Sterol biosynthetic enzymes

This application claims priority benefit of U.S. Provisional Application No. 60/112,555 filed Dec. 16, 1998, now pending.

This invention is in the field of plant molecular biology. More specifically, this invention pertains to nucleic acid fragments encoding sterol biosynthetic enzymes in plants and seeds.

The C-8 sterol isomerase enzyme is involved in plant, animal, and fungal sterol biosynthesis. The product of the ERG2 gene is also called delta 8-delta 7-sterol isomerase, C-8,7 sterol isomerase or D8AE7 isomerase and is non-essential in yeast. This membrane-bound enzyme is required for the isomerization of the delta 8 double bond to the delta 7 position in the distal portion of the ergosterol biosynthesis pathway (Ashman et al. (1991) Lipids 26:628-632).

Brassinosteroids are plant enzymes important in growth promotion. Steroid 22-alplha-hydroxylase (DWF4) is a brassinosteroid biosynthetic enzyme, a member of the cytochrome P450 superfamily. The Arabidopsis thaliana DWF4 is a cytochrome P450 monooxygenase with significant similarity to the brassinosteroid enzyme CPD. DWF4 contains the four domains characteristic of cytochrome non-A P450 enzymes and is involved in plant sterol biosynthesis functioning as a 22 alpha-hydroxylase in what is probably the rate limiting step in brassinosteroid biosynthesis (Choe t al. (1998) Plant Cell 10:231-243).

3-beta hydroxy-delta-5-steroid dehydrogenase (EC 1.1.1.145) is also called progesterone reductase. It is an oxidoreductase which acts on the CH—OH group of donors with NAD+ or NADP+ as acceptors in the C-21 steroid metabolism and the androgen and estrogen metabolisms. The enzyme converts 3 beta hydroxy-5-ene-steroids into 3-keto-4-ene derivatives and interconvers 3 beta-hydroxy and 3-keto-5 alpha-androstane steroids (Labrie et al. (1992) J. Steroid Biochem. Mol. Biol. 41:421-435).

The present invention relates to isolated polynucleotides comprising a nucleotide sequence encoding a polypeptide of at least 40 amino acids that has at least 80% identity based on the Clustal method of alignment when compared to a C-8,7 sterol isomerase polypeptide of SEQ ID NOs:2, 4, 6, 8, 22, 24, 26, and 28. The present invention also relates to an isolated polynucleotide comprising the complement of the nucleotide sequences described above.

The present invention relates to isolated polynucleotides comprising a nucleotide sequence encoding a polypeptide of at least 70 amino acids that has at least 80% identity based on the Clustal method of alignment when compared to a steroid 22-alpha-hydroxylase polypeptide of SEQ ID NOs:10, 12, 14, 30, 32, 34, 36, 38, and 40. The present invention also relates to an isolated polynucleotide comprising the complement of the nucleotide sequences described above.

The present invention relates to isolated polynucleotides comprising a nucleotide sequence encoding a polypeptide of at least 80 amino acids that has at least 80% identity based on the Clustal method of alignment when compared to a 3-beta-hydroxy-delta-5-steroid dehydrogenase polypeptide of SEQ ID NOs:16, 18, 20, 42, and 44. The present invention also relates to an isolated polynucleotide comprising the complement of the nucleotide sequences described above.

It is preferred that the isolated polynucleotides of the claimed invention consist of a nucleic acid sequence selected from the group consisting of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31 33, 35, 37, 39, 41, and 43 that codes for the polypeptide selected from the group consisting of SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, and 44. The present invention also relates to an isolated polynucleotide comprising a nucleotide sequences of at least 60 (preferably at least 40, most preferably at least 30) contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31 33, 35, 37, 39, 41, and 43 and the complement of such nucleotide sequences.

The present invention relates to a chimeric gene comprising an isolated polynucleotide of the present invention operably linked to suitable regulatory sequences.

The present invention relates to an isolated host cell comprising a chimeric gene of the present invention or an isolated polynucleotide of the present invention. The host cell may be eukaryotic, such as a yeast or a plant cell, or prokaryotic, such as a bacterial cell. The present invention also relates to a virus, preferably a baculovirus, comprising an isolated polynucleotide of the present invention or a chimeric gene of the present invention.

The present invention relates to a process for producing an isolated host cell comprising a chimeric gene of the present invention or an isolated polynucleotide of the present invention, the process comprising either transforming or transfecting an isolated compatible host cell with a chimeric gene or isolated polynucleotide of the present invention.

The present invention relates to a C-8,7 sterol isomerase polypeptide of at least 40 amino acids comprising at least 80% homology based on the Clustal method of alignment compared to a polypeptide selected from the group consisting of SEQ ID NOs:2, 4, 6, 8, 22, 24, 26, and 28.

The present invention relates to a Steroid 22-alpha-hydroxylase polypeptide of at least 70 amino acids comprising at least 80% homology based on the Clustal method of alignment compared to a polypeptide selected from the group consisting of SEQ ID NOs:10, 12, 14, 30, 32, 34, 36, 38, and 40.

The present invention relates to a 3-beta-Hydroxy-delta-5-steroid dehydrogenase polypeptide of at least 80 amino acids comprising at least 80% homology based on the Clustal method of alignment compared to a polypeptide selected from the group consisting of SEQ ID NOs:16, 18, 20, 42, and 44.

The present invention relates to a method of selecting an isolated polynucleotide that affects the level of expression of a sterol biosynthetic enzyme polypeptide in a host cell, preferably a plant cell, the method comprising the steps of: (a) constructing an isolated polynucleotide of the present invention or an isolated chimeric gene of the present invention; (b) introducing the isolated polynucleotide or the isolated chimeric gene into a host cell; (c) measuring the level of a C-8,7 sterol isomerase, a steroid 22-alpha-hydroxylase or a 3-beta-hydroxy-delta-5-steroid dehydrogenase polypeptide in the host cell containing the isolated polynucleotide; and (d) comparing the level of a sterol biosynthetic enzyme polypeptide in the host cell containing the isolated polynucleotide with the level of a sterol biosynthetic enzyme polypeptide in the host cell that does not contain the isolated polynucleotide.

The present invention relates to a method of obtaining a nucleic acid fragment encoding a substantial portion of a C-8,7 sterol isomerase, a steroid 22-alpha-hydroxylase or a 3-beta-hydroxy-delta-5-steroid dehydrogenase polypeptide gene, preferably a plant C-8,7 sterol isomerase, steroid 22-alpha-hydroxylase or 3-beta-hydroxy-delta-5-steroid dehydrogenase polypeptide gene, comprising the steps of: synthesizing an oligonucleotide primer comprising a nucleotide sequence of at least 60 (preferably at least 40, most preferably at least 30) contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31 33, 35, 37, 39, 41, and 43 and the complement of such nucleotide sequences; and amplifying a nucleic acid fragment (preferably a cDNA inserted in a cloning vector) using the oligonucleotide primer. The amplified nucleic acid fragment preferably will encode a portion of a C-8,7 sterol isomerase, a steroid 22-alpha-hydroxylase or a 3-beta-hydroxy-delta-5-steroid dehydrogenase amino acid sequence.

The present invention also relates to a method of obtaining a nucleic acid fragment encoding all or a substantial portion of the amino acid sequence encoding a C-8,7 sterol isomerase, a steroid 22-alpha-hydroxylase or a 3-beta-hydroxy-delta-5-steroid dehydrogenase polypeptide comprising the steps of: probing a cDNA or genomic library with an isolated polynucleotide of the present invention; identifying a DNA clone that hybridizes with an isolated polynucleotide of the present invention; isolating the identified DNA clone; and sequencing the cDNA or genomic fragment that comprises the isolated DNA clone.

A further embodiment of the instant invention is a method for evaluating at least one compound for its ability to inhibit the activity of a C-8,7 sterol isomerase, a steroid 22-alpha-hydroxylase or a 3-beta-hydroxy-delta-5-steroid dehydrogenase, the method comprising the steps of: (a) transforming a host cell with a chimeric gene comprising a nucleic acid fragment encoding a C-8,7 sterol isomerase, a steroid 22-alpha-hydroxylase or a 3-beta-hydroxy-delta-5-steroid dehydrogenase, operably linked to suitable regulatory sequences; (b) growing the transformed host cell under conditions that are suitable for expression of the chimeric gene wherein expression of the chimeric gene results in production of C-8,7 sterol isomerase, steroid 22-alpha-hydroxylase or 3-beta-hydroxy-delta-5-steroid dehydrogenase in the transformed host cell; (c) optionally purifying the C-8,7 sterol isomerase, the steroid 22-alpha-hydroxylase or the 3-beta-hydroxy-delta-5-steroid dehydrogenase expressed by the transformed host cell; (d) treating the C-8,7 sterol isomerase, the steroid 22-alpha-hydroxylase or the 3-beta-hydroxy-delta-5-steroid dehydrogenase with a compound to be tested; and (e) comparing the activity of the C-8,7 sterol isomerase, the steroid 22-alpha-hydroxylase or the 3-beta-hydroxy-delta-5-steroid dehydrogenase that has been treated with a test compound to the activity of an untreated C-8,7 sterol isomerase, steroid 22-alpha-hydroxylase or 3-beta-hydroxy-delta-5-steroid dehydrogenase, thereby selecting compounds with potential for inhibitory activity.

The present invention relates to a composition, such as a hybridization mixture, comprising an isolated polynucleotide of the present invention.

The present invention relates to an isolated polynucleotide of the present invention comprising at least 30 contiguous nucleotides derived from a nucleic acid sequence selected from the group consisting of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31 33, 35, 37, 39, 41, and 43.

The present invention relates to an expression cassette comprising an isolated polynucleotide of the present invention operably linked to a promoter.

The present invention relates to a method for positive selection of a transformed cell comprising: (a) transforming a host cell with the chimeric gene of the present invention or an expression cassette of the present invention; and (b) growing the transformed host cell, preferably plant cell, such as a monocot or a dicot, under conditions which allow expression of the C-8,7 sterol isomerase, the steroid 22-alpha-hydroxylase or the 3-beta-hydroxy-delta-5-steroid dehydrogenase polynucleotide in an amount sufficient to complement a null mutant to provide a positive selection means.

The invention can be more fully understood from the following detailed description and the accompanying Sequence Listing which form a part of this application.

Table 1 lists the polypeptides that are described herein, the designation of the cDNA clones that comprise the nucleic acid fragments encoding polypeptides representing all or a substantial portion of these polypeptides, and the corresponding identifier (SEQ ID NO:) as used in the attached Sequence Listing. The sequence descriptions and Sequence Listing attached hereto comply with the rules governing nucleotide and/or amino acid sequence disclosures in patent applications as set forth in 37 C.F.R. §1.821-1.825.

The Sequence Listing contains the one letter code for nucleotide sequence characters and the three letter codes for amino acids as defined in conformity with the IUPAC-IUBMB standards described in Nucleic Acids Res. 13:3021-3030 (1985) and in the Biochemical J. 219 (No. 2):345-373 (1984) which are herein incorporated by reference. The symbols and format used for nucleotide and amino acid sequence data comply with the rules set forth in 37 C.F.R. §1.822.

In the context of this disclosure, a number of terms shall be utilized. As used herein, a “polynucleotide” is a nucleotide sequence such as a nucleic acid fragment. A polynucleotide may be a polymer of RNA or DNA that is single- or double-stranded, that optionally contains synthetic, non-natural or altered nucleotide bases. A polynucleotide in the form of a polymer of DNA may be comprised of one or more segments of cDNA, genomic DNA, synthetic DNA, or mixtures thereof. An isolated polynucleotide of the present invention may include at least 60 contiguous nucleotides, preferably at least 40 contiguous nucleotides, most preferably one of at least 30 contiguous nucleotides derived from SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31 33, 35, 37, 39, 41, and 43, or the complement of such sequences.

As used herein, “contig” refers to a nucleotide sequence that is assembled from two or more constituent nucleotide sequences that share common or overlapping regions of sequence homology. For example, the nucleotide sequences of two or more nucleic acid fragments can be compared and aligned in order to identify common or overlapping sequences. Where common or overlapping sequences exist between two or more nucleic acid fragments, the sequences (and thus their corresponding nucleic acid fragments) can be assembled into a single contiguous nucleotide sequence.

As used herein, “substantially similar” refers to nucleic acid fragments wherein changes in one or more nucleotide bases results in substitution of one or more amino acids, but do not affect the functional properties of the polypeptide encoded by the nucleotide sequence. “Substantially similar” also refers to nucleic acid fragments wherein changes in one or more nucleotide bases does not affect the ability of the nucleic acid fragment to mediate alteration of gene expression by gene silencing through for example antisense or co-suppression technology. “Substantially similar” also refers to modifications of the nucleic acid fragments of the instant invention such as deletion or insertion of one or more nucleotides that do not substantially affect the functional properties of the resulting transcript vis-a-vis the ability to mediate gene silencing or alteration of the functional properties of the resulting protein molecule. It is therefore understood that the invention encompasses more than the specific exemplary nucleotide or amino acid sequences and includes functional equivalents thereof.

