AU780523B2 - Novel genes encoding wheat starch synthases and uses therefor - Google Patents

Description

WO 00/66745 PCT/AU00/00385 -1- NOVEL GENES ENCODING WHEAT STARCH SYNTHASES AND USES THEREFOR FIELD OF THE INVENTION The present invention relates generally to isolated nucleic acid molecules encoding wheat starch synthase enzymes and more particularly, to isolated nucleic acid molecules that encode wheat SS1II and SSIII enzyme activities. The isolated nucleic acid molecules provide the means for modifying starch content and composition in plants, for example the ratio of amylose:amylopectin in the starch granule of the endosperm during the grain-filling phase of endosperm development. The isolated nucleic acid molecules of the present invention also provide the means for screening plant lines to determine the presence of natural and/or induced mutations in starch synthase genes which affect starch content and/or composition. The isolated nucleic acid molecules of the present invention further provide for the screening-assisted breeding of plants having desirable starch content and/or composition, in addition to providing for the direct genetic manipulation of plant starch content and/or composition.

GENERAL

Bibliographic details of the publications numerically referred to in this specification are collected at the end of the description. Reference herein to any published document is not to be taken as an indication or admission that any such published document is part of the common general knowledge or background information of a skilled worker in the relevant field.

This specification contains nucleotide and amino acid sequence information (SEQ ID NOS:) prepared using the programme Patentln Version 2.0, presented herein at the end of the specification. Each nucleotide or amino acid sequence is identified in the sequence listing by the numeric indicator <210> followed by the sequence identifier <210>1, <210>2, etc). The length, type of sequence (DNA, protein (PRT), etc) and source organism for each nucleotide or amino acid sequence are indicated by information provided in the numeric indicator fields <211>, <212> and <213>, WO 00/66745 PCT/AU00/00385 -2respectively. Nucleotide and amino acid sequences (SEQ ID NOs:) referred to in the specification are defined by the information provided in numeric indicator field <400> followed by the sequence identifier (eg. SEQ ID NO: 1 is <400>1, etc).

The designation of nucleotide residues referred to herein are those recommended by the IUPAC-IUB Biochemical Nomenclature Commission, wherein A represents Adenine, C represents Cytosine, G represents Guanine, T represents thymine, Y represents a pyrimidine residue, R represents a purine residue, M represents Adenine or Cytosine, K represents Guanine or Thymine, S represents Guanine or Cytosine, W represents Adenine or Thymine, H represents a nucleotide other than Guanine, B represents a nucleotide other than Adenine, V represents a nucleotide other than Thymine, D represents a nucleotide other than Cytosine and N represents any nucleotide residue.

The designations for naturally-occurring amino acid residues referred to herein are set forth in Table I. The designations for a non-limiting set of non-naturally-occurring amino acids is listed in Table 2.

As used herein the term "derived from" shall be taken to indicate that a specified integer may be obtained from a particular source albeit not necessarily directly from that source.

Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated step or element or integer or group of steps or elements or integers but not the exclusion of any other step or element or integer or group of steps or elements or integers.

WO 00/66745 WO 0066745PCT/AUOO/00385 -3- TABLE 1 Amino Acid Three-letter Code One-letter Code Alanine Ala A Arginine Arg R Asparagine Asn N Aspartic acid Asp D Cysteine Cys C Glutamnine Gin Q Glutamic acid Glu E Glycine Gly G Histidine His H Isoleucine Ile 1 Leucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe F Proline- Pro P Serine Ser S Threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine Val V Aspartate/glutamate Baa. B Asparagine/glutamine Any amino acid as above Xaa X WO 00/66745 WO 0066745PCT/AUOO/00385 -4- TABLE 2 Non-conventional Code Non-conventional Code amino acid amino acid a-arninobutyric acid c-amino-a-methylbutyrate aminocyclopropanecarboxylate amninoisobutyric acid arninonorbornylcarboxylate cyclohexylalanine cyclopentylalanine D-alanine D-argmnine D-aspartic acid D-cysteine D-glutamine D-glutamic acid D-histidine D-isoleucine D-Ieucine D-lysine D-methionine D-ornithine D-phenylalanine D-proline D-serine D-threonine D-tryptophan Abu Mgabu Cpro Aib Norb Chexa Cpen Dal Darg Dasp Dcys Dgln Dglu Dhis Dule Dleu Dlys Dmet Domn Dphe Dpro Dser Dthr Dtrp L-N-methylalanine L-N-methylarginine L-N-methylasparagine L-N-methylaspartic acid L-N-methylcysteine L-N-methylglutamnine L-N-methylglutamic acid L-N-methylhistidine L-N-methylisolleucine L-N-methylleucine L-N-methyllysine L-N-methylniethionine L-N-methylnorleucine L-N-methylnorvaline L-N-methylornithine L-N-methylphenylalanine L-N-methylproline L-N-methylserine L-N-methylthreonine L-N-methyltryptophan L-N-methyltyrosine L-N-methylvaline L-N-.methylethylglycine L-N-methyl-t-butylglycine L-norleucine L-norvaline Nmala Nmarg Nmasn Nmasp Nmcys Nmgln Nmglu Nmnhis Nmile Nmnleu Nmlys Nmmnet Nrnnle Nmnnva Nmorn Nmphe Nmpro Nmser Nnflhr Nmtrp Nmtyr Nmval Nmetg Nmtbug NMe Nva WO 00/66745 WO 0066745PCT/AUOO/00385 D-tyrosine D-valine D-a-methylalanine D-a-methylarginine D-a-methylasparagine D-a-methylaspartate D-a-methylcysteine D-a-methylglutamine D-a-methylhistidine D-a-methylisoleucine D-a-methylleucine D-a-methyllysine D-a-methylmethionine D-a-methylornithine D-a-methylphenylalanine D-a-methylproline D-a-methylserine D-a-methylthreonine D-a-methyltryptophan D-cz-methyltyrosine D-a-methylvaline D-N-methylalanine D-N-methylarginine D-N-methylasparagine D-N-methylaspartate D-N-methylcysteine Dtyr a-methyl-aminoisobutyrate DvaI a-methyl-y-aminobutyrate Dmala c-methylcyclohexylalanine Dmarg a-methylcylcopentylalanine Dmasn a-methyl-cz-napdhylalanine Dmasp a-methylpenicillamine Dmcys N-(4-aminobutyl)glycine Dmgln N-(2-aminoedhyl)glycine Dmhis N-(3-aminopropyl)glycine Dmile N-amino-a-methylbutyrate Dmleu a-napthylalanine Dm-lys N-benzylglycine Dmmet N-(2-carbamylediyl)glycine Dmorn N-(carbamylmethyl)glycine Dmphe N-(2-carboxyethyl)glycine Dmpro N-(carboxymethyl)glycine Dmser N-cyclobutylglycine Dmthr N-cycloheptylglycine Dmtrp N-cyclohexylglycine Dmty N-cyclodecylglycine Dmval N-cylcododecylglycine Dnmala N-cyclooctylglycine Drnarg N-cyclopropylglycine Dminasn N-cycloundecylglycine Drnasp N-(2,2-diphenylethyl) glycine Dnmcys N-(3 ,3-diphenylpropyl) glycine Maib Mgabu Mchexa Mcpen Manap Mpen Nglu Naeg Norn Nmaabu Anap Nphe Ngln Nasn Nglu Nasp Ncbut Nchep Nchex Ncdec Ncdod Ncoct Ncpro Ncund Nbhm Nbhe WO 00/66745 WO 0066745PCT/AUOO/00385 D-N-methylglutamine D-N-methylglutamnate D-N-methylhistidine D-N-methylisoleucine D-N-methylleucine D-N-methyllysine N-methylcyclohexylalanine D-N-methylornithine N-methylglycine N-methylamninoisobutyrate 1-methylpropyl)glycine N-(2-methylpropyl)glycine D-N-methyltryptophan D-N-methyltyrosine D-N-methylvaline y-anxinobutyric acid L-t-butylglycine L-ethylglycine L-homnophenylalanine L-a-methylarginine L-a-methylaspartate L-a-methylcysteine L-a-methylglutamine L-ac-methylhistidine L-ca-methylisoleucine Drngln Dnglu Dnnihis Dnmile DnmlJeu Dnmlys Nmchexa Dnorn Nala Nmaib Nile Nleu Dntrp Dnmyr Dmnval Gabu Tbug Etg Hphe Marg Masp Mcys Mgln Mhis Mile N-(3-guanidinopropyl) glycine I-hydroxyethyl)glycine N-Qiydroxyethyl))glycine N-(imidazolylethyl)) glycine N-(3-indolylyethyl) glycine N-methyl-y-axninobutyrate D-N-methylmethionine N-methylcyclopentylalanine D-N-Methylphenylalanine D-N-methylproline D-N-methylserine D-N-methylthreonine 1-methylethyl)glycine N-methyla-napthylalanine N-methylpenicillamnine N-(p-hydroxyphenyl)glycine N-(ffiiomethyl)glycine penicillamnine L-a-methylalanine L-a-methylasparagine L-cc-methyl-t-butylglycine L-methylethylglycine L-a-methylglutamate L-cc-methylhomo phenylalanine N-(2-methylthioethyl) glycine L-a-methyllysine Narg Nthr Nser Nhis Nhtrp Nnigabu Dnmmnet Nmcpen Dninphe Dnmnpro Drnser Dnnnhr Nval Nmanap Nmpen Nhtyr Ncys Pen Mala Masn Mtbug Metg Mglu Mhphe Nmet Mlys L-ca-methylleucine Mleu WO 00/66745 PCT/AU00/00385 -7- L-e-methylmethionine L-a-methynorvaline L-a-methylphenylalanine L-a-methylserine L-c-methyltryptophan L-a-methylvaline N-(N-(2,2-diphenylethyl) carbamylmethyl)glycine 1-carboxy-l-(2,2-diphenylethylanino)cyclopropane Mmet Mnva Mphe Mser Mtrp Mval Nnbhm Nmbc L-a-methylnorleucine L-a-methylornithine L-a-methylproline L-a-methylthreonine L-a-methyltyrosine L-N-methylhomo phenylalanine N-(N-(3,3-diphenylpropyl) carbamylmethyl)glycine Mnle Morn Mpro Mthr Mtyr Nmhphe Nnbhe Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features.

The present invention is not to be limited in scope by the specific embodiments described herein, which are intended for the purposes of exemplification only.

Functionally-equivalent products, compositions and methods are clearly within the scope of the invention, as described herein.

BACKGROUND TO THE INVENTION The biosynthesis of the starch granule is a complex process which involves the action of an array of isoforms of enzymes involved in the starch biosynthesis. Following the formation of glucose-1-phosphate, the enzyme activities required for the synthesis of granular starch include ADP glucose pyrophosphorylase (EC 2.7.7.27), starch synthases (EC 2.4.1.21), branching enzymes (EC 2.4.1.18) and debranching enzymes WO 00/66745 PCT/AU00/00385 -8- (EC 3.2.1.41 and EC 3.2.1.68) (Mouille et al., 1996). Plants contain isozymes of each of these activities, and the definition of these isoforms and their roles has been conducted through investigation of the properties of the suite of soluble enzymes found in the stroma of the plastid, analysis of the proteins entrapped within the matrix of the starch granule, and mutational studies to identify genes and define linkages between individual genes and their specific roles.

Starch synthases extend regions of a-1,4 glucan through the transfer of the glucosyl moiety of ADPglucose to the non-reducing end of a pre-existing a-1,4 glucan. In addition to GBSS, 3 other classes of starch synthase have been identified in plants, SSI (wheat, Li et al., 1999 and GenBank Accession No. U48227; rice, Baba et al., 1993; potato, Genbank Accession No. STSTASYNT), SSII (pea, Dry et al. 1992; potato, Edwards et al., 1995; maize, Ham et al. 1998 and GenBank Accession No.

U66377) and SSIII (potato, Abel et 1996; maize, Gao et al., 1998). In the cereals, the most comprehensively studied species is maize, where in addition to GBSS, cDNAs encoding SSI, SSlla, and SSIIb have been isolated, and both cDNA and genomic clones for dull1 have been characterised (Knight et al., 1998; Harn et al., 1998; Gao et al., 1998). In maize, the product of the dul gene is known as maize SSII, however this gene is the homologue of potato SSIII.

The proteins within the matrix of the wheat starch granule have been extensively studied (Denyer et al., 1995; Rahman et al., 1995; Takaoka et al., 1997; Yamamori and Endo, 1996) and 60, 75, 85, 100, 104 and 105 kDa protein bands can be visualised following SDS-PAGE. The predominant 60 kDa protein is exclusively granule-bound and is analogous to the "waxy" granule bound starch synthase (GBSS) gene in maize (Rahman et al., 1995). The combination of three null alleles for this enzyme from each of the wheat genomes (Nakamura et al., 1995) results in the amylose-free "waxy" phenotype found in other species The 75 kDa starch synthase I (wSSI) is found in both the granule and the soluble fraction of wheat endosperm (Denyer et al., 1995; Li et al., 1999) and has been assigned to chromosomes 7A, 7B and 7D (Yamamori and Endo, 1996; Li et al., 1999). The 85 kDa band contains a WO 00/66745 PCT/AU00/00 3 8 -9class II branching enzyme and an unidentified polypeptide (Rahman et al., 1995). The 100, 104 and 105 kDa proteins of the wheat starch granule (designated Sgp-B 1 Sgp- D1 and Sgp-A1 by Yamamori and Endo, 1996) have been shown to be encoded by a homeologous set of genes on the short arm of chromosome 7B, 7A and 7D respectively (Yamamori and Endo, 1996; Takaoka et al., 1997). Denyer et al. (1995) concluded on the basis of enzyme activity assays that these proteins were also starch synthases. These genes are referred to hereinafter as the "wheat SSII genes".

While GBSS has been established to be essential for amylose synthesis, the remaining starch synthases are thought to be primarily responsible for the elongation of amylopectin chains, although this does not preclude them from also having nonessential roles in amylose biosynthesis. Differences in kinetic properties between isoforms, and the analysis of mutants lacking various isoforms, suggests that each isoenzyme contributes to the extension of specific subsets of the available nonreducing ends.

SUMMARY OF THE INVENTION The production of plants that produce improved starches that are modified for particular end-use applications, such as, for example, starches having high or low amylose:amylopectin ratios, requires the availability of genes encoding the various starch synthase isoforms. Because of species-specific codon usages, and variations in the kinetic parameters of the starch synthase isoforms between species, the production of modified starches may require the use of genes derived from particular species.

Furthermore, the screening-assisted breeding of plants having desirable starch content and/or composition requires specific gene sequences to be provided that can be used to distinguish between different homeologous genes encoding the various isoforms of wheat starch synthases, such as, for example, to identify and distinguish between naturally-occurring variant gene sequences. It is a particular object of the present invention to provide gene sequences to facilitate the screening-assisted selection of WO 00/66745 PCT/AU00/00385 wheat plants having starch traits which are associated with the presence and/or expression of one or more wheat SSI and/or SSIII genes.

Accordingly, the present invention provides isolated nucleotide sequences encoding the wheat SSII wSSII) and wheat SSIII wSSIII) isoenzymes, and DNA markers derived therefrom. The present invention further facilitates the production of transformed plants carrying these nucleotide sequences.

More particularly, the present invention provides isolated nucleic acid molecules encoding the 100, 104 and 105 kDa SSII (Sgp-1) polypeptides of the wheat starch granule matrix, as determined using the SDS/PAGE system of Rahman et al. (1995), which polypeptides are equivalent to the 100, 108 and 115 kDa polypeptides described by Yamamori and Endo (1996).

The present invention further provides isolated nucleic acid molecules encoding the soluble dul/1-type wheat starch synthase III polypeptide. Analysis of the polypeptides encoded by these nucleic acid molecules reveals several consensus amino acid sequence motifs that are highly conserved in wheat starch synthase isoenzymes, in addition to isoenzyme-specific sequences, which sequences possess utility in isolating related starch synthase-encoding sequences and in assaying plants for their expression of one or more starch synthase isoenzymes.

Accordingly, one aspect of the present invention provides an isolated nucleic acid molecule which comprises a sequence of nucleotides which encodes, or is complementary to a nucleic acid molecule which encodes a wheat starch synthase polypeptide, protein or enzyme molecule or a functional subunit thereof selected from the following: a wheat starch synthase II (wSSII) polypeptide, protein or enzyme or functional subunit thereof which comprises an amino acid sequence which is at least about 85% identical overall to an amino acid sequence set forth in any one of SEQ ID NOS: 2, 4, or 6; WO 00/66745 PCT/AU00/00385 11 (ii) a wheat starch synthase III (wSSIII) polypeptide, protein or enzyme or functional subunit thereof which comprises an amino acid sequence which is at least about 85% identical overall to an amino acid sequence set forth in any one of SEQ ID NOS: 8 or (iii) a wheat starch synthase polypeptide, protein or enzyme or functional subunit thereof which comprises a conserved amino acid sequence having at least 25% identity to an amino acid sequence selected from the group consisting of: KVGGLGDVVTS (SEQ ID NO: 39); GHTVEVILPKY (SEQ ID NO: HDWSSAPVAWLYKEHY (SEQ ID NO: 41); GILNGIDPDIWDPYTD (SEQ ID NO: 42); DVPIVGIITRLTAQKG (SEQ ID NO: 43); NGQWLLGSA (SEQ ID NO: 44); (g)AGSDFIIVPSIFEPCGLTQLVAMRYGS (SEQ ID NO: 45); and (h)TGGLVDTV (SEQ ID NO: 46); wherein said wheat starch synthase polypeptide further comprises an amino acid sequence having at least about 85% identity overall to an amino acid sequence set forth in any one of SEQ ID NOS: 2, 4, 6, 8 or 10; and (iv) a wheat starch synthase polypeptide, protein or enzyme or functional subunit thereof which comprises a conserved amino acid sequence having at least 25% identity to an amino acid sequence selected from the group consisting of: KTGGLGDVAGA (SEQ ID NO: 47); GHRVMVVVPRY (SEQ ID NO: 48); NDWHTALLPVYLKAYY (SEQ ID NO: 49); GIVNGIDNMEWNPEVD (SEQ ID NO: DVPLLGFIGRLDGQKG (SEQ ID NO: 51); DVQLVMLGTG (SEQ ID NO: 52); (g)AGADALLMPSRF(EN)PCGLNQLYAMAYGT (SEQ ID NO: 53); and (h)VGG(V/L)RDTV (SEQ ID NO: 54); WO 00/66745 PCT/AU00/00385 -12wherein said wheat starch synthase polypeptide further comprises an amino acid sequence having at least about 85% identity overall to an amino acid sequence set forth in any one of SEQ ID NOS: 2, 4, 6, 8 or In a preferred embodiment, the isolated nucleic acid molecule encodes a starch synthase polypeptide, protein or enzyme having at least about 90% amino acid sequence identity to any one of SEQ ID NOS: 2, 4, 6, 8 or 10, more preferably having at least about 95% or about 97% or about 99% identity to any one of said amino acid sequences.

In an alternative embodiment, the isolated nucleic acid molecule of the present invention encodes a wheat starch synthase polypeptide which comprises one or more amino acid sequences selected from the group consisting of:

GHTVEVILPKY;

HDWSSAPVAWLYKEHY;

DVPIVGIITRLTAQKG;

NGQWLLGSA;

(e)AGSDFIIVPSIFEPCGLTQLVAMRYGS; (f)TGGLVDTV; GIVNGIDNMEWNPEVD; and

AGADALLMPSRF(EN)PCGLNQLYAMAYGT.

in an alternative embodiment, the present invention provides an isolated nucleic acid molecule which encodes a wheat starch synthase polypeptide, protein or enzyme molecule or a functional subunit thereof, wherein said nucleic acid molecule comprises a nucleotide sequence having at least about 85% nucleotide sequence identity to any one of SEQ ID NOS: 1, 3, 5, 7, 9,11-16, 37 or 38 or a complementary nucleotide sequence thereto.

In a preferred embodiment, the isolated nucleic acid molecule comprises the nucleotide sequence set forth in any one of SEQ ID NOS: 1, 3, 5, 7, 9,11-16, 37 or 38, WO 00/66745 PCT/AU00/00385 13or is at least about 90% identical, more preferably at least about 95% or 97% or 99% identical to all or a protein-encoding part thereof.

In an alternative embodiment, the present invention provides an isolated nucleic acid molecule which encodes a wheat starch synthase polypeptide, protein or enzyme molecule or a functional subunit thereof, wherein said nucleic acid molecule comprises a nucleotide sequence that is capable of hybridising under at least moderate stringency hybridisation conditions to at least about 30 contiguous nucleotides derived from any one of SEQ ID NOS: 1, 3, 5, 7, 9,11-16, 37 or 38, or a complementary nucleotide sequence thereto.

A second aspect of the present invention provides a method of isolating a nucleic acid molecule that encodes a starch synthase polypeptide, protein or enzyme described supra, said method comprising: hybridising a probe or primer comprising at least about 15 contiguous nucleotides in length derived from any one of SEQ ID NOS: 1, 3, 5, 7, 9,11-16, 37 or 38, or a complementary nucleotide sequence thereto to single-stranded or double-stranded mRNA, cDNA or genomic DNA; and (ii) detecting the hybridised mRNA, cDNA or genomic DNA using a detecting means.

Preferably, the detecting means is a reporter molecule covalently attached to the probe or primer molecule or alternatively, a polymerase chain reaction format. Accordingly, the present invention clearly extends to the use of the nucleic acid molecules provided herein to isolate related starch synthase-encoding sequences using standard hybridisation and/or polymerase chain reaction techniques.

A third aspect of the invention provides an isolated probe or primer comprising at least about 15 contiguous nucleotides in length derived from any one of SEQ ID NOS: 1, 3, 5, 7, 9,11-16, 37 or 38, or a complementary nucleotide sequence thereto.

WO 00/66745 PCT/AU00/00385 -14- Preferably, the probe or primer comprises a nucleotide sequence set forth in any one of SEQ ID NOS: 25 to 34.

A fourth aspect of the present invention is directed to an isolated or recombinant starch synthase polypeptide, protein or enzyme, preferably substantially free of conspecific or non-specific proteins, which comprises an amino acid sequence selected from the following: a wheat starch synthase II (wSSII) polypeptide, protein or enzyme or functional subunit thereof which comprises an amino acid sequence which is at least about 85% identical overall to an amino acid sequence set forth in any one of SEQ ID NOS: 2, 4, or 6; (ii) a wheat starch synthase III (wSSIII) polypeptide, protein or enzyme or functional subunit thereof which comprises an amino acid sequence which is at least about 85% identical overall to an amino acid sequence set forth in any one of SEQ ID NOS: 8 or (iii) a wheat starch synthase polypeptide, protein or enzyme or functional subunit thereof which comprises a conserved amino acid sequence having at least 25% identity to an amino acid sequence selected from the group consisting of:

KVGGLGDVVTS;

GHTVEVILPKY;

HDWSSAPVAWLYKEHY;

GILNGIDPDIWDPYTD;

DVPIVGIITRLTAQKG;

NGQWLLGSA;

(g)AGSDFIIVPSIFEPCGLTQLVAMRYGS; and (h)TGGLVDTV wherein said wheat starch synthase polypeptide further comprises an amino acid sequence having at least about 85% identity overall to an amino acid sequence set forth in any one of SEQ ID NOS: 2, 4, 6, 8 or 10; and (iv) a wheat starch synthase polypeptide, protein or enzyme or functional WO 00/66745 PCT/AU00/00385 subunit thereof which comprises a conserved amino acid sequence having at least 25% identity to an amino acid sequence selected from the group consisting of:

KTGGLGDVAGA;

GHRVMVVVPRY;

NDWHTALLPVYLKAYY;

GIVNGIDNMEWNPEVD;

DVPLLGFIGRLDGQKG;

DVQLVMLGTG;

(g)AGADALLMPSRF(EN)PCGLNQLYAMAYGT; and (h)VGG(V/L)RDTV wherein said wheat starch synthase polypeptide further comprises an amino acid sequence having at least about 85% identity overall to an amino acid sequence set forth in any one of SEQ ID NOS: 2, 4, 6, 8 or The present invention clearly encompasses the mature protein region of a wheat starch synthase polypeptide which is obtained by removal of the N-terminal transit peptide sequence.

A further aspect of the invention provides a method of assaying for the presence or absence of a starch synthase isoenzyme or the copy number of a gene encoding same in a plant, comprising contacting a biological sample derived from said plant with an isolated nucleic acid molecule derived from any one of SEQ ID NOS 1, 3, 5, 7, 9,11- 16, 37 or 38, or any one of SEQ ID NOS: 25 to 34, or a complementary nucleotide sequence thereto for a time and under conditions sufficient for hybridisation to occur and then detecting said hybridisation using a detection means.

The detection means according to this aspect of the invention is any nucleic acid based hybridisation or amplification reaction.

A further aspect of the present invention utilises the above-mentioned assay method WO 00/66745 PCT/AU00/00385 -16in the breeding and/or selection of plants which express or do not express particular starch synthase isoenzymes or alternatively, which express a particular starch synthase isoenzyme at a particular level in one or more plant tissues. This aspect clearly extends to the selection of transformed plant material which contains one or more of the isolated nucleic acid molecules of the present invention.

A further aspect of the present invention provides a method of modifying the starch content and/or starch composition of one or more tissues or organs of a plant, comprising expressing therein a sense molecule, antisense molecule, ribozyme molecule, co-suppression molecule, or gene-targeting molecule having at least about nucleotide sequence identity to any one of any one of SEQ ID NOS: 1, 3, 5, 7, 9,11-16, 37 or 38, or a complementary nucleotide sequence thereto for a time and under conditions sufficient for the enzyme activity of one or more starch synthase isoenzymes to be modified. This aspect of the invention clearly extends to the introduction of the sense molecule, antisense molecule, ribozyme molecule, cosuppression molecule, or gene-targeting molecule to isolated plant cells, tissues or organs or organelles by cell fusion or transgenic means and the regeneration of intact plants therefrom.

A further aspect of the present invention provides an isolated promoter that is operable in the endosperm of a monocotyledonous plant cell, tissue or organ, and preferably in the endosperm of a monocotyledonous plant cell, tissue or organ. For example, the HMG promoter from wheat, or the maize zein gene promoter are particularly preferred, as is the promoter derived from a starch synthase gene of the present invention, such as a promoter that is linked in vivo to any one of SEQ ID NOS 1, 3, 5, 7, 9,11-16, 37 or 38, or a complementary nucleotide sequence thereto.

A still further aspect of the present invention contemplates a transgenic plant comprising an introduced sense molecule, antisense molecule, ribozyme molecule, cosuppression molecule, or gene-targeting molecule having at least about nucleotide sequence identity to any one of any one of SEQ ID NOS: 1,3, 5, 7, 9,11-16, WO 00/66745 PCT/AU00/00385 -17- 37 or 38, or a complementary nucleotide sequence thereto or a genetic construct comprising same, and to plant propagules, cells, tissues, organs or plant parts derived from said transgenic plant that also carry the introduced molecule(s).

