Maize Cellulose Synthases and Uses Thereof

Abstract

The invention provides isolated cellulose synthase nucleic acids and their encoded proteins. The present invention provides methods and compositions relating to altering cellulose synthase levels in plants. The invention further provides recombinant expression cassettes, host cells, and transgenic plants comprising said nucleic acids.

Description

RELATED APPLICATIONS

[0001] This application is a divisional of co-pending U.S. patent application Ser. No. 13/887,430 filed May 6, 2013 which is a continuation of U.S. patent application Ser. No. 13/459,370 filed Apr. 30, 2012, now issued as U.S. Pat. No. 8,481,682 which is a continuation of U.S. patent application Ser. No. 13/079,071 filed Apr. 4, 2011, now issued as U.S. Pat. No. 8,207,302, which is a divisional of U.S. patent application Ser. No. 12/903,472 filed Oct. 13, 2010, now issued as U.S. Pat. No. 7,982,009, which is a divisional of Ser. No. 12/782,738 filed May 19, 2010, now issued as U.S. Pat. No. 7,838,632, which is a divisional of Ser. No. 12/486,129 filed Jun. 17, 2009, now issued as U.S. Pat. No. 7,851,597, which is a divisional of U.S. patent application Ser. No. 12/277,418 filed Nov. 25, 2008, now issued as U.S. Pat. No. 7,579,443, which is a divisional of U.S. patent application Ser. No. 11/859,968 filed Sep. 24, 2007, now issued as U.S. Pat. No. 7,524,933, which is a divisional of U.S. patent application Ser. No. 10/963,217 filed Oct. 12, 2004, now issued as U.S. Pat. No. 7,307,149, which is a continuation-in-part of U.S. patent application Ser. No. 10/209,059, filed Jul. 31, 2002, now issued as U.S. Pat. No. 6,930,225, which is a continuation-in-part of U.S. patent application Ser. No. 09/550,483, filed Apr. 14, 2000, now abandoned, which is a continuation-in-part of U.S. patent application Ser. No. 09/371,383, filed Aug. 6, 1999, now abandoned, which claims benefit of U.S. Provisional Patent Application Ser. No. 60/096,822, filed Aug. 17, 1998, now abandoned, all of which are incorporated herein by reference. Also incorporated by reference are U.S. patent application Ser. No. 11/493,187, filed Jul. 26, 2006, now issued as U.S. Pat. No. 7,312,377 and U.S. patent application Ser. No. 10/961,254 filed Oct. 8, 2004, now issued as U.S. Pat. No. 7,214,852, U.S. patent application Ser. No. 10/160,719, filed Jun. 3, 2002, now issued as U.S. Pat. No. 6,803,498, which is a continuation of U.S. patent application Ser. No. 09/371,383, filed Aug. 6, 1999, now abandoned, which claims benefit of U.S. Provisional Patent Application Ser. No. 60/096,822, filed Aug. 17, 1998, now abandoned.

TECHNICAL FIELD

[0002] The present invention relates generally to plant molecular biology. More specifically, it relates to nucleic acids and methods for modulating their expression in plants.

BACKGROUND OF THE INVENTION

[0003] Polysaccharides constitute the bulk of the plant cell walls and have been traditionally classified into three categories: cellulose, hemicellulose and pectin. Fry, (1988) The growing plant cell wall: Chemical and metabolic analysis, New York: Longman Scientific & Technical. Whereas cellulose is made at the plasma membrane and directly laid down into the cell wall, hemicellulosic and pectic polymers are first made in the Golgi apparatus and then exported to the cell wall by exocytosis. Ray, et al., (1976) Ber. Deutsch. Bot. Ges. Bd. 89:121-146. The variety of chemical linkages in the pectic and hemicellulosic polysaccharides indicates that there must be tens of polysaccharide synthases in the Golgi apparatus. Darvill, et al., (1980) The primary cell walls of flowering plants. In The Plant Cell (N E Tolbert, ed.), Vol. 1 in Series: The biochemistry of plants: A comprehensive treatise, eds. Stumpf and Conn, (New York: Academic Press), pp. 91-162.

[0004] Even though sugar and polysaccharide compositions of the plant cell walls have been well characterized, very limited progress has been made toward identification of the enzymes involved in polysaccharides formation, the reason being their labile nature and recalcitrance to solubilization by available detergents. Sporadic claims for the identification of cellulose synthase from plant sources were made over the years. Callaghan and Benziman, (1984) Nature 311:165-167; Okuda, et al., (1993) Plant Physiol. 101:1131-1142. However, these claims were met with skepticism. Callaghan and Benziman, (1985), Nature 314:383-384; Delmer, et al., (1993) Plant Physiol. 103:307-308. It was only relatively recently that a putative gene for plant cellulose synthase (CesA) was cloned from the developing cotton fibers based on homology to the bacterial gene. Pear, et al., Proc. Natl. Acad. Sci. USA 93:12637-12642; Saxena, et al., (1990) Plant Molecular Biology 15:673-684; see also, WO 1998/18949; see also, Arioli, et al., (1998). Molecular analysis of cellulose biosynthesis in Arabidopsis. Science Washington D C. Jan. 279:717-720. A number of genes for cellulose synthase family were later isolated from other plant species based on sequence homology to the cotton gene (Richmond and Somerville, (2000) Plant Physiology 124:495-498.)

[0005] Cellulose, by virtue of its ability to form semicrystalline microfibrils, has a very high tensile strength which approaches that of some metals. Niklas, (1992), Plant Biomechanics: An engineering approach to plant form and function, The University of Chicago Press, p. 607. Bending strength of the culm of normal and brittle-culm mutants of barley has been found to be directly correlated with the concentration of cellulose in the cell wall. Kokubo, et al., (1989), Plant Physiology 91:876-882; Kokubo, et al., (1991) Plant Physiology 97:509-514.

[0006] Although stalk composition contributes to numerous quality factors important in maize breeding, little is known in the art about the impact of cellulose levels on such agronomically important traits as stalk lodging, silage digestibility or downstream processing. The present invention provides these and other advantages.

SUMMARY OF THE INVENTION

[0007] Generally, it is the object of the present invention to provide nucleic acids and proteins relating to cellulose synthases. It is an object of the present invention to provide transgenic plants comprising the nucleic acids of the present invention and methods for modulating, in a transgenic plant, expression of the nucleic acids of the present invention.

[0008] Therefore, in one aspect the present invention relates to an isolated nucleic acid comprising a member selected from the group consisting of (a) a polynucleotide having a specified sequence identity to a polynucleotide encoding a polypeptide of the present invention; (b) a polynucleotide which is complementary to the polynucleotide of (a) and (c) a polynucleotide comprising a specified number of contiguous nucleotides from a polynucleotide of (a) or (b). The isolated nucleic acid can be DNA.

[0009] In other aspects the present invention relates to: 1) recombinant expression cassettes, comprising a nucleic acid of the present invention operably linked to a promoter, 2) a host cell into which has been introduced the recombinant expression cassette, 3) a transgenic plant comprising the recombinant expression cassette and 4) a transgenic plant comprising a recombinant expression cassette containing more than one nucleic acid of the present invention each operably linked to a promoter. Furthermore, the present invention also relates to combining by crossing and hybridization recombinant cassettes from different transformants. The host cell and plant are optionally from maize, wheat, rice or soybean.

[0010] In other aspects the present invention relates to methods of altering stalk lodging and other standability traits, including, but not limited to brittle snap and improving stalk digestibility, through the introduction of one or more of the polynucleotides that encode the polypeptides of the present invention. Additional aspects of the present invention include methods and transgenic plants useful in the end use processing of compounds such ads cellulose or use of transgenic plants as end products either directly, such as silage, or indirectly following processing, for such uses known to those of skill in the art, such as, but not limited to, ethanol. Also, one of skill in the art would recognize that the polynucleotides and encoded polypeptides of the present invention can be introduced into an host cell or transgenic plant wither singly or in multiples, sometimes referred to in the art as "stacking" of sequences or traits. It is intended that these compositions and methods be encompassed in the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0011] FIG. 1: Stalk breaking strength of hybrids and its comparison with the lodging scores. The mechanical strength is very similar to the lodging scores that have been assigned based on field observations. The vertical light-colored bar in the upper right corner of the figure is the least significant difference (LSD) estimate at 5% level.

[0012] FIG. 2: Stalk strength of T.sub.0 transgenic plants. Plants overexpressing ("up") ZmCesA4 and ZmCesA8 had significantly stronger stalks than the controls. Overexpression of CesA5 did not alter stalk strength whereas the overexpression of CesA1 led to weaker and stunted stalks.

[0013] FIG. 3: Correlation between unit cellulose and stalk breaking strength. Stalk breaking strength was highly correlated with the amount of cellulose in a unit stalk length (correlation coefficient, r: 0.76; 0.63; 0.92; 0.86.) While these correlations are specifically related to the different CesA genes, it should be noted that in general the same correlation would apply. In other words, it is expected this would apply to low cellulose levels as well as higher cellulose levels.

[0014] FIG. 4: Unrooted cladogram of CesA proteins from different species. Sequences are labeled by prefixes. This cladogram demonstrates the relevance of the maize genes to those of Arabidopsis and rice. Prefixes: At, Arabidopsis thaliana; Gh, Gossypium herbaceum; Lj, Lotus japonicus; Mt, Medicago truncatula; Na, Nicotiana alata; Os, Oriza sativa; Pc, Populus canescens; Ptr, Populus tremula.times.tremuloides; Ze, Zinnia elegans; Zm, Zea mays.

[0015] FIG. 5: Expression pattern of CesA10, 11 and 12 in different maize tissues. All three genes are nearly synchronously expressed in tissues rich in secondary wall.

[0016] FIG. 6: Effect of overexpression of different CesA genes on plant height in corn. Whereas the overexpression of CesA8 led to an increase in height, CesA4 and CesA5 had not effect. Overexpression of CesA1 resulted in stunted plants.

[0017] FIG. 7: Effect of the overexpression of different CesA genes on the amount of cellulose in a unit length of the stalk tissue below ear in corn. CesA4 and CesA8, when overexpressed, resulted in an increased cellulose/length, CesA5 had no effect and CesA1 resulted in reduced cellulose/length.

[0018] FIG. 8: Contribution of different stalk components to dry matter, diameter, volume and stalk strength in maize hybrids. The data are derived from seven hybrids grown at three densities (27, 43 and 59 K per acre) in three replications each in 2001. Two stalks were sampled from each replication. Internodes 3 and 4 below the ear were broken with Instron model 4411 (Instron Corporation, 100 Royall Street, Canton, Mass. 02021). After breaking, the 3rd internode was separated into rind and inner tissue. Path coefficient analyses were performed using rind and inner tissue as independent variables (X.sub.1 and X.sub.2, respectively) and the whole stalk as the dependent variable (Y). The multiple regression equation: Y=a+b.sub.1X.sub.1+b.sub.2X.sub.2+e where a is the intercept and e error. Path coefficients were calculated as follows: .rho.YXn=b.sub.n*.delta..sub.n/.epsilon..sub.Y where n is 1 or 2. The contribution of each independent variable to whole stalk (Y) was calculated as follows: .rho.Yxn*rYxn where r is the correlation coefficient.

[0019] FIG. 9: Expression of the maize CesA genes in the pulvinal tissue of leaf derived from an elongating internode. The expression was studied by the Lynx MPSS technology.

DETAILED DESCRIPTION OF THE INVENTION

Overview

A. Nucleic Acids and Protein of the Present Invention

[0020] Unless otherwise stated, the polynucleotide and polypeptide sequences identified in Table 1 represent polynucleotides and polypeptides of the present invention. Table 1 cross-references these polynucleotide and polypeptides to their gene name and internal database identification number (SEQ ID NO.). A nucleic acid of the present invention comprises a polynucleotide of the present invention. A protein of the present invention comprises a polypeptide of the present invention.

TABLE-US-00001 TABLE 1 Database ID Polynucleotide Polypeptide SEQ Gene Name NO: SEQ ID NO: ID NO: Cellulose synthase CesA-1 1 2 Cellulose synthase CesA-2 45 46 Cellulose synthase CesA-3 5 6 Cellulose synthase CesA-4 9 10 Cellulose synthase CesA-5 13 14 Cellulose synthase CesA-6 41 42 Cellulose synthase CesA-7 49 50 Cellulose synthase CesA-8 17 18 Cellulose synthase CesA-9 21 22 Cellulose synthase CesA-10 25 26 Cellulose synthase CesA-11 27 28 Cellulose synthase CesA-12 29 30

[0021] Table 2 further provides a comparison detailing the homology as a percentage of the 12 CesA genes from maize that have been described herein (see, also, "Related Applications" above).

TABLE-US-00002 TABLE 2 CesA1 CesA2 CesA3 CesA4 CesA5 CesA6 CesA7 CesA8 CesA9 CesA10 CesA11 CesA12 CesA1 93 60 59 60 55 55 57 61 51 51 46 CesA2 60 59 61 55 55 57 61 51 51 47 CesA3 47 48 49 45 46 49 46 52 50 CesA4 77 54 52 58 86 54 53 52 CesA5 55 53 57 75 52 52 51 CesA6 74 73 56 56 55 53 CesA7 70 54 50 48 46 CesA8 59 55 52 51 CesA9 52 52 50 CesA10 53 64 CesA11 56 CesA12

[0022] Further characterization of the CesA group is provided in FIG. 4, as a consensus tree for plant Ces A proteins. It describes the relationship between Ces A from maize, rice and Arabidopsis sources.

B. Exemplary Utility of the Present Invention

[0023] The present invention provides utility in such exemplary applications as improvement of stalk quality for improved stand lodging or standability or silage digestibility. Further, the present invention provides for an increased concentration of cellulose in the pericarp, hardening the kernel and thus improving its handling ability. Stalk lodging at maturity can cause significant yield losses in corn. Environmental stresses from flowering to harvest, such as drought and nutrient deficiency, further worsen this problem. The effect of abiotic stresses is exacerbated by biotic factors, such as stalk rot resulting from the soil-living pathogens growing through the ground tissue.

[0024] Maize hybrids known to be resistant to stalk lodging have mechanically stronger stalks. At the compositional level, cellulose in a unit stalk length is highly correlated with breaking strength. The present invention provides for modulation of cellulose synthase composition leading to increased stalk strength.

DEFINITIONS

[0025] Units, prefixes and symbols may be denoted in their SI accepted form. Unless otherwise indicated, nucleic acids are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively. Numeric ranges recited within the specification are inclusive of the numbers defining the range and include each integer within the defined range. Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUBMB Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes. Unless otherwise provided for, software, electrical and electronics terms as used herein are as defined in The New IEEE Standard Dictionary of Electrical and Electronics Terms (5th edition, 1993). The terms defined below are more fully defined by reference to the specification as a whole. Section headings provided throughout the specification are not limitations to the various objects and embodiments of the present invention.

[0026] By "amplified" is meant the construction of multiple copies of a nucleic acid sequence or multiple copies complementary to the nucleic acid sequence using at least one of the nucleic acid sequences as a template. Amplification systems include the polymerase chain reaction (PCR) system, ligase chain reaction (LCR) system, nucleic acid sequence based amplification (NASBA, Cangene, Mississauga, Ontario), Q-Beta Replicase systems, transcription-based amplification system (TAS) and strand displacement amplification (SDA). See, e.g., Diagnostic Molecular Microbiology Principles and Applications, Persing, et al., Ed., American Society for Microbiology, Washington, D.C. (1993). The product of amplification is termed an amplicon.

[0027] As used herein, "antisense orientation" includes reference to a duplex polynucleotide sequence that is operably linked to a promoter in an orientation where the antisense strand is transcribed. The antisense strand is sufficiently complementary to an endogenous transcription product such that translation of the endogenous transcription product is often inhibited.

[0028] By "encoding" or "encoded", with respect to a specified nucleic acid, is meant comprising the information for translation into the specified protein. A nucleic acid encoding a protein may comprise non-translated sequences (e.g., introns) within translated regions of the nucleic acid or may lack such intervening non-translated sequences (e.g., as in cDNA). The information by which a protein is encoded is specified by the use of codons. Typically, the amino acid sequence is encoded by the nucleic acid using the "universal" genetic code. However, variants of the universal code, such as are present in some plant, animal and fungal mitochondria, the bacterium Mycoplasma capricolumn or the ciliate Macronucleus, may be used when the nucleic acid is expressed therein.

[0029] When the nucleic acid is prepared or altered synthetically, advantage can be taken of known codon preferences of the intended host where the nucleic acid is to be expressed. For example, although nucleic acid sequences of the present invention may be expressed in both monocotyledonous and dicotyledonous plant species, sequences can be modified to account for the specific codon preferences and GC content preferences of monocotyledons or dicotyledons as these preferences have been shown to differ (Murray, et al., (1989) Nucl. Acids Res. 17:477-498). Thus, the maize preferred codon for a particular amino acid may be derived from known gene sequences from maize. Maize codon usage for 28 genes from maize plants is listed in Table 4 of Murray, et al., supra.

[0030] As used herein "full-length sequence" in reference to a specified polynucleotide or its encoded protein means having the entire amino acid sequence of a native (non-synthetic), endogenous, biologically (e.g., structurally or catalytically) active form of the specified protein. Methods to determine whether a sequence is full-length are well known in the art, including such exemplary techniques as northern or western blots, primer extension, S1 protection and ribonuclease protection. See, e.g., Plant Molecular Biology: A Laboratory Manual, Clark, Ed., Springer-Verlag, Berlin (1997). Comparison to known full-length homologous (orthologous and/or paralogous) sequences can also be used to identify full-length sequences of the present invention. Additionally, consensus sequences typically present at the 5' and 3' untranslated regions of mRNA aid in the identification of a polynucleotide as full-length. For example, the consensus sequence ANNNNAUGG, where the underlined codon represents the N-terminal methionine, aids in determining whether the polynucleotide has a complete 5' end. Consensus sequences at the 3' end, such as polyadenylation sequences, aid in determining whether the polynucleotide has a complete 3' end.

[0031] As used herein, "heterologous" in reference to a nucleic acid is a nucleic acid 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 human intervention. For example, a promoter operably linked to a heterologous structural gene is from a species different from that from which the structural gene was derived, or, if from the same species, one or both are substantially modified from their original form. A heterologous protein may originate from a foreign species or, if from the same species, is substantially modified from its original form by human intervention.

[0032] By "host cell" is meant a cell which contains a vector and supports the replication and/or expression of the vector. Host cells may be prokaryotic cells such as E. coli, or eukaryotic cells such as yeast, insect, amphibian or mammalian cells. Preferably, host cells are monocotyledonous or dicotyledonous plant cells. A particularly preferred monocotyledonous host cell is a maize host cell.

[0033] The term "introduced" includes reference to the incorporation of a nucleic acid into a eukaryotic or prokaryotic cell where the nucleic acid may be incorporated into the genome of the cell (e.g., chromosome, plasmid, plastid or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (e.g., transfected mRNA). The term includes such nucleic acid introduction means as "transfection", "transformation" and "transduction".

[0034] The term "isolated" refers to material, such as a nucleic acid or a protein, which is: (1) substantially or essentially free from components which normally accompany or interact with it as found in its natural environment. The isolated material optionally comprises material not found with the material in its natural environment or (2) if the material is in its natural environment, the material has been synthetically altered or synthetically produced by deliberate human intervention and/or placed at a different location within the cell. The synthetic alteration or creation of the material can be performed on the material within or apart from its natural state. For example, a naturally-occurring nucleic acid becomes an isolated nucleic acid if it is altered or produced by non-natural, synthetic methods or if it is transcribed from DNA which has been altered or produced by non-natural, synthetic methods. The isolated nucleic acid may also be produced by the synthetic re-arrangement ("shuffling") of a part or parts of one or more allelic forms of the gene of interest. Likewise, a naturally-occurring nucleic acid (e.g., a promoter) becomes isolated if it is introduced to a different locus of the genome. Nucleic acids which are "isolated," as defined herein, are also referred to as "heterologous" nucleic acids. See, e.g., Compounds and Methods for Site Directed Mutagenesis in Eukaryotic Cells, Kmiec, U.S. Pat. No. 5,565,350; In Vivo Homologous Sequence Targeting in Eukaryotic Cells, Zarling, et al., WO 1993/22443 (PCT/US93/03868).

[0035] As used herein, "nucleic acid" includes reference to a deoxyribonucleotide or ribonucleotide polymer or chimeras thereof in either single- or double-stranded form and unless otherwise limited, encompasses known analogues having the essential nature of natural nucleotides in that they hybridize to single-stranded nucleic acids in a manner similar to naturally occurring nucleotides (e.g., peptide nucleic acids).

[0036] By "nucleic acid library" is meant a collection of isolated DNA or RNA molecules which comprise and substantially represent the entire transcribed fraction of a genome of a specified organism, tissue or of a cell type from that organism. Construction of exemplary nucleic acid libraries, such as genomic and cDNA libraries, is taught in standard molecular biology references such as Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology, Vol. 152, Academic Press, Inc., San Diego, Calif. (Berger); Sambrook, et al., Molecular Cloning--A Laboratory Manual, 2.sup.nd ed., Vol. 1-3 (1989) and Current Protocols in Molecular Biology, Ausubel, et al., Eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc. (1994).

[0037] As used herein "operably linked" includes reference to a functional linkage between a promoter and a second sequence, wherein the promoter sequence initiates and mediates transcription of the DNA sequence corresponding to the second sequence. Generally, operably linked means that the nucleic acid sequences being linked are contiguous and, where necessary to join two protein coding regions, contiguous and in the same reading frame.

[0038] As used herein, the term "plant" includes reference to whole plants, plant parts or organs (e.g., leaves, stems, roots, etc.), plant cells, seeds and progeny of same. Plant cell, as used herein, further includes, without limitation, cells obtained from or found in: seeds, suspension cultures, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen and microspores. Plant cells can also be understood to include modified cells, such as protoplasts, obtained from the aforementioned tissues. The class of plants which can be used in the methods of the invention is generally as broad as the class of higher plants amenable to transformation techniques, including both monocotyledonous and dicotyledonous plants. A particularly preferred plant is Zea mays.

[0039] As used herein, "polynucleotide" includes reference to a deoxyribopolynucleotide, ribopolynucleotide or chimeras or analogs thereof that have the essential nature of a natural deoxy- or ribo-nucleotide in that they hybridize, under stringent hybridization conditions, to substantially the same nucleotide sequence as naturally occurring nucleotides and/or allow translation into the same amino acid(s) as the naturally occurring nucleotide(s). A polynucleotide can be full-length or a subsequence of a native or heterologous structural or regulatory gene. Unless otherwise indicated, the term includes reference to the specified sequence as well as the complementary sequence thereof. Thus, DNAs or RNAs with backbones modified for stability or for other reasons are "polynucleotides" as that term is intended herein. Moreover, DNAs or RNAs comprising unusual bases, such as inosine, or modified bases, such as tritylated bases, to name just two examples, are polynucleotides as the term is used herein. It will be appreciated that a great variety of modifications have been made to DNA and RNA that serve many useful purposes known to those of skill in the art. The term polynucleotide as it is employed herein embraces such chemically, enzymatically or metabolically modified forms of polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including among other things, simple and complex cells.

[0040] The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. The essential nature of such analogues of naturally occurring amino acids is that, when incorporated into a protein, that protein is specifically reactive to antibodies elicited to the same protein but consisting entirely of naturally occurring amino acids. The terms "polypeptide", "peptide" and "protein" are also inclusive of modifications including, but not limited to, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation. Further, this invention contemplates the use of both the methionine-containing and the methionine-less amino terminal variants of the protein of the invention.

[0041] As used herein "promoter" includes reference to a region of DNA upstream from the start of transcription and involved in recognition and binding of RNA polymerase and other proteins to initiate transcription. A "plant promoter" is a promoter capable of initiating transcription in plant cells whether or not its origin is a plant cell. Exemplary plant promoters include, but are not limited to, those that are obtained from plants, plant viruses and bacteria which comprise genes expressed in plant cells such Agrobacterium or Rhizobium. Examples of promoters under developmental control include promoters that preferentially initiate transcription in certain tissues, such as leaves, roots or seeds. Such promoters are referred to as "tissue preferred". Promoters which initiate transcription only in certain tissue are referred to as "tissue specific". A "cell type" specific promoter primarily drives expression in certain cell types in one or more organs, for example, vascular cells in roots or leaves. An "inducible" or "repressible" promoter is a promoter which is under environmental control. Examples of environmental conditions that may effect transcription by inducible promoters include anaerobic conditions or the presence of light. Tissue specific, tissue preferred, cell type specific and inducible promoters constitute the class of "non-constitutive" promoters. A "constitutive" promoter is a promoter which is active under most environmental conditions.

[0042] As used herein "recombinant" includes reference to a cell or vector, that has been modified by the introduction of a heterologous nucleic acid or that the cell is derived from a cell so modified. Thus, for example, recombinant cells express genes that are not found in identical form within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under-expressed or not expressed at all as a result of human intervention. The term "recombinant" as used herein does not encompass the alteration of the cell or vector by naturally occurring events (e.g., spontaneous mutation, natural transformation/transduction/transposition) such as those occurring without human intervention.

[0043] As used herein, a "recombinant expression cassette" is a nucleic acid construct, generated recombinantly or synthetically, with a series of specified nucleic acid elements which permit transcription of a particular nucleic acid in a host cell. The recombinant expression cassette can be incorporated into a plasmid, chromosome, mitochondrial DNA, plastid DNA, virus or nucleic acid fragment. Typically, the recombinant expression cassette portion of an expression vector includes, among other sequences, a nucleic acid to be transcribed and a promoter.

[0044] The terms "residue" or "amino acid residue" or "amino acid" are used interchangeably herein to refer to an amino acid that is incorporated into a protein, polypeptide or peptide (collectively "protein"). The amino acid may be a naturally occurring amino acid and, unless otherwise limited, may encompass non-natural analogs of natural amino acids that can function in a similar manner as naturally occurring amino acids.

[0045] The term "selectively hybridizes" includes reference to hybridization, under stringent hybridization conditions, of a nucleic acid sequence to a specified nucleic acid target sequence to a detectably greater degree (e.g., at least 2-fold over background) than its hybridization to non-target nucleic acid sequences and to the substantial exclusion of non-target nucleic acids. Selectively hybridizing sequences typically have about at least 80% sequence identity, preferably 90% sequence identity and most preferably 100% sequence identity (i.e., complementary) with each other.

[0046] The term "stringent conditions" or "stringent hybridization conditions" includes reference to conditions under which a probe will selectively 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 can be identified which are 100% complementary to the probe (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, optionally less than 500 nucleotides in length.

[0047] 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.degree. C. for short probes (e.g., 10 to 50 nucleotides) and at least about 60.degree. 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.degree. C. and a wash in 1.times. to 2.times.SSC (20.times.SSC=3.0 M NaCl/0.3 M trisodium citrate) at 50 to 55.degree. C. Exemplary moderate stringency conditions include hybridization in 40 to 45% formamide, 1 M NaCl, 1% SDS at 37.degree. C. and a wash in 0.5.times. to 1.times.SSC at 55 to 60.degree. C. Exemplary high stringency conditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37.degree. C. and a wash in 0.1.times.SSC at 60 to 65.degree. C.

[0048] 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 T.sub.m can be approximated from the equation of Meinkoth and Wahl, (1984) Anal. Biochem., 138:267-284: T.sub.m=81.5.degree. 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 T.sub.m is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridizes to a perfectly matched probe. T.sub.m is reduced by about 1.degree. C. for each 1% of mismatching; thus, T.sub.m, 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 T.sub.m can be decreased 10.degree. C. Generally, stringent conditions are selected to be about 5.degree. C. lower than the thermal melting point ("T.sub.m") 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.degree. C. lower than the T.sub.m; moderately stringent conditions can utilize a hybridization and/or wash at 6, 7, 8, 9 or 10.degree. C. lower than the T.sub.m; low stringency conditions can utilize a hybridization and/or wash at 11, 12, 13, 14, 15 or 20.degree. C. lower than the T.sub.m. Using the equation, hybridization and wash compositions, and desired T.sub.m, 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 T.sub.m of less than 45.degree. C. (aqueous solution) or 32.degree. C. (formamide solution) it is preferred to increase the SSC concentration so that a higher temperature can be used. Hybridization and/or wash conditions can be applied for at least 10, 30, 60, 90, 120 or 240 minutes. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Laboratory Techniques in Biochemistry and Molecular Biology--Hybridization with Nucleic Acid Probes, Part I, Chapter 2 "Overview of principles of hybridization and the strategy of nucleic acid probe assays", Elsevier, New York (1993); and Current Protocols in Molecular Biology, Chapter 2, Ausubel, et al., Eds., Greene Publishing and Wiley-Interscience, New York (1995).

[0049] As used herein, "transgenic plant" includes reference to a plant which comprises within its genome a heterologous polynucleotide. Generally, the heterologous polynucleotide is stably integrated within the genome such that the polynucleotide is passed on to successive generations. The heterologous polynucleotide may be integrated into the genome alone or as part of a recombinant expression cassette. "Transgenic" is used herein to include any cell, cell line, callus, tissue, plant part or plant, the genotype of which has been altered by the presence of heterologous nucleic acid including those transgenics initially so altered as well as those created by sexual crosses or asexual propagation from the initial transgenic. The term "transgenic" as used herein does not encompass the alteration of the genome (chromosomal or extra-chromosomal) by conventional plant breeding methods or by naturally occurring events such as random cross-fertilization, non-recombinant viral infection, non-recombinant bacterial transformation, non-recombinant transposition or spontaneous mutation.

[0050] As used herein, "vector" includes reference to a nucleic acid used in introduction of a polynucleotide of the present invention into a host cell. Vectors are often replicons. Expression vectors permit transcription of a nucleic acid inserted therein.

[0051] The following terms are used to describe the sequence relationships between a polynucleotide/polypeptide of the present invention with a reference polynucleotide/polypeptide: (a) "reference sequence", (b) "comparison window", (c) "sequence identity" and (d) "percentage of sequence identity".

[0052] (a) As used herein, "reference sequence" is a defined sequence used as a basis for sequence comparison with a polynucleotide/polypeptide of the present invention. 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.

[0053] (b) As used herein, "comparison window" includes reference to a contiguous and specified segment of a polynucleotide/polypeptide sequence, wherein the polynucleotide/polypeptide sequence may be compared to a reference sequence and wherein the portion of the polynucleotide/polypeptide 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 sequences. Generally, the comparison window is at least 20 contiguous nucleotides/amino acids residues 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/polypeptide sequence, a gap penalty is typically introduced and is subtracted from the number of matches.

[0054] Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman, (1981) Adv. Appl. Math. 2:482; by the homology alignment algorithm of Needleman and Wunsch, (1970) J. Mol. Biol. 48:443; by the search for similarity method of Pearson and Lipman, (1988) Proc. Natl. Acad. Sci. 85:2444; by computerized implementations of these algorithms, including, but not limited to: CLUSTAL in the PC/Gene program by Intelligenetics, Mountain View, Calif.; GAP, BESTFIT, BLAST, FASTA and TFASTA in the Wisconsin Genetics Software Package.RTM., Genetics Computer Group (GCG.RTM.), 575 Science Dr., Madison, Wis., USA; the CLUSTAL program is well described by Higgins and Sharp, (1988) Gene 73:237-244; Higgins and Sharp, (1989) CABIOS 5:151-153; Corpet, et al., (1988) Nucleic Acids Research 16:10881-90; Huang, et al., (1992) Computer Applications in the Biosciences 8:155-65 and Pearson, et al., (1994) Methods in Molecular Biology 24:307-331.

[0055] The BLAST family of programs which can be used for database similarity searches includes: BLASTN for nucleotide query sequences against nucleotide database sequences; BLASTX for nucleotide query sequences against protein database sequences; BLASTP for protein query sequences against protein database sequences; TBLASTN for protein query sequences against nucleotide database sequences and TBLASTX for nucleotide query sequences against nucleotide database sequences. See, Current Protocols in Molecular Biology, Chapter 19, Ausubel, et al., Eds., Greene Publishing and Wiley-Interscience, New York (1995); Altschul, et al., (1990) J. Mol. Biol., 215:403-410 and Altschul, et al., (1997) Nucleic Acids Res. 25:3389-3402.

[0056] Software for performing BLAST analyses is publicly available, e.g., through the National Center for Biotechnology Information. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold. These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always>0) and N (penalty score for mismatching residues; always<0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, a cutoff of 100, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see, Henikoff and Henikoff, (1989) Proc. Natl. Acad. Sci. USA 89:10915).

[0057] In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul, (1993) Proc. Nat'l. Acad. Sci. USA 90:5873-5877). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.

[0058] BLAST searches assume that proteins can be modeled as random sequences. However, many real proteins comprise regions of nonrandom sequences which may be homopolymeric tracts, short-period repeats or regions enriched in one or more amino acids. Such low-complexity regions may be aligned between unrelated proteins even though other regions of the protein are entirely dissimilar. A number of low-complexity filter programs can be employed to reduce such low-complexity alignments. For example, the SEG (Wooten and Federhen, (1993) Comput. Chem. 17:149-163) and XNU (Claverie and States, (1993) Comput. Chem 17:191-201) low-complexity filters can be employed alone or in combination.

[0059] Unless otherwise stated, nucleotide and protein identity/similarity values provided herein are calculated using GAP (GCG.RTM. Version 10) under default values.

[0060] GAP (Global Alignment Program) can also be used to compare a polynucleotide or polypeptide of the present invention with a reference sequence. GAP uses the algorithm of Needleman and Wunsch, (J. Mol. Biol. 48: 443-453 (1970)) 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 Wisconsin Genetics Software Package.RTM. 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 100. Thus, for example, the gap creation and gap extension penalties can each independently be: 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60 or greater.

[0061] 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 Wisconsin Genetics Software Package.RTM. is BLOSUM62 (see, Henikoff and Henikoff, (1989) Natl. Acad. Sci. USA 89:10915).

[0062] Multiple alignment of the sequences can be 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 are KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5.

[0063] (c) As used herein, "sequence identity" or "identity" in the context of two nucleic acid or polypeptide sequences includes reference to the residues in the two sequences which 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. Where sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences which 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., according to the algorithm of Meyers and Miller, (1988) Computer Applic. Biol. Sci. 4:11-17 e.g., as implemented in the program PC/GENE (Intelligenetics, Mountain View, Calif., USA).

[0064] (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.

Utilities

[0065] The present invention provides, among other things, compositions and methods for modulating (i.e., increasing or decreasing) the level of polynucleotides and polypeptides of the present invention in plants. In particular, the polynucleotides and polypeptides of the present invention can be expressed temporally or spatially, e.g., at developmental stages, in tissues and/or in quantities, which are uncharacteristic of non-recombinantly engineered plants.

[0066] The present invention also provides isolated nucleic acids comprising polynucleotides of sufficient length and complementarity to a polynucleotide of the present invention to use as probes or amplification primers in the detection, quantitation or isolation of gene transcripts. For example, isolated nucleic acids of the present invention can be used as probes in detecting deficiencies in the level of mRNA in screenings for desired transgenic plants, for detecting mutations in the gene (e.g., substitutions, deletions or additions), for monitoring upregulation of expression or changes in enzyme activity in screening assays of compounds, for detection of any number of allelic variants (polymorphisms), orthologs or paralogs of the gene or for site directed mutagenesis in eukaryotic cells (see, e.g., U.S. Pat. No. 5,565,350). The isolated nucleic acids of the present invention can also be used for recombinant expression of their encoded polypeptides or for use as immunogens in the preparation and/or screening of antibodies. The isolated nucleic acids of the present invention can also be employed for use in sense or antisense suppression of one or more genes of the present invention in a host cell, tissue or plant. Attachment of chemical agents which bind, intercalate, cleave and/or crosslink to the isolated nucleic acids of the present invention can also be used to modulate transcription or translation.

[0067] The present invention also provides isolated proteins comprising a polypeptide of the present invention (e.g., preproenzyme, proenzyme or enzymes). The present invention also provides proteins comprising at least one epitope from a polypeptide of the present invention. The proteins of the present invention can be employed in assays for enzyme agonists or antagonists of enzyme function or for use as immunogens or antigens to obtain antibodies specifically immunoreactive with a protein of the present invention. Such antibodies can be used in assays for expression levels, for identifying and/or isolating nucleic acids of the present invention from expression libraries, for identification of homologous polypeptides from other species or for purification of polypeptides of the present invention.

[0068] The isolated nucleic acids and polypeptides of the present invention can be used over a broad range of plant types, particularly monocots such as the species of the family Gramineae including Hordeum, Secale, Oryza, Triticum, Sorghum (e.g., S. bicolor) and Zea (e.g., Z. mays) and dicots such as Glycine.

[0069] The isolated nucleic acid and proteins of the present invention can also be used in species from the genera: Cucurbita, Rosa, Vitis, Juglans, Fragaria, Lotus, Medicago, Onobrychis, Trifolium, Trigonella, Vigna, Citrus, Linum, Geranium, Manihot, Daucus, Arabidopsis, Brassica, Raphanus, Sinapis, Atropa, Capsicum, Datura, Hyoscyamus, Lycopersicon, Nicotiana, Solanum, Petunia, Digitalis, Majorana, Ciahorium, Helianthus, Lactuca, Bromus, Asparagus, Antirrhinum, Heterocallis, Nemesis, Pelargonium, Panieum, Pennisetum, Ranunculus, Senecio, Salpiglossis, Cucumis, Browallia, Pisum, Phaseolus, Lolium and Avena.

Nucleic Acids

[0070] The present invention provides, among other things, isolated nucleic acids of RNA, DNA and analogs and/or chimeras thereof, comprising a polynucleotide of the present invention.

[0071] A polynucleotide of the present invention is inclusive of those in Table 1 and:

[0072] (a) an isolated polynucleotide encoding a polypeptide of the present invention such as those referenced in Table 1, including exemplary polynucleotides of the present invention;

[0073] (b) an isolated polynucleotide which is the product of amplification from a plant nucleic acid library using primer pairs which selectively hybridize under stringent conditions to loci within a polynucleotide of the present invention;

[0074] (c) an isolated polynucleotide which selectively hybridizes to a polynucleotide of (a) or (b);

[0075] (d) an isolated polynucleotide having a specified sequence identity with polynucleotides of (a), (b) or (c);

[0076] (e) an isolated polynucleotide encoding a protein having a specified number of contiguous amino acids from a prototype polypeptide, wherein the protein is specifically recognized by antisera elicited by presentation of the protein and wherein the protein does not detectably immunoreact to antisera which has been fully immunosorbed with the protein;

[0077] (f) complementary sequences of polynucleotides of (a), (b), (c), (d) or (e);

[0078] (g) an isolated polynucleotide comprising at least a specific number of contiguous nucleotides from a polynucleotide of (a), (b), (c), (d), (e) or (f);

[0079] (h) an isolated polynucleotide from a full-length enriched cDNA library having the physico-chemical property of selectively hybridizing to a polynucleotide of (a), (b), (c), (d), (e), (f) or (g);

[0080] (i) an isolated polynucleotide made by the process of: 1) providing a full-length enriched nucleic acid library, 2) selectively hybridizing the polynucleotide to a polynucleotide of (a), (b), (c), (d), (e), (f), (g) or (h), thereby isolating the polynucleotide from the nucleic acid library.

A. Polynucleotides Encoding a Polypeptide of the Present Invention

[0081] As indicated in (a), above, the present invention provides isolated nucleic acids comprising a polynucleotide of the present invention, wherein the polynucleotide encodes a polypeptide of the present invention. Every nucleic acid sequence herein that encodes a polypeptide also, by reference to the genetic code, describes every possible silent variation of the nucleic acid. One of ordinary skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine and UGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Thus, each silent variation of a nucleic acid which encodes a polypeptide of the present invention is implicit in each described polypeptide sequence and is within the scope of the present invention. Accordingly, the present invention includes polynucleotides of the present invention and polynucleotides encoding a polypeptide of the present invention.

B. Polynucleotides Amplified from a Plant Nucleic Acid Library

[0082] As indicated in (b), above, the present invention provides an isolated nucleic acid comprising a polynucleotide of the present invention, wherein the polynucleotides are amplified, under nucleic acid amplification conditions, from a plant nucleic acid library. Nucleic acid amplification conditions for each of the variety of amplification methods are well known to those of ordinary skill in the art. The plant nucleic acid library can be constructed from a monocot such as a cereal crop. Exemplary cereals include maize, sorghum, alfalfa, canola, wheat or rice. The plant nucleic acid library can also be constructed from a dicot such as soybean. Zea mays lines B73, PHRE1, A632, BMS-P2#10, W23 and Mo17 are known and publicly available. Other publicly known and available maize lines can be obtained from the Maize Genetics Cooperation (Urbana, Ill.). Wheat lines are available from the Wheat Genetics Resource Center (Manhattan, Kans.).

[0083] The nucleic acid library may be a cDNA library, a genomic library or a library generally constructed from nuclear transcripts at any stage of intron processing. cDNA libraries can be normalized to increase the representation of relatively rare cDNAs. In optional embodiments, the cDNA library is constructed using an enriched full-length cDNA synthesis method. Examples of such methods include Oligo-Capping (Maruyama and Sugano, (1994) Gene 138:171-174,), Biotinylated CAP Trapper (Carninci, et al., (1996) Genomics 37:327-336) and CAP Retention Procedure (Edery, et al., (1995) Molecular and Cellular Biology 15:3363-3371). Rapidly growing tissues or rapidly dividing cells are preferred for use as an mRNA source for construction of a cDNA library. Growth stages of maize are described in "How a Corn Plant Develops," Special Report Number 48, Iowa State University of Science and Technology Cooperative Extension Service, Ames, Iowa, Reprinted February 1993.

[0084] A polynucleotide of this embodiment (or subsequences thereof) can be obtained, for example, by using amplification primers which are selectively hybridized and primer extended, under nucleic acid amplification conditions, to at least two sites within a polynucleotide of the present invention or to two sites within the nucleic acid which flank and comprise a polynucleotide of the present invention or to a site within a polynucleotide of the present invention and a site within the nucleic acid which comprises it. Methods for obtaining 5' and/or 3' ends of a vector insert are well known in the art. See, e.g., RACE (Rapid Amplification of Complementary Ends) as described in Frohman, in PCR Protocols: A Guide to Methods and Applications, Innis, et al., Eds. (Academic Press, Inc., San Diego), pp. 28-38 (1990)); see, also, U.S. Pat. No. 5,470,722 and Current Protocols in Molecular Biology, Unit 15.6, Ausubel, et al., Eds., Greene Publishing and Wiley-Interscience, New York (1995); Frohman and Martin, Techniques 1:165 (1989).

[0085] Optionally, the primers are complementary to a subsequence of the target nucleic acid which they amplify but may have a sequence identity ranging from about 85% to 99% relative to the polynucleotide sequence which they are designed to anneal to. As those skilled in the art will appreciate, the sites to which the primer pairs will selectively hybridize are chosen such that a single contiguous nucleic acid can be formed under the desired nucleic acid amplification conditions. The primer length in nucleotides is selected from the group of integers consisting of from at least 15 to 50. Thus, the primers can be at least 15, 18, 20, 25, 30, 40 or 50 nucleotides in length. Those of skill will recognize that a lengthened primer sequence can be employed to increase specificity of binding (i.e., annealing) to a target sequence. A non-annealing sequence at the 5' end of a primer (a "tail") can be added, for example, to introduce a cloning site at the terminal ends of the amplicon.