Substantially similar nucleic acid fragments may be selected by screening nucleic acid fragments representing subfragments or modifications of the nucleic acid fragments of the instant invention, wherein one or more nucleotides are substituted, deleted and/or inserted, for their ability to affect the level of the polypeptide encoded by the unmodified nucleic acid fragment in a plant or plant cell. For example, a substantially similar nucleic acid fragment representing at least 30 contiguous nucleotides derived from the instant nucleic acid fragment can be constructed and introduced into a plant or plant cell. The level of the polypeptide encoded by the unmodified nucleic acid fragment present in a plant or plant cell exposed to the substantially similar nucleic fragment can then be compared to the level of the polypeptide in a plant or plant cell that is not exposed to the substantially similar nucleic acid fragment.

For example, it is well known in the art that antisense suppression and co-suppression of gene expression may be accomplished using nucleic acid fragments representing less than the entire coding region of a gene, and by nucleic acid fragments that do not share 100% sequence identity with the gene to be suppressed. Moreover, alterations in a nucleic acid fragment which result in the production of a chemically equivalent amino acid at a given site, but do not effect the functional properties of the encoded polypeptide, are well known in the art. Thus, a codon for the amino acid alanine, a hydrophobic amino acid, may be substituted by a codon encoding another less hydrophobic residue, such as glycine, or a more hydrophobic residue, such as valine, leucine, or isoleucine. Similarly, changes which result in substitution of one negatively charged residue for another, such as aspartic acid for glutamic acid, or one positively charged residue for another, such as lysine for arginine, can also be expected to produce a functionally equivalent product. Nucleotide changes which result in alteration of the N-terminal and C-terminal portions of the polypeptide molecule would also not be expected to alter the activity of the polypeptide. Each of the proposed modifications is well within the routine skill in the art, as is determination of retention of biological activity of the encoded products. Consequently, an isolated polynucleotide comprising a nucleotide sequence of at least 60 (preferably at least 40, most preferably at least 30) contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31 33, 35, 37, 39, 41, and 43 and the complement of such nucleotide sequences may be used in methods of selecting an isolated polynucleotide that affects the expression of a sterol metabolism enzyme polypeptide such as aC-8,7 sterol isomerase, a steroid 22-alpha-hydroxylase or a 3-beta-hydroxy-delta-5-steroid dehydrogenase in a host cell. A method of selecting an isolated polynucleotide that affects the level of expression of a polypeptide in a host cell (eukaryotic, such as plant or yeast, prokaryotic such as bacterial, or viral) may comprise the steps of: constructing an isolated polynucleotide of the present invention or an isolated chimeric gene of the present invention; introducing the isolated polynucleotide or the isolated chimeric gene into a host cell; measuring the level a polypeptide in the host cell containing the isolated polynucleotide; and comparing the level of a polypeptide in the host cell containing the isolated polynucleotide with the level of a polypeptide in a host cell that does not contain the isolated polynucleotide.

Moreover, substantially similar nucleic acid fragments may also be characterized by their ability to hybridize. Estimates of such homology are provided by either DNA-DNA or DNA-RNA hybridization under conditions of stringency as is well understood by those skilled in the art (Hames and Higgins, Eds. (1985) Nucleic Acid Hybridisation, IRL Press, Oxford, U.K.). Stringency conditions can be adjusted to screen for moderately similar fragments, such as homologous sequences from distantly related organisms, to highly similar fragments, such as genes that duplicate functional enzymes from closely related organisms. Post-hybridization washes determine stringency conditions. One set of preferred conditions uses a series of washes starting with 6× SSC, 0.5% SDS at room temperature for 15 min, then repeated with 2× SSC, 0.5% SDS at 45° C. for 30 min, and then repeated twice with 0.2× SSC, 0.5% SDS at 50° C. for 30 min. A more preferred set of stringent conditions uses higher temperatures in which the washes are identical to those above except for the temperature of the final two 30 min washes in 0.2× SSC, 0.5% SDS was increased to 60° C. Another preferred set of highly stringent conditions uses two final washes in 0.1× SSC, 0.1% SDS at 65° C.

Substantially similar nucleic acid fragments of the instant invention may also be characterized by the percent identity of the amino acid sequences that they encode to the amino acid sequences disclosed herein, as determined by algorithms commonly employed by those skilled in this art. Suitable nucleic acid fragments (isolated polynucleotides of the present invention) encode polypeptides that are at least about 70% identical, preferably at least about 80% identical to the amino acid sequences reported herein. Preferred nucleic acid fragments encode amino acid sequences that are about 85% identical to the amino acid sequences reported herein. More preferred nucleic acid fragments encode amino acid sequences that are at least about 90% identical to the amino acid sequences reported herein. Most preferred are nucleic acid fragments that encode amino acid sequences that are at least about 95% identical to the amino acid sequences reported herein. Suitable nucleic acid fragments not only have the above homologies but typically encode a polypeptide having at least about 50 amino acids, preferably at least about 100 amino acids, more preferably at least about 150 amino acids, still more preferably at least about 200 amino acids, and most preferably at least about 250 amino acids. Sequence alignments and percent identity calculations were performed using the Megalign program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.). Multiple alignment of the sequences was performed using the Clustal method of alignment (Higgins and Sharp (1989) CABIOS. 5:151-153) with the default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Default parameters for pairwise alignments using the Clustal method were KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5.

A “substantial portion” of an amino acid or nucleotide sequence comprises an amino acid or a nucleotide sequence that is sufficient to afford putative identification of the protein or gene that the amino acid or nucleotide sequence comprises. Amino acid and nucleotide sequences can be evaluated either manually by one skilled in the art, or by using computer-based sequence comparison and identification tools that employ algorithms such as BLAST (Basic Local Alignment Search Tool; Altschul et al. (1993) J. Mol. Biol. 215:403-410). In general, a sequence of ten or more contiguous amino acids or thirty or more contiguous nucleotides is necessary in order to putatively identify a polypeptide or nucleic acid sequence as homologous to a known protein or gene. Moreover, with respect to nucleotide sequences, gene-specific oligonucleotide probes comprising 30 or more contiguous nucleotides may be used in sequence-dependent methods of gene identification (e.g., Southern hybridization) and isolation (e.g., in situ hybridization of bacterial colonies or bacteriophage plaques). In addition, short oligonucleotides of 12 or more nucleotides may be used as amplification primers in PCR in order to obtain a particular nucleic acid fragment comprising the primers. Accordingly, a “substantial portion” of a nucleotide sequence comprises a nucleotide sequence that will afford specific identification and/or isolation of a nucleic acid fragment comprising the sequence. The instant specification teaches amino acid and nucleotide sequences encoding polypeptides that comprise one or more particular plant proteins. The skilled artisan, having the benefit of the sequences as reported herein, may now use all or a substantial portion of the disclosed sequences for purposes known to those skilled in this art. Accordingly, the instant invention comprises the complete sequences as reported in the accompanying Sequence Listing, as well as substantial portions of those sequences as defined above.

“Codon degeneracy” refers to divergence in the genetic code permitting variation of the nucleotide sequence without affecting the amino acid sequence of an encoded polypeptide. Accordingly, the instant invention relates to any nucleic acid fragment comprising a nucleotide sequence that encodes all or a substantial portion of the amino acid sequences set forth herein. The skilled artisan is well aware of the “codon-bias” exhibited by a specific host cell in usage of nucleotide codons to specify a given amino acid. Therefore, when synthesizing a nucleic acid fragment for improved expression in a host cell, it is desirable to design the nucleic acid fragment such that its frequency of codon usage approaches the frequency of preferred codon usage of the host cell.

“Synthetic nucleic acid fragments” can be assembled from oligonucleotide building blocks that are chemically synthesized using procedures known to those skilled in the art. These building blocks are ligated and annealed to form larger nucleic acid fragments which may then be enzymatically assembled to construct the entire desired nucleic acid fragment. “Chemically synthesized”, as related to nucleic acid fragment, means that the component nucleotides were assembled in vitro. Manual chemical synthesis of nucleic acid fragments may be accomplished using well established procedures, or automated chemical synthesis can be performed using one of a number of commercially available machines. Accordingly, the nucleic acid fragments can be tailored for optimal gene expression based on optimization of nucleotide sequence to reflect the codon bias of the host cell. The skilled artisan appreciates the likelihood of successful gene expression if codon usage is biased towards those codons favored by the host. Determination of preferred codons can be based on a survey of genes derived from the host cell where sequence information is available.

“Gene” refers to a nucleic acid fragment that expresses a specific protein, including regulatory sequences preceding (5′ non-coding sequences) and following (3′ non-coding sequences) the coding sequence. “Native gene” refers to a gene as found in nature with its own regulatory sequences. “Chimeric gene” refers any gene that is not a native gene, comprising regulatory and coding sequences that are not found together in nature. Accordingly, a chimeric gene may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that found in nature. “Endogenous gene” refers to a native gene in its natural location in the genome of an organism. A “foreign” gene refers to a gene not normally found in the host organism, but that is introduced into the host organism by gene transfer. Foreign genes can comprise native genes inserted into a non-native organism, or chimeric genes. A “transgene” is a gene that has been introduced into the genome by a transformation procedure.

“Coding sequence” refers to a nucleotide sequence that codes for a specific amino acid sequence. “Regulatory sequences” refer to nucleotide sequences located upstream (5′ non-coding sequences), within, or downstream (3′ non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences may include promoters, translation leader sequences, introns, and polyadenylation recognition sequences.

“Promoter” refers to a nucleotide sequence capable of controlling the expression of a coding sequence or functional RNA. In general, a coding sequence is located 3′ to a promoter sequence. The promoter sequence consists of proximal and more distal upstream elements, the latter elements often referred to as enhancers. Accordingly, an “enhancer” is a nucleotide sequence which can stimulate promoter activity and may be an innate element of the promoter or a heterologous element inserted to enhance the level or tissue-specificity of a promoter. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic nucleotide segments. It is understood by those skilled in the art that different promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental conditions. Promoters which cause a nucleic acid fragment to be expressed in most cell types at most times are commonly referred to as “constitutive promoters”. New promoters of various types useful in plant cells are constantly being discovered; numerous examples may be found in the compilation by Okamuro and Goldberg (1989) Biochemistry of Plants 15:1-82. It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, nucleic acid fragments of different lengths may have identical promoter activity.

The “translation leader sequence” refers to a nucleotide sequence located between the promoter sequence of a gene and the coding sequence. The translation leader sequence is present in the fully processed mRNA upstream of the translation start sequence. The translation leader sequence may affect processing of the primary transcript to mRNA, mRNA stability or translation efficiency. Examples of translation leader sequences have been described (Turner and Foster (1995) Mol. Biotechnol. 3:225-236).

The “3′ non-coding sequences” refer to nucleotide sequences located downstream of a coding sequence and include polyadenylation recognition sequences and other sequences encoding regulatory signals capable of affecting mRNA processing or gene expression. The polyadenylation signal is usually characterized by affecting the addition of polyadenylic acid tracts to the 3′ end of the mRNA precursor. The use of different 3′ non-coding sequences is exemplified by Ingelbrecht et al. (1989) Plant Cell 1:671-680.

“RNA transcript” refers to the product resulting from RNA polymerase-catalyzed transcription of a DNA sequence. When the RNA transcript is a perfect complementary copy of the DNA sequence, it is referred to as the primary transcript or it may be a RNA sequence derived from post transcriptional processing of the primary transcript and is referred to as the mature RNA. “Messenger RNA (mRNA)” refers to the RNA that is without introns and that can be translated into polypeptide by the cell. “cDNA” refers to a double-stranded DNA that is complementary to and derived from mRNA. “Sense” RNA refers to an RNA transcript that includes the mRNA and so can be translated into a polypeptide by the cell. “Antisense RNA” refers to an RNA transcript that is complementary to all or part of a target primary transcript or mRNA and that blocks the expression of a target gene (see U.S. Pat. No. 5,107,065, incorporated herein by reference). The complementarity of an antisense RNA may be with any part of the specific nucleotide sequence, i.e., at the 5′ non-coding sequence, 3′ non-coding sequence, introns, or the coding sequence. “Functional RNA” refers to sense RNA, antisense RNA, ribozyme RNA, or other RNA that may not be translated but yet has an effect on cellular processes.

