggtt
taataagctt cttatgaggt tgtctggact taatggaact gattattcaa 300ggtatcctac
atacattcgg tctaaactaa ctaagatttt tcagatatat gaattgaaat 360ttggtgaagt
gtgcttgagt gcacaacaac ataagagtgc tggtacggca ggtaaggcta 420cagaggcatg
ggatgacata tatggggatg atatccttat gccttcccaa tctactagag 480ctactcctac
agctgtatca tctactgctg ctgctatatc tgagttgtca tcatatcttg 540atagtgatac
tgtcacccag tttgactctg atttcattct tctaaactgg tggcagcgac 600acaagttgac
atatcctgtg ctttctatac ttgctaaaga tgttataatt gtgcctgctt 660ccactgtatc
atcagagtcc actttcagtt tagctggcag ggtgcttgaa gaccgacggc 720ggcgcctaac
tcctgatatg gttgaagttt tgtcttgcat aaaggactgg gagcttgctg 780acttgcatag
tcagcacacg gtggagaaag ataccaaaga acttgaagtt gtttttgaag 840caatgtacct
agaagaaact ggtggaggca aagaaagaag aggtggagga tctggtggag 900cgggtagatc
ttgagcagct gaattattgc tattactata ctctgttctt ctgttgtaac 960ttgtgatgaa
ctattaaact ctggacttaa attgaaccta tataggagct ggctctactc 1020tttttcttcc
tagggttttc tcacgaggtg tgagttttta cctaggaagg tttttaatga 1080ggcagcattg
cactaaggct ccattagtat attgtttgca taaacttttg tgaactgtga 1140ttttgtttct
gagatgtttt gtgaactgtg tgaattgact gaaatctgat ataggaactg 1200tgtgaaatct
gatataggaa ctgtgtgaat tgactgaaat ttgatatatg aactgtgtga 1260aatttgattc
agctgtttat tgtgaaatta ctgtgcttcg ggtcagcccg gccctatggg 1320ctgaccgggc
cagaggcacg gcacgacaca acccgtttaa gccactttcg tgccgtgctt 1380gtgccaacag
tttagcccgc gggccagcac ggcacggcac ggaagtagga tcgtgccgtg 1440cccggcacgc
acagtaacgt gctgtgcttg gccgtgcccg tgccgtgccg gcccgacaca 1500cacgaatgga
catgtatagt cctgactgtc ccgtaaccaa acggacccca acatactcga 1560tgttgtttag
accgaccgac tgatcgtgcc acattgcact gcgcgtgaag aggtcgatac 1620cgatcgttta
gaccaccatg tcagctgatg gtactgtccc acgttggcat tggagcagct 1680tacctatcat
acatatcatc tattttttat ttaaaaattt actataaata gtgtagtata 1740caatataaaa
tagtatcata tgctcaatat gcttgagaca gctttaatag gatcaaacta 1800agattctcgg
gcccggcatc ggtagcgacg acaccggcta tatataatgc actcagtgag 1860cttcctggtg
gctcttgctg cttcttcctt gctgttccat ccgtccacag ttcttgtggg 1920aacccaagat
cgatcttgac ggggacgtga gcacggcacg tcgcgacctt attcttccgt 1980cttggccccg
tgcaccggca agcggcaacc aaatgcgcat gcccctgtga aagctaatag 2040tagctacatc
acacagcaag acactatagc cagctagcca 2080644365DNAZea
mays5'UTR(3125)...(3226)intron(3227)...(4365) 64tagttgtcgt tcgctgatgc
cttttgctgc aggggctgca cgtcagcctg atgcatttgt 60tttgagattc atggcatgtc
ttgcaggtca acttttgcct gattaataac atgctggtaa 120acacggaggc tgggccacaa
tgtctaagtt tagtaggtca aattgaagaa ctaactttag 180actaaaaatt aaatcaaaca
ggcctccaac ggtgcactaa atagcattcc taaccgtaca 240tacagtatga acagttcaat
gtaaggagtc ctcgtatcta tagggagaag gaatctccct 300gtatatatat agagtacgaa
gcttcctcta tactttaata gagtcgtttc tttacggtat 360taatcttatt taaatctcct
aatatagtaa taatatatta tgatagtaca aatattatat 420aactttttat ttttaaaaaa
tgtaaataga tgttaattag ttgaatttta taatacatat 480gaagaggtgt ataaggaaat
ggttggaaac cttgtatatg tacgaggaga attttttaaa 540atgagatagt aaaatatatt
agaacgtaat ataaggagag atgtatatat gaaaagttgt 600ataaagaaat agttggagac
gtcatatatg tgagaagata attttaagat gagatagtaa 660aatatattag aacgtagaga
tgtataggaa aaatggttgg gagggtcatg tcatccgtca 720accaccaacc ccggaggtag
gagcacctac caccactgcc acggccccat tttgtcctcc 780catgtgggcc ctaaagtggc
caagtggggc gcctcatgtc tagtagtttt atgaggatta 840tgatgggaga tcagctccaa
ctccaatcta tgagttcgaa ttacatttat ttggttgaac 900caggaagacg atgcgcatac
actcatgcag tgtgttttga gtgtgatgta agccagattc 960aagaaaaaaa aagtagctgg
atgggagctt tcatggttgg tgggggctgg tgggccgagg 1020agatgctcct actactccca
caccgtttga gggttggtgg cacaaaatat tttctcgatc 1080tgataatacc gtttttgaac
ataccataat attttagatt cattgacgtt tagaagcacg 1140tttaaaactt gtgtatttta
aaccatggtt ttactaatac catagtattt ttttgggata 1200aaaaactttg gtctaaacta
tttttttttg cttgcacgca gctgcagttt tctcttttcc 1260tacactaact aaaatattgt
atcttcaaat atgtgttgga ttagacacat gtaaaatata 1320ccctagtaay gtcacggtay
acaataaacc atgatattgt aaactacggt tttaaaaaat 1380agagttccta aacagacatg
tgtcatgatt ggctcgttgt ggaaaatcaa tttagacatc 1440tttgaaactc aggaatctca
tgagaatgct atagaaattt tacagaaatt agtttaaaaa 1500tacagatatc cttttgatcc
tgttcggact ttgggttgtc cgtagcttcg catgcaatta 1560gttgtagttt catatgacta
gccgctaaca atctttttaa tccccactga cctagctaat 1620tgttagctaa taactacttt
actagttaca tcaaactagc taataacagt taatattagc 1680tagtagctaa taattagcag
ccaatagatg accaaaaaat gaagcataca aacaatacta 1740caaactgaca tcggcttcat
ttccaagtaa atcggctttt aaggttatca taagctattt 1800ttttaaaaaa ataatcaaat
ttataggaaa aacaaacgta tttatgctac caaatcacag 1860taattagata aatcataaaa
tgtattttta cagtttattt acttagatta atagatattt 1920atattggtat tctataaatt
tggtcagaca taaaataaaa agcttcactc aaagacaatt 1980ctttcggtgc ggatggtgta
cctatcttta gtgtattcca tgaatatcaa ggcaatcaat 2040gaagagcgtg ctgtaaacaa
tcgtcatatc ttggccttat ttggttagaa ggaaattgcg 2100gtgcatcaag tgcttgtttg
gttagaaatg aattaagtag gatttgaaat ctcatactat 2160ttaaaaatta aataacaaga
gatttaattt tcacaatcct ctataatccc tatacaaccg 2220aacaagacat aagagctagt
ttgaaaattc aaattctctc cgtggaattt aagtttctaa 2280actagaatat atatcaacat
tatcaacatc accaacaacc ccacatctgt attctgccct 2340gctagctaag cacgtctcat
tagctggcgt aagcgccttt ttttaataca ccatttttct 2400acgatctgct gcttgccagt
tgggcctttg tgcattcccc tctgtaaaat aataaaatac 2460gaaatttccg tttccgtttc
attagttggc attcgctgtg tagactgcaa aagtcagcct 2520gttgctgttt tttttttctc
ttccatggat gcgacagcta ctagcacggt cgttcagatt 2580catcatatgg cgcactcgct
tgccattcta acccaaatct cctgattaaa acgccaagat 2640ttgtgccact cttattatag
aaaattgttt gtttcacgcg aaattgttaa ttccaagttt 2700ttagcaaagg cggagaggta
cgtgtagacg tttcattgtt gctagtattt gggtctgctc 2760attcgaacga attctgcaga
aaatctagat tgcataaatt ttctaggagt ttccgatgcc 2820gtagatccgg tttctttttg
cctataatca attcgtttaa aaactgtcat gaggtttttt 2880tttattcttg attttcgatc
gcactagctc aaaaaattta tgtagcaaga aaaagcagaa 2940ataatcaaaa caaacgtttt
tttttccaaa acaaaaaaga aagaaaacct caggcaccaa 3000cggatctggc agatgggaaa
tgggatctca ccaaatccca cgtactagcg cgcaccacct 3060aacgcagacg atacaccctt
ttataaatga aacccacgaa cccctcagat ttcccgtgct 3120catcatcacc agttcaccac
ccacctccca ctcccagttc accccgtcgt cctcggcgcc 3180accactcctc gtcccccggc
gctactcccc cgctccacgg tccaagggta agcgcgcctc 3240cccaccgctc ccttgctcta
tatagccctt cccactccac cgctcgccca ttccttcgct 3300tccgctgtct ccccgcgcct
cccggatcgc ctcggcgcgc ggtgagtctg gcgtgctgtt 3360gggccgcctg cctgcctgcc
cgtctctctt cggtctggat gcgtagccat tgtctccttc 3420ccggtcgggg ttgctttgct
gcgcgaggct gtgcggaatt ggtagttttt ttggtcgaga 3480atggctggtt cgattttcgg
gttccttttt gcacatgtcg ttgagatcgc cgctgggtca 3540ctacgggatt agagcctgtt
gccccctttt gtttctcgag gagatggttc gagtcgtaac 3600tatatgaaat tcaggcccca
gaattttgtt agcagcagaa cgggctttcc aaaactgttg 3660ttacatctgt tggaaaattt
agaatttctc catgtatgta tgtatgatcc gaaagttggg 3720tggcagtttc agtgaatgga
cagtgatatt ttttatattg atgttgtttt ctgtggctgt 3780agtttaatat atcatgcttc
ctgtccaaac tatagtttct tacggatgtt atttgtagca 3840tgatccctgc atgtctgaga
gggaccttac ttcccttccg accgatttta gctctcctgt 3900acccacatcc tggaaaggtt
aggttgcaac ctaaatggaa gactgtagtg catacagcat 3960acctccatgg tatggttaat
ccttaccagt ttaaagaaac agccttgatt gaccagaggt 4020atttctctgc atcgaatcat
ttagatctta tgggagcaac acatgcagta tgaattcaga 4080gtttcacatg gaaggataag
aattcagttc agtttatgtt tcagtgaaat atatagaata 4140tttttgtagc ttgtttgcag
ctttgttcag ataaatattc agttatctgt tgcagtgaat 4200caaagctgat tttaacattt
ttgctgttat atagaaaggt ggtgtcccat attgttggat 4260acacttgcat gagccccaag
agggagctct tttagcttat ttgcagcttt gttgaggcaa 4320atattcagct agcttctcta
tttctgtgaa tcaacctgat cttaa 4365654215DNAZea
mayspromoter(1)...(4215) 65gatccctgtg gagaaatttt tacgtcgcgg ggatggtatg
gggagttatt cccctgtagg 60aaatgggtga cgcctaagag ggagggtgaa gtaggacttc
taaaactttc actaaactag 120gccacaaata attccctaga gcaaaaccta tgcaaatagt
caaactagaa tgtgcaaacc 180aagttttgtc taagtgttgc tatctctacc gcaatggcta
agtttcaatc tacactatat 240aagtatgaat acaagaatga aacttaaata cttaatataa
atgcggaaac ttaaagagca 300aggtagagat gcaaattctc gtggatgacg cctgcatttt
tatcgaggta tccggaacca 360cgcaaggtcc cgactaatcc tcattggtgc ccctacgcaa
agggaagccc acgcgagggc 420caagcacctc ggtcgagtaa ctctatagag agccgtgggc
cttctccacg cgcaagtggt 480gctctgcttt cagctcctct cagaccctcc ccgctgtctc
cactatcgag cttccggctg 540aaaatgccat gggcctcgtt ccctccggta cacggtggcg
gccgtgacac aaatgcggtt 600atcacggtct cgcaagactc tcacccccac ttggtacaat
ttcaatggct cgcacaagag 660ccgaggggtt gatggtttat ctaatctcac tcaactaact
aggattcatc taaagcaagc 720gctagagcgg tctaactaac ctaagcactt cacaaagcac
ctacgctaat caccgagtga 780ttctatttag cacttgggtg caagagcact tgagaatgtc
tactatatgc cttgctatgt 840ctcttgggct cccaaacttg gaaatggccg gttggtggtg
tatttatagc ccccaacaca 900aaactagccg ttggaggaag ctgctgcttt ttgtggtgca
ccggacagtc cggtggggtc 960accagacagt ccgacgcccc tgtccggtgc ccctgtccga
tgcgcctagt tgttgggtct 1020gtcagcgtag gtgaccgttg gcgcgcaggc tttttgcacc
ggacagtccg gtggtcttcc 1080ctcgacagtg ccacctggag ctagccgtta gggctactgt
tcctggtgca ccggacagta 1140gtccggtgct cttgtctgga cagtccgact gtggcaacac
ttcttctttt cttggacttt 1200acttgatctt catgatgtct tcttttgagg tgttgctttc
ctaagtgcct tggtccaagt 1260aacttatcat cctgtgaact acaaacacaa atagtagcaa
acacattagt ccacaggtta 1320tgttgatcat caaataccaa aatctattaa gccaaatggc
ccagggtcca ttttccttac 1380atccccgacg aagaattctc cgttgccatc cctatctgtg
tacgcactac tggaatccgg 1440gtctttgctg agtaccgcac tcggcaaagt cctactctcg
gtaacgatgc cttttgccga 1500gagcaggact ctcggcacag gaatacactc ggcgaagggc
gggtctcggc aaaggccgtt 1560agccaccgtc caaagctgac ggtcgttacc tatgccgagt
ggtggaaaga tattgtgaag 1620gcctaaggcc gatttcgtcc taagcagggc ccaaaggaag
gaagtacttc agtggatcaa 1680gatgttgatg ttccctgatg ggtatgcagc taacctgagt
aggtggggtg aacttatcta 1740ctctgtgagt cttagggatg aagagtcatg acttccacat
atggattgaa cagattcttc 1800tctgtgcatg gacaatctgg ggcggcatcc aacaaccctc
atggatcgcc cggccaatcg 1860ccgcaccagt ccatccgccc acctcgatga gacttatgtt
cttagtgttg agacttcaga 1920acttattgat aatgctgtat tggatactta tgtttgtgtt
cgatacttat gtgagaactt 1980gagacttatg agacttatgt tcttgatact tatgtttgtg
ttgagaactt ggatatttat 2040gtttgtgttg gatacttatg tctgtgatga tatatgtgat