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a copy of a photographic representation showing the distribution of wheat endosperm starch synthases between the starch granule and soluble fractions. Lane 1, SDS-PAGE of wheat endosperm starch granule proteins revealed by silver staining; lanes 2-7, immunoblot of wheat endosperm soluble phase and starch granule proteins separated by SDS-PAGE from various developmental stages and probed with an anti- (wheat wSSII peptide) monoclonal antibody. Lanes 2-4 contain proteins from the soluble fraction of wheat endosperm at 15 days post anthesis (Lane 20 days post anthesis (Lane and at 25 days post anthesis (Lane Lanes 5-7 contain proteins from the starch granule of wheat endosperm at 15 days post anthesis (Lane days post anthesis (Lane and at 25 days post anthesis (Lane 7).

Figure 2 is a copy of a schematic representation comparing the nucleotide sequences of cDNA clones designated wSSIIA, wSSIIB and wSSIID, encoding the starch synthase II polypeptides from wheat, using the PILEUP programme of Devereaux et al. (1984).

Figure 3 is a copy of a schematic representation comparing the deduced amino acid sequences of starch synthase II from wheat (wSSIIA, wSSIIB and wSSIID), maize (maize SSIIa and maize SSIIb; Ham et al., 1998), pea (pea SSII; Dry et al., 1992) and potato (potato SSII; van der Leij et al., 1991). Identical amino acid residues among each of these sequences are indicated below the sequences with The alignments of maize SSIIla with maize SSIIb, and pea SSII and potato SSII are essentially as described in Ham et al. (1998) and Edwards et al. (1995). All sequences are aligned to position the transit peptide cleavage site below the arrow between residues 59 and 60 of the wSSIIA sequence. The wSSllpl sequence, the sequence of SGP-B1 (peptide3), and of eight conserved regions are annotated and underlined.

WO 00/66745 PCT/AU00/00385 -18- Figure 4 is a copy of a photographic representation of a northern blot showing the expression of wheat wSSII mRNA in wheat plants. Total RNAs were isolated from leaves pre-anthesis florets and endosperm of the wheat cultivar "Gabo", grown under 0 a photoperiod comprising 16 hours daylength, and at 18 C during the day, and at 0 13 C during the night cycle, and probed with the wSSIIp2 DNA fragment. The source of each RNA is indicated at the top of the Figure as follows: Lane 1, leaf; Lane 2, preanthesis florets; Lanes 3-11, endosperm at: 4 days post-anthesis (Lane 6 days post-anthesis (Lane 8 days post-anthesis (Lane 10 days post-anthesis (Lane 6);12 days post-anthesis (Lane 15 days post-anthesis (Lane 18 days postanthesis (Lane 21 days post-anthesis (Lane 10); and 25 days post-anthesis (Lane 11).

Figure 5 is a copy of a photographic representation showing the localization of wheat starch synthase II genes on the wheat genome by PCR, using the primers ssllc, sslld and sslle in the amplification reaction. The nullisomic-tetrasomic genomic DNA of wheat cv. Chinese Spring was used as template DNA. Lane D, Triticum tauschii; Lane AB, Accession line N7DT7B having no 7D chromosome and four copies of the 7B chromosome; Lane AD, Accession line N7BT7A having no 7B chromosome and four copies of the 7A chromosome; Lane BD, Accession line N7AT7B having no 7A chromosome and four copies of the 7B chromosome; Lane ABD, wheat cv. Chinese Spring. PCR products derived from each cDNA clone are labelled. The results indicate that the cDNA clones, wSSIIB, wSSIIA and wSSIID are derived from the A- and Dgenomes of wheat, respectively.

Figure 6 is a schematic representation showing the organisation of introns (lines) and exons (boxes) in the wheat SSII gene shown in SEQ ID NO: 37. The scale (bases), relative to the nucleotide sequence set forth in SEQ ID NO: 37, is provided at the bottom of the figure.

Figure 7 is a schematic representation comparing the deduced amino acid Sequences of the maize, potato and wheat SSIII polypeptides.

WO 00/66745 PCT/AU00/00385 -19- Figure 8 is a copy of a photographic representation showing the expression of wheat wSSIII mRNA in wheat. Total RNAs were isolated from the endosperm of the wheat cultivars Wyuna (Panel a) and Gabo (Panel b) leaves pre-anthesis florets and endosperm of the wheat cultivar "Gabo", grown under a photoperiod comprising 16 0 0 hours daylength, and at 18 C during the day cycle, and at 13 C during the night cycle, and probed with the wSSIIIpl DNA fragment derived from wSSIII.B3 cDNA. The source of each RNA is indicated at the top of the Figure as follows: Lane 1, endosperm at: 4 days post-anthesis; Lane 2, endosperm at 6 days post-anthesis; Lane 4, endosperm at 8 days post-anthesis; Lane 4, endosperm at 10 days post-anthesis; Lane 5, endosperm at 12 days post-anthesis; Lane 6, endosperm at 15 days postanthesis; Lane 7, endosperm at 18 days post-anthesis; Lane 8, endosperm at 21 days post-anthesis; Lane 9, endosperm at 25 days post-anthesis; and Lane 10, endosperm at 31 days post-anthesis (Panel a only). In panel L refers to leaf RNA, and P refers to RNA from pre-anthesis florets derived from the cultivar Gabo.

Figure 9 is a schematic representation showing the position of conserved amino acid sequences within four wheat starch synthase proteins. The eight highly-conserved regions between the wheat starch synthase polypeptides are underlined and annotated at the top of each group of amino acid sequences. The sequences included in the alignment are the wheat SSII-A1 and wheat SSIII polypeptides of the present invention; wheat GBSS (wGBSS; Yan et al., 1999); wheat SSI (wSS1; Li et al., 1999); wheat SSII (wSS2; SEQ ID NO: and wheat SSIII (wSS3; SEQ ID NO: 8).

Figure 10 is a schematic representation showing the relationships between the primary amino acid sequences of starch synthases (SS) and glycogen synthase of E.

coli The dendrogram was generated by the program PILEUP (Devereaux et al., 1984). The amino acid sequences used for the analysis are those of the wheat SSIIA, wheat SSIIB, wheat SSIID, and wheat SSIII polypeptides of the present invention compared to the deduced amino acid sequences of wheat GBSS (Clark et al., 1991), wheat SSI (Li et al., 1999), rice GBSS (Okagaki, 1992), rice SSI (Baba et al., 1993), maize GBSS (Kloesgen et al., 1986), maize SSI (Knight et al., 1998), maize SSlla and WO 00/66745 PCT/AU00/00385 maize SSIIb (Ham et al., 1998), maize SSIII (Gao et al., 1998), pea GBSS (Dry et al., 1992), pea SSII (Dry et al., 1992), potato GBSS (van der Leij et al., 1991), potato SSI (Genbank accession number: STSTASYNT), potato SSII (Edwards etal., 1995), potato SSIII (Abel et al., 1996), and E. coli glycogen synthase (GS) (Kumar et al., 1986). Five groups of enzymes included in the alignment are granule-bound starch synthase (GBSS), starch synthase-I (SSI), starch synthase-lI (SSII), starch synthase-llI (SSIII) and glycogen synthase (GS).

Figure 11 is a schematic representation showing the position of conserved regions within cereal starch synthase genes. Comparisons of cereal starch synthases were made based on their deduced amino acid sequences and 8 conserved regions identified. Conserved regions are shown in bold and transit peptides (where defined) in grey. The sequences included in the alignment are the wheat SSII-A1 and wheat SSIII polypeptides of the present invention; wheat GBSS (Ainsworth et al., 1993); wheat SSI (Li et 1999); maize SSlla (Harn et al., 1998); and maize dull-1(Gao et al., 1998).

Figure 12 is a copy of a schematic representation of a gene map showing the alignment of fragments 1 to 6 of the genomic SSIII gene (lower line) with the corresponding SSIII cDNA clone (upper line). Raised regions in the genomic clone fragments (lower line) represent protein-encoding regions of the gene.

Figure 13 is a schematic representation showing the organisation of introns (lines) and exons (boxes) in the wheat SSIII gene shown in SEQ ID NO: 38. The scale (bases), relative to the nucleotide sequence set forth in SEQ ID NO: 38, is provided at the bottom of the figure.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS One aspect of the present invention provides an isolated nucleic acid molecule which comprises a sequence of nucleotides which encodes, or is complementary to a nucleic WO 00/66745 PCT/AU00/00385 -21acid molecule which encodes a wheat starch synthase polypeptide, protein or enzyme molecule or a functional subunit thereof selected from the following: a wheat starch synthase II (wSSII) polypeptide, protein or enzyme or functional subunit thereof which comprises an amino acid sequence set forth in any one of SEQ ID NOS: 2,4, or 6; and (ii) a wheat starch synthase III (wSSIII) polypeptide, protein or enzyme or functional subunit thereof which comprises an amino acid sequence set forth in any one of SEQ ID NOS: 8 or Alternatively or in addition, the isolated nucleic acid molecule of the present invention encodes a wheat starch synthase II (wSSII) polypeptide, protein or enzyme or functional subunit thereof and comprises a nucleotide sequence set forth in any one of SEQ ID NOS: 1, 3, 5, or 37.

Altematively or in addition, the isolated nucleic acid molecule of the present invention encodes a wheat starch synthase III (wSSIII) polypeptide, protein or enzyme or functional subunit thereof and comprises a nucleotide sequence set forth in any one of SEQ ID NOS: 7, 9, or 38.

As used herein, the term "starch synthase" shall be taken to refer to any enzymaticallyactive peptide, polypeptide, oligopeptide, polypeptide, protein or enzyme molecule that is at least capable of transferring a glucosyl moiety from ADP-glucose to an a-1,4glucan molecule, or a peptide, polypeptide, oligopeptide or polypeptide fragment of such an enzymatically-active molecule.

The term "wheat starch synthase" refers to a starch synthase derived from hexaploid wheat or barley or a progenitor species, or a relative thereto such as the diploid Triticum tauschii or other diploid, tetraploid, aneuploid, polyploid, nullisomic, or a wheat/barley addition line, amongst others, the only requirement that the genomic DNA is at least about 80% identical to the genome of a wheat plant as determined by standard DNA melting curve analyses.

WO 00/66745 PCT/AU00/00385 -22- The term "starch synthase II" or "wSSII" or similar term shall be taken to refer to a starch synthase as hereinbefore defined that is detectable in the starch granule of a plant seed endosperm and possesses one or more properties selected from the group consisting of: it is immunologically cross-reactive with the wheat starch granule proteins designated Sgp-B1 and/or Sgp-D1 and/or Sgp-A1, having estimated molecular weights of about 85 kDa to about 115 kDa; (ii) it is encoded by one of a homeologous set of genes localised on wheat chromosomes 7B or 7A or 7D; (iii) it is encoded by a nudeotide sequence that comprises at least about nucleotides in length derived from any one or more of SEQ ID NOS: 1, 3, 5, or 37 or a complementary nucleotide sequence thereto; (iv) it is encoded by a nucleotide sequence that is at least about identical to one or more of the nucleotide sequences set forth in SEQ ID NOS: 1, 3, 5, or 37, or a complementary nucleotide sequence thereto; it comprises an amino acid sequence having at least about 85% identity to one or more of SEQ ID NOS: 2 or 4 or 6; (vi) it comprises at least about 5 contiguous amino acids, preferably at least about 10 contiguous amino acids, more preferably at least about 15 contiguous amino acids, even more preferably at least about 20 contiguous amino acids and still even more preferably at least about 25-50 contiguous amino acids of the amino acid sequences set forth in SEQ ID NOS: 2 or 4 or 6; (vii) it which comprises a conserved amino acid sequence having at least identity to an amino acid sequence selected from the group consisting of:

KVGGLGDVVTS;

GHTVEVILPKY;

HDWSSAPVAWLYKEHY;

GILNGIDPDIWDPYTD;

DVPIVGIITRLTAQKG;

NGQWLLGSA;

(g)AGSDFIIVPSIFEPCGLTQLVAMRYGS; and WO 00/66745 PCT/AU00/00385 -23- (h)TGGLVDTV, in addition to any one or more of to and (viii) it which comprises a conserved amino acid sequence having at least identity to an amino acid sequence selected from the group consisting of:

KTGGLGDVAGA;

GHRVMVVVPRY;

NDWHTALLPVYLKAYY;

GIVNGIDNMEWNPEVD;

DVPLLGFIGRLDGQKG;

DVQLVMLGTG;

(g)AGADALLMPSRF(EN)PCGLNQLYAMAYGT; and (h)VGG(V/L)RDTV, in addition to any one or more of to (vi).

The term "starch synthase III" or "wSSIII" or similar term shall be taken to refer to a starch synthase as hereinbefore defined that possesses one or more properties selected from the group consisting of: it is encoded by a nucleotide sequence that comprises at least about nucleotides in length derived from any one or more of SEQ ID NOS: 7, 9, 11- 16, or 38, or a complementary nucleotide sequence thereto; (ii) it is encoded by a nucleotide sequence that is at least about identical to one or more of the nucleotide sequences set forth in SEQ ID NOS: 7, 9, 11-16, or 38, or a complementary nucleotide sequence thereto; and (iii) it comprises an amino acid sequence having at least about 85% identity to one or more of SEQ ID NOS: 8 or (iv) it comprises at least about 5 contiguous amino acids, preferably at least about 10 contiguous amino acids, more preferably at least about 15 contiguous amino acids, even more preferably at least about 20 contiguous amino acids and still even more preferably at least about 25-50 contiguous amino acids of the amino acid sequences set forth in SEQ ID NOS: 8 or which comprises a conserved amino acid sequence having at least WO 00/66745 PCT/AU00/00385 -24identity to an amino acid sequence selected from the group consisting of:

KVGGLGDWTS;

GHTVEVILPKY;

HDWSSAPVAWLYKEHY;

GILNGIDPDIWDPYTD;

DVPIVGIITRLTAQKG;

NGQWLLGSA;

(g)AGSDFIIVPSIFEPCGLTQLVAMRYGS; and (h)TGGLVDTV in addition to any one or more of to and (vi) it which comprises a conserved amino acid sequence having at least identity to an amino acid sequence selected from the group consisting of:

KTGGLGDVAGA;

GHRVMVVVPRY;

NDWHTALLPVYLKAYY;

GIVNGIDNMEWNPEVD;

DVPLLGFIGRLDGQKG;

DVQLVMLGTG;

(g)AGADALLMPSRF(EN)PCGLNQLYAMAYGT; and (h)VGG(V/L)RDTV, in addition to any one or more of to (iv).

In a more preferred embodiment, the WSSII or WSSIII polypeptide encoded by the nucleic acid molecule of the present invention will comprise a substantial contiguous region of any one of SEQ ID NOS: 2, 4, 6, 8 or 10 or 17 sufficient to possess the biological activity of a starch synthase polypeptide.

For the purposes of nomenclature, the nucleotide sequence set forth in SEQ ID NO: 1 relates to the cDNA molecule encoding the WSSII Sgp-B1) polypeptide of wheat. The amino acid sequence of the corresponding polypeptide is set forth herein as SEQ ID NO:2. The nucleotide sequence set forth in SEQ ID NO: 3 relates to the WO 00/66745 PCT/AU00/00385 cDNA molecule encoding the WSSII Sgp-A1) polypeptide of wheat. The amino acid sequence of the corresponding polypeptide is set forth herein as SEQ ID NO:4.

The nucleotide sequence set forth in SEQ ID NO: 5 relates to the cDNA molecule encoding the WSSII Sgp-D1) polypeptide of wheat. The amino acid sequence of the corresponding polypeptide is set forth herein as SEQ ID NO:6. The nucleotide sequences set forth in SEQ ID NOs: 7 and 9 relate, respectively, to full-length and partial cDNA molecules encoding the WSSIII polypeptide of wheat. The amino acid sequences of the corresponding polypeptides are set forth herein as SEQ ID NOS: 8 and 10, respectively. The nucleotide sequences set forth in SEQ ID NOs: 11 to 16 relates to fragments of the genomic gene encoding the WSSIII polypeptide of wheat, significant protein-encoding regions of which are described by reference to Table 4 and Figure 11. The nucleotide sequence set forth in SEQ ID NO: 37 relates to the WSSII genomic gene of Triticum tauschii, corresponding to the WSSII gene of the Dgenome of wheat, which encodes the WSSIII polypeptide. The nucleotide sequence set forth in SEQ ID NO: 38 relates to the wheat WSSIII genomic gene.

Preferably, the isolated nucleic acid molecule of the present invention comprises a sequence of nucleotides which encodes, or is complementary to a nucleic acid molecule which encodes a wheat starch synthase polypeptide, protein or enzyme molecule or a functional subunit thereof which comprises an amino acid sequence which is at least about 85% identical overall to an amino acid sequence set forth in any one of SEQ ID NOS: 2, 4, 6, 8, or 10 and more preferably, which additionally comprises which comprises one or more amino acid sequences selected from the group consisting of:

KVGGLGDVVTS;

GHTVEVILPKY;

HDWSSAPVAWLYKEHY;

GILNGIDPDIWDPYTD;

DVPIVGIITRLTAQKG;

NGQWLLGSA;

(g)AGSDFIIVPSIFEPCGLTQLVAMRYGS; WO 00/66745 PCT/AU00/00385 -26- (h)TGGLVDTV;

KTGGLGDVAGA;

GHRVMVVVPRY;

NDWHTALLPVYLKAYY;

GIVNGIDNMEWNPEVD;

DVPLLGFIGRLDGQKG;

DVQLVMLGTG;

(o)AGADALLMPSRF(EN)PCGLNQLYAMAYGT; and (p)VGG(V/L)RDTV.

The present invention clearly extends to homologues, analogues and derivatives of the wheat starch synthase II and III genes exemplified by the nucleotide sequences set forth herein as SEQ ID NOs: 1, 3, 5, 7, 9,11-16, 37 or 38.

Preferred starch synthase genes may be derived from a naturally-occurring starch synthase gene by standard recombinant techniques. Generally, a starch synthase gene may be subjected to mutagenesis to produce single or multiple nucleotide substitutions, deletions and/or additions. Nucleotide insertional derivatives of the starch synthase gene of the present invention include 5' and 3' terminal fusions as well as intra-sequence insertions of single or multiple nucleotides. Insertional nucleotide sequence variants are those in which one or more nucleotides are introduced into a predetermined site in the nucleotide sequence although random insertion is also possible with suitable screening of the resulting product. Deletional variants are characterised by the removal of one or more nucleotides from the sequence. Substitutional nucleotide variants are those in which at least one nucleotide in the sequence has been removed and a different nucleotide inserted in its place.

Such a substitution may be "silent" in that the substitution does not change the amino acid defined by the codon. Altematively, substituents are designed to alter one amino acid for another similar acting amino acid, or amino acid of like charge, polarity, or hydrophobicity.

For the present purpose, "homologues" of a nucleotide sequence shall be taken to WO 00/66745 PCT/AU00/00385 -27refer to an isolated nucleic acid molecule which is substantially the same as the nucleic acid molecule of the present invention or its complementary nucleotide sequence, notwithstanding the occurrence within said sequence, of one or more nucleotide substitutions, insertions, deletions, or rearrangements.

"Analogues" of a nucleotide sequence set forth herein shall be taken to refer to an isolated nucleic acid molecule which is substantially the same as a nucleic acid molecule of the present invention or its complementary nucleotide sequence, notwithstanding the occurrence of any non-nucleotide constituents not normally present in said isolated nucleic acid molecule, for example carbohydrates, radiochemicals including radionucleotides, reporter molecules such as, but not limited to DIG, alkaline phosphatase or horseradish peroxidase, amongst others.

"Derivatives" of a nucleotide sequence set forth herein shall be taken to refer to any isolated nucleic acid molecule which contains significant sequence similarity to said sequence or a part thereof. Generally, the nucleotide sequence of the present invention may be subjected to mutagenesis to produce single or multiple nucleotide substitutions, deletions and/or insertions. Nucleotide insertional derivatives of the nucleotide sequence of the present invention include 5' and 3' terminal fusions as well as intra-sequence insertions of single or multiple nucleotides or nucleotide analogues.

Insertional nucleotide sequence variants are those in which one or more nucleotides or nucleotide analogues are introduced into a predetermined site in the nucleotide sequence of said sequence, although random insertion is also possible with suitable screening of the resulting product being performed. Deletional variants are characterised by the removal of one or more nucleotides from the nucleotide sequence. Substitutional nucleotide variants are those in which at least one nucleotide in the sequence has been removed and a different nucleotide or nucleotide analogue inserted in its place.

The present invention extends to the isolated nucleic acid molecule when integrated into the genome of a cell as an addition to the endogenous cellular complement of starch synthase genes, irrespective of whether or not the introduced nucleotide WO 00/66745 PCT/AU00/00385 -28sequence is translatable or non-translatable to produce a polypeptide. The present invention clearly contemplates the introduction of additional copies of starch synthase genes into plants, particularly wheat plants, in the antisense orientation to reduce the expression of particular wheat starch synthase genes. As will be known to those skilled in the art, such antisense genes are non-translatable, notwithstanding that they can be expressed to produce antisense mRNA molecules.

The said integrated nucleic acid molecule may, or may not, contain promoter sequences to regulate expression of the subject genetic sequence.

Accordingly, the present invention clearly encompasses preferred homologues, analogues and derivatives that comprise a sequence of nucleotides which encodes, or is complementary to a nucleic acid molecule which encodes a wheat starch synthase polypeptide, protein or enzyme molecule or a functional subunit thereof selected from the following: a wheat starch synthase II (wSSII) polypeptide, protein or enzyme or functional subunit thereof which comprises an amino acid sequence which is at least about 85% identical overall to an amino acid sequence set forth in any one of SEQ ID NOS: 2, 4, or 6; (ii) a wheat starch synthase III (wSSIII) polypeptide, protein or enzyme or functional subunit thereof which comprises an amino acid sequence which is at least about 85% identical overall to an amino acid sequence set forth in any one of SEQ ID NOS: 8 or (iii) a wheat starch synthase polypeptide, protein or enzyme or functional subunit thereof which comprises a conserved amino acid sequence having at least 25% identity to an amino acid sequence selected from the group consisting of:

KVGGLGDVVTS;

GHTVEVILPKY;

HDWSSAPVAWLYKEHY;

GILNGIDPDIWDPYTD;

WO 00/66745 PCT/AU00/00385 -29-

DVPIVGIITRLTAQKG;

NGQWLLGSA;

(g)AGSDFIIVPSIFEPCGLTQLVAMRYGS; and (h)TGGLVDTV and wherein said wheat starch synthase polypeptide further comprises an amino acid sequence having at least about 85% identity overall to an amino acid sequence set forth in any one of SEQ ID NOS: 2, 4, 6, 8 or 10; and (iv) a wheat starch synthase polypeptide, protein or enzyme or functional subunit thereof which comprises a conserved amino acid sequence having at least 25% identity to an amino acid sequence selected from the group consisting of:

KTGGLGDVAGA;

GHRVMVVVPRY;

NDWHTALLPVYLKAYY;

GIVNGIDNMEWNPEVD;

DVPLLGFIGRLDGQKG;

DVQLVMLGTG;

(g)AGADALLMPSRF(E/V)PCGLNQLYAMAYGT; and (h)VGG(V/L)RDTV, and wherein said wheat starch synthase polypeptide further comprises an amino acid sequence having at least about 85% identity overall to an amino acid sequence set forth in any one of SEQ ID NOS: 2, 4, 6, 8 or Preferably, the isolated nucleic acid molecule encodes a starch synthase polypeptide, protein or enzyme that comprises two, more preferably three, more preferably four, more preferably five, more preferably six, more preferably seven and even more preferably eight of the conserved amino acid motifs listed supra. Even more preferably, the said amino acid motifs are located in a relative configuration such as that shown for the wheat SSII or wheat SSIII polypeptides described herein.

In a preferred embodiment, the isolated nucleic acid molecule encodes a starch WO 00/66745 PCT/AU00/00385 synthase polypeptide, protein or enzyme having at least about 90% amino acid sequence identity to any one of SEQ ID NOS: 2, 4, 6, 8 or 10, more preferably having at least about 95% or about 97% or about 99% identity to any one of said amino acid sequences.

In an alternative embodiment, the present invention provides an isolated nucleic acid molecule which encodes a wheat starch synthase polypeptide, protein or enzyme molecule or a functional subunit thereof, wherein said nucleic acid molecule comprises a nucleotide sequence having at least about 85% nucleotide sequence identity to any one of SEQ ID NOS: 1, 3, 5, 7, 9,11-16, 37, or 38, or a degenerate nucleotide sequence thereto or a complementary nucleotide sequence thereto.

By "degenerate nucleotide sequence" is meant a nucleotide sequence that encodes a substantially identical amino acid sequence as a stated nucleotide sequence.

In a preferred embodiment, the isolated nucleic acid molecule comprises the nucleotide sequence set forth in any one of SEQ ID NOS: 1, 3, 5, 7, 9,11-16, 37, or 38, or is at least about 90% identical, more preferably at least about 95% or 97% or 99% identical to all or a protein-encoding part thereof.

In an alternative embodiment, preferred homologues, analogues and derivatives of the nucleic acid molecule of the present invention encodes a wheat starch synthase polypeptide, protein or enzyme molecule or a functional subunit thereof and comprises a -nucleotide sequence that is capable of hybridising under at least moderate stringency hybridisation conditions to at least about 30 contiguous nucleotides derived from any one of SEQ ID NOS: 1, 3, 5, 7, 9,11-16, 37, or 38, or a complementary nucleotide sequence thereto.

For the purposes of defining the level of stringency, a low stringency is defined herein as being a hybridisation and/or a wash carried out in 6xSSC buffer, 0.1% SDS at 28°C. Generally, the stringency is increased by reducing the concentration of SSC WO 00/66745 PCT/AU00/00385 -31 buffer, and/or increasing the concentration of SDS and/or increasing the temperature of the hybridisation and/or wash. A moderate stringency comprises a hybridisation and/or a wash carried out in 0.2 x SSC-2 x SSC buffer, 0.1% SDS at 42 0 C to 0 C, while a high stringency comprises a hybridisation and/or a wash carried out in 0.1xSSC-0.2 x SSC buffer, 0.1% SDS at a temperature of at least 55 0

C.

Conditions for hybridisations and washes are well understood by one normally skilled in the art. For the purposes of further clarification only, reference to the parameters affecting hybridisation between nucleic acid molecules is found in pages 2.10.8 to 2.10.16. of Ausubel et al. (1987), which is herein incorporated by reference.

Those skilled in the art will be aware of procedures for the isolation of further wheat starch synthase genes to those specifically described herein or homologues, analogues or derivatives of said genes, for example further cDNA sequences and genomic gene equivalents, when provided with one or more of the nucleotide sequences set forth in SEQ ID NOs: 1, 3, 5, 7, 9,11-16, 37, or 38. In particular, amplifications and/or hybridisations may be performed using one or more nucleic acid primers or hybridisation probes comprising at least 10 contiguous nucleotides and preferably at least about 20 contiguous nucleotides or 50 contiguous nucleotides derived from the nucleotide sequences set forth herein, to isolate cDNA clones, mRNA molecules, genomic clones from a genomic library (in particular genomic clones containing the entire 5' upstream region of the gene including the promoter sequence, and the entire coding region and 3'-untranslated sequences), and/or synthetic oligonucleotide molecules, amongst others. The present invention clearly extends to such related sequences.