The term “operably linked” refers to the association of two or more nucleic acid fragments on a single nucleic acid fragment so that the function of one is affected by the other. For example, a promoter is operably linked with a coding sequence when it is capable of affecting the expression of that coding sequence (i.e., that the coding sequence is under the transcriptional control of the promoter). Coding sequences can be operably linked to regulatory sequences in sense or antisense orientation.

The term “expression”, as used herein, refers to the transcription and stable accumulation of sense (mRNA) or antisense RNA derived from the nucleic acid fragment of the invention. Expression may also refer to translation of mRNA into a polypeptide. “Antisense inhibition” refers to the production of antisense RNA transcripts capable of suppressing the expression of the target protein. “Over expression” refers to the production of a gene product in transgenic organisms that exceeds levels of production in normal or non-transformed organisms. “Co-suppression” refers to the production of sense RNA transcripts capable of suppressing the expression of identical or substantially similar foreign or endogenous genes (U.S. Pat. No. 5,231,020, incorporated herein by reference).

“Altered levels” refers to the production of gene product(s) in transgenic organisms in amounts or proportions that differ from that of normal or non-transformed organisms.

“Mature” protein refers to a post-translationally processed polypeptide; i.e., one from which any pre- or propeptides present in the primary translation product have been removed. “Precursor” protein refers to the primary product of translation of mRNA; i.e., with pre- and propeptides still present. Pre- and propeptides may be but are not limited to intracellular localization signals.

A “chloroplast transit peptide” is an amino acid sequence which is translated in conjunction with a protein and directs the protein to the chloroplast or other plastid types present in the cell in which the protein is made. “Chloroplast transit sequence” refers to a nucleotide sequence that encodes a chloroplast transit peptide. A “signal peptide” is an amino acid sequence which is translated in conjunction with a protein and directs the protein to the secretory system (Chrispeels (1991) Ann. Rev. Plant Phys. Plant Mol. Biol. 42:21-53). If the protein is to be directed to a vacuole, a vacuolar targeting signal (supra) can further be added, or if to the endoplasmic reticulum, an endoplasmic reticulum retention signal (supra) may be added. If the protein is to be directed to the nucleus, any signal peptide present should be removed and instead a nuclear localization signal included (Raikhel (1992) Plant Phys. 100:1627-1632).

“Transformation” refers to the transfer of a nucleic acid fragment into the genome of a host organism, resulting in genetically stable inheritance. Host organisms containing the transformed nucleic acid fragments are referred to as “transgenic” organisms. Examples of methods of plant transformation include Agrobacterium-mediated transformation (De Blaere et al. (1987) Meth. Enzymol. 143:277) and particle-accelerated or “gene gun” transformation technology (Klein et al. (1987) Nature (London) 327:70-73; U.S. Pat. No. 4,945,050, incorporated herein by reference).

Standard recombinant DNA and molecular cloning techniques used herein are well known in the art and are described more fully in Sambrook et al. Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, 1989 (hereinafter “Maniatis”).

Nucleic acid fragments encoding at least a portion of several sterol biosynthetic enzymes have been isolated and identified by comparison of random plant cDNA sequences to public databases containing nucleotide and protein sequences using the BLAST algorithms well known to those skilled in the art. The nucleic acid fragments of the instant invention may be used to isolate cDNAs and genes encoding homologous proteins from the same or other plant species. Isolation of homologous genes using sequence-dependent protocols is well known in the art. Examples of sequence-dependent protocols include, but are not limited to, methods of nucleic acid hybridization, and methods of DNA and RNA amplification as exemplified by various uses of nucleic acid amplification technologies (e.g., polymerase chain reaction, ligase chain reaction).

For example, genes encoding other C-8,7 sterol isomerases, steroid 22-alpha-hydroxylases or 3-beta-hydroxy-delta-5-steroid dehydrogenases, either as cDNAs or genomic DNAs, could be isolated directly by using all or a portion of the instant nucleic acid fragments as DNA hybridization probes to screen libraries from any desired plant employing methodology well known to those skilled in the art. Specific oligonucleotide probes based upon the instant nucleic acid sequences can be designed and synthesized by methods known in the art (Maniatis). Moreover, the entire sequences can be used directly to synthesize DNA probes by methods known to the skilled artisan such as random primer DNA labeling, nick translation, or end-labeling techniques, or RNA probes using available in vitro transcription systems. In addition, specific primers can be designed and used to amplify a part or all of the instant sequences. The resulting amplification products can be labeled directly during amplification reactions or labeled after amplification reactions, and used as probes to isolate full length cDNA or genomic fragments under conditions of appropriate stringency.

In addition, two short segments of the instant nucleic acid fragments may be used in polymerase chain reaction protocols to amplify longer nucleic acid fragments encoding homologous genes from DNA or RNA. The polymerase chain reaction may also be performed on a library of cloned nucleic acid fragments wherein the sequence of one primer is derived from the instant nucleic acid fragments, and the sequence of the other primer takes advantage of the presence of the polyadenylic acid tracts to the 3′ end of the mRNA precursor encoding plant genes. Alternatively, the second primer sequence may be based upon sequences derived from the cloning vector. For example, the skilled artisan can follow the RACE protocol (Frohman et al. (1988) Proc. Natl. Acad. Sci. USA 85:8998-9002) to generate cDNAs by using PCR to amplify copies of the region between a single point in the transcript and the 3′ or 5′ end. Primers oriented in the 3′ and 5′ directions can be designed from the instant sequences. Using commercially available 3′ RACE or 5′ RACE systems (BRL), specific 3′ or 5′ cDNA fragments can be isolated (Ohara et al. (1989) Proc. Natl. Acad. Sci. USA 86:5673-5677; Loh et al. (1989) Science 243:217-220). Products generated by the 3′ and 5′ RACE procedures can be combined to generate full-length cDNAs (Frohman and Martin (1989) Techniques 1:165). Consequently, a polynucleotide comprising a nucleotide sequence of at least 60 (preferably at least 40, most preferably at least 30) contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31 33, 35, 37, 39, 41, and 43 and the complement of such nucleotide sequences may be used in such methods to obtain a nucleic acid fragment encoding a substantial portion of an amino acid sequence of a polypeptide. The present invention relates to a method of obtaining a nucleic acid fragment encoding a substantial portion of a sterol biosynthetic enzyme polypeptide such as a C-8,7 sterol isomerase, a steroid 22-alpha-hydroxylase or a 3-beta-hydroxy-delta-5-steroid dehydrogenase, preferably a substantial portion of a plant polypeptide of a gene, comprising the steps of: synthesizing an oligonucleotide primer comprising a nucleotide sequence of at least 60 (preferably at least 40, most preferably at least 30) contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31 33, 35, 37, 39, 41, and 43, and the complement of such nucleotide sequences; and amplifying a nucleic acid fragment (preferably a cDNA inserted in a cloning vector) using the oligonucleotide primer. The amplified nucleic acid fragment preferably will encode a portion of a C-8,7 sterol isomerase, a steroid 22-alpha-hydroxylase or a 3-beta-hydroxy-delta-5-steroid dehydrogenase polypeptide.

Availability of the instant nucleotide and deduced amino acid sequences facilitates immunological screening of cDNA expression libraries. Synthetic peptides representing portions of the instant amino acid sequences may be synthesized. These peptides can be used to immunize animals to produce polyclonal or monoclonal antibodies with specificity for peptides or proteins comprising the amino acid sequences. These antibodies can be then be used to screen cDNA expression libraries to isolate full-length cDNA clones of interest (Lerner (1984) Adv. Immunol. 36:1-34; Maniatis).

The nucleic acid fragments of the instant invention may be used to create transgenic plants in which the disclosed polypeptides are present at higher or lower levels than normal or in cell types or developmental stages in which they are not normally found. This would have the effect of altering the level of steroids or brassinosteroids in those cells. Manipulation of the expression of any one of these genes may affect the steroid hormone response enhancing biomass production and grain yield. Changes in the expression patterns of these genes may also result in differences in stress response.

Overexpression of the proteins of the instant invention may be accomplished by first constructing a chimeric gene in which the coding region is operably linked to a promoter capable of directing expression of a gene in the desired tissues at the desired stage of development. The chimeric gene may comprise promoter sequences and translation leader sequences derived from the same genes. 3′ Non-coding sequences encoding transcription termination signals may also be provided. The instant chimeric gene may also comprise one or more introns in order to facilitate gene expression.

Plasmid vectors comprising the isolated polynucleotide (or chimeric gene) may be constructed. The choice of plasmid vector is dependent upon the method that will be used to transform host plants. The skilled artisan is well aware of the genetic elements that must be present on the plasmid vector in order to successfully transform, select and propagate host cells containing the chimeric gene. The skilled artisan will also recognize that different independent transformation events will result in different levels and patterns of expression (Jones et al. (1985) EMBO J. 4:2411-2418; De Almeida et al. (1989) Mol. Gen. Genetics 218:78-86), and thus that multiple events must be screened in order to obtain lines displaying the desired expression level and pattern. Such screening may be accomplished by Southern analysis of DNA, Northern analysis of mRNA expression, Western analysis of protein expression, or phenotypic analysis.

For some applications it may be useful to direct the instant polypeptides to different cellular compartments, or to facilitate its secretion from the cell. It is thus envisioned that the chimeric gene described above may be further supplemented by directing the coding sequence to encode the instant polypeptides with appropriate intracellular targeting sequences such as transit sequences (Keegstra (1989) Cell 56:247-253), signal sequences or sequences encoding endoplasmic reticulum localization (Chrispeels (1991) Ann. Rev. Plant Phys. Plant Mol. Biol. 42:21-53), or nuclear localization signals (Raikhel (1992) Plant Phys. 100:1627-1632) with or without removing targeting sequences that are already present. While the references cited give examples of each of these, the list is not exhaustive and more targeting signals of use may be discovered in the future.

The instant polypeptides (or portions thereof may be produced in heterologous host cells, particularly in the cells of microbial hosts, and can be used to prepare antibodies to the these proteins by methods well known to those skilled in the art. The antibodies are useful for detecting the polypeptides of the instant invention in situ in cells or in vitro in cell extracts. Preferred heterologous host cells for production of the instant polypeptides are microbial hosts. Microbial expression systems and expression vectors containing regulatory sequences that direct high level expression of foreign proteins are well known to those skilled in the art. Any of these could be used to construct a chimeric gene for production of the instant polypeptides. This chimeric gene could then be introduced into appropriate microorganisms via transformation to provide high level expression of the encoded sterol metabolic enzymes. An example of a vector for high level expression of the instant polypeptides in a bacterial host is provided (Example 8).

Additionally, the instant polypeptides can be used as targets to facilitate design and/or identification of inhibitors of those enzymes that may be useful as herbicides. This is desirable because the polypeptides described herein catalyze various steps in steroid biosynthesis. Accordingly, inhibition of the activity of one or more of the enzymes described herein could lead to inhibition of plant growth. Thus, the instant polypeptides could be appropriate for new herbicide discovery and design.

All or a substantial portion of the nucleic acid fragments of the instant invention may also be used as probes for genetically and physically mapping the genes that they are a part of, and as markers for traits linked to those genes. Such information may be useful in plant breeding in order to develop lines with desired phenotypes. For example, the instant nucleic acid fragments may be used as restriction fragment length polymorphism (RFLP) markers. Southern blots (Maniatis) of restriction-digested plant genomic DNA may be probed with the nucleic acid fragments of the instant invention. The resulting banding patterns may then be subjected to genetic analyses using computer programs such as MapMaker (Lander et al. (1987) Genomics 1:174-181) in order to construct a genetic map. In addition, the nucleic acid fragments of the instant invention may be used to probe Southern blots containing restriction endonuclease-treated genomic DNAs of a set of individuals representing parent and progeny of a defined genetic cross. Segregation of the DNA polymorphisms is noted and used to calculate the position of the instant nucleic acid sequence in the genetic map previously obtained using this population (Botstein et al. (1980) Am. J Hum. Genet. 32:314-331).

The production and use of plant gene-derived probes for use in genetic mapping is described in Bernatzky and Tanksley (1986) Plant Mol. Biol. Reporter 4:37-41. Numerous publications describe genetic mapping of specific cDNA clones using the methodology outlined above or variations thereof. For example, F2 intercross populations, backcross populations, randomly mated populations, near isogenic lines, and other sets of individuals may be used for mapping. Such methodologies are well known to those skilled in the art.

Nucleic acid probes derived from the instant nucleic acid sequences may also be used for physical mapping (i.e., placement of sequences on physical maps; see Hoheisel et al. In: Nonmammalian Genomic Analysis: A Practical Guide, Academic press 1996, pp. 319-346, and references cited therein).