gtatatatgt gatgtatatg 2100tgacatatgt gatgtatatg tggtatcttt tgtttgtttg
gatggaatag agaaagcaaa 2160taaaaatgtg tatactggtc actttgtcga gtgtaacact
cggcaaaaag gtgctttgcc 2220gagtgttagg gccatagcac tcggtagaga accaatactt
aggcaccggt aaagcttttt 2280tgccgagtgt tgtggccctg gcactcagct ttgccgagtg
cctcacagag cactcgacaa 2340agaacctgac aaatggaccc gctggtaaat cctttaccga
gtgcaggtca gtagacactc 2400ggcaaaggta acttctttgc cgagtgccgc ttagaacatt
tgacaaaggg tcatctccgt 2460tacccggtgt cgtgacggcc gcttttcttt gccgagtgcc
tgatagaaag tactcggcaa 2520agaagtcgtt gccaatgtat tgttcgctga ggtctctttg
tcaagtatta cactcggcaa 2580agactgtgcc gagtgttttt cagactttgc cgagtggttt
aagcactcag caaagcgctc 2640gatttcggta gtgacggttg tttggcaata gtaaaatcca
gccctctccc gtggggaaaa 2700aactggtagg atctggctcg tggctaagat tctctttctt
ccctttgtaa aaaaagagaa 2760gaaaaaaaaa acgactgtca cggtgccttg tctggtaatg
atcgcgcggt cggctctgtc 2820ctaacccgta agatggacgg gagctgatga tagcgtgacc
tccaaataaa caacaagggc 2880gtgttccccg tggtcgaata ttttaagggc cactgattag
gtgcggttga atacatcaac 2940ttcacgaaca tcatctgatc tgatctgatt tggtctgata
tgatctgggt agtcatttct 3000gcaatgagca tctatcaggt gaaccaatta atattgatga
cattatgagt tcgaagatat 3060actctaaagt gttatctaaa tacagaagac attcgttcgt
tctttgccta taactctaaa 3120aggcttgtaa caccctcatt catcctctat atacgaagac
tctctcctat catttttatc 3180gatttatttt ttttatattt tagacaatgg aattaaatag
aactaaaata tatataagaa 3240tctgaggacc cgagatggta atggggactc gatcctcgat
tctccacgga gaattcctct 3300aggatatagg taatttgtcc ccacgaggat tgaaacgggg
taatttggtc cccatgtgcc 3360cgtcccgcga acttctcttg atctaaatta gtctatttcc
atgttaaaac tatactaaaa 3420atttaataca cagtctatta taaaatagca aactaaattc
taaagttgat gcatcttgta 3480attttaaatc tggtttgttc aagttatatt catttgatat
aataaatttg aatttgactc 3540ttaatatcgt attttttcct aacggggacg gattctccac
ggggataaat tccatgatac 3600agatgggatg aaagaaaaat ctcccgtatg aacttttgca
ggaatgggga tgggccagag 3660aaattttctc cctgcgggga cgggggagcc atatcctcgg
tggagaattt cccattatca 3720tccttatttg tggtacatat atatgcataa tctttttttt
ttgactgaca tgtgggaaag 3780tatcccatct caatagtaga aaatcttggg aacggtagga
tcgaacacaa agatcagcta 3840gcttgtaatc accgagccat atagctagag ggtaatagat
catgaatcaa atgttttttt 3900cataaattat taaggctcta aattattttt aatttaaaaa
taaataaaaa tatagttcga 3960ttcttacatt ttatagtgta aaactttaaa gtctattatt
acccctactt attgagttat 4020ggttcagttc ttgtcgacgg agagtaatga gatatagaat
aaggtaccct atagaataaa 4080gaatctttct ctgaaaagtc tgacgtacgt aaataagata
taataaaaaa aatacaaaga 4140gaagcgctgg actggagatg ctcctatatg cggcaatgcc
tgtgcttata aatagccacc 4200tcggtcggca aggac
421566419DNAZea mays3'UTR(1)...(419)ZmCkx2 TR1
66gttgtacaaa agtgtaggta aaaagtatcc cctgtaaaga caatatctac ggaaggtagc
60tagcctgaag aacacagcat agcgactttt tttatagtgg ccgaagacac ctcagagcaa
120tacttcaaag tggagcaatg tcacctgaac tctgacacct ttggaggcaa tcactggagg
180atcgtagcgg tccaagaaac ctgtatgttg ttacagcgtt gataattgag acgagctgtg
240atgatcaact gatcactaac cagtatcccg gttatcaaca gtgcagaaag ttgcttgagg
300gtagtagtgc cttgattaac aaataacact gcctgttatt tcacttgtaa ctagcatctc
360atcccactca ggagtgcctg cgtaaatgta gcaggtttac attactttcc atgagtcag
419672146DNAZea maysCDS(68)...(1645)ZmCkx2b or cko3; clone 2 67tctctctctc
tctctgcctt ctgtttccag gacgtcccaa ctgcccagcg ccgaccggcc 60ggccacc atg
aag ccg cca tca tca ctg gtg cac tac ttc aag ctg ctg 109 Met
Lys Pro Pro Ser Ser Leu Val His Tyr Phe Lys Leu Leu 1
5 10gtc ctg ctg gcg ctc gcc agg ctg acc atg cac gtc
ccc gac gag gac 157Val Leu Leu Ala Leu Ala Arg Leu Thr Met His Val
Pro Asp Glu Asp15 20 25
30gtg ctc ttg tcc ctc ggc gcg ctg cgc ctc gac ggc cat ttc agt ttc
205Val Leu Leu Ser Leu Gly Ala Leu Arg Leu Asp Gly His Phe Ser Phe
35 40 45cac gac gtc tcc gcc atg
gcg cgg gac ttc ggc aac cag tgc agc ttc 253His Asp Val Ser Ala Met
Ala Arg Asp Phe Gly Asn Gln Cys Ser Phe 50 55
60ctg ccg gcc gcc gtg ctc cac cct ggc tcg gtc tcc gac
atc gcc gcc 301Leu Pro Ala Ala Val Leu His Pro Gly Ser Val Ser Asp
Ile Ala Ala 65 70 75atc gtt agg
cac gtc ttc tcc ctg ggc gag ggc tcg ccg ctc acg gtc 349Ile Val Arg
His Val Phe Ser Leu Gly Glu Gly Ser Pro Leu Thr Val 80
85 90gcg gcg cgc ggg cac ggg cac tcc ctc atg ggc cag
tcc cag gcc gcc 397Ala Ala Arg Gly His Gly His Ser Leu Met Gly Gln
Ser Gln Ala Ala95 100 105
110cag ggg atc gtg gtc agg atg gag tcg ctc cgg ggt cct agg ctt cag
445Gln Gly Ile Val Val Arg Met Glu Ser Leu Arg Gly Pro Arg Leu Gln
115 120 125gtc aac gac gcc ggc
gtg tcg cca ccg tct gtc gat gct ccc gga gga 493Val Asn Asp Ala Gly
Val Ser Pro Pro Ser Val Asp Ala Pro Gly Gly 130
135 140gag ctc tgg atc aac gtg ctg cgt gag acg ctc aag
cac ggt ctg gca 541Glu Leu Trp Ile Asn Val Leu Arg Glu Thr Leu Lys
His Gly Leu Ala 145 150 155ccc aag
tcg tgg acg gac tac ctc cat ctc acg gtc ggt ggc acc ttg 589Pro Lys
Ser Trp Thr Asp Tyr Leu His Leu Thr Val Gly Gly Thr Leu 160
165 170tct aat gcg ggg gtc agc ggg cag gcg ttc cgc
cac gga ccg cag gtc 637Ser Asn Ala Gly Val Ser Gly Gln Ala Phe Arg
His Gly Pro Gln Val175 180 185
190agc aat gtc aat caa ctg gag att gtg aca gga aga gga gat gtc gtt
685Ser Asn Val Asn Gln Leu Glu Ile Val Thr Gly Arg Gly Asp Val Val
195 200 205acc tgc tca ccc gat
gat aac gct gat ctc ttc tat gct gct ctc ggt 733Thr Cys Ser Pro Asp
Asp Asn Ala Asp Leu Phe Tyr Ala Ala Leu Gly 210
215 220gat ctt ggt cag ttc ggg atc atc acc aga gca agg
att gca ctt gag 781Asp Leu Gly Gln Phe Gly Ile Ile Thr Arg Ala Arg
Ile Ala Leu Glu 225 230 235cct gct
cca aag atg gtg agg tgg ata aga gtt ctt tac tcg gat ttt 829Pro Ala
Pro Lys Met Val Arg Trp Ile Arg Val Leu Tyr Ser Asp Phe 240
245 250gaa agc ttc acc gag gac cag gag atg ctg ata
atg gca gag aac tcc 877Glu Ser Phe Thr Glu Asp Gln Glu Met Leu Ile
Met Ala Glu Asn Ser255 260 265
270ttt gac tac gtt gaa ggt ttt gtc atc ata aac agg aca ggc gtc ctc
925Phe Asp Tyr Val Glu Gly Phe Val Ile Ile Asn Arg Thr Gly Val Leu
275 280 285aac aac tgg agg gcg
tcc ttc aag cca caa gac cca gtc gaa gca agc 973Asn Asn Trp Arg Ala
Ser Phe Lys Pro Gln Asp Pro Val Glu Ala Ser 290
295 300cat ttt cag tcg gat gga aga gta cta tac tgc ctc
gag cta acc aag 1021His Phe Gln Ser Asp Gly Arg Val Leu Tyr Cys Leu
Glu Leu Thr Lys 305 310 315aac ttc
aat agt gac gac act gat acc atg gaa cag gaa gtt act gta 1069Asn Phe
Asn Ser Asp Asp Thr Asp Thr Met Glu Gln Glu Val Thr Val 320
325 330ctg cta tct cga ctt aga ttc ata cag tct act
cta ttc cac acc gat 1117Leu Leu Ser Arg Leu Arg Phe Ile Gln Ser Thr
Leu Phe His Thr Asp335 340 345
350gtc acg tac ctg gag ttc ttg gac agg gtg cac acc tct gag ttg aaa
1165Val Thr Tyr Leu Glu Phe Leu Asp Arg Val His Thr Ser Glu Leu Lys
355 360 365ctg agg gca caa ggc
ctc tgg gaa gtt cca cat cct tgg ctg aat ctt 1213Leu Arg Ala Gln Gly
Leu Trp Glu Val Pro His Pro Trp Leu Asn Leu 370
375 380cta ata ccg agg agc tcc atc cgc aga ttt gct aag
gaa gtc ttt ggc 1261Leu Ile Pro Arg Ser Ser Ile Arg Arg Phe Ala Lys
Glu Val Phe Gly 385 390 395aag atc
ctg aaa gat agc aac aat ggt ccc ata ttg ctt tat cca gtg 1309Lys Ile
Leu Lys Asp Ser Asn Asn Gly Pro Ile Leu Leu Tyr Pro Val 400
405 410aac aaa tca aag tgg gac aac aga acg tca gta
gtc ata cca gat gag 1357Asn Lys Ser Lys Trp Asp Asn Arg Thr Ser Val
Val Ile Pro Asp Glu415 420 425
430gaa att ttc tac cta gtg ggg ttc ctt tct tca gca ccg tct ctc tca
1405Glu Ile Phe Tyr Leu Val Gly Phe Leu Ser Ser Ala Pro Ser Leu Ser
435 440 445ggt tac ggc agc att
gca cat tca atg aac ctg aac aaa cag ata gtg 1453Gly Tyr Gly Ser Ile
Ala His Ser Met Asn Leu Asn Lys Gln Ile Val 450
455 460gag ttc tgt gaa gag gct ggt att ggg atg aaa cag
tat ctg gca ccc 1501Glu Phe Cys Glu Glu Ala Gly Ile Gly Met Lys Gln
Tyr Leu Ala Pro 465 470 475tac acc
aca cag cag cag tgg aaa gcc cac ttt gga gca agg tgg gag 1549Tyr Thr
Thr Gln Gln Gln Trp Lys Ala His Phe Gly Ala Arg Trp Glu 480
485 490aca ttt gaa cgg agg aaa cac aga tat gat ccc
cta gcc atc cta gcg 1597Thr Phe Glu Arg Arg Lys His Arg Tyr Asp Pro
Leu Ala Ile Leu Ala495 500 505
510cca gga cag aga ata ttc cca aag gcg tca ctg cca ttg cct ttg tga
1645Pro Gly Gln Arg Ile Phe Pro Lys Ala Ser Leu Pro Leu Pro Leu
515 520 525cagttcctgc tacttgaagg
attctgtaga gcatacgttt acgttgtaca aaagtgtagg 1705taaaaagtat cccctgtaaa
gacaatatct acggaaggta gctagcctga agaacacagc 1765atagcgactt tttttatagt
ggccgaagac acctcagagc aatacttcaa agtggggcaa 1825tgtcacctga actctgacac
ctttggaggc aatcactgga ggatcgtagc ggtccaagaa 1885acctgtatgt tgttacagcg
ttgataattg agacgagctg tgatgatcaa ctgatcacta 1945accagtatcc cggttatcaa
cagtgcagaa agttgcttga gggtagtagt gccttgatta 2005acaaataaca ctgcctgtta
tttcacttgt aactagcatc tcatcccact caggagtgcc 2065tgcgtaaatg tagcaggttt
acattacttt ccatgagtca gaatattata tgctcatgca 2125aaaacagtaa agtctcttat c
214668525PRTZea mays 68Met
Lys Pro Pro Ser Ser Leu Val His Tyr Phe Lys Leu Leu Val Leu1
5 10 15 Leu Ala Leu Ala Arg Leu
Thr Met His Val Pro Asp Glu Asp Val Leu 20 25
30 Leu Ser Leu Gly Ala Leu Arg Leu Asp Gly His
Phe Ser Phe His Asp 35 40 45
Val Ser Ala Met Ala Arg Asp Phe Gly Asn Gln Cys Ser Phe Leu Pro
50 55 60 Ala Ala Val
Leu His Pro Gly Ser Val Ser Asp Ile Ala Ala Ile Val65 70
75 80 Arg His Val Phe Ser Leu Gly Glu
Gly Ser Pro Leu Thr Val Ala Ala 85 90
95 Arg Gly His Gly His Ser Leu Met Gly Gln Ser Gln Ala
Ala Gln Gly 100 105 110
Ile Val Val Arg Met Glu Ser Leu Arg Gly Pro Arg Leu Gln Val Asn
115 120 125 Asp Ala Gly Val
Ser Pro Pro Ser Val Asp Ala Pro Gly Gly Glu Leu 130
135 140 Trp Ile Asn Val Leu Arg Glu Thr
Leu Lys His Gly Leu Ala Pro Lys145 150
155 160 Ser Trp Thr Asp Tyr Leu His Leu Thr Val Gly Gly
Thr Leu Ser Asn 165 170
175 Ala Gly Val Ser Gly Gln Ala Phe Arg His Gly Pro Gln Val Ser Asn
180 185 190 Val Asn Gln
Leu Glu Ile Val Thr Gly Arg Gly Asp Val Val Thr Cys 195
200 205 Ser Pro Asp Asp Asn Ala Asp Leu
Phe Tyr Ala Ala Leu Gly Asp Leu 210 215
220 Gly Gln Phe Gly Ile Ile Thr Arg Ala Arg Ile Ala Leu
Glu Pro Ala225 230 235
240 Pro Lys Met Val Arg Trp Ile Arg Val Leu Tyr Ser Asp Phe Glu Ser