[0086] The amplification products can be translated using expression systems well known to those of skill in the art. The resulting translation products can be confirmed as polypeptides of the present invention by, for example, assaying for the appropriate catalytic activity (e.g., specific activity and/or substrate specificity) or verifying the presence of one or more epitopes which are specific to a polypeptide of the present invention. Methods for protein synthesis from PCR derived templates are known in the art and available commercially. See, e.g., Amersham Life Sciences, Inc, Catalog '97, p. 354.

C. Polynucleotides which Selectively Hybridize to a Polynucleotide of (A) or (B)

[0087] As indicated in (c), above, the present invention provides isolated nucleic acids comprising polynucleotides of the present invention, wherein the polynucleotides selectively hybridize, under selective hybridization conditions, to a polynucleotide of sections (A) or (B) as discussed above. Thus, the polynucleotides of this embodiment can be used for isolating, detecting and/or quantifying nucleic acids comprising the polynucleotides of (A) or (B). For example, polynucleotides of the present invention can be used to identify, isolate or amplify partial or full-length clones in a deposited library. In some embodiments, the polynucleotides are genomic or cDNA sequences isolated or otherwise complementary to a cDNA from a dicot or monocot nucleic acid library. Exemplary species of monocots and dicots include, but are not limited to: maize, canola, soybean, cotton, wheat, sorghum, sunflower, alfalfa, oats, sugar cane, millet, barley and rice. The cDNA library comprises at least 50% to 95% full-length sequences (for example, at least 50%, 60%, 70%, 80%, 90% or 95% full-length sequences). The cDNA libraries can be normalized to increase the representation of rare sequences. See, e.g., U.S. Pat. No. 5,482,845. Low stringency hybridization conditions are typically, but not exclusively, employed with sequences having a reduced sequence identity relative to complementary sequences. Moderate and high stringency conditions can optionally be employed for sequences of greater identity. Low stringency conditions allow selective hybridization of sequences having about 70% to 80% sequence identity and can be employed to identify orthologous or paralogous sequences.

D. Polynucleotides Having a Specific Sequence Identity with the Polynucleotides of (A), (B) or (C)

[0088] As indicated in (d), above, the present invention provides isolated nucleic acids comprising polynucleotides of the present invention, wherein the polynucleotides have a specified identity at the nucleotide level to a polynucleotide as disclosed above in sections (A), (B) or (C), above. Identity can be calculated using, for example, the BLAST, CLUSTALW or GAP algorithms under default conditions. The percentage of identity to a reference sequence is at least 50% and, rounded upwards to the nearest integer, can be expressed as an integer selected from the group of integers consisting of from 50 to 99. Thus, for example, the percentage of identity to a reference sequence can be at least 60%, 70%, 75%, 80%, 85%, 90% or 95%.

[0089] Optionally, the polynucleotides of this embodiment will encode a polypeptide that will share an epitope with a polypeptide encoded by the polynucleotides of sections (A), (B) or (C). Thus, these polynucleotides encode a first polypeptide which elicits production of antisera comprising antibodies which are specifically reactive to a second polypeptide encoded by a polynucleotide of (A), (B) or (C). However, the first polypeptide does not bind to antisera raised against itself when the antisera has been fully immunosorbed with the first polypeptide. Hence, the polynucleotides of this embodiment can be used to generate antibodies for use in, for example, the screening of expression libraries for nucleic acids comprising polynucleotides of (A), (B) or (C), or for purification of, or in immunoassays for, polypeptides encoded by the polynucleotides of (A), (B) or (C). The polynucleotides of this embodiment comprise nucleic acid sequences which can be employed for selective hybridization to a polynucleotide encoding a polypeptide of the present invention.

[0090] Screening polypeptides for specific binding to antisera can be conveniently achieved using peptide display libraries. This method involves the screening of large collections of peptides for individual members having the desired function or structure. Antibody screening of peptide display libraries is well known in the art. The displayed peptide sequences can be from 3 to 5000 or more amino acids in length, frequently from 5-100 amino acids long, and often from about 8 to 15 amino acids long. In addition to direct chemical synthetic methods for generating peptide libraries, several recombinant DNA methods have been described. One type involves the display of a peptide sequence on the surface of a bacteriophage or cell. Each bacteriophage or cell contains the nucleotide sequence encoding the particular displayed peptide sequence. Such methods are described in PCT Patent Publication Numbers 1991/17271, 1991/18980, 1991/19818 and 1993/08278. Other systems for generating libraries of peptides have aspects of both in vitro chemical synthesis and recombinant methods. See, PCT Patent Publication Numbers 1992/05258, 1992/14843 and 1997/20078. See also, U.S. Pat. Nos. 5,658,754 and 5,643,768. Peptide display libraries, vectors and screening kits are commercially available from such suppliers as Invitrogen (Carlsbad, Calif.).

E. Polynucleotides Encoding a Protein Having a Subsequence from a Prototype Polypeptide and Cross-Reactive to the Prototype Polypeptide

[0091] As indicated in (e), above, the present invention provides isolated nucleic acids comprising polynucleotides of the present invention, wherein the polynucleotides encode a protein having a subsequence of contiguous amino acids from a prototype polypeptide of the present invention such as are provided in (a), above. The length of contiguous amino acids from the prototype polypeptide is selected from the group of integers consisting of from at least 10 to the number of amino acids within the prototype sequence. Thus, for example, the polynucleotide can encode a polypeptide having a subsequence having at least 10, 15, 20, 25, 30, 35, 40, 45 or 50, contiguous amino acids from the prototype polypeptide. Further, the number of such subsequences encoded by a polynucleotide of the instant embodiment can be any integer selected from the group consisting of from 1 to 20, such as 2, 3, 4 or 5. The subsequences can be separated by any integer of nucleotides from 1 to the number of nucleotides in the sequence such as at least 5, 10, 15, 25, 50, 100 or 200 nucleotides.

[0092] The proteins encoded by polynucleotides of this embodiment, when presented as an immunogen, elicit the production of polyclonal antibodies which specifically bind to a prototype polypeptide such as but not limited to, a polypeptide encoded by the polynucleotide of (a) or (b), above. Generally, however, a protein encoded by a polynucleotide of this embodiment does not bind to antisera raised against the prototype polypeptide when the antisera has been fully immunosorbed with the prototype polypeptide. Methods of making and assaying for antibody binding specificity/affinity are well known in the art. Exemplary immunoassay formats include ELISA, competitive immunoassays, radioimmunoassays, Western blots, indirect immunofluorescent assays and the like.

[0093] In a preferred assay method, fully immunosorbed and pooled antisera which is elicited to the prototype polypeptide can be used in a competitive binding assay to test the protein. The concentration of the prototype polypeptide required to inhibit 50% of the binding of the antisera to the prototype polypeptide is determined. If the amount of the protein required to inhibit binding is less than twice the amount of the prototype protein, then the protein is said to specifically bind to the antisera elicited to the immunogen. Accordingly, the proteins of the present invention embrace allelic variants, conservatively modified variants and minor recombinant modifications to a prototype polypeptide.

[0094] A polynucleotide of the present invention optionally encodes a protein having a molecular weight as the non-glycosylated protein within 20% of the molecular weight of the full-length non-glycosylated polypeptides of the present invention. Molecular weight can be readily determined by SDS-PAGE under reducing conditions. Optionally, the molecular weight is within 15% of a full length polypeptide of the present invention, more preferably within 10% or 5%, and most preferably within 3%, 2% or 1% of a full length polypeptide of the present invention.

[0095] Optionally, the polynucleotides of this embodiment will encode a protein having a specific enzymatic activity at least 50%, 60%, 80% or 90% of a cellular extract comprising the native, endogenous full-length polypeptide of the present invention. Further, the proteins encoded by polynucleotides of this embodiment will optionally have a substantially similar affinity constant (K.sub.m) and/or catalytic activity (i.e., the microscopic rate constant, k.sub.cat) as the native endogenous, full-length protein. Those of skill in the art will recognize that k.sub.cat/K.sub.m value determines the specificity for competing substrates and is often referred to as the specificity constant. Proteins of this embodiment can have a k.sub.cat/K.sub.m value at least 10% of a full-length polypeptide of the present invention as determined using the endogenous substrate of that polypeptide. Optionally, the k.sub.cat/K.sub.m value will be at least 20%, 30%, 40%, 50% and most preferably at least 60%, 70%, 80%, 90% or 95% the k.sub.cat/K.sub.m value of the full-length polypeptide of the present invention. Determination of k.sub.cat, K.sub.m and k.sub.cat/K.sub.m can be determined by any number of means well known to those of skill in the art. For example, the initial rates (i.e., the first 5% or less of the reaction) can be determined using rapid mixing and sampling techniques (e.g., continuous-flow, stopped-flow or rapid quenching techniques), flash photolysis or relaxation methods (e.g., temperature jumps) in conjunction with such exemplary methods of measuring as spectrophotometry, spectrofluorimetry, nuclear magnetic resonance or radioactive procedures. Kinetic values are conveniently obtained using a Lineweaver-Burk or Eadie-Hofstee plot.

F. Polynucleotides Complementary to the Polynucleotides of (A)-(E) As indicated in (f), above, the present invention provides isolated nucleic acids comprising polynucleotides complementary to the polynucleotides of paragraphs A-E, above. As those of skill in the art will recognize, complementary sequences base-pair throughout the entirety of their length with the polynucleotides of sections (A)-(E) (i.e., have 100% sequence identity over their entire length). Complementary bases associate through hydrogen bonding in double stranded nucleic acids. For example, the following base pairs are complementary: guanine and cytosine; adenine and thymine and adenine and uracil. G. Polynucleotides which are Subsequences of the Polynucleotides of (A)-(F)

[0096] As indicated in (g), above, the present invention provides isolated nucleic acids comprising polynucleotides which comprise at least 15 contiguous bases from the polynucleotides of sections (A) through (F) as discussed above. The length of the polynucleotide is given as an integer selected from the group consisting of from at least 15 to the length of the nucleic acid sequence from which the polynucleotide is a subsequence of. Thus, for example, polynucleotides of the present invention are inclusive of polynucleotides comprising at least 15, 20, 25, 30, 40, 50, 60, 75 or 100 contiguous nucleotides in length from the polynucleotides of (A)-(F). Optionally, the number of such subsequences encoded by a polynucleotide of the instant embodiment can be any integer selected from the group consisting of from 1 to 20, such as 2, 3, 4 or 5. The subsequences can be separated by any integer of nucleotides from 1 to the number of nucleotides in the sequence such as at least 5, 10, 15, 25, 50, 100 or 200 nucleotides.

[0097] Subsequences can be made by in vitro synthetic, in vitro biosynthetic or in vivo recombinant methods. In optional embodiments, subsequences can be made by nucleic acid amplification. For example, nucleic acid primers will be constructed to selectively hybridize to a sequence (or its complement) within, or co-extensive with, the coding region.

[0098] The subsequences of the present invention can comprise structural characteristics of the sequence from which it is derived. Alternatively, the subsequences can lack certain structural characteristics of the larger sequence from which it is derived such as a poly (A) tail. Optionally, a subsequence from a polynucleotide encoding a polypeptide having at least one epitope in common with a prototype polypeptide sequence as provided in (a), above, may encode an epitope in common with the prototype sequence. Alternatively, the subsequence may not encode an epitope in common with the prototype sequence but can be used to isolate the larger sequence by, for example, nucleic acid hybridization with the sequence from which it's derived. Subsequences can be used to modulate or detect gene expression by introducing into the subsequences compounds which bind, intercalate, cleave and/or crosslink to nucleic acids. Exemplary compounds include acridine, psoralen, phenanthroline, naphthoquinone, daunomycin or chloroethylaminoaryl conjugates.

H. Polynucleotides from a Full-Length Enriched cDNA Library Having the Physico-Chemical Property of Selectively Hybridizing to a Polynucleotide of (A)-(G)

[0099] As indicated in (h), above, the present invention provides an isolated polynucleotide from a full-length enriched cDNA library having the physico-chemical property of selectively hybridizing to a polynucleotide of paragraphs (A), (B), (C), (D), (E), (F) or (G) as discussed above. Methods of constructing full-length enriched cDNA libraries are known in the art and discussed briefly below. The cDNA library comprises at least 50% to 95% full-length sequences (for example, at least 50%, 60%, 70%, 80%, 90% or 95% full-length sequences). The cDNA library can be constructed from a variety of tissues from a monocot or dicot at a variety of developmental stages. Exemplary species include maize, wheat, rice, canola, soybean, cotton, sorghum, sunflower, alfalfa, oats, sugar cane, millet, barley and rice. Methods of selectively hybridizing, under selective hybridization conditions, a polynucleotide from a full-length enriched library to a polynucleotide of the present invention are known to those of ordinary skill in the art. Any number of stringency conditions can be employed to allow for selective hybridization. In optional embodiments, the stringency allows for selective hybridization of sequences having at least 70%, 75%, 80%, 85%, 90%, 95% or 98% sequence identity over the length of the hybridized region. Full-length enriched cDNA libraries can be normalized to increase the representation of rare sequences.

I. Polynucleotide Products Made by a cDNA Isolation Process

[0100] As indicated in (I), above, the present invention provides an isolated polynucleotide made by the process of: 1) providing a full-length enriched nucleic acid library, 2) selectively hybridizing the polynucleotide to a polynucleotide of paragraphs (A), (B), (C), (D), (E), (F), (G) or (H) as discussed above, and thereby isolating the polynucleotide from the nucleic acid library. Full-length enriched nucleic acid libraries are constructed as discussed in paragraph (G) and below. Selective hybridization conditions are as discussed in paragraph (G). Nucleic acid purification procedures are well known in the art. Purification can be conveniently accomplished using solid-phase methods; such methods are well known to those of skill in the art and kits are available from commercial suppliers such as Advanced Biotechnologies (Surrey, UK). For example, a polynucleotide of paragraphs (A)-(H) can be immobilized to a solid support such as a membrane, bead, or particle. See, e.g., U.S. Pat. No. 5,667,976. The polynucleotide product of the present process is selectively hybridized to an immobilized polynucleotide and the solid support is subsequently isolated from non-hybridized polynucleotides by methods including, but not limited to, centrifugation, magnetic separation, filtration, electrophoresis and the like.

Construction of Nucleic Acids

[0101] The isolated nucleic acids of the present invention can be made using (a) standard recombinant methods, (b) synthetic techniques or combinations thereof. In some embodiments, the polynucleotides of the present invention will be cloned, amplified or otherwise constructed from a monocot such as maize, rice or wheat or a dicot such as soybean.

[0102] The nucleic acids may conveniently comprise sequences in addition to a polynucleotide of the present invention. For example, a multi-cloning site comprising one or more endonuclease restriction sites may be inserted into the nucleic acid to aid in isolation of the polynucleotide. Also, translatable sequences may be inserted to aid in the isolation of the translated polynucleotide of the present invention. For example, a hexa-histidine marker sequence provides a convenient means to purify the proteins of the present invention. A polynucleotide of the present invention can be attached to a vector, adapter or linker for cloning and/or expression of a polynucleotide of the present invention. Additional sequences may be added to such cloning and/or expression sequences to optimize their function in cloning and/or expression, to aid in isolation of the polynucleotide, or to improve the introduction of the polynucleotide into a cell. Typically, the length of a nucleic acid of the present invention less the length of its polynucleotide of the present invention is less than 20 kilobase pairs, often less than 15 kb and frequently less than 10 kb. Use of cloning vectors, expression vectors, adapters, and linkers is well known and extensively described in the art. For a description of various nucleic acids see, for example, Stratagene Cloning Systems, Catalogs 1999 (La Jolla, Calif.) and Amersham Life Sciences, Inc, Catalog '99 (Arlington Heights, Ill.).

A. Recombinant Methods for Constructing Nucleic Acids

[0103] The isolated nucleic acid compositions of this invention, such as RNA, cDNA, genomic DNA or a hybrid thereof, can be obtained from plant biological sources using any number of cloning methodologies known to those of skill in the art. In some embodiments, oligonucleotide probes which selectively hybridize, under stringent conditions, to the polynucleotides of the present invention are used to identify the desired sequence in a cDNA or genomic DNA library. Isolation of RNA, and construction of cDNA and genomic libraries is well known to those of ordinary skill in the art. See, e.g., Plant Molecular Biology: A Laboratory Manual, Clark, Ed., Springer-Verlag, Berlin (1997); and, Current Protocols in Molecular Biology, Ausubel, et al., Eds., Greene Publishing and Wiley-Interscience, New York (1995).

A1. Full-Length Enriched cDNA Libraries

[0104] A number of cDNA synthesis protocols have been described which provide enriched full-length cDNA libraries. Enriched full-length cDNA libraries are constructed to comprise at least 600%, and more preferably at least 70%, 80%, 90% or 95% full-length inserts amongst clones containing inserts. The length of insert in such libraries can be at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more kilobase pairs. Vectors to accommodate inserts of these sizes are known in the art and available commercially. See, e.g., Stratagene's lambda ZAP Express (cDNA cloning vector with 0 to 12 kb cloning capacity). An exemplary method of constructing a greater than 95% pure full-length cDNA library is described by Carninci, et al., (1996) Genomics, 37:327-336. Other methods for producing full-length libraries are known in the art. See, e.g., Edery, et al., (1995) Mol. Cell Biol. 15(6):3363-3371 and PCT Application Number WO 1996/34981.

A2. Normalized or Subtracted cDNA Libraries

[0105] A non-normalized cDNA library represents the mRNA population of the tissue it was made from. Since unique clones are out-numbered by clones derived from highly expressed genes their isolation can be laborious. Normalization of a cDNA library is the process of creating a library in which each clone is more equally represented. Construction of normalized libraries is described in Ko, (1990) Nucl. Acids. Res. 18(19):5705-5711; Patanjali, et al., (1991) Proc. Natl. Acad. U.S.A. 88:1943-1947; U.S. Pat. Nos. 5,482,685, 5,482,845 and 5,637,685. In an exemplary method described by Soares, et al., normalization resulted in reduction of the abundance of clones from a range of four orders of magnitude to a narrow range of only 1 order of magnitude. Proc. Natl. Acad. Sci. USA, 91:9228-9232 (1994).

[0106] Subtracted cDNA libraries are another means to increase the proportion of less abundant cDNA species. In this procedure, cDNA prepared from one pool of mRNA is depleted of sequences present in a second pool of mRNA by hybridization. The cDNA:mRNA hybrids are removed and the remaining un-hybridized cDNA pool is enriched for sequences unique to that pool. See, Foote, et al., in, Plant Molecular Biology: A Laboratory Manual, Clark, Ed., Springer-Verlag, Berlin (1997); Kho and Zarbl, (1991) Technique 3(2):58-63; Sive and St. John, (1988) Nucl. Acids Res., 16(22):10937; Current Protocols in Molecular Biology, Ausubel, et al., Eds., Greene Publishing and Wiley-Interscience, New York (1995) and Swaroop, et al., (1991) Nucl. Acids Res., 19(8):1954. cDNA subtraction kits are commercially available. See, e.g., PCR-Select (Clontech, Palo Alto, Calif.).

[0107] To construct genomic libraries, large segments of genomic DNA are generated by fragmentation, e.g., using restriction endonucleases, and are ligated with vector DNA to form concatemers that can be packaged into the appropriate vector. Methodologies to accomplish these ends and sequencing methods to verify the sequence of nucleic acids are well known in the art. Examples of appropriate molecular biological techniques and instructions sufficient to direct persons of skill through many construction, cloning and screening methodologies are found in Sambrook, et al., Molecular Cloning A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Vols. 1-3 (1989), Methods in Enzymology, Vol. 152: Guide to Molecular Cloning Techniques, Berger and Kimmel, Eds., San Diego: Academic Press, Inc. (1987), Current Protocols in Molecular Biology, Ausubel, et al., Eds., Greene Publishing and Wiley-Interscience, New York (1995); Plant Molecular Biology: A Laboratory Manual, Clark, Ed., Springer-Verlag, Berlin (1997). Kits for construction of genomic libraries are also commercially available.

[0108] The cDNA or genomic library can be screened using a probe based upon the sequence of a polynucleotide of the present invention such as those disclosed herein. Probes may be used to hybridize with genomic DNA or cDNA sequences to isolate homologous genes in the same or different plant species. Those of skill in the art will appreciate that various degrees of stringency of hybridization can be employed in the assay and either the hybridization or the wash medium can be stringent.

[0109] The nucleic acids of interest can also be amplified from nucleic acid samples using amplification techniques. For instance, polymerase chain reaction (PCR) technology can be used to amplify the sequences of polynucleotides of the present invention and related genes directly from genomic DNA or cDNA libraries. PCR and other in vitro amplification methods may also be useful, for example, to clone nucleic acid sequences that code for proteins to be expressed, to make nucleic acids to use as probes for detecting the presence of the desired mRNA in samples, for nucleic acid sequencing or for other purposes. The T4 gene 32 protein (Boehringer Mannheim) can be used to improve yield of long PCR products.

[0110] PCR-based screening methods have been described. Wilfinger, et al., describe a PCR-based method in which the longest cDNA is identified in the first step so that incomplete clones can be eliminated from study. BioTechniques, 22(3):481-486 (1997). Such methods are particularly effective in combination with a full-length cDNA construction methodology, above.

B. Synthetic Methods for Constructing Nucleic Acids

[0111] The isolated nucleic acids of the present invention can also be prepared by direct chemical synthesis by methods such as the phosphotriester method of Narang, et al., (1979) Meth. Enzymol. 68: 90-99; the phosphodiester method of Brown, et al., (1979) Meth. Enzymol. 68:109-151; the diethylphosphoramidite method of Beaucage, et al., (1981) Tetra. Lett. 22:1859-1862; the solid phase phosphoramidite triester method described by Beaucage and Caruthers, (1981) Tetra. Letts. 22(20):1859-1862, e.g., using an automated synthesizer, e.g., as described in Needham-VanDevanter, et al., (1984) Nucleic Acids Res., 12:6159-6168 and the solid support method of U.S. Pat. No. 4,458,066. Chemical synthesis generally produces a single stranded oligonucleotide. This may be converted into double stranded DNA by hybridization with a complementary sequence or by polymerization with a DNA polymerase using the single strand as a template. One of skill will recognize that while chemical synthesis of DNA is best employed for sequences of about 100 bases or less, longer sequences may be obtained by the ligation of shorter sequences.

Recombinant Expression Cassettes

[0112] The present invention further provides recombinant expression cassettes comprising a nucleic acid of the present invention. A nucleic acid sequence coding for the desired polypeptide of the present invention, for example a cDNA or a genomic sequence encoding a full length polypeptide of the present invention, can be used to construct a recombinant expression cassette which can be introduced into the desired host cell. A recombinant expression cassette will typically comprise a polynucleotide of the present invention operably linked to transcriptional initiation regulatory sequences which will direct the transcription of the polynucleotide in the intended host cell, such as tissues of a transformed plant.

[0113] For example, plant expression vectors may include (1) a cloned plant gene under the transcriptional control of 5' and 3' regulatory sequences and (2) a dominant selectable marker. Such plant expression vectors may also contain, if desired, a promoter regulatory region (e.g., one conferring inducible or constitutive, environmentally- or developmentally-regulated, or cell- or tissue-specific/selective expression), a transcription initiation start site, a ribosome binding site, an RNA processing signal, a transcription termination site and/or a polyadenylation signal.

[0114] A plant promoter fragment can be employed which will direct expression of a polynucleotide of the present invention in all tissues of a regenerated plant. Such promoters are referred to herein as "constitutive" promoters and are active under most environmental conditions and states of development or cell differentiation. Examples of constitutive promoters include the cauliflower mosaic virus (CaMV) 35S transcription initiation region, the 1'- or 2'-promoter derived from T-DNA of Agrobacterium tumefaciens, the ubiquitin 1 promoter, the Smas promoter, the cinnamyl alcohol dehydrogenase promoter (U.S. Pat. No. 5,683,439), the Nos promoter, the pEmu promoter, the rubisco promoter and the GRP1-8 promoter.

[0115] Alternatively, the plant promoter can direct expression of a polynucleotide of the present invention in a specific tissue or may be otherwise under more precise environmental or developmental control. Such promoters are referred to here as "inducible" promoters. Environmental conditions that may effect transcription by inducible promoters include pathogen attack, anaerobic conditions or the presence of light. Examples of inducible promoters are the Adh1 promoter which is inducible by hypoxia or cold stress, the Hsp70 promoter which is inducible by heat stress and the PPDK promoter which is inducible by light.

[0116] Examples of promoters under developmental control include promoters that initiate transcription only, or preferentially, in certain tissues, such as leaves, roots, fruit, seeds or flowers. Exemplary promoters include the anther-specific promoter 5126 (U.S. Pat. Nos. 5,689,049 and 5,689,051), glb-1 promoter and gamma-zein promoter. Also see, for example, U.S. Patent Application Ser. Nos. 60/155,859 and 60/163,114. The operation of a promoter may also vary depending on its location in the genome. Thus, an inducible promoter may become fully or partially constitutive in certain locations.

[0117] Both heterologous and non-heterologous (i.e., endogenous) promoters can be employed to direct expression of the nucleic acids of the present invention. These promoters can also be used, for example, in recombinant expression cassettes to drive expression of antisense nucleic acids to reduce, increase or alter concentration and/or composition of the proteins of the present invention in a desired tissue. Thus, in some embodiments, the nucleic acid construct will comprise a promoter, functional in a plant cell, operably linked to a polynucleotide of the present invention. Promoters useful in these embodiments include the endogenous promoters driving expression of a polypeptide of the present invention.

[0118] In some embodiments, isolated nucleic acids which serve as promoter or enhancer elements can be introduced in the appropriate position (generally upstream) of a non-heterologous form of a polynucleotide of the present invention so as to up or down regulate expression of a polynucleotide of the present invention. For example, endogenous promoters can be altered in vivo by mutation, deletion and/or substitution (see, Kmiec, U.S. Pat. No. 5,565,350; Zarling, et al., PCT/US93/03868) or isolated promoters can be introduced into a plant cell in the proper orientation and distance from a cognate gene of a polynucleotide of the present invention so as to control the expression of the gene. Gene expression can be modulated under conditions suitable for plant growth so as to alter the total concentration and/or alter the composition of the polypeptides of the present invention in plant cell. Thus, the present invention provides compositions, and methods for making, heterologous promoters and/or enhancers operably linked to a native, endogenous (i.e., non-heterologous) form of a polynucleotide of the present invention.

[0119] If polypeptide expression is desired, it is generally desirable to include a polyadenylation region at the 3'-end of a polynucleotide coding region. The polyadenylation region can be derived from the natural gene, from a variety of other plant genes or from T-DNA. The 3' end sequence to be added can be derived from, for example, the nopaline synthase or octopine synthase genes or alternatively from another plant gene or less preferably from any other eukaryotic gene.

[0120] An intron sequence can be added to the 5' untranslated region or the coding sequence of the partial coding sequence to increase the amount of the mature message that accumulates in the cytosol. Inclusion of a spliceable intron in the transcription unit in both plant and animal expression constructs has been shown to increase gene expression at both the mRNA and protein levels up to 1000-fold. Buchman and Berg, (1988) Mol. Cell Biol. 8:4395-4405; Callis, et al., (1987) Genes Dev. 1:1183-1200. Such intron enhancement of gene expression is typically greatest when placed near the 5' end of the transcription unit. Use of maize introns Adh1-S intron 1, 2, and 6, the Bronze-1 intron are known in the art. See generally, The Maize Handbook, Chapter 116, Freeling and Walbot, Eds., Springer, New York (1994). The vector comprising the sequences from a polynucleotide of the present invention will typically comprise a marker gene which confers a selectable phenotype on plant cells. Typical vectors useful for expression of genes in higher plants are well known in the art and include vectors derived from the tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens described by Rogers, et al., (1987) Meth. in Enzymol. 153:253-277.

[0121] A polynucleotide of the present invention can be expressed in either sense or anti-sense orientation as desired. It will be appreciated that control of gene expression in either sense or anti-sense orientation can have a direct impact on the observable plant characteristics. Antisense technology can be conveniently used to inhibit gene expression in plants. To accomplish this, a nucleic acid segment from the desired gene is cloned and operably linked to a promoter such that the anti-sense strand of RNA will be transcribed. The construct is then transformed into plants and the antisense strand of RNA is produced. In plant cells, it has been shown that antisense RNA inhibits gene expression by preventing the accumulation of mRNA which encodes the enzyme of interest, see, e.g., Sheehy, et al., (1988) Proc. Nat'l. Acad. Sci. (USA) 85:8805-8809 and Hiatt, et al., U.S. Pat. No. 4,801,340.

[0122] Another method of suppression is sense suppression (i.e., co-supression). Introduction of nucleic acid configured in the sense orientation has been shown to be an effective means by which to block the transcription of target genes. For an example of the use of this method to modulate expression of endogenous genes see, Napoli, et al., (1990) The Plant Cell 2:279-289 and U.S. Pat. No. 5,034,323.

[0123] Catalytic RNA molecules or ribozymes can also be used to inhibit expression of plant genes. It is possible to design ribozymes that specifically pair with virtually any target RNA and cleave the phosphodiester backbone at a specific location, thereby functionally inactivating the target RNA. In carrying out this cleavage, the ribozyme is not itself altered, and is thus capable of recycling and cleaving other molecules, making it a true enzyme. The inclusion of ribozyme sequences within antisense RNAs confers RNA-cleaving activity upon them, thereby increasing the activity of the constructs. The design and use of target RNA-specific ribozymes is described in Haseloff, et al., (1988) Nature 334:585-591.

[0124] A variety of cross-linking agents, alkylating agents and radical generating species as pendant groups on polynucleotides of the present invention can be used to bind, label, detect and/or cleave nucleic acids. For example, Vlassov, et al., (1986) Nucleic Acids Res 14:4065-4076, describe covalent bonding of a single-stranded DNA fragment with alkylating derivatives of nucleotides complementary to target sequences. A report of similar work by the same group is that by Knorre, et al., (1985) Biochimie 67:785-789. Iverson and Dervan also showed sequence-specific cleavage of single-stranded DNA mediated by incorporation of a modified nucleotide which was capable of activating cleavage (J Am Chem Soc (1987) 109:1241-1243). Meyer, et al., (1989) J Am Chem Soc 111:8517-8519, effect covalent crosslinking to a target nucleotide using an alkylating agent complementary to the single-stranded target nucleotide sequence. A photoactivated crosslinking to single-stranded oligonucleotides mediated by psoralen was disclosed by Lee, et al., (1988) Biochemistry 27:3197-3203. Use of crosslinking in triple-helix forming probes was also disclosed by Home, et al., (1990) J Am Chem Soc 112:2435-2437. Use of N4,N4-ethanocytosine as an alkylating agent to crosslink to single-stranded oligonucleotides has also been described by Webb and Matteucci, (1986) J Am Chem Soc 108:2764-2765; Nucleic Acids Res (1986) 14:7661-7674; Feteritz, et al., (1991) J. Am. Chem. Soc. 113:4000. Various compounds to bind, detect, label and/or cleave nucleic acids are known in the art. See, for example, U.S. Pat. Nos. 5,543,507; 5,672,593; 5,484,908; 5,256,648 and 5,681,941.

Proteins

[0125] The isolated proteins of the present invention comprise a polypeptide having at least 10 amino acids from a polypeptide of the present invention (or conservative variants thereof) such as those encoded by any one of the polynucleotides of the present invention as discussed more fully above (e.g., Table 1). The proteins of the present invention or variants thereof can comprise any number of contiguous amino acid residues from a polypeptide of the present invention, wherein that number is selected from the group of integers consisting of from 10 to the number of residues in a full-length polypeptide of the present invention. Optionally, this subsequence of contiguous amino acids is at least 15, 20, 25, 30, 35 or 40 amino acids in length, often at least 50, 60, 70, 80 or 90 amino acids in length. Further, the number of such subsequences can be any integer selected from the group consisting of from 1 to 20, such as 2, 3, 4 or 5.

[0126] The present invention further provides a protein comprising a polypeptide having a specified sequence identity/similarity with a polypeptide of the present invention. The percentage of sequence identity/similarity is an integer selected from the group consisting of from 50 to 99. Exemplary sequence identity/similarity values include 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% and 95%. Sequence identity can be determined using, for example, the GAP, CLUSTALW or BLAST algorithms.

[0127] As those of skill will appreciate, the present invention includes, but is not limited to, catalytically active polypeptides of the present invention (i.e., enzymes). Catalytically active polypeptides have a specific activity of at least 20%, 30% or 40% and preferably at least 50%, 60% or 70% and most preferably at least 80%, 90% or 95% that of the native (non-synthetic), endogenous polypeptide. Further, the substrate specificity (k.sub.cat/K.sub.m) is optionally substantially similar to the native (non-synthetic), endogenous polypeptide. Typically, the K.sub.m will be at least 30%, 40%, or 50%, that of the native (non-synthetic), endogenous polypeptide; and more preferably at least 60%, 70%, 80% or 90%. Methods of assaying and quantifying measures of enzymatic activity and substrate specificity (k.sub.cat/K.sub.m) are well known to those of skill in the art.

[0128] Generally, the proteins of the present invention will, when presented as an immunogen, elicit production of an antibody specifically reactive to a polypeptide of the present invention. Further, the proteins of the present invention will not bind to antisera raised against a polypeptide of the present invention which has been fully immunosorbed with the same polypeptide. Immunoassays for determining binding are well known to those of skill in the art. A preferred immunoassay is a competitive immunoassay. Thus, the proteins of the present invention can be employed as immunogens for constructing antibodies immunoreactive to a protein of the present invention for such exemplary utilities as immunoassays or protein purification techniques.

Expression of Proteins in Host Cells

[0129] Using the nucleic acids of the present invention, one may express a protein of the present invention in a recombinantly engineered cell such as bacteria, yeast, insect, mammalian or preferably plant cells. The cells produce the protein in a non-natural condition (e.g., in quantity, composition, location and/or time), because they have been genetically altered through human intervention to do so.

[0130] It is expected that those of skill in the art are knowledgeable in the numerous expression systems available for expression of a nucleic acid encoding a protein of the present invention. No attempt to describe in detail the various methods known for the expression of proteins in prokaryotes or eukaryotes will be made.

[0131] In brief summary, the expression of isolated nucleic acids encoding a protein of the present invention will typically be achieved by operably linking, for example, the DNA or cDNA to a promoter (which is either constitutive or regulatable), followed by incorporation into an expression vector. The vectors can be suitable for replication and integration in either prokaryotes or eukaryotes. Typical expression vectors contain transcription and translation terminators, initiation sequences and promoters useful for regulation of the expression of the DNA encoding a protein of the present invention. To obtain high level expression of a cloned gene, it is desirable to construct expression vectors which contain, at the minimum, a strong promoter to direct transcription, a ribosome binding site for translational initiation and a transcription/translation terminator. One of skill would recognize that modifications can be made to a protein of the present invention without diminishing its biological activity. Some modifications may be made to facilitate the cloning, expression, or incorporation of the targeting molecule into a fusion protein. Such modifications are well known to those of skill in the art and include, for example, a methionine added at the amino terminus to provide an initiation site or additional amino acids (e.g., poly His) placed on either terminus to create conveniently located purification sequences. Restriction sites or termination codons can also be introduced.

Synthesis of Proteins

[0132] The proteins of the present invention can be constructed using non-cellular synthetic methods. Solid phase synthesis of proteins of less than about 50 amino acids in length may be accomplished by attaching the C-terminal amino acid of the sequence to an insoluble support followed by sequential addition of the remaining amino acids in the sequence. Techniques for solid phase synthesis are described by Barany and Merrifield, Solid-Phase Peptide Synthesis, pp. 3-284 in The Peptides: Analysis, Synthesis, Biology Vol. 2: Special Methods in Peptide Synthesis, Part A.; Merrifield, et al., (1963) J. Am. Chem. Soc. 85:2149-2156 and Stewart, et al., Solid Phase Peptide Synthesis, 2nd ed., Pierce Chem. Co., Rockford, Ill. (1984). Proteins of greater length may be synthesized by condensation of the amino and carboxy termini of shorter fragments. Methods of forming peptide bonds by activation of a carboxy terminal end (e.g., by the use of the coupling reagent N,N'-dicycylohexylcarbodiimide) are known to those of skill.

Purification of Proteins

[0133] The proteins of the present invention may be purified by standard techniques well known to those of skill in the art. Recombinantly produced proteins of the present invention can be directly expressed or expressed as a fusion protein. The recombinant protein is purified by a combination of cell lysis (e.g., sonication, French press) and affinity chromatography. For fusion products, subsequent digestion of the fusion protein with an appropriate proteolytic enzyme releases the desired recombinant protein.

[0134] The proteins of this invention, recombinant or synthetic, may be purified to substantial purity by standard techniques well known in the art, including detergent solubilization, selective precipitation with such substances as ammonium sulfate, column chromatography, immunopurification methods and others. See, for instance, Scopes, Protein Purification: Principles and Practice, Springer-Verlag: New York (1982); Deutscher, Guide to Protein Purification, Academic Press (1990). For example, antibodies may be raised to the proteins as described herein. Purification from E. coli can be achieved following procedures described in U.S. Pat. No. 4,511,503. The protein may then be isolated from cells expressing the protein and further purified by standard protein chemistry techniques as described herein. Detection of the expressed protein is achieved by methods known in the art and include, for example, radioimmunoassays, Western blotting techniques or immunoprecipitation.

Introduction of Nucleic Acids into Host Cells

[0135] The method of introducing a nucleic acid of the present invention into a host cell is not critical to the instant invention. Transformation or transfection methods are conveniently used. Accordingly, a wide variety of methods have been developed to insert a DNA sequence into the genome of a host cell to obtain the transcription and/or translation of the sequence to effect phenotypic changes in the organism. Thus, any method which provides for effective introduction of a nucleic acid may be employed.

A. Plant Transformation

[0136] A nucleic acid comprising a polynucleotide of the present invention is optionally introduced into a plant. Generally, the polynucleotide will first be incorporated into a recombinant expression cassette or vector. Isolated nucleic acid acids of the present invention can be introduced into plants according to techniques known in the art. Techniques for transforming a wide variety of higher plant species are well known and described in the technical, scientific, and patent literature. See, for example, Weising, et al., (1988) Ann. Rev. Genet. 22:421-477. For example, the DNA construct may be introduced directly into the genomic DNA of the plant cell using techniques such as electroporation, polyethylene glycol (PEG) poration, particle bombardment, silicon fiber delivery or microinjection of plant cell protoplasts or embryogenic callus. See, e.g., Tomes, et al., Direct DNA Transfer into Intact Plant Cells Via Microprojectile Bombardment. pp. 197-213 in Plant Cell, Tissue and Organ Culture, Fundamental Methods. eds. Gamborg and Phillips. Springer-Verlag Berlin Heidelberg New York, 1995; see, U.S. Pat. No. 5,990,387. The introduction of DNA constructs using PEG precipitation is described in Paszkowski, et al., (1984) Embo J. 3:2717-2722. Electroporation techniques are described in Fromm, et al., (1985) Proc. Natl. Acad. Sci. (USA) 82:5824. Ballistic transformation techniques are described in Klein, et al., (1987) Nature 327:70-73.

[0137] Agrobacterium tumefaciens-mediated transformation techniques are well described in the scientific literature. See, for example, Horsch, et al., (1984) Science 233:496-498; Fraley, et al., (1983) Proc. Natl. Acad. Sci. (USA) 80:4803 and Plant Molecular Biology: A Laboratory Manual, Chapter 8, Clark, Ed., Springer-Verlag, Berlin (1997). The DNA constructs may be combined with suitable T-DNA flanking regions and introduced into a conventional Agrobacterium tumefaciens host vector. The virulence functions of the Agrobacterium tumefaciens host will direct the insertion of the construct and adjacent marker into the plant cell DNA when the cell is infected by the bacteria. See, U.S. Pat. No. 5,591,616. Although Agrobacterium is useful primarily in dicots, certain monocots can be transformed by Agrobacterium. For instance, Agrobacterium transformation of maize is described in U.S. Pat. No. 5,550,318.

[0138] Other methods of transfection or transformation include (1) Agrobacterium rhizogenes-mediated transformation (see, e.g., Lichtenstein and Fuller In: Genetic Engineering, vol. 6, Rigby, Ed., London, Academic Press, 1987; and Lichtenstein, and Draper, In: DNA Cloning, Vol. II, Glover, Ed., Oxford, IRI Press, 1985), PCT Application Number PCT/US87/02512 (WO 1988/02405 published Apr. 7, 1988) describes the use of A. rhizogenes strain A4 and its Ri plasmid along with A. tumefaciens vectors pARC8 or pARC16 (2) liposome-mediated DNA uptake (see, e.g., Freeman, et al., (1984) Plant Cell Physiol. 25:1353), (3) the vortexing method (see, e.g., Kindle, (1990) Proc. Natl. Acad. Sci., (USA) 87:1228).

[0139] DNA can also be introduced into plants by direct DNA transfer into pollen as described by Zhou, et al., (1983) Methods in Enzymology 101:433; Hess, (1987) Intern Rev. Cytol. 107:367; Luo, et al., (1988) Plant Mol. Biol. Reporter 6:165. Expression of polypeptide coding genes can be obtained by injection of the DNA into reproductive organs of a plant as described by Pena, et al., (1987) Nature, 325.274. DNA can also be injected directly into the cells of immature embryos and the rehydration of desiccated embryos as described by Neuhaus, et al., (1987) Theor. Appl. Genet., 75:30 and Benbrook, et al., in Proceedings Bio Expo 1986, Butterworth, Stoneham, Mass., pp. 27-54 (1986). A variety of plant viruses that can be employed as vectors are known in the art and include cauliflower mosaic virus (CaMV), geminivirus, brome mosaic virus and tobacco mosaic virus.

B. Transfection of Prokaryotes, Lower Eukaryotes, and Animal Cells

[0140] 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 dextran, 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, Biochemical Methods in Cell Culture and Virology, Dowden, Hutchinson and Ross, Inc. (1977).

Transgenic Plant Regeneration

[0141] Plant cells which directly result or are derived from the nucleic acid introduction techniques can be cultured to regenerate a whole plant which possesses the introduced genotype. Such regeneration techniques often rely on manipulation of certain phytohormones in a tissue culture growth medium. Plants cells can be regenerated, e.g., from single cells, callus tissue or leaf discs according to standard plant tissue culture techniques. It is well known in the art that various cells, tissues, and organs from almost any plant can be successfully cultured to regenerate an entire plant. Plant regeneration from cultured protoplasts is described in Evans, et al., Protoplasts Isolation and Culture, Handbook of Plant Cell Culture, Macmillan Publishing Company, New York, pp. 124-176 (1983) and Binding, Regeneration of Plants, Plant Protoplasts, CRC Press, Boca Raton, pp. 21-73 (1985).

[0142] The regeneration of plants from either single plant protoplasts or various explants is well known in the art. See, for example, Methods for Plant Molecular Biology, Weissbach and Weissbach, eds., Academic Press, Inc., San Diego, Calif. (1988). This regeneration and growth process includes the steps of selection of transformant cells and shoots, rooting the transformant shoots and growth of the plantlets in soil. For maize cell culture and regeneration see generally, The Maize Handbook, Freeling and Walbot, Eds., Springer, New York (1994); Corn and Corn Improvement, 3.sup.rd edition, Sprague and Dudley Eds., American Society of Agronomy, Madison, Wis. (1988). For transformation and regeneration of maize see, Gordon-Kamm, et al., (1990) The Plant Cell 2:603-618.