Accordingly, a second aspect of the present invention provides a method of isolating a nucleic acid molecule that encodes a starch synthase polypeptide, protein or enzyme said method comprising: hybridising a probe or primer comprising at least about 15 contiguous nucleotides in length derived from any one of SEQ ID NOS 1, 3, 5, 7, 9,11-16, 37, or 38, or a complementary nucleotide sequence thereto to single-stranded or double-stranded mRNA, cDNA or genomic DNA; and WO 00/66745 PCT/AU00/00385 -32- (ii) detecting the hybridised mRNA, cDNA or genomic DNA using a detecting means.

Preferably, the detecting means is a reporter molecule covalently attached to the probe or primer molecule or alternatively, a polymerase chain reaction format.

An alternative method contemplated in the present invention involves hybridising two nucleic acid "primer molecules" to a nucleic acid "template molecule" which comprises a related starch synthase gene or related starch synthase genetic sequence or a functional part thereof, wherein the first of said primers comprises contiguous nucleotides derived from any one or more of SEQ ID NOS: 1, 3, 5, 7, 9,11-16, 37, or 38, and the second of said primers comprises contiguous nucleotides complementary to any one or more of SEQ ID NOS: 1, 3, 5, 7, 9,11-16, 37, or 38. Specific nucleic acid molecule copies of the template molecule are amplified enzymatically in a polymerase chain reaction, a technique that is well known to one skilled in the art.

In a preferred embodiment, each nucleic acid primer molecule is at least nucleotides in length, more preferably at least 20 nucleotides in length, even more preferably at least 30 nucleotides in length, still more preferably at least 40 nucleotides in length and even still more preferably at least 50 nucleotides in length.

Furthermore, the nucleic acid primer molecules consists of a combination of any of the nucleotides adenine, cytidine, guanine, thymidine, or inosine, or functional analogues or derivatives thereof which are at least capable of being incorporated into a polynucleotide molecule without having an inhibitory effect on the hybridisation of said primer to the template molecule in the environment in which it is used.

Furthermore, one or both of the nucleic acid primer molecules may be contained in an aqueous mixture of other nucleic acid primer molecules, for example a mixture of degenerate primer sequences which vary from each other by one or more nucleotide substitutions or deletions. Alternatively, one or both of the nucleic acid primer molecules may be in a substantially pure form.

WO 00/66745 PCT/AU00/00385 -33- The nucleic acid template molecule may be in a recombinant form, in a virus particle, bacteriophage particle, yeast cell, animal cell, or a plant cell. Preferably, the nucleic acid template molecule is derived from a plant cell, tissue or organ, in particular a cell, tissue or organ derived from a wheat or barley plant or a progenitor species, or a relative thereto such as the diploid Triticum tauschii or other diploid, tetraploid, aneuploid, polyploid, nullisomic, or a wheat/barley addition line, amongst others.

Those skilled in the art will be aware that there are many known variations of the basic polymerase chain reaction procedure, which may be employed to isolate a related starch synthase gene or related starch synthase genetic sequence when provided with the nucleotide sequences set forth herein. Such variations are discussed, for example, in McPherson et al (1991). The present invention extends to the use of all such variations in the isolation of related starch synthase genes or related starch synthase genetic sequences using the nucleotide sequences embodied by the present invention.

As exemplified herein, the present inventors have isolated several wheat starch synthase genes using both hybridisation and polymerase chain reaction approaches, employing novel probes and primer sequences to do so.

Accordingly, a third aspect of the invention provides an isolated probe or primer comprising at least about 15 contiguous nucleotides in length derived from any one of SEQ ID NOS: 1, 3, 5, 7, 9,11-16, 37, or 38, or a complementary nucleotide sequence thereto.

Preferably, the probe or primer comprises a nucleotide sequence set forth in any one of SEQ ID NOS: 25 to 34.

The isolated nucleic acid molecule of the present invention may be introduced into and expressed in any cell, for example a plant cell, fungal cell, insect cell. animal cell, yeast cell or bacterial cell. Those skilled in the art will be aware of any modifications which are required to the codon usage or promoter sequences or other regulatory WO 00/66745 PCT/AU00/00385 -34sequences, in order for expression to occur in such cells.

A further aspect of the invention provides a method of assaying for the presence or absence of a starch synthase isoenzyme or the copy number of a gene encoding same in a plant, comprising contacting a biological sample derived from said plant with an isolated nucleic acid molecule derived from any one of SEQ ID NOS 1, 3, 5, 7, 9,11- 16, 37, or 38, or any one of SEQ ID NOS: 25 to 34, or a complementary nucleotide sequence thereto for a time and under conditions sufficient for hybridisation to occur and then detecting said hybridisation using a detection means.

The detection means according to this aspect of the invention is any nucleic acid based hybridisation or amplification reaction.

The hexaploid nature of wheat prevents the straightforward identification of starch synthase allelic variants by hybridisation using the complete starch synthase-encoding sequence, because the similarities between the various alleles generally results in significant cross-hybridisation. Accordingly, sequence-specific hybridisation probes are required to distinguish between the various alleles. Similarly, wherein PCR is used to amplify specific allelic variants of a starch synthase gene, one or more sequencespecific amplification primers are generally required. As will be apparent from the amino acid sequence comparisons provided herein, such as in Figures 3 and 13, nonconserved regions of particular wheat starch synthase polypeptides are particularly useful for the design of probes and primers that are capable of distinguishing between one or more starch synthase polypeptide isoenzyme or allelic variant. The present invention clearly contemplates the design of such probes and primers based upon the sequence comparisons provided herein.

In the performance of this embodiment of the present invention, the present inventors particularly contemplate the identification of wheat starch synthase null alleles or alternatively, mutations wherein specific amino acids are inserted or deleted or substituted, compared to one or more of the wheat SSII or SSIII alleles disclosed WO 00/66745 PCT/AU00/00385 herein. Such null alleles and other allelic variants are readily identifiable using PCR screening which employs amplification primers based upon the nucleotide and amino acid sequences disclosed herein for SSII and/or SSIII. Once identified, the various mutations can be stacked or pyramided into one or more new wheat lines, such as by introgression and/or standard plant breeding and/or recombinant approaches (eg.

transformation, transfection, etc) thereby producing a novel germplasm which exhibits altered starch properties compared to existing lines. DNA markers based upon the nucleotide and amino acid sequences disclosed herein for SSII and/or SSIII can be employed to monitor the stacking of genes into the new lines and to correlate the presence of particular genes with starch phenotypes of said lines.

In this regard, a significant advantage conferred by the present invention is the design of new DNA markers that reveal polymorphisms such as, for example, length polymorphisms, restriction site polymorphisms, and single nucleotide polymorphisms, amongst others, between wheat starch synthases and, in particular, between wheat GBSS and/or SSI and/or SSII and/or SSIII, or between allelic variants of one or more of said starch synthases, that can be used to identify the three genomes of hexaploid wheats the A, B and D genomes).

Preferably, such DNA markers are derived from the intron region of a starch synthase gene disclosed herein, more preferably the wheat SSII and/or the wheat SSIII gene.

Those skilled in the art will be aware that such regions generally have a higher degree of variation than in the protein-ercoding regions and, as a consequence, are particularly useful in identifying specific allelic variants of a particular gene, such as allelic variants contained in any one of the three wheat genomes, or alternatively or in addition, for the purpose of distinguishing between wheat GBSS, SSI, SSII or SSIII genes.

A further approach contemplated by the present inventors is the design of unique isoenzyme-specific and/or allele-specific peptides based upon the amino acid sequence disclosed herein as SEQ ID NOS: 25 and/or SEQ ID NO: 4 and/or SEQ ID WO 00/66745 PCT/AU00/00385 -36- NO: 6 and/or SEQ ID NO: 8 and/or SEQ ID NO: 10, which peptides are then used to produce polyclonal or monoclonal antibodies by conventional means. Alternatively, the genes encoding these polypeptides or unique peptide regions thereof can be introduced in an expressible format into an appropriate prokaryotic or eukaryotic expression system, where they can be expressed to produce the isoenzyme-specific and/or allele-specific peptides for antibody production. Such antibodies may also be used as markers for the purpose of both identifying parental lines and germplasms and monitoring the stacking of genes in new lines, using conventional immunoassays such as, for example, ELISA and western blotting.

A further aspect of the present invention utilises the above-mentioned nucleic acid based assay method in the breeding and/or selection of plants which express or do not express particular starch synthase isoenzymes or alternatively, which express a particular starch synthase isoenzyme at a particular level in one or more plant tissues.

This aspect clearly extends to the selection of transformed plant material which contains one or. more of the isolated nucleic acid molecules of the present invention.

Yet another aspect of the present invention provides for the expression of the nucleic acid molecule of the present invention in a suitable host a prokaryote or eukaryote) to produce full length or non-full length recombinant starch synthase gene products.

Hereinafter the term "starch synthase gene product" shall be taken to refer to a recombinant product of a starch synthase gene of the present invention.

Preferably, the recombinant starch synthase gene product comprises an amino acid sequence having the catalytic activity of a starch synthase polypeptide or a functional mutant, derivative part, fragment, or analogue thereof.

In a particularly preferred embodiment of the invention, the recombinant starch synthase gene product is selected from the following: WO 00/66745 PCT/AU00/00385 -37a wheat starch synthase II (wSSII) polypeptide, protein or enzyme or functional subunit thereof which comprises an amino acid sequence which is at least about 85% identical overall to an amino acid sequence set forth in any one of SEQ ID NOS: 2,4, or 6; (ii) a wheat starch synthase III (wSSIII) polypeptide, protein or enzyme or functional subunit thereof which comprises an amino acid sequence which is at least about 85% identical overall to an amino acid sequence set forth in any one of SEQ ID NOS: 8 or 10; and (iii) a wheat starch synthase polypeptide, protein or enzyme or functional subunit thereof which comprises a conserved amino acid sequence having at least 25% identity to an amino acid sequence selected from the group consisting of:

KVGGLGDVVTS;

GHTVEVILPKY;

HDWSSAPVAWLYKEHY;

GILNGIDPDIWDPYTD;

DVPIVGIITRLTAQKG;

NGQWLLGSA;

(g)AGSDFIIVPSIFEPCGLTQLVAMRYGS; (h)TGGLVDTV; a wheat starch synthase II (wSSII) polypeptide, protein or enzyme or functional subunit thereof which comprises an amino acid sequence which is at least about 85% identical overall to an amino acid sequence set forth in any one of SEQ ID NOS: 2, 4, or 6; (ii) a wheat starch synthase III (wSSIII) polypeptide, protein or enzyme or functional subunit thereof which comprises an amino acid sequence which is at least about 85% identical overall to an amino acid sequence set forth in any one of SEQ ID NOS: 8 or (iii) a wheat starch synthase polypeptide, protein or enzyme or functional subunit thereof which comprises a conserved amino acid sequence having at WO 00/66745 PCT/AU00/00385 -38least 25% identity to an amino acid sequence selected from the group consisting of:

KVGGLGDVVTS;

GHTVEVILPKY;

HDWSSAPVAWLYKEHY;

GILNGIDPDIWDPYTD;

DVPIVGIITRLTAQKG;

NGQWLLGSA;

(g)AGSDFIIVPSIFEPCGLTQLVAMRYGS; and (h)TGGLVDTV;

KTGGLGDVAGA;

GHRVMVVVPRY;

NDWHTALLPVYLKAYY;

GIVNGIDNMEWNPEVD;

DVPLLGFIGRLDGQKG;

DVQLVMLGTG;

(o)AGADALLMPSRF(EN)PCGLNQLYAMAYGT; and (p)VGG(V/L)RDTV.

Accordingly, the present invention clearly extends to homologues, analogues and derivatives of the amino acid sequences set forth herein as SEQ ID NOS: 2, 4, 6, 8 and In the present context, "homologues" of an amino acid sequence refer to those polypeptides, enzymes or proteins which have a similar catalytic activity to the amino acid sequences described herein, notwithstanding any amino acid substitutions, additions or deletions thereto. A homologue may be isolated or derived from the same or another plant species as the species from which the polypeptides of the invention are derived.

"Analogues" encompass polypeptides of the invention notwithstanding the occurrence WO 00/66745 PCT/AU00/00385 -39of any non-naturally occurring amino acid analogues therein.

"Derivatives" include modified peptides in which ligands are attached to one or more of the amino acid residues contained therein, such as carbohydrates, enzymes, proteins, polypeptides or reporter molecules such as radionuclides or fluorescent compounds. Glycosylated, fluorescent, acylated or alkylated forms of the subject peptides are particularly contemplated by the present invention. Additionally, derivatives of an amino acid sequence described herein which comprises fragments or parts of the subject amino acid sequences are within the scope of the invention, as are homopolymers or heteropolymers comprising two or more copies of the subject polypeptides. Procedures for derivatizing peptides are well-known in the art.

Substitutions encompass amino acid alterations in which an amino acid is replaced with a different naturally-occurring or a non-conventional amino acid residue. Such substitutions may be classified as "conservative", in which an amino acid residue contained in a starch synthase gene product is replaced with another naturallyoccurring amino acid of similar character, for example Gly-Ala, Val-Ille-Leu, Asp--Glu, Lys*-Arg, Asn'-Gln or Phe-*Trp-Tyr.

Substitutions encompassed by the present invention may also be "non-conservative", in which an amino acid residue which is present in a starch synthase gene product described herein is substituted with an amino acid with different properties, such as a naturally-occurring amino acid from a different group (eg. substituted a charged or hydrophobic arnino acid with alanine), or alternatively, in which a naturally-occurring amino acid is substituted with a non-conventional amino acid.

Non-conventional amino acids encompassed by the invention include, but are not limited to those listed in Table 2.

Amino acid substitutions are typically of single residues, but may be of multiple residues, either clustered or dispersed.

WO 00/66745 PCT/AU00/00385 Amino acid deletions will usually be of the order of about 1-10 amino acid residues, while insertions may be of any length. Deletions and insertions may be made to the N-terminus, the C-terminus or be intemal deletions or insertions. Generally, insertions within the amino acid sequence will be smaller than amino- or carboxy-terminal fusions and of the order of 1-4 amino acid residues, A homologue, analogue or derivative of a starch synthase gene product as referred to herein may readily be made using peptide synthetic techniques well-known in the art, such as solid phase peptide synthesis and the like, or by recombinant DNA manipulations. Techniques for making substituent mutations at pre-determined sites using recombinant DNA technology, for example by M13 mutagenesis, are also wellknown. The manipulation of nucleic acid molecules to produce variant peptides, polypeptides or proteins which manifest as substitutions, insertions or deletions are well-known in the art.

The starch synthase gene products described herein may be derivatized further by the inclusion or attachment thereto of a protective group which prevents, inhibits or slows proteolytic or cellular degradative processes. Such derivatization may be useful where the half-life of the subject polypeptide is required to be extended, for example to increase the amount of starch produced in the endosperm or alternatively, to increase the amount of protein produced in a bacterial or eukaryotic expression system.

Examples of chemical groups suitable for this purpose include, but are not limited to, any of the non-conventional amino acid residues listed in Table 2, in particular a Dstereoisomer or a methylated form of a naturally-occurring amino acid listed in Table 1. Additional chemical groups which are useful for this purpose are selected from the list comprising aryl or heterocyclic N-acyl substituents, polyalkylene oxide moieties, desulphatohirudin muteins, alpha-muteins, alpha-aminophosphonic acids, watersoluble polymer groups such as polyethylene glycol attached to sugar residues using hydrazone or oxime groups, benzodiazepine dione derivatives, glycosyl groups such as beta-glycosylamine or a derivative thereof, isocyanate conjugated to a polyol functional group or polyoxyethylene polyol capped with diisocyanate, amongst others.

Similarly, a starch synthase gene product or a homologue, analogue or derivative WO 00/66745 PCT/AU00/00385 -41thereof may be cross-linked or fused to itself or to a protease inhibitor peptide, to reduce susceptibility of said molecule to proteolysis.

In a particularly preferred embodiment, the percentage similarity to in any one of SEQ ID NOS: 2, 4, 6, 8 or 10 is at least about 90%, more preferably at least about even more preferably at least about 97% and even more preferably at least about 98%, or about 99% or 100%.

In a related embodiment, the present invention provides a "sequencably pure" form of the amino acid sequence described herein. "Sequencably pure" is hereinbefore described as substantially homogeneous to facilitate amino acid determination.

In a further related embodiment, the present invention provides a "substantially homogeneous" form of the subject amino acid sequence, wherein the term "substantially homogeneous" is hereinbefore defined as being in a form suitable for interaction with an immunologically interactive molecule. Preferably, the polypeptide is at least 20% homogeneous, more preferably at least 50% homogeneous, still more preferably at least 75% homogeneous and yet still more preferably at least about 100% homogenous, in terms of activity per microgram of total protein in the protein preparation.

To produce the recombinant polypeptide of the present invention, the coding region of a starch synthase gene described herein or a functional homologue, analogue or derivative thereof is placed operably in connection with a promoter sequence in the sense orientation, such that a .starch synthase gene product is capable of being expressed under the control of said promoter sequence.

In the present context, the term "in operable connection with" means that expression of the isolated nucleotide sequence is under the control of the promoter sequence with which it is connected, regardless of the relative physical distance of the sequences from each other or their relative orientation with respect to each other.

WO 00/66745 PCT/AU00/00385 -42- Reference herein to a "promoter" is to be taken in its broadest context and includes the transcriptional regulatory sequences of a classical genomic gene, including the TATA box which is required for accurate transcription initiation, with or without a CCAAT box sequence and additional regulatory elements upstream activating sequences, enhancers and silencers) which alter gene expression in response to developmental and/or external stimuli, or in a tissue-specific manner. A promoter is usually, but not necessarily, positioned upstream or of a structural gene, the expression of which it regulates. Furthermore, the regulatory elements comprising a promoter are usually positioned within 2 kb of the start site of transcription of the gene.

In the present context, the term "promoter" is also used to describe a synthetic or fusion molecule, or derivative which confers, activates or enhances expression of a structural gene or other nucleic acid molecule, particularly in a plant cell and more preferably in a wheat plant or other monocotyledonous plant cell, tissue or organ.

Preferred promoters may contain additional copies of one or more specific regulatory elements, to further enhance expression and/or to alter the spatial expression and/or temporal expression. For example, regulatory elements which confer copper inducibility may be placed adjacent to a heterologous promoter sequence, thereby conferring copper inducibility on the expression of said molecule.

Those skilled in the art will be aware that in order to obtain optimum expression of the starch synthase gene of the present invention, it is necessary to position said gene in an appropriate configuration such that expression is controlled by the promoter sequence. Promoters are generally positioned 5' (upstream) to !he genes that they control. In the construction of heterologous promoter/structural gene combinations it is generally preferred to position the promoter at a distance from the gene transcription start site that is approximately the same as the distance between that promoter and the gene it controls in its natural setting, the gene from which the promoter is derived. As is known in the art, some variation in this distance can be accommodated without loss of promoter function. Similarly, the preferred positioning of a regulatory sequence element with respect to a heterologous gene to be placed under its control is defined by the positioning of the element in its natural setting, the genes from WO 00/66745 PCT/AU00/00385 -43which it is derived. Again, as is known in the art, some variation in this distance can also occur.

Examples of promoters suitable for expressing the starch synthase gene of the present invention include viral, fungal, bacterial, animal and plant derived promoters capable of functioning in prokaryotic or eukaryotic cells. Preferred promoters are those capable of regulating the expression of the subject starch synthase genes in plants cells, fungal cells, insect cells, yeast cells, animal cells or bacterial cells, amongst others.

Particularly preferred promoters are capable of regulating expression of the subject nucleic acid molecules in monocotyledonous plant cells. The promoter may regulate the expression of the said molecule constitutively, or differentially with respect to the tissue in which expression occurs or, with respect to the developmental stage at which expression occurs, or in response to external stimuli such as physiological stresses, or plant pathogens, or metal ions, amongst others.

Accordingly, strong constitutive promoters are particularly preferred for the purposes of the present invention.

Examples of preferred promoters include the bacteriophage T7 promoter, bacteriophage T3 promoter, SP6 promoter, lac operator-promoter, tac promoter, late promoter, SV40 early promoter, RSV-LTR promoter, CMV IE promoter, CaMV promoter, SCSV promoter, SCBV promoter and the like.

Particularly preierred promoters operable in plant cells include, for example the CaMV 35S promoter, and the SCBV promoter. Those skilled in the art will readily be aware of additional promoter sequences other than those specifically described.

In a particularly preferred embodiment, the promoter may be derived from a genomic starch synthase gene. Preferably, the promoter sequence comprises nucleotide sequences that are linked in vivo to nucleotide sequences set forth in any one of SEQ ID NOs: 1, 3, 5, 7, 9,11-16, 37, or 38. By "linked in vivo" means that the promoter is present in its native state in the genome of a wheat plant where it controls expression WO 00/66745 PCT/AU00/00385 -44of the starch synthase gene of the present invention.

Conveniently, genetic constructs are employed to facilitate expression of a starch synthase genetic sequence of the present invention or a functional derivative, part, homologue, or analogue thereof. To produce a genetic construct, the starch synthase gene of the invention is inserted into a suitable vector or episome molecule, such as a bacteriophage vector, viral vector or a plasmid, cosmid or artificial chromosome vector which is capable of being maintained and/or replicated and/or expressed in the host cell, tissue or organ into which it is subsequently introduced. The said genetic construct comprises the subject nucleic acid molecule placed operably under the control of a promoter sequence and optionally, a terminator sequence.

The term "terminator" refers to a DNA sequence at the end of a transcriptional unit which signals termination of transcription. Terminators are 3'-non-translated DNA sequences containing a polyadenylation signal, which facilitates the addition of polyadenylate sequences to the 3'-end of a primary transcript. Terminators active in bacteria, yeasts, animal cells and plant cells are known and described in the literature.

They may be isolated from bacteria, fungi, viruses, animals and/or plants.

Examples of terminators particularly suitable for use in expressing the nucleic acid molecule of the present invention in plant cells include the nopaline synthase (NOS) gene terminator of Agrobacterium tumefaciens, the terminator of the Cauliflower mosaic virus (CaMV) 35S gene, and the zein gene terminator from Zea mays.

Genetic constructs will generally further comprise one or more origins of replication and/or selectable marker gene sequences.

The origin of replication can be functional in a bacterial cell and comprise, for example, the pUC or the ColE1 origin. Altematively, the origin of replication is operable in a eukaryotic cell, tissue and more preferably comprises the 2 micron 2 origin of replication or the SV40 origin of replication.

WO 00/66745 PCT/AU00/00385 As used herein, the term "selectable marker gene" includes any gene which confers a phenotype on a cell in which it is expressed to facilitate the identification and/or selection of cells which are transfected or transformed with a genetic construct of the invention or a derivative thereof.

Suitable selectable marker genes contemplated herein include the ampicillin-resistance gene (Amp), tetracycline-resistance gene bacterial kanamycin-resistance gene (Kan), is the zeocin resistance gene (Zeocin is a drug of bleomycin family which is trademark of InVitrogen Corporation), the AURI-C gene which confers resistance to the antibiotic aureobasidin A, phosphinothricin-resistance gene, neomycin phosphotransferase gene (nptll), hygromycin-resistance gene, 3-glucuronidase (GUS) gene, chloramphenicol acetyltransferase (CAT) gene, green fluorescent proteinencoding gene or the luciferase gene, amongst others. Those skilled in the art will be aware of other selectable marker genes useful in the performance of the present invention and the subject invention is not limited by the nature of the selectable marker gene.

Usually, an origin of replication or a selectable marker gene suitable for use in bacteria is physically-separated from those genetic sequences contained in the genetic construct which are intended to be expressed or transferred to a eukaryotic cell, or integrated into the genome of a eukaryotic cell.

Standard methods can be used to introduce genetic constructs into a cell, tissue or organ for the purposes of modulating gene expression. Particularly preterred methods suited to the introduction of synthetic genes and genetic constructs comprising same to eukaryotic cells include liposome-mediated transfection or transformation, transformation of cells with attenuated virus particles or bacterial cells and standard procedures for the transformation of plant and animal cells, tissues, organs or organisms. Any standard means may be used for their introduction including cell mating, transformation or transfection procedures known to those skilled in the art or described by Ausubel et al. (1992).

WO 00/66745 PCT/AU00/00385 -46- In a further embodiment of the present invention, the starch synthase genes of the present invention and genetic constructs comprising same are adapted for integration into the genome of a cell in which it is expressed. Those skilled in the art will be aware that, in order to achieve integration of a genetic sequence or genetic construct into the genome of a host cell, certain additional genetic sequences may be required. In the case of plants, left and right border sequences from the T-DNA of the Agrobacterium tumefaciens Ti plasmid will generally be required.

The invention further contemplates increased starch and/or modified starch composition in transgenic plants expressing the nucleic acid molecule of the invention in the sense orientation such that the activity of one or more starch synthase isoenzymes is increased therein. By increasing the level of one or more starch synthase isoenzymes, the deposition of starch in the amyloplast or chloroplast is increased and/or a modified starch granule structure is produced and/or starch composition is modified and/or the amylose/amylopectin ratio is altered in the plant.

Wherein it is desired to increase the synthesis of a particular starch synthase isoenzyme in a plant cell, the coding region of a starch synthase gene is placed operably behind a promoter, in the sense orientation, such that said starch synthase is expressed under the control of said promoter sequence. In a preferred embodiment, the starch synthase genetic sequence is a starch synthase genomic sequence, cDNA molecule or protein-coding sequence.

Wherein it is desirable to reduce the level of a particular starch synthase isoenzyme in a plant cell, the nucleic acid molecule of the present invention can be expressed in the antisense orientation, as an antisense molecule or a ribozyme molecule, under the control of a suitable promoter.

Altematively, the nucleic acid molecule of the present invention may also be expressed in the sense orientation, in the form of a co-suppression molecule, to reduce the level of a particular starch synthase isoenzyme in a plant cell. As will be known to those skilled in the art, co-suppression molecules that comprise inverted repeat sequences WO 00/66745 PCT/AU00/00385 -47of a target nucleic acid molecule provide optimum efficiency at reducing expression of said target nucleic acid molecule and, as a consequence, the present invention clearly contemplates the use of inverted repeat sequences of any one or more of the starch synthase genetic sequences exemplified herein, or inverted repeat sequences of a homologue, analogue or derivative of said starch synthase genetic sequences, to reduce the level of a starch synthase isoenzyme in a plant.

The expression of an antisense, ribozyme or co-suppression molecule comprising a starch synthase gene in a cell such as a plant cell, fungal cell, insect cell. animal cell, yeast cell or bacterial cell, may also increase the availability of carbon as a precursor for a secondary metabolite other than starch sucrose or cellulose). By targeting the endogenous starch synthase gene, expression is diminished, reduced or otherwise lowered to a level that results in reduced deposition of starch in the amyloplast or chloroplast and/or leads to modified starch granule structure and/or composition and/or altered amylose/amylopectin ratio.

Accordingly, a further aspect of the present invention provides a method of modifying the starch content and/or starch composition of one or more tissues or organs of a plant, comprising expressing therein a sense molecule, antisense molecule, ribozyme molecule, co-suppression molecule, or gene-targeting molecule having at least about nucleotide sequence identity to any one of any one of SEQ ID NOS: 1, 3, 5, 7, 9,11-16, 37, or 38, or a complementary nucleotide sequence thereto for a time and under conditions sufficient for the enzyme activity of one or more starch synthase isoenzymes to be modified. This aspect of the invention clearly extends to the introduction of the sense molecule, antisense molecule, ribozyme molecule, cosuppression molecule, or gene-targeting molecule to isolated plant cells, tissues or organs or organelles by cell fusion or transgenic means and the regeneration of intact plants therefrom.