245 250 255 Phe Thr Glu Asp
Gln Glu Met Leu Ile Met Ala Glu Asn Ser Phe Asp 260
265 270 Tyr Val Glu Gly Phe Val Ile Ile Asn
Arg Thr Gly Val Leu Asn Asn 275 280
285 Trp Arg Ala Ser Phe Lys Pro Gln Asp Pro Val Glu Ala Ser
His Phe 290 295 300
Gln Ser Asp Gly Arg Val Leu Tyr Cys Leu Glu Leu Thr Lys Asn Phe305
310 315 320 Asn Ser Asp Asp Thr
Asp Thr Met Glu Gln Glu Val Thr Val Leu Leu 325
330 335 Ser Arg Leu Arg Phe Ile Gln Ser Thr Leu
Phe His Thr Asp Val Thr 340 345
350 Tyr Leu Glu Phe Leu Asp Arg Val His Thr Ser Glu Leu Lys Leu
Arg 355 360 365 Ala
Gln Gly Leu Trp Glu Val Pro His Pro Trp Leu Asn Leu Leu Ile 370
375 380 Pro Arg Ser Ser Ile Arg
Arg Phe Ala Lys Glu Val Phe Gly Lys Ile385 390
395 400 Leu Lys Asp Ser Asn Asn Gly Pro Ile Leu Leu
Tyr Pro Val Asn Lys 405 410
415 Ser Lys Trp Asp Asn Arg Thr Ser Val Val Ile Pro Asp Glu Glu Ile
420 425 430 Phe Tyr Leu
Val Gly Phe Leu Ser Ser Ala Pro Ser Leu Ser Gly Tyr 435
440 445 Gly Ser Ile Ala His Ser Met Asn
Leu Asn Lys Gln Ile Val Glu Phe 450 455
460 Cys Glu Glu Ala Gly Ile Gly Met Lys Gln Tyr Leu Ala
Pro Tyr Thr465 470 475
480 Thr Gln Gln Gln Trp Lys Ala His Phe Gly Ala Arg Trp Glu Thr Phe
485 490 495 Glu Arg Arg Lys
His Arg Tyr Asp Pro Leu Ala Ile Leu Ala Pro Gly 500
505 510 Gln Arg Ile Phe Pro Lys Ala Ser Leu
Pro Leu Pro Leu 515 520 525
693390DNAZea mayspromoter(1)...(3390)ZmCkx6 promoter 69aaaaactgtg
cagctagcta agagaagctg aaaaacagtt ttttttttta aaaaaaatct 60gtctactctt
agagcatctc caacaacgtg acctataaaa ttgccctata atttgaaaat 120aagtatattt
tatagaattt agggcaccaa caaaacacct cgctccaaca gtaaagtccc 180aaatctagat
tatagggcag accactacag tgtagtatat ttgagtcact tgagagggtg 240ctctatagtt
ttttgacaaa aaattatgaa atatggcact gttggagtag tttttcctgt 300gtagagccct
atatttcaat tttaggcact agtttaaggc attgttggag atgctcttat 360tttttaacga
aaagctgaaa aactggcctt cgattgataa aaaaacattc agattaataa 420tgttgtgagt
ggtacctatg ccttctctta tttttttctt aatgatttat gagaaactat 480aaattcttat
attaacatat agagaaaaag gctctttgtt ttgcgaccga gcgagggagt 540atacacggat
acaccggtac ctccgctccg cacgtacctg gaggctggag cagacgtttg 600actgggacgc
gccgagtgtc cggccaatga gagcgacgca cgtagcgcgg gggcgccgct 660gcggcggcac
atcatcacgt gcatgcggcc acgcgcgcgg gcgacagaca acgcgcgagc 720gacaggtcga
cccccgtggc cgaaccgaat cgcgtagggg atctcgacct atggcagcaa 780atttaacgcc
gcgttccggt ggcggtcccg ctccagcgat ggccgcgtac cgtacctacg 840gcgaccagac
cacgggataa tgcgtgcgat tgttcttttg ggtgggggag aatgctcgat 900cgatcgcaaa
tgccggtgct ccccggccgt tcgtcgtcgg ccggtcgatc acaggtacat 960actggcagta
aaaacagacg tgcaggttcc cgacctgtca tcgtattata ttcggcgtta 1020ctgacaccat
ggcaatggca tgcatggtac gaagccaagt aaggagcaga cgtgttcgta 1080cgcctgtcgt
cgtcttcgcg cgcgcgccca cgagcagcat gtctcacgcg cccagcaaat 1140tcgcgcgcgc
ggatgcagcc cgatcggtta tattcgatcg gttataatgc atcatcgtca 1200acggcgtcaa
aacaacgcga gagaggacac ctacattttt cccctccgga aattaatctt 1260aaaatttgcg
cctcttatgc tattaatata cgtattaaaa tttgtataat ttaaaactca 1320aaaaacattg
ccaaatgcat tgacgcgatt aaaaagttaa aaaaacaaaa aggataagaa 1380taagtgtagc
tacttttgaa ctttaaaacg tggtaaggct acagtgcagc tacctttgtc 1440tagttactgc
ctcgtgcgtg gaagattaga attccaccta gagtacgttt ttttccttct 1500ttttgttagt
tattactaac aataaagttc taactagaga caatttggct aattaaaaga 1560aggaaagcag
aggatgcaag ctgcctgttc tgtacagagc ctgaatatgc acgtcatctc 1620tgaagttact
aaccgtaatt taggagagaa aatatagcag agacaggaaa atcgttcggt 1680gtatctggaa
actcacgaat gagttatgtt ttcagagaaa cttgctcgag aagcatggag 1740ctgttactac
acacgcgata agcggacttt cacagaaatg gaaaacttta cgcccgccag 1800aaacgaaaga
gcaattggag atcagatcac cgtggagaaa aataatagcg tgtttggttt 1860gtaggttggg
ctgcttctgg agccatccag acctgtgtcc gagcctacat cagcgtttgg 1920tttgaatcgc
agaatgatgt cgtccgccac tgtattgttc taataataaa ctagcatgcg 1980ggttcaactc
actccacaag gaactgccgg acggctccat ccggagccaa gccacgacgg 2040atgagcgaaa
ccgccggacc aaacgcgctg taaaagaatg cagataggtt aggttttggg 2100agttgtgtga
tcttcagctt tctgccgata ggctgtctgt aagaggtctt tcagttttgt 2160ttggttctgt
ttctggttgg aaccagttcc cttggcctca ggcttcagca caagtctagg 2220tgtgatttaa
actgcactgt attgaatact ttagtctttt gacaatactg tagttaaaag 2280gccggggggt
tttgccttgg aactctaaaa aaatatacag tattaaccat ggactctgaa 2340ctctgtctgc
gtccacaggc aagtcatctt tcttccttgc actggttatc ttattgaaac 2400agaacggaaa
tcttttttgg aacaagagaa tttcgtcaca tcttgcctgc agtaaagttt 2460cccatctaga
tgcatactcc ctccgtccaa gtttaactgg cgttttagct tttctcagac 2520ataaatacca
gccaagagaa tagacgcatg tacccctgtg atctagcgtg aagtattaat 2580tgcaattact
gctgaggaca cgaaacggtt cacaacctcc agccctccac ggtggatgag 2640aggagaccaa
gagtccgttg gtgtgggaac gaagcgaacg ggtgtgtgaa acgaggagat 2700aactataatg
gcatcgaggt gtagaccacg aacgacacat aattctggac aaataaaatg 2760agctaaaacg
ccattaaact tggacggagg gagtatacta tcaacatttc gatcaaaagt 2820tactatacaa
aatttgcact gtccgaaaag cgatccttat caggaaggcg caggattcgt 2880cccagctaag
cgcaccggcc acaagtattc caccaccccc ggtcaatagc taaagaaatt 2940gggcggcaag
tgaaagtctc cgggatggga atgtgcatga gtcatgacgc gcctccgccc 3000tccggcctcc
gcagttgttt attcgcagcg cgcgggtggc ggcccgcccg tccgtgttct 3060ctgctccctg
tgttcggcac atcgtcaccc ccaccgtttc ctgtgcctct ctctcctatc 3120ttcctcggtc
tcctcccgta atcctttgcc tgataccccg ctctaccagg ccgccaccac 3180ctccctccag
gctccagcag cctataaata cgcccgcgtc gcccaccacc gcacaccact 3240tgaatactcc
atctcaactt cccttcctct cccgtgctgc gctgagctat atagctgctc 3300ctcgacctcc
aagaagcacg cgggcggagc ccggagcgag tgattagtga aaggcatagc 3360ataaggccgc
ccggccggga agtggtggca 3390701507DNAZea
mayspromoter(1)...(1507)ZmCkx7 promoter 70gctcggattg tcgcacgcga
gggtagtgta cgctacctgc cgcggtgact tcggaatggt 60cgaaatactg aagactgtcg
aaacggtctt tttttgacac gttgcgtctg agttttgttt 120ttgtgggggc ttttgttggt
atattacatg gctccgcctt gttaaaaacc tcacccccgg 180ggggaaaaga gtgcgggccg
gaataacatt gtttgctgga ttacaagggc gcatgggccc 240tgatgattaa aaaaattgcg
gtggttgtca atgttccagg agtgctctaa gtcttcacca 300gatgtcgtta ctagcctata
cgcgctgggg gatgccttcg acatgacgat gaatgggcct 360tctcatttcg gctctagatt
gccccgtgat tctgtctggg tggttcggac gagtacgagg 420tctcctttgt cgaactctgt
tgggacgacc gcgtggttgc gctaggcctt agtttgagcc 480tgatatttgt tgagggcttg
tagggcgagg acgcggtctc cgtcgatgag gtctttggac 540attggttcgt cgacatcggg
gaccgctgat gtgcttgttc ggggtgagcc atgctttatc 600tcctgcggta tcatgacctc
ggatccatac aatagacgaa aaggtgtgaa tccggttgcc 660cgacattctg tcgtgtttag
ggcccagacc acttcaggta gttggttggc ccatttgccc 720ttcttgtcat cgagatgtcg
tttcttgatg gcagtgaaga tcttgccgtt ggcgcgctcc 780accaatccaa cgcgcgcaac
agcaatttac gtggacttgc aggcttggag caaggaacaa 840caccaaaaca aaaaagaaac
atgcaacaag taatattgaa atttactttg aaacaggtat 900gcatgtttat ttaatatatt
ttgtacttga tgtttggact atttcatatt aacttgcata 960ctaaattatt tatgaaaaat
ttcttatggc atacctcatg aataaatcct agctacgcca 1020ctattaccag tggtttttgg
tttttatgat ttttttaaat cttttgaatt tagaacgaat 1080tttttaaaaa acggtgattt
atcgaaaccg tatcccgact ggtttaccgt cagtttttac 1140cggttttgta aaccatgcct
acgctacaca tatatacata cggtattacg gtgtatgtac 1200gtcgtatata tatcttagct
tatatatctt attgcatggt tctgtacgtg tccgacgagt 1260gacgacggct atcttagctt
atactctctc cctctgtttt tttagttgtt gctggatagt 1320ttaattttac actatccagc
gacaactaaa acgaaacgaa gggagtatat atcttactct 1380caatcgttcg taacaataat
aatggtaata ataacagcag tttaatctat atataggcca 1440ccacggctct ccactgctgc
gtgcgtgcgt gcgtacatcg tcaaaaacct ccatcaagca 1500actgatc
15077118DNAArtificial
Sequenceprimer 71ggtgcacggc gaggaggt
187224DNAArtificial Sequenceprimer 72tcgccgccga catgccgtcg
tccc 2473527PRTOryza
sativaPEPTIDE(1)...(527)Os CKX6 73Met Ala Ala Arg Cys Ser Ile Ala Phe Met
Val Met Ala Ser Cys Leu1 5 10
15 Ser Val Val Val Ser Gly Gly Leu Pro Gly Asp Leu Phe Ala His
Ser 20 25 30 Val
Ala Ser Lys Leu Arg Val Asp Arg Asp Thr Thr Ala Arg Ala Ser 35
40 45 Ser Asp Phe Gly Arg Ile
Val Ala Ala Ala Pro Glu Ala Val Leu His 50 55
60 Pro Ala Thr Pro Ala Glu Ile Ala Glu Leu Val
Arg Phe Ser Ala Ser65 70 75
80 Ser Pro Ser Pro Phe Pro Val Ala Pro Arg Gly Gln Gly His Ser Ala
85 90 95 Arg Gly Gln
Ser Leu Ala Pro Gly Gly Val Val Val Asp Met Arg Ala 100
105 110 Leu Ala Ala Arg Arg Gly Arg Val
Asn Val Ser Ala Gly Gly Ala Gly 115 120
125 Ala Ala Pro Tyr Val Asp Ala Gly Gly Glu Gln Leu Trp
Ala Asp Val 130 135 140
Leu Arg Ala Thr Leu Glu His Gly Leu Ala Pro Arg Val Trp Thr Asp145
150 155 160 Tyr Leu Arg Ile Thr
Val Ala Gly Thr Leu Ser Asn Ala Gly Ile Gly 165
170 175 Gly Gln Ala Phe Arg His Gly Pro Gln Ile
Ala Asn Val Leu Glu Leu 180 185
190 Asp Val Ile Thr Gly Arg Gly Asp Met Val Thr Cys Ser Arg Asp
Lys 195 200 205 Glu
Pro Asp Leu Phe Phe Ala Val Leu Gly Gly Leu Gly Gln Phe Gly 210
215 220 Ile Ile Thr Arg Ala Arg
Ile Gly Leu Glu Pro Ala Pro Lys Arg Val225 230
235 240 Arg Trp Val Arg Leu Ala Tyr Ser Asp Val Val
Thr Phe Thr Arg Asp 245 250
255 Gln Glu Leu Leu Ile Ser Lys Arg Ala Ser Glu Ala Gly Phe Asp Tyr
260 265 270 Val Glu Gly
Gln Val Gln Leu Asn Arg Thr Leu Thr Glu Gly Pro Lys 275
280 285 Ser Thr Pro Phe Phe Ser Arg Phe
Asp Ile Asp Arg Leu Ala Gly Leu 290 295
300 Ala Ser Glu Ser Val Ser Gly Val Ile Tyr Phe Ile Glu
Gly Ala Met305 310 315
320 Tyr Tyr Asn Glu Ser Thr Thr Ala Ser Val Asp Gln Lys Leu Thr Ser
325 330 335 Val Leu Glu Gln
Leu Ser Phe Asp Lys Gly Phe Val Phe Thr Lys Asp 340
345 350 Val Ser Tyr Val Gln Phe Leu Asp Arg
Val Arg Glu Glu Glu Arg Ile 355 360
365 Leu Arg Ser Ile Gly Met Trp Asp Val Pro His Pro Trp Leu
Asn Leu 370 375 380
Phe Val Pro Gln Ser Arg Ile Leu Asp Phe Asp Thr Gly Val Leu Lys385
390 395 400 Gly Val Phe Val Gly
Ala Asn Pro Val Gly Val Ile Leu Met Tyr Pro 405
410 415 Met Asn Arg Asn Met Trp Asp Asp Arg Met
Thr Ala Val Ser Gly Asn 420 425
430 Asp Asp Met Phe Tyr Val Val Gly Leu Leu Arg Ser Ala Val Val
Pro 435 440 445 Gly
Asp Val Glu Arg Leu Glu Arg Glu Asn Glu Ala Val Leu Ala Phe 450
455 460 Cys Asp Asn Glu Gly Ile
Gly Cys Lys Gln Tyr Leu Pro His Tyr Ala465 470
475 480 Ser Gln Asp Gly Trp Arg Ser His Phe Gly Ala
Lys Trp Ser Arg Val 485 490
495 Thr Glu Leu Lys Val Lys Tyr Asp Pro Tyr Gly Ile Leu Ser Pro Gly