[0143] The regeneration of plants containing the polynucleotide of the present invention and introduced by Agrobacterium from leaf explants can be achieved as described by Horsch, et al., (1985) Science, 227:1229-1231. In this procedure, transformants are grown in the presence of a selection agent and in a medium that induces the regeneration of shoots in the plant species being transformed as described by Fraley, et al., (1983) Proc. Natl. Acad. Sci. (U.S.A.) 80:4803. This procedure typically produces shoots within two to four weeks and these transformant shoots are then transferred to an appropriate root-inducing medium containing the selective agent and an antibiotic to prevent bacterial growth. Transgenic plants of the present invention may be fertile or sterile.

[0144] One of skill will recognize that after the recombinant expression cassette is stably incorporated in transgenic plants and confirmed to be operable, it can be introduced into other plants by sexual crossing. Any of a number of standard breeding techniques can be used, depending upon the species to be crossed. In vegetatively propagated crops, mature transgenic plants can be propagated by the taking of cuttings or by tissue culture techniques to produce multiple identical plants. Selection of desirable transgenics is made and new varieties are obtained and propagated vegetatively for commercial use. In seed propagated crops, mature transgenic plants can be self-crossed to produce a homozygous inbred plant. The inbred plant produces seed containing the newly introduced heterologous nucleic acid. These seeds can be grown to produce plants that would produce the selected phenotype. Parts obtained from the regenerated plant, such as flowers, seeds, leaves, branches, fruit and the like are included in the invention, provided that these parts comprise cells comprising the isolated nucleic acid of the present invention. Progeny and 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.

[0145] Transgenic plants expressing a polynucleotide of the present invention can be screened for transmission of the nucleic acid of the present invention by, for example, standard immunoblot and DNA detection techniques. Expression at the RNA level can be determined initially to identify and quantitate expression-positive plants. Standard techniques for RNA analysis can be employed and include PCR amplification assays using oligonucleotide primers designed to amplify only the heterologous RNA templates and solution hybridization assays using heterologous nucleic acid-specific probes. The RNA-positive plants can then analyzed for protein expression by Western immunoblot analysis using the specifically reactive antibodies of the present invention. In addition, in situ hybridization and immunocytochemistry according to standard protocols can be done using heterologous nucleic acid specific polynucleotide probes and antibodies, respectively, to localize sites of expression within transgenic tissue. Generally, a number of transgenic lines are usually screened for the incorporated nucleic acid to identify and select plants with the most appropriate expression profiles.

[0146] A preferred embodiment is a transgenic plant that is homozygous for the added heterologous nucleic acid; i.e., a transgenic plant that contains two added nucleic acid sequences, one gene at the same locus on each chromosome of a chromosome pair. A homozygous transgenic plant can be obtained by sexually mating (selfing) a heterozygous transgenic plant that contains a single added heterologous nucleic acid, germinating some of the seed produced and analyzing the resulting plants produced for altered expression of a polynucleotide of the present invention relative to a control plant (i.e., native, non-transgenic). Back-crossing to a parental plant and out-crossing with a non-transgenic plant are also contemplated.

Modulating Polypeptide Levels and/or Composition

[0147] The present invention further provides a method for modulating (i.e., increasing or decreasing) the concentration or ratio of the polypeptides of the present invention in a plant or part thereof. Modulation can be effected by increasing or decreasing the concentration and/or the ratio of the polypeptides of the present invention in a plant. The method comprises introducing into a plant cell a recombinant expression cassette comprising a polynucleotide of the present invention as described above to obtain a transgenic plant cell, culturing the transgenic plant cell under transgenic plant cell growing conditions and inducing or repressing expression of a polynucleotide of the present invention in the transgenic plant for a time sufficient to modulate concentration and/or the ratios of the polypeptides in the transgenic plant or plant part.

[0148] In some embodiments, the concentration and/or ratios of polypeptides of the present invention in a plant may be modulated by altering, in vivo or in vitro, the promoter of a gene to up- or down-regulate gene expression. In some embodiments, the coding regions of native genes of the present invention can be altered via substitution, addition, insertion or deletion to decrease activity of the encoded enzyme. (See, e.g., Kmiec, U.S. Pat. No. 5,565,350; Zarling, et al., PCT/US93/03868.) And in some embodiments, an isolated nucleic acid (e.g., a vector) comprising a promoter sequence is transfected into a plant cell. Subsequently, a plant cell comprising the promoter operably linked to a polynucleotide of the present invention is selected for by means known to those of skill in the art such as, but not limited to, Southern blot, DNA sequencing or PCR analysis using primers specific to the promoter and to the gene and detecting amplicons produced therefrom. 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 ratios of polypeptides of the present invention in the plant. Plant forming conditions are well known in the art and discussed briefly, supra.

[0149] In general, concentration or the ratios of the polypeptides is increased or decreased by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% relative to a native control plant, plant part or cell lacking the aforementioned recombinant expression cassette. Modulation in the present invention may occur during and/or subsequent to growth of the plant to the desired stage of development. Modulating nucleic acid expression temporally and/or in particular tissues can be controlled by employing the appropriate promoter operably linked to a polynucleotide of the present invention in, for example, sense or antisense orientation as discussed in greater detail, supra. Induction of expression of a polynucleotide of the present invention can also be controlled by exogenous administration of an effective amount of inducing compound. Inducible promoters and inducing compounds which activate expression from these promoters are well known in the art. In preferred embodiments, the polypeptides of the present invention are modulated in monocots, particularly maize.

UTRs and Codon Preference

[0150] In general, translational efficiency has been found to be regulated by specific sequence elements in the 5' non-coding or untranslated region (5' UTR) of the RNA. Positive sequence motifs include translational initiation consensus sequences (Kozak, (1987) Nucleic Acids Res. 15:8125) and the 7-methylguanosine cap structure (Drummond, et al., (1985) Nucleic Acids Res. 13:7375). Negative elements include stable intramolecular 5' UTR stem-loop structures (Muesing, et al., (1987) Cell 48:691) and AUG sequences or short open reading frames preceded by an appropriate AUG in the 5' UTR (Kozak, supra, Rao, et al., (1988) Mol. and Cell. Biol. 8:284). Accordingly, the present invention provides 5' and/or 3' untranslated regions for modulation of translation of heterologous coding sequences.

[0151] Further, the polypeptide-encoding segments of the polynucleotides of the present invention can be modified to alter codon usage. Altered codon usage can be employed to alter translational efficiency and/or to optimize the coding sequence for expression in a desired host such as to optimize the codon usage in a heterologous sequence for expression in maize. Codon usage in the coding regions of the polynucleotides of the present invention can be analyzed statistically using commercially available software packages such as "Codon Preference" available from the University of Wisconsin Genetics Computer Group (see, Devereaux, et al., (1984) Nucleic Acids Res. 12:387-395) or MacVector 4.1 (Eastman Kodak Co., New Haven, Conn.). Thus, the present invention provides a codon usage frequency characteristic of the coding region of at least one of the polynucleotides of the present invention. The number of polynucleotides that can be used to determine a codon usage frequency can be any integer from 1 to the number of polynucleotides of the present invention as provided herein. Optionally, the polynucleotides will be full-length sequences. An exemplary number of sequences for statistical analysis can be at least 1, 5, 10, 20, 50 or 100.

Sequence Shuffling

[0152] The present invention provides methods for sequence shuffling using polynucleotides of the present invention, and compositions resulting therefrom. Sequence shuffling is described in PCT Publication Number WO 1997/20078. See also, Zhang, et al., (1997) Proc. Natl. Acad. Sci. USA 94:4504-4509. Generally, sequence shuffling provides a means for generating libraries of polynucleotides having a desired characteristic which can be selected or screened for. Libraries of recombinant polynucleotides are generated from a population of related sequence polynucleotides which comprise sequence regions which have substantial sequence identity and can be homologously recombined in vitro or in vivo. The population of sequence-recombined polynucleotides comprises a subpopulation of polynucleotides which possess desired or advantageous characteristics and which can be selected by a suitable selection or screening method. The characteristics can be any property or attribute capable of being selected for or detected in a screening system and may include properties of: an encoded protein, a transcriptional element, a sequence controlling transcription, RNA processing, RNA stability, chromatin conformation, translation or other expression property of a gene or transgene, a replicative element, a protein-binding element or the like, such as any feature which confers a selectable or detectable property. In some embodiments, the selected characteristic will be a decreased K.sub.m and/or increased K.sub.cat over the wild-type protein as provided herein. In other embodiments, a protein or polynucleotide generated from sequence shuffling will have a ligand binding affinity greater than the non-shuffled wild-type polynucleotide. The increase in such properties can be at least 110%, 120%, 130%, 140% or at least 150% of the wild-type value.

Generic and Consensus Sequences

[0153] Polynucleotides and polypeptides of the present invention further include those having: (a) a generic sequence of at least two homologous polynucleotides or polypeptides, respectively, of the present invention and (b) a consensus sequence of at least three homologous polynucleotides or polypeptides, respectively, of the present invention. The generic sequence of the present invention comprises each species of polypeptide or polynucleotide embraced by the generic polypeptide or polynucleotide sequence, respectively. The individual species encompassed by a polynucleotide having an amino acid or nucleic acid consensus sequence can be used to generate antibodies or produce nucleic acid probes or primers to screen for homologs in other species, genera, families, orders, classes, phyla or kingdoms. For example, a polynucleotide having a consensus sequence from a gene family of Zea mays can be used to generate antibody or nucleic acid probes or primers to other Gramineae species such as wheat, rice or sorghum. Alternatively, a polynucleotide having a consensus sequence generated from orthologous genes can be used to identify or isolate orthologs of other taxa. Typically, a polynucleotide having a consensus sequence will be at least 9, 10, 15, 20, 25, 30 or 40 amino acids in length, or 20, 30, 40, 50, 100 or 150 nucleotides in length. As those of skill in the art are aware, a conservative amino acid substitution can be used for amino acids which differ amongst aligned sequence but are from the same conservative substitution group as discussed above. Optionally, no more than 1 or 2 conservative amino acids are substituted for each 10 amino acid length of consensus sequence.

[0154] Similar sequences used for generation of a consensus or generic sequence include any number and combination of allelic variants of the same gene, orthologous or paralogous sequences as provided herein. Optionally, similar sequences used in generating a consensus or generic sequence are identified using the BLAST algorithm's smallest sum probability (P(N)). Various suppliers of sequence-analysis software are listed in chapter 7 of Current Protocols in Molecular Biology, Ausubel et al., Eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc. (Supplement 30). A polynucleotide sequence is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.1, more preferably less than about 0.01, or 0.001 and most preferably less than about 0.0001 or 0.00001. Similar polynucleotides can be aligned and a consensus or generic sequence generated using multiple sequence alignment software available from a number of commercial suppliers such as the Genetics Computer Group's (Madison, Wis.) PILEUP software, Vector NTI's (North Bethesda, Md.) ALIGNX, or Genecode's (Ann Arbor, Mich.) SEQUENCHER. Conveniently, default parameters of such software can be used to generate consensus or generic sequences.

Machine Applications

[0155] The present invention provides machines, data structures, and processes for modeling or analyzing the polynucleotides and polypeptides of the present invention.

A. Machines: Data, Data Structures, Processes and Functions

[0156] The present invention provides a machine having a memory comprising: 1) data representing a sequence of a polynucleotide or polypeptide of the present invention, 2) a data structure which reflects the underlying organization and structure of the data and facilitates program access to data elements corresponding to logical sub-components of the sequence, 3) processes for effecting the use, analysis, or modeling of the sequence, and 4) optionally, a function or utility for the polynucleotide or polypeptide. Thus, the present invention provides a memory for storing data that can be accessed by a computer programmed to implement a process for affecting the use, analyses or modeling of a sequence of a polynucleotide, with the memory comprising data representing the sequence of a polynucleotide of the present invention.

[0157] The machine of the present invention is typically a digital computer. The term "computer" includes one or several desktop or portable computers, computer workstations, servers (including intranet or internet servers), mainframes and any integrated system comprising any of the above irrespective of whether the processing, memory, input or output of the computer is remote or local, as well as any networking interconnecting the modules of the computer. The term "computer" is exclusive of computers of the United States Patent and Trademark Office or the European Patent Office when data representing the sequence of polypeptides or polynucleotides of the present invention is used for patentability searches.

[0158] The present invention contemplates providing as data a sequence of a polynucleotide of the present invention embodied in a computer readable medium. As those of skill in the art will be aware, the form of memory of a machine of the present invention or the particular embodiment of the computer readable medium, are not critical elements of the invention and can take a variety of forms. The memory of such a machine includes, but is not limited to, ROM or RAM or computer readable media such as, but not limited to, magnetic media such as computer disks or hard drives or media such as CD-ROMs, DVDs and the like.

[0159] The present invention further contemplates providing a data structure that is also contained in memory. The data structure may be defined by the computer programs that define the processes (see below) or it may be defined by the programming of separate data storage and retrieval programs subroutines or systems. Thus, the present invention provides a memory for storing a data structure that can be accessed by a computer programmed to implement a process for affecting the use, analysis or modeling of a sequence of a polynucleotide. The memory comprises data representing a polynucleotide having the sequence of a polynucleotide of the present invention. The data is stored within memory. Further, a data structure, stored within memory, is associated with the data reflecting the underlying organization and structure of the data to facilitate program access to data elements corresponding to logical sub-components of the sequence. The data structure enables the polynucleotide to be identified and manipulated by such programs.

[0160] In a further embodiment, the present invention provides a data structure that contains data representing a sequence of a polynucleotide of the present invention stored within a computer readable medium. The data structure is organized to reflect the logical structuring of the sequence, so that the sequence is easily analyzed by software programs capable of accessing the data structure. In particular, the data structures of the present invention organize the reference sequences of the present invention in a manner which allows software tools to perform a wide variety of analyses using logical elements and sub-elements of each sequence.

[0161] An example of such a data structure resembles a layered hash table, where in one dimension the base content of the sequence is represented by a string of elements A, T, C, G and N. The direction from the 5' end to the 3' end is reflected by the order from the position 0 to the position of the length of the string minus one. Such a string, corresponding to a nucleotide sequence of interest, has a certain number of substrings, each of which is delimited by the string position of its 5' end and the string position of its 3' end within the parent string. In a second dimension, each substring is associated with or pointed to one or multiple attribute fields. Such attribute fields contain annotations to the region on the nucleotide sequence represented by the substring.

[0162] For example, a sequence under investigation is 520 bases long and represented by a string named SeqTarget. There is a minor groove in the 5' upstream non-coding region from position 12 to 38, which is identified as a binding site for an enhancer protein HM-A, which in turn will increase the transcription of the gene represented by SeqTarget. Here, the substring is represented as (12, 38) and has the following attributes: [upstream uncoded], [minor groove], [HM-A binding] and [increase transcription upon binding by HM-A]. Similarly, other types of information can be stored and structured in this manner, such as information related to the whole sequence, e.g., whether the sequence is a full length viral gene, a mammalian house keeping gene or an EST from clone X, information related to the 3' down stream non-coding region, e.g., hair pin structure and information related to various domains of the coding region, e.g., Zinc finger.

[0163] This data structure is an open structure and is robust enough to accommodate newly generated data and acquired knowledge. Such a structure is also a flexible structure. It can be trimmed down to a 1-D string to facilitate data mining and analysis steps, such as clustering, repeat-masking, and HMM analysis. Meanwhile, such a data structure also can extend the associated attributes into multiple dimensions. Pointers can be established among the dimensioned attributes when needed to facilitate data management and processing in a comprehensive genomics knowledgebase. Furthermore, such a data structure is object-oriented. Polymorphism can be represented by a family or class of sequence objects, each of which has an internal structure as discussed above. The common traits are abstracted and assigned to the parent object, whereas each child object represents a specific variant of the family or class. Such a data structure allows data to be efficiently retrieved, updated and integrated by the software applications associated with the sequence database and/or knowledgebase.

[0164] The present invention contemplates providing processes for effecting analysis and modeling, which are described in the following section.

[0165] Optionally, the present invention further contemplates that the machine of the present invention will embody in some manner a utility or function for the polynucleotide or polypeptide of the present invention. The function or utility of the polynucleotide or polypeptide can be a function or utility for the sequence data, per se, or of the tangible material. Exemplary function or utilities include the name (per International Union of Biochemistry and Molecular Biology rules of nomenclature) or function of the enzyme or protein represented by the polynucleotide or polypeptide of the present invention; the metabolic pathway of the protein represented by the polynucleotide or polypeptide of the present invention; the substrate or product or structural role of the protein represented by the polynucleotide or polypeptide of the present invention or the phenotype (e.g., an agronomic or pharmacological trait) affected by modulating expression or activity of the protein represented by the polynucleotide or polypeptide of the present invention.

B. Computer Analysis and Modeling

[0166] The present invention provides a process of modeling and analyzing data representative of a polynucleotide or polypeptide sequence of the present invention. The process comprises entering sequence data of a polynucleotide or polypeptide of the present invention into a machine having a hardware or software sequence modeling and analysis system, developing data structures to facilitate access to the sequence data, manipulating the data to model or analyze the structure or activity of the polynucleotide or polypeptide and displaying the results of the modeling or analysis. Thus, the present invention provides a process for affecting the use, analysis or modeling of a polynucleotide sequence or its derived peptide sequence through use of a computer having a memory. The process comprises: 1) placing into the memory data representing a polynucleotide having the sequence of a polynucleotide of the present invention, developing within the memory a data structure associated with the data and reflecting the underlying organization and structure of the data to facilitate program access to data elements corresponding to logical sub-components of the sequence, 2) programming the computer with a program containing instructions sufficient to implement the process for effecting the use, analysis or modeling of the polynucleotide sequence or the peptide sequence and 3) executing the program on the computer while granting the program access to the data and to the data structure within the memory.

[0167] A variety of modeling and analytic tools are well known in the art and available commercially. Included amongst the modeling/analysis tools are methods to: 1) recognize overlapping sequences (e.g., from a sequencing project) with a polynucleotide of the present invention and create an alignment called a "contig"; 2) identify restriction enzyme sites of a polynucleotide of the present invention; 3) identify the products of a T1 ribonuclease digestion of a polynucleotide of the present invention; 4) identify PCR primers with minimal self-complementarity; 5) compute pairwise distances between sequences in an alignment, reconstruct phylogentic trees using distance methods and calculate the degree of divergence of two protein coding regions; 6) identify patterns such as coding regions, terminators, repeats and other consensus patterns in polynucleotides of the present invention; 7) identify RNA secondary structure; 8) identify sequence motifs, isoelectric point, secondary structure, hydrophobicity and antigenicity in polypeptides of the present invention; 9) translate polynucleotides of the present invention and backtranslate polypeptides of the present invention and 10) compare two protein or nucleic acid sequences and identifying points of similarity or dissimilarity between them.

[0168] The processes for effecting analysis and modeling can be produced independently or obtained from commercial suppliers. Exemplary analysis and modeling tools are provided in products such as InforMax's (Bethesda, Md.) Vector NTI Suite (Version 5.5), Intelligenetics' (Mountain View, Calif.) PC/Gene program and Genetics Computer Group's (Madison, Wis.) Wisconsin Package.RTM. (Version 10.0); these tools, and the functions they perform, (as provided and disclosed by the programs and accompanying literature) are incorporated herein by reference and are described in more detail in section C which follows.

[0169] Thus, in a further embodiment, the present invention provides a machine-readable media containing a computer program and data, comprising a program stored on the media containing instructions sufficient to implement a process for affecting the use, analysis or modeling of a representation of a polynucleotide or peptide sequence. The data stored on the media represents a sequence of a polynucleotide having the sequence of a polynucleotide of the present invention. The media also includes a data structure reflecting the underlying organization and structure of the data to facilitate program access to data elements corresponding to logical sub-components of the sequence, the data structure being inherent in the program and in the way in which the program organizes and accesses the data.

C. Homology Searches

[0170] As an example of such a comparative analysis, the present invention provides a process of identifying a candidate homologue (i.e., an ortholog or paralog) of a polynucleotide or polypeptide of the present invention. The process comprises entering sequence data of a polynucleotide or polypeptide of the present invention into a machine having a hardware or software sequence analysis system, developing data structures to facilitate access to the sequence data, manipulating the data to analyze the structure the polynucleotide or polypeptide and displaying the results of the analysis. A candidate homologue has statistically significant probability of having the same biological function (e.g., catalyzes the same reaction, binds to homologous proteins/nucleic acids, has a similar structural role) as the reference sequence to which it is compared. Accordingly, the polynucleotides and polypeptides of the present invention have utility in identifying homologs in animals or other plant species, particularly those in the family Gramineae such as, but not limited to, sorghum, wheat or rice.

[0171] The process of the present invention comprises obtaining data representing a polynucleotide or polypeptide test sequence. Test sequences can be obtained from a nucleic acid of an animal or plant. Test sequences can be obtained directly or indirectly from sequence databases including, but not limited to, those such as: GenBank, EMBL, GenSeq, SWISS-PROT or those available on-line via the UK Human Genome Mapping Project (HGMP) GenomeWeb. In some embodiments the test sequence is obtained from a plant species other than maize whose function is uncertain but will be compared to the test sequence to determine sequence similarity or sequence identity. The test sequence data is entered into a machine, such as a computer, containing: i) data representing a reference sequence and ii) a hardware or software sequence comparison system to compare the reference and test sequence for sequence similarity or identity.

[0172] Exemplary sequence comparison systems are provided for in sequence analysis software such as those provided by the Genetics Computer Group (Madison, Wis.) or InforMax (Bethesda, Md.) or Intelligenetics (Mountain View, Calif.). Optionally, sequence comparison is established using the BLAST or GAP suite of programs. Generally, a smallest sum probability value (P(N)) of less than 0.1, or alternatively, less than 0.01, 0.001, 0.0001 or 0.00001 using the BLAST 2.0 suite of algorithms under default parameters identifies the test sequence as a candidate homologue (i.e., an allele, ortholog or paralog) of the reference sequence. Those of skill in the art will recognize that a candidate homologue has an increased statistical probability of having the same or similar function as the gene/protein represented by the test sequence.

[0173] The reference sequence can be the sequence of a polypeptide or a polynucleotide of the present invention. The reference or test sequence is each optionally at least 25 amino acids or at least 100 nucleotides in length. The length of the reference or test sequences can be the length of the polynucleotide or polypeptide described, respectively, above in the sections entitled "Nucleic Acids" (particularly section (g)) and "Proteins". As those of skill in the art are aware, the greater the sequence identity/similarity between a reference sequence of known function and a test sequence, the greater the probability that the test sequence will have the same or similar function as the reference sequence. The results of the comparison between the test and reference sequences are outputted (e.g., displayed, printed, recorded) via any one of a number of output devices and/or media (e.g., computer monitor, hard copy or computer readable medium).

Detection of Nucleic Acids

[0174] The present invention further provides methods for detecting a polynucleotide of the present invention in a nucleic acid sample suspected of containing a polynucleotide of the present invention, such as a plant cell lysate, particularly a lysate of maize. In some embodiments, a cognate gene of a polynucleotide of the present invention or portion thereof can be amplified prior to the step of contacting the nucleic acid sample with a polynucleotide of the present invention. The nucleic acid sample is contacted with the polynucleotide to form a hybridization complex. The polynucleotide hybridizes under stringent conditions to a gene encoding a polypeptide of the present invention. Formation of the hybridization complex is used to detect a gene encoding a polypeptide of the present invention in the nucleic acid sample. Those of skill will appreciate that an isolated nucleic acid comprising a polynucleotide of the present invention should lack cross-hybridizing sequences in common with non-target genes that would yield a false positive result. Detection of the hybridization complex can be achieved using any number of well known methods. For example, the nucleic acid sample, or a portion thereof, may be assayed by hybridization formats including but not limited to, solution phase, solid phase, mixed phase or in situ hybridization assays.

[0175] Detectable labels suitable for use in the present invention include any composition detectable by spectroscopic, radioisotopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Useful labels in the present invention include biotin for staining with labeled streptavidin conjugate, magnetic beads, fluorescent dyes, radiolabels, enzymes and colorimetric labels. Other labels include ligands which bind to antibodies labeled with fluorophores, chemiluminescent agents and enzymes. Labeling the nucleic acids of the present invention is readily achieved such as by the use of labeled PCR primers.

[0176] Although the present 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.

Example 1

[0177] This example describes the construction of a cDNA library.

[0178] Total RNA can be isolated from maize tissues with TRIzol Reagent (Life Technology Inc. Gaithersburg, Md.) using a modification of the guanidine isothiocyanate/acid-phenol procedure described by Chomczynski and Sacchi (Chomczynski and Sacchi, (1987) Anal. Biochem. 162:156). In brief, plant tissue samples is pulverized in liquid nitrogen before the addition of the TRIzol Reagent and then further homogenized with a mortar and pestle. Addition of chloroform followed by centrifugation is conducted for separation of an aqueous phase and an organic phase. The total RNA is recovered by precipitation with isopropyl alcohol from the aqueous phase.

[0179] The selection of poly(A)+ RNA from total RNA can be performed using PolyATact system (Promega Corporation. Madison, Wis.). Biotinylated oligo(dT) primers are used to hybridize to the 3' poly(A) tails on mRNA. The hybrids are captured using streptavidin coupled to paramagnetic particles and a magnetic separation stand. The mRNA is then washed at high stringency conditions and eluted by RNase-free deionized water. cDNA synthesis and construction of unidirectional cDNA libraries can be accomplished using the SuperScript Plasmid System (Life Technology Inc. Gaithersburg, Md.). The first strand of cDNA is synthesized by priming an oligo(dT) primer containing a Not I site. The reaction is catalyzed by SuperScript Reverse Transcriptase II at 45.degree. C. The second strand of cDNA is labeled with alpha-.sup.32P-dCTP and a portion of the reaction analyzed by agarose gel electrophoresis to determine cDNA sizes. cDNA molecules smaller than 500 base pairs and unligated adapters are removed by Sephacryl-S400 chromatography. The selected cDNA molecules are ligated into pSPORT1 vector in between of Not I and Sal I sites.

[0180] Alternatively, cDNA libraries can be prepared by any one of many methods available. For example, the cDNAs may be introduced into plasmid vectors by first preparing the cDNA libraries in Uni-ZAP.TM. XR vectors according to the manufacturer's protocol (Stratagene Cloning Systems, La Jolla, Calif.). The Uni-ZAP.TM. XR libraries are converted into plasmid libraries according to the protocol provided by Stratagene. Upon conversion, cDNA inserts will be contained in the plasmid vector pBluescript. In addition, the cDNAs may be introduced directly into precut Bluescript II SK(+) vectors (Stratagene) using T4 DNA ligase (New England Biolabs), followed by transfection into DH10B cells according to the manufacturer's protocol (GIBCO BRL Products). Once the cDNA inserts are in plasmid vectors, plasmid DNAs are prepared from randomly picked bacterial colonies containing recombinant pBluescript plasmids or the insert cDNA sequences are amplified via polymerase chain reaction using primers specific for vector sequences flanking the inserted cDNA sequences. Amplified insert DNAs or plasmid DNAs are sequenced in dye-primer sequencing reactions to generate partial cDNA sequences (expressed sequence tags or "ESTs"; see, Adams, et al., (1991) Science 252:1651-1656). The resulting ESTs are analyzed using a Perkin Elmer Model 377 fluorescent sequencer.

Example 2

[0181] This method describes construction of a full-length enriched cDNA library.

[0182] An enriched full-length cDNA library can be constructed using one of two variations of the method of Carninci, et al., (1996) Genomics 37:327-336. These variations are based on chemical introduction of a biotin group into the diol residue of the 5' cap structure of eukaryotic mRNA to select full-length first strand cDNA. The selection occurs by trapping the biotin residue at the cap sites using streptavidin-coated magnetic beads followed by RNase I treatment to eliminate incompletely synthesized cDNAs. Second strand cDNA is synthesized using established procedures such as those provided in Life Technologies' (Rockville, Md.) "SuperScript Plasmid System for cDNA Synthesis and Plasmid Cloning" kit. Libraries made by this method have been shown to contain 50% to 70% full-length cDNAs.

[0183] The first strand synthesis methods are detailed below. An asterisk denotes that the reagent was obtained from Life Technologies, Inc.

A. First Strand cDNA Synthesis Method 1 (with Trehalose)

TABLE-US-00003 mRNA (10 ug) 25 .mu.l *Not I primer (5 ug) 10 .mu.l *5x 1.sup.st strand buffer 43 .mu.l *0.1 m DTT 20 .mu.l *dNTP mix 10 mm 10 .mu.l BSA 10 ug/.mu.l 1 .mu.l Trehalose (saturated) 59.2 .mu.l RNase inhibitor (Promega) 1.8 .mu.l *Superscript II RT 200 u/.mu.l 20 .mu.l 100% glycerol 18 .mu.l Water 7 .mu.l

[0184] The mRNA and Not I primer are mixed and denatured at 65.degree. C. for 10 min. They are then chilled on ice and other components added to the tube. Incubation is at 45.degree. C. for 2 min. Twenty microliters of RT (reverse transcriptase) is added to the reaction and start program on the thermocycler (MJ Research, Waltham, Mass.):

TABLE-US-00004 Step 1 45.degree. C. 10 min Step 2 45.degree. C. -0.3.degree. C./cycle, 2 seconds/cycle Step 3 go to 2 for 33 cycles Step 4 35.degree. C. 5 min Step 5 45.degree. C. 5 min Step 6 45.degree. C. 0.2.degree. C./cycle, 1 sec/cycle Step 7 go to 7 for 49 cycles Step 8 55.degree. C. 0.1.degree. C./cycle, 12 sec/cycle Step 9 go to 8 for 49 cycles Step 10 55.degree. C. 2 min Step 11 60.degree. C. 2 min Step 12 go to 11 for 9 times Step 13 4.degree. C. forever Step 14 end

B. First Strand cDNA Synthesis Method 2

TABLE-US-00005 mRNA (10 .mu.g) 25 .mu.l water 30 .mu.l *Not I adapter primer (5 .mu.g) 10 .mu.l 65.degree. C. for 10 min, chill on ice, then add following reagents, *5x first buffer 20 .mu.l *0.1M DTT 10 .mu.l *10 mM dNTP mix 5 .mu.l

[0185] Incubate at 45.degree. C. for 2 min, then add 10 .mu.l of *Superscript II RT (200u/.mu.l), start the following program:

TABLE-US-00006 Step 1 45.degree. C. for 6 sec, -0.1.degree. C./cycle Step 2 go to 1 for 99 additional cycles Step 3 35.degree. C. for 5 min Step 4 45.degree. C. for 60 min Step 5 50.degree. C. for 10 min Step 6 4.degree. C. forever Step 7 end

[0186] After the 1.sup.st strand cDNA synthesis, the DNA is extracted by phenol according to standard procedures, and then precipitated in NaOAc and ethanol, and stored in -20.degree. C.

C. Oxidization of the Diol Group of mRNA for Biotin Labeling

[0187] First strand cDNA is spun down and washed once with 70% EtOH. The pellet resuspended in 23.2 .mu.l of DEPC treated water and put on ice. Prepare 100 mM of NalO4 freshly and then add the following reagents:

TABLE-US-00007 mRNA:1.sup.st cDNA (start with 20 .mu.g mRNA) 46.4 .mu.l 100 mM NaIO4 (freshly made) 2.5 .mu.l NaOAc 3M pH 4.5 1.1 .mu.l

[0188] To make 100 mM NalO4, use 21.39 .mu.g of NalO4 for 1 .mu.l of water.

[0189] Wrap the tube in a foil and incubate on ice for 45 min.

[0190] After the incubation, the reaction is then precipitated in:

TABLE-US-00008 5M NaCl 10 .mu.l 20% SDS 0.5 .mu.l isopropanol 61 .mu.l

[0191] Incubate on ice for at least 30 min, then spin it down at max speed at 4.degree. C. for 30 min and wash once with 70% ethanol and then 80% EtOH.

D. Biotinylation of the mRNA Diol Group

[0192] Resuspend the DNA in 110 .mu.l DEPC treated water, then add the following reagents:

TABLE-US-00009 20% SDS 5 .mu.l 2M NaOAc pH 6.1 5 .mu.l 10 mm biotin hydrazide (freshly made) 300 .mu.l

[0193] Wrap in a foil and incubate at room temperature overnight.

E. RNase I Treatment

[0194] Precipitate DNA in:

TABLE-US-00010 5M NaCl 10 .mu.l 2M NaOAc pH 6.1 75 .mu.l biotinylated mRNA:cDNA 420 .mu.l 100% EtOH (2.5 Vol) 1262.5 .mu.l

[0195] (Perform this precipitation in two tubes and split the 420 .mu.l of DNA into 210 .mu.l each, add 5 .mu.l of 5M NaCl, 37.5 .mu.l of 2M NaOAc pH 6.1 and 631.25 .mu.l of 100% EtOH).

[0196] Store at -20.degree. C. for at least 30 min. Spin the DNA down at 4.degree. C. at maximal speed for 30 min. and wash with 80% EtOH twice, then dissolve DNA in 70 .mu.l RNase free water. Pool two tubes and end up with 140 .mu.l.

[0197] Add the following reagents:

TABLE-US-00011 RNase One 10 U/.mu.l 40 .mu.l 1.sup.st cDNA:RNA 140 .mu.l 10X buffer 20 .mu.l Incubate at 37.degree. C. for 15 min.

[0198] Add 5 .mu.l of 40 .mu.g/.mu.l yeast tRNA to each sample for capturing.

F. Full Length 1.sup.st cDNA Capturing

[0199] Blocking the beads with yeast tRNA:

TABLE-US-00012 Beads 1 ml Yeast tRNA 40 .mu.g/.mu.l 5 .mu.l

[0200] Incubate on ice for 30 min with mixing, wash 3 times with 1 ml of 2M NaCl, 50 mmEDTA, pH 8.0.

[0201] Resuspend the beads in 800 .mu.l of 2M NaCl, 50 mm EDTA, pH 8.0, add RNase I treated sample 200 .mu.l, and incubate the reaction for 30 min at room temperature.

[0202] Capture the beads using the magnetic stand, save the supernatant, and start following washes:

[0203] 2 washes with 2M NaCl, 50 mm EDTA, pH 8.0, 1 ml each time,

[0204] 1 wash with 0.4% SDS, 50 .mu.g/ml tRNA,

[0205] 1 wash with 10 mm Tris-Cl pH 7.5, 0.2 mm EDTA, 10 mm NaCl, 20% glycerol,

[0206] 1 wash with 50 .mu.g/m tRNA,

[0207] 1 wash with 1.sup.st cDNA buffer

G. Second Strand cDNA Synthesis

[0208] Resuspend the beads in:

TABLE-US-00013 *5X first buffer 8 .mu.l *0.1 mM DTT 4 .mu.l *10 mm dNTP mix 8 .mu.l *5X 2nd buffer 60 .mu.l *E. coli Ligase 10 U/.mu.l 2 .mu.l *E. coli DNA polymerase 10 U/.mu.l 8 .mu.l *E. coli RNaseH 2 U/.mu.l 2 .mu.l P32 dCTP 10 .mu.ci/.mu.l 2 .mu.l Or water up to 300 .mu.l 208 .mu.l

[0209] Incubate at 16.degree. C. for 2 hr with mixing the reaction in every 30 min.

[0210] Add 4 .mu.l of T4 DNA polymerase and incubate for additional 5 min at 16.degree. C.

[0211] Elute 2.sup.nd cDNA from the beads.

[0212] Use a magnetic stand to separate the 2.sup.nd cDNA from the beads, then resuspend the beads in 200 .mu.l of water, and then separate again, pool the samples (about 500 .mu.l), Add 200 .mu.l of water to the beads, then 200 .mu.l of phenol:chloroform, vortex and spin to separate the sample with phenol.

[0213] Pool the DNA together (about 700 .mu.l) and use phenol to clean the DNA again, DNA is then precipitated in 2 .mu.g of glycogen and 0.5 vol of 7.5M NH4OAc and 2 vol of 100% EtOH. Precipitate overnight. Spin down the pellet and wash with 70% EtOH, air-dry the pellet.

TABLE-US-00014 DNA 250 .mu.l DNA 200 .mu.l 7.5M NH4OAc 125 .mu.l 7.5M NH4OAc 100 .mu.l 100% EtOH 750 .mu.l 100% EtOH 600 .mu.l glycogen 1 .mu.g/.mu.l 2 .mu.l glycogen 1 .mu.g/.mu.l 2 .mu.l

H. Sal I Adapter Ligation

[0214] Resuspend the pellet in 26 .mu.l of water and use 1 .mu.l for TAE gel.

[0215] Set up reaction as following:

TABLE-US-00015 2.sup.nd strand cDNA 25 .mu.l *5X T4 DNA ligase buffer 10 .mu.l *Sal I adapters 10 .mu.l *T4 DNA ligase 5 .mu.l

[0216] Mix gently, incubate the reaction at 16.degree. C. overnight.

[0217] Add 2 .mu.l of ligase second day and incubate at room temperature for 2 hrs (optional).

[0218] Add 50 .mu.l water to the reaction and use 100 .mu.l of phenol to clean the DNA, 90 .mu.l of the upper phase is transferred into a new tube and precipitate in:

TABLE-US-00016 Glycogen 1 .mu.g/.mu.l 2 .mu.l Upper phase DNA 90 .mu.l 7.5M NH4OAc 50 .mu.l 100% EtOH 300 .mu.l

precipitate at -20.degree. C. overnight

[0219] Spin down the pellet at 4.degree. C. and wash in 70% EtOH, dry the pellet.

I. Not I Digestion

TABLE-US-00017 [0220] 2.sup.nd cDNA 41 .mu.l *Reaction 3 buffer 5 .mu.l *Not I 15 u/.mu.l 4 .mu.l

[0221] Mix gently and incubate the reaction at 37.degree. C. for 2 hr.

[0222] Add 50 .mu.l of water and 100 .mu.l of phenol, vortex, and take 90 .mu.l of the upper phase to a new tube, then add 50 .mu.l of NH.sub.40Ac and 300 .mu.l of EtOH. Precipitate overnight at -20.degree. C.

[0223] Cloning, ligation and transformation are performed per the Superscript cDNA synthesis kit.

Example 3

[0224] This example describes cDNA sequencing and library subtraction.

[0225] Individual colonies can be picked and DNA prepared either by PCR with M13 forward primers and M13 reverse primers or by plasmid isolation. cDNA clones can be sequenced using M13 reverse primers.

[0226] cDNA libraries are plated out on 22.times.22 cm.sup.2 agar plate at density of about 3,000 colonies per plate. The plates are incubated in a 37.degree. C. incubator for 12-24 hours. Colonies are picked into 384-well plates by a robot colony picker, Q-bot (GENETIX Limited). These plates are incubated overnight at 37.degree. C. Once sufficient colonies are picked, they are pinned onto 22.times.22 cm.sup.2 nylon membranes using Q-bot. Each membrane holds 9,216 or 36,864 colonies. These membranes are placed onto an agar plate with an appropriate antibiotic. The plates are incubated at 37.degree. C. overnight.

[0227] After colonies are recovered on the second day, these filters are placed on filter paper prewetted with denaturing solution for four minutes, then incubated on top of a boiling water bath for an additional four minutes. The filters are then placed on filter paper prewetted with neutralizing solution for four minutes. After excess solution is removed by placing the filters on dry filter papers for one minute, the colony side of the filters is placed into Proteinase K solution, incubated at 37.degree. C. for 40-50 minutes. The filters are placed on dry filter papers to dry overnight. DNA is then cross-linked to nylon membrane by UV light treatment

[0228] Colony hybridization is conducted as described by Sambrook, et al., (in Molecular Cloning: A laboratory Manual, 2.sup.nd Edition). The following probes can be used in colony hybridization: [0229] 1. First strand cDNA from the same tissue as the library was made from to remove the most redundant clones. [0230] 2. 48-192 most redundant cDNA clones from the same library based on previous sequencing data. [0231] 3. 192 most redundant cDNA clones in the entire maize sequence database. [0232] 4. A Sal-A20 oligo nucleotide: TCG ACC CAC GCG TCC GAA AAA AAA AAA AAA AAA AAA, SEQ ID NO: 31, removes clones containing a poly A tail but no cDNA. [0233] 5. cDNA clones derived from rRNA.

[0234] The image of the autoradiography is scanned into computer and the signal intensity and cold colony addresses of each colony is analyzed. Re-arraying of cold-colonies from 384 well plates to 96 well plates is conducted using Q-bot.

Example 4

[0235] This example describes identification of the gene from a computer homology search.

[0236] Gene identities can be determined by conducting BLAST (Basic Local Alignment Search Tool; Altschul, et al., (1993) J. Mol. Biol. 215:403-410) searches under default parameters for similarity to sequences contained in the BLAST "nr" database (comprising all non-redundant GenBank CDS translations, sequences derived from the 3-dimensional structure Brookhaven Protein Data Bank, the last major release of the SWISS-PROT protein sequence database, EMBL and DDBJ databases). The cDNA sequences are analyzed for similarity to all publicly available DNA sequences contained in the "nr" database using the BLASTN algorithm. The DNA sequences are translated in all reading frames and compared for similarity to all publicly available protein sequences contained in the "nr" database using the BLASTX algorithm (Gish and States, (1993) Nature Genetics 3:266-272) provided by the NCBI. In some cases, the sequencing data from two or more clones containing overlapping segments of DNA are used to construct contiguous DNA sequences.

[0237] Sequence alignments and percent identity calculations can be performed using the Megalign program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.). Multiple alignments of the sequences can be 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 are KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5.

[0238] Other methods of sequence alignment and percent identity analysis known to those of skill in the art, including those disclosed herein, can also be employed.

Example 5

[0239] This example describes expression of transgenes in monocot cells.

[0240] A transgene comprising a cDNA encoding the instant polypeptides in sense orientation with respect to the maize 27 kD zein promoter that is located 5' to the cDNA fragment, and the 10 kD zein 3' end that is located 3' to the cDNA fragment, can be constructed. The cDNA fragment of this gene may be generated by polymerase chain reaction (PCR) of the cDNA clone using appropriate oligonucleotide primers. Cloning sites (NcoI or SmaI) can be incorporated into the oligonucleotides to provide proper orientation of the DNA fragment when inserted into the digested vector pML103 as described below. Amplification is then performed in a standard PCR. The amplified DNA is then digested with restriction enzymes NcoI and SmaI and fractionated on an agarose gel. The appropriate band can be isolated from the gel and combined with a 4.9 kb NcoI-SmaI fragment of the plasmid pML103. Plasmid pML103 has been deposited under the terms of the Budapest Treaty at ATCC (American Type Culture Collection, 10801 University Blvd., Manassas, Va. 20110-2209) and bears accession number ATCC 97366. The DNA segment from pML103 contains a 1.05 kb SalI-NcoI promoter fragment of the maize 27 kD zein gene and a 0.96 kb SmaI-SalI fragment from the 3' end of the maize 10 kD zein gene in the vector pGem9Zf(+) (Promega). Vector and insert DNA can be ligated at 15.degree. C. overnight, essentially as described (Maniatis). The ligated DNA may then be used to transform E. coli XL1-Blue (Epicurian Coli XL-1 Blue; Stratagene). Bacterial transformants can be screened by restriction enzyme digestion of plasmid DNA and limited nucleotide sequence analysis using the dideoxy chain termination method (Sequenase DNA Sequencing Kit; US Biochemical). The resulting plasmid construct would comprise a transgene encoding, in the 5' to 3' direction, the maize 27 kD zein promoter, a cDNA fragment encoding the instant polypeptides and the 10 kD zein 3' region.