Co-suppression is the reduction in expression of an endogenous gene that occurs when one or more copies of said gene, or one or more copies of a substantially similar WO 00/66745 PCT/AU00/00385 -48gene are introduced into the cell, preferably in the form of an inverted repeat structure.

The present inventors have discovered that the genetic sequences disclosed herein are capable of being used to modify the level of starch when expressed, particularly when expressed in plants cells. Accordingly, the present invention clearly extends to the modification of starch biosynthesis in plants, in particular wheat or barley plants or a progenitor plant species, or a relative thereto such as the diploid Triticum tauschii or other diploid, tetraploid, aneuploid, polyploid, nullisomic, or a wheat/barley addition line, amongst others.

In particular, the present invention contemplates decreased starch production and/or modified starch composition in transgenic plants expressing the nucleic acid molecule of the invention in the antisense orientation or alteratively, expressing a ribozyme or co-suppression molecule comprising the nucleic acid sequence of the invention such that the activity of one or more starch synthase isoenzymes is decreased therein.

In the context of the present invention, an antisense molecule is an RNA molecule which is transcribed from the complementary strand of a nuclear gene to that which is normally transcribed to produce a "sense" mRNA molecule capable of being translated into a starch synthase polypeptide. The antisense molecule is therefore complementary to the mRNA transcribed from a sense starch synthase gene or a part thereof. Although not limiting the mode of action of the antisense molecules of the present invention to any specific mechanism, the antisense RNA molecule possesses the capacity to form a double-stranded mRNA by base pairing with the sense mRNA, which may prevent translation of the sense mRNA and subsequent synthesis of a polypeptide gene product.

Ribozymes are synthetic RNA molecules which comprise a hybridising region complementary to two regions, each of at least 5 contiguous nucleotide bases in the target sense mRNA. In addition, ribozymes possess highly specific endoribonuclease WO 00/66745 PCT/AU00/00385 -49activity, which autocatalytically cleaves the target sense mRNA. A complete description of the function of ribozymes is presented by Haseloff and Gerlach (1988) and contained in International Patent Application No. WO89/05852.

The present invention extends to ribozyme which target a sense mRNA encoding a native starch synthase gene product, thereby hybridising to said sense mRNA and cleaving it, such that it is no longer capable of being translated to synthesise a functional polypeptide product.

According to this embodiment, the present invention provides a ribozyme or antisense molecule comprising at least 5 contiguous nucleotide bases derived from any one of SEQ ID NOS: 1, 3, 5, 7, 9,11-16, 37, or 38, or a complementary nucleotide sequence thereto or a homologue, analogue or derivative thereof, wherein said antisense or ribozyme molecule is able to form a hydrogen-bonded complex with a sense mRNA encoding a starch synthase gene product to reduce translation thereof.

In a preferred embodiment, the antisense or ribozyme molecule comprises at least to 20 contiguous nucleotides derived from any one of SEQ ID NOS: 1,3, 5, 7, 9,11-16, 37, or 38, or a complementary nucleotide sequence thereto or a homologue, analogue or derivative thereof. Although the preferred antisense and/or ribozyme molecules hybridise to at least about 10 to 20 nucleotides of the target molecule, the present invention extends to molecules capable of hybridising to at least about 50-100 nucleotide bases in length, or a molecule capable of hybridising to a full-length or substantially full-length mRNA encoded by a starch synthase gene.

Those skilled in the art will be aware of the necessary conditions, if any, for selecting or preparing the antisense or ribozyme molecules of the invention.

It is understood in the art that certain modifications, including nucleotide substitutions amongst others, may be made to the antisense and/or ribozyme molecules of the present invention, without destroying the efficacy of said molecules in inhibiting the expression of a starch synthase gene. It is therefore within the scope of the present WO 00/66745 PCT/AU00/00385 invention to include any nucleotide sequence variants, homologues, analogues, or fragments of the said gene encoding same, the only requirement being that said nucleotide sequence variant, when transcribed, produces an antisense and/or ribozyme molecule which is capable of hybridising to a sense mRNA molecule which encodes a starch synthase gene product .Gene targeting is the replacement of an endogenous gene sequence within a cell by a related DNA sequence to which it hybridises, thereby altering the form and/or function of the endogenous gene and the subsequent phenotype of the cell. According to this embodiment, at least a part of the DNA sequence defined by any one of SEQ ID NOS: 1, 3, 5, 7, 9,11-16, 37, or 38 may be introduced into target cells containing an endogenous gene that encodes a particular starch synthase isoenzyme, thereby replacing said endogenous gene. According to this embodiment, the polypeptide product of the gene targetting molecule generally encodes a starch synthase isoenzyme that possesses different catalytic activity to the polypeptide product of the endogenous gene, producing in tum modified starch content and/or composition in the target cell.

The present invention extends to genetic constructs designed to facilitate expression of a sense molecule, an antisense molecule, ribozyme molecule, co-suppression molecule, or gene targeting molecule of the present invention. The requirements for expressing such molecules are similar to those for expressing a recombinant polypeptide as described supra.

The present invention further extends to the production and use of starches and proteins produced using the novel genes described herein. Modified starches produced by plants which have been selected using marker-assisted selection, or alternatively, produced by transgenic plants carrying the introduced starch synthase genes, are particularly suitable for use in food products, such as, for example, flour and flour-based products, in particular those products selected from the group consisting of: flour-based sauce; leavened bread; unleavened bread; pasta, noodle; cereal; snack food; cake; and pastry. Modified proteins are also suitable for use in non- WO 00/66745 PCT/AU00/00385 -51food products, such as, for example, those non-food products selected from the group consisting of: films; coatings; adhesives; building materials; and packaging materials.

Additionally, starch hydrolysates or undegraded starches are both useful in industry and, as a consequence, the present invention is useful in applications relating tothe use of both starch hydrolysates and undegraded starches. By "starch hydrolysates" is meant the glucose and glucan components that are obtainable by the enzymatic or chemical degradation of starch in chemical modifications and processes, such as fermentation.

Starch produced by plants expressing the sense, antisense, co-suppression, genetargetting or ribozyme molecules of the present invention may exhibit modified viscosities and/or gelling properties of its glues when compared to starch derived from wild-type plants. Native starches produced by the performance of the inventive method are useful as an additive in the following: foodstuffs, for the purpose of increasing the viscosity or gelling properties of food; (ii) in non-foodstuffs, such as an adjuvant or additive in the paper and cardboard industries, for retention or as a size filler, or as a solidifying substance or for dehydration, or film coating, amongst others; (iii) in the adhesive industry as pure starch glue, as an additive to synthetic resins and polymer or-as -an -extenders-for-synthetic-adhesives;- (iv) in-the textile-and-textile care industries to strengthen woven products and reduce burring or to thicken dye pastes; in the building industry, such as a binding agent in the production of gypsum plaster boards, or for the deceleration of the sizing process; (vi) in ground stabilization or for the temporary protection of ground particles against water in artificial earth shifting; (vii) as a wetting agent in plant protectants and fertilizers; (viii) as a binding agent in drugs, pharmaceuticals and medicated foodstuff such as vitamins, etc; (ix) as an additive in coal and briquettes; (xi) as a flocculent in the processing of coal ore and slurries; (xii) as a binding agent in casting processes to increase flow resistance and improve binding strength; and (xiii) to improve the technical and optical quality of rubber and plastic products. Additional applications are not excluded.

A further aspect of the present invention provides an isolated promoter that is operable WO 00/66745 PCT/AU00/00385 -52in the endosperm of a monocotyledonous plant cell, tissue or organ, and preferably in the endosperm of a monocotyledonous plant cell, tissue or organ. According to this embodiment, it is preferred that the promoter is derived from a starch synthase gene of the present invention, such as a promoter that is linked in vivo to any one of SEQ ID NOS: 1, 3, 5, 7, 9,11-16, 37, or 38, or a complementary nucleotide sequence thereto.

In a particularly preferred embodiment, the promoter comprises a nucleotide sequence derivable from the 5'-upstream region of SEQ ID NO: 11 or SEQ ID NO: 37 or SEQ ID NO: 38, or a complementary nucleotide sequence thereto, an more preferably comprises nucleotides 1 to about 287 of SEQ ID NO: 11, or nucleotides 1 to about 1416 of SEQ ID NO: 37, or nucleotides 1 to about 973 of SEQ ID NO: 38, or a complementary nucleotide sequence thereto. The present invention clearly extends to promoter sequences that comprise further nucleotide sequences in the region upstream of the stated nucleotide sequence that are linked in vivo to said nucleotide sequence in the wheat genome.

In a related embodiment, the promoter sequence of the present invention will further comprise an exon sequence derived from a starch synthase gene, such as, for example, an intron I sequence described herein, or a complementary nucleotide sequence thereto. Those skilled in the art will be aware that the inclusion of such nucleotide sequences may increase the expression of a heterologous structural gene, the expression of which is controlled thereby. Preferred intron I sequences include, for example, nucleotide sequences in the region of about position 1744 to about 1847 of SEQ ID NO: 37, and/or about position 1100 to about position 2056 of SEQ ID NO: 38. Additional sequences comprising intron/exon junction boundary sequences which are readily determined by those skilled in the art are not excluded.

The present invention further extends to the expression of any structural gene operably under the control of the starch synthase promoter sequence exemplified herein or a functional homologue, analogue or derivative of said promoter sequence.

WO 00/66745 PCT/AU00/00385 -53- As with other embodiments described herein for expression in cells, a genetic construct may be employed to effect said expression and the present invention clearly extends to said genetic constructs.

The polypeptide encoded by the structural gene component may be a reporter molecule which is encoded by a gene such as the bacterial 1-glucuronidase gene or chloramphenicol acetyltransferase gene or alternatively, the firefly luciferase gene.

Alternatively, wherein it is desirable to alter carbon partitioning within the endosperm, the polypeptide may be an enzyme of the starch sucrose biosynthetic pathways.

Preferably, the promoter sequence is used to regulate the expression of one or more of the starch synthase genes of the present invention or a sense, antisense, ribozyme, co-suppression or gene-targetting molecule comprising or derived from same.

Recombinant DNA molecules carrying the aforesaid nucleic acid molecule of the present invention or a sense, antisense, ribozyme, gene-targetting or co-suppression molecule and/or genetic construct comprising same, may be introduced into plant tissue, thereby producing a "transgenic plant", by various techniques known to those skilled in the art. The technique used for a given plant species or specific type of plant tissue depends on the known successful techniques. Means for introducing recombinant DNA into plant tissue include, but are not limited to, transformation (Paszkowski et al., 1984), electroporation (Fromm et al., 1985), or microinjection of the DNA (Crossway et al., 1986), or T-DNA-mediated transfer from Agrobacterium to the plant tissue. Representative T-DNA vector systems are described in the following references: An et a.(1985); Herrera-Estrella et al. (1983a, Herrer--Estrella et al.

(1985). Once introduced into the plant tissue, the expression of the introduced gene may be assayed in a transient expression system, or it may be determined after selection for stable integration within the plant genome. Techniques are known for the in vitro culture of plant tissue, and in a number of cases, for regeneration into whole plants. Procedures for transferring the introduced gene from the originally transformed plant into commercially useful cultivars are known to those skilled in the art.

In general, plants are regenerated from transformed plant cells or tissues or organs on WO 00/66745 PCT/AU00/00385 -54hormone-containing media and the regenerated plants may take a variety of forms, such as chimeras of transformed cells and non-transformed cells; clonal transformants all cells transformed to contain the expression cassette); grafts of transformed and untransformed tissues transformed root stock grafted to an untransformed scion in citrus species). Transformed plants may be propagated by a variety of means, such as by clonal propagation or classical breeding techniques. For example, a first generation (or T1) transformed plants may be selfed to give homozygous second generation (or T2) transformed plants, and the T2 plants further propagated through classical breeding techniques.

Accordingly, a still further aspect of the present invention contemplates a transgenic plant comprising an introduced sense molecule, antisense molecule, ribozyme molecule, co-suppression molecule, or gene-targeting molecule having at least about nucleotide sequence identity to any one of any one of SEQ ID NOS: 1, 3, 5, 7, 9,11-16, 37, or 38, or a complementary nucleotide sequence thereto or a genetic construct comprising same. The present invention further extends to those plant parts, propagules and progeny of said transgenic plant or derived therefrom, the only requirement being that said propagules and progeny also carry the introduced nucleic acid molecule(s).

The present invention is further described by reference to the following non-limiting examples.

EXAMPLE 1 Plant material Genetic stocks of hexaploid bread wheat Triticum aestivum cv. Chinese Spring with various chromosome additions and deletions were kindly supplied by Dr E. Lagudah (CSIRO Plant Industry, Canberra) and derived from stocks described in Sears and Millet (1985). The hexaploid (Triticum aestivum) wheats cv Gabo and cv Wyuna were grown in controlled growth cabinet conditions (180C day and 13 C night, with a photoperiod of 16 Wheat leaves and florets prior to anthesis, and endosperm were collected over the grain filling period, immediately frozen in liquid nitrogen and stored WO 00/66745 PCT/AU00/00385 at -80 0 C until required.

EXAMPLE 2 Gel Electrophoresis, Antibodies and Immunoblotting Monoclonal antibodies against the Sgp-1 proteins, and their use in the immunoblotting of SDS-PAGE gels have been described previously (Rahman et al., 1995).

EXAMPLE 3 Preparation of total RNA from wheat Total RNA was isolated from the leaf, floret and endosperm tissues of wheat essentially as described by Higgins et al. (1976) or Rahman et al. (1998). RNA was quantified by UV absorption and by separation in 1.4% agarose-formaldehyde gels which were then visualised under UV light after staining with ethidium bromide.

EXAMPLE 4 Construction and screening of cDNA libraries A first cDNA library, an expression cDNA library of wheat endosperm, was constructed from mRNA isolated from wheat cv Chinese Spring. RNA from 5, 7, 9, 11 and 13 days after anthesis was pooled and random primers were used for the first strand of cDNA synthesis. Monoclonal antibodies against 100 -105 kDa proteins in wheat starch granules (Rahman et al., 1995) were used for immunoscreening of the expression cDNA library.

A second cDNA library was constructed from the endosperm mRNA of the hexaploid Triticum aestivum cultivar Wyuna, 8 12 days after anthesis, as described by Rahman et al. (1997). This library was screened with a 85-bp cDNA fragment, wSSllpl, which was obtained by immunoscreening of the expression cDNA library as described above.

The wSSllpl probe corresponded to nucleotide positions 988 to 1072 of wSSllB (SEQ ID NO:1) at the hybridisation conditions as described earlier (Rahman et al., 1998).

A third cDNA library was constructed from RNA from the endosperm of the hexaploid WO 00/66745 PCT/AU00/00385 -56- Triticum aestivum cultivar Rosella as described by Rahman et al. (1997). This library was screened with a 347-bp cDNA fragment, wSSIIIpl for the first screening, and a 478-bp cDNA fragment wSSlllp3 for the second screening using the hybridisation conditions described herein.

EXAMPLE Construction and screening of Triticum tauschii genomic library The genomic library used in this study, prepared from Triticum tauschii, var strangulata, (Accession Number CPI 110799), has been described in Rahman et al., (1997). Of all the accessions of T. tauschii surveyed, DNA marker analysis suggests that the genome of CPI 110799 is the most closely related to the D genome of hexaploid wheat (Lagudah et al., 1991).

Hybridisations were carried out in 25% formamide, 6 x SSC, 0.1% SDS at 42°C for 16 hours, then filters were washed 3 times using 2 x SSC containing 0.1% SDS at for 1 hour per wash.

For the isolation of a genomic wSSII clone, the probe comprised the PCR-derived DNA fragment wSSllp2 and positive-hybridising plaques were digested using the restriction enzyme BamHI, separated on a 1% agarose gel, transferred to nitrocellulose membrane and hybridised to probe wSSllp4 comprising nucleotides 1 to 367 of the wSSIIA cDNA clone, using the conditions described by Rahman et al. (1997).

For the isolation of a genomic wSSIII clone, plaques hybridising to the PCR-derived DNA fragment wSSlllpl from clone wSSIII.B3 nucleotides 3620 to 3966 of SEQ ID NO:7) were selected and re-screened until plaque-purified.

EXAMPLE 6 DNA sequencing and analysis DNA sequencing was performed using the automated ABI system with dye terminators as described by the manufacturers. DNA sequences were analysed using the GCG WO 00/66745 PCT/AU00/00385 -57suite of programs (Devereaux et al., 1984).

EXAMPLE 7 DNA and RNA analysis DNA was isolated and analysed as previously described (Maniatis et al., 1982; Rahman et al., 1998). Approximately 20 pg of DNA was digested with restriction enzymes BamHI, Dral and EcoRI, separated on a 1% agarose gel and transferred to reinforced nitrocellulose membranes (BioRad) and hybridised with "P-labelled DNA probe, either wSSIIIpl, corresponding to nucleotides 3620 to 3966 of the wheat SSIII gene, or altematively, with the entire wSSII cDNA clone. DNA fragment probes were labelled with the Rapid Multiprime DNA Probe Labelling Kit (Promega).

The hybridisation and wash conditions were performed as described in Rahman et al.

(1997). For RNA analysis, 10 /ug of total RNA was separated in a 1.4% agaroseformaldehyde gel and transferred to a Hybond N+ membrane (Amersham), and hybridised with cDNA probe at 42 0 C as previously described by Khandjian et al., (1987) or Rahman et al., (1998). After washing for 30 minutes at 65 0 C with 2x SSC, 0.1% SDS; followed by three washes of 40 minutes at 65 0 C with 0.2x SSC, 1% SDS, the membranes were visualised by ovemight exposure at -80 0 C with Kodak MR X-ray film.

EXAMPLE 8 Expression of wheat Sgp-1 polypeptides in the wheat endosperm The development and use of monoclonal antibodies to the Sgp-1 proteins has been described previously (Rahman et al., 1995). These antibodies were used by the present inventors to characterise the expression and localisation of the Sgp-1 proteins.

The proteins found in the matrix of the wheat starch granule are shown in Figure 1, lane 1. The remaining lanes show an immunoblot of proteins from the soluble phase (Figure 1; lanes 2-4) and the starch granule (Figure 1; lanes respectively, following SDS-PAGE. In addition to cross-reactivity with the 100-105 kDa proteins, a WO 00/66745 PCT/AU00/00385 -58weak cross-reaction with a 50 kDa protein in both the granule and the soluble fractions were observed (Figure The Sgp-1 polypeptides are present in the starch granule throughout endosperm development (Figure 1; lanes 5-7, also see Rahman et al., 1995). However, as the endosperms matures, there is a reduction in the amount of Sgp-1 protein found in the soluble fraction. Lane 4 shows that by 25 days after anthesis, the level of these proteins in the soluble fraction is substantially reduced.

This observation is consistent with previous results from Rahman et al., (1995), who suggested that the Sgp-1 proteins were exclusively granule bound based on studies of granules from endosperm in mid-late stages endosperm development, however, these results suggest that the partitioning of these proteins between the granule and the soluble phase changes during development EXAMPLE 9 Isolation of cDNA clones encoding wheat starch synthase II (wSSII) proteins Monoclonal antibodies against Sgp-1 polypeptides (Rahman et al., 1995) were used to probe the expression library described in Example 4 the first cDNA library).

Three immunoreactive plaques were identified and sequenced. One done, designated wSSIlpl, contained an 85-bp cDNA insert with homology to maize SSIIa (Har et al., 1998).

DNA from the wSSIIpl clone was used as a probe in the hybridisation screening of the second cDNA library, prepared from Triticum aestivum cultivarWyuna endosperm RNA as described in Example 4. Ten hybridising cDNA clones were selected and sequenced. On the basis of the DNA sequences obtained, the 10 cDNA clones can be classified into three groups. Group 1 contains 7 cDNA clones, group 2 contains 2 cDNA clones and group 3 contains 1 cDNA clone.

The longest clone from group 1 (designated wSSIIB) is 2939 bp in length (SEQ ID NO:1) and encodes a 798 -amino acid polypeptide in the region from nucleotide position 176 to nucleotide position 2569 (SEQ ID NO:2).

WO 00/66745 PCT/AU00/00385 -59- The longest clone from group 2 (designated wSSIIA) is 2842 bp in length (SEQ ID NO:3) and encodes a 799 -amino acid polypeptide in the region from nucleotide position 89 to nucleotide position 2485 (SEQ ID NO:4).

The cDNA from group 3 is a partial cDNA clone (designated wSSIID), which is 2107 bp in length (SEQ ID NO:5) and encodes a 597 -amino acid polypeptide in the region from nucleotide position 1 to nucleotide position 1791 (SEQ ID NO:6). The encoded polypeptide is approximately a 200 amino acid residues shorter than that of polypeptides encoded by longest clones of group 1 or 2 clones, respectively (Figure 2).

Comparison of the three cDNA clones, wSSIIB, wSSIIA and wSSIID shows that they share 95.7% to 96.6% identity at the amino acid level, with variation at 44 amino acid positions between the three sequences (Figure Of the 44 amino acid changes between these sequences, 31 changes occur in the N-terminal region (residues 1 to 300), 10 changes occur in.the central region (residues 301 to 729) and 3 changes occur in the C-terminal region (residues 730 to 799). The wSSIIA polypeptide (799 amino acid residues) and wSSIIB polypeptide (798 amino acid residues) sequences differ in length by a single amino acid residue, due to the deletion of Asp-69 from the wSSIIB polypeptide sequence.

A comparison of the nucleotide sequences of the wSSIA, wSSIIB and wSSIID cDNA clones with the nucleotide sequence of the wSSIIpl cDNA obtained by immunoscreening confirms that the wSSIIpl sequence is found in each cDNA (Figure The peptide encoded by the wSSIIpl cDNA clone corresponds to amino acid residues in the region from residue 272 to residue 298 of the wSSIIA polypeptide, and to amino acid residues in the region from residue 271 to residue 297 of the wSSIIB polypeptide (see Figure Thus, the peptide epitope encoded by wSSIIpl that reacts with the anti-Sgp-1 monoclonal antibodies can therefore be localised to this region of the wSSIIA and wSSIIB polypeptides and to the corresponding region of the wSSIID polypeptide.

WO 00/66745 PCT/AU00/00385 Notwithstanding that a region having about 63% amino acid sequence identity to the peptide epitope encoded by clone wSSIIpl is found in the maize SSIIla polypeptide (Figure the degree of amino acid conservation between maize and wheat sequences in this region of the polypeptide is insufficient for immunological crossreactivity to occur between these species using the monoclonal antibodies to the wheat Sgp-1 proteins described by Rahman et al. (1995). Additionally, this peptide epitope is not found in granule-bound starch synthases, SSI, or SSIII (data not shown).

The wSSIIB cDNA (SEQ ID NO:1) encodes an amino acid sequence comprising the peptide motif AAGKKDAGID (SEQ ID NO: 18) between residues 60 and 69 of SEQ ID NO:2 (Figure 3) which, with the exception of the second residue, is identical to the Nterminal of the 100 kDa (AT/LGKKDAGID: SEQ ID NOS:19 and 20) protein (Sgp-B1) from the wheat starch granule (note that the sequence given in Rahman et al., 1995 (AT/LGKKDAL: SEQ ID NOS: 21 and 22) has been revised following further amino acid sequence analysis).

The wSSIIA cDNA clone (SEQ ID NO:3) encodes an amino acid sequence comprising the peptide motif AAGKKDARVDDDAA (SEQ ID NO: 23) at residues 60 to 73 of SEQ ID NO:4, which is about 66% identical to the N-terminal amino acid sequence (i.e.

ALGKKDAGIVDGA: SEQ ID NO: 24) of the 104 kDa and 105 kDa starch granule proteins, Sgp-D1 and Sgp-A1 respectively, as determined by sequence analysis of isolated protein (Rahman et al., 1995).

Furthermore, Takaoka et al. (1997) reported the amino acid sequences of 3 polypeptides obtained from sequencing starch granule proteins derived from the Sgp-1 proteins. Peptide 3 described by Takaoka et al. (1997) corresponds to amino acid residues 378 to 387 of the amino acid sequence of the wSSIIA cDNA (SEQ ID NO:4; Figure Peptides 1 and 2 described by Takaoka et al. (1997) could not be detected in the amino acid sequences of the wSSII cDNA clones of the present invention, however peptide 1 of Takaoka et al. (1997) can be found in the amino acid sequences of SSI from maize, rice, wheat and potato (data not shown).

WO 00/66745 PCT/AU00/00385 -61 Denyer et al. (1995) demonstrated that the Sgp-1 proteins possess starch synthase activity and, as a consequence, the wSSIIB, wSSIA and wSSIID cDNA clones encode starch synthase enzymes that are differentially expressed in a developmentallyregulated manner in both the soluble and granule-bound fractions of the endosperm (Figure Based on the nomenclature suggested by Ham et al. (1998), it is appropriate to describe the Sgp-1 proteins as "starch synthases" rather than "granulebound starch synthases".

EXAMPLE Analysis of wheat starch synthase II mRNA expression The mRNA for wheat starch synthase II could be detected in leaves, pre-anthesis florets and endosperm of wheat when total RNAs isolated from these tissue were probed with a PCR probe, wSSIIp2, corresponding to nucleotide positions 1435 to 1835 bp of wSSIIB-cDNA (SEQ ID NO:1; Figure Unlike wSSI, which could not be detected in wheat leaves derived from plants grown under the same conditions, wSSII genes are highly-expressed in the leaves (Figure 4, lane and expressed at an intermediate level in pre-anthesis florets (Figure 4, lane and at much lower levels in developing wheat endosperm cells (Figure 4, lanes 3-11). In contrast, the maize SSlla is expressed predominantly in the endosperm, whilst the maize SSIIb is detected mainly in the leaf, albeit at low levels (Ham et al., 1998).

The wSSII mRNA was detectable in the endosperm 6 days after anthesis and mRNA levels increase between 8 and 18 days post-anthesis, after which time levels of mRNA decline.

Southern blotting experiments in wheat demonstrated that the wSSIIp2 probe used detected only a single copy of the SSII gene in each genome (data not shown). Thus, it is unlikely that this probe cross-hybridised with mRNAs encoded by genes other than wSSII.

WO 00/66745 PCT/AU00/00385 -62- EXAMPLE 11 Chromosomal localization of the wheat wSSII genes.

I. Amplification of specific cDNA regions of wheat starch synthase II using PCR Two PCR products, wSSIIp2 and wSSllp3 were amplified from the cDNA clone wSSIIB and used for the northem hybridisation and Southern hybridisation, respectively.

The primers sslla TGTTGAGGTTCCATGGCACGTTC SEQ ID NO: 25) and ssllb AGTCGTTCTGCCGTATGATGTCG SEQ ID NO: 26) were used to amplify the cDNA fragment wSSIIp2 nucleotide positions 1435 to 1835 of SEQ ID NO:1).

The primers ssllc CCAAGTACCAGTGGTGAACGC SEQ ID NO: 27) and sslld CGGTGGGATCCAACGGCCC SEQ ID NO: 28) were used to amplify the cDNA fragment wSSIlp3 nucleotide positions 2556 to 2921 of SEQ ID NO:1).