In another embodiment, nucleic acid probes derived from the instant nucleic acid sequences may be used in direct fluorescence in situ hybridization (FISH) mapping (Trask (1991) Trends Genet. 7:149-154). Although current methods of FISH mapping favor use of large clones (several to several hundred KB; see Laan et al. (1995) Genome Res. 5:13-20), improvements in sensitivity may allow performance of FISH mapping using shorter probes.

A variety of nucleic acid amplification-based methods of genetic and physical mapping may be carried out using the instant nucleic acid sequences. Examples include allele-specific amplification (Kazazian (1989) J. Lab. Clin. Med. 11:95-96), polymorphism of PCR-amplified fragments (CAPS; Sheffield et al. (1993) Genomics 16:325-332), allele-specific ligation (Landegren et al. (1988) Science 241:1077-1080), nucleotide extension reactions (Sokolov (1990) Nucleic Acid Res. 18:3671), Radiation Hybrid Mapping (Walter et al. (1997) Nat. Genet. 7:22-28) and Happy Mapping (Dear and Cook (1989) Nucleic Acid Res. 17:6795-6807). For these methods, the sequence of a nucleic acid fragment is used to design and produce primer pairs for use in the amplification reaction or in primer extension reactions. The design of such primers is well known to those skilled in the art. In methods employing PCR-based genetic mapping, it may be necessary to identify DNA sequence differences between the parents of the mapping cross in the region corresponding to the instant nucleic acid sequence. This, however, is generally not necessary for mapping methods.

Loss of function mutant phenotypes may be identified for the instant cDNA clones either by targeted gene disruption protocols or by identifying specific mutants for these genes contained in a maize population carrying mutations in all possible genes (Ballinger and Benzer (1989) Proc. Natl. Acad. Sci USA 86:9402-9406; Koes et al. (1995) Proc. Natl. Acad. Sci USA 92:8149-8153; Bensen et al. (1995) Plant Cell 7:75-84). The latter approach may be accomplished in two ways. First, short segments of the instant nucleic acid fragments may be used in polymerase chain reaction protocols in conjunction with a mutation tag sequence primer on DNAs prepared from a population of plants in which Mutator transposons or some other mutation-causing DNA element has been introduced (see Bensen, supra). The amplification of a specific DNA fragment with these primers indicates the insertion of the mutation tag element in or near the plant gene encoding the instant polypeptides. Alternatively, the instant nucleic acid fragment may be used as a hybridization probe against PCR amplification products generated from the mutation population using the mutation tag sequence primer in conjunction with an arbitrary genomic site primer, such as that for a restriction enzyme site-anchored synthetic adaptor. With either method, a plant containing a mutation in the endogenous gene encoding the instant polypeptides can be identified and obtained. This mutant plant can then be used to determine or confirm the natural function of the instant polypeptides disclosed herein.

The present invention is further defined in the following Examples, in which all parts and percentages are by weight and degrees are Celsius, unless otherwise stated. It should be understood that these Examples, while indicating preferred embodiments of the invention, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions.