[0241] The transgene described above can then be introduced into maize cells by the following procedure. Immature maize embryos can be dissected from developing caryopses derived from crosses of the inbred maize lines H99 and LH132. The embryos are isolated 10 to 11 days after pollination when they are 1.0 to 1.5 mm long. The embryos are then placed with the axis-side facing down and in contact with agarose-solidified N6 medium (Chu, et al., (1975) Sci. Sin. Peking 18:659-668). The embryos are kept in the dark at 27.degree. C. Friable embryogenic callus consisting of undifferentiated masses of cells with somatic proembryoids and embryoids borne on suspensor structures proliferates from the scutellum of these immature embryos. The embryogenic callus isolated from the primary explant can be cultured on N6 medium and sub-cultured on this medium every 2 to 3 weeks.

[0242] The plasmid, p35S/Ac (Hoechst Ag, Frankfurt, Germany) or equivalent may be used in transformation experiments in order to provide for a selectable marker. This plasmid contains the Pat gene (see, EP Patent Publication Number 0 242 236) which encodes phosphinothricin acetyl transferase (PAT). The enzyme PAT confers resistance to herbicidal glutamine synthetase inhibitors such as phosphinothricin. The pat gene in p35S/Ac is under the control of the 35S promoter from Cauliflower Mosaic Virus (Odell, et al., (1985) Nature 313:810-812) and the 3' region of the nopaline synthase gene from the T-DNA of the Ti plasmid of Agrobacterium tumefaciens.

[0243] The particle bombardment method (Klein, et al., (1987) Nature 327:70-73) may be used to transfer genes to the callus culture cells. According to this method, gold particles (1 .mu.m in diameter) are coated with DNA using the following technique. Ten .mu.g of plasmid DNAs are added to 50 .mu.L of a suspension of gold particles (60 mg per mL). Calcium chloride (50 .mu.L of a 2.5 M solution) and spermidine free base (20 .mu.L of a 1.0 M solution) are added to the particles. The suspension is vortexed during the addition of these solutions. After 10 minutes, the tubes are briefly centrifuged (5 sec at 15,000 rpm) and the supernatant removed. The particles are resuspended in 200 .mu.L of absolute ethanol, centrifuged again and the supernatant removed. The ethanol rinse is performed again and the particles resuspended in a final volume of 30 .mu.L of ethanol. An aliquot (5 .mu.L) of the DNA-coated gold particles can be placed in the center of a Kapton flying disc (Bio-Rad Labs). The particles are then accelerated into the maize tissue with a Biolistic PDS-1000/He (Bio-Rad Instruments, Hercules Calif.), using a helium pressure of 1000 psi, a gap distance of 0.5 cm and a flying distance of 1.0 cm.

[0244] For bombardment, the embryogenic tissue is placed on filter paper over agarose-solidified N6 medium. The tissue is arranged as a thin lawn and covers a circular area of about 5 cm in diameter. The petri dish containing the tissue can be placed in the chamber of the PDS-1000/He approximately 8 cm from the stopping screen. The air in the chamber is then evacuated to a vacuum of 28 inches of Hg. The macrocarrier is accelerated with a helium shock wave using a rupture membrane that bursts when the He pressure in the shock tube reaches 1000 psi.

[0245] Seven days after bombardment the tissue can be transferred to N6 medium that contains gluphosinate (2 mg per liter) and lacks casein or proline. The tissue continues to grow slowly on this medium. After an additional 2 weeks the tissue can be transferred to fresh N6 medium containing gluphosinate. After 6 weeks, areas of about 1 cm in diameter of actively growing callus can be identified on some of the plates containing the glufosinate-supplemented medium. These calli may continue to grow when sub-cultured on the selective medium.

[0246] Plants can be regenerated from the transgenic callus by first transferring clusters of tissue to N6 medium supplemented with 0.2 mg per liter of 2,4-D. After two weeks the tissue can be transferred to regeneration medium (Fromm, et al., (1990) Bio/Technology 8:833-839).

Example 6

[0247] This example describes expression of transgenes in dicot cells.

[0248] A seed-specific expression cassette composed of the promoter and transcription terminator from the gene encoding the .beta. subunit of the seed storage protein phaseolin from the bean Phaseolus vulgaris (Doyle, et al., (1986) J. Biol. Chem. 261:9228-9238) can be used for expression of the instant polypeptides in transformed soybean. The phaseolin cassette includes about 500 nucleotides upstream (5') from the translation initiation codon and about 1650 nucleotides downstream (3') from the translation stop codon of phaseolin. Between the 5' and 3' regions are the unique restriction endonuclease sites Nco I (which includes the ATG translation initiation codon), SmaI, KpnI and XbaI. The entire cassette is flanked by Hind III sites.

[0249] The cDNA fragment of this gene may be generated by polymerase chain reaction (PCR) of the cDNA clone using appropriate oligonucleotide primers. Cloning sites can be incorporated into the oligonucleotides to provide proper orientation of the DNA fragment when inserted into the expression vector. Amplification is then performed as described above, and the isolated fragment is inserted into a pUC18 vector carrying the seed expression cassette.

[0250] Soybean embryos may then be transformed with the expression vector comprising sequences encoding the instant polypeptides. To induce somatic embryos, cotyledons, 3-5 mm in length dissected from surface sterilized, immature seeds of the soybean cultivar A2872, can be cultured in the light or dark at 26.degree. C. on an appropriate agar medium for 6-10 weeks. Somatic embryos which produce secondary embryos are then excised and placed into a suitable liquid medium. After repeated selection for clusters of somatic embryos which multiplied as early, globular staged embryos, the suspensions are maintained as described below.

[0251] Soybean embryogenic suspension cultures can maintained in 35 mL liquid media on a rotary shaker, 150 rpm, at 26.degree. 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.

[0252] 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 DuPont Biolistic PDS1000/HE instrument (helium retrofit) can be used for these transformations.

[0253] A selectable marker gene which 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 seed expression cassette comprising the phaseolin 5' region, the fragment encoding the instant polypeptides and the phaseolin 3' region 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.

[0254] To 50 .mu.L of a 60 mg/mL 1 .mu.mgold particle suspension is added (in order): 5 .mu.L DNA (1 .mu.g/4), 20 .mu.l spermidine (0.1 M), and 50 .mu.L CaCl.sub.2 (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 .mu.L 70% ethanol and resuspended in 40 .mu.L 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.

[0255] Approximately 300-400 mg of a two-week-old suspension culture is placed in an empty 60.times.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.

[0256] 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 7

[0257] This example describes expression of a transgene in microbial cells.

[0258] The cDNAs encoding the instant polypeptides can be inserted into the T7 E. coli expression vector pBT430. This vector is a derivative of pET-3a (Rosenberg, et al., (1987) Gene 56:125-135) which employs the bacteriophage T7 RNA polymerase/T7 promoter system. Plasmid pBT430 was constructed by first destroying the EcoR I and Hind III sites in pET-3a at their original positions. An oligonucleotide adaptor containing EcoR I and Hind III sites was inserted at the BamH I site of pET-3a. This created pET-3aM with additional unique cloning sites for insertion of genes into the expression vector. Then, the Nde I site at the position of translation initiation was converted to an Nco I site using oligonucleotide-directed mutagenesis. The DNA sequence of pET-3aM in this region, 5'-CATATGG, was converted to 5'-CCCATGG in pBT430.

[0259] Plasmid DNA containing a cDNA may be appropriately digested to release a nucleic acid fragment encoding the protein. This fragment may then be purified on a 1% NuSieve GTG low melting agarose gel (FMC). Buffer and agarose contain 10 .mu.g/ml ethidium bromide for visualization of the DNA fragment. The fragment can then be purified from the agarose gel by digestion with GELase (Epicentre Technologies) according to the manufacturer's instructions, ethanol precipitated, dried and resuspended in 20 .mu.L of water. Appropriate oligonucleotide adapters may be ligated to the fragment using T4 DNA ligase (New England Biolabs, Beverly, Mass.). The fragment containing the ligated adapters can be purified from the excess adapters using low melting agarose as described above. The vector pBT430 is digested, dephosphorylated with alkaline phosphatase (NEB) and deproteinized with phenol/chloroform as described above. The prepared vector pBT430 and fragment can then be ligated at 16.degree. C. for 15 hours followed by transformation into DH5 electrocompetent cells (GIBCO BRL). Transformants can be selected on agar plates containing LB media and 100 .mu.g/mL ampicillin. Transformants containing the gene encoding the instant polypeptides are then screened for the correct orientation with respect to the T7 promoter by restriction enzyme analysis.

[0260] For high level expression, a plasmid clone with the cDNA insert in the correct orientation relative to the T7 promoter can be transformed into E. coli strain BL21(DE3) (Studier, et al., (1986) J. Mol. Biol. 189:113-130). Cultures are grown in LB medium containing ampicillin (100 mg/L) at 25.degree. C. At an optical density at 600 nm of approximately 1, IPTG (isopropylthio-.beta.-galactoside, the inducer) can be added to a final concentration of 0.4 mM and incubation can be continued for 3 h at 25.degree. C. Cells are then harvested by centrifugation and re-suspended in 50 .mu.L of 50 mM Tris-HCl at pH 8.0 containing 0.1 mM DTT and 0.2 mM phenyl methylsulfonyl fluoride. A small amount of 1 mm glass beads can be added and the mixture sonicated 3 times for about 5 seconds each time with a microprobe sonicator. The mixture is centrifuged and the protein concentration of the supernatant determined. One microgram of protein from the soluble fraction of the culture can be separated by SDS-polyacrylamide gel electrophoresis. Gels can be observed for protein bands migrating at the expected molecular weight.

Example 8

[0261] Isolation of the CesA10, 11 and 12 genes and their relevance to cell wall synthesis and stalk strength in maize.

[0262] All three genes were isolated from a library made from the zone of an elongating corn stalk internode between the elongation zone and the most mature part of the internode, the "transition zone". The library was made, subtracted, and sequenced as described in the preceding Examples 1, 2 and 3. A genomic database search was conducted as described in Example 4. Derived polypeptide sequences of all the Expressed Tag Sequences (ESTs) showing homology to the 1 kb 5'-end of any of the 9 previously known ZmCesA genes were aligned with the protein sequences of the latter. The sequences that did not fully match any of the known genes were sequenced from both ends of the respective cDNA clones. Three new, full-length genes, ZmCesA10 (SEQ ID NO: 25), ZmCesA11 (SEQ ID NO: 27) and ZmCesA12 (SEQ ID NO: 29) were isolated by this method.

[0263] The polypeptide sequences of the three genes derived from the cDNA sequences (SEQ ID NOS: 26, 28 and 30, respectively) clustered with the CesA genes from other species where they are known to be involved in secondary wall formation (FIG. 4). AtCesA7 and AtCesA8 have been found to make secondary wall in the vascular bundles (Taylor, et al., (2000). Multiple cellulose synthase catalytic subunits are required for cellulose synthesis in Arabidopsis. Plant Cell, (2000) 12:2529-2539.). Retrotransposon insertions into OsCesA4 and OsCesA7 resulted in a brittle culm phenotype in rice (Katsuyuki Tanaka, Akio Miyao, Kazumasa Murata, Katsura Onosato, Naoko Kojima, Yumiko Yamashita, Mayuko Harada, Takuji Sasaki, Hirohiko Hirochika, 2002, Analysis of rice brittle mutants caused by disruption of cellulose synthase genes OsCesA4 and OsCesA11 with the retrotransposon tos17. Plant, Animal & Microbe Genomes X. San Diego, Calif. Abs. Number 324). Each of the genes, ZmCesA 10, 11 or 12, groups with one or the other CesA gene from Arabidopsis or rice known to be involved in secondary wall formation and thus in determining tissue strength (FIG. 4). The CesA genes derived from the tissues specializing in secondary wall formation from other species (Gossypium, Zinnia, Populus) also group into the same clades with the aforementioned genes.

[0264] Further evidence that the maize genes are involved in secondary wall formation and thus in determining stalk strength was obtained from their expression pattern using the Massively Parallel Signature Sequencing (MPSS) technology (Brenner, et al., (2000), In vitro cloning of complex mixtures of DNA on microbeads: Physical separation of differentially expressed cDNAs. Proceedings-of-the-National-Academy-of-Sciences-of-the-United-States-of-A- merica (Feb. 15, 2000) 97:1665-1670; see also, Brenner, et al., (2000) Gene expression analysis by massively parallel signature sequencing (MPSS) on microbead arrays. Nature-Biotechnolog, [print] (June 2000) 18:630-634; see also, Dhugga, (2001), Building the wall: genes and enzyme complexes for polysaccharide synthases, Curr. Opin. Plant Biol. 4:488-493). All three genes are expressed in the tissues rich in cell wall content, supporting their involvement in secondary wall formation as deduced from their relationship to the genes from the other, aforementioned species know to play this role (FIG. 5). All three genes are expressed nearly identically across multiple tissues as seen from the correlation coefficient matrix (Table 3), further strengthening the argument that they are involved in secondary wall formation in the vascular bundles and thus in determining tissue strength.

[0265] Correlation among the expression level of the different CesA genes from maize as studied from Lynx are shown in Table 3.

TABLE-US-00018 TABLE 3 CesA1 CesA2 CesA3 CesA4 CesA5 CesA6 CesA7 CesA8 CesA10 CesA11 CesA12 CesA1 1 CesA2 0.59 1.00 CesA3 0.07 -0.15 1.00 CesA4 0.44 0.55 -0.10 1.00 CesA5 -0.20 -0.29 0.45 -0.33 1.00 CesA6 0.56 0.14 0.14 0.08 -0.13 1.00 CesA7 0.68 0.76 -0.06 0.57 -0.29 0.32 1.00 CesA8 0.59 0.73 -0.16 0.58 -0.36 0.26 0.61 1.00 CesA10 0.27 0.37 -0.27 0.33 -0.26 0.02 0.33 0.36 1.00 CesA11 0.39 0.47 -0.22 0.38 -0.28 0.11 0.40 0.42 0.95 1.00 CesA12 0.34 0.49 -0.27 0.37 -0.31 0.08 0.44 0.45 0.95 0.95 1

[0266] The correlation matrix was derived from the expression, measured in PPM, from 65 different tissue libraries. Note the nearly perfect correlation among the expression pattern of the CesA10, 11 and 12 genes.

Example 9

[0267] This example describes a procedure to identify plants containing Mu inserted into genes of interest and a strategy to identify the function of those genes. This procedure was also described in U.S. patent application Ser. No. 09/371,383 which disclosed members of the same gene family as the present application. One of skill in the art could readily conceive of use of this procedure with the any of the Cellulose Synthase (CesA) sequences disclosed in the current application. The current example is based on work with the CesA11 gene, identified as SEQ ID NO: 27 herein.

[0268] The Trait Utility System for Corn (TUSC) is a method that employs genetic and molecular techniques to facilitate the study of gene function in maize. Studying gene function implies that the gene's sequence is already known, thus the method works in reverse: from sequence to phenotype. This kind of application is referred to as "reverse genetics", which contrasts with "forward" methods that are designed to identify and isolate the gene(s) responsible for a particular trait (phenotype).

[0269] Pioneer Hi-Bred International, Inc., has a proprietary collection of maize genomic DNA from approximately 42,000 individual F.sub.1 plants (Reverse genetics for maize, Meeley and Briggs, (1995) Maize Genet. Coop. Newslett. 69:67-82). The genome of each of these individuals contains multiple copies of the transposable element family, Mutator (Mu). The Mu family is highly mutagenic; in the presence of the active element Mu-DR, these elements transpose throughout the genome, inserting into genic regions, and often disrupting gene function. By collecting genomic DNA from a large number (42,000) of individuals, Pioneer has assembled a library of the mutagenized maize genome.

[0270] Mu insertion events are predominantly heterozygous; given the recessive nature of most insertional mutations, the F.sub.1 plants appear wild-type. Each of the F.sub.1 plants is selfed to produce F.sub.2 seed, which is collected. In generating the F.sub.2 progeny, insertional mutations segregate in a Mendelian fashion so are useful for investigating a mutant allele's effect on the phenotype. The TUSC system has been successfully used by a number of laboratories to identify the function of a variety of genes (Cloning and characterization of the maize An1 gene, Bensen, et al., (1995) Plant Cell 7:75-84; Diversification of C-function activity in maize flower development, Mena, et al., (1996) Science 274:1537-1540; Analysis of a chemical plant defense mechanism in grasses, Frey, et al., (1997) Science 277:696-699; The control of maize spikelet meristem fate by the APETALA2-like gene Indeterminate spikelet 1, Chuck, et al., (1998) Genes and Development 12:1145-1154; A SecY homologue is required for the elaboration of the chloroplast thylakoid membrane and for normal chloroplast gene expression, Roy and Barkan, (1998) J. Cell Biol. 141:1-11).

[0271] PCR Screening for Mu insertions in CesA11:

[0272] Two primers were designed from within the CesA11 cDNA and designated as gene-specific primers (GSPs):

TABLE-US-00019 Forward primer (GSP1/SEQ ID NO. 32): 5'- TACGATGAGTACGAGAGGTCCATGCTCA -3' Reverse primer (GSP2/SEQ ID NO. 33): 5'- GGCAAAAGCCCAGATGCGAGATAGAC -3' Mu TIR primer (SEQ ID NO. 34): 5'- AGAGAAGCCAACGCCAWCGCCTCYATTTCGTC -3'

[0273] Pickoligo was used to select primers for PCR. This program chooses the Tm according to the following equation:

Tm=[((GC*3+AT*2)*37-562)/length]-5

[0274] PCR reactions were run with an annealing temperature of 62.degree. C. and a thermocycling profile as follows:

##STR00001##

[0275] Gel electrophoresis of the PCR products confirmed that there was no false priming in single primer reactions and that only one fragment was amplified in paired GSP reactions.

[0276] The genomic DNA from 42,000 plants, combined into pools of 48 plants each, was subjected to PCR with either GSP1 or GSP2 and Mu TIR. The pools that were confirmed to be positive by dot-blot hybridization using CesA11 cDNA as a probe were subjected to gel-blot analysis in order to determine the size of fragments amplified. The pools in which clean fragments were identified were subjected to further analysis to identify the individual plants within those pools that contained Mu insertion(s).

[0277] Seed from F.sub.1 plants identified in this manner was planted in the field. Leaf discs from twenty plants in each F.sub.2 row were collected and genomic DNA was isolated. The same twenty plants were selfed and the F.sub.3 seed saved. Pooled DNA (from 20 plants) from each of twelve rows was subjected to PCR using GSP1 or GSP2 and Mu TIR primer as mentioned above. Three pools identified to contain Mu insertions were subjected to individual plant analysis and homozygotes identified. The Mu insertion sites with the surrounding signature sequences are identified below:

TABLE-US-00020 Allele 1: 5'-TGGCGGCCG (SEQ ID NO: 35)- Mu-TCTGAAATG (SEQ ID NO: 36)-3' Allele 2: 5'-GCCCACAAG (SEQ ID NO: 37)- Mu-CATCCTGGT (SEQ ID NO: 38)-3' Allele 3: 5'-GTGTTCTTC (SEQ ID NO: 39)- Mu-GCCATGTGG (SEQ ID NO: 40)-3'

[0278] All three insertions are within 500 nucleotides of each other in the open reading frame, suggesting that this region in the gene might represent a hot spot for Mu insertion. One of the insertions, allele 1, is in the region upstream of the predicted six transmembrane domains near the C-terminal end of the protein. Each of these insertions is expected to inactivate the gene since they are all in the exonic regions of the gene.

Example 10

[0279] This example describes the method used to measure mechanical strength of the maize stalks as well as the effect of the overexpression of different CesA genes on stalk strength. The mechanical strength of the mature corn stalks was measured with an electromechanical test system. The internodes below the ear were subjected to a 3-point bend test using an Instron, model 4411 (Instron Corporation, 100 Royall Street, Canton, Mass. 02021), with a span-width of 200 mm between the anchoring points and a speed of 200 mm/min of the 3.sup.rd point attached to a load cell. For measuring rind puncture strength, a needle was mounted on the load cell of the Instron and the load taken to puncture the rind was used as a measure of rind puncture strength.

[0280] Load needed to break the internode was used as a measure of mechanical strength. The internodes are stronger toward the base of the stalk. This mechanical stalk breaking strength or the "load to break" was used to classify the hybrids with known stalk characteristics into respective categories based on the internodal breaking strength. The load to break the internodal zone was very similar to the lodging score that had been assigned to the hybrids based on field observations (see, FIG. 1). Approximately 90% of the variation for internodal breaking strength was explained by unit stalk dry matter below the ear (47%), stalk diameter (30%) and rind puncture resistance (10%). Moisture levels above 30% in the stalk tissue masked the contribution of the rind tissue to breaking strength. The internodal breaking strength was highly correlated with the amount of cellulose per unit length of the stalk.

[0281] Four of the CesA genes were expressed under the control of a weak constitutive promoter, F3.7 (see, Coughlin, et al., U.S. patent application Ser. No. 09/387,720, filed Aug. 30, 1999). Table 4 discloses the construct numbers, corresponding sequence IDs from the patent, promoters, and the gene names. In2 is an inducible promoter from the In2 gene from maize. The In2 promoter responds to benzenesulfonamide herbicide safeners (see, Hershey, et al., (1991) Mol. Gen. Genetics 227:229-237 and Gatz, et al., (1994) Mol. Gen Genetics 243:32-38).

TABLE-US-00021 TABLE 4 Construct CesA SEQ ID NO. Promoter Gene name 1 1 F3.7 CesA1 2 9 F3.7 CesA4 3 13 F3.7 CesA5 4 17 F3.7 CesA8 5 Control IN2 GUSINT

[0282] Twenty-five individual T.sub.0 events for each construct were generated in a hybrid maize background using Agrobacterium-mediated transformation. Data for various traits, such as plant height, stalk mass below ear, stalk diameter, internodal breaking strength and structural material and cellulose percentages in the internodal tissue were collected.

[0283] The plants from the transgenic events generated using the CesA8 gene were significantly taller in comparison to the control plants containing a GUS gene. Interestingly, a reduction in height was observed when the CesA1 gene was introduced. The other two genes, CesA4 and CesA5, did not differ from the control plants. (See, FIG. 6.) It has long been known that cellulose synthase occurs as a terminal rosette complex consisting of multiple functional cellulose synthase polypeptides that are organized in a ring with a hexagonal symmetry. Each of the six members of the ring is believed to contain six or more functional enzyme units. In general, 36 or more cellulose chains are extruded simultaneous to their synthesis through the plasma membrane into the apoplast. These chains are crystallized into a microfibril right as they come in contact with each other after extrusion through the rosette complex. A functional cellulose synthase is believed to consist of two polypeptides derived form different CesA genes, forming a heterodimer, resulting in a total of 72 or more CesA polypeptides in each rosette.

[0284] While not intending to be limited to a single theory, it is possible that a homodimer could also form a functional enzyme. Therefore, the possible reasons for a reduction in plant height in the events where CesA1 was overexpressed are: 1) the other CesA gene with which its polypeptide forms a heterodimer is down-regulated and 2) the expression of the other gene is not affected but the CESA1 homodimer forms a nonfunctional enzyme, in which case the functional dimers are competed out of the rosette complex. In the latter case, the overexpressed gene behaves as a dominant repressor of cellulose synthesis. This should manifest in the form of microfibrils with fewer cellulose chains. This could be detected by some physical techniques such as differential scanning calorimetry (DSC). The reverse could be true for the CesA8 gene whose homodimers may be functional, and/or whose overexpression might induce the expression of its partner gene the product of which it uses to make a functional enzyme. The fact that an increase in height is observed may result from stalk becoming an active sink when CesA8 is overexpressed. Stalk is usually considered to be a passive sink which cannot compete well with the developing ear. This argument is supported by the observation that the plants containing CesA8 as a transgene had smaller ears.

[0285] Cellulose content and stalk length below the ear is highly correlated with the breaking strength of the stalk (see, FIG. 3). An increase in cellulose production can be accommodated by the following alterations: 1) synthesis of the other cell wall constituents stays constant, leading to an increased cellulose percentage in the wall and 2) increase in cellulose synthesis upregulates the synthesis of the other cell wall constituents as well, in which case the percentage of cellulose does not change in the wall but the amount of cellulose in a unit length does. Two of the CesA genes, CesA4 and CesA8, showed an increase in the amount of cellulose in a unit length of the stalk below the ear (see, FIG. 7). One of the genes, CesA5, did not have any effect on the amount of cellulose in the stalk. It was recently suggested, based on its expression pattern in different tissues, that CesA5 might actually be involved in the formation of some non-cellulosic polysaccharide, most probably mixed-linked glucan (Dhugga, (2001) Curr. Opin. Plant Biol. 4:488-493). The data in the accompanying figure seem to support this argument.

[0286] The internodes were subjected to breakage with a 3-point Instron and the load to break plotted as a function of unit cellulose amount. (See, FIG. 2.) A high correlation between these two traits is observed from the multiple events, particularly for CesA8 (FIG. 3). We have found from other studies that this gene is involved in cellulose synthesis in the vascular bundles in the elongating cells (Holland, et al., (2000) Plant Physiol. 123:1313-1323). These data support our previous observations and supports the observation that the amount of cellulose in a unit length of stalk below the ear results in an increased stalk strength.

Example 11

[0287] This example describes the method used to overexpress CesA genes which will increase the quality of harvested stover, leading to an increase in ethanol yield per unit stover.

[0288] Transgenic plants expressing the Ces A gene of interest could be produced by the method outlined in Example 5 or other suitable methods. These plants containing increased quantities of cellulose would then be used to produce higher quality stover.

[0289] The following is an example of the applications of the present invention in applications of ethanol biorefineries. In addition the cellulose biosynthetic pathway's role as primary determinant of tissue strength, a trait that is of significant interest in agriculture, where cellulose constitutes the most abundant renewable energy resource. More than 200 million metric tons of stover is produced just from maize in the United States every year. About one-third of this could potentially be utilized in ethanol biorefineries (Kadam and McMillan, 2003). The worldwide production of lignocellulosic wastes from cereal stover and straw is estimated to be .about.3 billion tons per year (Kuhad and Singh, 1993). Stover material containing higher amounts of cellulose and lower amounts of lignin is expected to increase ethanol production in the biorefineries. Lignin is a target for reduction because it is an undesirable constituent in paper industry as well as in silage digestibility (Hu, et al., 1999; Li, et al., 2003).

[0290] Corn stover alone offers a significant target as a feedstock for the ethanol biorefineries (see, the World Wide Web at ctic.purdue.edu/Core4/ctic-dc.ppt; and_bioproducts-bioenergy.gov/pdfs/bcota/abstracts/31/z263.pdf.) Aside from its use in biorefineries, it can substitute hardwood fiber for paper production. With rapid progress being made in streamlining the process of fermentation of stover material and increasing cost of imported oil, corn stover is expected to become a key feedstock in ethanol and paper production (Wheals, et al., 1999; Atistidou and Penttila, 2000). In addition to supplying 5-8 billion gallons of ethanol per year with no additional land use, it is expected to contribute to an annual farm income of $2.3 billion and reduce the greenhouse gases by 60-95 million metric tons, which is 12-20% of the US-Kyoto commitment (see, the World Wide Web at ctic.purdue.edu/Core4/ctic-dc.ppt; and bioproducts--bioenergy.gov/pdfs/bcota/abstracts/31/z263.pdf). Ethanol combustion results in carbon dioxide and water, the same molecules plant primarily uses to make biomass.

[0291] The concern about there being an effect on the soil organic matter by the removal of the aboveground biomass is mitigated by the findings that over a 30-year period, no significant difference was observed in the soil organic matter between a field where the aboveground stover was removed for silage and the one where the stover was ploughed into the ground after grain harvest. Most of the ploughed stover is lost as carbon dioxide into the atmosphere.

[0292] During pretreatment of the corn stover for enzymatic digestion, the soluble sugars are discarded. Also, pentose sugars are not as well fermented as the hexose sugars despite the progress made in the fermentation process, which involves using Zymomonas bacteria instead of the traditional yeast (Atistidou and Penttila, 2000; Badger, 2002). The polysaccharide fraction of the corn stalk contains .about.20% pentose sugars, the remainder being hexose sugars (Dhugga, unpublished). Also, the free sugar concentration ranges from 4-12%. Lignin content averages .about.19% and ranges from 18-23%. By overexpressing the CesA genes of the present invention, the free sugars can be converted into polymeric (cellulosic) form, which will increase ethanol yield per unit of the harvested stover. The claimed invention also teaches an increase cellulose at the expense of pentose-containing polymers (e.g., arabinoxylan) and lignin.

Example 12

[0293] This example discusses the application of CesA genes in late season stalk strength.

[0294] Stalk lodging results in significant yield losses in crop plants, particularly in cereals (Duvick and Cassman, 1999). Stalk standability is dependent upon the amount of dry matter per unit length of the stalk and is thus a function of resource partitioning and allocation. Harvest index, the ratio of the grain to total aboveground biomass, is an indicator of dry matter partitioning efficiency. It has remained around 50% for over a hundred years in maize (Sinclair, 1998). In comparison to maize, harvest index acquired a different role in increasing plant standability in small grain cereals where it was significantly increased with the introduction of dwarfing genes. Reduced stature made these cereals less likely to lodge by reducing torque on the top-heavy straw, which allowed for higher inputs such as fertilizers and irrigation, resulting in increased biomass production per unit land area. Whereas yield increases in small grain cereals have resulted from an increase in both harvest index and total biomass production per unit land area, those in maize have been the consequence of mainly an increase in total biomass. Increased planting density as a means of increasing grain yield in maize has affected changes in leaf angle and shape as adaptations to this environment and has in general resulted in increased plant and ear heights (Duvick and Cassman, 1999). The stalk becomes mechanically weaker with increasing planting density because of reduction in individual plant vigor that results from a nonlinear relationship between planting density and biomass increase.

[0295] The understanding that cellulose in a unit length of the stalk is indeed the main determinant of mechanical strength had been proposed. (Appenzeller, et al., 2004). Most of the dry matter and thus cellulose in the stalk is concentrated in the outer layers, collectively referred to as rind, which is composed of densely packed vascular bundles. Vascular bundles are surrounded by sclerenchymatous cells. Although vascular bundles are also sparsely distributed in the internal tissue, a great majority of them are present in the outer layers as judged from the dry matter distribution (FIG. 8). FIG. 8 describes the contribution of different stalk components to dry matter, diameter, volume and stalk strength in maize hybrids. The data are derived from seven hybrids grown at three densities (27, 43 and 59 K per acre) in three replications each in 2001. Two stalks were sampled from each replication. Internodes 3 and 4 below the ear were broken with Instron. After breaking, the 3rd internode was separated into rind and inner tissue. Path coefficient analyses were performed using rind and inner tissue as independent variables (X.sub.1 and X.sub.2, respectively) and the whole stalk as the dependent variable (Y). The multiple regression equation: Y=a+b.sub.1X.sub.1+b.sub.2X.sub.2+e where a is the intercept and e error. Path coefficients were calculated as follows: .SIGMA.YXn=b.sub.n*.delta..sub.n/.delta..sub.Y where n is 1 or 2. The contribution of each independent variable to whole stalk (Y) was calculated as follows: .rho.Yxn*rYxn where r is the correlation coefficient. Note: the unexplained variation for diameter is attributable to the corn stalk not being perfectly round and the difficulty thus associated with determining the cross-sectional area accurately. Some other variable, like size and number of vascular bundles and their density, may account for the remaining variation in strength. The introduced transgene, by removing the limitation of the particular step it encodes the enzyme for catalyzing, may either lead to an increase in the percentage of that particular polysaccharide (composition changed) or of the whole cell wall (composition not changed). In the latter case, the additional dry matter could be accommodated in enlarged vascular bundles which could, in turn, result in an increased diameter.

[0296] Isolation of genes that affect cellulose formation has made it possible to test their respective roles in stalk strength by transgenic and reverse genetics approaches (Appenzeller, et al., 2004). Transgenic plants expressing the Ces A gene of interest are produced by the method outlined in Example 5 or other suitable methods. Expression of the cellulose synthase gene is measured Three of the twelve cellulose synthase genes are preferentially expressed in the secondary wall-forming cells (Appenzeller, et al., 2004). Whereas two of these genes, CesA10 and CesA11, are expressed more highly in the vascular bundles, CesA12 appears to be more highly expressed in the surrounding, sclerenchymatous cells (Appenzeller, et al., 2004). Overexpression of the three CesA genes individually and in combinations is used to increase cellulose production in the rind cells as well as the internal tissue cells. Internal tissue cells, as shown in FIG. 8, account for a majority of the volume but only a small amount of biomass and thus offer suitable targets for making more cellulose. Isolated promoters for each of these genes are used to drive the expression of these genes in different cell types. In addition, promoters from other genes can be used to express the CesA genes in other cell types.

Example 13

[0297] This example discusses the application of the CesA genes in improving nodal strength to reduce mid-season green snap.

[0298] Mid-season green snap is a significant problem in the Western plains, e.g., Nebraska, North and South Dakota and Western Minnesota, whereby the stalk snaps at the nodal plate before flowering in a severe windstorm at or below the ear node, resulting in yield losses of up to 80%. The underlying reason for this lesion is the disparity in the rates of elongation growth and dry matter deposition in the corn plant before flowering. Whereas a plant doubles in height in approximately two weeks before flowering, the most rapid rate of elongation growth, it accumulates only 30% additional dry matter during the same period, resulting in a 3-fold disparity (Dhugga, unpublished data). The plant thus becomes susceptible to breakage. It has been determined that the breakage occurs through the pulvinal zone at the base of the leaf sheath.

[0299] Transgenic plants expressing the CesA genes are produced by the method outlined in Example 5 or other suitable methods. The expression pattern of eleven of the twelve maize CesA genes in the pulvinal zone tissue is shown in FIG. 9. The twelfth, CesA9, had the same tag at CesA4 to which it is very highly related. Seven of the genes, CesA1, 4, 7, 8, 10, 11 and 12 are expressed at a higher level than the remaining genes. CesA8 shows the highest expression in this tissue. The expression of the various CesA genes, particularly CesA8, in this tissue can be used to increase its strength.

[0300] The above examples are provided to illustrate the invention but not to limit its scope. Other variants of the invention will be readily apparent to one of ordinary skill in the art and are encompassed by the appended claims. All publications, patents, patent applications, and computer programs cited herein are hereby incorporated by reference.