The amplification reactions were performed using a FTS-1 thermal sequencer (Corbett, Australia) for 1 cycle of 95 0 C for 2 minutes; 35 cycles of 95 0 C for 30 seconds, 60 0 C for 1 minutes, 720C for 2 minutes and 1 cycle of 250C for 1 minute.

II. PCR and nucleotide sequence analysis of 3' sequences of wheat SSII genes Genomic DNA was extracted from wild-type Chinese Spring wheat, and from three nullisomic-tetrasomic lines of chromosome 7 of Chinese Spring wheat, and from Triticum tauschii (var strangulata, accession number CPI 100799), and used as a template for the amplification and nucleotide sequence analysis of wheat SSII genes.

RFLP analysis of BamHI and EcoRI restricted DNA from each wheat or T. Tauschii line was carried out using the wSSIlp3 fragment as a probe. Three hybridising bands were obtained which could be assigned to chromosomes 7A, 7B and 7D, respectively (data not shown). This analysis indicates that there is a single copy of the wSSII gene in each genome in hexaploid wheat, consistent with the findings of Yamamori and Endo (1996) who located the SGP-A1, B1 and D1 proteins to the short arm of chromosome 7.

WO 00/66745 PCT/AU00/00385 -63- PCR analysis was used to assign each of the cDNA clones to the individual wheat genomes. A single 365 bp PCR fragment was obtained from nullisomic-tetrasomic genomic DNA of Chinese Spring when primers ssllc and sslld were used for the PCR amplification (Figure 5, right panel). This PCR product is obtained only from lines bearing the B genome. The fragment was cloned and sequenced and shown to be identical to a 365 bp region of the wSSIIB cDNA. An identical fragment is obtained by PCR amplification of the wSSIIB cDNA clone, but not by amplification of the wSSIIA or wSSIID clones, supporting the conclusion that the wSSIIB cDNA is the product of a gene located on chromosome 7 of the B genome of hexaploid wheat.

Two PCR products were also amplified from nullisomic-tetrasomic genomic DNA of Chinese Spring using the primers ssllc and sslle (Figure 5, left panel). One PCR fragment, approximately 350 bp is only amplified when the A genome is present, and a second 322 bp product is only amplified when the D-genome is present. The 350 and 322 bp PCR products were also cloned and sequenced and shown to be identical to the wSSIIA and wSSIID cDNAs, respectively, supporting the conclusion that the wSSIIA and wSSIID cDNAs are the products of genes located on chromosomes 7A and 7D, respectively.

EXAMPLE 12 Isolation of genomic wSSII clones Screening of a genomic library from the D-genome donor of wheat, T. tauschii, was performed as described in Example 5, using the PCR-derived DNA fragment wSSIlp2 as a hybridisation probe. A positive-hybridising clone, designated wSSII-8, and comprising a putative T. tauschiihomologue of the wSSII gene, was isolated.

Positive-hybridising plaques were digested using the restriction enzyme BamHI, separated on a 1% agarose gel, transferred to nitrocellulose membrane and hybridised to probe wSSllp4 comprising nucleotides 1 to 367 of the wSSIIA cDNA clone, using the conditions described by Rahman et al. (1997). Clone wSSII-8 also hybridises strongly to the wSSIIp4 probe, confirming its identity as a genomic wSSII gene.

WO 00/66745 PCT/AU00/00385 -64- The complete nucleotide sequence of the wSSII gene was determined and is presented herein as SEQ ID NO: 37. The structural features of this gene are present in Table 3. A schematic representation of the intron/exon organisation of this gene is also presented in Figure 6.

TABLE 3 Structural features of the wheat starch synthase II genomic gene Nucleotide Position Feature Length (bases) in SEQ ID NO: 37 1- 1416 5'-untranscribed region and 1416 promoter sequence 1417 1743 exon 1 327 1480-1482 translation start codon (ATG) 3 1744 1847 intron 1 104 1848 2553 exon 2 706 2554 2641 intron 2 88 2642 2706 exon 3 2707 3606 intron 3 900 3607 3684 exon 4 78 3685 3773 intron 4 89 3774 -3884 exon 5 111 3885 3981 intron 5 97 3982 4026 exon 6 4027 4406 intron 6 380 4407 -4580 exon 7 174 4581 7296 intron 7 2716 7297 8547 exon 8 1251 8251 8253 translation stop codon (TGA) 3 8548 -9024 3'-untranscribed region 477 WO 00/66745 PCT/AU00/00385 EXAMPLE 13 Cloning of specific cDNA regions of wheat starch synthase III using RT-PCR PCR primers were used to amplify sequences of starch synthase III from wheat endosperm cDNA. The design of PCR primers was based on the sequences of starch synthase III from potato and the dul starch synthase III gene of maize.

First-strand cDNAs were synthesised from 1 pg of total RNA (derived from endosperm of the cultivar Rosella, 12 days after anthesis) as described by Maniatis et al (1982), and then used as templates to amplify two specific cDNA regions, wSSIIIpl and wSSIIIp2, of wheat starch synthase III by PCR.

The primers used to obtain the cDNA clone wSSIIIpl were as follows: Primer wSS3pa GGAGGTCTTGGTGATGTTGT SEQ ID NO: 29); and Primer wSS3pb CTTGACCAATCATGGCAATG SEQ ID NO: The primers used to obtain the cDNA clone wSSIIIp2 were as follows: Primer wSS3pc CATTGCCATGATTGGTCAAG SEQ ID NO: 31); and Primer wSS3pd ACCACCTGTCCGTTCCGTTGC SEQ ID NO: 32).

The amplified clones wSSIIpl and wSSIlp2 were used as probes to screen the third cDNA library and T. tauschii genomic DNA library as described in Example 4.

A further probe designated wSSlllp3 was used for screening the third cDNA library, as described in Example 4. Probe wSSlllp3 was amplified by PCR from a cDNA clone produced from the first screening using the following amplification primers: Primer wSS3pe GCACGGTCTATGAGAACAATGGC SEQ ID NO: 33); and Primer wSS3pf TCTGCATACCACCAATCGCCG SEQ ID NO: 34).

The amplification reactions were performed using a FTS-1 or FTS4000 thermal sequencer (Corbett, Australia) for 1 cycle of 95°C for 2 minutes; 35 cycles of 95°C for seconds, 60°C for 1 minutes, 72°C for 2 minutes and 1 cycle of 25 0 C for 1 minute.

WO 00/66745 PCT/AU00/00385 -66- Amplified sequences of the expected length were obtained, cloned and sequenced, and shown to contain DNA sequences highly homologous to the maize and potato SSIII genes. PCR fragments were subsequently used to probe a wheat cDNA library by DNA hybridisation and 8 positive clones were obtained, including one 3 kb cDNA.

A region from the 5' end of this cDNA was amplified by PCR and used a probe for a second round of screening the cDNA library, obtaining 8 cDNA clones. Of these, one cDNA was demonstrated to be full length (wSSIII.B3, 5.36 kb insert). The sequence of the 5,346 bp wSSIII.B3 cDNA clone is given in SEQ ID NO:7.

Sequencing of the 8 cDNA clones obtained from the second round screening of the wheat cDNA library revealed that there were at least 2 classes of cDNA encoding SSIII present, possibly being encoded by homeologous genes on different wheat genomes.

The sequence of a representative of this second class of cDNA clones, wSSIII.B1, is shown in SEQ ID NO:9. The 3261 bp clone wSSIII.B1 is not full length, however it is similar to nucleotides 1739 to 5346 of the homeologous clone wSSIII.B3 (SEQ ID NO: Clone wSSIII.BI has an open reading frame between nucleotide positions 1 and 3177.

An open reading frame is found in the cDNA clone wSSIII.B3 (SEQ ID NO:7), in the region between position 29, commencing the ATG start codon, and nucleotide position 4912. The amino acid sequence deduced from this open reading frame is shown in SEQ ID NO:8.

An alignment of the deduced amino acid sequences of SSIII from maize, potato and wheat is shown in Figure 7. There is about 56.6% identity between the maize SSIII and wheat wSSIII.B3 sequence at the amino acid level.

The C-terminal domain of starch synthases comprise the catalytic domain, and a characteristic amino acid sequence motif KVGGLGDVVTSLSRAVQDLGHNVEV (SEQ ID NO: 35) in maize, or alternatively KVGGLGDVVTSLSRAIQDLGHTVEV (SEQ ID WO 00/66745 PCT/AU00/00385 -67- NO: 36) in wheat, marking the first conserved region in the C-terminal domain. This amino acid sequence is present at amino acid residues 1194 to 1218 of SEQ ID NO: 8.

The amino acid identity between maize dull1 and wSSIII.B3 in the N-terminal region amino acids 1 to 600 in Figure 7) is only 32.2%; whilst the amino acid identity in the central region amino acids 601 to 1248 in Figure 7) is 68.4%; and in the Cterminal region amino acids 1249 to 1631 in Figure 7) is 84.6%. Accordingly, the SSIII starch synthases are much more highly conserved between maize and wheat in the region comprising the catalytic domain of the proteins.

EXAMPLE 14 Analysis of wheat starch synthase III mRNA expression Figure 8 shows the expression of wSSIII mRNA during endosperm development in two wheat varieties grown under defined environmental conditions. The expression of the gene is seen very early in endosperm development in both cultivars, 4 days after anthesis (Figure 8, panels a and Expression in the leaf of the variety Gabo is very weak (Figure 8, panel c, Lane L) whereas strong expression is seen in pre-anthesis florets (Figure 8, panel c, Lane P).

EXAMPLE Amino acid sequence comparisons between wheat SSII and SSIII polypeptides Amino acid sequence comparisons between wheat BSSS, SSI, SSII and SSIII polypeptides reveals eight highly-conserved domains (Figure The amino acid sequences of these domains are represented in the wheat SSIII amino acid sequence by the following sequence motifs: Region 1: KVGGLGDVVTS; Region 2: GHTVEVILPKY; Region 3: HDWSSAPVAWLYKEHY; Region 4: GILNGIDPDIWDPYTD; WO 00/66745 PCT/AU00/00385 -68- Region 5: DVPIVGIITRLTAQKG; Region 5a: NGQWLLGSA; Region 6: AGSDFIIVPSIFEPCGLTQLVAMRYGS; and Region 7: TGGLVDTV.

These conserved amino acid sequences are summarised in Table 4. As shown in Table 4 below, there is at least about 25% amino acid sequence identity, preferably at least about 30% amino acid sequence identity, more preferably at least about amino acid sequence identity, more preferably at least about 40% amino acid sequence identity, more preferably at least about 45% amino acid sequence identity, more preferably at least about 50% amino acid sequence identity, more preferably at least about 55% amino acid sequence identity, more preferably at least about amino acid sequence identity, more preferably at least about 65% amino acid sequence identity, more preferably at least about 70% amino acid sequence identity, more preferably at least about 75% amino acid sequence identity, more preferably at least about 80% amino acid sequence identity, more preferably at least about amino acid sequence identity, more preferably at least about 90% amino acid sequence identity and even more preferably at least about 95% amino acid sequence identity between the amino acid sequences of plant starch synthase enzymes, in particular wheat starch synthases.

From the data presented in Table 4, the most conserved regions of the wheat SSII and SSIII polypeptides are a region of 6 or 7 identical amino acids in Region 1 and a region of 8 or 9 identical amino acids in Region 6. The lowest regions of identity are found in regions 3 and For each of the amino acid sequences presented in the first column of Table 4, which are specific for wSSIII polypeptides, corresponding signature motifs which are specific for wSSII-A, wSSII-B, and wSSII-D polypeptides can be derived from the alignment, as follows: Region 1: KTGGLGDVAGA; WO 00/66745 PCT/AU00/00385 -69- Region 2: GHRVMVVVPRY; Region 3: NDWHTALLPVYLKAYY; Region 4: GIVNGIDNMEWNPEVD; Region 5: DVPLLGFIGRLDGQKG; Region 5a: DVQLVMLGTG; Region 6: AGADALLMPSRF(EN)PCGLNQLYAMAYGT; and Region 7: VGG(V/L)RDTV.

Comparison of the amino acid sequences of all available starch synthases with the deduced amino acid sequences of the three wSSII cDNA clones of the present invention wSSIIB, wSSIIA and wSSIID) was conducted using PILEUP analysis (Devereaux et al., 1984) and data are presented herein as a dendrogram (Figure The sequence of the glycogen synthase of E. coli was also included. Based upon their amino acid similarities, four classes of plant starch synthases can be defined: GBSS, SSI, SSII and SSIII.

Table 5 shows that levels of identity at the amino acid level between the wSSII sequences, as determined using the BESTFIT programme in GCG (Devereaux et al., 1984), and other class II starch synthases range from 70% identity with potato SSII to 85% identity with maize SSIlla. Both wSSIIB and wSSIID showed significantly higher homology to maize SSIIa than wSSIIA. Based upon sequence identities and the function of the Sgp-1 proteins in wheat, the wSSIIB, wSSIIA and wSSID cDNA clones are members of the starch synthase II (SSII) group and are more similar in sequence to maize SSIIa than maize SSIlb.

WO 00/66745 PCT/AU00/00385 70 TABLE 4 Identities between conserved motifs of plant starch synthases Sequence in wSSIII Number of conserved Number of conserved polypeptide residues between wheat residues between starch synthases wheat SSII and SSIII polypeptides Region 1: KVGGLGDWTS 6/11 residues 6/11 residues Region 2: GHTVEVILPKY 6/11 residues 6/11 residues Region 3: HDWSSAPVAWLYKEHY 4/16 residues 5/16 residues Region 4: GILNGIDPDIWDPYTD 7/16 residues 8/16 residues Region DVPIVGIITRLTAQKG 8/16 residues 10/16 residues Region NGQWLLGSA 4/10 residues 4/10 residues Region 6: AGSDFIIVPSIFEPCGLT 15/27 residues 17/27 residues

QLVAMRYGS

Region 7: TGGLVDTV 5/9 residues 5/9 residues WO 00/66745 PCT/AU00/00385 -71 TABLE wSSII-A wSSII-B wSSII-D wSSI-A 100% wSSII-B 95.9% 100% wSSII-D 96.3% 96.7% 100% maize SSIIla 76.1% 85.2% 84.7% maize SSIIb 76.3% 76.7% 75.9% pea SSII 72.0% 72.2% 71.8% potato SSII 70.9% 71.1% 70.3% Figure 11 shows a schematic representation of an alignment of plant starch synthase sequences, including wheat GBSS, wheat SSI, wheat SSII-A1, maize SSIla, and maize dull-1 polypeptides, in which the position of the first homologous region, comprising the consensus motif KXGG, is used as the basis of the alignment. The major differences in structure between the classes of genes are found in the length of the N-terminal region between the transit peptide and the first conserved region. At one extreme, the GBSS genes have a very short N-terminal arm, whereas the dul starch synthase contains a very long N-terminal extension containing several distinct regions. The wSSII genes contain an N-terminal extension which is longer than either GBSS, SSI, or SSIIb, and slightly longer than the maize SSlla gene.

EXAMPLE 16 Isolation of genomic clones for SSIII Screening of a genomic library from the D-genome donor of wheat, T. tauschii, identified a number of clones which hybridised to the wSSIII PCR fragment. Positive plaques in the genomic library were selected as those hybridising with a probe that had been generated by PCR (amplifying between nucleotide positions 3620 to 3966) from the SSIII cDNA as template. The primer sequences used were as follows: wSS3pa GGAGGTCTTGGTGATGTTGT SEQ ID NO: 29); and wSS3pb CTTGACCAATCATGGCAATG 3' SEQ ID NO: WO 00/66745 PCT/AU00/00385 -72- Hybridisation was carried out in 25% formamide, 6 x SSC, 0.1% SDS at 42 OC for 16 hour, then washed three times with 2 x SSC containing 0.1% SDS at 65 for 1 hour per wash. shows an example of a plaque lift showing positive and negative hybridisations for plaques containing the T. tauschii homologue of the wSSIII.B3 gene.

DNA was isolated from positive-hybridising A clones using methods described by Maniatis et al. Briefly, DNA was digested using BamHI or Bg/l and sub-cloned in to the vector pJKKmfm. DNA sequencing was performed using the automated ABI system with dye terminators as described by the manufacturers. DNA sequences were analysed using the GCG suite of programs (Devereaux et al., 1984).

Nucleotide sequences of the genomic SSIII clone from T. tauschii are provided herein as 6 contiguous sequences designated fragments 1 to 6 (SEQ ID NOs: 11 to 16, respectively). Table 6 defines the relative positions of these fragments with respect to the SSIII cDNA and describes the positions of exons. Figure 11 shows this information schematically.

The complete nucleotide sequence of a wheat SSIII genomic gene is presented herein as SEQ ID NO: 38. The structural features of this gene are presented in Table 7. A schematic representation of the intron/exon organisation of this gene is also presented in Figure 12.

EXAMPLE 17 Discussion Early work on the Sgp-1 starch synthase proteins (Denyer et al., 1995; Rahman et al., 1995) was based on the localisation of these proteins in the wheat starch granule, and no definitive conclusion conceming their presence or absence in soluble extracts of the wheat endosperm was presented.

We have now demonstrated that a monoclonal antibody against the Sgp-1 proteins cross reacts strongly with those starch synthase proteins having apparent molecular WO 00/66745 PCT/AU00/00385 -73weights of 100-105 kDa in soluble extracts, however, the appearance of these proteins in soluble extracts is dependant on the developmental stage of the endosperm material. Whilst the proteins can be detected in the soluble phase in early to mid endosperm development, little or no soluble protein remains in late endosperm development (Figure This observation accounts for the failure of Rahman et al.

(1995) to detect the protein in soluble extracts in a previous report.

Based upon the localisation of the Sgp-1 starch synthase proteins in the wheat endosperm, the following nomenclature is suggested for wheat starch synthase enzymes: wGBSS for the 60 kDa granule bound starch synthase wSSI for the kDa starch synthase I (Sgp-3); wSSII for the 100 105 kDa proteins (Sgp-1); and wSSIII for a soluble high molecular starch synthase.

The present invention provides cDNA and genomic clones encoding the wSSII and wSSIII polypeptides and the corresponding genomic clones. Whilst the evidence is compelling that the wSSIIA, wSSIIB and wSSIID cDNAs encode the Sgp-A1, Sgp-B1 and Sgp-D1 proteins of the wheat starch granule, molecular weights calculated from the deduced amino acid sequences of the clones are considerably lower than estimates obtained from SDS-PAGE. The molecular weight of the precursor wSSIIA protein is 87,229 Da, and the mature protein 81,164 Da, yet the estimated molecular weight in our experience is 105 kDa. The assignment of the wSSIIA cDNA to the Agenome of wheat is demonstrated in Figure 5, and the assignment of the 105 kDa protein to the A-genome in Denyer et al. (1995) and Yamamori and Endo (1996).

Similarly, the molecular weight of the wSSliB protein is 86,790 Da and the mature protein 80,759 Da, yet the molecular weight of the Sgp-B1 protein is estimated to be 100 kDa. No comparison can be made of the wSSIID sequences as a full length cDNA clone was not obtained. The wSSIIA and wSSIIB amino acid sequences differ by just a single amino acid residue, yet there is an apparent difference of 5 kDa in molecular weight when estimated by SDS-PAGE. Several possibilities can be advanced to account for this apparent discrepancy in molecular weights. Firstly, the wSSII proteins may not migrate in SDS-PAGE in accordance with their molecular weight because they WO 00/66745 PCT/AU00/00385 -74retain some conformation under the denaturing conditions used. Secondly, the proteins may be glycosylated. It is also possible that the proteins may be non-covalently linked to starch through a high affinity starch binding site which survives denaturation and SDS-PAGE. Differences between the apparent molecular weights and those calculated from the deduced amino acid sequences will have to be defined in establishing the relationship between the wSSII proteins and proteins encoded by the analogous SSII genes of other species.

The catalytic domain of the starch synthases is found at the C-terminal end of the protein (Gao et al., 1998; Ham et al., 1998). Ham et al. (1998) identified 7 conserved regions among SSIIa, SSIIb, SSI and GBSS sequences. We have identified an additional conserved region (designated region 5a in Table 4 and Figure comprising the amino acid sequence motif DVQLVMLGTG, by a comparison of the wSSII and wSSIII sequences of the present invention with differing isoforms of other plant starch synthases (GBSS, SS1, SSII and SSIII). The conservation of eight peptide regions among the 4 classes of starch synthases is striking, in terms of their sequence homologies and their alignment.

Analysis of the wheat SSII genes shows that there is a motif, PVNGENK, which is repeated. The area surrounding the repeated PVNGENK motif is not homologous to maize SSIIa and the insertion of this region is responsible for the difference in length between the wheat SSII and maize SSIIa genes. In pea and potato SSII polypeptides, a PPP motif (Figure 3; residues 251-253 and 287-289 respectively) has been suggested to mark the end of the N-terminal region and to facilitate the flexibility of an "N-terminal arm". This motif is not found in either the maize or wheat SSII sequences.

The generation of a wheat line combining null alleles at each of the three wSSII loci, wSSIIA, wSSIIB and wSSIID, has been reported recently by Yamamori (1998). In this triple null line, the large starch granules were reported to be mostly deformed and a novel starch with high blue value was observed when stained with iodine, indicating that wSSII is a key enzyme for the synthesis of starch in wheat. Further analysis of the WO 00/66745 PCT/AU00/00385 starch derived from this triple null mutant is in progress.

Mutations in starch synthases are known in three other species. In pea, mutation in SSII gives rise to starch with altered granule morphology and an amylopectin which yields an oligosaccharide distribution with reduced chain length on debranching, compared to the wild type (Craig et al., 1998). A similar mutation in a gene designated SSII is known in Chlamydomonas (the sta-3 mutation) and similar effects on granule morphology and amylopectin structure are observed (Fontaine et al., 1993). In maize, two mutations affecting starch synthases are known. First, the dull1 mutation has been shown to be caused by a lesion within the dul SSIII-type starch synthase gene (Gao et al., 1998). A second mutation, the sugary-2 mutation yields a starch with reduced amylopectin chain lengths on debranching (this mutation co-segregates with the SSIIa locus (Ham et al., 1998) although direct evidence that the sugary-2 mutation is caused by a lesion in the SSIIla gene is lacking). In the SSII mutants of each of these species, amylose biosynthesis capacity is retained, suggesting different roles in amylose and amylopectin synthesis for the GBSS and SSII genes. Given the conservation in overall organisation of the GBSS and SSII genes (see Figures 12 and 13), when an alignment is made based on the KTGGL motif of the first conserved region, this focuses attention on the role(s) of the N-terminal region in defining substrate specificity and the localisation of the proteins as the N-terminal region is the major area of divergence between the 4 classes of starch synthases. However, it is premature to exclude the influence of more subtle mutations in central and C-terminal regions of the gene.

The cloning of the wSSII and wSSIII cDNAs and genomic clones described herein provides useful tools for the further study of the roles of the starch synthases in wheat.

Firstly, they provide a source of markers which can be used to recover and combine null or divergent alleles. Secondly, genetic manipulation of wheat by gene suppression or over-expression can be carried out, and the genes may be used for over expression in other species. The promoter regions of these genes are also useful in regulating the expression of starch synthase genes and other heterologous genes in the developing wheat endosperm and in pre-anthesis florets of wheat.

TABLE 6 Summary of the Wheat Starch Synthase III Genomic Sequence Fragment in genomic DNA Length Features In SEQ ID NOS:11 to 16 Corresponding region In cDNA sequence clone Fragment 1 728 Translation start codon (nucleotides 287 to 289); (SEQ ID NO: 11) Exon 1.1 (nucleotides 260 to 385). Exon 1.1: nucleotides I to 126 Fragment 2 2446 Exon 2.1 nucleotides I to 1938); Exon 2.1: nucleotides 1008 to 2948; (SEQ ID NO: 12) Exon 2.2 (nucleotides 2197 to 2418). Exon 2.2: nucleotides 2949 to 3171 Fragment 3 1032 Exon 3.1 (nucleotides 310 to 580) Exon 3.1: nucleotides 3172 to 3440 (SEQ ID NO: 13) Fragment 4 892 Exon 4.1 (nucleotides 678 to 853) Exon 4. 1: nucleotides 3441 to 3616 (SEQ ID NO: 14) Fragment 5 871 Partial Exon 5.1 (nucleotides I to 29) Exon 5.1: nucleotides 3908 to 3937 (partial) (SEQ ID NO: 15) Exon 5.2 (nucleotides 293 to 463) Exon 5.2: nucleotides 3938 to 4108 Exon 5.3 (nucleotides 589 to 695) Exon 5.3: nucleotides 4109 to 4215 Fragment 6 1583 Exon 6.1 (nucleotides 471 to 653); Exon 6.1: nucleotides 4238 to 4420 (SEQ ID NO: 16) Exon 6.2 (nucleotides 770 to 902); Exon 6.2: nucleotides 4421 to 4552 Exon 6.3 (nucleotides 999 to 1110); Exon 6.3: nucleotides 4553 to 4664 Exon 6.4 (nucleotides 1201 to 1328); Exon 6.4: nucleotides 4665 to 4793 Partial Exon 6.5 (nucleotides 1408 to 1583); Exon 6.5: nucleotides 4794 to 4966 (partial) stop codon (nucleotides 1536 to 1538) WO 00/66745 WO 0066745PCTAUOOIOO385 77 TABLE 7 Structural features of the wheat starch synthase III genomic gene Nucleotide Position Feature Length (bases) in SEQ ID NO: 38 1- 973 5'-u ntra nscribed region and 973 sequence 974-1099 exoni1 126 1001-1003 translation start codon (ATG) 3 1100-2056 intronl1 957 2057-2120 exon 2 64 2121-2588 intron 2 468 2589-5291 exon 3 2703 5292 -5549 intron 3 258 5550-5767 exon 4 218 5768-6103 intron 4 336 6104-6374 exon 5 271 .6375 -7148 intron 5 774 7149-7324 exon 6 176 7325-7438 intron 6 114 7439-7546 exon 7 108 7547 -7792 intron 7 246 7793-7902 exon 8 110 7903-8797 intron 8 895 8798-8900 exon 9 103 8901-9164 intron 9 264 9165-9335 exonl10 171 9336-9460 intronl10 125 9461-9589 exonl11 129 9590-9677 intronl11 88 WO 00/66745 PCT/AU00/00385 -78- 9678-9860 exon 12 183 9861 -9977 intron 12 117 9978-10109 exon 13 132 10110-10205 intron 13 96 10206 -10317 exon 14 112 10318- 10407 intron 14 10408 10536 exon 15 129 10537-10618 intron 15 82 10619 -11146 exon 16 128 10744 10746 translation stop codon (TGA) 3 11147 -11611 3'-untranscribed region 465

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EDITORIAL NOTE APPLICATION NUMBER 40924/00 The following Sequence Listing pages 1 to 59 are part of the description. The claims pages follow on pages "82" to "98".