500 505 510 Gln Arg Ile
Phe Ser Ser Leu Thr Pro Met Ala Leu Val Ala Met 515
520 525 74524PRTOryza
sativaPEPTIDE(1)...(524)OsCKX7 74Met Ala Ala Arg Cys Ser Ile Ala Phe Met
Ile Met Ala Ser Cys Leu1 5 10
15 Ser Val Val Val Ser Gly Gly Leu Pro Gly Asp Leu Phe Ala Leu
Ser 20 25 30 Val
Ala Ser Lys Leu Arg Val Asp Arg Asn Ser Thr Ala Arg Ala Ser 35
40 45 Ser Asp Phe Gly Arg Ile
Val Ala Ala Ala Pro Glu Ala Val Leu His 50 55
60 Pro Ala Thr Pro Ala Glu Ile Ala Glu Leu Val
Arg Phe Ser Ala Ser65 70 75
80 Ser Pro Ser Pro Phe Pro Val Ala Pro Arg Gly Gln Gly His Ser Ala
85 90 95 Arg Gly Gln
Ser Leu Ala Pro Gly Gly Val Val Val Asp Met Arg Ala 100
105 110 Leu Ala Ser Arg Arg Gly Arg Val
Asn Val Ser Ala Gly Ala Ala Pro 115 120
125 Tyr Val Asp Ala Gly Gly Glu Gln Leu Trp Ala Asp Val
Leu Arg Ala 130 135 140
Thr Leu Glu His Gly Leu Ala Pro Arg Val Trp Thr Asp Tyr Leu Arg145
150 155 160 Ile Thr Val Ala Gly
Thr Leu Ser Asn Ala Gly Ile Gly Gly Gln Ala 165
170 175 Phe Arg His Gly Pro Gln Ile Ala Asn Val
Leu Glu Leu Asp Val Ile 180 185
190 Thr Gly Thr Gly Asp Met Val Thr Cys Ser Arg Asp Lys Asp Ser
Asp 195 200 205 Leu
Phe Phe Ala Val Leu Gly Gly Leu Gly Gln Phe Gly Ile Ile Thr 210
215 220 Arg Ala Arg Ile Gly Leu
Met Pro Ala Pro Lys Arg Val Arg Trp Val225 230
235 240 Arg Leu Ala Tyr Ser Asp Val Ala Thr Phe Thr
Lys Asp Gln Glu Leu 245 250
255 Leu Ile Ser Lys Arg Ala Ser Glu Ala Gly Phe Asp Tyr Val Glu Gly
260 265 270 Gln Val Gln
Leu Asn Arg Thr Leu Thr Glu Gly Pro Lys Ser Thr Pro 275
280 285 Phe Phe Ser Ser Ser Asp Ile Gly
Arg Leu Ala Gly Leu Ala Ser Lys 290 295
300 Ser Val Ser Gly Val Ile Tyr Val Ile Glu Gly Thr Met
Tyr Tyr Asn305 310 315
320 Glu Ser Thr Ser Thr Thr Met Asp Gln Lys Leu Glu Ser Ile Leu Gly
325 330 335 Gln Leu Ser Phe
Glu Glu Gly Phe Val Phe Thr Lys Asp Val Arg Tyr 340
345 350 Val Gln Phe Leu Asp Arg Val Arg Glu
Glu Glu Arg Val Leu Arg Ser 355 360
365 Ile Gly Met Trp Asp Val Pro His Pro Trp Leu Asn Leu Phe
Val Pro 370 375 380
Arg Ser Arg Ile Leu Asp Phe Asp Ala Gly Val Phe Lys Gly Val Phe385
390 395 400 Ala Gly Ala Asn Pro
Val Gly Val Ile Leu Met Tyr Pro Met Asn Thr 405
410 415 Asn Met Trp Asp Asp Cys Met Met Ala Val
Ala Ser Asp Asp Asp Val 420 425
430 Phe Tyr Ala Val Gly Leu Leu Arg Ser Ala Ala Val Ile Gly Asp
Val 435 440 445 Glu
Arg Leu Glu Lys Glu Asn Glu Ala Val Leu Ala Phe Cys His Asn 450
455 460 Glu Asp Ile Gly Cys Lys
Gln Tyr Leu Pro Tyr Tyr Thr Ser Gln Asp465 470
475 480 Gly Trp Gln Arg His Phe Gly Ala Lys Trp Ser
Arg Val Ala Asp Leu 485 490
495 Lys Ala Lys Tyr Asp Pro His Arg Ile Leu Ser Pro Gly Gln Arg Ile
500 505 510 Phe Ser Ser
Pro Ala Ser Met Val Val Val Ser Met 515 520
75532PRTOryza sativaPEPTIDE(1)...(532)OsCKX8 75Met Glu Leu Lys
Ala Met Tyr Leu Tyr Ala Ala Val Leu Ala Val Leu1 5
10 15 Leu Cys Ser Ser Val Asn Phe Ile Gln
Ser Pro Thr Asp Val Leu Gly 20 25
30 Pro Val Ala Leu Leu Glu Pro Thr Pro Ser Ser Ala Arg Asp
Phe Gly 35 40 45
Ala Val Val Ser Asp Ala Pro Phe Ala Val Met Arg Pro Glu Ser Pro 50
55 60 Asp Asp Ile Ala Leu
Leu Leu Gly Ala Leu Ser Ser Thr Ala Pro Ser65 70
75 80 Pro Arg Ala Thr Val Ala Ala Val Gly Ala
Gly His Ser Leu His Gly 85 90
95 Gln Ala Gln Ala Arg Asp Gly Ile Val Val Glu Thr Arg Ala Leu
Pro 100 105 110 Arg
Asp Val His Val Val Ser Ala Arg Ala His Gly Gly Asp Asp Asp 115
120 125 Ala Thr Val Arg Ala Tyr
Ala Asp Val Gly Ala Gly Ala Leu Trp Val 130 135
140 Glu Val Leu Glu Glu Cys Leu Lys Leu Gly Leu
Ala Pro Pro Ser Trp145 150 155
160 Thr Asp Tyr Leu Tyr Leu Thr Val Gly Gly Thr Leu Ser Asn Gly Gly
165 170 175 Ile Ser Gly
Gln Thr Phe Lys His Gly Pro Gln Ile Ser Asn Val Leu 180
185 190 Gln Leu Glu Val Val Thr Gly Lys
Gly Glu Val Val Thr Cys Ser Pro 195 200
205 Thr Glu Ile Pro Glu Leu Phe Phe Ala Val Leu Gly Gly
Leu Gly Gln 210 215 220
Phe Gly Ile Ile Thr Arg Ala Arg Ile Pro Leu Gln Leu Ala Pro Pro225
230 235 240 Lys Val Arg Trp Val
Arg Ala Phe Tyr Asp Ser Phe Glu Thr Phe Thr 245
250 255 Gly Asp Gln Glu Leu Leu Val Ser Met Pro
Glu Gln Val Asp Tyr Val 260 265
270 Glu Gly Phe Met Val Leu Asn Glu Gln Ser Leu His Ser Ser Ser
Val 275 280 285 Ala
Phe Pro Ala Gln Leu Asn Phe Ser Pro Asp Phe Gly Ser Lys Gly 290
295 300 Arg Lys Lys Val Tyr Tyr
Cys Ile Glu Phe Ala Val His Asp Phe Gln305 310
315 320 Gln Asp Ser Ser Arg Ala Asp His Val Val Lys
Leu Val Ser Ala Lys 325 330
335 Leu Ser Tyr Leu Arg Pro His Val Tyr Ser Val Glu Val Ser Tyr Phe
340 345 350 Asp Phe Leu
Asn Arg Val Arg Met Glu Glu Glu Ser Leu Arg Ser Arg 355
360 365 Gly Leu Trp Asp Val Pro His Pro
Trp Leu Asn Val Phe Val Pro Lys 370 375
380 His Gly Ile Thr Gln Phe Lys Gly Leu Leu Met Asp Thr
Val Ser Ala385 390 395
400 Asp Asp Phe Glu Gly Pro Ile Leu Val Tyr Pro Leu Leu Thr Asp Lys
405 410 415 Trp Asp Gly Asn
Thr Ser Ala Val Val Pro Ala Ala Pro Asp Gly Val 420
425 430 Met Tyr Ile Phe Gly Val Leu Arg Ser
Thr Asp Pro Ala Arg Cys Gly 435 440
445 Arg Ala Cys Val Asp Ser Ile Met Ala Arg His Arg Arg Val
Ala Asp 450 455 460
Glu Ala Cys Arg Asp Gly Gly Gly Gly Gly Arg Gly Ile Gly Ala Lys465
470 475 480 Gln Tyr Leu Ala Arg
Gln Pro Ser Pro Ala Arg Trp Arg Asp His Phe 485
490 495 Gly Ala Gly Trp Gly Arg Phe Ala Ala Arg
Lys Ala Arg Phe Asp Pro 500 505
510 Leu His Val Leu Gly Pro Gly Gln Gly Ile Phe Pro Arg Thr Asp
Ser 515 520 525 Ala
Gly Ser Met 530 76521PRTOryza sativaPEPTIDE(1)...(521)OsCKX9
76Met Arg Pro Ser Leu Leu Gln Tyr Leu Lys Leu Leu Leu Leu Leu Ala1
5 10 15 Leu Gly Gly Val
Thr Thr Met His Val Pro Lys Gln Asp Val Pro Ser 20
25 30 Ser Leu Glu Glu Leu Thr Leu Asp Gly
His Phe Ser Phe His Asp Val 35 40
45 Ser Ala Ala Ala Gln Asp Phe Gly Asn Leu Ser Ser Phe Pro
Pro Val 50 55 60
Ala Val Leu His Pro Gly Ser Val Ala Asp Ile Ala Thr Thr Ile Arg65
70 75 80 His Val Phe Leu Met
Gly Glu His Ser Thr Leu Thr Val Ala Ala Arg 85
90 95 Gly His Gly His Ser Leu Tyr Gly Gln Ser
Gln Ala Ala Glu Gly Ile 100 105
110 Ile Ile Ser Met Glu Ser Leu Gln Ser Asn Thr Met Arg Val Asn
Pro 115 120 125 Gly
Val Ser Pro Tyr Val Asp Ala Ser Gly Gly Glu Leu Trp Ile Asn 130
135 140 Val Leu His Glu Thr Leu
Lys Tyr Gly Leu Ala Pro Lys Ser Trp Thr145 150
155 160 Asp Tyr Leu His Leu Thr Val Gly Gly Thr Leu
Ser Asn Ala Gly Val 165 170
175 Ser Gly Gln Thr Phe Arg His Gly Pro Gln Ile Ser Asn Val Asn Glu
180 185 190 Leu Glu Ile
Val Thr Gly Arg Gly Asp Val Ile Thr Cys Ser Pro Glu 195
200 205 Gln Asn Ser Asp Leu Phe His Ala
Ala Leu Gly Gly Leu Gly Gln Phe 210 215
220 Gly Val Ile Thr Arg Ala Arg Ile Pro Leu Glu Pro Ala
Pro Lys Met225 230 235
240 Val Arg Trp Leu Arg Val Leu Tyr Leu Asp Phe Thr Ser Phe Thr Glu
245 250 255 Asp Gln Glu Met
Leu Ile Ser Ala Glu Lys Thr Phe Asp Tyr Ile Glu 260
265 270 Gly Phe Val Ile Ile Asn Arg Thr Gly
Ile Leu Asn Asn Trp Arg Ser 275 280
285 Ser Phe Asn Pro Gln Asp Pro Val Arg Ser Ser Gln Phe Glu
Ser Asp 290 295 300
Gly Lys Val Leu Phe Cys Leu Glu Met Thr Lys Asn Phe Asn Pro Asp305
310 315 320 Glu Ala Asp Val Met
Glu Gln Glu Val Asn Thr Leu Leu Ser Gln Leu 325
330 335 Arg Tyr Met Pro Ser Ser Leu Phe His Thr
Asp Val Thr Tyr Ile Glu 340 345
350 Phe Leu Asp Arg Val His Ser Ser Glu Met Lys Leu Arg Ala Lys
Gly 355 360 365 Met
Trp Glu Val Pro His Pro Trp Leu Asn Ile Ile Ile Pro Arg Ser 370
375 380 Met Ile His Lys Phe Ala
Lys Glu Val Phe Gly Lys Ile Leu Lys Asp385 390
395 400 Ser Asn Asn Gly Pro Ile Leu Leu Tyr Pro Val
Asn Lys Ser Arg Trp 405 410
415 Asp Asn Arg Thr Ser Val Val Ile Pro Asp Glu Glu Val Phe Tyr Leu
420 425 430 Val Ala Phe
Leu Ser Ser Ala Leu Gly Pro His Asn Ile Lys His Thr 435
440 445 Leu Asp Leu Asn Tyr Arg Ile Ile
Glu Phe Ser Asp Lys Ala Gly Ile 450 455
460 Gly Val Lys Gln Tyr Leu Pro Asn Tyr Thr Thr Glu Gln
Glu Trp Gln465 470 475
480 Ser His Phe Gly Ala Arg Trp Asp Thr Phe Gln Gln Arg Lys Lys Ala
485 490 495 Tyr Asp Pro Leu
Ala Ile Leu Ala Pro Gly Gln Arg Ile Phe Gln Lys 500
505 510 Ala Ser Ala Ser Leu Pro Leu Pro Ser
515 520 77550PRTOryza
sativaPEPTIDE(1)...(550)OsCKX10 77Met Met Pro Arg Ala Gln Leu Thr Thr Phe
Leu Ile Val Thr Ser Phe1 5 10
15 Leu Ser Thr Val Pro Tyr Leu Arg Ala Pro Val His Gly Gly Val
Leu 20 25 30 Thr
Ser Tyr Asp Val Ser Ser Leu Asp Ile Met Ser Lys Ile His Thr 35
40 45 Asp His Asp Ala Thr Thr
Lys Ala Ser Ser Asp Phe Gly His Ile Val 50 55
60 His Ala Thr Pro Asn Gly Val Phe Arg Pro Thr
Phe Pro Ala Asp Ile65 70 75
80 Ala Ala Leu Ile Arg Leu Ser Leu Ser Gln Pro Thr Pro Phe Thr Val
85 90 95 Ala Pro Arg
Gly Lys Gly His Ser Ser Arg Gly Gln Ala Phe Ala Pro 100
105 110 Gly Gly Ile Val Val Asp Met Ser
Ala Leu Gly Asp His Gly His His 115 120
125 Thr Ser His Arg Ile Asp Val Ser Val Asp Arg Met Tyr
Val Asp Ala 130 135 140
Gly Gly Glu Gln Leu Trp Ile Asp Val Leu His Thr Ala Leu Lys His145
150 155 160 Gly Leu Thr Pro Arg
Val Trp Thr Asp Tyr Leu Arg Ile Thr Val Gly 165
170 175 Gly Thr Leu Ser Asn Ala Gly Ile Gly Gly
Gln Ala Phe Arg His Gly 180 185
190 Pro Gln Ile Ser Asn Val His Glu Leu Asp Val Val Thr Gly Met
Gly 195 200 205 Glu
Met Ile Thr Cys Ser Pro Glu Val Asn Ser Ala Leu Phe Phe Ala 210
215 220 Val Leu Gly Gly Leu Gly
Gln Phe Gly Val Ile Thr Arg Ala Arg Ile225 230
235 240 Arg Leu Glu Pro Ala Pro Lys Arg Val Lys Trp
Val Arg Ile Ala Tyr 245 250
255 Ser Asp Val His Pro Phe Thr Thr Asp Gln Glu Leu Leu Ile Ser Lys
260 265 270 Trp Ala Ser
Gly Ser Gly Phe Asp Tyr Val Glu Gly Gln Val Gln Leu 275
280 285 Asn Arg Thr Leu Thr Gln Gly Arg
Arg Ser Ser Ser Phe Phe Ser Ala 290 295
300 Thr Asp Leu Ala Arg Leu Thr Gly Leu Ala Ile Asp Thr
Gly Ser Val305 310 315
320 Ala Ile Tyr Tyr Ile Glu Gly Ala Met Tyr Tyr Asp Asp Asn Thr Ala
325 330 335 Ala Ser Val Asp
Gln Lys Leu Asp Ala Leu Leu Glu Glu Leu Ser Phe 340
345 350 Val Arg Gly Phe Val Phe Val Arg Asp
Ala Ser Tyr Val Glu Phe Leu 355 360
365 Asp Arg Val Gly Arg Glu Glu Gln Asn Leu Arg Ser Ala Gly
Ala Trp 370 375 380
Asp Val Pro His Pro Trp Leu Asn Leu Phe Val Pro Arg Ser Arg Ile385
390 395 400 Leu His Phe Asp Ala