Sequence CWU 1

1

5213780DNAZea mays 1gtcgacccac gcgtccgcag cagcagaagc actgcgcggc attgcagcga tcgagcggga 60ggaatttggg gcatggtggt cgccaacgcc gctcggatct agaggcccgc acgggccgat 120tggtctccgc ccgcctcgtc ggtgttggtg tcgttggcgt gtggagccgt ctcggtggga 180gcagcgggga gggagcggag atggcggcca acaaggggat ggtggcgggc tcgcacaacc 240gcaacgagtt cgtcatgatc cgccacgacg gcgatgtgcc gggctcggct aagcccacaa 300agagtgcgaa tggacaggtc tgccagattt gcggtgactc tgtgggtgtt tcagccactg 360gtgatgtctt tgttgcctgc aatgagtgtg ccttccctgt ctgccgccca tgctatgagt 420atgagcgcaa ggaggggaac caatgctgcc cccagtgcaa gactagatac aagagacaga 480aaggtagccc tcgagttcat ggtgatgagg atgaggaaga tgttgatgac ctagacaatg 540aattcaacta caagcaaggc agtgggaaag gcccagagtg gcaactgcaa ggagatgatg 600ctgatctgtc ttcatctgct cgccatgagc cacatcatcg gattccacgc ctgacaagcg 660gtcaacagat atctggagag attcctgatg cttcccctga ccgtcattct atccgcagtc 720caacatcgag ctatgttgat ccaagcgtcc cagttcctgt gaggattgtg gacccctcga 780aggacttgaa ttcctatggg cttaatagtg ttgactggaa ggaaagagtt gagagctgga 840gggttaaaca ggacaaaaat atgatgcaag tgactaataa atatccagag gctagaggag 900gagacatgga ggggactggc tcaaatggag aagatatgca aatggttgat gatgcacggc 960tacctttgag ccgtatcgtg ccaatttcct caaaccagct caacctttac cgggtagtga 1020tcattctccg tcttatcatc ctgtgcttct tcttccagta tcgtgtcagt catccagtgc 1080gtgatgctta tggattatgg ctagtatctg ttatctgcga ggtctggttt gccttgtctt 1140ggcttctaga tcagttccca aaatggtatc caatcaaccg tgagacatat cttgacaggc 1200ttgcattgag gtatgataga gagggagagc catcacagct ggctcccatt gatgtcttcg 1260tcagtacagt ggatccattg aaggaacctc cactgatcac agccaacact gttttgtcca 1320ttctttctgt ggattaccct gttgacaaag tgtcatgcta tgtttctgat gatggttcag 1380ctatgctgac ttttgagtct ctctcagaaa ccgcagaatt tgctagaaag tgggttccct 1440tttgtaagaa gcacaatatt gaaccaagag ctccagaatt ttactttgct caaaaaatag 1500attacctgaa ggacaaaatt caaccttcat ttgttaagga aagacgcgca atgaagaggg 1560agtatgaaga attcaaagta agaatcaatg cccttgttgc caaagcacag aaagtgcctg 1620aagaggggtg gaccatggct gatggaactg catggcctgg gaataatcct agggaccatc 1680ctggcatgat tcaggttttc ttggggcaca gtggtgggct cgacactgat ggaaatgagt 1740taccacgtct tgtctatgtc tctcgtgaaa agagaccagg ctttcagcat cacaagaagg 1800ctggtgcaat gaatgcgctg attcgtgtat ctgctgtgct gacaaatggt gcctatcttc 1860tcaatgtgga ttgcgaccat tacttcaata gcagcaaagc tcttagagaa gcaatgtgct 1920tcatgatgga tccggctcta ggaaggaaaa cttgttatgt acaatttcca cagagatttg 1980atggcattga cttgcacgat cgatatgcta atcggaacat agttttcttt gatatcaaca 2040tgaaaggtct ggatggcatt cagggtccag tttacgtggg aacaggatgc tgtttcaata 2100gacaggcttt gtatggatac gatcctgttt tgactgaagc tgatctggag ccaaacattg 2160ttattaagag ctgctgtggt agaaggaaga aaaagaacaa gagttatatg gatagtcaaa 2220gccgtattat gaagagaaca gaatcttcag ctcccatctt caatatggaa gacatcgaag 2280agggtattga aggttacgag gatgaaaggt cagtgcttat gtcccagagg aaattggaga 2340aacgctttgg tcagtctcct attttcattg catccacctt tatgacacaa ggtggcatac 2400caccttcaac aaacccagct tctctactaa aggaagctat ccatgtcatc agttgtggat 2460atgaggacaa aactgaatgg ggaaaagaga ttggctggat ctatggttca gtaacggagg 2520atattctgac tgggtttaaa atgcatgcaa ggggctggca atcaatctac tgcatgccac 2580cacgaccttg tttcaagggt tctgcaccaa tcaatctttc cgatcgtctt aatcaggtgc 2640tccgttgggc tcttgggtca gtggaaattc tgcttagtag acattgtcct atctggtatg 2700gttacaatgg acgattgaag cttttggaga ggctggctta catcaacact attgtatatc 2760caatcacatc cattccgctt attgcctatt gtgtgcttcc cgctatctgc ctccttacca 2820ataaatttat cattcctgag attagcaatt atgctgggat gttcttcatt cttcttttcg 2880cctccatttt tgccactggt atattggagc ttagatggag tggtgttggc attgaagatt 2940ggtggagaaa tgagcagttt tgggttattg gtggcacctc tgcccatctc ttcgcagtgt 3000tccagggtct gctgaaagtg ttggctggga ttgataccaa cttcacagtt acctcaaagg 3060catctgatga ggatggcgac tttgctgagc tatatgtgtt caagtggacc agtttgctca 3120ttcctccgac cactgttctt gtcattaacc tggtcggaat ggtggcagga atttcttatg 3180ccattaacag tggctaccaa tcctggggtc cgctctttgg aaagctgttc ttctcgatct 3240gggtgatcct ccatctctac cccttcctca agggtctcat gggaaggcag aaccgcacac 3300caacaatcgt cattgtctgg tccatccttc ttgcatctat cttctccttg ctgtgggtga 3360agatcgatcc tttcatctcc ccgacacaga aagctgctgc cttggggcaa tgtggcgtca 3420actgctgatc gagacagtga ctcttatttg aagaggctca atcaagatct gccccctcgt 3480gtaaatacct gaggaggcta gatgggaatt ccttttgttg taggtgagga tggatttgca 3540tctaagttat gcctctgttc attagcttct tccgtgccgg tgctgctgcg gactaagaat 3600cacggagcct ttctaccttc catgtagcgc cagccagcag cgtaagatgt gaattttgaa 3660gttttgttat gcgtgcagtt tattgtttta gagtaaatta tcatttgttt gtgggaactg 3720ttcacacgag cttataatgg caatgctgtt atttaaaaaa aaaaaaaaaa gggcggccgc 378021075PRTZea mays 2Met Ala Ala Asn Lys Gly Met Val Ala Gly Ser His Asn Arg Asn Glu1 5 10 15 Phe Val Met Ile Arg His Asp Gly Asp Val Pro Gly Ser Ala Lys Pro 20 25 30 Thr Lys Ser Ala Asn Gly Gln Val Cys Gln Ile Cys Gly Asp Ser Val 35 40 45 Gly Val Ser Ala Thr Gly Asp Val Phe Val Ala Cys Asn Glu Cys Ala 50 55 60 Phe Pro Val Cys Arg Pro Cys Tyr Glu Tyr Glu Arg Lys Glu Gly Asn65 70 75 80 Gln Cys Cys Pro Gln Cys Lys Thr Arg Tyr Lys Arg Gln Lys Gly Ser 85 90 95 Pro Arg Val His Gly Asp Glu Asp Glu Glu Asp Val Asp Asp Leu Asp 100 105 110 Asn Glu Phe Asn Tyr Lys Gln Gly Ser Gly Lys Gly Pro Glu Trp Gln 115 120 125 Leu Gln Gly Asp Asp Ala Asp Leu Ser Ser Ser Ala Arg His Glu Pro 130 135 140 His His Arg Ile Pro Arg Leu Thr Ser Gly Gln Gln Ile Ser Gly Glu145 150 155 160 Ile Pro Asp Ala Ser Pro Asp Arg His Ser Ile Arg Ser Pro Thr Ser 165 170 175 Ser Tyr Val Asp Pro Ser Val Pro Val Pro Val Arg Ile Val Asp Pro 180 185 190 Ser Lys Asp Leu Asn Ser Tyr Gly Leu Asn Ser Val Asp Trp Lys Glu 195 200 205 Arg Val Glu Ser Trp Arg Val Lys Gln Asp Lys Asn Met Met Gln Val 210 215 220 Thr Asn Lys Tyr Pro Glu Ala Arg Gly Gly Asp Met Glu Gly Thr Gly225 230 235 240 Ser Asn Gly Glu Asp Met Gln Met Val Asp Asp Ala Arg Leu Pro Leu 245 250 255 Ser Arg Ile Val Pro Ile Ser Ser Asn Gln Leu Asn Leu Tyr Arg Val 260 265 270 Val Ile Ile Leu Arg Leu Ile Ile Leu Cys Phe Phe Phe Gln Tyr Arg 275 280 285 Val Ser His Pro Val Arg Asp Ala Tyr Gly Leu Trp Leu Val Ser Val 290 295 300 Ile Cys Glu Val Trp Phe Ala Leu Ser Trp Leu Leu Asp Gln Phe Pro305 310 315 320 Lys Trp Tyr Pro Ile Asn Arg Glu Thr Tyr Leu Asp Arg Leu Ala Leu 325 330 335 Arg Tyr Asp Arg Glu Gly Glu Pro Ser Gln Leu Ala Pro Ile Asp Val 340 345 350 Phe Val Ser Thr Val Asp Pro Leu Lys Glu Pro Pro Leu Ile Thr Ala 355 360 365 Asn Thr Val Leu Ser Ile Leu Ser Val Asp Tyr Pro Val Asp Lys Val 370 375 380 Ser Cys Tyr Val Ser Asp Asp Gly Ser Ala Met Leu Thr Phe Glu Ser385 390 395 400 Leu Ser Glu Thr Ala Glu Phe Ala Arg Lys Trp Val Pro Phe Cys Lys 405 410 415 Lys His Asn Ile Glu Pro Arg Ala Pro Glu Phe Tyr Phe Ala Gln Lys 420 425 430 Ile Asp Tyr Leu Lys Asp Lys Ile Gln Pro Ser Phe Val Lys Glu Arg 435 440 445 Arg Ala Met Lys Arg Glu Tyr Glu Glu Phe Lys Val Arg Ile Asn Ala 450 455 460 Leu Val Ala Lys Ala Gln Lys Val Pro Glu Glu Gly Trp Thr Met Ala465 470 475 480 Asp Gly Thr Ala Trp Pro Gly Asn Asn Pro Arg Asp His Pro Gly Met 485 490 495 Ile Gln Val Phe Leu Gly His Ser Gly Gly Leu Asp Thr Asp Gly Asn 500 505 510 Glu Leu Pro Arg Leu Val Tyr Val Ser Arg Glu Lys Arg Pro Gly Phe 515 520 525 Gln His His Lys Lys Ala Gly Ala Met Asn Ala Leu Ile Arg Val Ser 530 535 540 Ala Val Leu Thr Asn Gly Ala Tyr Leu Leu Asn Val Asp Cys Asp His545 550 555 560 Tyr Phe Asn Ser Ser Lys Ala Leu Arg Glu Ala Met Cys Phe Met Met 565 570 575 Asp Pro Ala Leu Gly Arg Lys Thr Cys Tyr Val Gln Phe Pro Gln Arg 580 585 590 Phe Asp Gly Ile Asp Leu His Asp Arg Tyr Ala Asn Arg Asn Ile Val 595 600 605 Phe Phe Asp Ile Asn Met Lys Gly Leu Asp Gly Ile Gln Gly Pro Val 610 615 620 Tyr Val Gly Thr Gly Cys Cys Phe Asn Arg Gln Ala Leu Tyr Gly Tyr625 630 635 640 Asp Pro Val Leu Thr Glu Ala Asp Leu Glu Pro Asn Ile Val Ile Lys 645 650 655 Ser Cys Cys Gly Arg Arg Lys Lys Lys Asn Lys Ser Tyr Met Asp Ser 660 665 670 Gln Ser Arg Ile Met Lys Arg Thr Glu Ser Ser Ala Pro Ile Phe Asn 675 680 685 Met Glu Asp Ile Glu Glu Gly Ile Glu Gly Tyr Glu Asp Glu Arg Ser 690 695 700 Val Leu Met Ser Gln Arg Lys Leu Glu Lys Arg Phe Gly Gln Ser Pro705 710 715 720 Ile Phe Ile Ala Ser Thr Phe Met Thr Gln Gly Gly Ile Pro Pro Ser 725 730 735 Thr Asn Pro Ala Ser Leu Leu Lys Glu Ala Ile His Val Ile Ser Cys 740 745 750 Gly Tyr Glu Asp Lys Thr Glu Trp Gly Lys Glu Ile Gly Trp Ile Tyr 755 760 765 Gly Ser Val Thr Glu Asp Ile Leu Thr Gly Phe Lys Met His Ala Arg 770 775 780 Gly Trp Gln Ser Ile Tyr Cys Met Pro Pro Arg Pro Cys Phe Lys Gly785 790 795 800 Ser Ala Pro Ile Asn Leu Ser Asp Arg Leu Asn Gln Val Leu Arg Trp 805 810 815 Ala Leu Gly Ser Val Glu Ile Leu Leu Ser Arg His Cys Pro Ile Trp 820 825 830 Tyr Gly Tyr Asn Gly Arg Leu Lys Leu Leu Glu Arg Leu Ala Tyr Ile 835 840 845 Asn Thr Ile Val Tyr Pro Ile Thr Ser Ile Pro Leu Ile Ala Tyr Cys 850 855 860 Val Leu Pro Ala Ile Cys Leu Leu Thr Asn Lys Phe Ile Ile Pro Glu865 870 875 880 Ile Ser Asn Tyr Ala Gly Met Phe Phe Ile Leu Leu Phe Ala Ser Ile 885 890 895 Phe Ala Thr Gly Ile Leu Glu Leu Arg Trp Ser Gly Val Gly Ile Glu 900 905 910 Asp Trp Trp Arg Asn Glu Gln Phe Trp Val Ile Gly Gly Thr Ser Ala 915 920 925 His Leu Phe Ala Val Phe Gln Gly Leu Leu Lys Val Leu Ala Gly Ile 930 935 940 Asp Thr Asn Phe Thr Val Thr Ser Lys Ala Ser Asp Glu Asp Gly Asp945 950 955 960 Phe Ala Glu Leu Tyr Val Phe Lys Trp Thr Ser Leu Leu Ile Pro Pro 965 970 975 Thr Thr Val Leu Val Ile Asn Leu Val Gly Met Val Ala Gly Ile Ser 980 985 990 Tyr Ala Ile Asn Ser Gly Tyr Gln Ser Trp Gly Pro Leu Phe Gly Lys 995 1000 1005 Leu Phe Phe Ser Ile Trp Val Ile Leu His Leu Tyr Pro Phe Leu Lys 1010 1015 1020 Gly Leu Met Gly Arg Gln Asn Arg Thr Pro Thr Ile Val Ile Val Trp1025 1030 1035 1040Ser Ile Leu Leu Ala Ser Ile Phe Ser Leu Leu Trp Val Lys Ile Asp 1045 1050 1055 Pro Phe Ile Ser Pro Thr Gln Lys Ala Ala Ala Leu Gly Gln Cys Gly 1060 1065 1070 Val Asn Cys 1075325DNAZea mays 3atggcggcca acaaggggat ggtgg 25425DNAZea mays 4tcagcagttg acgccacatt gcccc 2552830DNAZea maysmisc_feature2809, 2818, 2824, 2826, 2829n = A,T,C or G 5tacctctaag tcgcatagtt ccgatatctc caaacgagct taacctttat cggatcgtga 60ttgttctccg gcttatcatc ctatgtttct tctttcaata tcgtataact catccagtgg 120aagatgctta tgggttgtgg cttgtatctg ttatttgtga agtttggttt gccttgtctt 180ggcttctaga tcagttccca aagtggtatc ctatcaaccg tgaaacttac ctcgatagac 240ttgcattgag atatgatagg gagggtgagc catcccagtt ggctccaatc gatgtctttg 300ttagtacagt ggatccactt aaggaacctc ctctaattac tggcaacact gtcctgtcca 360ttcttgctgt ggattaccct gttgacaaag tatcatgtta tgtttctgat gacggttcag 420ctatgttgac ttttgaagcg ctatctgaaa ccgcagagtt tgcaaggaaa tgggttccct 480tttgcaagaa acacaatatt gaacctaggg ctccagagtt ttactttgct cgaaagatag 540attacctaaa ggacaaaata caaccttctt ttgtgaaaga aaggcgggct atgaagaggg 600agtgtgaaga gttcaaagta cggatcgatg cccttgttgc aaaagcgcaa aaaatacctg 660aggagggctg gaccatggct gatggcactc cttggcctgg gaataaccct agagatcatc 720caggaatgat ccaagtattc ttgggccaca gtggtgggct tgacacggat gggaatgagt 780tgccacggct tgtttatgtt tctcgtgaaa agaggccagg cttccagcac cacaagaagg 840ctggtgccat gaatgctttg attcgcgtat cagctgtcct gacgaatggt gcttatcttc 900ttaatgtgga ttgtgatcac tacttcaata gcagcaaagc tcttagagag gctatgtgtt 960tcatgatgga tccagcacta ggaaggaaaa cttgctatgt tcagtttcca caaagatttg 1020atggtataga cttgcatgat cgatatgcaa accggaacat tgtcttcttt gatattaata 1080tgaagggtct agatggcatt caaggacctg tttatgtggg aacaggatgc tgtttcaata 1140ggcaggcctt gtatggctat gatcctgtat tgacagaagc tgatttggag cctaacatta 1200tcattaaaag ttgctgtggc ggaagaaaaa agaaggacaa gagctatatt gattccaaaa 1260accgtgatat gaagagaaca gaatcttcgg ctcccatctt caacatggaa gatatagaag 1320agggatttga aggttacgag gatgaaaggt cactgcttat gtctcagaag agcttggaga 1380aacgctttgg ccagtctcca atttttattg catccacctt tatgactcaa ggtggcatac 1440ccccttcaac aaacccaggt tccctgctaa aggaagctat acatgtcatt agttgtggat 1500atgaggataa aacagaatgg gggaaagaga tcggatggat atatggctct gttactgaag 1560atattttaac tggtttcaag atgcatgcaa gaggttggat atccatctac tgcatgccac 1620ttcggccttg cttcaagggt tctgctccaa ttaatctttc tgatcgtctc aaccaagtgt 1680tacgctgggc tcttggttca gttgaaattc tacttagcag acactgtcct atctggtatg 1740gttacaatgg aaggctaaag cttctggaga gactggcata catcaacacc attgtttatc 1800caattacatc tatcccacta gtagcatact gcgtccttcc tgctatctgt ttactcacca 1860acaaatttat tattcctgcg attagcaatt atgctggggc gttcttcatc ctgctttttg 1920cttccatctt cgccactggt attttggagc ttcgatggag tggtgttggc attgaggatt 1980ggtggagaaa tgagcagttt tgggtcattg gtggcacctc tgcacatctc tttgctgtgt 2040tccaaggtct cttaaaagtg ctagcaggga tcgacacaaa cttcacggtc acatcaaagg 2100caaccgatga tgatggtgat tttgctgagc tgtatgtgtt caagtggaca actcttctga 2160tcccccccac cactgtgctt gtgattaacc tggttggtat agtggctgga gtgtcgtatg 2220ctatcaacag tggctaccaa tcatggggtc cactattcgg gaagctgttc tttgcaatct 2280gggtgatcct ccacctctac cctttcctga agggtctcat ggggaagcag aaccgcacac 2340cgaccatcgt catcgtttgg tccgtccttc ttgcttccat attctcgctg ctgtgggtga 2400agatcgaccc cttcatatcc cctacccaga aggctctttc ccgtgggcag tgtggtgtaa 2460actgctgaaa tgatccgaac tgcctgctga ataacattgc tccggcacaa tcatgatcta 2520ccccttcgtg taaataccag aggttaggca agacttttct tggtaggtgg cgaagatgtg 2580tcgtttaagt tcactctact gcatttgggg tgggcagcat gaaactttgt caacttatgt 2640cgtgctactt atttgtagct aagtagcagt aagtagtgcc tgtttcatgt tgactgtcgt 2700gactacctgt tcaccgtggg ctctggactg tcgtgatgta acctgtatgt tggaacttca 2760agtactgatt gagctgtttg gtcaatgaca ttgagggatt ctctctctng aaattaanac 2820aaantnggnt 28306821PRTZea mays 6Pro Leu Ser Arg Ile Val Pro Ile Ser Pro Asn Glu Leu Asn Leu Tyr1 5 10 15 Arg Ile Val Ile Val Leu Arg Leu Ile Ile Leu Cys Phe Phe Phe Gln 20 25 30 Tyr Arg Ile Thr His Pro Val Glu Asp Ala Tyr Gly Leu Trp Leu Val 35 40 45 Ser Val Ile Cys Glu Val Trp Phe Ala Leu Ser Trp Leu Leu Asp Gln 50 55 60 Phe Pro Lys Trp Tyr Pro Ile Asn Arg Glu Thr Tyr Leu Asp Arg Leu65 70 75 80 Ala Leu Arg Tyr Asp Arg Glu Gly Glu Pro Ser Gln Leu Ala Pro Ile 85 90 95 Asp Val Phe Val Ser Thr Val Asp Pro Leu Lys Glu Pro Pro Leu Ile 100 105 110 Thr Gly Asn Thr Val Leu Ser Ile Leu Ala Val Asp Tyr Pro Val Asp 115 120 125 Lys Val Ser Cys Tyr Val Ser Asp Asp Gly Ser Ala Met Leu Thr Phe 130 135 140 Glu Ala Leu Ser Glu Thr Ala Glu Phe Ala Arg Lys Trp Val Pro Phe145 150 155 160 Cys Lys Lys His Asn Ile Glu Pro Arg Ala Pro Glu Phe Tyr Phe Ala 165 170 175 Arg Lys Ile Asp

Tyr Leu Lys Asp Lys Ile Gln Pro Ser Phe Val Lys 180 185 190 Glu Arg Arg Ala Met Lys Arg Glu Cys Glu Glu Phe Lys Val Arg Ile 195 200 205 Asp Ala Leu Val Ala Lys Ala Gln Lys Ile Pro Glu Glu Gly Trp Thr 210 215 220 Met Ala Asp Gly Thr Pro Trp Pro Gly Asn Asn Pro Arg Asp His Pro225 230 235 240 Gly Met Ile Gln Val Phe Leu Gly His Ser Gly Gly Leu Asp Thr Asp 245 250 255 Gly Asn Glu Leu Pro Arg Leu Val Tyr Val Ser Arg Glu Lys Arg Pro 260 265 270 Gly Phe Gln His His Lys Lys Ala Gly Ala Met Asn Ala Leu Ile Arg 275 280 285 Val Ser Ala Val Leu Thr Asn Gly Ala Tyr Leu Leu Asn Val Asp Cys 290 295 300 Asp His Tyr Phe Asn Ser Ser Lys Ala Leu Arg Glu Ala Met Cys Phe305 310 315 320 Met Met Asp Pro Ala Leu Gly Arg Lys Thr Cys Tyr Val Gln Phe Pro 325 330 335 Gln Arg Phe Asp Gly Ile Asp Leu His Asp Arg Tyr Ala Asn Arg Asn 340 345 350 Ile Val Phe Phe Asp Ile Asn Met Lys Gly Leu Asp Gly Ile Gln Gly 355 360 365 Pro Val Tyr Val Gly Thr Gly Cys Cys Phe Asn Arg Gln Ala Leu Tyr 370 375 380 Gly Tyr Asp Pro Val Leu Thr Glu Ala Asp Leu Glu Pro Asn Ile Ile385 390 395 400 Ile Lys Ser Cys Cys Gly Gly Arg Lys Lys Lys Asp Lys Ser Tyr Ile 405 410 415 Asp Ser Lys Asn Arg Asp Met Lys Arg Thr Glu Ser Ser Ala Pro Ile 420 425 430 Phe Asn Met Glu Asp Ile Glu Glu Gly Phe Glu Gly Tyr Glu Asp Glu 435 440 445 Arg Ser Leu Leu Met Ser Gln Lys Ser Leu Glu Lys Arg Phe Gly Gln 450 455 460 Ser Pro Ile Phe Ile Ala Ser Thr Phe Met Thr Gln Gly Gly Ile Pro465 470 475 480 Pro Ser Thr Asn Pro Gly Ser Leu Leu Lys Glu Ala Ile His Val Ile 485 490 495 Ser Cys Gly Tyr Glu Asp Lys Thr Glu Trp Gly Lys Glu Ile Gly Trp 500 505 510 Ile Tyr Gly Ser Val Thr Glu Asp Ile Leu Thr Gly Phe Lys Met His 515 520 525 Ala Arg Gly Trp Ile Ser Ile Tyr Cys Met Pro Leu Arg Pro Cys Phe 530 535 540 Lys Gly Ser Ala Pro Ile Asn Leu Ser Asp Arg Leu Asn Gln Val Leu545 550 555 560 Arg Trp Ala Leu Gly Ser Val Glu Ile Leu Leu Ser Arg His Cys Pro 565 570 575 Ile Trp Tyr Gly Tyr Asn Gly Arg Leu Lys Leu Leu Glu Arg Leu Ala 580 585 590 Tyr Ile Asn Thr Ile Val Tyr Pro Ile Thr Ser Ile Pro Leu Val Ala 595 600 605 Tyr Cys Val Leu Pro Ala Ile Cys Leu Leu Thr Asn Lys Phe Ile Ile 610 615 620 Pro Ala Ile Ser Asn Tyr Ala Gly Ala Phe Phe Ile Leu Leu Phe Ala625 630 635 640 Ser Ile Phe Ala Thr Gly Ile Leu Glu Leu Arg Trp Ser Gly Val Gly 645 650 655 Ile Glu Asp Trp Trp Arg Asn Glu Gln Phe Trp Val Ile Gly Gly Thr 660 665 670 Ser Ala His Leu Phe Ala Val Phe Gln Gly Leu Leu Lys Val Leu Ala 675 680 685 Gly Ile Asp Thr Asn Phe Thr Val Thr Ser Lys Ala Thr Asp Asp Asp 690 695 700 Gly Asp Phe Ala Glu Leu Tyr Val Phe Lys Trp Thr Thr Leu Leu Ile705 710 715 720 Pro Pro Thr Thr Val Leu Val Ile Asn Leu Val Gly Ile Val Ala Gly 725 730 735 Val Ser Tyr Ala Ile Asn Ser Gly Tyr Gln Ser Trp Gly Pro Leu Phe 740 745 750 Gly Lys Leu Phe Phe Ala Ile Trp Val Ile Leu His Leu Tyr Pro Phe 755 760 765 Leu Lys Gly Leu Met Gly Lys Gln Asn Arg Thr Pro Thr Ile Val Ile 770 775 780 Val Trp Ser Val Leu Leu Ala Ser Ile Phe Ser Leu Leu Trp Val Lys785 790 795 800 Ile Asp Pro Phe Ile Ser Pro Thr Gln Lys Ala Leu Ser Arg Gly Gln 805 810 815 Cys Gly Val Asn Cys 820 725DNAZea mays 7cctctaagtc gcatagttcc gatat 25825DNAZea mays 8tcagcagttt acaccacact gccca 2593773DNAZea mays 9gtcgacccac gcgtccgcta ggatcaaaac cgtctcgccg ctgcaataat cttttgtcaa 60ttcttaatcc ctcgcgtcga cagcgacagc ggaaccaact cacgttgccg cggcttcctc 120catcggtgcg gtgccctgtc cttttctctc gtccctcctc cccccgtata gttaagcccc 180gccccgctac tactactact agcagcagca gcgctctcgc agcgggagat gcggtgttga 240tccgtgcccc gctcggatct cgggactggt gccggctctg cccaggcccc aggctccagg 300ccagctccct cgacgtttct cggcgagctc gcttgccatg gagggcgacg cggacggcgt 360gaagtcgggg aggcgcggtg gcggacaggt gtgccagatc tgcggcgacg gcgtgggcac 420cacggcggag ggggacgtct tcgccgcctg cgacgtctgc gggtttccgg tgtgccgccc 480ctgctacgag tacgagcgca aggacggcac gcaggcgtgc ccccagtgca agaccaagta 540caagcgccac aaggggagcc cggcgatccg tggggaggaa ggagacgaca ctgatgccga 600tagcgacttc aattaccttg catctggcaa tgaggaccag aagcagaaga ttgccgacag 660aatgcgcagc tggcgcatga acgttggggg cagcggggat gttggtcgcc ccaagtatga 720cagtggcgag atcgggctta ccaagtatga cagtggcgag attcctcggg gatacatccc 780atcagtcact aacagccaga tctcaggaga aatccctggt gcttcccctg accatcatat 840gatgtcccca actgggaaca ttggcaagcg tgctccattt ccctatgtga accattcgcc 900aaatccgtca agggagttct ctggtagcat tgggaatgtt gcctggaaag agagggttga 960tggctggaaa atgaagcagg acaaggggac gattcccatg acgaatggca caagcattgc 1020tccctctgag ggtcggggtg ttggtgatat tgatgcatca actgattaca acatggaaga 1080tgccttattg aacgacgaaa ctcgacagcc tctatctagg aaagttccac ttccttcctc 1140caggataaat ccatacagga tggtcattgt gctgcgattg attgttctaa gcatcttctt 1200gcactaccgt atcacaaatc ctgtgcgcaa tgcataccca ttatggcttc tatctgttat 1260atgtgagatc tggtttgctc tttcgtggat attggatcag ttccctaagt ggtttccaat 1320caaccgggag acgtaccttg ataggctggc attaaggtat gaccgggaag gtgagccatc 1380tcagttggct gctgttgaca ttttcgtcag tacagtcgac ccaatgaagg agcctcctct 1440tgtcactgcc aataccgtgc tatccattct tgctgtggat taccctgtgg ataaggtctc 1500ttgctatgta tctgatgatg gagctgcgat gctgacattt gatgcactag ctgagacttc 1560agagtttgct agaaaatggg taccatttgt taagaagtac aacattgaac ctagagctcc 1620tgaatggtac ttctcccaga aaattgatta cttgaaggac aaagtgcacc cttcatttgt 1680taaagaccgc cgggccatga agagagaata tgaagaattc aaagttaggg taaatggcct 1740tgttgctaag gcacagaaag ttcctgagga aggatggatc atgcaagatg gcacaccatg 1800gccaggaaac aataccaggg accatcctgg aatgattcag gttttccttg gtcacagtgg 1860tggccttgat actgagggca atgagctacc ccgtttggtc tatgtttctc gtgaaaagcg 1920tcctggattc cagcatcaca agaaagctgg tgccatgaat gctcttgttc gtgtctcagc 1980tgtgcttacc aatggacaat acatgttgaa tcttgattgt gatcactaca ttaacaacag 2040taaggctctc agggaagcta tgtgcttcct tatggaccct aacctaggaa ggagtgtctg 2100ctacgtccag tttccccaga gattcgatgg cattgacagg aatgatcgat atgccaacag 2160gaacaccgtg tttttcgata ttaacttgag aggtcttgat ggcatccaag gaccagttta 2220tgtcggaact ggctgtgttt tcaaccgaac agctctatat ggttatgagc ccccaattaa 2280gcagaagaag ggtggtttct tgtcatcact atgtggcggt aggaagaagg caagcaaatc 2340aaagaagggc tcggacaaga agaagtcgca gaagcatgtg gacagttctg tgccagtatt 2400caaccttgaa gatatagagg agggagttga aggcgctgga tttgacgacg agaaatcact 2460tcttatgtct caaatgagcc tggagaagag atttggccag tccgcagcgt ttgttgcctc 2520cactctgatg gagtatggtg gtgttcctca gtccgcaact ccggagtctc ttctgaaaga 2580agctatccat gttataagct gtggctatga ggacaagact gaatggggaa ctgagatcgg 2640gtggatctac ggttctgtga cagaagacat tctcaccgga ttcaagatgc acgcgcgagg 2700ctggcggtcg atctactgca tgcccaagcg gccagctttc aaggggtctg cccccatcaa 2760tctttcggac cgtctgaacc aggtgctccg gtgggctctt gggtccgtgg agatcctctt 2820cagccggcac tgccccctgt ggtacggcta cggagggcgg ctcaagttcc tggagagatt 2880cgcgtacatc aacaccacca tctacccgct cacgtccatc ccgcttctca tctactgcat 2940cctgcccgcc atctgtctgc tcaccggaaa gttcatcatt ccagagatca gcaacttcgc 3000cagcatctgg ttcatctccc tcttcatctc gatcttcgcc acgggcatcc tggagatgag 3060gtggagcggg gtgggcatcg acgagtggtg gaggaacgag cagttctggg tgatcggggg 3120catctccgcg cacctcttcg ccgtgttcca gggcctgctc aaggtgctgg ccggcatcga 3180caccaacttc accgtcacct ccaaggcctc ggacgaggac ggcgacttcg cggagctgta 3240catgttcaag tggacgacgc tcctgatccc gcccaccacc atcctgatca tcaacctggt 3300cggcgtcgtc gccggcatct cctacgccat caacagcgga taccagtcgt ggggcccgct 3360cttcggcaag ctcttcttcg ccttctgggt catcgtccac ctgtacccgt tcctcaaggg 3420cctcatgggc aggcagaacc gcaccccgac catcgtcgtc gtctgggcca tcctgctggc 3480gtccatcttc tccttgctgt gggttcgcat cgaccccttc accacccgcg tcactggccc 3540ggatacccag acgtgtggca tcaactgcta gggaagtgga aggtttgtac tttgtagaaa 3600cggaggaata ccacgtgcca tctgttgtct gttaagttat atatatataa gcagcaagtg 3660gcgttattta cagctacgta cagaccagtg gatattgttt accacaaagt tttacttgtg 3720ttaatatgca ttcttttgtt gatataaaaa aaaaaaaaaa aaagggcggc cgc 3773101077PRTZea mays 10Met Glu Gly Asp Ala Asp Gly Val Lys Ser Gly Arg Arg Gly Gly Gly1 5 10 15 Gln Val Cys Gln Ile Cys Gly Asp Gly Val Gly Thr Thr Ala Glu Gly 20 25 30 Asp Val Phe Ala Ala Cys Asp Val Cys Gly Phe Pro Val Cys Arg Pro 35 40 45 Cys Tyr Glu Tyr Glu Arg Lys Asp Gly Thr Gln Ala Cys Pro Gln Cys 50 55 60 Lys Thr Lys Tyr Lys Arg His Lys Gly Ser Pro Ala Ile Arg Gly Glu65 70 75 80 Glu Gly Asp Asp Thr Asp Ala Asp Ser Asp Phe Asn Tyr Leu Ala Ser 85 90 95 Gly Asn Glu Asp Gln Lys Gln Lys Ile Ala Asp Arg Met Arg Ser Trp 100 105 110 Arg Met Asn Val Gly Gly Ser Gly Asp Val Gly Arg Pro Lys Tyr Asp 115 120 125 Ser Gly Glu Ile Gly Leu Thr Lys Tyr Asp Ser Gly Glu Ile Pro Arg 130 135 140 Gly Tyr Ile Pro Ser Val Thr Asn Ser Gln Ile Ser Gly Glu Ile Pro145 150 155 160 Gly Ala Ser Pro Asp His His Met Met Ser Pro Thr Gly Asn Ile Gly 165 170 175 Lys Arg Ala Pro Phe Pro Tyr Val Asn His Ser Pro Asn Pro Ser Arg 180 185 190 Glu Phe Ser Gly Ser Ile Gly Asn Val Ala Trp Lys Glu Arg Val Asp 195 200 205 Gly Trp Lys Met Lys Gln Asp Lys Gly Thr Ile Pro Met Thr Asn Gly 210 215 220 Thr Ser Ile Ala Pro Ser Glu Gly Arg Gly Val Gly Asp Ile Asp Ala225 230 235 240 Ser Thr Asp Tyr Asn Met Glu Asp Ala Leu Leu Asn Asp Glu Thr Arg 245 250 255 Gln Pro Leu Ser Arg Lys Val Pro Leu Pro Ser Ser Arg Ile Asn Pro 260 265 270 Tyr Arg Met Val Ile Val Leu Arg Leu Ile Val Leu Ser Ile Phe Leu 275 280 285 His Tyr Arg Ile Thr Asn Pro Val Arg Asn Ala Tyr Pro Leu Trp Leu 290 295 300 Leu Ser Val Ile Cys Glu Ile Trp Phe Ala Leu Ser Trp Ile Leu Asp305 310 315 320 Gln Phe Pro Lys Trp Phe Pro Ile Asn Arg Glu Thr Tyr Leu Asp Arg 325 330 335 Leu Ala Leu Arg Tyr Asp Arg Glu Gly Glu Pro Ser Gln Leu Ala Ala 340 345 350 Val Asp Ile Phe Val Ser Thr Val Asp Pro Met Lys Glu Pro Pro Leu 355 360 365 Val Thr Ala Asn Thr Val Leu Ser Ile Leu Ala Val Asp Tyr Pro Val 370 375 380 Asp Lys Val Ser Cys Tyr Val Ser Asp Asp Gly Ala Ala Met Leu Thr385 390 395 400 Phe Asp Ala Leu Ala Glu Thr Ser Glu Phe Ala Arg Lys Trp Val Pro 405 410 415 Phe Val Lys Lys Tyr Asn Ile Glu Pro Arg Ala Pro Glu Trp Tyr Phe 420 425 430 Ser Gln Lys Ile Asp Tyr Leu Lys Asp Lys Val His Pro Ser Phe Val 435 440 445 Lys Asp Arg Arg Ala Met Lys Arg Glu Tyr Glu Glu Phe Lys Val Arg 450 455 460 Val Asn Gly Leu Val Ala Lys Ala Gln Lys Val Pro Glu Glu Gly Trp465 470 475 480 Ile Met Gln Asp Gly Thr Pro Trp Pro Gly Asn Asn Thr Arg Asp His 485 490 495 Pro Gly Met Ile Gln Val Phe Leu Gly His Ser Gly Gly Leu Asp Thr 500 505 510 Glu Gly Asn Glu Leu Pro Arg Leu Val Tyr Val Ser Arg Glu Lys Arg 515 520 525 Pro Gly Phe Gln His His Lys Lys Ala Gly Ala Met Asn Ala Leu Val 530 535 540 Arg Val Ser Ala Val Leu Thr Asn Gly Gln Tyr Met Leu Asn Leu Asp545 550 555 560 Cys Asp His Tyr Ile Asn Asn Ser Lys Ala Leu Arg Glu Ala Met Cys 565 570 575 Phe Leu Met Asp Pro Asn Leu Gly Arg Ser Val Cys Tyr Val Gln Phe 580 585 590 Pro Gln Arg Phe Asp Gly Ile Asp Arg Asn Asp Arg Tyr Ala Asn Arg 595 600 605 Asn Thr Val Phe Phe Asp Ile Asn Leu Arg Gly Leu Asp Gly Ile Gln 610 615 620 Gly Pro Val Tyr Val Gly Thr Gly Cys Val Phe Asn Arg Thr Ala Leu625 630 635 640 Tyr Gly Tyr Glu Pro Pro Ile Lys Gln Lys Lys Gly Gly Phe Leu Ser 645 650 655 Ser Leu Cys Gly Gly Arg Lys Lys Ala Ser Lys Ser Lys Lys Gly Ser 660 665 670 Asp Lys Lys Lys Ser Gln Lys His Val Asp Ser Ser Val Pro Val Phe 675 680 685 Asn Leu Glu Asp Ile Glu Glu Gly Val Glu Gly Ala Gly Phe Asp Asp 690 695 700 Glu Lys Ser Leu Leu Met Ser Gln Met Ser Leu Glu Lys Arg Phe Gly705 710 715 720 Gln Ser Ala Ala Phe Val Ala Ser Thr Leu Met Glu Tyr Gly Gly Val 725 730 735 Pro Gln Ser Ala Thr Pro Glu Ser Leu Leu Lys Glu Ala Ile His Val 740 745 750 Ile Ser Cys Gly Tyr Glu Asp Lys Thr Glu Trp Gly Thr Glu Ile Gly 755 760 765 Trp Ile Tyr Gly Ser Val Thr Glu Asp Ile Leu Thr Gly Phe Lys Met 770 775 780 His Ala Arg Gly Trp Arg Ser Ile Tyr Cys Met Pro Lys Arg Pro Ala785 790 795 800 Phe Lys Gly Ser Ala Pro Ile Asn Leu Ser Asp Arg Leu Asn Gln Val 805 810 815 Leu Arg Trp Ala Leu Gly Ser Val Glu Ile Leu Phe Ser Arg His Cys 820 825 830 Pro Leu Trp Tyr Gly Tyr Gly Gly Arg Leu Lys Phe Leu Glu Arg Phe 835 840 845 Ala Tyr Ile Asn Thr Thr Ile Tyr Pro Leu Thr Ser Ile Pro Leu Leu 850 855 860 Ile Tyr Cys Ile Leu Pro Ala Ile Cys Leu Leu Thr Gly Lys Phe Ile865 870 875 880 Ile Pro Glu Ile Ser Asn Phe Ala Ser Ile Trp Phe Ile Ser Leu Phe 885 890 895 Ile Ser Ile Phe Ala Thr Gly Ile Leu Glu Met Arg Trp Ser Gly Val 900 905 910 Gly Ile Asp Glu Trp Trp Arg Asn Glu Gln Phe Trp Val Ile Gly Gly 915 920 925 Ile Ser Ala His Leu Phe Ala Val Phe Gln Gly Leu Leu Lys Val Leu 930 935 940 Ala Gly Ile Asp Thr Asn Phe Thr Val Thr Ser Lys Ala Ser Asp Glu945 950 955 960 Asp Gly Asp Phe Ala Glu Leu Tyr Met Phe Lys Trp Thr Thr Leu Leu 965 970 975 Ile Pro Pro Thr Thr Ile Leu Ile Ile Asn Leu Val Gly Val Val Ala 980 985 990 Gly Ile Ser Tyr Ala Ile Asn Ser Gly Tyr Gln Ser Trp Gly Pro Leu 995 1000 1005 Phe Gly Lys Leu Phe Phe Ala Phe Trp Val Ile Val His Leu Tyr Pro 1010 1015 1020 Phe Leu Lys Gly Leu Met Gly Arg Gln Asn Arg Thr Pro Thr Ile Val1025 1030 1035 1040Val Val Trp Ala Ile Leu Leu Ala Ser Ile Phe Ser Leu Leu Trp Val 1045 1050 1055 Arg Ile Asp Pro Phe Thr Thr Arg Val Thr Gly Pro Asp Thr Gln Thr