Zea mays

1

gccctgttgc ggcgacgact ccgtgactcc tagtccctac ccagatcggc gcccgacgca 60

aggccgagca ggcgatggcc gcagcggcgt caatggggca tccttacgcg ccagccgagc 120

tggaccttcc gggtttcgtg ccgctgaagc tgtcccaggt cgagatcctc gtgtcctacc 180

tcggcgcctc cgtcttcgtt ttcctcgccg tctggctcgt ctccggaaga tgtgtcagat 240

tatccaagac cgaccgtctg ctcatgtgct ggtgggcatt cacagggttg acccacataa 300

tgatcgaggg gcccttcgtc ttcactcctg atttcttcaa gaaggagaac cccaatttct 360

ttgatgaagt ttggaaagag tatagtaagg gagactctag gtatgttgct agggacactg 420

caactgttac ggtcgaaggg atcac 445

2

Met Gly His Pro Tyr Ala Pro Ala Glu Leu Asp Leu Pro Gly Phe Val

1 5 10 15

Pro Leu Lys Leu Ser Gln Val Glu Ile Leu Val Ser Tyr Leu Gly Ala

20 25 30

Ser Val Phe Val Phe Leu Ala Val Trp Leu Val Ser Gly Arg Xaa Xaa

35 40 45

Ser Lys Thr Asp Arg Leu Leu Met Cys Trp Trp Ala Phe Thr Gly Leu

50 55 60

Thr His Ile Met Ile Glu Gly Pro Phe Val Phe Thr Pro Asp Phe Phe

65 70 75 80

Lys Lys Glu Asn Pro Asn Phe Phe Asp Glu Val Trp Lys Glu Tyr Ser

85 90 95

Lys Gly Asp Ser Arg Tyr Val Ala Arg Asp Thr Ala Thr Val Thr Val

100 105 110

Glu Gly Ile

115

3

ctgctcgatc cgatccaatc cgctcctctc cagtccagat cggaaggaag ccaggaatgg 60

ggcaccccca cccccaccct tacgcgccgg cggagcttca cctcccgggc ttcgtgcctc 120

tcnaactgtc cnaggccnaa atcctcgtgc cctacctcgn nacntccctc ttcancatca 180

tcgnngtggg gctaatctcg ggaaaaat 208

4

His Pro Tyr Ala Pro Ala Glu Leu His Leu Pro Gly Phe Val Pro Leu

1 5 10 15

Xaa Leu Ser Xaa Ala Xaa Ile Leu Val Pro Tyr Leu Xaa Thr Ser Leu

20 25 30

Phe Xaa Ile Ile Xaa Val Gly Leu Ile Ser Gly Lys

35 40

5

gtccaactca gtagtcaaca ctaggcactc tctattccca ttcattgcag aagaagatgg 60

aggctcaccc ctacgtccca cgcgatttgc acttacccgg ctacgctccc tgcttccttt 120

ccatgtccaa cattctttcc gtcttcgcct cctcctcatt gctcatcgtc actctcgtct 180

ggatcttctc tggacgcttt aagaaaacca aagttgatag agtgctgatg tgctggtggg 240

ccttcacagg tctcacacac attattcttg agggttattt tgttttctct cctgagtttt 300

tcaaggataa aactggcttc tacctggctg aagtttggaa ggaatatagc aaaggggatt 360

caaggtatgc aggaagggat gcaggggtag ttactgttga aggaataaca gcggttttgg 420

agggtccagc tagccttcta gcagtatacg ctatagctac tgggaagtca tatagctaca 480

tacttcagtt tgcatttctt tgggccagct atacggaact gctgtttatt agataacagc 540

aanccttggg anggtganaa ttttccacaa accnngttta caataagcaa actacattgg 600

ggcnantg 608

6

155

Glycine max

6

Ala His Pro Tyr Val Pro Arg Asp Leu His Leu Pro Gly Tyr Ala Pro

1 5 10 15

Cys Phe Leu Ser Met Ser Asn Ile Leu Ser Val Phe Ala Ser Ser Ser

20 25 30

Leu Leu Ile Val Thr Leu Val Trp Ile Phe Ser Gly Arg Lys Lys Thr

35 40 45

Lys Val Asp Arg Val Leu Met Cys Trp Trp Ala Phe Thr Gly Leu Thr

50 55 60

His Ile Ile Leu Glu Gly Tyr Phe Val Phe Ser Pro Glu Phe Phe Lys

65 70 75 80

Asp Lys Thr Gly Phe Tyr Leu Ala Glu Val Trp Lys Glu Tyr Ser Lys

85 90 95

Gly Asp Ser Arg Tyr Ala Gly Arg Asp Ala Gly Val Val Thr Val Glu

100 105 110

Gly Ile Thr Ala Val Leu Glu Gly Pro Ala Ser Leu Leu Ala Val Tyr

115 120 125

Ala Ile Ala Thr Gly Lys Ser Tyr Ser Tyr Ile Leu Gln Val Cys Ile

130 135 140

Ser Leu Gly Gln Leu Tyr Gly Thr Ala Val Tyr

145 150 155

7

gccgtctggc tcctctccgg gagatgccgc aggttgtccg ggaccgaccg cctgctcatg 60

tgctggtggg cgttcaccgg cctcacccac atactcatcg aggggccctt cgtctttacc 120

cccgatttct tcaccaagac caaccccaac ttcttcgacg aagtctggaa ggagtacagc 180

aagggtgact ccaggtacgt cgccagggac accgccaccg tcaccgtcga gggaatcacg 240

gccgtgctga aaggccctgc ctcgctgctc gcagtctatg ctatcgcatc gcgcaagtcc 300

tacagccaca tcctccagtt cgccgtatgc ctcggtcagc tctacggatg catcgtctac 360

ttcaccaccg cctacttgga cggcttcaac ttctgggcca gtccgttcta cttctgggca 420

tatttcatcg gcgcaaacag ttcgtggatt gtgataccgt tgctgattgc cacgangagc 480

tggaagaaga tatgtgcggc cgttcatcaa agcgagaagg tcaagacaaa taattctgca 540

acagcatata nggttggtgc aacaagacac actgaacttc ttgnttatca cagaaattgc 600

tccctgtatt gtacccttan aacacattgt cgaacaagta tcngtacaat aacattgtnc 660

cctctgccta aatgtagaca ntttgcacan acgnggatnt taccacttga g 711

8

Arg Arg Leu Ser Gly Thr Asp Arg Leu Leu Met Cys Trp Trp Ala Phe

1 5 10 15

Thr Gly Leu Thr His Ile Leu Ile Glu Gly Pro Phe Val Phe Thr Pro

20 25 30

Asp Phe Phe Thr Lys Thr Asn Pro Asn Phe Phe Asp Glu Val Trp Lys

35 40 45

Glu Tyr Ser Lys Gly Asp Ser Arg Tyr Val Ala Arg Asp Thr Ala Thr

50 55 60

Val Thr Val Glu Gly Ile Thr Ala Val Leu Lys Gly Pro Ala Ser Leu

65 70 75 80

Leu Ala Val Tyr Ala Ile Ala Ser Arg Lys Ser Tyr Ser His Ile Leu

85 90 95

Gln Phe Ala Val Cys Leu Gly Gln Leu Tyr Gly Cys Ile Val Tyr Phe

100 105 110

Thr Thr Ala Tyr Leu Asp Gly Phe Asn Phe Trp Ala Ser Pro Phe Tyr

115 120 125

Phe Trp Ala Tyr Phe Ile Gly Ala Asn Ser Ser Trp Ile Val Ile Pro

130 135 140

Leu Leu Ile Ala Thr Xaa Ser Trp Lys Lys Ile Cys Ala Ala

145 150 155

9

gagcacgaca gcataagatc caacaaaggc aaggaggagt gcttgacttc agaagactac 60

aagaagatgg aatataccca acaagtcatc aacgaggcgc tgagatgcgg caacatcgtc 120

aagttcgtcc accggaaggc gctgaaagac gtcnaatacn aagagtatct gattccatcn 180

ggctggaagg ncctaccggt cntcactgcc gttcatctga acccctcact tcatggagac 240

gcgcanagtt tcagccctgt aggtgggagg gcacaagcca anggacaagc nannggttta 300

cacccttggt ggtggncccg ggntgcccag gtnagagtcc taaanngggc tgctttttcn 360

ccatannttg ccncattata gtngngagtg tggg 394

10

Glu Glu Cys Leu Thr Ser Glu Asp Tyr Lys Lys Met Glu Tyr Thr Gln

1 5 10 15

Gln Val Ile Asn Glu Ala Leu Arg Cys Gly Asn Ile Val Lys Phe Val

20 25 30

His Arg Lys Ala Leu Lys Asp Val Xaa Tyr Xaa Glu Tyr Leu Ile Pro

35 40 45

Ser Gly Trp Lys Xaa Leu Pro Val Xaa Thr Ala Val His Leu Asn Pro

50 55 60

Ser Leu His Gly Asp Ala Xaa Ser Phe Ser Pro Val Gly Gly Arg Ala

65 70 75 80

Gln Ala Xaa Gly

11

gcagaacgag gggaggctgt tcgagtgcag ctacccgcgc agcatcggcg gcatcctggg 60

caagtggtcc atgctggtcc tcgtcgggga cccgcaccgc gagatgcgcg ccatctccct 120

caacttcctc tcctccgtcc gcctccgcgc cgtcctcctc cccgaggtcg agcgccacac 180

cctcctcgtc ctccgcgcct ggnccccttc ctccaccttc tccgctcaag caccaagcca 240

agaagttcac gttcaacctg atggcgaaga acataatgag catggacccg ggggagggag 300

aaacggngcg gctgcggcgg ggagtacatc accttcatna agggggtggt ctccgcgccg 360

ctcaanctgg ccgggacgcc ctactggaan ggtctcaant ccgtgctggc aattcncggg 420

ggtaataana ggnaaattgg nangaagcng gtt 453

12

Gln Asn Glu Gly Arg Leu Phe Glu Cys Ser Tyr Pro Arg Ser Ile Gly

1 5 10 15

Gly Ile Leu Gly Lys Trp Ser Met Leu Val Leu Val Gly Asp Pro His

20 25 30

Arg Glu Met Arg Ala Ile Ser Leu Asn Phe Leu Ser Ser Val Arg Leu

35 40 45

Arg Ala Val Leu Leu Pro Glu Val Glu Arg His Thr Leu Leu Val Leu

50 55 60

Arg Ala Trp Xaa Pro Ser Ser Thr Phe Ser Ala Gln

65 70 75

13

436

Triticum aestivum

13

cttcaaccct tggagatgga agggcaacgc atccggcgtg gcgcagaaca gcaacttcat 60

gccctacggc ggcggcacca ggctctgcgc cgggtcggag ctcgccaagc tcgagatggc 120

catcttcctg caccacctgg tgctcaactt ccggtgggag ctcgccgagc cggaccaggc 180

gttcgtctac ccgttcgtcg acttccccaa gggcctgccc atcagggtcc ataggattgc 240

acaggaggaa gaaggagaag agtaaagcgt tttgaccgtg gacatatatg atcggtgctt 300

cagtctagcg tctaggggag aagtatacag aggaaatgta cacatgtccg tccttgtttt 360

ctttcccttt ggggtttgtg ttatgtagat gggataaaca aagatgctaa gggtattacc 420

ataaagaaga aatgtt 436

14

72

Triticum aestivum

14

Gly Asn Ala Ser Gly Val Ala Gln Asn Ser Asn Phe Met Pro Tyr Gly

1 5 10 15

Gly Gly Thr Arg Leu Cys Ala Gly Ser Glu Leu Ala Lys Leu Glu Met

20 25 30

Ala Ile Phe Leu His His Leu Val Leu Asn Phe Arg Trp Glu Leu Ala

35 40 45

Glu Pro Asp Gln Ala Phe Val Tyr Pro Phe Val Asp Phe Pro Lys Gly

50 55 60

Leu Pro Ile Arg Val His Arg Ile

65 70

15

caaagatctg aataataatg ggttcgctcc gtgatggcaa gaccaacagc agcagtggcc 60

ggcggtggtg cgcggtgacc ggcggccggg gcttcatggc gaggcacctg gtggccgcgc 120

tgctgcgctc cggcgagtgg cttgtgcggg tcaccgacct cgccccggat gtcgtgctcg 180

gcctcggcga caccgaggac gtcctcgatg acgccctccg tgatggccgc gccgtctatg 240

cctcagcgga tgtctgcaac ctagaccagc ttattcaact tttgaagggg ttgaggttgt 300

tttcacacag ctgctcggat caancaagaa cgaccagcaa cttcactata aggtaacgtt 360

gaggggnaaa gacgtggtta tcgtcatatt 390

16

82

Zea mays

16

Arg Trp Cys Ala Val Thr Gly Gly Arg Gly Phe Met Ala Arg His Leu

1 5 10 15

Val Ala Ala Leu Leu Arg Ser Gly Glu Trp Leu Val Arg Val Thr Asp

20 25 30

Leu Ala Pro Asp Val Val Leu Gly Leu Gly Asp Thr Glu Asp Val Leu

35 40 45

Asp Asp Ala Leu Arg Asp Gly Arg Ala Val Tyr Ala Ser Ala Asp Val

50 55 60

Cys Asn Leu Asp Gln Leu Ile Gln Leu Leu Lys Gly Leu Arg Leu Phe

65 70 75 80

Ser His

17

gttggaatga ggtgagcgtt gtgtttgagt tancttgact atggaagcaa aagataagtg 60

gtgcgtggtg ancggaggtc gcggcttcgc tgctcggcat ttggtggaaa tgctaattcg 120

tcacaaggag tactgcgttc gcatcgcnga tttggaagtc ancattgttc tcgagcccgc 180

cgagcagtta ggccttctcg gccaggccct gcactctggc cgagcccaat atgtctccct 240

cgatcttcgc aacaaggccc aagttctaaa agcgttggag ggagttgagg tggtgttcca 300

catggtctgc tccaaactct tccattaaca agctaccagc ttcancattc cgtcaatgtg 360

caagggacta ataatgtcat cgatgcttgc gtggagctgn cacgtgaagc gtcncgntta 420

cactaagctg tctcgtttac aacaagctct cccaacgtct tcctccgacn atggttcaag 480

ggaattcana atgggaaacn anaanaatgc cttatgcnca ttcgcctaaa tgatcaa 537

18

Asp Lys Trp Cys Val Val Xaa Gly Gly Arg Gly Phe Ala Ala Arg His

1 5 10 15

Leu Val Glu Met Leu Ile Arg His Lys Glu Tyr Cys Val Arg Ile Ala

20 25 30

Asp Leu Glu Val Xaa Ile Val Leu Glu Pro Ala Glu Gln Leu Gly Leu

35 40 45

Leu Gly Gln Ala Leu His Ser Gly Arg Ala Gln Tyr Val Ser Leu Asp

50 55 60

Leu Arg Asn Lys Ala Gln Val Leu Lys Ala Leu Glu Gly Val Glu Val

65 70 75 80

Val Phe His Met

19

ctggtgccga attcggcacg aggccgcgcc gcctacttct ctgtcgacgt ctgcgaatta 60

gcccagctta caaaagcttt ggaaggggta gatactgttt tccacactgc tgcggcggat 120

cataccaaca acaacttcca acttcattac aaggttaatg tcgagggtac aaggaatgtc 180

atcgaggctt gtaacacatg caaggttaaa acactcatat atactagttc cagtggagtt 240

gtattcgatg gagttcatgg cctctttggc gtagacgaat ctacaccgta tccagataag 300

tttcccgacg catacacaga gacaaaggca agaagcagaa aagatggtga taaagtccaa 360

cggaagaaat gagcttctca cttgctgtat acgtcctggc agcatttttg ggtcctggag 420

acacgatagt gccaatttta gtatcttatg gaggaatgat gatcantgtt ggtgatggca 480

agaantgtga tgattttgta natgttgaag aatgtaccca aggtc 525

20

130

Triticum aestivum

20

Gly Arg Ala Ala Tyr Phe Ser Val Asp Val Cys Glu Leu Ala Gln Leu

1 5 10 15

Thr Lys Ala Leu Glu Gly Val Asp Thr Val Phe His Thr Ala Ala Ala

20 25 30

Asp His Thr Asn Asn Asn Phe Gln Leu His Tyr Lys Val Asn Val Glu

35 40 45

Gly Thr Arg Asn Val Ile Glu Ala Cys Asn Thr Cys Lys Val Lys Thr

50 55 60

Leu Ile Tyr Thr Ser Ser Ser Gly Val Val Phe Asp Gly Val His Gly

65 70 75 80

Leu Phe Gly Val Asp Glu Ser Thr Pro Tyr Pro Asp Lys Phe Pro Asp

85 90 95

Ala Tyr Thr Glu Thr Arg Gln Glu Ala Glu Lys Met Val Ile Lys Ser

100 105 110

Asn Gly Arg Asn Glu Leu Leu Thr Cys Cys Ile Arg Pro Gly Ser Ile

115 120 125

Phe Gly

130

21

895

Zea mays

21

ccacgcgtcc gccctgttgc ggcgacgact ccgtgactcc tagtccctac ccagatcggc 60

gcccgacgca aggccgagca ggcgatggcc gcagcggcgt caatggggca tccttacgcg 120

ccagccgagc tggaccttcc gggtttcgtg ccgctgaagc tgtcccaggt cgagatcctc 180

gtgtcctacc tcggcgcctc cgtcttcgtt ttcctcgccg tctggctcgt ctccggaaga 240

tgtgtcagat tatccaagac cgaccgtctg ctcatgtgct ggtgggcatt cacagggttg 300

acccacataa tgatcgaggg gcccttcgtc ttcactcctg atttcttcaa gaaggagaac 360

cccaatttct ttgatgaagt ttggaaagag tatagtaagg gagactctag gtatgttgct 420

agggacactg caactgttac ggtcgaaggg atcaccgctg tattggaagg ccctgcatca 480

ctacttgctg tctacgctat tgcatccagg aagtccttca gccacattct ccagttcgcc 540

gtctgtctgg gccagctcta cggatgcctg gtttacttca tcaccgcgta cttggacagc 600

ttcaacttct gggtcggccc gttctacttc tgggcgtatt tcattggcgc aaacagcttc 660

tggatctgga taccgatgct catcgccata aggtcctgga agaaaacttg cgccgcgttt 720

caagctgaga aggtgaagaa gaccaaataa aagctgtctt tagctaccgt tttattggag 780

accgtcattt tgatggctta aattcttgtg ttggttggta ctggaaactg catgatgtac 840

tctttgctca aacaaatgaa gaccggttta caaattcaaa aaaaaaaaaa aaaag 895

22

221

Zea mays

22

Met Ala Ala Ala Ala Ser Met Gly His Pro Tyr Ala Pro Ala Glu Leu

1 5 10 15

Asp Leu Pro Gly Phe Val Pro Leu Lys Leu Ser Gln Val Glu Ile Leu

20 25 30

Val Ser Tyr Leu Gly Ala Ser Val Phe Val Phe Leu Ala Val Trp Leu

35 40 45

Val Ser Gly Arg Cys Val Arg Leu Ser Lys Thr Asp Arg Leu Leu Met

50 55 60

Cys Trp Trp Ala Phe Thr Gly Leu Thr His Ile Met Ile Glu Gly Pro

65 70 75 80

Phe Val Phe Thr Pro Asp Phe Phe Lys Lys Glu Asn Pro Asn Phe Phe

85 90 95

Asp Glu Val Trp Lys Glu Tyr Ser Lys Gly Asp Ser Arg Tyr Val Ala

100 105 110

Arg Asp Thr Ala Thr Val Thr Val Glu Gly Ile Thr Ala Val Leu Glu

115 120 125

Gly Pro Ala Ser Leu Leu Ala Val Tyr Ala Ile Ala Ser Arg Lys Ser

130 135 140

Phe Ser His Ile Leu Gln Phe Ala Val Cys Leu Gly Gln Leu Tyr Gly

145 150 155 160

Cys Leu Val Tyr Phe Ile Thr Ala Tyr Leu Asp Ser Phe Asn Phe Trp

165 170 175

Val Gly Pro Phe Tyr Phe Trp Ala Tyr Phe Ile Gly Ala Asn Ser Phe

180 185 190

Trp Ile Trp Ile Pro Met Leu Ile Ala Ile Arg Ser Trp Lys Lys Thr

195 200 205

Cys Ala Ala Phe Gln Ala Glu Lys Val Lys Lys Thr Lys

210 215 220

23

1039

Oryza sativa

23

ctgctcgatc cgatccaatc cgctcctctc cagtccagat cggaaggaag ccaggaatgg 60

ggcaccccca cccccaccct tacgcgccgg cggagcttca cctcccgggc ttcgtgcctc 120

tccaactgtc ccaggcccaa atcctcgtgc cctacctcgc cacctccctc ttcctcctcc 180

tcgccgtctg gctcatctcc gggagatgca gtcgtaggct ttccgacacc gaccgctggc 240

tcatgtgctg gtgggccttc accggcctca cccacattat catcgaggga acctttgtct 300

ttgctcctaa tttcttctcc aaccaaaacc cttcttactt cgatgaagtt tggaaagagt 360

acagcaaagg tgactccaga tatgtcgcca gagaccctgc tactgttaca gttgaaggaa 420

ttacagctgt cttggaaggc cctgcttcac tccttgctgt ctatgccatc gcatcgggca 480

agtcctacag ccatatcctc cagttcactg tctgtcttgg tcagctctat ggatgcctgg 540

tgtactttat tacagcctac ttggatggct tcaacttctg gactagcccg ttctacttct 600

gggcttattt cattggtgca aacagctcgt gggttgttat accaactatg atcgccataa 660

ggagctggaa gaagatttgt gcagcatttc aaggtgaaaa ggtgaagact aaataggcaa 720

agggtgcacc ttagtcacag tttggttgga aattaataga catttgtgct atgaacaaca 780

tcattgttcc tgcaattgaa acccttgtgt tactgaaaaa ttgcattgta cccttggagg 840

gaacatcagt gttaattgga tgtgacttta tgcagctatt atgtattgct gcaaaagaaa 900

ttgggttttg tttatcctct atcattcaag ctcgctcttc tttacccttg aagaaataaa 960

agggcaggga gttgccattt tgaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1020