WO 00/66745 WO 0066745PCT/AUOO/00385 -1I- SEQUENCE LISTING <110> COMMONWEALTH SCIENTIFIC AND INDUSTRIAL RESEARCH ORGANISATION GOODMAN FIELDER LIMITED GROUPE LIMAGRAIN PACIFIC PTY LTD <120> NOVEL GENES ENCODING WHEAT STARCH SYN

THEREFOR

<130> p: \oper\mro\pi-wss.pct <140> TO BE ADVISED <141> 2000-04-28 <150> AU PQ0052/99 <151> 1999-04-29 <160> 54 <170> Patentln Ver. <210> 1 <211> 2939 <212> DNA <213> Triticum aestivum <22 0> <221> CDS <222> (176) (2569) <400> 1 atttcctcgg cctgaccccg tgcgtttacc ccacacagag cccactgccg cgctactccc cactcccact gccaccacct gcggaccaac ccgcgcatcg tatcacgatc acccaccccg THASES AND USES cacactccag ccgcctgcgc atcccggccg tcg Ser tcc Sex c ac His acg Thr gcg Ala ggt Gly ctc Leu tccagtccag cgcgctctgg ccgcc atg Met rtcc gcc tSer Ala cca ccc Pro Pro fcag cgc )Gin Arg faag gac Lys Asp ictc cgc LLeu Arg aag acg *Lys Thr rgca ccg )Ala Pro WO 00/66745 WO 0066745PCT/AUOO/00385 -2a gg Arg ggt Gly 130 ctg Leu ceg Pro ata Ile gae Asp tee Se r 210 gte Val1 gaa Giu caa Gin gtg Vali tte Phe 290 gte Val1 aca Th r aag Lys cag Gin 115 gaa Giu ec Pro gtg Val1 gc Ala aag Lys 195 ggc Gly agc Se r get Ala caa Gin gag G iu 275 gaa Glu atg Met ggt Giy aga.

Arg 100 gag Giu aae Asn gca Ala aat As n gag Glu 180 gcg Ala tea Ser g ac Asp cca Pro gac Asp 260 gee Ala eae His aae Asn ggt Gly gga Gly 340 gae Asp a aa Lys cee Pro ggt Gly 165 gte Val e ea Pro aat Asn g tg Val aae Asn 245 e tt Leu aag Lys eae His gtg Val ett Leu 325 eat His gee Ala tet Se r gea.

Ala 150 gaa.

Giu geg Ala gag Glu tte Phe gaa Giu 230 eea.

Pro tgg rrp gat Asp cag Gln gte Val 310 gga Gly cgt Arg eg t Arg aee Th r 135 ege Arg aae Asn get Ala tee Se r gtg Val1 215 ett Leu aag Lys gae Asp ga t Asp aat Asn 295 gte Val1 gat Asp gtt Val1 etg Leu 120 gge Gly geg Ala aaa Lys eeg Pro gtt Val1 200 ec Pro gaa.

Glu get Ala t te Phe gge Gly 280 e ae His gtg Val gtt Val atg Met 105 eeg Pro ggC G ly eee Pro get Ala gat Asp 185 gte Val1 teg Se r etg Leu ett Leu aag Lys 265 egg Arg ga t Asp get Al a gee Ala gtt Val1 345 age atg aae Ser Met Asn gge Gly eag Gin aae Asn 170 eee Pro eea Pro get Ala aag Lys teg Se r 250 aaa Lys get Ala tee Se r get Ala ggt Gly 330 g tg Val gge Gly eeg Pro 155 gte Val1 gea Ala gee Ala tet Se r aag Lys 235 eeg Pro tae Tyr gtt Val1 ggg Giy gaa Giu 315 get Ala gta Val1 geg Ala 140 teg Ser gee Ala get Ala gag Giu get Ala 220 ggt Giy eee Pro att Ile gea Ala eet Pro 300 tgt Cys t tg Leu eea Pro gge Gly 125 act Thr age Ser teg Se r aee Thr aag Lys 205 eee Pro geg Ala gea Ala ggt Gly gat Asp 285 ttg Leu tet Ser ec Pro agg Arg 110 a tg Met aaa Lys cag Gin ceg Pro a tt Ile 190 geg Ala ggg Gly gte Val gea Ala tte Phe 270 gat Asp gea Ala zee Pro aag Lys tat Tyr 350 ceg gtg aae Pro Val Asn gac Asp aa e Asn ceg Pro 175 tee Ser ceg Pro tet Se r att Ile c Pro 255 gag Glu geg Ala ggg Gly tgg T rp get Ala 335 ggg Gly age Se r aga Arg 160 aeg Th r ate Ile ceg Pro gac Asp gte Val1 240 get Ala gag Giu gge Gly gag Giu tge Cys 320 ttg Leu gac Asp ggg Gly 145 gta.

Val1 age Se r agt Se r teg Se r act Thr 225 aaa Lys gta Val1 ccc Pro tee Ser aae Asn 305 aaa.

Lys geg Ala tat Tyr 562 610 658- 706 754 802 850 898 946 994 1042 1090 1138 1186 1234 1282 gag g~a gee tac gat gte gga Glu Giu Ala Tyr Asp Val Gly 355 360 gte ega aaa tae Val Arg Lys Tyr aag get get gga.

Lys Ala Ala Gly WO 00/66745 WO 0066745PCT/AUOO/00385 -3cag gat atg Gin Asp Met 370 ttt gtg ttc Phe Val Phe tat ggg ggc Tyr Gly Gly aag gcc get Lys Ala Ala 420 tat ggg gat Tyr Gly Asp 435 ctc ctg cct Leu Leu Pro 450 cag tac act Gin Tyr Thr cgt ggc cca Arg Gly Pro ctg gaa cac Leu Giu His 500 tac ttc gcc Tyr Phe Ala 515 ccg ggg tac Pro Gly Tyr 530 cac gac ate His Asp Ile ggc atc gac Gly Ile Asp tcg gac ggc Ser Asp Gly 580 cgg cag tgc Arg Gin Cys 595 ggc gac gtg Gly Asp Val get tat Ala Tyr 380 cga cac Arg His 395 aag cgc Lys Arg gtt cca Val Pro gca aat Ala Asn tac agg Tyr Arg 460 cat aae His Asn 475 ace gag Thr Giu gtg ggt Val Gly gac cag Asp Gin gtg gag Val Giu 540 aag ace Lys Thr 555 gag gtg Giu Val ggg aeg Gly Thr gag ctg Giu Leu ate gat gga gtt Ile Asp Gly Vai 1330 1378 1426 1474 1522 1570 1618 1666 1714 17 62 1810 1858 1906 1954 2002 2050 ccg ctg etc Pro Leu Leu ggc ttc ate ggg Gly Phe Ile Gly cgc etg gac ggg cag aag Arg Leu Asp Giy Gin Lys 620 625 610 615 WO 00/66745 WO 0066745PCT/AUOO/00385 -4ggc gtg gag atc atc Gly Val Giu Ile Ile 630 geg gac geg atg Ala Asp Ala Met gtg cag ctg Val Gin Leu ctg cgg cac Leu Arg His 660 ggg ttc tee Giy Phe Ser 675 cte ctc atg Leu Leu Met 690 gee atg gcc Ala Met Aia agg gac ace Arg Asp Thr tgg acg tte Trp Thr Phe 740 eac tgc etc His Cys Leu 755 cag gag cgc Gin Giu Arg 770 etc tac gag Leu Tyr Glu gte atg Vai Met 645 ttc gag Phe Glu gtg egg Val Arg eec tee Pro Ser tae gge Tyr Gly 710 gtg eeg Vai Pro 725 gac ege Asp Arg ege ace Arg Thr ggc atg Giy Met gac gte Asp Vai 790 etg gge Leu Giy egg gag Arg Glu ctg geg Leu Aia 680 egg tte Arg Phe 695 ace gte Thr Val ccg ttc Pro Phe gca gag Aia Giu tae egg Tyr Arg 760 tcg cag Ser Gin 775 etc gte Leu Val aee ggg Thr Giy 650 cac eae His His 665 eac egg His Arg gag ceg Glu Pro eee gte Pro Val gac ec Asp Pro 730 geg cag Ala Gin 745 gae tae Asp Tyr gae ttc Asp Phe aag gee Lys Ala ccc Pro 635 cgc Arg gac Asp ate Ile tge Cys gtg Val 715 ttc Phe aag Lys aag Lys age Se r aag tgg ate gtg agc cag gac Trp Ile Vai Ser Gin Asp 640 eae His aag Lys acg Tb r gga Gly 700 eat His a ac Asn ctg Leu gag Giu tgg Trp 780 tae gac Asp gtg Val1 gee Ala 685 etg Leu gcc Ala eac His atc Ile agc Se r 765 gag Giu eag Gin etg gag gge atg Leu Giu Gly Met 655 egc ggg tgg gtg Arg Gly Trp Val 670 gge gee gac geg Gly Ala Asp Ala aac eag etc tae Asn Gin Leu Tyr 705 gte ggt gge etg Val Gly Giy Leu 720 tec ggg cte ggg Ser Gly Leu Gly 735 gag geg etc ggg Giu Ala Leu Gly 750 tgg agg ggg ete Trp Arg Gly Leu eat gee gcc aag His Ala Ala Lys 785 tgg tgaaegetag Trp 2098 2146 2194 2242 2290 2338 2386 2434 2482 2530 2579 2639 2699 2759 2819 2879 2939 Lys Tyr 795 ctgetageeg gtceagceec geatgegtge atgacaggat ggaattgcge attgcgcaeg eaggaaggtg ccatggageg ceggeateeg egaagtacag tgaeatgagg tgtgtgtggt tgagaegetg atteegatet ggteegtage agagtagage ggaggtaggg aagcgetect tgttacaggt atatgggaat gttgttaact tggtattgta atttgttatg ttgtgtgeat tattacagag ggcaaegate tgegceggeg eaeeggccca aetgttggge eggtegcaea geagecgttg gateegaccg eetgggccgt tggatcecac egaaaaaaaa aaaaaaaaaa (210> 2 <211> 798 <212> PRT <213> Tritieum aestivum <400> 2 Met Ser Ser Ala Vai Ala Ser Ala Ala Ser Phe Leu Ala Leu Ala Ser 1 5 10 WO 00/66745 PCT/AU00/00385 Ala Pro Arg Asp Arg Thr Pro Asn Gly 145 Val Ser Ser Ser Thr 225 Lys Val Pro Ser Asn 305 Lys Ala Ser His Thr Ala Gly Leu Arg Gly 130 Leu Pro Ile Asp Ser 210 Val Glu Gin Val Phe 290 Val Thr Lys Gly Arg Gly Ala Arg Asp Ile Asp Ala Ala Arg Asp 100 Glu Asp Asn Lys Ala Pro Asn Gly 165 Glu Val 180 Ala Pro Ser Asn Asp Val Pro Asn 245 Asp Leu 260 Ala Lys His His Asn Val Gly Leu 325 Gly His 340 Arg Arg Ala 55 Ala Lys Ala Arg Thr 135 Arg Asn Ala Ser Val 215 Leu Lys Asp Asp Asn 295 Val Asp Val Arg His Ala Pro Ala Gly 105 Pro Gly Pro Ala Asp 185 Val Ser Leu Leu Lys 265 Arg Asp Ala Ala Val 345 Arg Val Pro Pro Arg Ala Arg Gin Arg Arg Ala Pro Met Asn Gly Ala 140 Pro Ser 155 Val Ala Ala Ala Ala Glu Ser Ala 220 Lys Gly 235 Pro Pro Tyr Ile Val Ala Gly Pro 300 Glu Cys 315 Ala Leu Val Pro Ser Pro Lys Ala Val Pro Pro Asp Asn Pro 175 Ser Pro Ser Ile Pro 255 Glu Ala Gly Trp Ala 335 Gly Tyr Glu Glu Ala Tyr Asp Val Gly Val Arg Lys Tyr Tyr Lys Ala Ala WO 00166745 WO 0066745PCT/AUOO/00385 -6- Gly Gin 370 Asp Phe 385 Ile Tyr Cys Lys Pro Tyr Ala Leu 450 Met Gin 465 Gly Arg Tyr Leu Asn Tyr Ser Pro 530 Leu His 545 Asn Giy Lys Ser Lys Arg Arg Gly 610 Lys Gly 625 Asp Val Met Leu Vai Gly Ala Leu 690 Met Giu Phe .1Ile Giy Ser 405 Aia Val 420 Asp Giy Pro Vai Thr Arg Pro Val 485 His Phe 500.

Ala Ala Tyr Leu Ile Ile Asp Asn 565 Gly Tyr 580 Cys Lys Va' Pro Glu Ile Leu Val 645 His Phe 660 Ser Val Met Pro 360 Asn Tyr 375 Ala Pro Gin Giu Val Pro Leu Val 440 Leu Lys 455 Ile Met Giu Phe Leu Tyr Leu Lys 520 Glu Leu 535 Gin Asn Giu Trp Asn Phe Aia Leu 600 Leu Gly 615 Ala Asp Leu Gly Arg Giu Leu Ala 680 Arg Phe 695 His Phe Met 410 His Ile Tyr Ile Phe 490 Pro Ala Thr Trp Pro 570 Leu Arg Ile Met Gly 650 His Arg Pro Ala Arg 395 Lys Val Ala Tyr His 475 Thr Val1 Asp Val Lys 555 Giu Gly Glu Gly Pro 635 Arg Asp Ile Cys Tyr 380 His Arg Pro Asn Arg 460 Asn Giu Gly Gin Glu 540 Thr Val1 Th r Leu Arg 620 Trp His Lys Thr Gly 700 365 Ile Asp Arg Gin Met Ile Cys Gly 430 Asp Trp 445 Asp His Ile Ala Leu Pro Gly Giu 510 Val Val 525 Gly Gly Arg Gly Asp Val Leu Asp 590 Gly Leu 605 Leu Asp Ile Val Asp Leu Val Arg 670 Ala Gly 685 Leu Asn Gly Vai Glu Asp 400 Leu Phe 415 Gly Val His Thr Gly Leu His Gin 480 Glu His 495 His Ala Val Vai Trp Giy Ile Val 560 His Leu 575 Ser Gly Gin Val Gly Gin Ser Gin 640 Giu Gly 655 Gly Trp Ala Asp Gin Leu WO 00/66745 WO 0066745PCT/AUOO/00385 -7- Tyr Ala Met Ala Tyr Gly Thr Val Pro Val Val His Ala Val Gly Giy 705 710 715 .720 Leu Arg Asp Thr Val Pro Pro Phe Asp Pro Phe Asn His Ser Gly Leu 725 730 735 Gly Trp Thr Phe Asp Arg Ala Glu Ala Gin Lys Leu Ile Glu Ala Leu 740 745 750 Gly His Cys Leu Arg Thr Tyr Arg Asp Tyr Lys Glu Ser Trp Arg Gly 755 760 765 Leu Gin Giu Arg Gly Met Ser Gin Asp Phe Ser Trp Giu His Ala Ala 770 775 780 Lys Leu Tyr Glu Asp Val Leu Val Lys Ala Lys Tyr Gin Trp 785 790 795 <210> 3 <211> 2842 <212> DNA <213> Triticum aestivum <220> <221> CDS <222> (2485) <400> 3 gctgccacca cctccgcctg cgccgcgctc tgggcggagg accaacccgc gcatcgtacc atcgcccgcc ccgatcccgg ccgccgcc atg tcg tcg gcg gtc gcg tcc gcc 112 Met Ser Ser Ala Val Ala Ser Ala 1 gcg tcc ttc ctc gcg cic gcc tcc gcc tcc ccc ggg aga tca cgc agg 160 Ala Ser Phe Leu Ala Leu Ala Ser Ala Ser Pro Gly Arg Ser Arg Arg 15 cgg gcg agg gtg agc gcg ccg cca ccc cac gcc ggg gcc ggc agg ctg 208 Arg Ala Arg Val Ser Ala Pro Pro Pro His Ala Gly Ala Gly Arg Leu 30 35 cac tgg ccg ccg tgg ccg ccg cag cgc acg gct cgc gac gga ggt gtg 256 His Trp Pro Pro Trp Pro Pro Gin Arg Thr Ala Arg Asp Gly Gly Val 50 gcc gcg cgc gcc gcc ggg aag aag gac gcg agg gtc gac gac gac gee 304 Ala Ala Arg Ala Ala Gly Lys Lys Asp Ala Arg Va'. Asp Asp Asp Ala 65 gcg tcc gcg agg cag ccc cgc gca cgc cgc ggt ggc gcc gcc acc aag 352 Ala Ser Ala Arg Gin Pro Arg Ala Arg Arg Gly Gly Ala Ala Thr Lys 80 gtc gcg gag cgg agg gat ccc gte aag acg ctc gat cgc gac gcc gcg 400 Val Ala Giu Arg Arg Asp Pro Val Lys Thr Leu Asp Arg Asp Ala Ala 95 100 gaa ggt ggc gcg ccg gca ceg ccg gca ccg agg cag gac gcc gcc egt 448 Glu Gly Gly Ala Pro Ala Pro Pro Ala Pro Arg Gin Asp Ala Ala Arg 105 110 115 120 cca ccg agt atg aac ggc acg ccg gtg aac ggt gag aac aaa tct acc 496 Pro Pro Ser Met Asn Gly Thr Pro Val Asn Gly Giu Asn Lys Ser Thr WO 00/66745 PCT/AUOO/00385 -8ggc Gly gcg Ala aaa Lys ccg Pro 185 gtt Val gtc Val gaa Glu gct Ala ttc Phe 265 ggc Gly cat His gtg Val gtt Va1 atg Met 345 gga Gly ggc Gly ccc Pro gct Ala 170 gat Asp gtc Va1 tcg Ser ctg Leu ctt Leu 250 aag Lys tgg Trp gat Asp gct Ala gcc Ala 330 gtt Va1 gtc Val ggc Gly cat His 155 aac Asn tcc Ser cca Pro gct Ala aag Lys 235 tcg Ser aaa Lys gct Ala tcC Ser gct Ala 315 ggt Gly gtg Va1 cga Arg ggc Gly 140 ccg Pro gtc Val gca Ala gcc Ala tct Ser 220 aag Lys ccg Pro tac Tyr gtt Val gga Gly 300 gaa Glu gct Ala gta Val aaa Lys acc Thr acc Thr tcg Ser acc Thr 190 aag Lys ccc Pro gcg Ala gca Ala ggc Gly 270 gat Asp ttg Leu tct Ser ccc Pro agg Arg 350 tac Tyr aaa Lys cag Gin ccg Pro 175 att Ile ccg Pro agg Arg gtc Val gcc Ala 255 ttc Phe gat Asp gca Ala ccc Pro aag Lys 335 tat Tyr aag Lys gac Asp aac Asn 160 ccg Pro tcc Ser ccg Pro ctg Leu atc Ile 240 ccc Pro gag Glu gcg Ala ggg Gly tgg Trp 320 gct Ala ggg Gly gct Ala 130 agc ggg Ser Gly 145 aga gta Arg Val *acg agc Thr Ser atc agt Ile Ser ccg tcg Pro Ser 210 gac att Asp Ile 225 gtc gaa Val Glu gct gta Ala Val gag ccc Glu Pro ggc tcc Gly Ser 290 gag aac Glu Asn 305 tgc aaa Cys Lys ttg gcg Leu Ala gac tat Asp Tyr gct gga Ala Gly 370 ccc Pro gtg Val gcc Ala 180 aag Lys ggc Gly agc Ser gct Ala gaa Glu 260 gag Glu gaa Glu atg Met ggt Gly aga Arg 340 gaa Glu gat Asp gca Ala aac Asn 165 gag Glu gcg Ala tca Ser gat Asp cca Pro 245 gac Asp gcc Ala cat His aac Asn ggt Gly 325 gga Gly gcc Ala atg Met ccc Pro 150 ggt Gly gtc Va1 ccg Pro aat Asn gtt Va1 230 aac Asn ctt Leu aag Lys cac His gtg Va1 310 ctt Leu cat His tac Tyr gaa Glu 135 gca Ala gaa Glu gtg Va1 gag Glu ttc Phe 215 gaa Glu cca Pro tgg Trp gat Asp cag Gln 295 gtc Va1 gga Gly cgt Arg gat Asp gtg Va1 375 cgc Arg aac Asn gct Ala tcc Ser 200 gtg Val cct Pro aag Lys gac Asp gat Asp 280 aac Asn gtc Val gat Asp gtt Va1 gtc Val 360 aat Asn 544 592 640 688 736 784 832 880 928 976 1024 1072 1120 1168 1216 1264 tat ttc cat gct tat atc gat Tyr Phe His Tyr Ile Asp gga gtt gat ttt gtg ttc att gac gct Gly Val Asp Phe Val Phe Ile Asp Ala 385 390 WO 00/66745 WO 0066745PCT/AUOO/00385 -9cc t Pro gaa Glu cc t Pro 425 gtg Val1 aaa Lys atg Met ttc Phe tac Tyr 505 aag Lys ctc Leu aac Asn tgg Trp ttc Phe 585 ctg Leu ggc Gly gac Asp c tc Leu att Ile 410 tgg T rp ttt Phe gca Ala gtg Val1 ccg Pro 490 gac Asp atg Met aag Lys gac Asp aa c Asn 570 tcc Ser cag Gin ttc Phe gcc Ala cga Arg aag Lys gtt Val gca Ala tac Tyr 460 cat His a cc Th r gtg Val gac Asp gtg Val 540 aag Lys gag Giu ggg Gly gag Glu ggc Gly 620 ccc Pro gac Asp ttc Pheg tc Val1 acg Thr t tg Leu 465 cag Gin cac His gcc Ala g tg Val ggg Gly 545 gtc Val ctc Leu ggc Gly gtc ValI cag Gin 625 cag Gin ggg ggc Gly Gly 405 gcc gct Ala Ala 420 ggg gat Gly Asp ctg cct Leu Pro tac act Tyr Thr ggc cca Gly Pro 485 gaa cac Giu His 500 ttc gcc Phe Ala ggg tac Gly Tyr gac atc Asp Ile atc gac Ile Asp 565 gac ggc Asp Gly 580 cag tgc Gin Cys gac gtg Asp Val gtg gag Val Giu cag ctg Gin Leu 645 1312 1360 1408 1456 1504 1552 1600 1648 1696 1744 1792 1840 1888 1936 1984 2032 WO 00/66745 WO 0066745PCT/AUOO/00385 ggc acc ggc cgc cae gac etg gag agc Gly Thr Gly Arg His Asp Leu Glu Ser 650 655 gag Glu 665 gcg Ala ttc Phe gte Val1 ttc Phe gag Glu 745 egg Arg.

cag Gin ctc cac His c9gg Arg ceg Pro gte Val 715 ccc Pro eac His tac Tyr ttc Phe gcc aag gtg Lys Val 670 aeg geg Thr Ala 685 ggg ttg Gly Leu cac gee His Ala aae eae Asn His etg ate Leu Ile 750 gag age Glu Ser 765 tgg gag Trp Glu tae eag ege ggg tgg Arg Gly Trp gge gee gae Gly Ala Asp aae eag ett Asn Gin Leu 705 gte gge ggg Val Gly Gly 720 tee gge etc Ser Gly Leu 735 gag geg etc Glu Ala Leu tgg agg gge Trp Arg Gly eat gee gee His Ala Ala 785 tgg tgaaeget tte gag cgg Phe Glu Arg gtg ege etg Val Arg Leu 680 eee tce egg Pro Ser Arg 695 tae gge ace Tyr Gly Thr 710 gtg ceg eeg Val Pro Pro gae cge gee Asp Arg Ala ege aec tac Arg Thr Tyr 760 ggc atg teg Gly Met Ser 775 gae gte etc Asp Val Leu 790 2080 2128 2176 2224 2272 2320 2368 2416 2464 2515 2575 2635 2695 2755 2815 2842 Lys Leu Tyr Glu ~ag ctgctagceg etecagcee Leu Lys Ala Lys Tyr Gin Trp 795 geatgcgtge atgcatgaga gggtggaact gegeattgeg eecgcaggaa egtgccatcc ttctegatgg gagegeegge ateegegagg tgeagtgaea tgagaggtgt gtgtggttga gaegetgatt eegatetega tctggtcegt ageagagtag agcggaegta gggaagegcZt eettgttgea ggtatatggg aatgttgtea aettggtatt gtagtttget atgttgtatg cgttattaea atgttgttac ttattettgt taagteggag geaaagggcg aaagetaget caeatgaaaa aaaaaaaaaa aaaaaaa (210> 4 (211> 799 (212> PRT <213> Triticum aestivum <400> 4 Met Ser Ser Ala Val Ala Ser Ala Ala Ser Phe Leu Ala Leu Ala Ser 1 5 10 Ala Ser Pro Gly Arg Ser Arg Arg Arg Ala Arg Val Ser Ala Pro Pro 25 WO 00/66745 WO 0066745PCT/AUOO/00385 -1I1I- Pro His Arg Thr Asp Ala Arg Arg Lys Thr Ala Pro Val Asn 130 Ser Giy 145 Arg Vai Thr Ser Ile Ser Pro Ser 210 Asp Ile 225 Val Glu Ala Vai Glu Pro Gly Ser 290 Glu Asn 305 Cys Lys Leu Ala Asp Tyr Ala Gly 370 Ala Al a Arg Gly Leu Arg 115 Gly Leu Pro Ile Asp 195 Se r Asp Glu Gin Val1 275 Phe Val1 Thr Lys Glu 355 Gin Gly Ala Arg Asp Val Asp Giy Ala Asp Arg 100 Gin Asp Giu Asn Pro Ala Val Asn 165 Ala Glu 180 Lys Ala Giy Ser Ser Asp Aia Pro 245 Giu Asp 260 Giu Ala Glu His Met Asn Gly Gly 325 Arg Gly 340 Giu Ala Asp Met Gly Gly Asp Ala Asp Ala Lys Pro 150 Gly Val1 Pro Asn Val1 230 As n Leu Lys His Val1 310 Leu His Ty r Glu Arg Gly 55 Asp Th r Ala Ala Se r 135 Ala Giu Val Glu Phe 215 Giu Pro Trp Asp Gin 295 Val Gly Arg Asp Val1 375 Leu 40 Val1 Ala Lys Ala Arg 120 Thr Arg Asn Ala Se r 200 Val1 Pro Lys Asp Asp 280 Asn Val1 Asp Val Val 360 Asn Pro Pro Trp Pro Pro Gin Ala Arg Arg Ala Met Gly 140 Pro Val Ala Ala Se r 220 Lys Pro Tyr Val Gly 300 Glu Ala Val1 Lys Ala 380 Ala Gin Arg Pro Asn 125 Ala Ser Ala Ala Giu 205 Ala Gly Pro Ile Ala 285 Pro Cys Le u Pro Tyr 365 Tyr Gly Pro Asp Ala 110 Gly Thr Thr Ser Th r 190 Lys Pro Ala Ala Gly 270 Asp Leu Se r Pro Arg 350 Tyr Ile WO 00/66745 PCT/AU00/00385 -12- Val Asp Phe Val Phe Ile Asp Ala Pro Leu Phe Arg His Arg Gin Glu 385 390 395 400 Asp Ile Tyr Gly Gly Ser Arg Gin Glu Ile Met Lys Arg Met Ile Leu 405 410 415 Phe Cys Lys Ala Ala Val Glu Val Pro Trp His Val Pro Cys Gly Gly 420 425 430 Val Pro Tyr Gly Asp Gly Asn Leu Val Phe Ile Ala Asn Asp Trp His 435 440 445 Thr Ala Leu Leu Pro Val Tyr Leu Lys Ala Tyr Tyr Arg Asp His Gly 450 455 460 Leu Met Gin Tyr Thr Arg Ser Ile Met Val Ile His Asn Ile Ala His 465 470 475 480 Gin Gly Arg Gly Pro Val Asp Glu Phe Pro Phe Thr Glu Leu Pro Glu 485 490 495 His Tyr Leu Glu His Phe Arg Leu Tyr Asp Pro Val Gly Gly Glu His 500 505 510 Ala Asn Tyr Phe Ala Ala Gly Leu Lys Met Ala Asp Gin Val Val Val 515 520 525 Val Ser Pro Gly Tyr Leu Trp Glu Leu Lys Thr Val Glu Gly Gly Trp 530 535 540 Gly Leu His Asp Ile Ile Arg Gin Asn Asp Trp Lys Thr Arg Gly Ile 545 550 555 560 Val Asn Gly Ile Asp Asn Met Glu Trp Asn Pro Glu Val Asp Val His 565 570 575 Leu Lys Ser Asp Gly Tyr Thr Asn Phe Ser Leu Gly Thr Leu Asp Ser 580 585 590 Gly Lys Arg Gin Cys Lys Glu Ala Leu Gin Arg Glu Leu Gly Leu Gin 595 600 605 Val Arg Ala Asp Val Pro Leu Leu Gly Phe Ile Gly Arg Leu Asp Gly 610 615 620 Gin Lys Gly Val Glu Ile Ile Ala Asp Ala Met Pro Trp Ile Val Ser 625 630 635 640 Gin Asp Val Gin Leu Val Met Leu Gly Thr Gly Arg His Asp Leu Glu 645 650 655 Ser Met Leu Arg His Phe Glu Arg Glu His His Asp Lys Val Arg Gly 660 665 670 Trp Val Gly Phe Ser Val Arg Leu Ala His Arg Ile Thr Ala Gly Ala 675 680 685 Asp Ala Leu Leu Met Pro Ser Arg Phe Glu Pro Cys Gly Leu Asn Gin 690 695 700 Leu Tyr Ala Met Ala Tyr Gly Thr Val Pro Val Val His Ala Val Gly 705 710 715 720 Gly Val Arg Asp Thr Val Pro Pro Phe Asp Pro Phe Asn His Ser Gly WO 00/66745 WO 0066745PCT/AUOO/00385 13 725 Leu Gly Trp Thr Phe Asp Arg Ala Giu 740 745 Leu Gly His Cys Leu Arg Thr Tyr Arg 755 760 Gly Leu Gin Glu Arg Gly Met Ser Gin 770 775 Ala Lys Leu Tyr Glu Asp Val Leu Leu 785 790 <210> <211> 2107 <212> DNA <213> Triticum aestivum 735 His Lys Leu Ile Glu Ala 750 Tyr Lys Glu Ser Trp Arg 765.