Ala Val Phe Lys Gly Ile Leu Arg Asn Ala Asn 405
410 415 Pro Val Gly Leu Ile Leu Met Tyr Pro Met
Asn Lys Asp Met Trp Asp 420 425
430 Asp Arg Met Thr Ala Met Thr Pro Asp Glu Asp Val Phe Tyr Ala
Val 435 440 445 Gly
Leu Leu Arg Ser Ala Val Ala Gly Gly Ser Gly Gly Asp Val Glu 450
455 460 Gln Leu Glu Arg Glu Asn
Ala Ala Val Leu Glu Leu Cys Asp Leu Ala465 470
475 480 Gly Gly Gly Ile Gly Cys Arg Gln Tyr Leu Pro
His His Ala Ser Arg 485 490
495 Asp Gly Trp Arg Arg His Phe Gly Ala Lys Trp Gly Arg Val Ala Asp
500 505 510 Leu Lys Ala
Arg Tyr Asp Pro Arg Ala Ile Leu Ser Pro Gly Gln Gly 515
520 525 Ile Phe Pro Pro Pro Pro Pro Pro
Ser Pro Pro Pro Pro Ala Ala Gly 530 535
540 Glu Pro Ile Thr Ala Ser545 550
78518PRTOryza sativaPEPTIDE(1)...(518)OsCKX11 78Met Met Leu Ala Tyr Met
Asp His Ala Ala Ala Ala Ala Glu Pro Asp1 5
10 15 Ala Gly Ala Glu Pro Ala Val Ala Ala Val Asp
Ala Ala Glu Phe Ala 20 25 30
Ala Ala Met Asp Phe Gly Gly Leu Val Ser Ala Arg Pro Ala Ala Val
35 40 45 Val Arg Pro
Ala Ser Ser Asp Asp Val Ala Ser Ala Ile Arg Ala Ala 50
55 60 Ala Arg Thr Ala His Leu Thr Val
Ala Ala Arg Gly Asn Gly His Ser65 70 75
80 Val Ala Gly Gln Ala Met Ala Arg Gly Gly Leu Val Leu
Asp Met Arg 85 90 95
Ala Leu Pro Arg Arg Met Gln Leu Val Val Ala Pro Ser Gly Glu Lys
100 105 110 Phe Ala Asp Val Pro
Gly Gly Ala Leu Trp Glu Glu Val Leu His Trp 115
120 125 Ala Val Ser Lys His Gly Leu Ala Pro
Ala Ser Trp Thr Asp Tyr Leu 130 135
140 Arg Leu Thr Val Gly Gly Thr Leu Ser Asn Gly Gly Val
Ser Gly Gln145 150 155
160 Ser Phe Arg Tyr Gly Pro Gln Val Ser Asn Val Ala Gln Leu Glu Val
165 170 175 Val Thr Gly Asp
Gly Glu Cys His Val Cys Ser Arg Ser Ala Asp Pro 180
185 190 Asp Leu Phe Phe Ala Val Leu Gly Gly
Leu Gly Gln Phe Gly Val Ile 195 200
205 Thr Arg Ala Arg Ile Pro Leu Ser Pro Ala Pro Gln Thr Val
Arg Trp 210 215 220
Thr Arg Val Val Tyr Ala Ser Phe Ala Asp Tyr Ala Ala Asp Ala Glu225
230 235 240 Trp Leu Val Thr Arg
Pro Pro His Glu Ala Phe Asp Tyr Val Glu Gly 245
250 255 Phe Ala Phe Val Arg Ser Asp Asp Pro Val
Asn Gly Trp Pro Thr Val 260 265
270 Pro Ile Pro Asp Gly Ala His Phe Asp Ala Ser Leu Leu Pro Ala
Asn 275 280 285 Ala
Gly Pro Val Leu Tyr Cys Leu Glu Val Ala Leu Tyr Gln Arg Gly 290
295 300 Gly Gly Gly Asp Gly Gly
Gly Asp Asp Met Asp Lys Arg Val Gly Glu305 310
315 320 Met Met Arg Gln Leu Lys Tyr Val Arg Gly Leu
Glu Phe Ala Ala Gly 325 330
335 Val Gly Tyr Val Asp Phe Leu Ser Arg Val Asn Arg Val Glu Asp Glu
340 345 350 Ala Arg Arg
Asn Gly Ser Trp Ala Ala Pro His Pro Trp Leu Asn Leu 355
360 365 Phe Ile Ser Ser Arg Asp Ile Ala
Ala Phe Asp Arg Ala Val Leu Asn 370 375
380 Gly Met Leu Ala Asp Gly Val Asp Gly Pro Met Leu Ile
Tyr Pro Met385 390 395
400 Leu Lys Ser Lys Trp Asp Pro Ala Thr Ser Val Ala Leu Pro Asn Gly
405 410 415 Glu Ile Phe Tyr
Leu Val Ala Leu Leu Arg Phe Cys Arg Pro Tyr Pro 420
425 430 Gly Gly Gly Pro Pro Val Asp Glu Leu
Val Ala Gln Asn Asn Ala Ile 435 440
445 Ile Asp Ala Cys Arg Ser Asn Gly Tyr Asp Tyr Lys Ile Tyr
Phe Pro 450 455 460
Ser Tyr His Ala Gln Ser Asp Trp Ser Arg His Phe Gly Ala Lys Trp465
470 475 480 Ser Arg Phe Val Asp
Arg Lys Ala Arg Tyr Asp Pro Leu Ala Ile Leu 485
490 495 Ala Pro Gly Gln Asn Ile Phe Ala Arg Thr
Pro Ser Ser Val Ala Ala 500 505
510 Ala Ala Ala Val Ile Val 515 79142PRTZea
maysDOMAIN(1)...(142)ZmCkx2a PF01565.13.ls domain per Figure 9 79Pro Ala
Ala Val Val Arg Pro Glu Ser Glu Glu Glu Val Ala Ala Ile1 5
10 15 Val Arg Leu Ala Arg Glu His
Gly Ile Pro Val Thr Pro Arg Gly Gly 20 25
30 Gly His Ser Leu Ser Phe Gly Gly Ala Val Pro Leu
Asn Thr Gly Gly 35 40 45
Val Val Leu Asp Leu Ser Arg Lys Leu Asn Arg Ile Ile Leu Glu Ile
50 55 60 Asp Pro Glu
Thr Asp Gly Thr Ala Thr Val Glu Ala Gly Val Thr Leu65 70
75 80 Asp Leu Asn Arg Ala Leu Ala Ala
Lys Gly Leu Phe Leu Pro Leu Asp 85 90
95 Pro Gly Ser Gly Ile Pro Gly Thr Val Gly Gly Ala Ile
Ala Thr Asn 100 105 110
Ala Gly Gly Tyr Gly Ser Glu Lys Tyr Gly Leu Thr Arg Asp Asn Val
115 120 125 Leu Gly Leu Glu
Val Val Leu Ala Asp Gly Glu Val Val Arg 130 135
140 80120PRTZea maysDOMAIN(1)...(120)ZmCkx2a
PF01565.13.fs domain per Figure 9 80Ile Pro Val Thr Pro Arg Gly Gly Gly
His Ser Leu Ser Phe Gly Gly1 5 10
15 Ala Val Pro Leu Asn Thr Gly Gly Val Val Leu Asp Leu Ser
Arg Lys 20 25 30
Leu Asn Arg Ile Ile Leu Glu Ile Asp Pro Glu Thr Asp Gly Thr Ala 35
40 45 Thr Val Glu Ala Gly
Val Thr Leu Asp Leu Asn Arg Ala Leu Ala Ala 50 55
60 Lys Gly Leu Phe Leu Pro Leu Asp Pro Gly
Ser Gly Ile Pro Gly Thr65 70 75
80 Val Gly Gly Ala Ile Ala Thr Asn Ala Gly Gly Tyr Gly Ser Glu
Lys 85 90 95 Tyr
Gly Leu Thr Arg Asp Asn Val Leu Gly Leu Glu Val Val Leu Ala
100 105 110 Asp Gly Glu Val Val
Arg Leu Ser 115 120 81297PRTZea
maysDOMAIN(1)...(297)ZmCkx2a PF09265.1.fs domain per Figure 9 81Pro Lys
Arg Val Arg Trp Val Arg Val Leu Tyr Ser Asp Phe Ala Ala1 5
10 15 Phe Thr Lys Asp Gln Glu Arg
Leu Ile Ser Lys Glu Asn Gly Gly Gly 20 25
30 Gly Ala Lys Val Gly Phe Asp Tyr Val Glu Gly Phe
Val Ile Leu Asn 35 40 45
Arg Thr Gly Leu Val Asn Asn Trp Arg Ser Ser Phe Phe Ser Pro Ser
50 55 60 Asp Pro Ala
Arg Ile Ala Ser Leu Ala Ser Lys Asn Asn Gly Gly Val65 70
75 80 Leu Tyr Cys Leu Glu Val Ala Lys
Tyr Tyr Asp Tyr Ala Asp Ser Asp 85 90
95 Ala Ala Thr Val Asp Gln Glu Val Glu Glu Leu Leu Arg
Gln Leu Ser 100 105 110
Phe Val Pro Gly Phe Leu Phe Ser Thr Asp Val Ser Tyr Val Asp Phe
115 120 125 Leu Asp Arg Val
His Arg Glu Glu Leu Lys Leu Arg Ser Lys Gly Leu 130
135 140 Trp Asp Val Pro His Pro Trp Leu
Asn Leu Phe Val Pro Lys Ser Arg145 150
155 160 Ile Leu Asp Phe Asp Arg Gly Val Phe Lys Gly Ile
Leu Leu Lys Asn 165 170
175 Thr Asn Asn Ser Gly Pro Ile Leu Val Tyr Pro Met Asn Arg Ser Lys
180 185 190 Trp Asp Asp
Arg Met Ser Ala Val Ile Pro Asp Glu Asp Glu Asp Val 195
200 205 Phe Tyr Leu Val Gly Leu Leu Arg
Ser Ala Val Pro Tyr Ser Ala Gly 210 215
220 Pro Gly Asp Leu Glu Glu Leu Glu Asn Gln Asn Arg Arg
Ile Leu Glu225 230 235
240 Phe Cys Glu Lys Ala Gly Ile Gly Tyr Lys Gln Tyr Leu Pro His Tyr
245 250 255 Leu Thr Ser Gln
Glu Asp Asn Tyr Trp Lys Arg His Phe Gly Ala Ala 260
265 270 Lys Trp Asp Arg Phe Val Asp Arg Lys
Ala Arg Tyr Asp Pro Lys Ala 275 280
285 Ile Leu Ser Pro Gly Gln Gly Ile Phe 290
295 82297PRTZea maysDOMAIN(1)...(297)ZmCkx2a PF09265.1.ls
domain per Figure 9 82Pro Lys Arg Val Arg Trp Val Arg Val Leu Tyr Ser Asp
Phe Ala Ala1 5 10 15
Phe Thr Lys Asp Gln Glu Arg Leu Ile Ser Lys Glu Asn Gly Gly Gly
20 25 30 Gly Ala Lys Val Gly
Phe Asp Tyr Val Glu Gly Phe Val Ile Leu Asn 35 40
45 Arg Thr Gly Leu Val Asn Asn Trp Arg Ser
Ser Phe Phe Ser Pro Ser 50 55 60
Asp Pro Ala Arg Ile Ala Ser Leu Ala Ser Lys Asn Asn Gly Gly
Val65 70 75 80 Leu
Tyr Cys Leu Glu Val Ala Lys Tyr Tyr Asp Tyr Ala Asp Ser Asp
85 90 95 Ala Ala Thr Val Asp Gln
Glu Val Glu Glu Leu Leu Arg Gln Leu Ser 100
105 110 Phe Val Pro Gly Phe Leu Phe Ser Thr Asp
Val Ser Tyr Val Asp Phe 115 120
125 Leu Asp Arg Val His Arg Glu Glu Leu Lys Leu Arg Ser Lys
Gly Leu 130 135 140
Trp Asp Val Pro His Pro Trp Leu Asn Leu Phe Val Pro Lys Ser Arg145
150 155 160 Ile Leu Asp Phe Asp
Arg Gly Val Phe Lys Gly Ile Leu Leu Lys Asn 165
170 175 Thr Asn Asn Ser Gly Pro Ile Leu Val Tyr
Pro Met Asn Arg Ser Lys 180 185
190 Trp Asp Asp Arg Met Ser Ala Val Ile Pro Asp Glu Asp Glu Asp
Val 195 200 205 Phe
Tyr Leu Val Gly Leu Leu Arg Ser Ala Val Pro Tyr Ser Ala Gly 210
215 220 Pro Gly Asp Leu Glu Glu
Leu Glu Asn Gln Asn Arg Arg Ile Leu Glu225 230
235 240 Phe Cys Glu Lys Ala Gly Ile Gly Tyr Lys Gln
Tyr Leu Pro His Tyr 245 250
255 Leu Thr Ser Gln Glu Asp Asn Tyr Trp Lys Arg His Phe Gly Ala Ala
260 265 270 Lys Trp Asp
Arg Phe Val Asp Arg Lys Ala Arg Tyr Asp Pro Lys Ala 275
280 285 Ile Leu Ser Pro Gly Gln Gly Ile
Phe 290 295 83144PRTZea
maysDOMAIN(1)...(144)ZmCkx2b PF01565.13.fs domain per Figure 9 83Pro Ala
Ala Val Val Arg Pro Glu Ser Glu Glu Glu Val Ala Ala Ile1 5
10 15 Val Arg Leu Ala Arg Glu His
Gly Ile Pro Val Thr Pro Arg Gly Gly 20 25
30 Gly His Ser Leu Ser Phe Gly Gly Ala Val Pro Leu
Asn Thr Gly Gly 35 40 45
Val Val Leu Asp Leu Ser Arg Lys Leu Asn Arg Ile Ile Leu Glu Ile
50 55 60 Asp Pro Glu
Thr Asp Gly Thr Ala Thr Val Glu Ala Gly Val Thr Leu65 70
75 80 Asp Leu Asn Arg Ala Leu Ala Ala
Lys Gly Leu Phe Leu Pro Leu Asp 85 90
95 Pro Gly Ser Gly Ile Pro Gly Thr Val Gly Gly Ala Ile
Ala Thr Asn 100 105 110
Ala Gly Gly Tyr Gly Ser Glu Lys Tyr Gly Leu Thr Arg Asp Asn Val
115 120 125 Leu Gly Leu Glu
Val Val Leu Ala Asp Gly Glu Val Val Arg Leu Ser 130
135 140 84144PRTZea
maysDOMAIN(1)...(144)ZmCkx2b PF01565.13.ls domain per Figure 9 84Pro Ala
Ala Val Val Arg Pro Glu Ser Glu Glu Glu Val Ala Ala Ile1 5
10 15 Val Arg Leu Ala Arg Glu His
Gly Ile Pro Val Thr Pro Arg Gly Gly 20 25
30 Gly His Ser Leu Ser Phe Gly Gly Ala Val Pro Leu
Asn Thr Gly Gly 35 40 45
Val Val Leu Asp Leu Ser Arg Lys Leu Asn Arg Ile Ile Leu Glu Ile
50 55 60 Asp Pro Glu
Thr Asp Gly Thr Ala Thr Val Glu Ala Gly Val Thr Leu65 70
75 80 Asp Leu Asn Arg Ala Leu Ala Ala
Lys Gly Leu Phe Leu Pro Leu Asp 85 90
95 Pro Gly Ser Gly Ile Pro Gly Thr Val Gly Gly Ala Ile
Ala Thr Asn 100 105 110
Ala Gly Gly Tyr Gly Ser Glu Lys Tyr Gly Leu Thr Arg Asp Asn Val
115 120 125 Leu Gly Leu Glu
Val Val Leu Ala Asp Gly Glu Val Val Arg Leu Ser 130
135 140 85297PRTZea
maysDOMAIN(1)...(297)ZmCkx2b PF09265.1.fs domain per Figure 9 85Pro Lys
Arg Val Arg Trp Val Arg Val Leu Tyr Ser Asp Phe Ala Ala1 5
10 15 Phe Thr Lys Asp Gln Glu Arg
Leu Ile Ser Lys Glu Asn Gly Gly Gly 20 25
30 Gly Ala Lys Val Gly Phe Asp Tyr Val Glu Gly Phe
Val Ile Leu Asn 35 40 45
Arg Thr Gly Leu Val Asn Asn Trp Arg Ser Ser Phe Phe Ser Pro Ser
50 55 60 Asp Pro Ala
Arg Ile Ala Ser Leu Ala Ser Lys Asn Asn Gly Gly Val65 70
75 80 Leu Tyr Cys Leu Glu Val Ala Lys
Tyr Tyr Asp Tyr Ala Asp Ser Asp 85 90
95 Ala Ala Thr Val Asp Gln Glu Val Glu Glu Leu Leu Arg
Gln Leu Ser 100 105 110
Phe Val Pro Gly Phe Leu Phe Ser Thr Asp Val Ser Tyr Val Asp Phe
115 120 125 Leu Asp Arg Val