aaaaaaaaaa aaaaaaaaa 1039

24

219

Oryza sativa

24

Met Gly His Pro His Pro His Pro Tyr Ala Pro Ala Glu Leu His Leu

1 5 10 15

Pro Gly Phe Val Pro Leu Gln Leu Ser Gln Ala Gln Ile Leu Val Pro

20 25 30

Tyr Leu Ala Thr Ser Leu Phe Leu Leu Leu Ala Val Trp Leu Ile Ser

35 40 45

Gly Arg Cys Ser Arg Arg Leu Ser Asp Thr Asp Arg Trp Leu Met Cys

50 55 60

Trp Trp Ala Phe Thr Gly Leu Thr His Ile Ile Ile Glu Gly Thr Phe

65 70 75 80

Val Phe Ala Pro Asn Phe Phe Ser Asn Gln Asn Pro Ser Tyr Phe Asp

85 90 95

Glu Val Trp Lys Glu Tyr Ser Lys Gly Asp Ser Arg Tyr Val Ala Arg

100 105 110

Asp Pro Ala Thr Val Thr Val Glu Gly Ile Thr Ala Val Leu Glu Gly

115 120 125

Pro Ala Ser Leu Leu Ala Val Tyr Ala Ile Ala Ser Gly Lys Ser Tyr

130 135 140

Ser His Ile Leu Gln Phe Thr Val Cys Leu Gly Gln Leu Tyr Gly Cys

145 150 155 160

Leu Val Tyr Phe Ile Thr Ala Tyr Leu Asp Gly Phe Asn Phe Trp Thr

165 170 175

Ser Pro Phe Tyr Phe Trp Ala Tyr Phe Ile Gly Ala Asn Ser Ser Trp

180 185 190

Val Val Ile Pro Thr Met Ile Ala Ile Arg Ser Trp Lys Lys Ile Cys

195 200 205

Ala Ala Phe Gln Gly Glu Lys Val Lys Thr Lys

210 215

25

993

Glycine max

25

gcacgaggtc caactcagta gtcaacacta ggcactctct attcccattc attgcagaag 60

aagatggagg ctcaccccta cgtcccacgc gatttgcact tacccggcta cgctccctgc 120

ttcctttcca tgtccaacat tctttccgtc ttcgcctcct cctcattgct catcgtcact 180

ctcgtctgga tcttctctgg acgctttaag aaaaccaaag ttgatagagt gctgatgtgc 240

tggtgggcct tcacaggtct cacacacatt attcttgagg gttattttgt tttctctcct 300

gagtttttca aggataaaac tggcttctac ctggctgaag tttggaagga atatagcaaa 360

ggggattcaa ggtatgcagg aagggatgca ggggtagtta ctgttgaagg aataacagcg 420

gttttggagg gtccagctag ccttctagca gtatacgcta tagctactgg gaagtcatat 480

agctacatac ttcagtttgc catttctttg ggccagctat acggaactgc tgtttattat 540

ataacagcaa tcttggaagg tgataatttt tctacaaact cgttttacta ttatgcatac 600

tacattggag caaatgcctc ctggattgta atacccttga tcattgccat ccgctgctgg 660

aggaagatct gtgcagcatt tcgagttcaa ggtggccaga caaaaaagcc taaagttcgt 720

tgaaggaaac attttcaggt tattggtctt agagatcctg agctaataat gatgttttgg 780

ggtagttgag ataggagaga ttgttgtaag aattgtgaca ttgattctct taaactgcat 840

atctcaggga gaaaaaaaaa atcccagtgt catttgagaa ggtgatgttt ttaattttaa 900

gttttgaata agtattttcc tatcattgat ttgaaattga atattgaatg ttagctggca 960

gatgctagaa tataaaaaaa aaaaaaaaaa aaa 993

26

219

Glycine max

26

Met Glu Ala His Pro Tyr Val Pro Arg Asp Leu His Leu Pro Gly Tyr

1 5 10 15

Ala Pro Cys Phe Leu Ser Met Ser Asn Ile Leu Ser Val Phe Ala Ser

20 25 30

Ser Ser Leu Leu Ile Val Thr Leu Val Trp Ile Phe Ser Gly Arg Phe

35 40 45

Lys Lys Thr Lys Val Asp Arg Val Leu Met Cys Trp Trp Ala Phe Thr

50 55 60

Gly Leu Thr His Ile Ile Leu Glu Gly Tyr Phe Val Phe Ser Pro Glu

65 70 75 80

Phe Phe Lys Asp Lys Thr Gly Phe Tyr Leu Ala Glu Val Trp Lys Glu

85 90 95

Tyr Ser Lys Gly Asp Ser Arg Tyr Ala Gly Arg Asp Ala Gly Val Val

100 105 110

Thr Val Glu Gly Ile Thr Ala Val Leu Glu Gly Pro Ala Ser Leu Leu

115 120 125

Ala Val Tyr Ala Ile Ala Thr Gly Lys Ser Tyr Ser Tyr Ile Leu Gln

130 135 140

Phe Ala Ile Ser Leu Gly Gln Leu Tyr Gly Thr Ala Val Tyr Tyr Ile

145 150 155 160

Thr Ala Ile Leu Glu Gly Asp Asn Phe Ser Thr Asn Ser Phe Tyr Tyr

165 170 175

Tyr Ala Tyr Tyr Ile Gly Ala Asn Ala Ser Trp Ile Val Ile Pro Leu

180 185 190

Ile Ile Ala Ile Arg Cys Trp Arg Lys Ile Cys Ala Ala Phe Arg Val

195 200 205

Gln Gly Gly Gln Thr Lys Lys Pro Lys Val Arg

210 215

27

889

Triticum aestivum

27

gttgactagc gctgatttgc tccctctcca gctccggctc ccagatcgag acggcggcgg 60

cggcggcgat gggcgcccac ccttacgtgc cggcgagcct ggacctcccg ggctacgtgc 120

cgctgcgcct cacccagctc gagatcctcg gggcctacct cggcacctcc ctcttcgtcc 180

tcgtcgccgt ctggctcctc tccgggagat gccgcaggtt gtccgggacc gaccgcctgc 240

tcatgtgctg gtgggcgttc accggcctca cccacatact catcgagggg cccttcgtct 300

ttacccccga tttcttcacc aagaccaacc ccaacttctt cgacgaagtc tggaaggagt 360

acagcaaggg tgactccagg tacgtcgcca gggacaccgc caccgtcacc gtcgagggaa 420

tcacggccgt gctgaaaggc cctgcctcgc tgctcgcagt ctatgctatc gcatcgcgca 480

agtcctacag ccacatcctc cagttcgccg tatgcctcgg tcagctctac ggatgcatcg 540

tctacttcac caccgcctac ttggacggct tcaacttctg ggccagtccg ttctacttct 600

gggcatattt catcggcgca aacagttcgt ggattgtgat accgttgctg atcgccacga 660

ggagctggaa gaagatatgt gcggccgttc atcaaagcga gaaggtcaag accaaataat 720

tcctgcaaca ggcatatagg gtttggctgc aaccaagaca cactgaactt cttgttttat 780

caccagaaat ttgcttcctt gtattgtacc ctttacaaac tacattgtct gaaacaagta 840

tgcggtacta catacacatt gtaccccctc tgtcctaaaa aaaaaaaaa 889

28

216

Triticum aestivum

28

Met Gly Ala His Pro Tyr Val Pro Ala Ser Leu Asp Leu Pro Gly Tyr

1 5 10 15

Val Pro Leu Arg Leu Thr Gln Leu Glu Ile Leu Gly Ala Tyr Leu Gly

20 25 30

Thr Ser Leu Phe Val Leu Val Ala Val Trp Leu Leu Ser Gly Arg Cys

35 40 45

Arg Arg Leu Ser Gly Thr Asp Arg Leu Leu Met Cys Trp Trp Ala Phe

50 55 60

Thr Gly Leu Thr His Ile Leu Ile Glu Gly Pro Phe Val Phe Thr Pro

65 70 75 80

Asp Phe Phe Thr Lys Thr Asn Pro Asn Phe Phe Asp Glu Val Trp Lys

85 90 95

Glu Tyr Ser Lys Gly Asp Ser Arg Tyr Val Ala Arg Asp Thr Ala Thr

100 105 110

Val Thr Val Glu Gly Ile Thr Ala Val Leu Lys Gly Pro Ala Ser Leu

115 120 125

Leu Ala Val Tyr Ala Ile Ala Ser Arg Lys Ser Tyr Ser His Ile Leu

130 135 140

Gln Phe Ala Val Cys Leu Gly Gln Leu Tyr Gly Cys Ile Val Tyr Phe

145 150 155 160

Thr Thr Ala Tyr Leu Asp Gly Phe Asn Phe Trp Ala Ser Pro Phe Tyr

165 170 175

Phe Trp Ala Tyr Phe Ile Gly Ala Asn Ser Ser Trp Ile Val Ile Pro

180 185 190

Leu Leu Ile Ala Thr Arg Ser Trp Lys Lys Ile Cys Ala Ala Val His

195 200 205

Gln Ser Glu Lys Val Lys Thr Lys

210 215

29

804

Zea mays

29

gcacgaggag cacgacagca taagatccaa caaaggcaag gaggagtgct tgacttcaga 60

agactacaag aagatggaat atacccaaca agtcatcaac gaggcgctga gatgcggcaa 120

catcgtcaag ttcgtccacc ggaaggcgct gaaagacgtc aaatacaaag agtatctgat 180

tccatctggc tggaaggtcc taccggtctt cactgccgtt catctgaacc cctcacttca 240

tggagacgcg cagcagtttc agccctgtag gtgggagggc acaagccaag ggacaagcaa 300

gaggtttaca ccgttcggtg gtggcccccg gctctgccca ggatcagagc tcgctaaagt 360

ggagactgct ttcttcctcc atcaccttgt cctcaattat agatggagaa ttgatggcga 420

tgacattcca atggcatacc cgtatgtgga gtttcagaga ggtctgccaa tagaaatcga 480

gccaacgtcc cctgaatctt gactgtcctg gagctacagc catcagttat cacaccagag 540

agaaaagggg aaggtgcatg gagtatacat gaatggtcag tgacagatct cacaagtgaa 600

ggaacactga gggcgcgtgc tagtagctag catatgaggc agctgagact gtaatttaat 660

gtacatggtg tagatatatt ttgtccatgg caattgcttg aagtggctga ttcacttcac 720

cctgtaaaac attctccagt ggtttcaact gctatcctat aaaaaagaag ggcccctgtg 780

tttagtaaaa aaaaaaaaaa aaaa 804

30

164

Zea mays

30

Glu His Asp Ser Ile Arg Ser Asn Lys Gly Lys Glu Glu Cys Leu Thr

1 5 10 15

Ser Glu Asp Tyr Lys Lys Met Glu Tyr Thr Gln Gln Val Ile Asn Glu

20 25 30

Ala Leu Arg Cys Gly Asn Ile Val Lys Phe Val His Arg Lys Ala Leu

35 40 45

Lys Asp Val Lys Tyr Lys Glu Tyr Leu Ile Pro Ser Gly Trp Lys Val

50 55 60

Leu Pro Val Phe Thr Ala Val His Leu Asn Pro Ser Leu His Gly Asp

65 70 75 80

Ala Gln Gln Phe Gln Pro Cys Arg Trp Glu Gly Thr Ser Gln Gly Thr

85 90 95

Ser Lys Arg Phe Thr Pro Phe Gly Gly Gly Pro Arg Leu Cys Pro Gly

100 105 110

Ser Glu Leu Ala Lys Val Glu Thr Ala Phe Phe Leu His His Leu Val

115 120 125

Leu Asn Tyr Arg Trp Arg Ile Asp Gly Asp Asp Ile Pro Met Ala Tyr

130 135 140

Pro Tyr Val Glu Phe Gln Arg Gly Leu Pro Ile Glu Ile Glu Pro Thr

145 150 155 160

Ser Pro Glu Ser

31

ggncacgagg ggaagactac aaggaaatgg ttttcacgca ngtggttata aacgagacat 60

tgcggctcgg caacgtggtc aggttcctgc accgngaagt catccgagat gtacactaca 120

atgggtacga cataccgcgg gggtggaaaa tcctgccggt tctagcggcg gtgcacctgg 180

actcgtcgct gtacgaggac cccagccggt tcaacccttg ggagatngga agctggcaga 240

gcaacaacgc gccaagcagc ttcatgccgt acggcggcgg gccgcggctg tgcgccgggt 300

cggagctggc caagctggag atggccatct tcctgcacca cctggtgctc aacttccggt 360

gggagctggc ggacgaccag gcctncgncn accctttcgt cgacttcccc aagggcctcc 420