1060 1065 1070 Cys Gly Ile Asn Cys 1075 1125DNAZea mays 11atggagggcg acgcggacgg cgtga 251225DNAZea mays 12ctagcagttg atgccacacg tctgg 25133704DNAZea mays 13gtcgacccac gcttccggtc ggttccgcgt cccttttccc ctcccccctc cgtcgccgcc 60tcgagcgagc tccaccactt gctcctgcgc gaggtgaaca ctgggttagg gccactgcca 120ccgctgggct gcctctgctt ctgcctctcc cgccagcgcg cgagcccggg ggcgattcgg 180cgccggcacg cgggagggga agccgaggaa tgcggtgagt cggcgggggt ccggcgtttg 240tgaactcgtg gagggctcgg attggtgcgc catggacggc ggcgacgcca cgaattcggg 300gaagcatgtg gccgggcagg tgtgccagat ctgcggcgac ggcgtgggca ccgcggcgga 360cggcgacctc ttcaccgcct gcgacgtctg cggcttcccc gtgtgccgcc catgctacga 420gtacgagcgc aaggacggca cccaggcgtg cccgcagtgc aagactaagt acaagcgcca 480caaagggagc ccaccagtac acggtgagga aaatgaggat gtggatgctg acgatgtgag 540tgactacaac taccaagcat ctggcaacca ggatcagaag caaaagattg ctgagagaat 600gctcacttgg cggacaaact cacgtggcag tgatattggc ctggctaagt atgacagcgg 660tgaaattggg catgggaagt atgacagtgg tgagatccct cgtggatata tcccgtcact 720aactcatagc cagatctcag gagagattcc tggagcttcc cctgatcata tgatgtctcc 780tgttgggaac attggcaggc gtggacatca atttccttat gtaaatcatt ctccaaaccc 840atcgagggag ttctccggta gccttggcaa tgttgcatgg aaagagaggg tggatggatg 900gaaaatgaag gataaaggtg caattcctat gaccaatgga acaagcattg ctccatcaga 960agggcgtgga gttgctgata ttgatgcttc tactgattat aacatggaag atgccttact 1020gaatgatgaa actcggcaac ctctatctag aaaagtgcca attccttcat ccagaataaa 1080tccgtacaga atggtcattg tgctacgttt ggctgttcta tgcatattct tgcgctaccg 1140tatcacacat cctgtgaaca atgcatatcc actgtggctt ttatccgtca tatgtgagat 1200ctggtttgct ttgtcctgga ttttggatca gttcccaaag tggtccccaa tcaaccgtga 1260aacatacctt gatagactgg ctttaaggta tgaccgagaa ggtgaaccat ctcaattagc 1320tcctgttgat atttttgtca gtactgtgga tccaatgaag gagcctcctc ttgtcactgc 1380aaatactgtg ctttccatcc ttgctgtcga ttatccggtt gacaaggtat cttgctatgt 1440ttcggatgat ggagctgcta tgctgacttt tgatgctctc tctgaaactt cagagtttgc 1500tagaaaatgg gttccgttct gtaagaagta caacatagag cctagggccc cggaatggta 1560ctttgctcag aaaattgatt acttgaaaga caaagttcaa acctcatttg tgaaagaacg 1620ccgggccatg aagagagaat atgaagaatt caaagttcgt atcaatggtc ttgtagccaa 1680ggcacaaaaa gttcccgagg agggatggat catgcaagat ggtacacctt ggcctgggaa 1740caatactagg gaccatcctg gaatgattca ggttttcctg ggtcacagtg gagggcttga 1800cgttgaaggc aatgaacttc ctcgtttggt ttatgtgtct cgtgaaaaac gtcctggatt 1860ccaacatcac aagaaggctg gtgccatgaa tgcacttgtt cgtgtatcag ctgtccttac 1920taatgggcaa tacatgttga atcttgattg tgaccactac atcaataata gcaaggctct 1980tcgagaagct atgtgcttcc ttatggaccc aaacctagga aggaatgtct gttatgtcca 2040atttcctcag aggtttgatg gtattgatag gaatgaccga tatgcaaaca ggaacactgt 2100gtttttcgat attaacttga gaggtcttga cggcattcaa gggccagttt atgtgggaac 2160tggttgtgtg tttaacagaa cggccttata tggttatgag cctccagtca agaaaaaaaa 2220gccaggcttc ttctcttcgc tttgtggggg aaggaaaaag acgtcaaaat ctaagaagag 2280ctcggaaaag aagaagtcac atagacacgc agacagttct gtaccagtat ttaatctcga 2340agatatagag gaagggattg aaggttctca gtttgatgat gagaaatcgc tgattatgtc 2400tcaaatgagc ttggagaaga gatttggcca gtccagtgtt tttgtagcct ctactctgat 2460ggaatatggt ggtgttccac aatctgcaac tccagagtct cttctgaaag aagctattca 2520tgtcatcagc tgtggctatg aggacaaaac tgactgggga actgagattg ggtggatcta 2580tggttctgtt acagaagaca ttctcaccgg attcaagatg catgctcgag gctggcgatc 2640aatctactgc atgcctaagc gaccagcttt caagggatct gctcctatca acctttcgga 2700tcgtttgaat caagtgcttc ggtgggctct tggttccatt gaaattcttt tcagcaggca 2760ttgtcccata tggtatggct atggaggccg gcttaaattc ctggagagat ttgcttatat 2820caacacaaca atttatccac tcacatcaat cccgctcctc ctgtactgca tattgccagc 2880agtttgtctt ctcactggga agttcatcat cccaaagatt agtaacctag agagtgtttg 2940gtttatatcg ctctttatct caatctttgc cactggtatc cttgagatga ggtggagtgg 3000tgttggcatt gatgaatggt ggaggaacga gcagttctgg gtcattggtg gtatttctgc 3060gcatttattt gccgtcttcc agggtctcct gaaggtgctt gctggtatcg acacgagctt 3120cactgtcacc tctaaggcca ctgacgaaga aggtgatttt gccgagctct acatgttcaa 3180gtggacaacg cttctgatcc caccaaccac tattttgatc atcaacctgg tcggcgtggt 3240cgctggcatt tcctacgcaa tcaatagcgg ttaccagtca tggggacctc ttttcgggaa 3300gctcttcttt gcgttctggg tgattgtcca cctgtacccc ttcctcaagg gcctcatggg 3360gaagcagaac cgcacgccga ccattgtcgt tgtctgggct atcctccttg cgtcgatctt 3420ttccctgatg tgggttcgta tcgatccatt caccacccgg gtcactggcc ctgatatcgc 3480gaaatgtggc atcaactgct aggatgagct gaagatagtt aaagagtgga actagacgca 3540ttgtgcatcg taagttatca gtgggtggct ctttttatag tatggtagga acttggtcgg 3600gagacgttaa ttacatatgc tatatgtacc tccgctggtc tttatccgta agttaatata 3660tatactgctt tgagaattaa aaaaaaaaaa aaaagggcgg ccgc 3704141076PRTZea mays 14Met Asp Gly Gly Asp Ala Thr Asn Ser Gly Lys His Val Ala Gly Gln1 5 10 15 Val Cys Gln Ile Cys Gly Asp Gly Val Gly Thr Ala Ala Asp Gly Asp 20 25 30 Leu Phe Thr Ala Cys Asp Val Cys Gly Phe Pro Val Cys Arg Pro Cys 35 40 45 Tyr Glu Tyr Glu Arg Lys Asp Gly Thr Gln Ala Cys Pro Gln Cys Lys 50 55 60 Thr Lys Tyr Lys Arg His Lys Gly Ser Pro Pro Val His Gly Glu Glu65 70 75 80 Asn Glu Asp Val Asp Ala Asp Asp Val Ser Asp Tyr Asn Tyr Gln Ala 85 90 95 Ser Gly Asn Gln Asp Gln Lys Gln Lys Ile Ala Glu Arg Met Leu Thr 100 105 110 Trp Arg Thr Asn Ser Arg Gly Ser Asp Ile Gly Leu Ala Lys Tyr Asp 115 120 125 Ser Gly Glu Ile Gly His Gly Lys Tyr Asp Ser Gly Glu Ile Pro Arg 130 135 140 Gly Tyr Ile Pro Ser Leu Thr His Ser Gln Ile Ser Gly Glu Ile Pro145 150 155 160 Gly Ala Ser Pro Asp His Met Met Ser Pro Val Gly Asn Ile Gly Arg 165 170 175 Arg Gly His Gln Phe Pro Tyr Val Asn His Ser Pro Asn Pro Ser Arg 180 185 190 Glu Phe Ser Gly Ser Leu Gly Asn Val Ala Trp Lys Glu Arg Val Asp 195 200 205 Gly Trp Lys Met Lys Asp Lys Gly Ala Ile Pro Met Thr Asn Gly Thr 210 215 220 Ser Ile Ala Pro Ser Glu Gly Arg Gly Val Ala Asp Ile Asp Ala Ser225 230 235 240 Thr Asp Tyr Asn Met Glu Asp Ala Leu Leu Asn Asp Glu Thr Arg Gln 245 250 255 Pro Leu Ser Arg Lys Val Pro Ile Pro Ser Ser Arg Ile Asn Pro Tyr 260 265 270 Arg Met Val Ile Val Leu Arg Leu Ala Val Leu Cys Ile Phe Leu Arg 275 280 285 Tyr Arg Ile Thr His Pro Val Asn Asn Ala Tyr Pro Leu Trp Leu Leu 290 295 300 Ser Val Ile Cys Glu Ile Trp Phe Ala Leu Ser Trp Ile Leu Asp Gln305 310 315 320 Phe Pro Lys Trp Ser Pro Ile Asn Arg Glu Thr Tyr Leu Asp Arg Leu 325 330 335 Ala Leu Arg Tyr Asp Arg Glu Gly Glu Pro Ser Gln Leu Ala Pro Val 340 345 350 Asp Ile Phe Val Ser Thr Val Asp Pro Met Lys Glu Pro Pro Leu Val 355 360 365 Thr Ala Asn Thr Val Leu Ser Ile Leu Ala Val Asp Tyr Pro Val Asp 370 375 380 Lys Val Ser Cys Tyr Val Ser Asp Asp Gly Ala Ala Met Leu Thr Phe385 390 395 400 Asp Ala Leu Ser Glu Thr Ser Glu Phe Ala Arg Lys Trp Val Pro Phe 405 410 415 Cys Lys Lys Tyr Asn Ile Glu Pro Arg Ala Pro Glu Trp Tyr Phe Ala 420 425 430 Gln Lys Ile Asp Tyr Leu Lys Asp Lys Val Gln Thr Ser Phe Val Lys 435 440 445 Glu Arg Arg Ala Met Lys Arg Glu Tyr Glu Glu Phe Lys Val Arg Ile 450 455 460 Asn Gly Leu Val Ala Lys Ala Gln Lys Val Pro Glu Glu Gly Trp Ile465 470 475 480 Met Gln Asp Gly Thr Pro Trp Pro Gly Asn Asn Thr Arg Asp His Pro 485 490 495 Gly Met Ile Gln Val Phe Leu Gly His Ser Gly Gly Leu Asp Val Glu 500 505 510 Gly Asn Glu Leu Pro Arg Leu Val Tyr Val Ser Arg Glu Lys Arg Pro 515 520 525 Gly Phe Gln His His Lys Lys Ala Gly Ala Met Asn Ala Leu Val Arg 530 535 540 Val Ser Ala Val Leu Thr Asn Gly Gln Tyr Met Leu Asn Leu Asp Cys545 550 555 560 Asp His Tyr Ile Asn Asn Ser Lys Ala Leu Arg Glu Ala Met Cys Phe 565 570 575 Leu Met Asp Pro Asn Leu Gly Arg Asn Val Cys Tyr Val Gln Phe Pro 580 585 590 Gln Arg Phe Asp Gly Ile Asp Arg Asn Asp Arg Tyr Ala Asn Arg Asn 595 600 605 Thr Val Phe Phe Asp Ile Asn Leu Arg Gly Leu Asp Gly Ile Gln Gly 610 615 620 Pro Val Tyr Val Gly Thr Gly Cys Val Phe Asn Arg Thr Ala Leu Tyr625 630 635 640 Gly Tyr Glu Pro Pro Val Lys Lys Lys Lys Pro Gly Phe Phe Ser Ser 645 650 655 Leu Cys Gly Gly Arg Lys Lys Thr Ser Lys Ser Lys Lys Ser Ser Glu 660 665 670 Lys Lys Lys Ser His Arg His Ala Asp Ser Ser Val Pro Val Phe Asn 675 680 685 Leu Glu Asp Ile Glu Glu Gly Ile Glu Gly Ser Gln Phe Asp Asp Glu 690 695 700 Lys Ser Leu Ile Met Ser Gln Met Ser Leu Glu Lys Arg Phe Gly Gln705 710 715 720 Ser Ser Val Phe Val Ala Ser Thr Leu Met Glu Tyr Gly Gly Val Pro 725 730 735 Gln Ser Ala Thr Pro Glu Ser Leu Leu Lys Glu Ala Ile His Val Ile 740 745 750 Ser Cys Gly Tyr Glu Asp Lys Thr Asp Trp Gly Thr Glu Ile Gly Trp 755 760 765 Ile Tyr Gly Ser Val Thr Glu Asp Ile Leu Thr Gly Phe Lys Met His 770 775 780 Ala Arg Gly Trp Arg Ser Ile Tyr Cys Met Pro Lys Arg Pro Ala Phe785 790 795 800 Lys Gly Ser Ala Pro Ile Asn Leu Ser Asp Arg Leu Asn Gln Val Leu 805 810 815 Arg Trp Ala Leu Gly Ser Ile Glu Ile Leu Phe Ser Arg His Cys Pro 820 825 830 Ile Trp Tyr Gly Tyr Gly Gly Arg Leu Lys Phe Leu Glu Arg Phe Ala 835 840 845 Tyr Ile Asn Thr Thr Ile Tyr Pro Leu Thr Ser Ile Pro Leu Leu Leu 850 855 860 Tyr Cys Ile Leu Pro Ala Val Cys Leu Leu Thr Gly Lys Phe Ile Ile865 870 875 880 Pro Lys Ile Ser Asn Leu Glu Ser Val Trp Phe Ile Ser Leu Phe Ile 885 890 895 Ser Ile Phe Ala Thr Gly Ile Leu Glu Met Arg Trp Ser Gly Val Gly 900 905 910 Ile Asp Glu Trp Trp Arg Asn Glu Gln Phe Trp Val Ile Gly Gly Ile 915 920 925 Ser Ala His Leu Phe Ala Val Phe Gln Gly Leu Leu Lys Val Leu Ala 930 935 940 Gly Ile Asp Thr Ser Phe Thr Val Thr Ser Lys Ala Thr Asp Glu Glu945 950 955 960 Gly Asp Phe Ala Glu Leu Tyr Met Phe Lys Trp Thr Thr Leu Leu Ile 965 970 975 Pro Pro Thr Thr Ile Leu Ile Ile Asn Leu Val Gly Val Val Ala Gly 980 985 990 Ile Ser Tyr Ala Ile Asn Ser Gly Tyr Gln Ser Trp Gly Pro Leu Phe 995 1000 1005 Gly Lys Leu Phe Phe Ala Phe Trp Val Ile Val His Leu Tyr Pro Phe 1010 1015 1020 Leu Lys Gly Leu Met Gly Lys Gln Asn Arg Thr Pro Thr Ile Val Val1025 1030 1035 1040Val Trp Ala Ile Leu Leu Ala Ser Ile Phe Ser Leu Met Trp Val Arg 1045 1050 1055 Ile Asp Pro Phe Thr Thr Arg Val Thr Gly Pro Asp Ile Ala Lys Cys 1060 1065 1070 Gly Ile Asn Cys 1075 1525DNAZea mays 15atggacggcg gcgacgccac gaatt 251625DNAZea mays 16ctagcagttg atgccacatt tcgcg 25173813DNAZea mays 17ccacagctca tataccaaga gccggagcag cttagcgcag cccagagcgg cgccgcgcca 60agcacaaccc ccacccgcca cagccgcgtg cgcatgtgag cggtcgccgc ggccgggaga 120ccagaggagg ggaggactac gtgcatttcg ctgtgccgcc gccgcggggt tcgtgcgcga 180gcgagatccg gcggggcggg gcggggggcc tgagatggag gctagcgcgg ggctggtggc 240cggctcgcat aaccggaacg agctggtggt gatccgccgc gaccgcgagt cgggagccgc 300gggcggcggc gcggcgcgcc gggcggaggc gccgtgccag atatgcggcg acgaggtcgg 360ggtgggcttc gacggggagc ccttcgtggc gtgcaacgag tgcgccttcc ccgtctgccg 420cgcctgctac gagtacgagc gccgcgaggg ctcgcaagcg tgcccgcagt gcaggacccg 480ctacaagcgc ctcaagggct gcccgcgggt ggccggcgac gaggaggagg acggcgtcga 540cgacctggag ggcgagttcg gcctgcagga cggcgccgcc cacgaggacg acccgcagta 600cgtcgccgag tccatgctca gggcgcagat gagctacggc cgcggcggcg acgcgcaccc 660cggcttcagc cccgtcccca acgtgccgct cctcaccaac ggccagatgg ttgatgacat 720cccgccggag cagcacgcgc tcgtgccgtc ctacatgagc ggcggcggcg gcgggggcaa 780gaggatccac ccgctccctt tcgcagatcc caaccttcca gtgcaaccga gatccatgga 840cccgtccaag gatctggccg cctacggata tggcagcgtg gcctggaagg agagaatgga 900gggctggaag cagaagcagg agcgcctgca gcatgtcagg agcgagggtg gcggtgattg 960ggatggcgac gatgcagatc tgccactaat ggatgaagct aggcagccat tgtccagaaa 1020agtccctata tcatcaagcc gaattaatcc ctacaggatg attatcgtta tccggttggt 1080ggttttgggt ttcttcttcc actaccgagt gatgcatccg gcgaaagatg catttgcatt 1140gtggctcata tctgtaatct gtgaaatctg gtttgcgatg tcctggattc ttgatcagtt 1200cccaaagtgg cttccaatcg agagagagac ttacctggac cgtttgtcac taaggtttga 1260caaggaaggt caaccctctc agcttgctcc aatcgacttc tttgtcagta cggttgatcc 1320cacaaaggaa cctcccttgg tcacagcgaa cactgtcctt tccatccttt ctgtggatta 1380tccggttgag aaggtctcct gctatgtttc tgatgatggt gctgcaatgc ttacgtttga 1440agcattgtct gaaacatctg aatttgcaaa gaaatgggtt cctttcagca aaaagtttaa 1500tatcgagcct cgtgctcctg agtggtactt ccaacagaag atagactacc tgaaagacaa 1560ggttgctgct tcatttgtta gggagaggag ggcgatgaag agagaatacg aggaattcaa 1620ggtaaggatc aatgccttgg ttgcaaaagc ccaaaaggtt cctgaggaag gatggacaat 1680gcaagatgga agcccctggc ctggaaacaa cgtacgcgat catcctggaa tgattcaggt 1740attccttggc caaagtggcg gtcgtgatgt ggaaggaaat gagttgcctc gcctggttta 1800tgtctcgaga gaaaagaggc caggttataa ccatcacaag aaggctggtg ccatgaatgc 1860actggtccgt gtctctgctg tcttatcaaa tgctgcatac ctattgaact tggactgtga 1920tcactacatc aacaatagca aggccataaa agaggctatg tgtttcatga tggatccttt 1980ggtggggaag aaagtgtgct atgtacagtt ccctcagagg tttgatggta ttgacaaaaa 2040tgatcgatac gctaacagga acgttgtctt ttttgacatc aacatgaaag gtttggacgg 2100tattcaagga cccatttatg tgggtactgg atgtgttttc agacggcagg cactgtatgg 2160ttatgatgct cctaaaacga agaagccacc atcaagaact tgcaactgct ggcccaagtg 2220gtgcctctct tgctgctgca gcaggaacaa gaataaaaag aagactacaa aaccaaagac 2280ggagaagaag aaaagattat ttttcaagaa agcagaaaac ccatctcctg catatgcttt 2340gggtgaaatt gatgaaggtg ctccaggtgc tgatatcgag aaggccggaa tcgtaaatca 2400acagaaacta gagaagaaat ttgggcagtc ttctgttttt gtcgcatcaa cacttcttga 2460gaacggaggg accctgaaga gcgcaagtcc agcttctctt ctgaaggaag ctatacatgt 2520tatcagctgc ggctacgaag acaagaccga ctggggaaaa gagattggct ggatttacgg 2580atcgatcaca gaggatatct tgactggatt taagatgcac tgccatggct ggcggtctat 2640ttactgcatc ccgaagcggc ctgcattcaa aggttctgcg cctctgaacc tttccgaccg 2700tcttcaccag gtccttcgct gggcccttgg gtccgtcgaa attttcttca gcaagcactg 2760cccactttgg tacggatacg gcggcgggct aaaattcctg gaaaggtttt cttatatcaa 2820ctccatcgtt tatccctgga cgtccattcc tctcctggct tactgtacct tgcctgccat 2880ctgcctgctc acggggaagt ttatcacacc agagcttacc aatgtcgcca gtatctggtt 2940catggcactt ttcatctgca tctccgtgac cggcatcctg gaaatgaggt ggagtggcgt 3000ggccatcgac gactggtgga ggaacgagca gttctgggtc atcggaggcg tttcggcgca 3060tctgttcgcg gtgttccagg gcctgctgaa ggtgttcgcc ggcatcgaca cgagcttcac 3120cgtgacgtcg aaggccgggg acgacgagga gttctcggag ctgtacacgt tcaagtggac 3180caccctgctg atacccccga ccacgctcct cctgctgaac ttcatcgggg tggtggccgg 3240gatctcgaac gcgatcaaca acgggtacga gtcgtggggc cccctgttcg ggaagctctt 3300cttcgccttc tgggtgatcg tccacctgta cccgttcctc aagggtctgg tggggaggca 3360gaacaggacg ccgacgatcg tcatcgtctg gtccatcctg ctggcctcga tcttctcgct 3420cctgtgggtc cgcgtcgacc cgttcctcgc caagagcaac ggcccgctcc tggaggagtg 3480tggcctggac tgcaactgaa gtgggggccc cctgtcactc gaagttctgt cacgggcgaa 3540ttacgcctga ttttttgttg ttgttgttgt tggaattctt tgctgtagat agaaaccaca 3600tgtccacggc atctctgctg tgtccattgg agcaggagag aggtgcctgc tgctgtttgt 3660tgagtaaatt aaaagtttta aagttataca gtgatgcaca ttccagtgcc cagtgtattc 3720cctttttaca gtctgtatat tagcgacaaa ggacatattg gttaggagtt tgattctttt 3780gtaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaa 3813181094PRTZea mays 18Met Glu Ala Ser Ala Gly Leu Val Ala Gly Ser His Asn Arg Asn Glu1

5 10 15 Leu Val Val Ile Arg Arg Asp Arg Glu Ser Gly Ala Ala Gly Gly Gly 20 25 30 Ala Ala Arg Arg Ala Glu Ala Pro Cys Gln Ile Cys Gly Asp Glu Val 35 40 45 Gly Val Gly Phe Asp Gly Glu Pro Phe Val Ala Cys Asn Glu Cys Ala 50 55 60 Phe Pro Val Cys Arg Ala Cys Tyr Glu Tyr Glu Arg Arg Glu Gly Ser65 70 75 80 Gln Ala Cys Pro Gln Cys Arg Thr Arg Tyr Lys Arg Leu Lys Gly Cys 85 90 95 Pro Arg Val Ala Gly Asp Glu Glu Glu Asp Gly Val Asp Asp Leu Glu 100 105 110 Gly Glu Phe Gly Leu Gln Asp Gly Ala Ala His Glu Asp Asp Pro Gln 115 120 125 Tyr Val Ala Glu Ser Met Leu Arg Ala Gln Met Ser Tyr Gly Arg Gly 130 135 140 Gly Asp Ala His Pro Gly Phe Ser Pro Val Pro Asn Val Pro Leu Leu145 150 155 160 Thr Asn Gly Gln Met Val Asp Asp Ile Pro Pro Glu Gln His Ala Leu 165 170 175 Val Pro Ser Tyr Met Ser Gly Gly Gly Gly Gly Gly Lys Arg Ile His 180 185 190 Pro Leu Pro Phe Ala Asp Pro Asn Leu Pro Val Gln Pro Arg Ser Met 195 200 205 Asp Pro Ser Lys Asp Leu Ala Ala Tyr Gly Tyr Gly Ser Val Ala Trp 210 215 220 Lys Glu Arg Met Glu Gly Trp Lys Gln Lys Gln Glu Arg Leu Gln His225 230 235 240 Val Arg Ser Glu Gly Gly Gly Asp Trp Asp Gly Asp Asp Ala Asp Leu 245 250 255 Pro Leu Met Asp Glu Ala Arg Gln Pro Leu Ser Arg Lys Val Pro Ile 260 265 270 Ser Ser Ser Arg Ile Asn Pro Tyr Arg Met Ile Ile Val Ile Arg Leu 275 280 285 Val Val Leu Gly Phe Phe Phe His Tyr Arg Val Met His Pro Ala Lys 290 295 300 Asp Ala Phe Ala Leu Trp Leu Ile Ser Val Ile Cys Glu Ile Trp Phe305 310 315 320 Ala Met Ser Trp Ile Leu Asp Gln Phe Pro Lys Trp Leu Pro Ile Glu 325 330 335 Arg Glu Thr Tyr Leu Asp Arg Leu Ser Leu Arg Phe Asp Lys Glu Gly 340 345 350 Gln Pro Ser Gln Leu Ala Pro Ile Asp Phe Phe Val Ser Thr Val Asp 355 360 365 Pro Thr Lys Glu Pro Pro Leu Val Thr Ala Asn Thr Val Leu Ser Ile 370 375 380 Leu Ser Val Asp Tyr Pro Val Glu Lys Val Ser Cys Tyr Val Ser Asp385 390 395 400 Asp Gly Ala Ala Met Leu Thr Phe Glu Ala Leu Ser Glu Thr Ser Glu 405 410 415 Phe Ala Lys Lys Trp Val Pro Phe Ser Lys Lys Phe Asn Ile Glu Pro 420 425 430 Arg Ala Pro Glu Trp Tyr Phe Gln Gln Lys Ile Asp Tyr Leu Lys Asp 435 440 445 Lys Val Ala Ala Ser Phe Val Arg Glu Arg Arg Ala Met Lys Arg Glu 450 455 460 Tyr Glu Glu Phe Lys Val Arg Ile Asn Ala Leu Val Ala Lys Ala Gln465 470 475 480 Lys Val Pro Glu Glu Gly Trp Thr Met Gln Asp Gly Ser Pro Trp Pro 485 490 495 Gly Asn Asn Val Arg Asp His Pro Gly Met Ile Gln Val Phe Leu Gly 500 505 510 Gln Ser Gly Gly Arg Asp Val Glu Gly Asn Glu Leu Pro Arg Leu Val 515 520 525 Tyr Val Ser Arg Glu Lys Arg Pro Gly Tyr Asn His His Lys Lys Ala 530 535 540 Gly Ala Met Asn Ala Leu Val Arg Val Ser Ala Val Leu Ser Asn Ala545 550 555 560 Ala Tyr Leu Leu Asn Leu Asp Cys Asp His Tyr Ile Asn Asn Ser Lys 565 570 575 Ala Ile Lys Glu Ala Met Cys Phe Met Met Asp Pro Leu Val Gly Lys 580 585 590 Lys Val Cys Tyr Val Gln Phe Pro Gln Arg Phe Asp Gly Ile Asp Lys 595 600 605 Asn Asp Arg Tyr Ala Asn Arg Asn Val Val Phe Phe Asp Ile Asn Met 610 615 620 Lys Gly Leu Asp Gly Ile Gln Gly Pro Ile Tyr Val Gly Thr Gly Cys625 630 635 640 Val Phe Arg Arg Gln Ala Leu Tyr Gly Tyr Asp Ala Pro Lys Thr Lys 645 650 655 Lys Pro Pro Ser Arg Thr Cys Asn Cys Trp Pro Lys Trp Cys Leu Ser 660 665 670 Cys Cys Cys Ser Arg Asn Lys Asn Lys Lys Lys Thr Thr Lys Pro Lys 675 680 685 Thr Glu Lys Lys Lys Arg Leu Phe Phe Lys Lys Ala Glu Asn Pro Ser 690 695 700 Pro Ala Tyr Ala Leu Gly Glu Ile Asp Glu Gly Ala Pro Gly Ala Asp705 710 715 720 Ile Glu Lys Ala Gly Ile Val Asn Gln Gln Lys Leu Glu Lys Lys Phe 725 730 735 Gly Gln Ser Ser Val Phe Val Ala Ser Thr Leu Leu Glu Asn Gly Gly 740 745 750 Thr Leu Lys Ser Ala Ser Pro Ala Ser Leu Leu Lys Glu Ala Ile His 755 760 765 Val Ile Ser Cys Gly Tyr Glu Asp Lys Thr Asp Trp Gly Lys Glu Ile 770 775 780 Gly Trp Ile Tyr Gly Ser Ile Thr Glu Asp Ile Leu Thr Gly Phe Lys785 790 795 800 Met His Cys His Gly Trp Arg Ser Ile Tyr Cys Ile Pro Lys Arg Pro 805 810 815 Ala Phe Lys Gly Ser Ala Pro Leu Asn Leu Ser Asp Arg Leu His Gln 820 825 830 Val Leu Arg Trp Ala Leu Gly Ser Val Glu Ile Phe Phe Ser Lys His 835 840 845 Cys Pro Leu Trp Tyr Gly Tyr Gly Gly Gly Leu Lys Phe Leu Glu Arg 850 855 860 Phe Ser Tyr Ile Asn Ser Ile Val Tyr Pro Trp Thr Ser Ile Pro Leu865 870 875 880 Leu Ala Tyr Cys Thr Leu Pro Ala Ile Cys Leu Leu Thr Gly Lys Phe 885 890 895 Ile Thr Pro Glu Leu Thr Asn Val Ala Ser Ile Trp Phe Met Ala Leu 900 905 910 Phe Ile Cys Ile Ser Val Thr Gly Ile Leu Glu Met Arg Trp Ser Gly 915 920 925 Val Ala Ile Asp Asp Trp Trp Arg Asn Glu Gln Phe Trp Val Ile Gly 930 935 940 Gly Val Ser Ala His Leu Phe Ala Val Phe Gln Gly Leu Leu Lys Val945 950 955 960 Phe Ala Gly Ile Asp Thr Ser Phe Thr Val Thr Ser Lys Ala Gly Asp 965 970 975 Asp Glu Glu Phe Ser Glu Leu Tyr Thr Phe Lys Trp Thr Thr Leu Leu 980 985 990 Ile Pro Pro Thr Thr Leu Leu Leu Leu Asn Phe Ile Gly Val Val Ala 995 1000 1005 Gly Ile Ser Asn Ala Ile Asn Asn Gly Tyr Glu Ser Trp Gly Pro Leu 1010 1015 1020 Phe Gly Lys Leu Phe Phe Ala Phe Trp Val Ile Val His Leu Tyr Pro1025 1030 1035 1040Phe Leu Lys Gly Leu Val Gly Arg Gln Asn Arg Thr Pro Thr Ile Val 1045 1050 1055 Ile Val Trp Ser Ile Leu Leu Ala Ser Ile Phe Ser Leu Leu Trp Val 1060 1065 1070 Arg Val Asp Pro Phe Leu Ala Lys Ser Asn Gly Pro Leu Leu Glu Glu 1075 1080 1085 Cys Gly Leu Asp Cys Asn 1090 1925DNAZea mays 19atggaggcta gcgcggggct ggtgg 252025DNAZea mays 20tcagttgcag tccaggccac actcc 25213799DNAZea maysmisc_feature3757, 3775, 3777, 3782n = A,T,C or G 21caactcacgt tgccgcggct tcctccatcg gtgcggtgcc ctgtcctttt ctctcctcca 60cctccctagt ccctcctccc ccccgcatac atagctacta ctagtagcac cacgctcgca 120gcgggagatg cggtgctgat ccgtgcccct gctcggatct cgggagtggt gccgacttgt 180gtcgcttcgg ctctgcctag gccagctcct tgtcggttct gggcgagctc gcctgccatg 240gagggcgacg cggacggcgt gaagtcgggg aggcgcgggg gagggcaggt gtgccagatc 300tgcggcgatg gcgtgggcac tacggcggag ggagacgtct tcaccgcctg cgacgtctgc 360gggttcccgg tgtgccgccc ctgctacgag tacgagcgca aggacggcac acaagcgtgc 420ccccagtgca aaaacaagta caagcgccac aaggggagtc cagcgatccg aggggaggaa 480ggagacgata ctgatgccga tgatgctagc gacttcaact accctgcatc tggcaatgac 540gaccagaagc agaagattgc tgacaggatg cgcagctggc gcatgaatgc tgggggcagc 600ggggatgttg gccgccccaa gtatgacagt ggtgagatcg ggcttaccaa gtacgacagt 660ggtgagatcc ctcggggata catcccgtca gtcactaaca gccagatttc gggagaaatc 720cctggtgctt cccctgacca tcatatgatg tctcctactg ggaacattgg caggcgcgcc 780ccatttccct atatgaatca ttcatcaaat ccgtcgaggg aattctctgg tagcgttggg 840aatgttgcct ggaaagagag ggttgatggc tggaaaatga agcaggacaa gggaacaatt 900cccatgacga atggcacaag cattgctccc tctgagggcc ggggtgttgg tgatattgat 960gcatcaactg attacaacat ggaagatgcc ttattaaacg atgaaactcg ccagcctcta 1020tctaggaaag ttccacttcc ttcctccagg ataaatccat acaggatggt cattgtgcta 1080cgattgattg ttctaagcat cttcttgcac taccggatca caaatcctgt gcgtaatgca 1140tacccactgt ggcttctatc tgttatatgt gagatctggt ttgctctttc ctggatattg 1200gatcagtttc caaagtggtt tccaatcaac cgcgagactt accttgatag actcgcatta 1260aggtatgacc gggaaggtga gccatctcag ttggctgctg ttgacatttt tgtcagtact 1320gtcgacccaa tgaaggagcc tcctcttgtc actgccaata ccgtgctatc cattctcgct 1380gtggactatc ctgtggataa ggtctcttgc tatgtatctg atgatggagc tgctatgctg 1440acatttgatg cactagctga gacttcagag tttgctagaa aatgggtgcc atttgttaag 1500aagtacaaca ttgaacctag agctcctgaa tggtacttct cccagaaaat tgattacttg 1560aaggacaaag tgcacccttc atttgttaaa gaccgccggg ccatgaagag agaatatgaa 1620gaattcaaaa ttagggtaaa tggccttgtt gctaaggcac aaaaagtccc tgaggaagga 1680tggatcatgc aagatggcac accatggcca ggaaacaata ccagggacca tcctggaatg 1740attcaggttt tccttggtca cagtggtggt cttgatactg agggtaatga gctaccccgt 1800ttggtctatg tttctcgtga aaaacgtcct ggattccagc atcacaagaa agctggtgcc 1860atgaatgctc ttgtccgcgt ctcagctgtg cttaccaatg gacaatacat gttgaatctt 1920gattgtgatc actacatcaa caacagtaag gctctcaggg aagctatgtg cttccttatg 1980gatcctaacc taggaaggag tgtctgctat gttcagtttc cccagaggtt cgatggtatt 2040gataggaatg atcgatatgc caacaggaac accgtgtttt tcgatattaa cttgagaggt 2100cttgatggca tccaaggacc agtttatgtg ggcactggct gtgttttcaa cagaacagct 2160ctatatggtt atgagccccc aattaagcaa aagaagggtg gtttcttgtc atcactatgt 2220ggtggcagga agaagggaag caaatcaaag aagggctcag acaagaaaaa gtcacagaag 2280catgtggaca gttctgtgcc agtattcaat cttgaagata tagaggaggg agttgaaggc 2340gctggatttg atgatgagaa atcacttctt atgtctcaaa tgagcttgga gaagagattt 2400ggccaatctg cagcttttgt tgcgtccact ctgatggaat atggtggtgt tcctcagtct 2460gcgactccag aatctcttct gaaagaagct atccatgtca taagttgtgg ctacgaggac 2520aagattgaat ggggaactga gattgggtgg atctatggtt ctgtgacgga agatattctc 2580actgggttca agatgcacgc acgaggctgg cggtcgatct actgcatgcc taagcggccg 2640gccttcaagg gatcggctcc catcaatctc tcagaccgtc tgaaccaggt gctccggtgg 2700gctctcggtt cagtggaaat ccttttcagc cggcattgcc ccctatggta cgggtacgga 2760ggacgcctga agttcttgga gagattcgcc tacatcaaca ccaccatcta cccgctcacg 2820tccctcccgc tcctcattta ctgtatcctg cctgccatct gcctgctcac ggggaagttc 2880atcatcccag agatcagcaa cttcgctagt atctggttca tctctctctt catctcgatc 2940ttcgccacgg gtatcctgga gatgaggtgg agcggcgtgg gcatcgacga gtggtggagg 3000aacgagcagt tctgggtcat cggaggcatc tccgcccacc tcttcgccgt cttccagggc 3060ctcctcaagg tgcttgccgg catcgacacc aacttcaccg tcacctccaa ggcctcggat 3120gaagacggcg acttcgcgga gctgtacatg ttcaagtgga cgacacttct gatcccgccc 3180accaccatcc tgatcatcaa cctggtcggc gttgttgccg gcatctccta cgccatcaac 3240agcgggtacc agtcgtgggg tccgctcttc ggcaagctct tcttcgcctt ctgggtgatc 3300gttcacctgt acccgttcct caagggtctc atgggtcggc agaaccgcac cccgaccatc 3360gtggttgtct gggcgatcct gctggcgtcg atcttctcct tgctgtgggt tcgcatcgat 3420ccgttcacca accgcgtcac tggcccggat actcgaacgt gtggcatcaa ctgctaggga 3480ggtggaaggt ttgtagaaac agagagatac cacgaatgtg ccgctgccac aaattgtctg 3540ttagtaagtt atataggcag gtggcgttat ttacagctac gtacacacaa ggggatactc 3600cgtttatcac tggtgtgcat tcttttgttg atataagtta ctatatatac gtattgcttc 3660tactttgtgg agagtggctg acaggaccag ttttgtaatg ttatgaacag caaagaaata 3720agttagtttc caaaaaaaaa aaaaaaaaaa aaaaaanaaa aaaaaaaaaa aaaananaaa 3780anaaaaaaaa aaaaacccc 3799221079PRTZea mays 22Met Glu Gly Asp Ala Asp Gly Val Lys Ser Gly Arg Arg Gly Gly Gly1 5 10 15 Gln Val Cys Gln Ile Cys Gly Asp Gly Val Gly Thr Thr Ala Glu Gly 20 25 30 Asp Val Phe Thr Ala Cys Asp Val Cys Gly Phe Pro Val Cys Arg Pro 35 40 45 Cys Tyr Glu Tyr Glu Arg Lys Asp Gly Thr Gln Ala Cys Pro Gln Cys 50 55 60 Lys Asn Lys Tyr Lys Arg His Lys Gly Ser Pro Ala Ile Arg Gly Glu65 70 75 80 Glu Gly Asp Asp Thr Asp Ala Asp Asp Ala Ser Asp Phe Asn Tyr Pro 85 90 95 Ala Ser Gly Asn Asp Asp Gln Lys Gln Lys Ile Ala Asp Arg Met Arg 100 105 110 Ser Trp Arg Met Asn Ala Gly Gly Ser Gly Asp Val Gly Arg Pro Lys 115 120 125 Tyr Asp Ser Gly Glu Ile Gly Leu Thr Lys Tyr Asp Ser Gly Glu Ile 130 135 140 Pro Arg Gly Tyr Ile Pro Ser Val Thr Asn Ser Gln Ile Ser Gly Glu145 150 155 160 Ile Pro Gly Ala Ser Pro Asp His His Met Met Ser Pro Thr Gly Asn 165 170 175 Ile Gly Arg Arg Ala Pro Phe Pro Tyr Met Asn His Ser Ser Asn Pro 180 185 190 Ser Arg Glu Phe Ser Gly Ser Val Gly Asn Val Ala Trp Lys Glu Arg 195 200 205 Val Asp Gly Trp Lys Met Lys Gln Asp Lys Gly Thr Ile Pro Met Thr 210 215 220 Asn Gly Thr Ser Ile Ala Pro Ser Glu Gly Arg Gly Val Gly Asp Ile225 230 235 240 Asp Ala Ser Thr Asp Tyr Asn Met Glu Asp Ala Leu Leu Asn Asp Glu 245 250 255 Thr Arg Gln Pro Leu Ser Arg Lys Val Pro Leu Pro Ser Ser Arg Ile 260 265 270 Asn Pro Tyr Arg Met Val Ile Val Leu Arg Leu Ile Val Leu Ser Ile 275 280 285 Phe Leu His Tyr Arg Ile Thr Asn Pro Val Arg Asn Ala Tyr Pro Leu 290 295 300 Trp Leu Leu Ser Val Ile Cys Glu Ile Trp Phe Ala Leu Ser Trp Ile305 310 315 320 Leu Asp Gln Phe Pro Lys Trp Phe Pro Ile Asn Arg Glu Thr Tyr Leu 325 330 335 Asp Arg Leu Ala Leu Arg Tyr Asp Arg Glu Gly Glu Pro Ser Gln Leu 340 345 350 Ala Ala Val Asp Ile Phe Val Ser Thr Val Asp Pro Met Lys Glu Pro 355 360 365 Pro Leu Val Thr Ala Asn Thr Val Leu Ser Ile Leu Ala Val Asp Tyr 370 375 380 Pro Val Asp Lys Val Ser Cys Tyr Val Ser Asp Asp Gly Ala Ala Met385 390 395 400 Leu Thr Phe Asp Ala Leu Ala Glu Thr Ser Glu Phe Ala Arg Lys Trp 405 410 415 Val Pro Phe Val Lys Lys Tyr Asn Ile Glu Pro Arg Ala Pro Glu Trp 420 425 430 Tyr Phe Ser Gln Lys Ile Asp Tyr Leu Lys Asp Lys Val His Pro Ser 435 440 445 Phe Val Lys Asp Arg Arg Ala Met Lys Arg Glu Tyr Glu Glu Phe Lys 450 455 460 Ile Arg Val Asn Gly Leu Val Ala Lys Ala Gln Lys Val Pro Glu Glu465 470 475 480 Gly Trp Ile Met Gln Asp Gly Thr Pro Trp Pro Gly Asn Asn Thr Arg 485 490 495 Asp His Pro Gly Met Ile Gln Val Phe Leu Gly His Ser Gly Gly Leu 500 505 510 Asp Thr Glu Gly Asn Glu Leu Pro Arg Leu Val Tyr Val Ser Arg Glu 515 520 525 Lys Arg Pro Gly Phe Gln His His Lys Lys Ala Gly Ala Met Asn Ala 530 535 540 Leu Val Arg Val Ser Ala Val Leu Thr Asn Gly Gln Tyr Met Leu Asn545 550 555 560 Leu Asp Cys Asp His Tyr Ile Asn Asn Ser Lys Ala Leu Arg Glu Ala 565 570 575 Met Cys Phe Leu Met Asp Pro Asn Leu Gly Arg Ser Val Cys Tyr Val 580 585 590 Gln Phe Pro Gln Arg Phe Asp Gly Ile Asp Arg Asn Asp Arg Tyr Ala 595 600