His Arg Glu Glu Leu Lys Leu Arg Ser Lys Gly Leu 130
135 140 Trp Asp Val Pro His Pro Trp Leu
Asn Leu Phe Val Pro Lys Ser Arg145 150
155 160 Ile Leu Asp Phe Asp Arg Gly Val Phe Lys Gly Ile
Leu Leu Lys Asn 165 170
175 Thr Asn Asn Ser Gly Pro Ile Leu Val Tyr Pro Met Asn Arg Ser Lys
180 185 190 Trp Asp Asp
Arg Met Ser Ala Val Ile Pro Asp Glu Asp Glu Asp Val 195
200 205 Phe Tyr Leu Val Gly Leu Leu Arg
Ser Ala Val Pro Tyr Ser Ala Gly 210 215
220 Pro Gly Asp Leu Glu Glu Leu Glu Asn Gln Asn Arg Arg
Ile Leu Glu225 230 235
240 Phe Cys Glu Lys Ala Gly Ile Gly Tyr Lys Gln Tyr Leu Pro His Tyr
245 250 255 Leu Thr Ser Gln
Glu Asp Asn Tyr Trp Lys Arg His Phe Gly Ala Ala 260
265 270 Lys Trp Asp Arg Phe Val Asp Arg Lys
Ala Arg Tyr Asp Pro Lys Ala 275 280
285 Ile Leu Ser Pro Gly Gln Gly Ile Phe 290
295 86297PRTZea maysDOMAIN(1)...(297)ZmCkx2b PF09265.1.ls
domain per Figure 9 86Pro Lys Arg Val Arg Trp Val Arg Val Leu Tyr Ser Asp
Phe Ala Ala1 5 10 15
Phe Thr Lys Asp Gln Glu Arg Leu Ile Ser Lys Glu Asn Gly Gly Gly
20 25 30 Gly Ala Lys Val Gly
Phe Asp Tyr Val Glu Gly Phe Val Ile Leu Asn 35 40
45 Arg Thr Gly Leu Val Asn Asn Trp Arg Ser
Ser Phe Phe Ser Pro Ser 50 55 60
Asp Pro Ala Arg Ile Ala Ser Leu Ala Ser Lys Asn Asn Gly Gly
Val65 70 75 80 Leu
Tyr Cys Leu Glu Val Ala Lys Tyr Tyr Asp Tyr Ala Asp Ser Asp
85 90 95 Ala Ala Thr Val Asp Gln
Glu Val Glu Glu Leu Leu Arg Gln Leu Ser 100
105 110 Phe Val Pro Gly Phe Leu Phe Ser Thr Asp
Val Ser Tyr Val Asp Phe 115 120
125 Leu Asp Arg Val His Arg Glu Glu Leu Lys Leu Arg Ser Lys
Gly Leu 130 135 140
Trp Asp Val Pro His Pro Trp Leu Asn Leu Phe Val Pro Lys Ser Arg145
150 155 160 Ile Leu Asp Phe Asp
Arg Gly Val Phe Lys Gly Ile Leu Leu Lys Asn 165
170 175 Thr Asn Asn Ser Gly Pro Ile Leu Val Tyr
Pro Met Asn Arg Ser Lys 180 185
190 Trp Asp Asp Arg Met Ser Ala Val Ile Pro Asp Glu Asp Glu Asp
Val 195 200 205 Phe
Tyr Leu Val Gly Leu Leu Arg Ser Ala Val Pro Tyr Ser Ala Gly 210
215 220 Pro Gly Asp Leu Glu Glu
Leu Glu Asn Gln Asn Arg Arg Ile Leu Glu225 230
235 240 Phe Cys Glu Lys Ala Gly Ile Gly Tyr Lys Gln
Tyr Leu Pro His Tyr 245 250
255 Leu Thr Ser Gln Glu Asp Asn Tyr Trp Lys Arg His Phe Gly Ala Ala
260 265 270 Lys Trp Asp
Arg Phe Val Asp Arg Lys Ala Arg Tyr Asp Pro Lys Ala 275
280 285 Ile Leu Ser Pro Gly Gln Gly Ile
Phe 290 295 87144PRTZea
maysDOMAIN(1)...(144)ZmCkx3 PF01565.13.fs domain per Figure 9 87Pro Ala
Ala Val Val Arg Pro Glu Ser Glu Glu Glu Val Ala Ala Ile1 5
10 15 Val Arg Leu Ala Arg Glu His
Gly Ile Pro Val Thr Pro Arg Gly Gly 20 25
30 Gly His Ser Leu Ser Phe Gly Gly Ala Val Pro Leu
Asn Thr Gly Gly 35 40 45
Val Val Leu Asp Leu Ser Arg Lys Leu Asn Arg Ile Ile Leu Glu Ile
50 55 60 Asp Pro Glu
Thr Asp Gly Thr Ala Thr Val Glu Ala Gly Val Thr Leu65 70
75 80 Asp Leu Asn Arg Ala Leu Ala Ala
Lys Gly Leu Phe Leu Pro Leu Asp 85 90
95 Pro Gly Ser Gly Ile Pro Gly Thr Val Gly Gly Ala Ile
Ala Thr Asn 100 105 110
Ala Gly Gly Tyr Gly Ser Glu Lys Tyr Gly Leu Thr Arg Asp Asn Val
115 120 125 Leu Gly Leu Glu
Val Val Leu Ala Asp Gly Glu Val Val Arg Leu Ser 130
135 140 88144PRTZea
maysDOMAIN(1)...(144)ZmCkx3 PF01565.13.ls domain per Figure 9 88Pro Ala
Ala Val Val Arg Pro Glu Ser Glu Glu Glu Val Ala Ala Ile1 5
10 15 Val Arg Leu Ala Arg Glu His
Gly Ile Pro Val Thr Pro Arg Gly Gly 20 25
30 Gly His Ser Leu Ser Phe Gly Gly Ala Val Pro Leu
Asn Thr Gly Gly 35 40 45
Val Val Leu Asp Leu Ser Arg Lys Leu Asn Arg Ile Ile Leu Glu Ile
50 55 60 Asp Pro Glu
Thr Asp Gly Thr Ala Thr Val Glu Ala Gly Val Thr Leu65 70
75 80 Asp Leu Asn Arg Ala Leu Ala Ala
Lys Gly Leu Phe Leu Pro Leu Asp 85 90
95 Pro Gly Ser Gly Ile Pro Gly Thr Val Gly Gly Ala Ile
Ala Thr Asn 100 105 110
Ala Gly Gly Tyr Gly Ser Glu Lys Tyr Gly Leu Thr Arg Asp Asn Val
115 120 125 Leu Gly Leu Glu
Val Val Leu Ala Asp Gly Glu Val Val Arg Leu Ser 130
135 140 89297PRTZea
maysDOMAIN(1)...(297)ZmCkx3 PF09265.1.fs domain per Figure 9 89Pro Lys
Arg Val Arg Trp Val Arg Val Leu Tyr Ser Asp Phe Ala Ala1 5
10 15 Phe Thr Lys Asp Gln Glu Arg
Leu Ile Ser Lys Glu Asn Gly Gly Gly 20 25
30 Gly Ala Lys Val Gly Phe Asp Tyr Val Glu Gly Phe
Val Ile Leu Asn 35 40 45
Arg Thr Gly Leu Val Asn Asn Trp Arg Ser Ser Phe Phe Ser Pro Ser
50 55 60 Asp Pro Ala
Arg Ile Ala Ser Leu Ala Ser Lys Asn Asn Gly Gly Val65 70
75 80 Leu Tyr Cys Leu Glu Val Ala Lys
Tyr Tyr Asp Tyr Ala Asp Ser Asp 85 90
95 Ala Ala Thr Val Asp Gln Glu Val Glu Glu Leu Leu Arg
Gln Leu Ser 100 105 110
Phe Val Pro Gly Phe Leu Phe Ser Thr Asp Val Ser Tyr Val Asp Phe
115 120 125 Leu Asp Arg Val
His Arg Glu Glu Leu Lys Leu Arg Ser Lys Gly Leu 130
135 140 Trp Asp Val Pro His Pro Trp Leu
Asn Leu Phe Val Pro Lys Ser Arg145 150
155 160 Ile Leu Asp Phe Asp Arg Gly Val Phe Lys Gly Ile
Leu Leu Lys Asn 165 170
175 Thr Asn Asn Ser Gly Pro Ile Leu Val Tyr Pro Met Asn Arg Ser Lys
180 185 190 Trp Asp Asp
Arg Met Ser Ala Val Ile Pro Asp Glu Asp Glu Asp Val 195
200 205 Phe Tyr Leu Val Gly Leu Leu Arg
Ser Ala Val Pro Tyr Ser Ala Gly 210 215
220 Pro Gly Asp Leu Glu Glu Leu Glu Asn Gln Asn Arg Arg
Ile Leu Glu225 230 235
240 Phe Cys Glu Lys Ala Gly Ile Gly Tyr Lys Gln Tyr Leu Pro His Tyr
245 250 255 Leu Thr Ser Gln
Glu Asp Asn Tyr Trp Lys Arg His Phe Gly Ala Ala 260
265 270 Lys Trp Asp Arg Phe Val Asp Arg Lys
Ala Arg Tyr Asp Pro Lys Ala 275 280
285 Ile Leu Ser Pro Gly Gln Gly Ile Phe 290
295 90297PRTZea maysDOMAIN(1)...(297)ZmCkx3 PF09265.1.ls domain
per Figure 9 90Pro Lys Arg Val Arg Trp Val Arg Val Leu Tyr Ser Asp Phe
Ala Ala1 5 10 15
Phe Thr Lys Asp Gln Glu Arg Leu Ile Ser Lys Glu Asn Gly Gly Gly
20 25 30 Gly Ala Lys Val Gly
Phe Asp Tyr Val Glu Gly Phe Val Ile Leu Asn 35 40
45 Arg Thr Gly Leu Val Asn Asn Trp Arg Ser
Ser Phe Phe Ser Pro Ser 50 55 60
Asp Pro Ala Arg Ile Ala Ser Leu Ala Ser Lys Asn Asn Gly Gly
Val65 70 75 80 Leu
Tyr Cys Leu Glu Val Ala Lys Tyr Tyr Asp Tyr Ala Asp Ser Asp
85 90 95 Ala Ala Thr Val Asp Gln
Glu Val Glu Glu Leu Leu Arg Gln Leu Ser 100
105 110 Phe Val Pro Gly Phe Leu Phe Ser Thr Asp
Val Ser Tyr Val Asp Phe 115 120
125 Leu Asp Arg Val His Arg Glu Glu Leu Lys Leu Arg Ser Lys
Gly Leu 130 135 140
Trp Asp Val Pro His Pro Trp Leu Asn Leu Phe Val Pro Lys Ser Arg145
150 155 160 Ile Leu Asp Phe Asp
Arg Gly Val Phe Lys Gly Ile Leu Leu Lys Asn 165
170 175 Thr Asn Asn Ser Gly Pro Ile Leu Val Tyr
Pro Met Asn Arg Ser Lys 180 185
190 Trp Asp Asp Arg Met Ser Ala Val Ile Pro Asp Glu Asp Glu Asp
Val 195 200 205 Phe
Tyr Leu Val Gly Leu Leu Arg Ser Ala Val Pro Tyr Ser Ala Gly 210
215 220 Pro Gly Asp Leu Glu Glu
Leu Glu Asn Gln Asn Arg Arg Ile Leu Glu225 230
235 240 Phe Cys Glu Lys Ala Gly Ile Gly Tyr Lys Gln
Tyr Leu Pro His Tyr 245 250
255 Leu Thr Ser Gln Glu Asp Asn Tyr Trp Lys Arg His Phe Gly Ala Ala
260 265 270 Lys Trp Asp
Arg Phe Val Asp Arg Lys Ala Arg Tyr Asp Pro Lys Ala 275
280 285 Ile Leu Ser Pro Gly Gln Gly Ile
Phe 290 295 9157PRTZea
maysDOMAIN(1)...(57)ZmCkx4 PF01565.13.fs domain per Figure 9 91Pro Ala
Ala Val Val Arg Pro Glu Ser Glu Glu Glu Val Ala Ala Ile1 5
10 15 Val Arg Leu Ala Arg Glu His
Gly Ile Pro Val Thr Pro Arg Gly Gly 20 25
30 Gly His Ser Leu Ser Phe Gly Gly Ala Val Pro Leu
Asn Thr Gly Gly 35 40 45
Val Val Leu Asp Leu Ser Arg Lys Leu 50 55
92144PRTZea maysDOMAIN(1)...(144)ZmCkx4 PF01565.13.ls domain per Figure
9 92Pro Ala Ala Val Val Arg Pro Glu Ser Glu Glu Glu Val Ala Ala Ile1
5 10 15 Val Arg Leu Ala
Arg Glu His Gly Ile Pro Val Thr Pro Arg Gly Gly 20
25 30 Gly His Ser Leu Ser Phe Gly Gly Ala
Val Pro Leu Asn Thr Gly Gly 35 40
45 Val Val Leu Asp Leu Ser Arg Lys Leu Asn Arg Ile Ile Leu
Glu Ile 50 55 60
Asp Pro Glu Thr Asp Gly Thr Ala Thr Val Glu Ala Gly Val Thr Leu65
70 75 80 Asp Leu Asn Arg Ala
Leu Ala Ala Lys Gly Leu Phe Leu Pro Leu Asp 85
90 95 Pro Gly Ser Gly Ile Pro Gly Thr Val Gly
Gly Ala Ile Ala Thr Asn 100 105
110 Ala Gly Gly Tyr Gly Ser Glu Lys Tyr Gly Leu Thr Arg Asp Asn
Val 115 120 125 Leu
Gly Leu Glu Val Val Leu Ala Asp Gly Glu Val Val Arg Leu Ser 130
135 140 9373PRTZea
maysDOMAIN(1)...(73)ZmCkx4 PF01565.13.fs domain per Figure 9 93Ala Thr
Val Glu Ala Gly Val Thr Leu Asp Leu Asn Arg Ala Leu Ala1 5
10 15 Ala Lys Gly Leu Phe Leu Pro
Leu Asp Pro Gly Ser Gly Ile Pro Gly 20 25
30 Thr Val Gly Gly Ala Ile Ala Thr Asn Ala Gly Gly
Tyr Gly Ser Glu 35 40 45
Lys Tyr Gly Leu Thr Arg Asp Asn Val Leu Gly Leu Glu Val Val Leu
50 55 60 Ala Asp Gly
Glu Val Val Arg Leu Ser65 70 94297PRTZea
maysDOMAIN(1)...(297)ZmCKx4 PF09265.1.fs domain per Figure 9 94Pro Lys
Arg Val Arg Trp Val Arg Val Leu Tyr Ser Asp Phe Ala Ala1 5
10 15 Phe Thr Lys Asp Gln Glu Arg
Leu Ile Ser Lys Glu Asn Gly Gly Gly 20 25
30 Gly Ala Lys Val Gly Phe Asp Tyr Val Glu Gly Phe
Val Ile Leu Asn 35 40 45
Arg Thr Gly Leu Val Asn Asn Trp Arg Ser Ser Phe Phe Ser Pro Ser
50 55 60 Asp Pro Ala
Arg Ile Ala Ser Leu Ala Ser Lys Asn Asn Gly Gly Val65 70
75 80 Leu Tyr Cys Leu Glu Val Ala Lys
Tyr Tyr Asp Tyr Ala Asp Ser Asp 85 90
95 Ala Ala Thr Val Asp Gln Glu Val Glu Glu Leu Leu Arg
Gln Leu Ser 100 105 110
Phe Val Pro Gly Phe Leu Phe Ser Thr Asp Val Ser Tyr Val Asp Phe
115 120 125 Leu Asp Arg Val
His Arg Glu Glu Leu Lys Leu Arg Ser Lys Gly Leu 130
135 140 Trp Asp Val Pro His Pro Trp Leu
Asn Leu Phe Val Pro Lys Ser Arg145 150
155 160 Ile Leu Asp Phe Asp Arg Gly Val Phe Lys Gly Ile
Leu Leu Lys Asn 165 170
175 Thr Asn Asn Ser Gly Pro Ile Leu Val Tyr Pro Met Asn Arg Ser Lys
180 185 190 Trp Asp Asp
Arg Met Ser Ala Val Ile Pro Asp Glu Asp Glu Asp Val 195
200 205 Phe Tyr Leu Val Gly Leu Leu Arg
Ser Ala Val Pro Tyr Ser Ala Gly 210 215
220 Pro Gly Asp Leu Glu Glu Leu Glu Asn Gln Asn Arg Arg
Ile Leu Glu225 230 235
240 Phe Cys Glu Lys Ala Gly Ile Gly Tyr Lys Gln Tyr Leu Pro His Tyr
245 250 255 Leu Thr Ser Gln
Glu Asp Asn Tyr Trp Lys Arg His Phe Gly Ala Ala 260
265 270 Lys Trp Asp Arg Phe Val Asp Arg Lys
Ala Arg Tyr Asp Pro Lys Ala 275 280
285 Ile Leu Ser Pro Gly Gln Gly Ile Phe 290