cgatcagggt ccagcgggtc gccgacgacc aaggccatcg tagcgttttg accgagagca 480

caagagntga nagggaggag cagttcaatt tttcgttgca ttttagggct gtgtctcgtg 540

ttatgagatt gtantantan attatacata ctaaacaaga tagaagcact atgacagagc 600

aaagagagat gtcaaaatgt ttgggngata aatngactca taggtttaan agccgtcatg 660

ccantaanat cta 673

32

Xaa Arg Gly Glu Asp Tyr Lys Glu Met Val Phe Thr Xaa Val Val Ile

1 5 10 15

Asn Glu Thr Leu Arg Leu Gly Asn Val Val Arg Phe Leu His Arg Glu

20 25 30

Val Ile Arg Asp Val His Tyr Asn Gly Tyr Asp Ile Pro Arg Gly Trp

35 40 45

Lys Ile Leu Pro Val Leu Ala Ala Val His Leu Asp Ser Ser Leu Tyr

50 55 60

Glu Asp Pro Ser Arg Phe Asn Pro Trp Glu Xaa Gly Ser Trp Gln Ser

65 70 75 80

Asn Asn Ala Pro Ser Ser Phe Met Pro Tyr Gly Gly Gly Pro Arg Leu

85 90 95

Cys Ala Gly Ser Glu Leu Ala Lys Leu Glu Met Ala Ile Phe Leu His

100 105 110

His Leu Val Leu Asn Phe Arg Trp Glu Leu Ala Asp Asp Gln Ala Xaa

115 120 125

Xaa Xaa Pro Phe Val Asp Phe Pro Lys Gly Leu Pro Ile Arg Val Gln

130 135 140

Arg

145

33

gagacaaagg ctaagggggg cgtccaaatt gagctgggaa gactacaagg aaatggtttt 60

cacgcagtgt gttataaacg agacattgcg gctcggcaac gtggtcaggt tcctgcaccg 120

gaaggtcatc cgagatgtac actacaatgg gtacgacata ccgcgggggt ggaaaatcct 180

gccggttcta gcggcggtgc acctggactc gtcgctgtac gaggacccca gccggttcaa 240

cccttggaga tggaagagca acaacgcgcc aagcagcttc atgccgtacg gcggcgggcc 300

gcggctgtgc gccgggtcgg agctggcaag ctggagatgg catcttcctg cacacctggt 360

gctcaacttc cggtgggact ggcggancgg acaagccttc gtcaaccttt cgtcnattcc 420

caaggctcca taggtcaacg gtcccgacac aagcacgtac gtttgccgag acacagagtg 480

aaggagagna ntaatttctt catttgaggg tgtggcctgt ttagtaatgg atatatattc 540

tcanacagat nacacttnaa gncaagngng tgcaaatttg ggaaaatttc cncaggttan 600

agcgcagcca ntatan 616

34

126

Zea mays

34

Arg Gln Arg Leu Arg Gly Ala Ser Lys Leu Ser Trp Glu Asp Tyr Lys

1 5 10 15

Glu Met Val Phe Thr Gln Cys Val Ile Asn Glu Thr Leu Arg Leu Gly

20 25 30

Asn Val Val Arg Phe Leu His Arg Lys Val Ile Arg Asp Val His Tyr

35 40 45

Asn Gly Tyr Asp Ile Pro Arg Gly Trp Lys Ile Leu Pro Val Leu Ala

50 55 60

Ala Val His Leu Asp Ser Ser Leu Tyr Glu Asp Pro Ser Arg Phe Asn

65 70 75 80

Pro Trp Arg Trp Lys Ser Asn Asn Ala Pro Ser Ser Phe Met Pro Tyr

85 90 95

Gly Gly Gly Pro Arg Leu Cys Ala Gly Ser Glu Leu Ala Ser Trp Arg

100 105 110

Trp His Leu Pro Ala His Leu Val Leu Asn Phe Arg Trp Asp

115 120 125

35

1181

Oryza sativa

35

gcacgaggca gaacgagggg aggctgttcg agtgcagcta cccgcgcagc atcggcggca 60

tcctgggcaa gtggtccatg ctggtcctcg tcggggaccc gcaccgcgag atgcgcgcca 120

tctccctcaa cttcctctcc tccgtccgcc tccgcgccgt cctcctcccc gaggtcgagc 180

gccacaccct cctcgtcctc cgcgcctggc ccccttcctc caccttctcc gctcagcacc 240

aagccaagaa gttcacgttc aacctgatgg cgaagaacat aatgagcatg gacccggggg 300

aggaagagac ggagcggctg cggcgggagt acatcacctt catgaagggc gtggtctccg 360

cgccgctcaa cctgcccggg acgccctact ggaaggctct caagtcgcgt gctgccattc 420

tcggagtaat agagaggaaa atggaagagc gggttgagaa gctgagcaag gaggatgcaa 480

gcgtagagca agacgatctt ctcggatggg ctctgaaaca atctaacctt tcaaaagagc 540

aaatcctgga cctcttgctg agcttgctct tcgccgggca cgagacgtcg tccatggcgc 600

tcgccctcgc catcttcttc cttgaaggct gccccaaggc tgtccaagaa ctgaggaagg 660

agcatcttgg gattgcaagg agacaaaggc taagagggga gtgcaaattg agctgggaag 720

actacaaaga gatggttttc acgcaatgtg tcataaacga gacgttgcgg ctaggaaacg 780

tggtcaggtt cctgcaccgg aaggtcatca aggacgtgca ctacaagggt tatgacattc 840

caagcggatg gaagatcctg ccggtgttag ccgcggtgca tctggactcg tccctgtacg 900

aggaccccca gcgcttcaat ccctggagat ggaagagtag cggatcatcc ggcggcttgg 960

ctcagagcag cagcttcatg ccgtacggcg gcgggacgcg gctgtgcgcc gggtcggagc 1020

tcgcgaagct ggagatggcc gtgttcttgc accacctggt gctcaacttc aggtgggagc 1080

tcgccgagcc ggaccaagcc ttcgtcttcc ccttcgtcga cttccccaag ggccttccca 1140

ttagggttca tagaattgca caggatgatg agcaggagta a 1181

36

392

Oryza sativa

36

Thr Arg Gln Asn Glu Gly Arg Leu Phe Glu Cys Ser Tyr Pro Arg Ser

1 5 10 15

Ile Gly Gly Ile Leu Gly Lys Trp Ser Met Leu Val Leu Val Gly Asp

20 25 30

Pro His Arg Glu Met Arg Ala Ile Ser Leu Asn Phe Leu Ser Ser Val

35 40 45

Arg Leu Arg Ala Val Leu Leu Pro Glu Val Glu Arg His Thr Leu Leu

50 55 60

Val Leu Arg Ala Trp Pro Pro Ser Ser Thr Phe Ser Ala Gln His Gln

65 70 75 80

Ala Lys Lys Phe Thr Phe Asn Leu Met Ala Lys Asn Ile Met Ser Met

85 90 95

Asp Pro Gly Glu Glu Glu Thr Glu Arg Leu Arg Arg Glu Tyr Ile Thr

100 105 110

Phe Met Lys Gly Val Val Ser Ala Pro Leu Asn Leu Pro Gly Thr Pro

115 120 125

Tyr Trp Lys Ala Leu Lys Ser Arg Ala Ala Ile Leu Gly Val Ile Glu

130 135 140

Arg Lys Met Glu Glu Arg Val Glu Lys Leu Ser Lys Glu Asp Ala Ser

145 150 155 160

Val Glu Gln Asp Asp Leu Leu Gly Trp Ala Leu Lys Gln Ser Asn Leu

165 170 175

Ser Lys Glu Gln Ile Leu Asp Leu Leu Leu Ser Leu Leu Phe Ala Gly

180 185 190

His Glu Thr Ser Ser Met Ala Leu Ala Leu Ala Ile Phe Phe Leu Glu

195 200 205

Gly Cys Pro Lys Ala Val Gln Glu Leu Arg Lys Glu His Leu Gly Ile

210 215 220

Ala Arg Arg Gln Arg Leu Arg Gly Glu Cys Lys Leu Ser Trp Glu Asp

225 230 235 240

Tyr Lys Glu Met Val Phe Thr Gln Cys Val Ile Asn Glu Thr Leu Arg

245 250 255

Leu Gly Asn Val Val Arg Phe Leu His Arg Lys Val Ile Lys Asp Val

260 265 270

His Tyr Lys Gly Tyr Asp Ile Pro Ser Gly Trp Lys Ile Leu Pro Val

275 280 285

Leu Ala Ala Val His Leu Asp Ser Ser Leu Tyr Glu Asp Pro Gln Arg

290 295 300

Phe Asn Pro Trp Arg Trp Lys Ser Ser Gly Ser Ser Gly Gly Leu Ala

305 310 315 320

Gln Ser Ser Ser Phe Met Pro Tyr Gly Gly Gly Thr Arg Leu Cys Ala

325 330 335

Gly Ser Glu Leu Ala Lys Leu Glu Met Ala Val Phe Leu His His Leu

340 345 350

Val Leu Asn Phe Arg Trp Glu Leu Ala Glu Pro Asp Gln Ala Phe Val

355 360 365

Phe Pro Phe Val Asp Phe Pro Lys Gly Leu Pro Ile Arg Val His Arg

370 375 380

Ile Ala Gln Asp Asp Glu Gln Glu

385 390

37

gaggtaccaa ccactctttt ttacttatgg catctttcat cttcacacct gtactctttc 60

ttcttatcat ctccgccgtc cttctcttcc tccaccgccg atcccgctgc cggcgcttcc 120

gcctaccgcc tggtacactc gggctcccct tcgtcggcga aaccttacag ctcatatcag 180

cttacaagag tgacaacccg gaacccttca tggaccagcg cgtgaaangg tacggtccaa 240

tcttcaccac ccatgtgttc ggcgaaccca ccgtgttctc gaccgaccca gaaacgaacc 300

ggttcatttt gctcaacgaa ggnaagctct tcgaatgcag ctaccccggt tcgatatcga 360

acctccttgg gaaacactct ctgctcctca tgaaaggttt cttcacaaga gaatgatcct 420

actatgagnt gcaactctcc atcataagga cattattggn gcatgacgct atcggatact 480

tggattcngg tcgccgggnc tttatgagan caagaantan gttngtgcag taanantgtg 540

anttgncagg gnatggctng nctaggagag acggctg 577

38

Met Ala Ser Phe Ile Phe Thr Pro Val Leu Phe Leu Leu Ile Ile Ser

1 5 10 15

Ala Val Leu Leu Phe Leu His Arg Arg Ser Arg Cys Arg Arg Phe Arg

20 25 30

Leu Pro Pro Gly Thr Leu Gly Leu Pro Phe Val Gly Glu Thr Leu Gln

35 40 45

Leu Ile Ser Ala Tyr Lys Ser Asp Asn Pro Glu Pro Phe Met Asp Gln

50 55 60

Arg Val Lys Xaa Tyr Gly Pro Ile Phe Thr Thr His Val Phe Gly Glu

65 70 75 80

Pro Thr Val Phe Ser Thr Asp Pro Glu Thr Asn Arg Phe Ile Leu Leu

85 90 95

Asn Glu Gly Lys Leu Phe Glu Cys Ser Tyr Pro Gly Ser Ile Ser Asn

100 105 110

Leu Leu Gly Lys His Ser Leu Leu Leu Met Lys Gly

115 120

39

600

Triticum aestivum

39

gcacgagctt caacccttgg agatggaagg gcaacgcatc cggcgtggcg cagaacagca 60

acttcatgcc ctacggcggc ggcaccaggc tctgcgccgg gtcggagctc gccaagctcg 120

agatggccat cttcctgcac cacctggtgc tcaacttccg gtgggagctc gccgagccgg 180

accaggcgtt cgtctacccg ttcgtcgact tccccaaggg cctgcccatc agggtccata 240

ggattgcaca ggaggaagaa ggagaagagt aaagcgtttt gaccgtggac atatatgatc 300

ggtgcttcag tctagcgtct aggggagagt atacagagga aatgtacaca tgtccgtcct 360

tgttttcttt ccctttgggg tttgtgttat gtagatggga taaacaaaga tgctagggta 420

ttaccataag aggaaatgtt ggtaggatca gcaagtagag ttgtaataag gccggcccac 480

agcccactag taagtgattc caaagagtag gcctagcggt cttgctatct tcttgcctcg 540

ttcctccagc caatccatat ttgattctta aaaaaaaggc aattcatatt ttgcctaaaa 600

40

89

Triticum aestivum

40

Thr Ser Phe Asn Pro Trp Arg Trp Lys Gly Asn Ala Ser Gly Val Ala

1 5 10 15