605 Asn Arg Asn Thr Val Phe Phe Asp Ile Asn Leu Arg Gly Leu Asp Gly 610 615 620 Ile Gln Gly Pro Val Tyr Val Gly Thr Gly Cys Val Phe Asn Arg Thr625 630 635 640 Ala Leu Tyr Gly Tyr Glu Pro Pro Ile Lys Gln Lys Lys Gly Gly Phe 645 650 655 Leu Ser Ser Leu Cys Gly Gly Arg Lys Lys Gly Ser Lys Ser Lys Lys 660 665 670 Gly Ser Asp Lys Lys Lys Ser Gln Lys His Val Asp Ser Ser Val Pro 675 680 685 Val Phe Asn Leu Glu Asp Ile Glu Glu Gly Val Glu Gly Ala Gly Phe 690 695 700 Asp Asp Glu Lys Ser Leu Leu Met Ser Gln Met Ser Leu Glu Lys Arg705 710 715 720 Phe Gly Gln Ser Ala Ala Phe Val Ala Ser Thr Leu Met Glu Tyr Gly 725 730 735 Gly Val Pro Gln Ser Ala Thr Pro Glu Ser Leu Leu Lys Glu Ala Ile 740 745 750 His Val Ile Ser Cys Gly Tyr Glu Asp Lys Ile Glu Trp Gly Thr Glu 755 760 765 Ile Gly Trp Ile Tyr Gly Ser Val Thr Glu Asp Ile Leu Thr Gly Phe 770 775 780 Lys Met His Ala Arg Gly Trp Arg Ser Ile Tyr Cys Met Pro Lys Arg785 790 795 800 Pro Ala Phe Lys Gly Ser Ala Pro Ile Asn Leu Ser Asp Arg Leu Asn 805 810 815 Gln Val Leu Arg Trp Ala Leu Gly Ser Val Glu Ile Leu Phe Ser Arg 820 825 830 His Cys Pro Leu Trp Tyr Gly Tyr Gly Gly Arg Leu Lys Phe Leu Glu 835 840 845 Arg Phe Ala Tyr Ile Asn Thr Thr Ile Tyr Pro Leu Thr Ser Leu Pro 850 855 860 Leu Leu Ile Tyr Cys Ile Leu Pro Ala Ile Cys Leu Leu Thr Gly Lys865 870 875 880 Phe Ile Ile Pro Glu Ile Ser Asn Phe Ala Ser Ile Trp Phe Ile Ser 885 890 895 Leu Phe Ile Ser Ile Phe Ala Thr Gly Ile Leu Glu Met Arg Trp Ser 900 905 910 Gly Val Gly Ile Asp Glu Trp Trp Arg Asn Glu Gln Phe Trp Val Ile 915 920 925 Gly Gly Ile Ser Ala His Leu Phe Ala Val Phe Gln Gly Leu Leu Lys 930 935 940 Val Leu Ala Gly Ile Asp Thr Asn Phe Thr Val Thr Ser Lys Ala Ser945 950 955 960 Asp Glu Asp Gly Asp Phe Ala Glu Leu Tyr Met Phe Lys Trp Thr Thr 965 970 975 Leu Leu Ile Pro Pro Thr Thr Ile Leu Ile Ile Asn Leu Val Gly Val 980 985 990 Val Ala Gly Ile Ser Tyr Ala Ile Asn Ser Gly Tyr Gln Ser Trp Gly 995 1000 1005 Pro Leu Phe Gly Lys Leu Phe Phe Ala Phe Trp Val Ile Val His Leu 1010 1015 1020 Tyr Pro Phe Leu Lys Gly Leu Met Gly Arg Gln Asn Arg Thr Pro Thr1025 1030 1035 1040Ile Val Val Val Trp Ala Ile Leu Leu Ala Ser Ile Phe Ser Leu Leu 1045 1050 1055 Trp Val Arg Ile Asp Pro Phe Thr Asn Arg Val Thr Gly Pro Asp Thr 1060 1065 1070 Arg Thr Cys Gly Ile Asn Cys 1075 2325DNAZea mays 23atggagggcg acgcggacgg cgtga 252425DNAZea mays 24ctagcagttg atgccacacg ttcga 25253470DNAZea mays 25gcccccggtc gatcgctcgg caatcggcat ggacgccggc tcggtcaccg gtggcctcgc 60cgcgggctcg cacatgcggg acgagctgca tgtcatgcgc gcccgcgagg agccgaacgc 120caaggtccgg agcgccgacg tgaagacgtg ccgcgtgtgc gccgacgagg tcgggacgcg 180ggaggacggg cagcccttcg tggcgtgcgc cgagtgcggc ttccccgtct gccggccctg 240ctacgagtac gagcgcagcg agggcacgca gtgctgcccg cagtgcaaca cccgctacaa 300gcgccagaaa gggtgcccga gggtggaagg ggacgaggag gagggcccgg agatggacga 360cttcgaggac gagttccccg ccaagagccc caagaagcct cacgagcctg tcgcgttcga 420cgtctactcg gagaacggcg agcacccggc gcagaaatgg cggacgggtg gccagacgct 480gtcgtccttc accggaagcg tcgccgggaa ggacctggag gcggagaggg agatggaggg 540gagcatggag tggaaggacc ggatcgacaa gtggaagacc aagcaggaga agaggggcaa 600gctcaaccac gacgacagcg acgacgacga cgacaagaac gaagacgagt acatgctgct 660tgccgaggcc cgacagccgc tgtggcgcaa ggttccgatc ccgtcgagca tgatcaaccc 720gtaccgcatc gtcatcgtgc tccgcctggt ggtgctctgc ttcttcctca agttccggat 780cacgacgccc gccacggacg ccgtgcctct gtggctggcg tccgtcatct gcgagctctg 840gttcgccttc tcctggatcc tggaccagct gccaaagtgg gcgccggtga cgcgggagac 900gtacctggac cgcctggcgc tgcggtacga ccgtgagggc gaggcgtgcc ggctgtcccc 960catcgacttc ttcgtcagca cggtggaccc gctcaaggag ccgcccatca tcaccgccaa 1020caccgtgctg tccatcctcg ccgtcgacta ccccgtggac cgcgtcagct gctacgtctc 1080cgacgacggc gcgtccatgc tgctcttcga cgcgctgtcc gagaccgccg agttcgcgcg 1140ccgctgggtg cccttctgca agaagttcgc cgtggagccg cgcgccccgg agttctactt 1200ctcgcagaag atcgactacc tcaaggacaa ggtgcagccg acgttcgtca aggagcgccg 1260cgccatgaag agggagtacg aggagttcaa ggtgcgcatc aacgcgctgg tggccaaggc 1320gcagaagaag cccgaggagg ggtgggtcat gcaggacggc acgccgtggc ccgggaacaa 1380cacgcgcgac cacccgggta tgatccaggt ctacctcggc aaccagggcg cgctggacgt 1440ggagggccac gagctgccgc gcctcgtcta cgtgtcccgt gagaagcgcc ccgggtacaa 1500ccaccacaag aaggcgggcg ccatgaacgc gctggtgcgc gtctccgccg tgctcaccaa 1560cgcgcccttc atcctcaacc tcgactgcga ccactacgtc aacaacagca aggccgtgcg 1620cgaggccatg tgcttcctca tggacccgca gctggggaag aagctctgct acgtccagtt 1680cccgcagcgc ttcgatggca tcgatcgcca cgaccgatac gccaaccgca acgtcgtctt 1740cttcgacatc aacatgaagg ggctggacgg catccagggc ccggtgtacg tcggcacggg 1800gtgcgtgttc aaccgccagg cgctgtacgg ctacgacccg ccgcggcccg agaagcggcc 1860caagatgacg tgcgactgct ggccgtcgtg gtgctgctgc tgctgctgct tcggcggcgg 1920caagcgcggc aaggcgcgca aggacaagaa gggcgacggc ggcgaggagc cgcgccgggg 1980cctgctcggc ttctacagga agcggagcaa gaaggacaag ctcggcggcg ggtcggtggc 2040cggcagcaag aagggcggcg ggctgtacaa gaagcaccag cgcgcgttcg agctggagga 2100gatcgaggag gggctggagg ggtacgacga gctggagcgc tcctcgctca tgtcgcagaa 2160gagcttcgag aagcggttcg gccagtcgcc cgtgttcatc gcctccacgc tcgtcgagga 2220cggcggcctg ccgcagggcg ccgccgccga ccccgccgcg ctcatcaagg aggccatcca 2280cgtcatcagc tgcggatacg aggagaagac cgagtggggc aaggagattg ggtggatcta 2340tgggtcggtg acagaggata tcctgacggg gttcaagatg cactgccggg ggtggaagtc 2400cgtgtactgc acgccgacac ggccggcgtt caaggggtcg gcgcccatca acttgtctga 2460tcgtctccac caggtgctgc gctgggcgct ggggtccgtg gagatcttca tgagccgcca 2520ctgcccgctc cggtacgcct acggcggccg gctcaagtgg ctggagcgct tcgcctacac 2580caacaccatc gtgtacccct tcacctccat cccgctcctc gcctactgca ccatccccgc 2640cgtctgcctg ctcaccggca agttcatcat tcccacgctg aacaacctcg ccagcatctg 2700gttcatcgcg ctcttcctgt ccatcatcgc gacgagcgtc ctggagctgc ggtggagcgg 2760ggtgagcatc gaggactggt ggcgcaacga gcagttctgg gtcatcggcg gcgtgtccgc 2820gcatctcttc gccgtgttcc agggcttcct caaggttctg ggcggcgtgg acaccagctt 2880caccgtcacc tccaaggcgg ccggcgacga ggccgacgcc ttcggggacc tctacctctt 2940caagtggacc accctgctgg tgccccccac cacgctcatc atcatcaaca tggtgggcat 3000cgtggccggc gtgtccgacg ccgtcaacaa cggctacggc tcctggggcc cgctcttcgg 3060caagctcttc ttctccttct gggtcatcgt ccacctctac ccgttcctca aggggctcat 3120ggggaggcag aaccggacgc ccaccatcgt cgtgctctgg tccatcctcc tcgcctccat 3180cttctcgctc gtctgggtca ggatcgaccc gtttatcccg aaggccaagg gccccatcct 3240caagccatgc ggagtcgagt gctgagctca cctagctacc ttcttgttgc atgtacggac 3300gccgccgtgc gtttggacat acaggcactt ttgggccagg ctactcatgt tcgacttttt 3360ttttaatttt gtacaagatt tgtgatcgag tgactgagtg agacagagtg ttgggtgtaa 3420gaactgtgat ggaattcact caaattaatg gacatttttt ttcttcaaaa 3470261078PRTZea mays 26Met Asp Ala Gly Ser Val Thr Gly Gly Leu Ala Ala Gly Ser His Met1 5 10 15 Arg Asp Glu Leu His Val Met Arg Ala Arg Glu Glu Pro Asn Ala Lys 20 25 30 Val Arg Ser Ala Asp Val Lys Thr Cys Arg Val Cys Ala Asp Glu Val 35 40 45 Gly Thr Arg Glu Asp Gly Gln Pro Phe Val Ala Cys Ala Glu Cys Gly 50 55 60 Phe Pro Val Cys Arg Pro Cys Tyr Glu Tyr Glu Arg Ser Glu Gly Thr65 70 75 80 Gln Cys Cys Pro Gln Cys Asn Thr Arg Tyr Lys Arg Gln Lys Gly Cys 85 90 95 Pro Arg Val Glu Gly Asp Glu Glu Glu Gly Pro Glu Met Asp Asp Phe 100 105 110 Glu Asp Glu Phe Pro Ala Lys Ser Pro Lys Lys Pro His Glu Pro Val 115 120 125 Ala Phe Asp Val Tyr Ser Glu Asn Gly Glu His Pro Ala Gln Lys Trp 130 135 140 Arg Thr Gly Gly Gln Thr Leu Ser Ser Phe Thr Gly Ser Val Ala Gly145 150 155 160 Lys Asp Leu Glu Ala Glu Arg Glu Met Glu Gly Ser Met Glu Trp Lys 165 170 175 Asp Arg Ile Asp Lys Trp Lys Thr Lys Gln Glu Lys Arg Gly Lys Leu 180 185 190 Asn His Asp Asp Ser Asp Asp Asp Asp Asp Lys Asn Glu Asp Glu Tyr 195 200 205 Met Leu Leu Ala Glu Ala Arg Gln Pro Leu Trp Arg Lys Val Pro Ile 210 215 220 Pro Ser Ser Met Ile Asn Pro Tyr Arg Ile Val Ile Val Leu Arg Leu225 230 235 240 Val Val Leu Cys Phe Phe Leu Lys Phe Arg Ile Thr Thr Pro Ala Thr 245 250 255 Asp Ala Val Pro Leu Trp Leu Ala Ser Val Ile Cys Glu Leu Trp Phe 260 265 270 Ala Phe Ser Trp Ile Leu Asp Gln Leu Pro Lys Trp Ala Pro Val Thr 275 280 285 Arg Glu Thr Tyr Leu Asp Arg Leu Ala Leu Arg Tyr Asp Arg Glu Gly 290 295 300 Glu Ala Cys Arg Leu Ser Pro Ile Asp Phe Phe Val Ser Thr Val Asp305 310 315 320 Pro Leu Lys Glu Pro Pro Ile Ile Thr Ala Asn Thr Val Leu Ser Ile 325 330 335 Leu Ala Val Asp Tyr Pro Val Asp Arg Val Ser Cys Tyr Val Ser Asp 340 345 350 Asp Gly Ala Ser Met Leu Leu Phe Asp Ala Leu Ser Glu Thr Ala Glu 355 360 365 Phe Ala Arg Arg Trp Val Pro Phe Cys Lys Lys Phe Ala Val Glu Pro 370 375 380 Arg Ala Pro Glu Phe Tyr Phe Ser Gln Lys Ile Asp Tyr Leu Lys Asp385 390 395 400 Lys Val Gln Pro Thr Phe Val Lys Glu Arg Arg Ala Met Lys Arg Glu 405 410 415 Tyr Glu Glu Phe Lys Val Arg Ile Asn Ala Leu Val Ala Lys Ala Gln 420 425 430 Lys Lys Pro Glu Glu Gly Trp Val Met Gln Asp Gly Thr Pro Trp Pro 435 440 445 Gly Asn Asn Thr Arg Asp His Pro Gly Met Ile Gln Val Tyr Leu Gly 450 455 460 Asn Gln Gly Ala Leu Asp Val Glu Gly His Glu Leu Pro Arg Leu Val465 470 475 480 Tyr Val Ser Arg Glu Lys Arg Pro Gly Tyr Asn His His Lys Lys Ala 485 490 495 Gly Ala Met Asn Ala Leu Val Arg Val Ser Ala Val Leu Thr Asn Ala 500 505 510 Pro Phe Ile Leu Asn Leu Asp Cys Asp His Tyr Val Asn Asn Ser Lys 515 520 525 Ala Val Arg Glu Ala Met Cys Phe Leu Met Asp Pro Gln Leu Gly Lys 530 535 540 Lys Leu Cys Tyr Val Gln Phe Pro Gln Arg Phe Asp Gly Ile Asp Arg545 550 555 560 His Asp Arg Tyr Ala Asn Arg Asn Val Val Phe Phe Asp Ile Asn Met 565 570 575 Lys Gly Leu Asp Gly Ile Gln Gly Pro Val Tyr Val Gly Thr Gly Cys 580 585 590 Val Phe Asn Arg Gln Ala Leu Tyr Gly Tyr Asp Pro Pro Arg Pro Glu 595 600 605 Lys Arg Pro Lys Met Thr Cys Asp Cys Trp Pro Ser Trp Cys Cys Cys 610 615 620 Cys Cys Cys Phe Gly Gly Gly Lys Arg Gly Lys Ala Arg Lys Asp Lys625 630 635 640 Lys Gly Asp Gly Gly Glu Glu Pro Arg Arg Gly Leu Leu Gly Phe Tyr 645 650 655 Arg Lys Arg Ser Lys Lys Asp Lys Leu Gly Gly Gly Ser Val Ala Gly 660 665 670 Ser Lys Lys Gly Gly Gly Leu Tyr Lys Lys His Gln Arg Ala Phe Glu 675 680 685 Leu Glu Glu Ile Glu Glu Gly Leu Glu Gly Tyr Asp Glu Leu Glu Arg 690 695 700 Ser Ser Leu Met Ser Gln Lys Ser Phe Glu Lys Arg Phe Gly Gln Ser705 710 715 720 Pro Val Phe Ile Ala Ser Thr Leu Val Glu Asp Gly Gly Leu Pro Gln 725 730 735 Gly Ala Ala Ala Asp Pro Ala Ala Leu Ile Lys Glu Ala Ile His Val 740 745 750 Ile Ser Cys Gly Tyr Glu Glu Lys Thr Glu Trp Gly Lys Glu Ile Gly 755 760 765 Trp Ile Tyr Gly Ser Val Thr Glu Asp Ile Leu Thr Gly Phe Lys Met 770 775 780 His Cys Arg Gly Trp Lys Ser Val Tyr Cys Thr Pro Thr Arg Pro Ala785 790 795 800 Phe Lys Gly Ser Ala Pro Ile Asn Leu Ser Asp Arg Leu His Gln Val 805 810 815 Leu Arg Trp Ala Leu Gly Ser Val Glu Ile Phe Met Ser Arg His Cys 820 825 830 Pro Leu Arg Tyr Ala Tyr Gly Gly Arg Leu Lys Trp Leu Glu Arg Phe 835 840 845 Ala Tyr Thr Asn Thr Ile Val Tyr Pro Phe Thr Ser Ile Pro Leu Leu 850 855 860 Ala Tyr Cys Thr Ile Pro Ala Val Cys Leu Leu Thr Gly Lys Phe Ile865 870 875 880 Ile Pro Thr Leu Asn Asn Leu Ala Ser Ile Trp Phe Ile Ala Leu Phe 885 890 895 Leu Ser Ile Ile Ala Thr Ser Val Leu Glu Leu Arg Trp Ser Gly Val 900 905 910 Ser Ile Glu Asp Trp Trp Arg Asn Glu Gln Phe Trp Val Ile Gly Gly 915 920 925 Val Ser Ala His Leu Phe Ala Val Phe Gln Gly Phe Leu Lys Val Leu 930 935 940 Gly Gly Val Asp Thr Ser Phe Thr Val Thr Ser Lys Ala Ala Gly Asp945 950 955 960 Glu Ala Asp Ala Phe Gly Asp Leu Tyr Leu Phe Lys Trp Thr Thr Leu 965 970 975 Leu Val Pro Pro Thr Thr Leu Ile Ile Ile Asn Met Val Gly Ile Val 980 985 990 Ala Gly Val Ser Asp Ala Val Asn Asn Gly Tyr Gly Ser Trp Gly Pro 995 1000 1005 Leu Phe Gly Lys Leu Phe Phe Ser Phe Trp Val Ile Val His Leu Tyr 1010 1015 1020 Pro Phe Leu Lys Gly Leu Met Gly Arg Gln Asn Arg Thr Pro Thr Ile1025 1030 1035 1040Val Val Leu Trp Ser Ile Leu Leu Ala Ser Ile Phe Ser Leu Val Trp 1045 1050 1055 Val Arg Ile Asp Pro Phe Ile Pro Lys Ala Lys Gly Pro Ile Leu Lys 1060 1065 1070 Pro Cys Gly Val Glu Cys 1075 273231DNAZea mays 27ccacgcgtcc gggaggggcc atgatggagt cggcggcggc ccagtcctgc gcggcgtgcg 60gggacgacgc gcgcgctgcc tgccgcgcgt gcagctacgc gctctgcagg gcgtgcctcg 120acgaggacgc cgccgagggc cgcaccacat gcgcgcgctg cggaggggac tacgccgcta 180tcaacccagc gcgcgccagc gagggaaccg aggcggagga ggaggtggtg gagaaccacc 240acaccgccgg tggcctgcgt gagagggtca ccatgggcag ccacctcaat gatcgccagg 300atgaagtaag ccacgccagg accatgagca gcttgtcggg aattggtagt gaattgaatg 360atgaatctgg taagcccatc tggaagaaca gggtggagag ttggaaggaa aagaagaatg 420agaagaaagc ctcggccaaa aagactgcag ctaaagcaca gcctccgcct gtcgaagaac 480agatcatgga tgaaaaagac ttgacagatg catatgagcc actctcccgg gtcatcccaa 540tatcaaagaa caagctcaca ccttacagag cagtgatcat tatgcggtta attgttcttg 600ggctcttctt tcactaccgt atcaccaatc ctgttaacag tgcctttggt ctctggatga 660catcagttat atgtgagatc tggtttggtt tctcctggat attggatcaa ttcccgaagt 720ggtatcctat caatcgtgag acttatgttg ataggctgat tgcacgatat ggagatggtg 780aagaatctgg gttagcacct gtagatttct ttgtcagtac agtggatcca ttgaaagagc 840ctccactaat cactgcaaac actgtgctgt ctattcttgc tgtggactat cccgttgaga 900agatctcatg ctatgtatct gatgatggtt ctgctatgct cacatttgaa tcgctcgcag 960agactgcaga atatgctaga aagtgggtgc cgttttgcaa gaagtacgcc attgagccac 1020gagctcctga gttctacttc tcacagaaaa ttgactactt gaaggacaag atacacccat 1080cttttgtcaa ggagcgtagg gctatgaaga gagactatga agagtacaag gtgaggataa 1140atgctttggt tgccaaggct caaaagacac ctgatgaagg ctggatcatg caagacggta 1200caccatggcc tgggaacaat cctcgtgacc accctggcat gatccaggtt ttcctgggtg 1260agactggtgc acgggacttt gatggaaatg aacttcctcg

gttagtgtat gtgtcaagag 1320agaaaagacc aggctaccaa caccacaaga aggcaggggc tatgaatgct ctggtccgag 1380tgtctgctgt tctgacaaat gccccttaca ttcttaatct tgattgtgat cactatgtta 1440acaacagcaa agctgttcgt gaagcaatgt gcttcatgat ggaccctact gttggcagag 1500atgtctgcta tgtacaattc ccccagaggt tcgatggcat tgatcgcagt gatcgatatg 1560ccaataggaa cgttgtgttc tttgatgtta atatgaaagg acttgatggc ctccaaggcc 1620cagtttatgt gggaactggt tgttgtttca ataggcaagc actttatggt tatgggcctc 1680catctctgcc cgcacttcca aagtcttcga tttgttcctg gtgttgctgc tgctgtccca 1740agaaaaaggt tgaaagaagt gagagggaaa tcaacagaga ctctcggcga gaagacctcg 1800agtctgccat ttttaacctt cgcgaaattg acaactacga tgagtacgag aggtccatgc 1860tcatctctca gatgagcttc gagaagtctt ttgggctgtc ctcggtcttt attgaatcga 1920cccttatgga gaatgggggc gtccctgaat ctgcaaaccc atctacccta attaaagaag 1980ccattcatgt cattagctgt ggatatgaag agaaaactga atggggaaaa gagattggct 2040ggatctatgg ttcagttaca gaggatattc tgactgggtt taagatgcac tgccgtggct 2100ggagatccat ctactgcatg ccggtgagac ctgcattcaa gggatcagcc ccaatcaatc 2160tttccgatcg tcttcaccaa gttctccggt gggctcttgt ttctgtcgag atcttcttca 2220gtcggcactg cccgctgtgg tacggttacg gtggcggccg tctgaaatgg ctccagaggc 2280tctcctacat caacaccatc gtgtacccgt tcacttctct tcctctcgtt gcctactgtt 2340gcctgcctgc catttgcctg ctcacaggaa agttcattat acctacgctg tccaacgctg 2400caacgatatg gtttcttggc ctcttcatgt ccatcatcgt gacgagcgtg ttggagctgc 2460ggtggagtgg catcgggatc gaggactggt ggcgcaacga gcagttctgg gtcatcggag 2520gcgtgtccgc gcacctgttc gccgtgttcc agggtatcct caagatgatt gccgggctgg 2580acaccaactt cacggtcacg gcaaaggcca cggacgacac tgagttcggg gagctgtacc 2640tgttcaagtg gacgacggtg ctgatcccgc ccacaagcat cctggtgctg aacctggtgg 2700gcgtggtggc tgggttctcg gccgcgctca acagcggcta cgagtcctgg ggcccgctct 2760tcggtaaggt gttcttcgcc atgtgggtga tcatgcacct gtacccgttc ctcaagggtc 2820tcatgggccg ccagaaccgc acgccgacca tcgtggtgct ctggtccgtc ctcctcgcct 2880ccgtcttctc cctcctgtgg gtcaagatcg acccattcgt tggaggaacc gagaccgtca 2940acaccaacaa ctgcaacaca catctgctga ttcaccatcg gtcagctgct gtcgtgccgc 3000ggcggacgtg tttctggtgt tgcaaacgtg ggttgcctgc ctgatgcggg tctcctctgt 3060ctatctcgca tctgggcttt tgccccagga tctgaagcgg gtggtgtagg ttagctttat 3120tttgcgtcca agtgttgatt gatgttgtct gtgttatgaa aagttttggt ggtgaaacct 3180gaaatgttaa aattcggctc aattgtgaga aaaaaaaaaa aaaaaaaaaa a 3231281007PRTZea mays 28Met Met Glu Ser Ala Ala Ala Gln Ser Cys Ala Ala Cys Gly Asp Asp1 5 10 15 Ala Arg Ala Ala Cys Arg Ala Cys Ser Tyr Ala Leu Cys Arg Ala Cys 20 25 30 Leu Asp Glu Asp Ala Ala Glu Gly Arg Thr Thr Cys Ala Arg Cys Gly 35 40 45 Gly Asp Tyr Ala Ala Ile Asn Pro Ala Arg Ala Ser Glu Gly Thr Glu 50 55 60 Ala Glu Glu Glu Val Val Glu Asn His His Thr Ala Gly Gly Leu Arg65 70 75 80 Glu Arg Val Thr Met Gly Ser His Leu Asn Asp Arg Gln Asp Glu Val 85 90 95 Ser His Ala Arg Thr Met Ser Ser Leu Ser Gly Ile Gly Ser Glu Leu 100 105 110 Asn Asp Glu Ser Gly Lys Pro Ile Trp Lys Asn Arg Val Glu Ser Trp 115 120 125 Lys Glu Lys Lys Asn Glu Lys Lys Ala Ser Ala Lys Lys Thr Ala Ala 130 135 140 Lys Ala Gln Pro Pro Pro Val Glu Glu Gln Ile Met Asp Glu Lys Asp145 150 155 160 Leu Thr Asp Ala Tyr Glu Pro Leu Ser Arg Val Ile Pro Ile Ser Lys 165 170 175 Asn Lys Leu Thr Pro Tyr Arg Ala Val Ile Ile Met Arg Leu Ile Val 180 185 190 Leu Gly Leu Phe Phe His Tyr Arg Ile Thr Asn Pro Val Asn Ser Ala 195 200 205 Phe Gly Leu Trp Met Thr Ser Val Ile Cys Glu Ile Trp Phe Gly Phe 210 215 220 Ser Trp Ile Leu Asp Gln Phe Pro Lys Trp Tyr Pro Ile Asn Arg Glu225 230 235 240 Thr Tyr Val Asp Arg Leu Ile Ala Arg Tyr Gly Asp Gly Glu Glu Ser 245 250 255 Gly Leu Ala Pro Val Asp Phe Phe Val Ser Thr Val Asp Pro Leu Lys 260 265 270 Glu Pro Pro Leu Ile Thr Ala Asn Thr Val Leu Ser Ile Leu Ala Val 275 280 285 Asp Tyr Pro Val Glu Lys Ile Ser Cys Tyr Val Ser Asp Asp Gly Ser 290 295 300 Ala Met Leu Thr Phe Glu Ser Leu Ala Glu Thr Ala Glu Tyr Ala Arg305 310 315 320 Lys Trp Val Pro Phe Cys Lys Lys Tyr Ala Ile Glu Pro Arg Ala Pro 325 330 335 Glu Phe Tyr Phe Ser Gln Lys Ile Asp Tyr Leu Lys Asp Lys Ile His 340 345 350 Pro Ser Phe Val Lys Glu Arg Arg Ala Met Lys Arg Asp Tyr Glu Glu 355 360 365 Tyr Lys Val Arg Ile Asn Ala Leu Val Ala Lys Ala Gln Lys Thr Pro 370 375 380 Asp Glu Gly Trp Ile Met Gln Asp Gly Thr Pro Trp Pro Gly Asn Asn385 390 395 400 Pro Arg Asp His Pro Gly Met Ile Gln Val Phe Leu Gly Glu Thr Gly 405 410 415 Ala Arg Asp Phe Asp Gly Asn Glu Leu Pro Arg Leu Val Tyr Val Ser 420 425 430 Arg Glu Lys Arg Pro Gly Tyr Gln His His Lys Lys Ala Gly Ala Met 435 440 445 Asn Ala Leu Val Arg Val Ser Ala Val Leu Thr Asn Ala Pro Tyr Ile 450 455 460 Leu Asn Leu Asp Cys Asp His Tyr Val Asn Asn Ser Lys Ala Val Arg465 470 475 480 Glu Ala Met Cys Phe Met Met Asp Pro Thr Val Gly Arg Asp Val Cys 485 490 495 Tyr Val Gln Phe Pro Gln Arg Phe Asp Gly Ile Asp Arg Ser Asp Arg 500 505 510 Tyr Ala Asn Arg Asn Val Val Phe Phe Asp Val Asn Met Lys Gly Leu 515 520 525 Asp Gly Leu Gln Gly Pro Val Tyr Val Gly Thr Gly Cys Cys Phe Asn 530 535 540 Arg Gln Ala Leu Tyr Gly Tyr Gly Pro Pro Ser Leu Pro Ala Leu Pro545 550 555 560 Lys Ser Ser Ile Cys Ser Trp Cys Cys Cys Cys Cys Pro Lys Lys Lys 565 570 575 Val Glu Arg Ser Glu Arg Glu Ile Asn Arg Asp Ser Arg Arg Glu Asp 580 585 590 Leu Glu Ser Ala Ile Phe Asn Leu Arg Glu Ile Asp Asn Tyr Asp Glu 595 600 605 Tyr Glu Arg Ser Met Leu Ile Ser Gln Met Ser Phe Glu Lys Ser Phe 610 615 620 Gly Leu Ser Ser Val Phe Ile Glu Ser Thr Leu Met Glu Asn Gly Gly625 630 635 640 Val Pro Glu Ser Ala Asn Pro Ser Thr Leu Ile Lys Glu Ala Ile His 645 650 655 Val Ile Ser Cys Gly Tyr Glu Glu Lys Thr Glu Trp Gly Lys Glu Ile 660 665 670 Gly Trp Ile Tyr Gly Ser Val Thr Glu Asp Ile Leu Thr Gly Phe Lys 675 680 685 Met His Cys Arg Gly Trp Arg Ser Ile Tyr Cys Met Pro Val Arg Pro 690 695 700 Ala Phe Lys Gly Ser Ala Pro Ile Asn Leu Ser Asp Arg Leu His Gln705 710 715 720 Val Leu Arg Trp Ala Leu Val Ser Val Glu Ile Phe Phe Ser Arg His 725 730 735 Cys Pro Leu Trp Tyr Gly Tyr Gly Gly Gly Arg Leu Lys Trp Leu Gln 740 745 750 Arg Leu Ser Tyr Ile Asn Thr Ile Val Tyr Pro Phe Thr Ser Leu Pro 755 760 765 Leu Val Ala Tyr Cys Cys Leu Pro Ala Ile Cys Leu Leu Thr Gly Lys 770 775 780 Phe Ile Ile Pro Thr Leu Ser Asn Ala Ala Thr Ile Trp Phe Leu Gly785 790 795 800 Leu Phe Met Ser Ile Ile Val Thr Ser Val Leu Glu Leu Arg Trp Ser 805 810 815 Gly Ile Gly Ile Glu Asp Trp Trp Arg Asn Glu Gln Phe Trp Val Ile 820 825 830 Gly Gly Val Ser Ala His Leu Phe Ala Val Phe Gln Gly Ile Leu Lys 835 840 845 Met Ile Ala Gly Leu Asp Thr Asn Phe Thr Val Thr Ala Lys Ala Thr 850 855 860 Asp Asp Thr Glu Phe Gly Glu Leu Tyr Leu Phe Lys Trp Thr Thr Val865 870 875 880 Leu Ile Pro Pro Thr Ser Ile Leu Val Leu Asn Leu Val Gly Val Val 885 890 895 Ala Gly Phe Ser Ala Ala Leu Asn Ser Gly Tyr Glu Ser Trp Gly Pro 900 905 910 Leu Phe Gly Lys Val Phe Phe Ala Met Trp Val Ile Met His Leu Tyr 915 920 925 Pro Phe Leu Lys Gly Leu Met Gly Arg Gln Asn Arg Thr Pro Thr Ile 930 935 940 Val Val Leu Trp Ser Val Leu Leu Ala Ser Val Phe Ser Leu Leu Trp945 950 955 960 Val Lys Ile Asp Pro Phe Val Gly Gly Thr Glu Thr Val Asn Thr Asn 965 970 975 Asn Cys Asn Thr His Leu Leu Ile His His Arg Ser Ala Ala Val Val 980 985 990 Pro Arg Arg Thr Cys Phe Trp Cys Cys Lys Arg Gly Leu Pro Ala 995 1000 1005 293028DNAZea mays 29cacgagttca acatcgacga cgagaatcag cagaggcagc tggagggcaa catgcagaac 60agccagatca ccgaggcgat gctgcacggc aggatgagct acgggagggg ccccgacgac 120ggcgacggca acaacacccc gcagatcccg cccatcatca ccggctcccg ctccgtgccg 180gtgagcggtg agtttccgat taccaacggg tatggccacg gcgaggtctc gtcttccctg 240cacaagcgca tccatccgta ccctgtgtct gagccaggga gtgccaagtg ggacgagaag 300aaagaagtga gctggaagga gaggatggac gactggaagt ccaagcaggg catcctcggc 360ggcggcgccg atcccgaaga catggacgcc gacgtggcac tgaacgacga ggcgaggcag 420ccgctgtcga ggaaggtgtc gatcgcgtcg agcaaggtga acccgtaccg gatggtgatc 480gtggtgcgtc tcgttgtgct cgccttcttc ctccggtacc gtatcctgca ccccgtcccg 540gacgccatcg ggctgtggct cgtctccatc atctgcgaga tctggttcgc catctcctgg 600atcctcgacc agttccccaa gtggttcccc atcgaccgcg agacgtacct cgaccgcctc 660tccctcaggt acgagaggga aggggagccg tcgctgctgt cggcggtgga cctgttcgtg 720agcacggtgg acccgctcaa ggagccgccg ctggtgaccg ccaacaccgt gctctccatc 780ctcgccgtag actaccccgt ggacaaggtc tcctgctacg tctccgacga cggcgcgtcg 840atgctgacgt tcgagtcgct gtcggagacg gccgagttcg cgcgcaagtg ggtgcccttc 900tgcaagaagt tcggcatcga gccccgcgcc ccggagttct acttctcgct caaggtcgac 960tacctcaagg acaaggtgca gcccaccttc gtgcaggagc gccgcgccat gaagagagag 1020tatgaggagt tcaaggtccg gatcaacgcg ctggtggcca aggccatgaa ggtgccggca 1080gaggggtgga tcatgaagga cggcacgccg tggcccggga acaacacccg cgaccacccc 1140ggcatgatcc aggtgttcct gggccacagc ggcggccacg acaccgaggg caacgagctg 1200ccccgcctcg tgtacgtctc ccgtgagaag cgcccgggat tccagcacca caagaaggcc 1260ggcgccatga acgctctgat tcgcgtctcc gccgtgctga ccaacgcgcc attcatgctc 1320aacttggact gtgatcacta catcaacaac agcaaggcca tccgggaggc catgtgcttc 1380ctcatggacc ctcaggtcgg ccggaaggtc tgctacgttc agttcccgca gaggttcgac 1440ggcatcgacg tgcacgaccg atacgctaac aggaacaccg tcttcttcga catcaacatg 1500aaggggctgg acggcatcca aggcccggtg tacgtcggga cagggtgcgt gttccggcgc 1560caggcgctct acggctacaa ccctcccaag ggacccaaga ggcccaagat ggtgacctgc 1620gactgctgcc cgtgcttcgg ccgcaagaag cggaaacacg ccaaggacgg gctgccggag 1680ggcaccgctg atatgggagt agatagcgac aaggagatgc tcatgtccca catgaacttc 1740gagaagcggt tcgggcagtc cgcggcgttc gtcacgtcga cgctgatgga ggaaggcggc 1800gtccctcctt cgtcgagccc cgccgcgctc ctcaaggagg ccatccatgt catcagctgc 1860ggctacgagg acaagaccga ctgggggctg gagctggggt ggatctacgg gtcgatcacg 1920gaggacatcc tgacggggtt caagatgcac tgccgcgggt ggcgctccgt gtactgcatg 1980ccgaagcggg cggcgttcaa ggggtcggcg ccgatcaatc tatcggaccg tctcaaccag 2040gtgctccggt gggcgctggg gtccgtcgag atcttcttca gccggcacag ccccctgctg 2100tacggctaca agaacggcaa cctcaagtgg ctggagcgct tcgcctacat caacaccacc 2160atctacccct tcacctcgct cccgctgctc gcctactgca ccctccccgc cgtctgcctc 2220ctcaccggca agttcatcat gccgtcgatt agcacgttcg ccagcctctt cttcatcgcc 2280ctcttcatgt ccatcttcgc gacgggcatc ctggagatgc ggtggagcgg ggtgagcatc 2340gaggagtggt ggaggaacga gcagttctgg gtcatcggcg gcgtgtccgc gcatctcttc 2400gccgtcgtgc agggcctgct caaggtcctc gccgggatcg acaccaactt caccgtcacc 2460tccaaggcca ccggcgacga ggacgacgag ttcgccgagc tctacgcctt caagtggacc 2520acgctcctca tcccgcccac cacgctgctc atcattaacg tcatcggcgt cgtggccggc 2580atctccgacg ccatcaacaa cgggtaccag tcctgggggc ccctcttcgg caagctcttc 2640ttcgccttct gggtcatcgt ccacctctac ccgttcctca aggggctcat ggggcgccag 2700aacaggacgc ccaccgttgt tgtcatctgg tccattctgc tggcctccat cttctccctg 2760ctctgggtca ggatcgaccc tttcatcgtc aggaccaagg gcccggacgt caggcagtgt 2820ggcatcaatt gctgagctgt ttattaaggt tcaaaattct ggagcttgtg catagggaga 2880aaaaaacaat ttagaaattt tgtaaggttg ttgtgtctgt aatgttatgg tacccagaat 2940tgtcggacga ggaattgaac aaaggacaag gtttgattgt taaatggcaa aaaaaaaaaa 3000aaaaaaaaaa aaaaaaaaaa aaaaaaaa 302830927PRTZea mays 30Met Gln Asn Ser Gln Ile Thr Glu Ala Met Leu His Gly Arg Met Ser1 5 10 15 Tyr Gly Arg Gly Pro Asp Asp Gly Asp Gly Asn Asn Thr Pro Gln Ile 20 25 30 Pro Pro Ile Ile Thr Gly Ser Arg Ser Val Pro Val Ser Gly Glu Phe 35 40 45 Pro Ile Thr Asn Gly Tyr Gly His Gly Glu Val Ser Ser Ser Leu His 50 55 60 Lys Arg Ile His Pro Tyr Pro Val Ser Glu Pro Gly Ser Ala Lys Trp65 70 75 80 Asp Glu Lys Lys Glu Val Ser Trp Lys Glu Arg Met Asp Asp Trp Lys 85 90 95 Ser Lys Gln Gly Ile Leu Gly Gly Gly Ala Asp Pro Glu Asp Met Asp 100 105 110 Ala Asp Val Ala Leu Asn Asp Glu Ala Arg Gln Pro Leu Ser Arg Lys 115 120 125 Val Ser Ile Ala Ser Ser Lys Val Asn Pro Tyr Arg Met Val Ile Val 130 135 140 Val Arg Leu Val Val Leu Ala Phe Phe Leu Arg Tyr Arg Ile Leu His145 150 155 160 Pro Val Pro Asp Ala Ile Gly Leu Trp Leu Val Ser Ile Ile Cys Glu 165 170 175 Ile Trp Phe Ala Ile Ser Trp Ile Leu Asp Gln Phe Pro Lys Trp Phe 180 185 190 Pro Ile Asp Arg Glu Thr Tyr Leu Asp Arg Leu Ser Leu Arg Tyr Glu 195 200 205 Arg Glu Gly Glu Pro Ser Leu Leu Ser Ala Val Asp Leu Phe Val Ser 210 215 220 Thr Val Asp Pro Leu Lys Glu Pro Pro Leu Val Thr Ala Asn Thr Val225 230 235 240 Leu Ser Ile Leu Ala Val Asp Tyr Pro Val Asp Lys Val Ser Cys Tyr 245 250 255 Val Ser Asp Asp Gly Ala Ser Met Leu Thr Phe Glu Ser Leu Ser Glu 260 265 270 Thr Ala Glu Phe Ala Arg Lys Trp Val Pro Phe Cys Lys Lys Phe Gly 275 280 285 Ile Glu Pro Arg Ala Pro Glu Phe Tyr Phe Ser Leu Lys Val Asp Tyr 290 295 300 Leu Lys Asp Lys Val Gln Pro Thr Phe Val Gln Glu Arg Arg Ala Met305 310 315 320 Lys Arg Glu Tyr Glu Glu Phe Lys Val Arg Ile Asn Ala Leu Val Ala 325 330 335 Lys Ala Met Lys Val Pro Ala Glu Gly Trp Ile Met Lys Asp Gly Thr 340 345 350 Pro Trp Pro Gly Asn Asn Thr Arg Asp His Pro Gly Met Ile Gln Val 355 360 365 Phe Leu Gly His Ser Gly Gly His Asp Thr Glu Gly Asn Glu Leu Pro 370 375 380 Arg Leu Val Tyr Val Ser Arg Glu Lys Arg Pro Gly Phe Gln His His385 390 395 400 Lys Lys Ala Gly Ala Met Asn Ala Leu Ile Arg Val Ser Ala Val Leu 405 410 415 Thr Asn Ala Pro Phe Met Leu Asn Leu Asp Cys Asp His Tyr Ile Asn 420 425 430 Asn Ser Lys Ala Ile Arg Glu Ala Met Cys Phe Leu Met Asp Pro Gln 435 440 445 Val Gly Arg Lys Val Cys Tyr Val Gln Phe Pro Gln Arg Phe Asp Gly 450 455 460 Ile Asp Val His Asp Arg Tyr Ala Asn Arg Asn Thr Val Phe Phe Asp465 470 475 480 Ile Asn Met Lys Gly Leu Asp Gly Ile Gln Gly Pro Val Tyr Val Gly 485 490 495 Thr Gly Cys Val Phe Arg Arg Gln Ala Leu Tyr Gly Tyr Asn Pro Pro 500 505 510 Lys Gly Pro Lys Arg Pro Lys Met Val Thr Cys Asp Cys Cys Pro Cys 515 520 525 Phe Gly Arg Lys Lys Arg Lys His