Phe Ser Trp Glu His Ala 780 Ala Lys Tyr Gin Trp 795 <220> <221> CDS <222> (1791) <400> eca get. gag aag acg Pro Ala Glu Lys Thr 1 5 gec tct gct ccc ggg Ala Ser Ala Pro Gly aag aag ggt gcg gtc Lys Lys Gly Ala Val teg ecg cet gca gcc Ser Pro Pro Ala Ala aaa tac att ggt ttc Lys Tyr Ile Gly Phe get gtc gca gat gat Ala Val Ala Asp Asp tcc gga cct ttg gca Ser Gly Pro Leu Ala 100 gct gag tgt tet ccc Ala Glu Cys Ser Pro 115 ggt get ctg ccc aag Gly Ala Leu Pro Lys 130 gtg gta eca agg tat Val Val Pro Arg Tyr 145 ccg ccg tcg tce ggc tea aat ttc gag tee teg Pro Pro Ser Ser Ser Asn Phe Glu Ser Ser WO 00/66745 WO 0066745PCT/AUOO/00385 -14cga Arg cat His ttc Phe atg Met cac His 225 att Ile tat Tyr ata Ile ttc Phe ccc Pro 305 gcg Ala acg Th r tgg Trp ccc Pro ctg Leu 385 cgc Arg atc gc t Ala gga Gly gaa Giu ttg Leu ggt Gly 230 cac His gg t Gly cac His gag Glu cac His 310 gtg Val1 tgg T rp atc Ile cac His tcc Se r 390 cag Gin ggg gga Gly gat Asp att Ile 200 tgc Cys cct Pro gca Ala atg Met ggc Gly 280 tac Tyr aac Asn a gc Se r ctt Leu aac Asn 360 aag Lys aag Lys cgc Arg a ag gaa Glu att Ile agc Se r gt t Vali 220 gga Gly gtc Vali Arg gta Val ttc Phe 300 gcc Ala ctg Leu ata Ile aac Asn tac Tyr 380 aag Lys ccg Pro atc 528 576 624 672 720 768 816 864 912 960 1008 1056 1104 1152 1200 1248 1296 WO 00/66745 WO 0066745PCTAUOOOO385 Ile Gly Arg Leu Asp Gly Gin Lys Gly Val Glu Ile Ile Ala Asp Ala 420 425 430 atg Met ggg Gly c ac His 465 egg Arg ccg Pro gte Val ccc Pro cac His 545 tte Phe tte Phe gee tgg ate Trp Ile 435 eae gae His Asp aag gtg Lys Val aeg geg Thr Ala ggg etg Giy Leu 500 eae gee His Ala 515 aae eae Asn His ctg ate Leu Ile gag age Glu Ser tgg gag Trp Glu 580 tae eag age Se r gag Glu ggg Gly 470 geg Ala eag Gin gge Gly ggg Gly geg Ala 550 agg Arg gee eag Gin age Se r 455 tgg Trp gae Asp ete Leu gge Gly etc Leu 535 etc Leu gee Ala gee eag etg Gin Leu cag eac Gin His tte tee Phe Ser 475 etc atg Leu Met 490 atg gee Met Ala gac ace Asp Thr aeg tte Thr Phe tge etc Cys Leu 555 gag ege Glu Arg 570 tac gag ctg Leu egg Arg etg Leu egg Arg ace Thr 510 ceg Pro gee Ala tac Tyr teg Se r etc ggc ace Gly Thr gag cac Glu His geg cac Ala His 480 tte gtg Phe Val 495 gte ccc Val Pro ttc gac Phe Asp gag geg Glu Ala ega. gac Arg Asp 560 cag gae Gin Asp 575 gte aag 1344 1392 1440 1488 1536 1584 1632 1680 1728 1776 1831 1891 1951 2011 2071 2107 Ala Ala Lys Leu Tyr Glu Asp Val Leu Val Lys 585 590 tgaaegctag ctgetagecg etecagccec gcatgcgtgc Ala Lys Tyr Gin Trp 595 atgacaggat ggaaetgeat tgcgeaegea ggaaagtgcc atggagegee ggcateegeg aagtacagtg aeatgaggtg tgtgtggttg agacgctgat teeaateegg ccgtagcag agtagagcgg aggtatatgg gaatettaae ttggtattgt aatttgttat gttgtgtgea ttattaeaat gttgttaett attcttgtta agteggagge caagggegaa agetagetea eatgtetgat ggatgeaaaa aaaaaaaaaa aaaaaa <210> 6 <211> 597 <212> PRT <213> Triticum aestivwn <400> 6 Pro Ala Glu Lys Thr Pro Pro Ser Ser Gly Ser Asn Phe Glu. Ser Ser WO 00/66745 PCT/AU00/00385 -16- 1 5 10 Ala Ser Ala Pro Gly Ser Asp Thr Val Ser Asp Val Glu Gin Glu Leu 25 Lys Lys Gly Ala Val Val Val Glu Glu Ala Pro Lys Pro Lys Ala Leu 40 Ser Pro Pro Ala Ala Pro Ala Val Gin Glu Asp Leu Trp Asp Phe Lys 55 Lys Tyr Ile Gly Phe Glu Glu Pro Val Glu Ala Lys Asp Asp Gly Arg 70 75 Ala Val Ala Asp Asp Ala Gly Ser Phe Glu His His Gin Asn His Asp 90 Ser Gly Pro Leu Ala Gly Glu Asn Val Met Asn Val Val Val Val Ala 100 105 110 Ala Glu Cys Ser Pro Trp Cys Lys Thr Gly Gly Leu Gly Asp Val Ala 115 120 125 Gly Ala Leu Pro Lys Ala Leu Ala Lys Arg Gly His Arg Val Met Val 130 135 140 Val Val Pro Arg Tyr Gly Asp Tyr Glu Glu Pro Thr Asp Val Gly Val 145 150 155 160 Arg Lys Tyr Tyr Lys Ala Ala Gly Gin Asp Met Glu Val Asn Tyr Phe 165 170 175 His Ala Tyr Ile Asp Gly Val Asp Phe Val Phe Ile Asp Ala Pro Leu 180 185 190 Phe Arg His Arg Glu Glu Asp Ile Tyr Gly Gly Ser Arg Gin Glu lle 195 200 205 Met Lys Arg Met lie Leu Phe Cys Lys Ala Ala Val Glu Val Pro Trp 210 215 220 His Val Pro Cys Gly Gly Val Pro Tyr Gly Asp Gly Asn Leu Val Phe 225 230 235 240 lie Ala Asn Asp Trp His Thr Ala Leu Leu Pro Val Tyr Leu Lys Ala 245 250 255 Tyr Tyr Arg Asp His Gly Leu Met Gin Tyr Thr Arg Ser Ile Met Val 260 265 270 Ile His Asn Ile Ala His Gin Gly Arg Gly Pro Val Asp Glu Phe Pro 275 280 285 Phe Thr Glu Leu Pro Glu His Tyr Leu Glu His Phe Arg Leu Tyr Asp 290 295 300 Pro Val Gly Gly Glu His Ala Asn Tyr Phe Ala Ala Gly Leu Lys Met 305 310 315 320 Ala Asp Gin Val Val Val Val Ser Pro Gly Tyr Leu Trp Glu Leu Lys 325 330 335 Thr Val Glu Gly Gly Trp Gly Leu His Asp Ile lie Arg Gin Asn Asp 340 345 350 WO 00/66745 WO 0066745PCT/AUOO/00385 -17- Trp Lys Thr Arg Gly Ile Val Asn Gly Ile Asp Asn Met Giu Trp Asn 355 365 Pro Leu 385 Arg Ile Met Gly His 465 Arg Pro Val1 Pro His 545 Phe Phe Ala Giu 370 Arg Giu Gly Pro Arg 450 Asp Ile Cys Val Phe 530 Lys Lys Se r Lys Val1 Thr Leu Arg Trp 435 His Lys Th r Gly His 515 Asn Leu Glu Trp Tyr 595 Asp Leu Gly Leu 420 Ile Asp Val Ala Leu 500 Ala His Ile Se r Glu 580 Gln Ala Asp Leu 405 Asp Val1 Leu Arg Gly 485 Asn Val Se r Giu Trp 565 His Trp His Ser 390 Gin Gly Se r Giu Gly 470 Ala Gin Gly Gly Ala 550 Arg Ala Leu 375 Gly Val Gin Gin Se r 455 Trp Asp Leu Gly Leu 535 Leu Ala Lys Lys Arg Lys Asp 440 Met Val1 Ala Tyr Leu 520 Gly Gly Leu Ser Asp Gly Tyr Thr Asn Phe Ser Arg Ala Gly 425 Val1 Leu Gly Leu Ala 505 Arg Trp His Gin Gin Asp 410 Val Gin Gin Phe Leu 490 Met Asp Thr Cys Glu 570 380 Lys Pro Ile Val Phe 460 Val Pro Tyr Val1 Asp 540 Arg Gly Giu Le u Ile Met 445 Giu Arg Se r Gly Pro 525 Arg Thr Met Ala Leu Ala 430 Leu Arg Le u Arg Th r 510 Pro Al a ryr Se r Leu Gly 415 Asp Gly Giu Ala Phe 495 Val1 Phe Giu Arg Gin 575 Gin 400 Phe Ala Thr His His 480 Val Pro Asp Ala Asp 560 A'sp Ala Lys Leu 585 Tyr Glu Asp Vai Leu Val Lys 590 <210> 7 <211> 5346 <212> DNA <213> Triticum aestivum <220> <221> CDS <222> (29) (4912) <400> 7 cggeacgagg tttagtaggt tcegggaa atg gag atg tct etc tgg eca egg Met Glu Met Ser Leu Trp Pro Arg 1 age ccc ctg tgc ect cgg age agg cag Ccg etc gte gte gte egg ccg Ser Pro Leu Cys Pro Arg Ser Arg Gin Pro Leu Val Val Val Arg Pro WO 00/66745 WO 0066745PCT/AUOO/00385 18gcc Ala act Thr aat Asn tct Se r aca Thr eta Leu 105 act Thr ggg Gly g tg Val1 aaa Lys aca Thr 185 caa Gin act Thr gcg Ala tca Se r gac Asp 265 cgc Arg agc Se r aaa Lys gga Gly cac His agt Se r aaa Lys gtg Val1 tca Ser 155 aga Arg aga Arg aca Thr eaa Gin gac Asp 235 aat Asn gte Val ggC etc Gly Leu 30 ace ett Thr Leu aga agg Arg Arg aeg aca Thr Thr aat egg Asn Arg gag aca Giu Thr 110 gac teg Asp Ser 125 gtg geg Val Ala age agt Ser Ser aaa gaa Lys Glu gtg ata Val Ile 190 aga agt Arg Ser 205 aca ttg Thr Leu get aga Ala Arg tea age Ser Ser cet gtt Pro Val 270 eag Gin tge Cys g ta Val1 etc Leu 80 gaa Giu gaa Glu eaa Gin gaa Glu atg Met 160 gca Ala gat Asp ata Ile agt Se r gaa Giu 240 geg Ala gae Asp ttt ttg Phe Leu 35 gta gea Vai Ala ect eag Pro Gin gtt gaa Val Giu aet ctt Thr Leu aea gat Thr Asp 115 get tta Ala Leu 130 ata ctt Ile Leu aag gaa Lys Giu gag etg Giu Leu atg gat Met Asp 195 gat gtg Asp Val 210 ata gta Ile Val gac gta Asp Val aec gtg Thr Val ttt gag Phe Giu 275 aat gge Asn Gly tea gat Ser Asp aaa gte Lys Val age aae Ser Asn aca tac Thr Tyr aga gaa Arg Giu agt tet Ser Ser get gat Ala Asp 150 gat gea Asp Ala 165 ttg eea Leu Pro aat ggg Asn Gly gat cat Asp His gtg atg Val Met 230 gag ctg Giu Leu 245 eta gat Leu Asp aee tea Thr Ser ttt Phe ee t Pro tet Se r aat Asn geg Ala gag Giu 120 att Ile aea Thr gac Asp act Thr gta Val1 200 ggg Gly gat Asp t tg Leu gtt Val1 gga Gly 280 148 196 244 292 340 388 436 484 532 580 628 676 724 772 '820 868 WO 00/66745 WO 0066745PCT/AUOO0/00385 19~ aat gtt tca aac agt gca acg gta cgg Asn Val Ser Asn Ser Ala Thr Val Arg 285 gct ggg Ala Gly ttt tea Phe Ser tct ata Ser Ile 330 agt gcg Ser Ala 345 aca ttt Thr Phe aga gaa Arg Giu ttt gca Phe Ala gca gtg Ala Val 410 cag caa Gin Gin 425 get aaa Ala Lys gaa gaa Giu Giu gat gat Asp Asp gat aaa Asp Lys 490 tct agt Ser Ser 505 aat gat Asn Asp 300 agc agt Ser Ser 315 aag gac Lys Asp ccg atg Pro Met gag gcg Glu Ala gtg gat Val Asp 380 atg gat Met Asp 395 gat tat Asp Tyr tat cca Tyr Pro aca ggt Thr Gly caa ggc Gin Gly 460 tta cca Leu Pro 475 tca att Ser Ile aaa caa Lys Gin ggc Gly aca Thr ttt Phe gat Asp 350 ttg Leu gtg Val1 ttt Phe ggt Gly ceg Pro 430 agt Ser gta Val caa Gin gat Asp cgg Arg 510 ata Ile gtg Val gag Giu 335 gca Ala tcg Se r gtg Val1 gca Ala gaa Giu 415 tct Se r ttg Leu aat Asn aac Asn gtt Val1 495 tea Se r ttt Phe gaa Giu 320 acg Thr att Ile gga Gly gat Asp agt Ser 400 get Ala tea Se r aat Asn ttt Phe caa Gin 480 gcg Ala at t Ile aga Arg 305 g tg Val1 gat Asp gat Asp aat Asn gaa Giu 385 gaa Giu ace Thr ttc Phe cc t Pro agt Se r 465 tcg Se r gga Gly gtt Val1 gaa G iu 290 gca Ala ggt Gly tcg Ser gaa Giu gct Ala 370 act Thr tea Se r gat Asp tet Se r gag Giu 450 gat Asp atc Ile ccg Pro get Ala gtg gat gca agt gat gaa Val Asp Ala Ser Asp Giu 295 gat Asp gca Ala tea Se r ace Thr 355 tea Se r aga Arg ggc Gly gaa Giu atg Met 435 etg Leu aaa Lys att Ile ace Th r t te Phe 515 ttg Leu gtg Val gga Gly 340 gtg Val age Ser tea Ser cat His gaa Giu 420 tgg Trp cga Arg aaa Lys ggt Gly eaa Gin 500 ccc Pro tca Se r gat Asp 325 aat Asn get Ala tgC Cys gaa Giu gag Giu 405 gag Giu gac Asp ctt Leu gac Asp tee Ser 485 tea Ser aaa Lys gga Gly 310 gaa Giu gtt Val1 gat Asp gca Ala gag Giu 390 aaa Lys act Th r aag Lys gte Val1 etg Leu 470 tat Tyr att Ile caa Gin aat Asn get Ala tea Ser eaa Gin aca Th r 375 gaa Giu cat His tac Tyr get Ala agg Arg 455 tea Se r aaa Lys ttt Phe aae Asn gtt Val1 ggg Gly aca Thr gac Asp 360 tac Tyr aca Thr atg Met caa Gin att Ile 440 gtt Val1 att Ile e aa Gin ggt Gly eag Gin 520 916 964 1012 1060 1108 1156 1204 1252 1300 1348 1396 1444 1492 1540 1588 1636 tea att gtt Ser Ile Val agt gte act gag caa aag cag tee ata Ser Val Thr Giu Gin Lys Gin Ser Ile gtt gga ttc egt Val Gly Phe Arg WO 00/66745 WO 0066745PCT/AUOO/00385 agt Se r gtt Val aga Arg cac His 585 gtt Val1 gaa Glu gaa Giu gag Giu cat H is 665 tta Leu gtt Val ccg Pro tcg Se r tca Se r 745 gaa Giu ctt Leu tct caa Gin ggt Gly cag Gin 570 ac a Thr gac Asp aag Lys cat His ct t Leu 650 g tg Val1 att Ile aac Asn caa Gin atg Met 730 gtt Val1 ccc Pro ttc Phe tgc gat Asp acg Thr 555 gat Asp tcc Se r a at Asn aaa Lys cag Gin 635 tct Se r ct t Leu gag Giu gtt Val1 gca Ala 715 agg Arg S tt Ile gat A~sp act rhr aaa ctt Leu 540 tcg Se r gca Ala gag Giu gtg Vai act Thr 620 a aa Lys ata Ile tct Se r gat Asp gaa Giu 700 c ta Leu aac Asn gat Asp gtc Val gaa Giu 780 ctg tcg Ser aga Arg ttg Leu a aa Lys ttg Leu 605 tgg Trp cgt Arg act Thr gag Giu gat Asp 685 caa.