295 95297PRTZea maysDOMAIN(1)...(297)ZmCkx4 PF09265.1.ls domain
per Figure 9 95Pro Lys Arg Val Arg Trp Val Arg Val Leu Tyr Ser Asp Phe
Ala Ala1 5 10 15
Phe Thr Lys Asp Gln Glu Arg Leu Ile Ser Lys Glu Asn Gly Gly Gly
20 25 30 Gly Ala Lys Val Gly
Phe Asp Tyr Val Glu Gly Phe Val Ile Leu Asn 35 40
45 Arg Thr Gly Leu Val Asn Asn Trp Arg Ser
Ser Phe Phe Ser Pro Ser 50 55 60
Asp Pro Ala Arg Ile Ala Ser Leu Ala Ser Lys Asn Asn Gly Gly
Val65 70 75 80 Leu
Tyr Cys Leu Glu Val Ala Lys Tyr Tyr Asp Tyr Ala Asp Ser Asp
85 90 95 Ala Ala Thr Val Asp Gln
Glu Val Glu Glu Leu Leu Arg Gln Leu Ser 100
105 110 Phe Val Pro Gly Phe Leu Phe Ser Thr Asp
Val Ser Tyr Val Asp Phe 115 120
125 Leu Asp Arg Val His Arg Glu Glu Leu Lys Leu Arg Ser Lys
Gly Leu 130 135 140
Trp Asp Val Pro His Pro Trp Leu Asn Leu Phe Val Pro Lys Ser Arg145
150 155 160 Ile Leu Asp Phe Asp
Arg Gly Val Phe Lys Gly Ile Leu Leu Lys Asn 165
170 175 Thr Asn Asn Ser Gly Pro Ile Leu Val Tyr
Pro Met Asn Arg Ser Lys 180 185
190 Trp Asp Asp Arg Met Ser Ala Val Ile Pro Asp Glu Asp Glu Asp
Val 195 200 205 Phe
Tyr Leu Val Gly Leu Leu Arg Ser Ala Val Pro Tyr Ser Ala Gly 210
215 220 Pro Gly Asp Leu Glu Glu
Leu Glu Asn Gln Asn Arg Arg Ile Leu Glu225 230
235 240 Phe Cys Glu Lys Ala Gly Ile Gly Tyr Lys Gln
Tyr Leu Pro His Tyr 245 250
255 Leu Thr Ser Gln Glu Asp Asn Tyr Trp Lys Arg His Phe Gly Ala Ala
260 265 270 Lys Trp Asp
Arg Phe Val Asp Arg Lys Ala Arg Tyr Asp Pro Lys Ala 275
280 285 Ile Leu Ser Pro Gly Gln Gly Ile
Phe 290 295 96144PRTZea
maysDOMAIN(1)...(144)ZmCkx5 PF01565.13.fs domain per Figure 9 96Pro Ala
Ala Val Val Arg Pro Glu Ser Glu Glu Glu Val Ala Ala Ile1 5
10 15 Val Arg Leu Ala Arg Glu His
Gly Ile Pro Val Thr Pro Arg Gly Gly 20 25
30 Gly His Ser Leu Ser Phe Gly Gly Ala Val Pro Leu
Asn Thr Gly Gly 35 40 45
Val Val Leu Asp Leu Ser Arg Lys Leu Asn Arg Ile Ile Leu Glu Ile
50 55 60 Asp Pro Glu
Thr Asp Gly Thr Ala Thr Val Glu Ala Gly Val Thr Leu65 70
75 80 Asp Leu Asn Arg Ala Leu Ala Ala
Lys Gly Leu Phe Leu Pro Leu Asp 85 90
95 Pro Gly Ser Gly Ile Pro Gly Thr Val Gly Gly Ala Ile
Ala Thr Asn 100 105 110
Ala Gly Gly Tyr Gly Ser Glu Lys Tyr Gly Leu Thr Arg Asp Asn Val
115 120 125 Leu Gly Leu Glu
Val Val Leu Ala Asp Gly Glu Val Val Arg Leu Ser 130
135 140 97144PRTZea
maysDOMAIN(1)...(144)ZmCkx5 PF01565.13.ls domain per Figure 9 97Pro Ala
Ala Val Val Arg Pro Glu Ser Glu Glu Glu Val Ala Ala Ile1 5
10 15 Val Arg Leu Ala Arg Glu His
Gly Ile Pro Val Thr Pro Arg Gly Gly 20 25
30 Gly His Ser Leu Ser Phe Gly Gly Ala Val Pro Leu
Asn Thr Gly Gly 35 40 45
Val Val Leu Asp Leu Ser Arg Lys Leu Asn Arg Ile Ile Leu Glu Ile
50 55 60 Asp Pro Glu
Thr Asp Gly Thr Ala Thr Val Glu Ala Gly Val Thr Leu65 70
75 80 Asp Leu Asn Arg Ala Leu Ala Ala
Lys Gly Leu Phe Leu Pro Leu Asp 85 90
95 Pro Gly Ser Gly Ile Pro Gly Thr Val Gly Gly Ala Ile
Ala Thr Asn 100 105 110
Ala Gly Gly Tyr Gly Ser Glu Lys Tyr Gly Leu Thr Arg Asp Asn Val
115 120 125 Leu Gly Leu Glu
Val Val Leu Ala Asp Gly Glu Val Val Arg Leu Ser 130
135 140 98297PRTZea
maysDOMAIN(1)...(297)ZmCkx5 PF09265.1.fs domain per Figure 9 98Pro Lys
Arg Val Arg Trp Val Arg Val Leu Tyr Ser Asp Phe Ala Ala1 5
10 15 Phe Thr Lys Asp Gln Glu Arg
Leu Ile Ser Lys Glu Asn Gly Gly Gly 20 25
30 Gly Ala Lys Val Gly Phe Asp Tyr Val Glu Gly Phe
Val Ile Leu Asn 35 40 45
Arg Thr Gly Leu Val Asn Asn Trp Arg Ser Ser Phe Phe Ser Pro Ser
50 55 60 Asp Pro Ala
Arg Ile Ala Ser Leu Ala Ser Lys Asn Asn Gly Gly Val65 70
75 80 Leu Tyr Cys Leu Glu Val Ala Lys
Tyr Tyr Asp Tyr Ala Asp Ser Asp 85 90
95 Ala Ala Thr Val Asp Gln Glu Val Glu Glu Leu Leu Arg
Gln Leu Ser 100 105 110
Phe Val Pro Gly Phe Leu Phe Ser Thr Asp Val Ser Tyr Val Asp Phe
115 120 125 Leu Asp Arg Val
His Arg Glu Glu Leu Lys Leu Arg Ser Lys Gly Leu 130
135 140 Trp Asp Val Pro His Pro Trp Leu
Asn Leu Phe Val Pro Lys Ser Arg145 150
155 160 Ile Leu Asp Phe Asp Arg Gly Val Phe Lys Gly Ile
Leu Leu Lys Asn 165 170
175 Thr Asn Asn Ser Gly Pro Ile Leu Val Tyr Pro Met Asn Arg Ser Lys
180 185 190 Trp Asp Asp
Arg Met Ser Ala Val Ile Pro Asp Glu Asp Glu Asp Val 195
200 205 Phe Tyr Leu Val Gly Leu Leu Arg
Ser Ala Val Pro Tyr Ser Ala Gly 210 215
220 Pro Gly Asp Leu Glu Glu Leu Glu Asn Gln Asn Arg Arg
Ile Leu Glu225 230 235
240 Phe Cys Glu Lys Ala Gly Ile Gly Tyr Lys Gln Tyr Leu Pro His Tyr
245 250 255 Leu Thr Ser Gln
Glu Asp Asn Tyr Trp Lys Arg His Phe Gly Ala Ala 260
265 270 Lys Trp Asp Arg Phe Val Asp Arg Lys
Ala Arg Tyr Asp Pro Lys Ala 275 280
285 Ile Leu Ser Pro Gly Gln Gly Ile Phe 290
295 99297PRTZea maysDOMAIN(1)...(297)ZmCkx5 PF09265.1.ls domain
per Figure 9 99Pro Lys Arg Val Arg Trp Val Arg Val Leu Tyr Ser Asp Phe
Ala Ala1 5 10 15
Phe Thr Lys Asp Gln Glu Arg Leu Ile Ser Lys Glu Asn Gly Gly Gly
20 25 30 Gly Ala Lys Val Gly
Phe Asp Tyr Val Glu Gly Phe Val Ile Leu Asn 35 40
45 Arg Thr Gly Leu Val Asn Asn Trp Arg Ser
Ser Phe Phe Ser Pro Ser 50 55 60
Asp Pro Ala Arg Ile Ala Ser Leu Ala Ser Lys Asn Asn Gly Gly
Val65 70 75 80 Leu
Tyr Cys Leu Glu Val Ala Lys Tyr Tyr Asp Tyr Ala Asp Ser Asp
85 90 95 Ala Ala Thr Val Asp Gln
Glu Val Glu Glu Leu Leu Arg Gln Leu Ser 100
105 110 Phe Val Pro Gly Phe Leu Phe Ser Thr Asp
Val Ser Tyr Val Asp Phe 115 120
125 Leu Asp Arg Val His Arg Glu Glu Leu Lys Leu Arg Ser Lys
Gly Leu 130 135 140
Trp Asp Val Pro His Pro Trp Leu Asn Leu Phe Val Pro Lys Ser Arg145
150 155 160 Ile Leu Asp Phe Asp
Arg Gly Val Phe Lys Gly Ile Leu Leu Lys Asn 165
170 175 Thr Asn Asn Ser Gly Pro Ile Leu Val Tyr
Pro Met Asn Arg Ser Lys 180 185
190 Trp Asp Asp Arg Met Ser Ala Val Ile Pro Asp Glu Asp Glu Asp
Val 195 200 205 Phe
Tyr Leu Val Gly Leu Leu Arg Ser Ala Val Pro Tyr Ser Ala Gly 210
215 220 Pro Gly Asp Leu Glu Glu
Leu Glu Asn Gln Asn Arg Arg Ile Leu Glu225 230
235 240 Phe Cys Glu Lys Ala Gly Ile Gly Tyr Lys Gln
Tyr Leu Pro His Tyr 245 250
255 Leu Thr Ser Gln Glu Asp Asn Tyr Trp Lys Arg His Phe Gly Ala Ala
260 265 270 Lys Trp Asp
Arg Phe Val Asp Arg Lys Ala Arg Tyr Asp Pro Lys Ala 275
280 285 Ile Leu Ser Pro Gly Gln Gly Ile
Phe 290 295 100144PRTZea
maysDOMAIN(1)...(144)ZmCkx6 PF01565.13.fs domain per Figure 9 100Pro Ala
Ala Val Val Arg Pro Glu Ser Glu Glu Glu Val Ala Ala Ile1 5
10 15 Val Arg Leu Ala Arg Glu His
Gly Ile Pro Val Thr Pro Arg Gly Gly 20 25
30 Gly His Ser Leu Ser Phe Gly Gly Ala Val Pro Leu
Asn Thr Gly Gly 35 40 45
Val Val Leu Asp Leu Ser Arg Lys Leu Asn Arg Ile Ile Leu Glu Ile
50 55 60 Asp Pro Glu
Thr Asp Gly Thr Ala Thr Val Glu Ala Gly Val Thr Leu65 70
75 80 Asp Leu Asn Arg Ala Leu Ala Ala
Lys Gly Leu Phe Leu Pro Leu Asp 85 90
95 Pro Gly Ser Gly Ile Pro Gly Thr Val Gly Gly Ala Ile
Ala Thr Asn 100 105 110
Ala Gly Gly Tyr Gly Ser Glu Lys Tyr Gly Leu Thr Arg Asp Asn Val
115 120 125 Leu Gly Leu Glu
Val Val Leu Ala Asp Gly Glu Val Val Arg Leu Ser 130
135 140 101144PRTZea
maysDOMAIN(1)...(144)ZmCkx6 PF01565.13.ls domain per Figure 9 101Pro Ala
Ala Val Val Arg Pro Glu Ser Glu Glu Glu Val Ala Ala Ile1 5
10 15 Val Arg Leu Ala Arg Glu His
Gly Ile Pro Val Thr Pro Arg Gly Gly 20 25
30 Gly His Ser Leu Ser Phe Gly Gly Ala Val Pro Leu
Asn Thr Gly Gly 35 40 45
Val Val Leu Asp Leu Ser Arg Lys Leu Asn Arg Ile Ile Leu Glu Ile
50 55 60 Asp Pro Glu
Thr Asp Gly Thr Ala Thr Val Glu Ala Gly Val Thr Leu65 70
75 80 Asp Leu Asn Arg Ala Leu Ala Ala
Lys Gly Leu Phe Leu Pro Leu Asp 85 90
95 Pro Gly Ser Gly Ile Pro Gly Thr Val Gly Gly Ala Ile
Ala Thr Asn 100 105 110
Ala Gly Gly Tyr Gly Ser Glu Lys Tyr Gly Leu Thr Arg Asp Asn Val
115 120 125 Leu Gly Leu Glu
Val Val Leu Ala Asp Gly Glu Val Val Arg Leu Ser 130
135 140 102297PRTZea
maysDOMAIN(1)...(297)ZmCkx6 PF09265.1.fs domain per Figure 9 102Pro Lys
Arg Val Arg Trp Val Arg Val Leu Tyr Ser Asp Phe Ala Ala1 5
10 15 Phe Thr Lys Asp Gln Glu Arg
Leu Ile Ser Lys Glu Asn Gly Gly Gly 20 25
30 Gly Ala Lys Val Gly Phe Asp Tyr Val Glu Gly Phe
Val Ile Leu Asn 35 40 45
Arg Thr Gly Leu Val Asn Asn Trp Arg Ser Ser Phe Phe Ser Pro Ser
50 55 60 Asp Pro Ala
Arg Ile Ala Ser Leu Ala Ser Lys Asn Asn Gly Gly Val65 70
75 80 Leu Tyr Cys Leu Glu Val Ala Lys
Tyr Tyr Asp Tyr Ala Asp Ser Asp 85 90
95 Ala Ala Thr Val Asp Gln Glu Val Glu Glu Leu Leu Arg
Gln Leu Ser 100 105 110
Phe Val Pro Gly Phe Leu Phe Ser Thr Asp Val Ser Tyr Val Asp Phe
115 120 125 Leu Asp Arg Val
His Arg Glu Glu Leu Lys Leu Arg Ser Lys Gly Leu 130
135 140 Trp Asp Val Pro His Pro Trp Leu
Asn Leu Phe Val Pro Lys Ser Arg145 150
155 160 Ile Leu Asp Phe Asp Arg Gly Val Phe Lys Gly Ile
Leu Leu Lys Asn 165 170
175 Thr Asn Asn Ser Gly Pro Ile Leu Val Tyr Pro Met Asn Arg Ser Lys
180 185 190 Trp Asp Asp
Arg Met Ser Ala Val Ile Pro Asp Glu Asp Glu Asp Val 195
200 205 Phe Tyr Leu Val Gly Leu Leu Arg
Ser Ala Val Pro Tyr Ser Ala Gly 210 215
220 Pro Gly Asp Leu Glu Glu Leu Glu Asn Gln Asn Arg Arg
Ile Leu Glu225 230 235
240 Phe Cys Glu Lys Ala Gly Ile Gly Tyr Lys Gln Tyr Leu Pro His Tyr
245 250 255 Leu Thr Ser Gln
Glu Asp Asn Tyr Trp Lys Arg His Phe Gly Ala Ala 260
265 270 Lys Trp Asp Arg Phe Val Asp Arg Lys
Ala Arg Tyr Asp Pro Lys Ala 275 280
285 Ile Leu Ser Pro Gly Gln Gly Ile Phe 290
295 103297PRTZea maysDOMAIN(1)...(297)ZmCkx6 PF09265.1.ls
domain per Figure 9 103Pro Lys Arg Val Arg Trp Val Arg Val Leu Tyr Ser
Asp Phe Ala Ala1 5 10 15
Phe Thr Lys Asp Gln Glu Arg Leu Ile Ser Lys Glu Asn Gly Gly Gly
20 25 30 Gly Ala Lys Val
Gly Phe Asp Tyr Val Glu Gly Phe Val Ile Leu Asn 35
40 45 Arg Thr Gly Leu Val Asn Asn Trp Arg
Ser Ser Phe Phe Ser Pro Ser 50 55 60
Asp Pro Ala Arg Ile Ala Ser Leu Ala Ser Lys Asn Asn Gly
Gly Val65 70 75 80
Leu Tyr Cys Leu Glu Val Ala Lys Tyr Tyr Asp Tyr Ala Asp Ser Asp
85 90 95 Ala Ala Thr Val Asp
Gln Glu Val Glu Glu Leu Leu Arg Gln Leu Ser 100
105 110 Phe Val Pro Gly Phe Leu Phe Ser Thr Asp
Val Ser Tyr Val Asp Phe 115 120
125 Leu Asp Arg Val His Arg Glu Glu Leu Lys Leu Arg Ser Lys
Gly Leu 130 135 140
Trp Asp Val Pro His Pro Trp Leu Asn Leu Phe Val Pro Lys Ser Arg145