Ala Lys Asp Gly Leu Pro Glu Gly 530 535 540 Thr Ala Asp Met Gly Val Asp Ser Asp Lys Glu Met Leu Met Ser His545 550 555 560 Met Asn Phe Glu Lys Arg Phe Gly Gln Ser Ala Ala Phe Val Thr Ser 565 570 575 Thr Leu Met Glu Glu Gly Gly Val Pro Pro Ser Ser Ser Pro Ala Ala 580 585 590 Leu Leu Lys Glu Ala Ile His Val Ile Ser Cys Gly Tyr Glu Asp Lys 595 600 605 Thr Asp Trp Gly Leu Glu Leu Gly Trp Ile Tyr Gly Ser Ile Thr Glu 610 615 620 Asp Ile Leu Thr Gly Phe Lys Met His Cys Arg Gly Trp Arg Ser Val625 630 635 640 Tyr Cys Met Pro Lys Arg Ala Ala Phe Lys Gly Ser Ala Pro Ile Asn 645 650 655 Leu Ser Asp Arg Leu Asn Gln Val Leu Arg Trp Ala Leu Gly Ser Val 660 665 670 Glu Ile Phe Phe Ser Arg His Ser Pro Leu Leu Tyr Gly Tyr Lys Asn 675 680 685 Gly Asn Leu Lys Trp Leu Glu Arg Phe Ala Tyr Ile Asn Thr Thr Ile 690 695 700 Tyr Pro Phe Thr Ser Leu Pro Leu Leu Ala Tyr Cys Thr Leu Pro Ala705 710 715 720 Val Cys Leu Leu Thr Gly Lys Phe Ile Met Pro Ser Ile Ser Thr Phe 725 730 735 Ala Ser Leu Phe Phe Ile Ala Leu Phe Met Ser Ile Phe Ala Thr Gly 740 745 750 Ile Leu Glu Met Arg Trp Ser Gly Val Ser Ile Glu Glu Trp Trp Arg 755 760 765 Asn Glu Gln Phe Trp Val Ile Gly Gly Val Ser Ala His Leu Phe Ala 770 775 780 Val Val Gln Gly Leu Leu Lys Val Leu Ala Gly Ile Asp Thr Asn Phe785 790 795 800 Thr Val Thr Ser Lys Ala Thr Gly Asp Glu Asp Asp Glu Phe Ala Glu 805 810 815 Leu Tyr Ala Phe Lys Trp Thr Thr Leu Leu Ile Pro Pro Thr Thr Leu 820 825 830 Leu Ile Ile Asn Val Ile Gly Val Val Ala Gly Ile Ser Asp Ala Ile 835 840 845 Asn Asn Gly Tyr Gln Ser Trp Gly Pro Leu Phe Gly Lys Leu Phe Phe 850 855 860 Ala Phe Trp Val Ile Val His Leu Tyr Pro Phe Leu Lys Gly Leu Met865 870 875 880 Gly Arg Gln Asn Arg Thr Pro Thr Val Val Val Ile Trp Ser Ile Leu 885 890 895 Leu Ala Ser Ile Phe Ser Leu Leu Trp Val Arg Ile Asp Pro Phe Ile 900 905 910 Val Arg Thr Lys Gly Pro Asp Val Arg Gln Cys Gly Ile Asn Cys 915 920 925 3136DNAArtificial SequenceSal-A20 oligonucleotide 31tcgacccacg cgtccgaaaa aaaaaaaaaa aaaaaa 363228DNAArtificial SequenceGSP1 forward primer 32tacgatgagt acgagaggtc catgctca 283326DNAArtificial SequenceGSP2 reverse primer 33ggcaaaagcc cagatgcgag atagac 263432DNAArtificial SequenceMu TIR primer 34agagaagcca acgccawcgc ctcyatttcg tc 32359DNAZea mays 35tggcggccg 9369DNAZea mays 36tctgaaatg 9379DNAZea mays 37gcccacaag 9389DNAZea mays 38catcctggt 9399DNAZea mays 39gtgttcttc 9409DNAZea mays 40gccatgtgg 9413568DNAZea maysmisc_feature3487n = A,T,C or G 41gtcgacccac gcgtccggag ctcgtcgtca tccgccgcga tggcgagcca gggccgaagc 60ccatggacca gcggaacggc caggtgtgcc agatttgcgg cgacgacgtg gggcgcaacc 120ccgacgggga gcctttcgtg gcctgcaacg agtgcgcctt ccccatctgc cgggactgct 180acgagtacga gcgccgcgag ggcacgcaga actgccccca gtgcaagacc cgcttcaagc 240gcttcaaggg gtgcgcgcgc gtgcccgggg acgaggagga ggacggcgtc gacgacctgg 300agaacgagtt caactggagc gacaagcacg actcccagta cctcgccgag tccatgctcc 360acgcccacat gagctacggc cgcggcgccg acctcgacgg cgtgccgcag ccattccacc 420ccatccccaa tgttcccctc ctcaccaacg gacagatggt cgatgacatc ccgccggacc 480agcacgccct tgtgccctcg ttcgtgggtg gcggggggaa gaggattcac cctctcccgt 540acgcggatcc caaccttcct gtgcaaccga ggtctatgga cccttccaag gatctcgccg 600catatggcta cgggagcgta gcatggaagg agaggatgga gagctggaag cagaagcagg 660agaggatgca ccagacgagg aacgatggcg gcggcgatga tggtgatgat gcagatctac 720cactaatgga tgaagctaga cagccattgt ccagaaagat cccgcttcct tcaagccaaa 780tcaaccccta taggatgatt ataataattc ggctagtggt tttgtgtttc ttcttccact 840accgagtgat gcatccggtg cctgatgcat ttgctttatg gctcatatct gtgatctgtg 900aaatttggtt tgccatgtct tggattcttg accagtttcc aaagtggttt cctatcgaga 960gggaaaccta tcttgaccgg ctgagtttaa ggtttgacaa ggaagggcat ccttctcaac 1020tcgcccctgt tgatttcttt gtcagtacgg ttgatccctt gaaggaacct ccattggtca 1080ctgctaatac tgttctatct atcctttcgg tggattatcc agttgataag gtttcatgct 1140acgtttctga tgatggtgct gccatgctga catttgaagc attgtctgaa acatctgaat 1200ttgcaaagaa atgggttcct ttctgcaaaa gatatagcct tgagcctcgt gctccagagt 1260ggtacttcca acagaagata gactacctga aagacaaggt ggcgccaaac tttgttagag 1320aacggagagc aatgaagaga gagtatgagg aattcaaggt cagaatcaat gccttggttg 1380ctaaagccca aaaggttcct gaggaaggat ggacaatgca ggatggaact ccatggcccg 1440gaaataatgt ccgtgatcat cctggaatga ttcaggtttt ccttggtcaa agtggtggcc 1500atgatgtgga aggaaatgag ctgcctcgat tggtttatgt ttcaagagaa aaacggccag 1560gctacaacca tcacaagaag gctggtgcta tgaatgcatt ggtccgagtc tctgctgtac 1620taactaatgc tccttatttg ctgaacttgg attgtgatca ctatatcaat aatagtaagg 1680ctataaagga agcaatgtgt tttatgatgg atcctttgct tggaaagaaa gtttgctatg 1740tgcagtttcc tcaaagattt gatgggattg atcgccatga tcgatatgct aacagaaatg 1800ttgtcttttt cgatatcaac atgaaaggtt tggatggtat ccagggccca atttatgtgg 1860gtactggatg tgtcttcaga aggcaggcat tatatggcta cgatgctccc aaaacaaaga 1920agccaccatc aagaacttgc aactgctggc caaagtggtg catttgctgt tgctgttttg 1980gtaacaggaa gaccaagaag aagaccaaga cctctaaacc taaatttgag aagataaaga 2040aactttttaa gaaaaaggaa aatcaagccc ctgcatatgc tcttggtgaa attgatgaag 2100ccgctccagg agctgaaaat gaaaaggcta gtattgtaaa tcaacagaag ttggaaaaga 2160aatttggcca gtcttcagtt tttgttgcat ccacacttct tgagaatggt ggaaccctga 2220agagtgccag tccagcttct cttctgaagg aagctataca tgtcatcagt tgtggatatg 2280aagacaaaac aggctgggga aaagatattg gttggattta tggatcagtc acagaagata 2340ttcttactgg gtttaagatg cactgccatg gttggcggtc aatttactgc atacctaaac 2400gggccgcctt caaaggttcc gcacctctca atctttccga tcgttttcac caggttcttc 2460ggtgggctct tggttcaatt gaaattttgt tcagcaacca ctgccctctc tggtatgggt 2520atggtggtgg actaaagttc ctggaaaggt tttcgtacat taactccatc gtataccctt 2580ggacatctat cccgctcttg gcctattgca cattgcctgc catctgcttg ctgacaggga 2640aatttatcac gccagagctt aacaatgttg ccagcctctg gttcatgtca cttttcatct 2700gcatttttgc tacgagcatc ctggaaatga gatggagtgg tgtaggcatc gatgactggt 2760ggagaaacga gcagttttgg gtcattggag gcgtgtcttc acatctcttt gctgtgttcc 2820agggactcct caaggtcata gctggtgtag acacgagctt cactgtgaca tccaagggcg 2880gagacgacga ggagttctca gagctgtaca cattcaaatg gacgaccctt ctgatacctc 2940cgacaaccct gctcctactg aacttcattg gagtggtagc tggcatctcc aatgcgatca 3000acaacggata tgaatcatgg ggccccctgt tcgggaagct cttctttgca ttttgggtga 3060tcgtccatct ttacccgttc ctcaagggtc tggttgggag gcagaacagg acgccaacga 3120ttgtcattgt ctggtccatc ctcctggctt cgatcttctc gctgctttgg gtccggatcg 3180acccgttcct tgcgaaggat gatggtcccc tgttggagga gtgtggtctg gattgcaact 3240aggaggtcag cacgtggact tccccgtcag tgtgtggtcg aagaagtatt tttgcagatg 3300ttttgtgccc atatttcttt actcaatttt tgtccctctg tagattgaaa caaggggtga 3360aggggaaaaa aagtacttgt atttcttttg ttccatggtg gtggtggtgg tgggcggctc 3420agcctcgtga gtgcaatatt gggcaaaccg gaggttgcgg caaccttgtg cagttcgtcc 3480acgaatntac tagggatgat cgcgaccaat caatcaatcg atgaccgagt tcaattgttc 3540aaaaaaaaaa aaaaaaaagg gcggccgc 3568421059PRTZea mays 42Met Asp Gln Arg Asn Gly Gln Val Cys Gln Ile Cys Gly Asp Asp Val1 5 10 15 Gly Arg Asn Pro Asp Gly Glu Pro Phe Val Ala Cys Asn Glu Cys Ala 20 25 30 Phe Pro Ile Cys Arg Asp Cys Tyr Glu Tyr Glu Arg Arg Glu Gly Thr 35 40 45 Gln Asn Cys Pro Gln Cys Lys Thr Arg Phe Lys Arg Phe Lys Gly Cys 50 55 60 Ala Arg Val Pro Gly Asp Glu Glu Glu Asp Gly Val Asp Asp Leu Glu65 70 75 80 Asn Glu Phe Asn Trp Ser Asp Lys His Asp Ser Gln Tyr Leu Ala Glu 85 90 95 Ser Met Leu His Ala His Met Ser Tyr Gly Arg Gly Ala Asp Leu Asp 100 105 110 Gly Val Pro Gln Pro Phe His Pro Ile Pro Asn Val Pro Leu Leu Thr 115 120 125 Asn Gly Gln Met Val Asp Asp Ile Pro Pro Asp Gln His Ala Leu Val 130 135 140 Pro Ser Phe Val Gly Gly Gly Gly Lys Arg Ile His Pro Leu Pro Tyr145 150 155 160 Ala Asp Pro Asn Leu Pro Val Gln Pro Arg Ser Met Asp Pro Ser Lys 165 170 175 Asp Leu Ala Ala Tyr Gly Tyr Gly Ser Val Ala Trp Lys Glu Arg Met 180 185 190 Glu Ser Trp Lys Gln Lys Gln Glu Arg Met His Gln Thr Arg Asn Asp 195 200 205 Gly Gly Gly Asp Asp Gly Asp Asp Ala Asp Leu Pro Leu Met Asp Glu 210 215 220 Ala Arg Gln Pro Leu Ser Arg Lys Ile Pro Leu Pro Ser Ser Gln Ile225 230 235 240 Asn Pro Tyr Arg Met Ile Ile Ile Ile Arg Leu Val Val Leu Cys Phe 245 250 255 Phe Phe His Tyr Arg Val Met His Pro Val Pro Asp Ala Phe Ala Leu 260 265 270 Trp Leu Ile Ser Val Ile Cys Glu Ile Trp Phe Ala Met Ser Trp Ile 275 280 285 Leu Asp Gln Phe Pro Lys Trp Phe Pro Ile Glu Arg Glu Thr Tyr Leu 290 295 300 Asp Arg Leu Ser Leu Arg Phe Asp Lys Glu Gly His Pro Ser Gln Leu305 310 315 320 Ala Pro Val Asp Phe Phe Val Ser Thr Val Asp Pro Leu Lys Glu Pro 325 330 335 Pro Leu Val Thr Ala Asn Thr Val Leu Ser Ile Leu Ser Val Asp Tyr 340 345 350 Pro Val Asp Lys Val Ser Cys Tyr Val Ser Asp Asp Gly Ala Ala Met 355 360 365 Leu Thr Phe Glu Ala Leu Ser Glu Thr Ser Glu Phe Ala Lys Lys Trp 370 375 380 Val Pro Phe Cys Lys Arg Tyr Ser Leu Glu Pro Arg Ala Pro Glu Trp385 390 395 400 Tyr Phe Gln Gln Lys Ile Asp Tyr Leu Lys Asp Lys Val Ala Pro Asn 405 410 415 Phe Val Arg Glu Arg Arg Ala Met Lys Arg Glu Tyr Glu Glu Phe Lys 420 425 430 Val Arg Ile Asn Ala Leu Val Ala Lys Ala Gln Lys Val Pro Glu Glu 435 440 445 Gly Trp Thr Met Gln Asp Gly Thr Pro Trp Pro Gly Asn Asn Val Arg 450 455 460 Asp His Pro Gly Met Ile Gln Val Phe Leu Gly Gln Ser Gly Gly His465 470 475 480 Asp Val Glu Gly Asn Glu Leu Pro Arg Leu Val Tyr Val Ser Arg Glu 485 490 495 Lys Arg Pro Gly Tyr Asn His His Lys Lys Ala Gly Ala Met Asn Ala 500 505 510 Leu Val Arg Val Ser Ala Val Leu Thr Asn Ala Pro Tyr Leu Leu Asn 515 520 525 Leu Asp Cys Asp His Tyr Ile Asn Asn Ser Lys Ala Ile Lys Glu Ala 530 535 540 Met Cys Phe Met Met Asp Pro Leu Leu Gly Lys Lys Val Cys Tyr Val545 550 555 560 Gln Phe Pro Gln Arg Phe Asp Gly Ile Asp Arg His Asp Arg Tyr Ala 565 570 575 Asn Arg Asn Val Val Phe Phe Asp Ile Asn Met Lys Gly Leu Asp Gly 580 585 590 Ile Gln Gly Pro Ile Tyr Val Gly Thr Gly Cys Val Phe Arg Arg Gln 595 600 605 Ala Leu Tyr Gly Tyr Asp Ala Pro Lys Thr Lys Lys Pro Pro Ser Arg 610 615 620 Thr Cys Asn Cys Trp Pro Lys Trp Cys Ile Cys Cys Cys Cys Phe Gly625 630 635 640 Asn Arg Lys Thr Lys Lys Lys Thr Lys Thr Ser Lys Pro Lys Phe Glu 645 650 655 Lys Ile Lys Lys Leu Phe Lys Lys Lys Glu Asn Gln Ala Pro Ala Tyr 660 665 670 Ala Leu Gly Glu Ile Asp Glu Ala Ala Pro Gly Ala Glu Asn Glu Lys 675 680 685 Ala Ser Ile Val Asn Gln Gln Lys Leu Glu Lys Lys Phe Gly Gln Ser 690 695 700 Ser Val Phe Val Ala Ser Thr Leu Leu Glu Asn Gly Gly Thr Leu Lys705 710 715 720 Ser Ala Ser Pro Ala Ser Leu Leu Lys Glu Ala Ile His Val Ile Ser 725 730 735 Cys Gly Tyr Glu Asp Lys Thr Gly Trp Gly Lys Asp Ile Gly Trp Ile 740 745 750 Tyr Gly Ser Val Thr Glu Asp Ile Leu Thr Gly Phe Lys Met His Cys 755 760 765 His Gly Trp Arg Ser Ile Tyr Cys Ile Pro Lys Arg Ala Ala Phe Lys 770 775 780 Gly Ser Ala Pro Leu Asn Leu Ser Asp Arg Phe His Gln Val Leu Arg785 790 795 800 Trp Ala Leu Gly Ser Ile Glu Ile Leu Phe Ser Asn His Cys Pro Leu 805 810 815 Trp Tyr Gly Tyr Gly Gly Gly Leu Lys Phe Leu Glu Arg Phe Ser Tyr 820 825 830 Ile Asn Ser Ile Val Tyr Pro Trp Thr Ser Ile Pro Leu Leu Ala Tyr 835 840 845 Cys Thr Leu Pro Ala Ile Cys Leu Leu Thr Gly Lys Phe Ile Thr Pro 850 855 860 Glu Leu Asn Asn Val Ala Ser Leu Trp Phe Met Ser Leu Phe Ile Cys865 870 875 880 Ile Phe Ala Thr Ser Ile Leu Glu Met Arg Trp Ser Gly Val Gly Ile 885 890 895 Asp Asp Trp Trp Arg Asn Glu Gln Phe Trp Val Ile Gly Gly Val Ser 900 905 910 Ser His Leu Phe Ala Val Phe Gln Gly Leu Leu Lys Val Ile Ala Gly 915 920 925 Val Asp Thr Ser Phe Thr Val Thr Ser Lys Gly Gly Asp Asp Glu Glu 930 935 940 Phe Ser Glu Leu Tyr Thr Phe Lys Trp Thr Thr Leu Leu Ile Pro Pro945 950 955 960 Thr Thr Leu Leu Leu Leu Asn Phe Ile Gly Val Val Ala Gly Ile Ser 965 970 975 Asn Ala Ile Asn Asn Gly Tyr Glu Ser Trp Gly Pro Leu Phe Gly Lys 980 985 990 Leu Phe Phe Ala Phe Trp Val Ile Val His Leu Tyr Pro Phe Leu Lys 995 1000 1005 Gly Leu Val Gly Arg Gln Asn Arg Thr Pro Thr Ile Val Ile Val Trp 1010 1015 1020 Ser Ile Leu Leu Ala Ser Ile Phe Ser Leu Leu Trp Val Arg Ile Asp1025 1030 1035 1040Pro Phe Leu Ala Lys Asp Asp Gly Pro Leu Leu Glu Glu Cys Gly Leu 1045 1050 1055 Asp Cys Asn4325DNAArtificial Sequenceamplicon 43atggaccagc ggaacggcca ggtgt 254425DNAArtificial Sequenceamplicon 44ctagttgcaa tccagaccac actcc 25453725DNAZea mays 45gcagcagcag caccaccact gcgcggcatt gcagcgagca agcgggaggg atctggggca 60tggtggcggt cgctgccgct gccgctcgga tctagagggc cgcacgggct gattgccctc 120cgccggcctc gtcggtgtcg gtggagtgtg aatcggtgtg tgtaggagga gcgcggagat 180ggcggccaac aaggggatgg tggcaggctc tcacaaccgc aacgagttcg tcatgatccg 240ccacgacggc gacgcgcctg tcccggctaa gcccacgaag agtgcgaatg ggcaggtctg 300ccagatttgt ggcgacactg ttggcgtttc agccactggt gatgtctttg ttgcctgcaa 360tgagtgtgcc ttccctgtct gccgcccttg ctatgagtac gagcgcaagg aagggaacca 420atgctgccct cagtgcaaga ctagatacaa gagacagaaa ggtagccctc gagttcatgg 480tgatgatgag gaggaagatg ttgatgacct ggacaatgaa ttcaactata agcaaggcaa 540tgggaagggc ccagagtggc agcttcaagg agatgacgct gatctgtctt catctgctcg 600ccatgaccca caccatcgga ttccacgcct tacaagtgga caacagatat ctggagagat 660ccctgatgca tcccctgacc gtcattctat ccgcagtcca acatcgagct atgttgatcc 720aagcgttcca gttcctgtga ggattgtgga cccctcgaag gacttgaatt cctatgggct 780taatagtgtt gactggaagg aaagagttga gagctggagg gttaaacagg acaaaaatat 840gttgcaagtg actaataaat atccagaggc tagaggagac atggagggga ctggctcaaa

900tggagaagat atgcaaatgg ttgatgatgc acgcctacct ttgagccgca ttgtgccaat 960ttcctcaaac cagctcaacc tttaccggat agtaatcatt ctccgtctta tcatcctgtg 1020cttcttcttc caatatcgta tcagtcatcc agtgcgtaat gcttatggat tgtggctagt 1080atctgttatc tgtgaggtct ggtttgcctt gtcctggctt ctagatcagt tcccaaaatg 1140gtatccaatc aaccgtgaga catatctcga caggcttgca ttgaggtatg atagagaggg 1200agagccatca cagctggctc ccattgatgt ctttgtcagt acagtggatc cattgaagga 1260acctccactg atcacagcca acactgtttt gtccattctt gctgtggatt accctgttga 1320caaagtgtca tgctatgttt ctgatgatgg ctcagctatg ctgacttttg agtctctctc 1380tgaaactgcc gaatttgcta gaaagtgggt tcccttttgt aagaagcaca atattgaacc 1440aagagctcca gaattttact ttgctcaaaa aatagattac ctgaaggaca aaattcaacc 1500ttcatttgtt aaggaaagac gagcaatgaa gagagagtat gaagaattca aaataagaat 1560caatgccctt gttgccaaag cacagaaagt gcctgaagag gggtggacca tggctgatgg 1620aactgcttgg cctgggaata accctaggga ccatcctggc atgattcagg tgttcttggg 1680gcacagtggt gggcttgaca ctgatggaaa tgaattacca cgtcttgtct atgtctctcg 1740tgaaaagaga ccaggctttc agcatcacaa gaaggctggt gcaatgaatg cactgattcg 1800tgtatctgct gtgctgacaa atggtgccta tcttctcaat gtggattgtg accattactt 1860caatagcagc aaagctctta gagaagcaat gtgcttcatg atggatccag ctctaggaag 1920gaaaacttgt tatgtacaat ttccacaaag atttgatggc attgacttgc acgatcgata 1980tgctaatagg aacatagtct tctttgatat caacatgaaa ggtctagatg gcattcaggg 2040tccagtctat gtgggaacag gatgctgttt caataggcag gctttgtatg gatatgatcc 2100tgttttgact gaagctgatc tggaacctaa cattgttgtt aagagctgct gtggtagaag 2160gaagagaaag aacaagagtt atatggatag tcaaagccgt attatgaaga gaacagaatc 2220ttcagctccc atctttaaca tggaagacat cgaggagggt attgaaggtt atgaggatga 2280aaggtcagtg cttatgtccc agaggaaatt ggagaaacgc tttggtcagt ctccaatctt 2340cattgcatcc acctttatga ctcaaggtgg cataccacct tcaacaaacc cagcttctct 2400actgaaggaa gctatccatg ttatcagctg tgggtacgag gacaaaactg aatggggaaa 2460agagattggc tggatctatg gttcagttac agaggatatt ctgactgggt ttaaaatgca 2520tgcaagaggc tggcaatcaa tctactgcat gccaccacga ccttgtttca agggttctgc 2580accaatcaat ctttctgatc gtcttaatca ggtgctccgt tgggctcttg ggtcagtgga 2640aattctgctt agcagacatt gtcctatatg gtatggctac aatgggcgat tgaagctttt 2700ggagaggctg gcttacatta acaccattgt ttatccaatc acatctgttc cgcttatcgc 2760ctattgtgtg cttcctgcta tctgtcttct taccaataaa tttatcattc ctgagattag 2820taattatgct ggaatgttct tcattcttct ttttgcctcc attttcgcaa ctggtatatt 2880ggagctcaga tggagtggtg ttggcattga agattggtgg agaaatgagc agttttgggt 2940tattggtggc acctctgccc atctcttcgc ggtgttccag ggtctgctga aagtgttggc 3000tgggattgat accaacttca cagttacctc aaaggcatct gatgaggatg gcgactttgc 3060tgagctatat gtgttcaagt ggaccagttt gctcatccct ccgaccactg ttcttgtcat 3120taacctggtc ggaatggtgg caggaatttc gtatgccatt aacagcggct accaatcctg 3180gggtccgctc tttggaaagc tgttcttctc gatctgggtg atcctccatc tctacccctt 3240cctcaagggt ctcatgggca ggcagaaccg cacgccaaca atcgtcatcg tttggtccat 3300cctccttgcg tctatcttct ccttgctgtg ggtgaagatc gatcctttca tctccccgac 3360acagaaagct gccgccttgg ggcaatgtgg tgtgaactgc tgatccagat tgtgactctt 3420atctgaagag gctcagccaa agatctgccc cctcgtgtaa atacctgagg gggctagatg 3480ggaatttttt gttgtagatg aggatggatc tgcatccaag ttatgcctct gtttattagc 3540ttcttcggtg ccggtgctgc tgcagacaat catggagcct ttctaccttg cttgtagtgc 3600tggccagcag cgtaaattgt gaattctgca tttttttata cgtggtgttt attgttttag 3660agtaaattat catttgtttg aggtaactat tcacacgaac tatatggcaa tgctgttatt 3720taaaa 3725461074PRTZea mays 46Met Ala Ala Asn Lys Gly Met Val Ala Gly Ser His Asn Arg Asn Glu1 5 10 15 Phe Val Met Ile Arg His Asp Gly Asp Ala Pro Val Pro Ala Lys Pro 20 25 30 Thr Lys Ser Ala Asn Gly Gln Val Cys Gln Ile Cys Gly Asp Thr Val 35 40 45 Gly Val Ser Ala Thr Gly Asp Val Phe Val Ala Cys Asn Glu Cys Ala 50 55 60 Phe Pro Val Cys Arg Pro Cys Tyr Glu Tyr Glu Arg Lys Glu Gly Asn65 70 75 80 Gln Cys Cys Pro Gln Cys Lys Thr Arg Tyr Lys Arg Gln Lys Gly Ser 85 90 95 Pro Arg Val His Gly Asp Asp Glu Glu Glu Asp Val Asp Asp Leu Asp 100 105 110 Asn Glu Phe Asn Tyr Lys Gln Gly Asn Gly Lys Gly Pro Glu Trp Gln 115 120 125 Leu Gln Gly Asp Asp Ala Asp Leu Ser Ser Ser Ala Arg His Asp Pro 130 135 140 His His Arg Ile Pro Arg Leu Thr Ser Gly Gln Gln Ile Ser Gly Glu145 150 155 160 Ile Pro Asp Ala Ser Pro Asp Arg His Ser Ile Arg Ser Pro Thr Ser 165 170 175 Ser Tyr Val Asp Pro Ser Val Pro Val Pro Val Arg Ile Val Asp Pro 180 185 190 Ser Lys Asp Leu Asn Ser Tyr Gly Leu Asn Ser Val Asp Trp Lys Glu 195 200 205 Arg Val Glu Ser Trp Arg Val Lys Gln Asp Lys Asn Met Leu Gln Val 210 215 220 Thr Asn Lys Tyr Pro Glu Ala Arg Gly Asp Met Glu Gly Thr Gly Ser225 230 235 240 Asn Gly Glu Asp Met Gln Met Val Asp Asp Ala Arg Leu Pro Leu Ser 245 250 255 Arg Ile Val Pro Ile Ser Ser Asn Gln Leu Asn Leu Tyr Arg Ile Val 260 265 270 Ile Ile Leu Arg Leu Ile Ile Leu Cys Phe Phe Phe Gln Tyr Arg Ile 275 280 285 Ser His Pro Val Arg Asn Ala Tyr Gly Leu Trp Leu Val Ser Val Ile 290 295 300 Cys Glu Val Trp Phe Ala Leu Ser Trp Leu Leu Asp Gln Phe Pro Lys305 310 315 320 Trp Tyr Pro Ile Asn Arg Glu Thr Tyr Leu Asp Arg Leu Ala Leu Arg 325 330 335 Tyr Asp Arg Glu Gly Glu Pro Ser Gln Leu Ala Pro Ile Asp Val Phe 340 345 350 Val Ser Thr Val Asp Pro Leu Lys Glu Pro Pro Leu Ile Thr Ala Asn 355 360 365 Thr Val Leu Ser Ile Leu Ala Val Asp Tyr Pro Val Asp Lys Val Ser 370 375 380 Cys Tyr Val Ser Asp Asp Gly Ser Ala Met Leu Thr Phe Glu Ser Leu385 390 395 400 Ser Glu Thr Ala Glu Phe Ala Arg Lys Trp Val Pro Phe Cys Lys Lys 405 410 415 His Asn Ile Glu Pro Arg Ala Pro Glu Phe Tyr Phe Ala Gln Lys Ile 420 425 430 Asp Tyr Leu Lys Asp Lys Ile Gln Pro Ser Phe Val Lys Glu Arg Arg 435 440 445 Ala Met Lys Arg Glu Tyr Glu Glu Phe Lys Ile Arg Ile Asn Ala Leu 450 455 460 Val Ala Lys Ala Gln Lys Val Pro Glu Glu Gly Trp Thr Met Ala Asp465 470 475 480 Gly Thr Ala Trp Pro Gly Asn Asn Pro Arg Asp His Pro Gly Met Ile 485 490 495 Gln Val Phe Leu Gly His Ser Gly Gly Leu Asp Thr Asp Gly Asn Glu 500 505 510 Leu Pro Arg Leu Val Tyr Val Ser Arg Glu Lys Arg Pro Gly Phe Gln 515 520 525 His His Lys Lys Ala Gly Ala Met Asn Ala Leu Ile Arg Val Ser Ala 530 535 540 Val Leu Thr Asn Gly Ala Tyr Leu Leu Asn Val Asp Cys Asp His Tyr545 550 555 560 Phe Asn Ser Ser Lys Ala Leu Arg Glu Ala Met Cys Phe Met Met Asp 565 570 575 Pro Ala Leu Gly Arg Lys Thr Cys Tyr Val Gln Phe Pro Gln Arg Phe 580 585 590 Asp Gly Ile Asp Leu His Asp Arg Tyr Ala Asn Arg Asn Ile Val Phe 595 600 605 Phe Asp Ile Asn Met Lys Gly Leu Asp Gly Ile Gln Gly Pro Val Tyr 610 615 620 Val Gly Thr Gly Cys Cys Phe Asn Arg Gln Ala Leu Tyr Gly Tyr Asp625 630 635 640 Pro Val Leu Thr Glu Ala Asp Leu Glu Pro Asn Ile Val Val Lys Ser 645 650 655 Cys Cys Gly Arg Arg Lys Arg Lys Asn Lys Ser Tyr Met Asp Ser Gln 660 665 670 Ser Arg Ile Met Lys Arg Thr Glu Ser Ser Ala Pro Ile Phe Asn Met 675 680 685 Glu Asp Ile Glu Glu Gly Ile Glu Gly Tyr Glu Asp Glu Arg Ser Val 690 695 700 Leu Met Ser Gln Arg Lys Leu Glu Lys Arg Phe Gly Gln Ser Pro Ile705 710 715 720 Phe Ile Ala Ser Thr Phe Met Thr Gln Gly Gly Ile Pro Pro Ser Thr 725 730 735 Asn Pro Ala Ser Leu Leu Lys Glu Ala Ile His Val Ile Ser Cys Gly 740 745 750 Tyr Glu Asp Lys Thr Glu Trp Gly Lys Glu Ile Gly Trp Ile Tyr Gly 755 760 765 Ser Val Thr Glu Asp Ile Leu Thr Gly Phe Lys Met His Ala Arg Gly 770 775 780 Trp Gln Ser Ile Tyr Cys Met Pro Pro Arg Pro Cys Phe Lys Gly Ser785 790 795 800 Ala Pro Ile Asn Leu Ser Asp Arg Leu Asn Gln Val Leu Arg Trp Ala 805 810 815 Leu Gly Ser Val Glu Ile Leu Leu Ser Arg His Cys Pro Ile Trp Tyr 820 825 830 Gly Tyr Asn Gly Arg Leu Lys Leu Leu Glu Arg Leu Ala Tyr Ile Asn 835 840 845 Thr Ile Val Tyr Pro Ile Thr Ser Val Pro Leu Ile Ala Tyr Cys Val 850 855 860 Leu Pro Ala Ile Cys Leu Leu Thr Asn Lys Phe Ile Ile Pro Glu Ile865 870 875 880 Ser Asn Tyr Ala Gly Met Phe Phe Ile Leu Leu Phe Ala Ser Ile Phe 885 890 895 Ala Thr Gly Ile Leu Glu Leu Arg Trp Ser Gly Val Gly Ile Glu Asp 900 905 910 Trp Trp Arg Asn Glu Gln Phe Trp Val Ile Gly Gly Thr Ser Ala His 915 920 925 Leu Phe Ala Val Phe Gln Gly Leu Leu Lys Val Leu Ala Gly Ile Asp 930 935 940 Thr Asn Phe Thr Val Thr Ser Lys Ala Ser Asp Glu Asp Gly Asp Phe945 950 955 960 Ala Glu Leu Tyr Val Phe Lys Trp Thr Ser Leu Leu Ile Pro Pro Thr 965 970 975 Thr Val Leu Val Ile Asn Leu Val Gly Met Val Ala Gly Ile Ser Tyr 980 985 990 Ala Ile Asn Ser Gly Tyr Gln Ser Trp Gly Pro Leu Phe Gly Lys Leu 995 1000 1005 Phe Phe Ser Ile Trp Val Ile Leu His Leu Tyr Pro Phe Leu Lys Gly 1010 1015 1020 Leu Met Gly Arg Gln Asn Arg Thr Pro Thr Ile Val Ile Val Trp Ser1025 1030 1035 1040Ile Leu Leu Ala Ser Ile Phe Ser Leu Leu Trp Val Lys Ile Asp Pro 1045 1050 1055 Phe Ile Ser Pro Thr Gln Lys Ala Ala Ala Leu Gly Gln Cys Gly Val 1060 1065 1070 Asn Cys4725DNAArtificial Sequenceamplicon 47atggcggcca acaaggggat ggtgg 254825DNAArtificial Sequenceamplicon 48tcagcagttc acaccacatt gcccc 25493969DNAZea mays 49cttctccctc gtcggtgcgg cgtggcgcgg ctcggcgttc ggtgagaaac cactcggggg 60atgaggatct gctgctagag tgagaggagc tacggtcagt atcctctgcc ttcgtcggcg 120gcggaagtgg aggggaggaa gcgatggagg cgagcgccgg gctggtggcc ggctcccaca 180accgcaacga gctcgtcgtc atccgccgcg acggcgatcc cgggccgaag ccgccgcggg 240agcagaacgg gcaggtgtgc cagatttgcg gcgacgacgt cggccttgcc cccggcgggg 300accccttcgt ggcgtgcaac gagtgcgcct tccccgtctg ccgggactgc tacgaatacg 360agcgccggga gggcacgcag aactgccccc agtgcaagac tcgatacaag cgcctcaagg 420gctgccaacg tgtgaccggt gacgaggagg aggacggcgt cgatgacctg gacaacgagt 480tcaactggga cggccatgac tcgcagtctg tggccgagtc catgctctac ggccacatga 540gctacggccg tggaggtgac cctaatggcg cgccacaagc tttccagctc aaccccaatg 600ttccactcct caccaacggg caaatggtgg atgacatccc accggagcag cacgcgctgg 660tgccttcttt catgggtggt gggggaaaga ggatacatcc ccttccttat gcggatccca 720gcttacctgt gcaacccagg tctatggacc catccaagga tcttgctgca tatgggtatg 780gtagtgttgc ttggaaggaa cggatggaga attggaagca gagacaagag aggatgcacc 840agacggggaa tgatggtggt ggtgatgatg gtgacgatgc tgatctacca ctaatggatg 900aagcaagaca acaactgtcc aggaaaattc cacttccatc aagccagatt aatccatata 960ggatgattat cattattcgg cttgtggttt tggggttctt cttccactac cgagtgatgc 1020atccggtgaa tgatgcattt gctttgtggc tcatatctgt tatctgtgaa atctggtttg 1080ccatgtcttg gattcttgat caattcccaa agtggttccc tattgagaga gagacttacc 1140tagaccggct gtcactgagg ttcgacaagg aaggccagcc atctcaactt gctccaattg 1200atttctttgt cagtacggtt gatcccttaa aggaacctcc tttggtcaca acaaatactg 1260ttctatctat cctttcggtg gattatcctg ttgataaggt ttcttgctat gtttctgatg 1320atggtgctgc aatgctaacg tttgaagcat tatctgaaac atctgaattt gcaaagaaat 1380gggttccttt ctgcaaacgg tacaatattg aacctcgcgc tccagagtgg tacttccaac 1440agaagataga ctacttgaaa gacaaggtgg cagcaaactt tgttagggag aggagagcaa 1500tgaagagaga gtatgaggaa ttcaaggtga gaatcaatgc cttagttgcc aaagcccaga 1560aagttcctga agaaggatgg acaatgcaag atggaacccc ctggcctgga aacaatgttc 1620gtgatcatcc tggaatgatt caggtcttcc ttggccaaag cggaggcctt gactgtgagg 1680gaaatgaact gccacgattg gtttatgttt ctagagagaa acgaccaggc tataaccatc 1740ataagaaagc tggtgctatg aatgcattgg tccgagtctc tgctgtacta acaaatgctc 1800catatttgtt aaacttggat tgtgatcact acatcaacaa cagcaaggct ataaaggaag 1860caatgtgttt tatgatggac cctttactag gaaagaaggt ttgctatgta cagttccctc 1920aaagatttga tgggattgat cgccatgacc gatatgctaa ccggaatgtt gtcttttttg 1980atatcaacat gaaaggtttg gatggtattc agggtccaat ttatgttggt actggatgtg 2040tatttagaag gcaggcatta tatggttatg atgcccccaa aacaaagaag ccaccatcaa 2100ggacttgcaa ctgctggccc aagtggtgct tttgctgttg ctgctttggc aataggaagc 2160aaaagaagac taccaaaccc aaaacagaga agaaaaagtt attatttttc aagaaagaag 2220agaaccaatc ccctgcatat gctcttggtg aaattgacga agctgctcca ggagctgaga 2280atgaaaaggc cggtattgta aatcaacaaa aattagaaaa gaaatttggc caatcttctg 2340tttttgttac atccacactt ctcgagaatg gtggaacctt gaagagtgca agtcctgctt 2400ctcttttgaa agaagctata catgtcatta gttgtggtta tgaagacaag acagactggg 2460gaaaagagat tggctggatc tatggatcag ttacagaaga tattctaact ggtttcaaga 2520tgcattgtca tggttggcgg tcaatttact gcatacctaa acgggttgca ttcaaaggtt 2580ctgcacctct gaatctttca gatcgtcttc accaggtgct tcggtgggct cttgggtcta 2640ttgagatctt cttcagcaat cattgccctc tttggtatgg gtatggtggc ggtctgaaat 2700ttttggaaag attttcctac atcaactcca tcgtgtatcc ttggacatct attcccctct 2760tggcttactg tacattgcct gccatctgtt tattgacagg gaaatttatc actccagagc 2820tgaataatgt tgccagcctg tggttcatgt cactttttat ctgcattttt gctacgagca 2880tcctagaaat gagatggagt ggtgttggaa ttgatgactg gtggaggaat gagcagttct 2940gggtcattgg aggtgtgtcc tcacacctct ttgctgtgtt ccagggactt ctcaaggtca 3000tagctggtgt tgatacaagc ttcaccgtga catcaaaggg tggagatgat gaggagttct 3060cagagctata tacattcaaa tggactacct tattgatacc tcctaccacc ttgcttctat 3120tgaacttcat tggtgtggtc gctggcgttt caaatgcgat caataacgga tatgagtcat 3180ggggccccct ctttgggaag ctattctttg cattttgggt gattgtccat ctttatccct 3240ttctcaaagg tttggttgga aggcaaaaca ggacaccaac gattgtcatc gtctggtcca 3300ttctgctggc ttcaatcttc tcgctccttt gggttcggat tgatcctttc cttgcgaagg 3360atgatggtcc gcttcttgag gagtgtggtt tggattgcaa ctaggatgtc agtgcatcag 3420ctcccccaat ctgcatatgc ttgaagtata ttttctggtg tttgtcccca tattcagtgt 3480ctgtagataa gagacatgaa atgtcccaag tttcttttga tccatggtga acctacttaa 3540tatctgagag atatactggg ggaaaatgga ggctgcggca atccttgtgc agttgggccg 3600tggaatacag catatgcaag tgtttgattg tgcagcattc tttattactt ggtcgcaata 3660tagatgggct gagccgaaca gcaaggtatt ttgattctgc actgctcccg tgtacaaact 3720tggttctcaa taaggcaggc aggaatgcat ctgccagtgg aacagagcaa cctgcacatt 3780atttatgtat gcctgttcat tggagggctt gttcattaca tgttcgtcta tactagaaaa 3840aacagaatat tagcattaat ctatagttaa ttaaagtatg taaatgcgcc tgttttttgt 3900tgtgtactgt aatcatctga gttggttttg tgaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3960aaaaaaaaa 3969501086PRTZea mays 50Met Glu Ala Ser Ala Gly Leu Val Ala Gly Ser His Asn Arg Asn Glu1 5 10 15 Leu Val Val Ile Arg Arg Asp Gly Asp Pro Gly Pro Lys Pro Pro Arg 20 25 30 Glu Gln Asn Gly Gln Val Cys Gln Ile Cys Gly Asp Asp Val Gly Leu 35 40 45 Ala Pro Gly Gly Asp Pro Phe Val Ala Cys Asn Glu Cys Ala Phe Pro 50 55 60 Val Cys Arg Asp Cys Tyr Glu Tyr Glu Arg Arg Glu Gly Thr Gln Asn65 70 75 80 Cys Pro Gln Cys Lys Thr Arg Tyr Lys Arg Leu Lys Gly Cys Gln Arg 85 90 95 Val Thr Gly Asp Glu Glu Glu Asp Gly Val Asp Asp Leu Asp Asn Glu 100 105 110 Phe Asn Trp Asp Gly His Asp Ser Gln Ser Val Ala Glu Ser Met Leu 115 120 125 Tyr Gly His Met Ser Tyr Gly Arg Gly Gly Asp Pro Asn Gly Ala Pro 130 135 140

Gln Ala Phe Gln Leu Asn Pro Asn Val Pro Leu Leu Thr Asn Gly Gln145 150 155 160 Met Val Asp Asp Ile Pro Pro Glu Gln His Ala Leu Val Pro Ser Phe 165 170 175 Met Gly Gly Gly Gly Lys Arg Ile His Pro Leu Pro Tyr Ala Asp Pro 180 185 190 Ser Leu Pro Val Gln Pro Arg Ser Met Asp Pro Ser Lys Asp Leu Ala 195 200 205 Ala Tyr Gly Tyr Gly Ser Val Ala Trp Lys Glu Arg Met Glu Asn Trp 210 215 220 Lys Gln Arg Gln Glu Arg Met His Gln Thr Gly Asn Asp Gly Gly Gly225 230 235 240 Asp Asp Gly Asp Asp Ala Asp Leu Pro Leu Met Asp Glu Ala Arg Gln 245 250 255 Gln Leu Ser Arg Lys Ile Pro Leu Pro Ser Ser Gln Ile Asn Pro Tyr 260 265 270 Arg Met Ile Ile Ile Ile Arg Leu Val Val Leu Gly Phe Phe Phe His 275 280 285 Tyr Arg Val Met His Pro Val Asn Asp Ala Phe Ala Leu Trp Leu Ile 290 295 300 Ser Val Ile Cys Glu Ile Trp Phe Ala Met Ser Trp Ile Leu Asp Gln305 310 315 320 Phe Pro Lys Trp Phe Pro Ile Glu Arg Glu Thr Tyr Leu Asp Arg Leu 325 330 335 Ser Leu Arg Phe Asp Lys Glu Gly Gln Pro Ser Gln Leu Ala Pro Ile 340 345 350 Asp Phe Phe Val Ser Thr Val Asp Pro Leu Lys Glu Pro Pro Leu Val 355 360 365 Thr Thr Asn Thr Val Leu Ser Ile Leu Ser Val Asp Tyr Pro Val Asp 370 375 380 Lys Val Ser Cys Tyr Val Ser Asp Asp Gly Ala Ala Met Leu Thr Phe385 390 395 400 Glu Ala Leu Ser Glu Thr Ser Glu Phe Ala Lys Lys Trp Val Pro Phe 405 410 415 Cys Lys Arg Tyr Asn Ile Glu Pro Arg Ala Pro Glu Trp Tyr Phe Gln 420 425 430 Gln Lys Ile Asp Tyr Leu Lys Asp Lys Val Ala Ala Asn Phe Val Arg 435 440 445 Glu Arg Arg Ala Met Lys Arg Glu Tyr Glu Glu Phe Lys Val Arg Ile 450 455 460 Asn Ala Leu Val Ala Lys Ala Gln Lys Val Pro Glu Glu Gly Trp Thr465 470 475 480 Met Gln Asp Gly Thr Pro Trp Pro Gly Asn Asn Val Arg Asp His Pro 485 490 495 Gly Met Ile Gln Val Phe Leu Gly Gln Ser Gly Gly Leu Asp Cys Glu 500 505 510 Gly Asn Glu Leu Pro Arg Leu Val Tyr Val Ser Arg Glu Lys Arg Pro 515 520 525 Gly Tyr Asn His His Lys Lys Ala Gly Ala Met Asn Ala Leu Val Arg 530 535 540 Val Ser Ala Val Leu Thr Asn Ala Pro Tyr Leu Leu Asn Leu Asp Cys545 550 555 560 Asp His Tyr Ile Asn Asn Ser Lys Ala Ile Lys Glu Ala Met Cys Phe 565 570 575 Met Met Asp Pro Leu Leu Gly Lys Lys Val Cys Tyr Val Gln Phe Pro 580 585 590 Gln Arg Phe Asp Gly Ile Asp Arg His Asp Arg Tyr Ala Asn Arg Asn 595 600 605 Val Val Phe Phe Asp Ile Asn Met Lys Gly Leu Asp Gly Ile Gln Gly 610 615 620 Pro Ile Tyr Val Gly Thr Gly Cys Val Phe Arg Arg Gln Ala Leu Tyr625 630 635 640 Gly Tyr Asp Ala Pro Lys Thr Lys Lys Pro Pro Ser Arg Thr Cys Asn 645 650 655 Cys Trp Pro Lys Trp Cys Phe Cys Cys Cys Cys Phe Gly Asn Arg Lys 660 665 670 Gln Lys Lys Thr Thr Lys Pro Lys Thr Glu Lys Lys Lys Leu Leu Phe 675 680 685 Phe Lys Lys Glu Glu Asn Gln Ser Pro Ala Tyr Ala Leu Gly Glu Ile 690 695 700 Asp Glu Ala Ala Pro Gly Ala Glu Asn Glu Lys Ala Gly Ile Val Asn705 710 715 720 Gln Gln Lys Leu Glu Lys Lys Phe Gly Gln Ser Ser Val Phe Val Thr 725 730 735 Ser Thr Leu Leu Glu Asn Gly Gly Thr Leu Lys Ser Ala Ser Pro Ala 740 745 750 Ser Leu Leu Lys Glu Ala Ile His Val Ile Ser Cys Gly Tyr Glu Asp 755 760 765 Lys Thr Asp Trp Gly Lys Glu Ile Gly Trp Ile Tyr Gly Ser Val Thr 770 775 780 Glu Asp Ile Leu Thr Gly Phe Lys Met His Cys His Gly Trp Arg Ser785 790 795 800 Ile Tyr Cys Ile Pro Lys Arg Val Ala Phe Lys Gly Ser Ala Pro Leu 805 810 815 Asn Leu Ser Asp Arg Leu His Gln Val Leu Arg Trp Ala Leu Gly Ser 820 825 830 Ile Glu Ile Phe Phe Ser Asn His Cys Pro Leu Trp Tyr Gly Tyr Gly 835 840 845 Gly Gly Leu Lys Phe Leu Glu Arg Phe Ser Tyr Ile Asn Ser Ile Val 850 855 860 Tyr Pro Trp Thr Ser Ile Pro Leu Leu Ala Tyr Cys Thr Leu Pro Ala865 870 875 880 Ile Cys Leu Leu Thr Gly Lys Phe Ile Thr Pro Glu Leu Asn Asn Val 885 890 895 Ala Ser Leu Trp Phe Met Ser Leu Phe Ile Cys Ile Phe Ala Thr Ser 900 905 910 Ile Leu Glu Met Arg Trp Ser Gly Val Gly Ile Asp Asp Trp Trp Arg 915 920 925 Asn Glu Gln Phe Trp Val Ile Gly Gly Val Ser Ser His Leu Phe Ala 930 935 940 Val Phe Gln Gly Leu Leu Lys Val Ile Ala Gly Val Asp Thr Ser Phe945 950 955 960 Thr Val Thr Ser Lys Gly Gly Asp Asp Glu Glu Phe Ser Glu Leu Tyr 965 970 975 Thr Phe Lys Trp Thr Thr Leu Leu Ile Pro Pro Thr Thr Leu Leu Leu 980 985 990 Leu Asn Phe Ile Gly Val Val Ala Gly Val Ser Asn Ala Ile Asn Asn 995 1000 1005 Gly Tyr Glu Ser Trp Gly Pro Leu Phe Gly Lys Leu Phe Phe Ala Phe 1010 1015 1020 Trp Val Ile Val His Leu Tyr Pro Phe Leu Lys Gly Leu Val Gly Arg1025 1030 1035 1040Gln Asn Arg Thr Pro Thr Ile Val Ile Val Trp Ser Ile Leu Leu Ala 1045 1050 1055 Ser Ile Phe Ser Leu Leu Trp Val Arg Ile Asp Pro Phe Leu Ala Lys 1060 1065 1070 Asp Asp Gly Pro Leu Leu Glu Glu Cys Gly Leu Asp Cys Asn 1075 1080 1085 5125DNAArtificial Sequenceamplicon 51atggaggcga gcgccgggct ggtgg 255225DNAArtificial Sequenceamplicon 52ctagttgcaa tccaaaccac actcc 25

Claims

1. A cellulose synthase product derived from the method of processing of plant tissues expressing an isolated polynucleotide encoding a functional cellulose synthase, the method comprising: a) transforming a plant cell with a recombinant expression cassette comprising a polynucleotide having at least 90% sequence identity to the full length sequence of a polynucleotide selected from the group consisting of SEQ ID NO: 13, 17, 21, 25, 27, 29, 45 and 49, operably linked to a promoter; and b) culturing the transformed plant cell under plant cell growing conditions; wherein the level of cellulose synthase in said transformed plant cell is modulated; c) growing the plant cell under plant-forming conditions to express the polynucleotide in the plant tissue; and d) processing the plant tissue to obtain a cellulose synthase product.

2. A cellulose synthase product according to claim 1, wherein the polynucleotide further encodes a polypeptide selected from the group consisting of SEQ ID NO: 14, 18, 22, 26, 28, 30, 46 and 50.

3. The transgenic plant of claim 1, wherein the plant is a monocot.

4. The transgenic plant of claim 1, wherein the plant is selected from the group consisting of: maize, soybean, sunflower, sorghum, canola, wheat, alfalfa, cotton, rice, barley and millet.

5. A cellulose synthase product according to claim 1, which improves stalk strength of a plant by overexpression of the polynucleotide.

6. A cellulose synthase product according to claim 1, which reduces green snap by improving nodal strength.

7. A cellulose synthase product according to claim 1, which is a constituent of ethanol.