Gln Asn Ser Asn Phe Met Pro Tyr Gly Gly Gly Thr Arg Leu Cys Ala

20 25 30

Gly Ser Glu Leu Ala Lys Leu Glu Met Ala Ile Phe Leu His His Leu

35 40 45

Val Leu Asn Phe Arg Trp Glu Leu Ala Glu Pro Asp Gln Ala Phe Val

50 55 60

Tyr Pro Phe Val Asp Phe Pro Lys Gly Leu Pro Ile Arg Val His Arg

65 70 75 80

Ile Ala Gln Glu Glu Glu Gly Glu Glu

85

41

1426

Zea mays

41

gcacgagcaa agatctgaat aataatgggt tcgctccgtg atggcaagac caacagcagc 60

agtggccggc ggtggtgcgc ggtgaccggc ggccggggct tcatggcgag gcacctggtg 120

gccgcgctgc tgcgctccgg cgagtggctt gtgcgggtca ccgacctcgc cccggatgtc 180

gtgctcggcc tcggcgacac cgaggacgtc ctcgatgacg ccctccgtga tggccgcgcc 240

gtctatgcct cagcggatgt ctgcaaccta gaccagctta ttcaagcttt tgaaggggtt 300

gaggttgttt tccacacagc tgctgcggat ccaagcaaga acgaccagca acttcactat 360

aaggtcaacg ttgaggggac aaagaacgtg gttgatgcgt gcatgatttg caaggtgaaa 420

aggcttatcc acaccagctc tattgctgtt gtgttcgacg gagttaatgg gcttctcgat 480

gcaaatgaat cattgccata cccagacaag tttcctgatg cgtatggaca aacaaaggca 540

gaagcagaaa agatagtcat gaaggctaat ggcattagtg gccttctaac ttgttgcata 600

cgtcctggta gcatttttgg ccctggtgac atagttatac taccaactct ggaccaatgt 660

ggaaaaacac actttgtttt tggtgatggg aagaattgtg atgattttgt atatgttgaa 720

aatgtggtac atggccacat ttgtgctgaa aaaactcttt ctacaatgga aggcgcaaaa 780

accagtggtg gaaaagccta ctttataacc aatacggaac caatgaacat gtgggacttt 840

ctatatctgc ttcaagaaga acttggatac aaaaggttgt tcaagataag aatacctttg 900

attgtcatcc aggcagtaag ctatttggta gagtggggat acaaggttct acaccattat 960

ggaatgtgcc agcctcaagt gctaacacca gcaaggatca agtatctgac agttcataga 1020

acattcagtt gtaacaaagc tgctgaagaa cttggctaca aaccaattgt gacacttatg 1080

gatggtatga agctagcagt caaatcatat attcggttga gaaatcatgc agatttatct 1140

tacaaacata tataaaagat tatcattatg gcataggatg cctcatgctg acgatataca 1200

acagcttttg agaattgtca taagcgaagg tctgccgtga ttatgtttcc aataaataaa 1260

aaattcggtg tttacaactc gcatcaatgt cctgatcaaa aacccagcat gttgtattct 1320

gatttagtag caaagataga ttgtaactcc ttaacaatga tcaggcatgt atttcagata 1380

tgttaattgg aacacaaata atattactaa aaaaaaaaaa aaaaaa 1426

42

376

Zea mays

42

Met Gly Ser Leu Arg Asp Gly Lys Thr Asn Ser Ser Ser Gly Arg Arg

1 5 10 15

Trp Cys Ala Val Thr Gly Gly Arg Gly Phe Met Ala Arg His Leu Val

20 25 30

Ala Ala Leu Leu Arg Ser Gly Glu Trp Leu Val Arg Val Thr Asp Leu

35 40 45

Ala Pro Asp Val Val Leu Gly Leu Gly Asp Thr Glu Asp Val Leu Asp

50 55 60

Asp Ala Leu Arg Asp Gly Arg Ala Val Tyr Ala Ser Ala Asp Val Cys

65 70 75 80

Asn Leu Asp Gln Leu Ile Gln Ala Phe Glu Gly Val Glu Val Val Phe

85 90 95

His Thr Ala Ala Ala Asp Pro Ser Lys Asn Asp Gln Gln Leu His Tyr

100 105 110

Lys Val Asn Val Glu Gly Thr Lys Asn Val Val Asp Ala Cys Met Ile

115 120 125

Cys Lys Val Lys Arg Leu Ile His Thr Ser Ser Ile Ala Val Val Phe

130 135 140

Asp Gly Val Asn Gly Leu Leu Asp Ala Asn Glu Ser Leu Pro Tyr Pro

145 150 155 160

Asp Lys Phe Pro Asp Ala Tyr Gly Gln Thr Lys Ala Glu Ala Glu Lys

165 170 175

Ile Val Met Lys Ala Asn Gly Ile Ser Gly Leu Leu Thr Cys Cys Ile

180 185 190

Arg Pro Gly Ser Ile Phe Gly Pro Gly Asp Ile Val Ile Leu Pro Thr

195 200 205

Leu Asp Gln Cys Gly Lys Thr His Phe Val Phe Gly Asp Gly Lys Asn

210 215 220

Cys Asp Asp Phe Val Tyr Val Glu Asn Val Val His Gly His Ile Cys

225 230 235 240

Ala Glu Lys Thr Leu Ser Thr Met Glu Gly Ala Lys Thr Ser Gly Gly

245 250 255

Lys Ala Tyr Phe Ile Thr Asn Thr Glu Pro Met Asn Met Trp Asp Phe

260 265 270

Leu Tyr Leu Leu Gln Glu Glu Leu Gly Tyr Lys Arg Leu Phe Lys Ile

275 280 285

Arg Ile Pro Leu Ile Val Ile Gln Ala Val Ser Tyr Leu Val Glu Trp

290 295 300

Gly Tyr Lys Val Leu His His Tyr Gly Met Cys Gln Pro Gln Val Leu

305 310 315 320

Thr Pro Ala Arg Ile Lys Tyr Leu Thr Val His Arg Thr Phe Ser Cys

325 330 335

Asn Lys Ala Ala Glu Glu Leu Gly Tyr Lys Pro Ile Val Thr Leu Met

340 345 350

Asp Gly Met Lys Leu Ala Val Lys Ser Tyr Ile Arg Leu Arg Asn His

355 360 365

Ala Asp Leu Ser Tyr Lys His Ile

370 375

43

gcacgaggtt ggaatgaggt gagcgttgtg tttgagttag cttgactatg gaagcaaaag 60

ataagtggtg cgtggtgacc ggaggtcgcg gcttcgctgc tcggcatttg gtggaaatgc 120

taattcgtca caaggagtac tgcgttcgca tcgccgattt ggaagtcagc attgttctcg 180

agcccgccga gcagttaggc cttctcggcc aggccctgca ctctggccga gcccaatatg 240

tctccctcga tcttcgcaac aaggcccaag ttctaaaagc gttggaggga gttgaggtgg 300

tgttccacat ggctgctcca aactcttcca ttaacaacta ccagcttcat cattccgtca 360

atgtgcaagg gacgaataat gtcatcgatg cttgcgtgga gctgaacgtg aagcgtctcg 420

tttacactag ctgtctcgtt tacaccagct ctcccagcgt cttcttcgac gatgttcatg 480

gaattcataa tggaaacgaa acaatgcctt atgcgcattc gcctaatgat cattattcag 540

caacgaaagc cgaggctgag gcattggtta ttaaagctaa tgggactaat gggctcctaa 600

cgtgctgcat acgccctagc agcatttttg ggcctggtga taggctgtcg gtgccttcac 660

tagttgatgc tgccagaaaa ggggaatcta agtttcttat tggtgatggc aataacgttt 720

atgatttcac atatgttgaa aatgtggctc atgcccatat atgtgctgat cgagctctag 780

cttcagaagg accggtttca gaaaaagctg cgggagaggc atatttcata acaaatatgg 840

agcctatgaa attctgggag ttcgtgtcat tggtagtgga aggtcttgga tatgaaaggc 900

caaggataaa gatccccacc tttgttatca tgcccattgc acatttggtg gagtggatat 960

ataagctgct aggcccatat gggatgaagc tgcctcagtt aattccttcg agaataagac 1020

tcatatcttg cagcagaact tttgattgct caaaagcaaa ggatcgcctt ggctatgcac 1080

ccatcgtaac actacaggag ggtctgcgaa ggacaattga atcatacaca cacttgaggg 1140

cggataatga acctaaaact aaaagagaag gtccctcaaa agcttccaaa tatcttggaa 1200

gtggaagagg tgtgaataat caactatatc tgtctaactt ctttgcttgg tgatgaggat 1260

caaacaagga ataaataaat tgaagaagac taatttttta ttaaataaag tttgtaatat 1320

ttagtgagac ataataaaag tgactgttgc ctcactaaaa aaaaaaaaaa aaaaaaaaaa 1380

aaaacacaac aaaaaaaaaa aaaaaanaac aaaaaaaaaa acaacaacna aaaaaaaaac 1440

ccccccgggg gggggggccg ggaccccaaa ttccccccaa aaagtgggct ctttataacc 1500

cccgccccaa tgggcctgtc ttttttaaaa actctcggtg agtggggaaa aacccttggg 1560

ggtaacccaa ctttaaatcc ccctttggaa aaaaatcccc cttttttccc aaaggtgggg 1620

ttataaaaaa aaaaagagcc cccacacttt tttccccttt cca 1663

44

401

Glycine max

44

Met Glu Ala Lys Asp Lys Trp Cys Val Val Thr Gly Gly Arg Gly Phe

1 5 10 15

Ala Ala Arg His Leu Val Glu Met Leu Ile Arg His Lys Glu Tyr Cys

20 25 30

Val Arg Ile Ala Asp Leu Glu Val Ser Ile Val Leu Glu Pro Ala Glu

35 40 45

Gln Leu Gly Leu Leu Gly Gln Ala Leu His Ser Gly Arg Ala Gln Tyr

50 55 60

Val Ser Leu Asp Leu Arg Asn Lys Ala Gln Val Leu Lys Ala Leu Glu

65 70 75 80

Gly Val Glu Val Val Phe His Met Ala Ala Pro Asn Ser Ser Ile Asn

85 90 95

Asn Tyr Gln Leu His His Ser Val Asn Val Gln Gly Thr Asn Asn Val

100 105 110

Ile Asp Ala Cys Val Glu Leu Asn Val Lys Arg Leu Val Tyr Thr Ser

115 120 125

Cys Leu Val Tyr Thr Ser Ser Pro Ser Val Phe Phe Asp Asp Val His

130 135 140

Gly Ile His Asn Gly Asn Glu Thr Met Pro Tyr Ala His Ser Pro Asn

145 150 155 160

Asp His Tyr Ser Ala Thr Lys Ala Glu Ala Glu Ala Leu Val Ile Lys

165 170 175

Ala Asn Gly Thr Asn Gly Leu Leu Thr Cys Cys Ile Arg Pro Ser Ser

180 185 190

Ile Phe Gly Pro Gly Asp Arg Leu Ser Val Pro Ser Leu Val Asp Ala

195 200 205

Ala Arg Lys Gly Glu Ser Lys Phe Leu Ile Gly Asp Gly Asn Asn Val

210 215 220

Tyr Asp Phe Thr Tyr Val Glu Asn Val Ala His Ala His Ile Cys Ala

225 230 235 240

Asp Arg Ala Leu Ala Ser Glu Gly Pro Val Ser Glu Lys Ala Ala Gly

245 250 255

Glu Ala Tyr Phe Ile Thr Asn Met Glu Pro Met Lys Phe Trp Glu Phe

260 265 270

Val Ser Leu Val Val Glu Gly Leu Gly Tyr Glu Arg Pro Arg Ile Lys

275 280 285

Ile Pro Thr Phe Val Ile Met Pro Ile Ala His Leu Val Glu Trp Ile

290 295 300

Tyr Lys Leu Leu Gly Pro Tyr Gly Met Lys Leu Pro Gln Leu Ile Pro

305 310 315 320

Ser Arg Ile Arg Leu Ile Ser Cys Ser Arg Thr Phe Asp Cys Ser Lys

325 330 335

Ala Lys Asp Arg Leu Gly Tyr Ala Pro Ile Val Thr Leu Gln Glu Gly

340 345 350

Leu Arg Arg Thr Ile Glu Ser Tyr Thr His Leu Arg Ala Asp Asn Glu

355 360 365

Pro Lys Thr Lys Arg Glu Gly Pro Ser Lys Ala Ser Lys Tyr Leu Gly

370 375 380

Ser Gly Arg Gly Val Asn Asn Gln Leu Tyr Leu Ser Asn Phe Phe Ala

385 390 395 400

Trp