150 155 160 Ile Leu Asp Phe Asp
Arg Gly Val Phe Lys Gly Ile Leu Leu Lys Asn 165
170 175 Thr Asn Asn Ser Gly Pro Ile Leu Val Tyr
Pro Met Asn Arg Ser Lys 180 185
190 Trp Asp Asp Arg Met Ser Ala Val Ile Pro Asp Glu Asp Glu Asp
Val 195 200 205 Phe
Tyr Leu Val Gly Leu Leu Arg Ser Ala Val Pro Tyr Ser Ala Gly 210
215 220 Pro Gly Asp Leu Glu Glu
Leu Glu Asn Gln Asn Arg Arg Ile Leu Glu225 230
235 240 Phe Cys Glu Lys Ala Gly Ile Gly Tyr Lys Gln
Tyr Leu Pro His Tyr 245 250
255 Leu Thr Ser Gln Glu Asp Asn Tyr Trp Lys Arg His Phe Gly Ala Ala
260 265 270 Lys Trp Asp
Arg Phe Val Asp Arg Lys Ala Arg Tyr Asp Pro Lys Ala 275
280 285 Ile Leu Ser Pro Gly Gln Gly Ile
Phe 290 295 10457PRTZea
maysDOMAIN(1)...(57)ZmCKx7 PF01565.13.fs domain per Figure 9 104Pro Ala
Ala Val Val Arg Pro Glu Ser Glu Glu Glu Val Ala Ala Ile1 5
10 15 Val Arg Leu Ala Arg Glu His
Gly Ile Pro Val Thr Pro Arg Gly Gly 20 25
30 Gly His Ser Leu Ser Phe Gly Gly Ala Val Pro Leu
Asn Thr Gly Gly 35 40 45
Val Val Leu Asp Leu Ser Arg Lys Leu 50 55
105144PRTZea maysDOMAIN(1)...(144)ZmCkx7 PF01565.13.ls domain per
Figure 9 105Pro Ala Ala Val Val Arg Pro Glu Ser Glu Glu Glu Val Ala Ala
Ile1 5 10 15 Val
Arg Leu Ala Arg Glu His Gly Ile Pro Val Thr Pro Arg Gly Gly 20
25 30 Gly His Ser Leu Ser Phe
Gly Gly Ala Val Pro Leu Asn Thr Gly Gly 35 40
45 Val Val Leu Asp Leu Ser Arg Lys Leu Asn Arg
Ile Ile Leu Glu Ile 50 55 60
Asp Pro Glu Thr Asp Gly Thr Ala Thr Val Glu Ala Gly Val Thr
Leu65 70 75 80 Asp
Leu Asn Arg Ala Leu Ala Ala Lys Gly Leu Phe Leu Pro Leu Asp
85 90 95 Pro Gly Ser Gly Ile Pro
Gly Thr Val Gly Gly Ala Ile Ala Thr Asn 100
105 110 Ala Gly Gly Tyr Gly Ser Glu Lys Tyr Gly
Leu Thr Arg Asp Asn Val 115 120
125 Leu Gly Leu Glu Val Val Leu Ala Asp Gly Glu Val Val Arg
Leu Ser 130 135 140
10683PRTZea maysDOMAIN(1)...(83)ZmCkx7 PF01565.13.fs domain per Figure 9
106Leu Glu Ile Asp Pro Glu Thr Asp Gly Thr Ala Thr Val Glu Ala Gly1
5 10 15 Val Thr Leu Asp
Leu Asn Arg Ala Leu Ala Ala Lys Gly Leu Phe Leu 20
25 30 Pro Leu Asp Pro Gly Ser Gly Ile Pro
Gly Thr Val Gly Gly Ala Ile 35 40
45 Ala Thr Asn Ala Gly Gly Tyr Gly Ser Glu Lys Tyr Gly Leu
Thr Arg 50 55 60
Asp Asn Val Leu Gly Leu Glu Val Val Leu Ala Asp Gly Glu Val Val65
70 75 80 Arg Leu
Ser107297PRTZea maysDOMAIN(1)...(297)ZmCkx7 PF09265.1.fs domain per
Figure 9 107Pro Lys Arg Val Arg Trp Val Arg Val Leu Tyr Ser Asp Phe Ala
Ala1 5 10 15 Phe
Thr Lys Asp Gln Glu Arg Leu Ile Ser Lys Glu Asn Gly Gly Gly 20
25 30 Gly Ala Lys Val Gly Phe
Asp Tyr Val Glu Gly Phe Val Ile Leu Asn 35 40
45 Arg Thr Gly Leu Val Asn Asn Trp Arg Ser Ser
Phe Phe Ser Pro Ser 50 55 60
Asp Pro Ala Arg Ile Ala Ser Leu Ala Ser Lys Asn Asn Gly Gly
Val65 70 75 80 Leu
Tyr Cys Leu Glu Val Ala Lys Tyr Tyr Asp Tyr Ala Asp Ser Asp
85 90 95 Ala Ala Thr Val Asp Gln
Glu Val Glu Glu Leu Leu Arg Gln Leu Ser 100
105 110 Phe Val Pro Gly Phe Leu Phe Ser Thr Asp
Val Ser Tyr Val Asp Phe 115 120
125 Leu Asp Arg Val His Arg Glu Glu Leu Lys Leu Arg Ser Lys
Gly Leu 130 135 140
Trp Asp Val Pro His Pro Trp Leu Asn Leu Phe Val Pro Lys Ser Arg145
150 155 160 Ile Leu Asp Phe Asp
Arg Gly Val Phe Lys Gly Ile Leu Leu Lys Asn 165
170 175 Thr Asn Asn Ser Gly Pro Ile Leu Val Tyr
Pro Met Asn Arg Ser Lys 180 185
190 Trp Asp Asp Arg Met Ser Ala Val Ile Pro Asp Glu Asp Glu Asp
Val 195 200 205 Phe
Tyr Leu Val Gly Leu Leu Arg Ser Ala Val Pro Tyr Ser Ala Gly 210
215 220 Pro Gly Asp Leu Glu Glu
Leu Glu Asn Gln Asn Arg Arg Ile Leu Glu225 230
235 240 Phe Cys Glu Lys Ala Gly Ile Gly Tyr Lys Gln
Tyr Leu Pro His Tyr 245 250
255 Leu Thr Ser Gln Glu Asp Asn Tyr Trp Lys Arg His Phe Gly Ala Ala
260 265 270 Lys Trp Asp
Arg Phe Val Asp Arg Lys Ala Arg Tyr Asp Pro Lys Ala 275
280 285 Ile Leu Ser Pro Gly Gln Gly Ile
Phe 290 295 108297PRTZea
maysDOMAIN(1)...(297)ZmCkx7 PF09265.1.ls domain per Figure 9 108Pro Lys
Arg Val Arg Trp Val Arg Val Leu Tyr Ser Asp Phe Ala Ala1 5
10 15 Phe Thr Lys Asp Gln Glu Arg
Leu Ile Ser Lys Glu Asn Gly Gly Gly 20 25
30 Gly Ala Lys Val Gly Phe Asp Tyr Val Glu Gly Phe
Val Ile Leu Asn 35 40 45
Arg Thr Gly Leu Val Asn Asn Trp Arg Ser Ser Phe Phe Ser Pro Ser
50 55 60 Asp Pro Ala
Arg Ile Ala Ser Leu Ala Ser Lys Asn Asn Gly Gly Val65 70
75 80 Leu Tyr Cys Leu Glu Val Ala Lys
Tyr Tyr Asp Tyr Ala Asp Ser Asp 85 90
95 Ala Ala Thr Val Asp Gln Glu Val Glu Glu Leu Leu Arg
Gln Leu Ser 100 105 110
Phe Val Pro Gly Phe Leu Phe Ser Thr Asp Val Ser Tyr Val Asp Phe
115 120 125 Leu Asp Arg Val
His Arg Glu Glu Leu Lys Leu Arg Ser Lys Gly Leu 130
135 140 Trp Asp Val Pro His Pro Trp Leu
Asn Leu Phe Val Pro Lys Ser Arg145 150
155 160 Ile Leu Asp Phe Asp Arg Gly Val Phe Lys Gly Ile
Leu Leu Lys Asn 165 170
175 Thr Asn Asn Ser Gly Pro Ile Leu Val Tyr Pro Met Asn Arg Ser Lys
180 185 190 Trp Asp Asp
Arg Met Ser Ala Val Ile Pro Asp Glu Asp Glu Asp Val 195
200 205 Phe Tyr Leu Val Gly Leu Leu Arg
Ser Ala Val Pro Tyr Ser Ala Gly 210 215
220 Pro Gly Asp Leu Glu Glu Leu Glu Asn Gln Asn Arg Arg
Ile Leu Glu225 230 235
240 Phe Cys Glu Lys Ala Gly Ile Gly Tyr Lys Gln Tyr Leu Pro His Tyr
245 250 255 Leu Thr Ser Gln
Glu Asp Asn Tyr Trp Lys Arg His Phe Gly Ala Ala 260
265 270 Lys Trp Asp Arg Phe Val Asp Arg Lys
Ala Arg Tyr Asp Pro Lys Ala 275 280
285 Ile Leu Ser Pro Gly Gln Gly Ile Phe 290
295 109144PRTZea maysDOMAIN(1)...(144)ZmCkx8 PF01565.13.fs
domain per Figure 9 109Pro Ala Ala Val Val Arg Pro Glu Ser Glu Glu Glu
Val Ala Ala Ile1 5 10 15
Val Arg Leu Ala Arg Glu His Gly Ile Pro Val Thr Pro Arg Gly Gly
20 25 30 Gly His Ser Leu
Ser Phe Gly Gly Ala Val Pro Leu Asn Thr Gly Gly 35
40 45 Val Val Leu Asp Leu Ser Arg Lys Leu
Asn Arg Ile Ile Leu Glu Ile 50 55 60
Asp Pro Glu Thr Asp Gly Thr Ala Thr Val Glu Ala Gly Val
Thr Leu65 70 75 80
Asp Leu Asn Arg Ala Leu Ala Ala Lys Gly Leu Phe Leu Pro Leu Asp
85 90 95 Pro Gly Ser Gly Ile
Pro Gly Thr Val Gly Gly Ala Ile Ala Thr Asn 100
105 110 Ala Gly Gly Tyr Gly Ser Glu Lys Tyr Gly
Leu Thr Arg Asp Asn Val 115 120
125 Leu Gly Leu Glu Val Val Leu Ala Asp Gly Glu Val Val Arg
Leu Ser 130 135 140
110144PRTZea maysDOMAIN(1)...(144)ZmCkx8 PF01565.13.ls domain per Figure
9 110Pro Ala Ala Val Val Arg Pro Glu Ser Glu Glu Glu Val Ala Ala Ile1
5 10 15 Val Arg Leu Ala
Arg Glu His Gly Ile Pro Val Thr Pro Arg Gly Gly 20
25 30 Gly His Ser Leu Ser Phe Gly Gly Ala
Val Pro Leu Asn Thr Gly Gly 35 40
45 Val Val Leu Asp Leu Ser Arg Lys Leu Asn Arg Ile Ile Leu
Glu Ile 50 55 60
Asp Pro Glu Thr Asp Gly Thr Ala Thr Val Glu Ala Gly Val Thr Leu65
70 75 80 Asp Leu Asn Arg Ala
Leu Ala Ala Lys Gly Leu Phe Leu Pro Leu Asp 85
90 95 Pro Gly Ser Gly Ile Pro Gly Thr Val Gly
Gly Ala Ile Ala Thr Asn 100 105
110 Ala Gly Gly Tyr Gly Ser Glu Lys Tyr Gly Leu Thr Arg Asp Asn
Val 115 120 125 Leu
Gly Leu Glu Val Val Leu Ala Asp Gly Glu Val Val Arg Leu Ser 130
135 140 111297PRTZea
maysDOMAIN(1)...(297)ZmCkx8 PF09265.1.fs domain per Figure 9 111Pro Lys
Arg Val Arg Trp Val Arg Val Leu Tyr Ser Asp Phe Ala Ala1 5
10 15 Phe Thr Lys Asp Gln Glu Arg
Leu Ile Ser Lys Glu Asn Gly Gly Gly 20 25
30 Gly Ala Lys Val Gly Phe Asp Tyr Val Glu Gly Phe
Val Ile Leu Asn 35 40 45
Arg Thr Gly Leu Val Asn Asn Trp Arg Ser Ser Phe Phe Ser Pro Ser
50 55 60 Asp Pro Ala
Arg Ile Ala Ser Leu Ala Ser Lys Asn Asn Gly Gly Val65 70
75 80 Leu Tyr Cys Leu Glu Val Ala Lys
Tyr Tyr Asp Tyr Ala Asp Ser Asp 85 90
95 Ala Ala Thr Val Asp Gln Glu Val Glu Glu Leu Leu Arg
Gln Leu Ser 100 105 110
Phe Val Pro Gly Phe Leu Phe Ser Thr Asp Val Ser Tyr Val Asp Phe
115 120 125 Leu Asp Arg Val
His Arg Glu Glu Leu Lys Leu Arg Ser Lys Gly Leu 130
135 140 Trp Asp Val Pro His Pro Trp Leu
Asn Leu Phe Val Pro Lys Ser Arg145 150
155 160 Ile Leu Asp Phe Asp Arg Gly Val Phe Lys Gly Ile
Leu Leu Lys Asn 165 170
175 Thr Asn Asn Ser Gly Pro Ile Leu Val Tyr Pro Met Asn Arg Ser Lys
180 185 190 Trp Asp Asp
Arg Met Ser Ala Val Ile Pro Asp Glu Asp Glu Asp Val 195
200 205 Phe Tyr Leu Val Gly Leu Leu Arg
Ser Ala Val Pro Tyr Ser Ala Gly 210 215
220 Pro Gly Asp Leu Glu Glu Leu Glu Asn Gln Asn Arg Arg
Ile Leu Glu225 230 235
240 Phe Cys Glu Lys Ala Gly Ile Gly Tyr Lys Gln Tyr Leu Pro His Tyr
245 250 255 Leu Thr Ser Gln
Glu Asp Asn Tyr Trp Lys Arg His Phe Gly Ala Ala 260
265 270 Lys Trp Asp Arg Phe Val Asp Arg Lys
Ala Arg Tyr Asp Pro Lys Ala 275 280
285 Ile Leu Ser Pro Gly Gln Gly Ile Phe 290
295 112297PRTZea maysDOMAIN(1)...(297)ZmCkx8 PF09265.1.ls
domain per Figure 9 112Pro Lys Arg Val Arg Trp Val Arg Val Leu Tyr Ser
Asp Phe Ala Ala1 5 10 15
Phe Thr Lys Asp Gln Glu Arg Leu Ile Ser Lys Glu Asn Gly Gly Gly
20 25 30 Gly Ala Lys Val
Gly Phe Asp Tyr Val Glu Gly Phe Val Ile Leu Asn 35
40 45 Arg Thr Gly Leu Val Asn Asn Trp Arg
Ser Ser Phe Phe Ser Pro Ser 50 55 60
Asp Pro Ala Arg Ile Ala Ser Leu Ala Ser Lys Asn Asn Gly
Gly Val65 70 75 80
Leu Tyr Cys Leu Glu Val Ala Lys Tyr Tyr Asp Tyr Ala Asp Ser Asp
85 90 95 Ala Ala Thr Val Asp
Gln Glu Val Glu Glu Leu Leu Arg Gln Leu Ser 100
105 110 Phe Val Pro Gly Phe Leu Phe Ser Thr Asp
Val Ser Tyr Val Asp Phe 115 120
125 Leu Asp Arg Val His Arg Glu Glu Leu Lys Leu Arg Ser Lys
Gly Leu 130 135 140
Trp Asp Val Pro His Pro Trp Leu Asn Leu Phe Val Pro Lys Ser Arg145
150 155 160 Ile Leu Asp Phe Asp
Arg Gly Val Phe Lys Gly Ile Leu Leu Lys Asn 165
170 175 Thr Asn Asn Ser Gly Pro Ile Leu Val Tyr
Pro Met Asn Arg Ser Lys 180 185
190 Trp Asp Asp Arg Met Ser Ala Val Ile Pro Asp Glu Asp Glu Asp
Val 195 200 205 Phe
Tyr Leu Val Gly Leu Leu Arg Ser Ala Val Pro Tyr Ser Ala Gly 210
215 220 Pro Gly Asp Leu Glu Glu
Leu Glu Asn Gln Asn Arg Arg Ile Leu Glu225 230
235 240 Phe Cys Glu Lys Ala Gly Ile Gly Tyr Lys Gln
Tyr Leu Pro His Tyr 245 250
255 Leu Thr Ser Gln Glu Asp Asn Tyr Trp Lys Arg His Phe Gly Ala Ala
260 265 270 Lys Trp Asp
Arg Phe Val Asp Arg Lys Ala Arg Tyr Asp Pro Lys Ala 275
280 285 Ile Leu Ser Pro Gly Gln Gly Ile
Phe 290 295
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