Gin aag Lys aag Lys ctt Leu gtc Val1 765 aga Arg tac gct Ala gag Giu tat Tyr act Thr 590 cgg Arg aag Lys gct Ala gaa Giu gaa Giu 670 gga Gly gat Asp gtg Val ctg Leu tat Tyr 750 atc Ile ttg Leu ata gtt Val1 ggt Gly gtg Vali 575 gat Asp aag Lys aaa Lys gcc Ala att Ile 655 gag Giu ca a Gin atc Ile atg Met ttt Phe 735 tta Leu aaa Lys cac His ccc agt Ser caa Gin 560 aat Asn gag Giu cat His gtt Val gaa Giu 640 gga Gly ctt Leu tat Tyr cag Gin ctg Leu 720 gtt Val aat Asn gga Giy aag Lys aag ctc Leu 545 aca Thr gga Gly ga t Asp cag Gin gat Asp 625 gga Giy a tg Met tca Se r gaa Giu ggg Gly 705 c aa Gin t tt Phe cgt Arg gca Ala agt Ser 785 gag cct Pro aag Lys ctg Leu gcg Ala gca Ala 610 gag Giu cag Gin ggg Gly tgg Trp gtt Val1 690 tca Se r gaa Giu cca Pro gac Asp ttc Phe 770 gac Asp gcc aaa Lys caa Gin gaa Giu ctt Leu 595 ga t Asp gaa Giu atg Met aga Arg tct Ser 675 gac Asp cca Pro ctc Leu ag Giu cta Leu 755 aat Asn ctt Leu tac caa Gin gtt Val gct Al a 580 cat His aga Arg cat His gta Val ggt Giy 660 gaa Giu gag Giu cag Gin gct Ala gta Val1 740 aca Thr gg t Gly gga Gly aga aac Asn cct Pro 565 aag Lys gta Val1 acc Thr ctt Leu gtt Val 645 gat Asp gat Asp a cc Thr gat Asp gag Giu 725 gtg Val gct Ala tgg Trp ggg Gly tta g ta Val 550 gtt Val1 gag Giu aag Lys caa Gin tac Tyr 630 aac Asn aaa Lys gaa Giu tct Se r gtt Val 710 aaa Lys aaa Lys t tg Leu aaa Lys gtt Val 790 gac cca Pro gtt Val g ga Giy ttt Phe gca Aia 615 atg Met gag Giu att Ile g tg Val gtg Val1 695 gtg Val1 aat As n gct Ala gcg Ala tgg Trp 775 tgg Trp ttt att Ile gat Asp gat Asp aat Asn 600 gtg Vai act Th r gat Asp cag Gin cag Gin 680 tcc Se r gat Asp tat Tyr gat Asp aa t Asn 760 agg Arg tgg Trp gtg 1684 1732 1780 1828 1876 1924 1972 2020 2068 2116 2164 2212 2260 2308 2356 2404 2452 WO 00/66745 WO 0066745PCT/AUOO/00385 -21- Se r ttc Phe tgt Cys 825 t tg Leu gct Ala agg Arg atc Ile gtt Val 905 act Thr act Thr tct Ser tgg Trp gtt Val1 985 gct Ala gaa Glu ca a Gin agt Se r Cys Lys Leu Tyr 795 ttc aac ggt cgc Phe Asn Gly Arg 810 ata gga ata gaa Ile Giy Ile Giu gtt aaa gaa aag Vai Lys Giu Lys 845 gaa agg agg aca Giu Arg Arg Thr 860 gct gca gat gaa Ala Ala Asp Glu 875 aag aag aaa aaa Lys Lys Lys Lys 890 gat aat ttg tgg Asp Asn Leu Trp atc agg tta tat- Ile Arg Leu Tyr 925 gag att tgg atg Giu Ile Trp Met 940 att gtt gaa agc Ile Val Giu Ser 955 tat gca gat gtt Tyr Ala Asp Val 970 ttt gct gat ggg Phe Ala Asp Gly cga caa gat ttc Arg Gin Asp Phe 1005 ggc ttc tgg gcg Gly Phe Trp Ala 1020 gaa agg aga gaa Giu Arg Arg Giu 1035 gca aat atc aaa Ala Asn Ile Lys Ile Pro Lys Giu Ala 800 acg gtc tat gag aac Thr Val Tyr Glu Asn 815 ggc act atg aat gaa Gly Thr Met Asn Giu 830 caa agg gag ctt gag Gin Arg Giu Leu Giu 850 cag act gaa gaa cag Gin Thr Giu Giu Gin 865 gct gtc agg gca caa Ala Val Arg Ala Gin 880 ttg caa agt atg ttg Leu Gin Ser Met Leu 895 tac ata gag gct agc Tyr Ile Giu Ala Ser 910 tat aac aga aac tcg Tyr Asn Arg Asn Ser 930 cat ggt ggt tac aac His Gly Gly Tyr Asn 945 ttt gtc aag tgc aat Phe Val Lys Cys Asn 960 att cca cct gaa aag Ile Pro Pro Giu Lys 975 cca gct ggg aat gca Pro Ala Gly Asn Ala 990 cat gct att ctt ccg His Ala Ile Leu Pro 1010 caa gag gag caa aac Gin Giu Giu Gin Asn 1025 aag gaa gaa acc atg Lys Giu Giu Thr Met 1040 gct gag atg aag gca Ala Giu Met Lys Ala Arg Leu Asp Phe Val 805 ggc aac aat gat ttc Gly Asn Asn Asp Phe 820 ctg ttt gag gat ttc Leu Phe Giu Asp Phe 840 ctt gcc atg gaa gaa Leu Ala Met Giu Giu 855 cga aga aag gaa gca Arg Arg Lys Giu Ala 870 aag gcc gag ata gag Lys Ala Giu Ile Giu 885 ttg gcc aga aca tgt Leu Ala Arg Thr Cys 900 gat aca aga gga gat Asp Thr'Arg Gly Asp 920 cca ctt gcg cat agt Pro Leu Ala His Ser 935 tgg aca gat gga ctc Trp Thr Asp Gly Leu 950 aaa gac ggc gat tgg Lys Asp Gly Asp Trp 965 ctt gtg ttg gac tgg Leu Val Leu Asp Trp 980 aac tat gac aac aat Asn Tyr Asp Asn Asn 1000 aac aat gta acc gag Asn Asn Val Thr Glu 1015 tat aca agg ctt ctg Tyr Thr Arg Leu Leu 1030 aga aag gct gag aga Arg Lys Ala Giu Arg 1045 act atg cga agg ttt Thr Met Arg Arg Phe 2500 2548 2596 2644 2692 2740 2788 2836 2884 2932 2980 3028 3076 3124 3172 3220 WO 00/66745 PCT/AU00/00385 -22- 1050 1055 1060 ctg ctt tcc cag aaa cac att gtt tat acc gaa ccg ctt gaa ata cgt 3268 Leu Leu Ser Gin Lys His Ile Val Tyr Thr Glu Pro Leu Glu Ile Arg 1065 1070 1075 1080 gcc gga acc aca gtg gat gtg cta tac aat ccc tct aac aca gtg cta 3316 Ala Gly Thr Thr Val Asp Val Leu Tyr Asn Pro Ser Asn Thr Val Leu 1085 1090 1095 aat gga aag tcg gag ggt tgg ttt aga tgc tcc ttt aac ctt tgg atg 3364 Asn Gly Lys Ser Glu Gly Trp Phe Arg Cys Ser Phe Asn Leu Trp Met 1100 1105 1110 cat tca agt ggg gca ttg cca ccc cag aag atg gtg aaa tca ggg gat 3412 His Ser Ser Gly Ala Leu Pro Pro Gin Lys Met Val Lys Ser Gly Asp 1115 1120 1125 ggg ccg ctc tta aaa gca aca gtt gat gtt cca ccg gat gcc tat atg 3460 Gly Pro Leu Leu Lys Ala Thr Val Asp Val Pro Pro Asp Ala Tyr Met 1130 1135 1140 atg gac ttt gtt ttc tcc gag tgg gaa gaa gat ggg atc tat gac aac 3508 Met Asp Phe Val Phe Ser Glu Trp Glu Glu Asp Gly Ile Tyr Asp Asn 1145 1150 1155 1160 agg aat ggg atg gac tat cat att cct gtt tct gat tca att gaa aca 3556 Arg Asn Gly Met Asp Tyr His Ile Pro Val Ser Asp Ser Ile Glu Thr 1165 1170 1175 gag aat tac atg cgt att atc cac att gcc gtt gag atg gcc ccc gtt 3604 Glu Asn Tyr Met Arg Ile Ile His lie Ala Val Glu Met Ala Pro Val 1180 1185 1190 gca aag gtt gga ggt ctt ggg gat gtt gtt aca agt ctt tea cgt gcc 3652 Ala Lys Val Gly Gly Leu Gly Asp Val Val Thr Ser Leu Ser Arg Ala 1195 1200 1205 att caa gat cta gga cat act gtc gag gtt att ctc ccg aag tac gac 3700 lie Gin Asp Leu Gly His Thr Val Glu Val Ile Leu Pro Lys Tyr Asp 1210 1215 1220 tgt ttg aac caa age agt gtc aag gat tta cat tta tat.caa agt ttt 3748 Cys Leu Asn Gin Ser Ser Val Lys Asp Leu His Leu Tyr Gin Ser Phe 1225 1230 1235 1240 tct tgg ggt ggt aca gaa ata aaa gta tgg gtt gga cga gtc gaa gac 3796 Ser Trp Gly Gly Thr Glu lie Lys Val Trp Val Gly Arg Val Glu Asp 1245 1250 1255 ctg acc gtt tac ttc ctg gaa cct caa aat ggg atg ttt ggc gtt gga 3844 Leu Thr Val Tyr Phe Leu Glu Pro Gin Asn Gly Met Phe Gly Val Gly 1260 1265 1270 tgt gta tat gga agg aat gat gac cgc aga ttt ggg ttc ttc tgt cat 3892 Cys Val Tyr Gly Arg Asn Asp Asp Arg Arg Phe Gly Phe Phe Cys His 1275 1280 1285 tct gct cta gag ttt ate etc cag aat gaa ttt tct cca cat ata ata 3940 Ser Ala Leu Glu Phe lie Leu Gin Asn Glu Phe Ser Pro His Ile lie 1290 1295 1300 cat tgc cat gat tgg tea agt get ccg gtc gcc tgg cta tat aag gaa 3988 His Cys His Asp Trp Set Ser Ala Pro Val Ala Trp Leu Tyr Lys Glu 1305 1310 1315 1320 WO 00/66745 PCT/AU00/00385 -23cac tat tcc caa tcc aga atg gca age act cgg gtt gta ttt acc ate 4036 His Tyr Ser Gin Ser Arg Met Ala Ser Thr Arg Val Val Phe Thr Ile 1325 1330 1335 cac aat ctt gaa ttt gga gca cat tat att ggt aaa gca atg aca tac 4084 His Asn Leu Glu Phe Gly Ala His Tyr Ile Gly Lys Ala Met Thr Tyr 1340 1345 1350 tgt gat aaa gcc aca act gtt tct cct aca tat tca agg gac gtg gca 4132 Cys Asp Lys Ala Thr Thr Val Ser Pro Thr Tyr Ser Arg Asp Val Ala 1355 1360 1365 ggc cat ggc gcc att get cct cat cgt gag aaa ttc tac ggc att ctc 4180 Gly His Gly Ala Ile Ala Pro His Arg Glu Lys Phe Tyr Gly Ile Leu 1370 1375 1380 aat gga att gat cca gat atc tgg gat ccg tac act gac aat ttt ate 4228 Asn Gly Ile Asp Pro Asp Ile Trp Asp Pro Tyr Thr Asp Asn Phe Ile 1385 1390 1395 1400 ccg gtc cct tat act tgt gag aat gtt gtc gaa ggc aag aga gct gca 4276 Pro Val Pro Tyr Thr Cys Glu Asn Val Val Glu Gly Lys Arg Ala Ala 1405 1410 1415 aaa agg gcc ttg cag cag aag ttt gga tta cag caa act gat gtc cct 4324 Lys Arg Ala Leu Gin Gin Lys Phe Gly Leu Gin Gin Thr Asp Val Pro 1420 1425 1430 att gtc gga ate atc acc cgt ctg-aca gcc cag aag gga ate cac ctc 4372 Ile Val Gly Ile Ile Thr Arg Leu Thr Ala Gin Lys Gly Ile His Leu 1435 1440 1445 ate aag cac gca att cac cga act ctc gaa age aac gga cat gtg gtt 4420 Ile Lys His Ala Ile His Arg Thr Leu Glu Ser Asn Gly His Val Val 1450 1455 1460 ttg ctt ggt tca gct cca gat cat cga ata caa ggc gat ttt tgc aga 4468 Leu Leu Gly Ser Ala Pro Asp His Arg Ile Gin Gly Asp Phe Cys Arg 1465 1470 1475 1480 ttg gcc gat gct ctt cat ggt gtt tac cat ggt agg gtg aag ctt gtt 4516 Leu Ala Asp Ala Leu His Gly Val Tyr His Gly Arg Val Lys Leu Val 1485 1490 1495 cta acc tat gat gag cct ctt tct cac ctg ata tac gct ggc tcg gac 4564 Leu Thr Tyr Asp Glu Pro Leu Ser His Leu Ile Tyr Ala Gly Ser Asp 1500 1505 1510 ttc ata att gtt cct tca ate ttc gaa ccc tgt ggc tta aca caa ctt 4612 Phe Ile Ile Val Pro Ser Ile Phe Glu Pro Cys Gly Leu Thr Gin Leu 1515 1520 1525 gtt gcc atg cgt tat gga tcg atc cct ata gtt cgg aaa act gga gga 4660 Val Ala Met Arg Tyr Gly Ser Ile Pro Ile Val Arg Lys Thr Gly Gly 1530 1535 1540 ctt cac gac aca gtc ttc gac gta gac aat gat aag gac cgg gct cgg 4708 Leu His Asp Thr Val Phe Asp Val Asp Asn Asp Lys Asp Arg Ala Arg 1545 1550 1555 1560 tct ctt ggt ctt gaa cca aat ggg ttc agt ttc gac gga gcc gac age 4756 Ser Leu Gly Leu Glu Pro Asn Gly Phe Ser Phe Asp Gly Ala Asp Ser 1565 1570 1575 WO 00/66745 PCTAUOO/00385 24 aat ggc gtg gat tat gcc etc aac aga gca atc ggc get tgg ttc gat Asn Gly Val Asp Tyr Ala Leu Asn Arg Ala Ile Gly Ala Trp Phe Asp 1580 1585 1590 gcc cgt gat tgg ttc cac tcc ctg tgt aag agg gte atg gag caa gac Ala Arg Asp Trp. Phe His Ser Leu Cys Lys Arg Val Met Glu Gin Asp 1595 1600 1605 tgg tcg tgg aac egg ccc gca ctg gac tac att gaa ttg tac cat gcc Trp Ser Trp Asn Arg Pro Ala Leu Asp Tyr Ile Glu Leu Tyr His Ala 1610 1615 1620 get cga aaa ttc tgacacccaa ctgaaccaat gacaagaaca agcgcattgt Ala Arg Lys Phe 1625 gggatcgact agtcatacag ggetgtgcag atcgtcttgc ttcagttagt gccctcttca gttagtteca agcgcactac agtcgtacat agetgaggat cctcttgcct cetaecaggg ggaacaaagc agaaatgcat gagtgcattg ggaagaettt tatgtatatt gttaaaaaaa tttccttttc ttttccttce ctgcacctgg aaatggttaa gcgcategcc gagataagaa ccgcagtgac attctgtgag tagctttgta tattctctca tcttgtgaaa actaatgttc atgttaggct gtctgatcat gtggaagctt tgttatatgt tacttatggt atacatcaat gatatttaca tttgtggaaa aaaaaaaaaa aaaa <210> 8 <211> 1628 <212> PRT <213> Triticum aestivum <400> 8 Met Glu Met Ser Leu Trp Pro Arg Ser Pro Leu Cys Pro Arg Ser Arg 1 5 10 Gin Pro Leu Val Val Val Arg Pro Ala Gly Arg Gly Gly Leu Thr Gin 25 Pro Phe Leu Met Asn Gly Arg Phe Thr Arg Ser Arg Thr Leu Arg Cys 40 Met Val Ala Ser Ser Asp Pro Pro Asn Arg Lys Ser Arg Arg Met Val 55 Pro Pro Gin Val Lys Val Ile Ser Ser Arg Gly Tyr Thr Thr Arg Leu 70 75 Ile Val Glu Pro Ser Asn Glu Asn Thr Glu His Asn Asn Arg Asp Glu 90 Giu Thr Leu Asp Thr Tyr Asn Ala Leu Leu Ser Thr Giu Thr Ala Giu 100 105 110 Trp Thr Asp Asn Arg Giu Ala Glu Thr Al-a Lys Ala Asp Ser Ser Gin 115 120 125 Asn Ala Leu Ser Ser Ser Ile Ile Gly Glu Val Asp Val Ala Asp Glu 130 135 140 Asp Ile Leu Ala Ala Asp Leu Thr Val Tyr Ser Leu Ser Ser Val Met 4804 4852 4900 4952 5012 5072 5132 5192 5252 5312 5346 WO 00/66745 WO 0066745PCT/AUOO/00385 145 Lys Phe ValI Val1 Val1 225 Giu Th r Lys Arg Arg 305 Val1 Asp Asp Asn Glu 385 Giu Th r Phe Pro Se r 465 Se r Ala Asp Ala Thr Thr' Val 200 Asn Gly 215 Asp Asp Asp Leu Ala Val Ser Gly 280 Asp Glu 295 Asn Val Ala Gly Ser Thr Gin Asp 360 Thr Tyr 375 Glu Thr His Met Tyr Gin Ala Ile 440 Arg Val 455 Ser Ile Lys Gin 160 Asp Ala 175 Val Asp Val Ile Arg Ser Val Giu 240 Ser Ala 255 Gin Asp Thr Vai Ile Phe Val Glu 320 Giu Thr 335 Ala Ile Ser Giy Vai Asp Ala Ser 400 Giu Aia 415 Ser Ser Leu Asn Asn Phe Asn Gin 480 Vai Ala 495 WO 00/66745 WO 0066745PCT/AUOO/00385 26 Gly Pro Thr Gin Ser Ile Phe Giy Ser Ser 500 Asn Gin 520 Phe Arg 535 Pro Ile Val Asp Giy Asp Phe Asn 600 Aia Vai 6i15 Met Thr Giu Asp Ile Gin Vai Gin 680 Val Ser 695 Vai Asp Asn Tyr Aia Asp Aia Asn 760 Trp Arg 775 Trp Trp Phe Vai Asp Phe Asp Phe 840 505 Ser Ile Ser Gin Vai Giy Arg Gin 570 His Thr 585 Vai Asp Giu Lys Giu His Giu Leu 650 His Vai 665 Leu Ile Vai Asn Pro Gin Ser Met 730 Ser Vai 745 Giu Pro Leu Phe Ser Cys Phe Phe 810 Cys Ile 825 Leu Val Gin Se r Leu 540 Se r Ala Giu Vali Thr 620 Lys Ile Se r Asp Giu 700 Leu Asn Asp Vali Giu 780 Leu Giy Ile Giu His Vali 525 Se r Arg Leu Lys Leu 605 Trp Arg Th r Glu Asp 685 Gin Lys Lys Leu Vali 765 Arg Tyr Arg Giu L~s 845 Ile Gin Se r Gin 560 Asn Giu His Vali Giu 640 Gly Leu Tyr Gin Leu 720 Vali Asn Giy Lys Lys 800 Tyr Met Giu WO 00/66745 WO 0066745PCT/AUOO/00385 -27- Leu Glu Lys Leu Ala Met Glu Glu 850 855 Giu Gin Arg Arg Arg Lys Giu Ala 865 870 Ala Gin Ala Lys Ala Glu Ile Glu 885 Met Leu Ser Leu Ala Arg Thr Cys 900 Ala Ser Thr Asp Thr Arg Giy Asp 915 920 Asn Ser Arg Pro Leu Ala Hius Ser 930 935 Tyr Asn Asn Trp Thr Asp Giy Leu 945 950 Cys Asn Asp Lys Asp Gly Asp Trp 965 Giu Lys Ala Leu Val Leu Asp Trp 980 Asn Ala Arg Asn Tyr Asp Asn Asn 995 1000 Leu Pro Asn Asn Asn Val Thr Giu 1010 1015 Gin Asn Ile Tyr Thr Arg Leu Leu 025 1030 Thr Met Lys Arg Lys Ala Giu Arg 1045 Lys Aia Lys Thr Met Arg Arg Phe 1060 Tyr Thr Giu Pro Leu Glu Ile Arg 1075 1080 Tyr Asn Pro Ser Asn Thr Val Leu 1090 1095 Arg Cys Ser Phe Asn Leu Trp Met 105 1110 Gin Lys Met Val Lys Ser Gly Asp 1125 Asp Val Pro Pro Asp Ala Tyr Met 1140 Giu Giu Asp Giy Ile Tyr Asp Asn 1155 1160 Pro Val Ser Asp Ser Ile Giu Thr Ala Giu Arg Arg Thr Gin Thr Giu 860 Arg Ala Ala Asp Giu Ala Vai Arg 875 880 Ile Lys Lys Lys Lys Leu Gin Ser 890 895 Val Asp Asn Leu Trp Tyr Ile Glu 905 910 Thr Ile Arg Leu Tyr Tyr Asn Arg 925 Thr Giu Ile Trp Met His Gly Gly 940 Ser Ile Val Giu Ser Phe Val Lys 955 960 Trp Tyr Ala Asp Val Ile Pro Pro 970 975 Val Phe Ala Asp Gly Pro Ala Gly 985 990 Ala Arg Gin Asp Phe His Ala Ile 1005 Giu Gly Phe Trp Ala Gin Giu Giu 1020 Gin Giu Arg Arg Giu Lys Giu Giu 1035 1040 Ser Ala Asn Ile Lys Ala Glu Met 1050 1055 Leu Leu Ser Gin Lys His Ile Val 1065 1070 Ala Gly Thr Thr Val Asp Val Leu 1085 Asn Gly Lys Ser Giu Gly Trp Phe 1100 His Ser Ser Gly Ala Leu Pro Pro 1115 1120 Gly Pro Leu Leu Lys Ala Thr Val 1130 1135 Met Asp Phe Val Phe Ser Giu Trp 1145 1150 Arg Asn Gly Met Asp Tyr His Ile 1165 Giu Asn Tyr Met Arg Ile Ile His 1180 Ala Lys Val Gly Giy Leu Gly Asp 1170 Ile Ala Val 1175 Giu Met Ala Pro Val WO 00/66745 PCT/AU00/00385 -28- 1190 1195 1200 Val Val Thr Ser Leu Ser Arg Ala Ile Gin Asp Leu Gly His Thr Val 1205 Glu Val Ile Leu Pro Lys Tyr Asp 1220 Asp Leu His Leu Tyr Gin Ser Phe 1235 1240 Val Trp Val Gly Arg Val Glu Asp 1250 1255 Gin Asn Gly Met Phe Gly Val Gly 265 1270 Arg Arg Phe Gly Phe Phe Cys His 1285 Asn Glu Phe Ser Pro His Ile Ile 1300 Pro Val Ala Trp Leu Tyr Lys Glu 1315 1320 Ser Thr Arg Val Val Phe Thr Ile 1330 1335 Tyr Ile Gly Lys Ala Met Thr Tyr 345 1350 Pro Thr Tyr Ser Arg Asp Val Ala 1365 Arg Glu Lys Phe Tyr Gly Ile Leu 1380 Asp Pro Tyr Thr Asp Asn Phe Ile 1395 1400 Val Val Glu Gly Lys Arg Ala Ala 1410 1415 Gly Leu Gin Gin Thr Asp Val Pro 425 1430 Thr Ala Gin Lys Gly Ile His Leu 1445 Leu Glu Ser Asn Gly His Val Val 1460 Arg Ile Gin Gly Asp Phe Cys Arg 1475 1480 Tyr His Gly Arg Val Lys Leu Val 1490 1495 His Leu Ile Tyr Ala Gly Ser Asp 505 1510 1210 1215 Cys Leu Asn Gin Ser Set Val Lys 1225 1230 Ser Trp Gly Gly Thr Glu Ile Lys 1245 Leu Thr.Val Tyr Phe Leu Glu Pro 1260 Cys Val Tyr Gly Arg Asn Asp Asp 1275 1280 Ser Ala Leu Glu Phe Ile Leu Gin 1290 1295 His Cys His Asp Trp Ser Ser Ala 1305 1310 His Tyr Ser Gin Ser Arg Met Ala 1325 His Asn Leu Glu Phe Gly Ala His 1340 Cys Asp Lys Ala Thr Thr Val Ser 1355 1360 Gly His Gly Ala Ile Ala Pro His 1370 1375 Asn Gly Ile Asp Pro Asp Ile Trp 1385 1390 Pro Val Pro Tyr Thr Cys Glu Asn 1405 Lys Arg Ala Leu Gin Gin Lys Phe 1420 Ile Val Gly Ile lle Thr Arg Leu 1435 1440 Ile Lys His Ala Ile His Arg Thr 1450 1455 Leu Leu Gly Ser Ala Pro Asp His 1465 1470 Leu Ala Asp Ala Leu His Gly Val 1485 Leu Thr Tyr Asp Glu Pro Leu Ser 1500 Phe Ile Ile Val Pro Ser Ile Phe 1515 1520 Glu Pro Cys Gly Leu Thr Gin Leu Val Ala Met Arg Tyr Gly Ser Ile 1525 1530 1535 WO 00/66745 WO 0066745PCT/AUOO/00385 29 Pro Ile Val Arg Lys Thr Gly Gly 1540 Asp Asn Asp Lys Asp Arg Ala Arg 1555 1560 Phe Ser Phe Asp Gly Ala Asp Ser 1570 1575 Arg Ala Ile Gly Ala Trp Phe Asp 585 1590 Cys Lys Arg Val Met Glu Gin Asp 1605 Asp Tyr Ile Giu Leu Tyr His Ala 1620* Leu His Asp Thr Val Phe Asp Val 1545 1550 Ser Leu Gly Leu Giu Pro Asn Gly 1565 Asn Gly Val Asp Tyr Ala Leu Asn 1580 Ala Arg Asp Trp Phe His Ser Leu 1595 1600 Trp Ser Trp Asn Arg Pro Ala Leu 1610 1615 Ala Arg Lys Phe 1625 <210> 9 <211> 3621 <212> DNA <213> Triticum aestivum <220> <221> CDS <222> (1)..(3177) <400> 9 gat gca ttg tat g Asp Ala Leu Tyr V 1 tcc gag aaa act g Ser Giu Lys Thr A aat gtg ttg cgg a Asn Val Leu Arg L aaa act tgg aag a Lys Thr Trp Lys L cag aaa cgt gct g Gin Lys Arg Ala A tct ata act gaa a Ser Ile Thr Giu I ctt tct gag gaa g Leu Ser Giu Giu G 100 gag gat gat gga c Giu Asp Asp Gly G 115 gtt gaa caa gat a Val Glu Gin Asp I 130 a ca Th r gac Asp aag Lys cat His ctt Leu gtg Val1 att Ile aac Asn caa Gin WO 00/66745 WO 0066745PCT/AUOO/00385 30 gca cta aag gtg atg ctg caa gaa ctc gct Ala Leu Lys Val Met Leu Gin Glu Leu Ala 145 agg Arg att Ile gat Asp act Thr aaa Lys 225 aac Asn gga Gly aaa Lys agg Arg gca Ala 305 aag Lys aa t Asn agg Arg att Ile gtt Val 385 gca 150 aac Asn gat Asp gtc Val gaa Glu 210 ctg Leu ggt Gly ata Ile gaa Giu agg Arg 290 gat Asp aaa Lys ttg Leu tta Leu tgg Trp 370 gaa Giu gat aag Lys ct t Le u gtc Val1 195 aga Arg tac Tyr cgc Arg gaa Giu aag Lys 275 aca Thr gaa Glu aaa Lys tgg rrp tat ryr 355 atg Met agc Se r gtt ctg Leu tat Tyr 180 atc Ile ttg Leu at a Ile acg Thr ggc Gly 260 caa Gin cag Gin gct Ala t tg Leu tac Tyr 340 tat Tyr cat His ttt Phe att ttt Phe 165 tta Leu aaa Lys cac His ccc Pro gtc Val1 245 act Thr agg Arg act Th r gtc Val caa Gin 325 ata Ile aac Asn ggt Gly gtc Val1 cca gt t Val1 aat As n gga Gly aag Lys aag Lys 230 tat Tyr atg Met gag Giu gaa Giu agg Arg 310 agt Se r gag Giu aga Arg ggt Gly aag Lys 390 cct ttt Phe cgt Arg gca Ala agt Ser 215 gag Giu gag Glu aa t Asn ctt Leu gaa Giu 295 gca Ala atg Met gct Ala a ac Asn tac Tyr 375 tgc Cys gaa cca Pro gac Asp ttc Phe 200 gac Asp gcc Ala a ac Asn gaa Glu gag Giu 280 cag Gin caa Gin ttg Leu agc Se r tcg Se r 360 aac Asn aat Asn aag gag Giu cta Leu 185 aat Asn ct t Leu tac Tyr aat Asn gat Asp 265 aaa Lys cgg Arg gcg Ala agt Ser aca Thr 345 agg Arg aat Asn gac Asp gca gta Val 170 aca Thr ggt Gly gga Gly aga Arg ggc Gly 250 ctg Leu ctt Leu cga Arg aag Lys L tg Leu 330 gat Asp cca Pro tgg rrp aaa Lys -tt gag *Glu 155 *gtg Val1 gct Ala tgg Trp ggg Gly tta Leu 235 aac Asn ttt Phe gcc Ala aga Arg gcc Ala 315 gcc Ala aca Thr ctt Leu tca Ser gac Asp 395 gtg aaa Lys aaa Lys ttg Leu aaa Lys gtt Val 220 gac Asp aat Asn gag Giu atg Met aag Lys 300 gag Gi u aga Arg aga Arg gcg Ala gat Asp 380 9gc Gly t tg aat Asn gct Ala gcg Ala tgg Trp 205 tgg Trp ttt Phe gat Asp gat Asp gaa Giu 285 gaa Giu ata Ile aca Thr gga Gly cat His 365 gga Gly gat Asp gacI tat Tyr gat Asp aat Asn 190 agg Arg tgg Trp gt g Val ttc Phe t tc Phe 270 gaa Giu gca Ala gag Giu tg t Cys gat Asp 350 ag t Ser ctc Leu tgg T'rp :gg tcg Se r tca Ser 175 gaa Giu ctt Leu tct Se r ttc Phe tgt Cys 255 ttg Leu gct Ala agg Arg atc Ile gtt Val 335 act Thr act Thr tct Ser tgg Trp gttI a tg Met 160 gtt Val1 ccc Pro t tc Phe tgc Cys t tc Phe 240 ata Ile gtt Val1 gaa Giu gct Ala sag Lys 320 gat Asp atc Ile gag Giu att Ile tat Tyr 4 00 ttt 480 528 576 624 672 720 768 816 864 912 960 1008 1056 1104 1152 1200 1248 WO 00/66745 WO 0066745PCT/AUOO/00385 -31 Ala Asp Val Ile Pro Pro Giu Lys Ala Leu Val Leu Asp Trp Val Phe 405 410 415 gct Ala caa Gin ttc Phe agg Arg 465 aat Asn tcc Se r gga Gly gga Giy tca Se r 545 ccg Pro gac Asp aat Asn aat Asn aag Lys 625 caa Gin gat Asp gat Asp tgg Trp 450 aga Arg a tc Ile cag Gin acc Thr aag Lys 530 agt Ser ctc Leu ttt Phe ggg Giy t ac Tyr 610 gtt Val1 gat Asp ggg Giy ttc Phe 435 gcg Aia gaa Giu aaa Lys aaa Lys aca Thr 515 tcg Se r ggg Giy tta Leu gt t Vali a tg Met 595 atg Met gga Gi y cta Leu cca Pro 420 cat His caa Gin aag Lys gct Ala cac His 500 g tg Val1 gag Giu gca Ala aaa Lys t tc Phe 580 gac Asp cgt Arg ggi Giy gga Giy gct Ala gct Ala gag Giu gaa Glu gag Glu 485 att Ile gat Asp ggt Giy ttg Leu gca Ala 565 tcc Se r tat Tyr att Ile ctt Leu cat His 645 ggg Gly att Ile gag Glu gaa Giu 470 atg Met gtt Val1 gtg Val1 tgg Trp cca Pro 550 aca Thr gag Giu cat His a tc Ile ggg Gly 630 act Thr aat Asn ctt Leu ca a Gin 455 acc Thr aag Lys tat Tyr cta Leu ttt Phe 535 ccc Pro gtt Val1 tgg Trp att Ile cac His 615 gat Asp gtc Val gca Aia ccg Pro 440 aac Asn a tg Met gca Ala acc Thr tac Tyr 520 aga Arg cag Gin ga t Asp ga a Giu cct Pro 600 att Ile gti va 1 gag Glu agg Arg 425 aac Asn atc Ile aa a Lys aaa Lys cga Arg 505 aat Asn tgc Cys aag Lys gtt Val1 gaa Giu 585 gtt Val gcc Ala gtt Val1 gtt Val1 aac Asn aac Asn tat Tyr aga Arg act Thr 490 acc Th r ccc Pro tcc Ser atg Met cca Pro 570 gat Asp tct Se r gtt ValI a ca Thr ati Ile 650 tat Tyr aat Asn aca Thr aag Lys 475 atg Met gnc Xaa tct Ser ttt Phe gig Val1 555 ccg Pro ggg Gly gat Asp gag Glu agt Se r 635 cic Leu gac Asp gia Val agg Arg 460 gct Ala cga Arg tg Leu aac Asn aac Asn 540 aaa Lys gat Asp a tc Ile tca Se r a tg Met 620 cii Leu ccg Pro aac Asn acc Thr 445 ct t Leu gag Giu agg Arg aaa Lys aca Th r 525 ct Leu tca Se r gcc Ala tat Tyr at Ile 605 gcc Ala ica Ser aag Lys aat Asn 430 gag Glu ctg Le u aga Arg tt Phe tac Tyr 510 gig Val1 tgg Trp ggg Gly tat Tyr gac Asp 590 gaa Glu ccc Pro cgt Arg tac Tyr gct Ala gaa Glu caa Gin ag t Ser cig Leu 495 gtg Val1 cta Leu atg Met ga t Asp aig Met 575 aac Asn aca Thr gtt Val1 gcc Ala gac Asp 655 cga Arg ggc Gly gaa Giu gca Al a 480 ctt Leu ccc Pro aat Asn cat His ggg Gly 560 a tg Met agg Arg gag Glu gca Al a att Ile 640 tgt Cys 1296 1344 1392 1440 1488 1536 1584 1632 1680 1728 1776 1824 1872 1920 1968 2016 ttg aac caa Leu Asn Gin agc agt gtc aag Ser Ser Val Lys agc at gicaag ta cat iia tat caa agi tit ict Ser Sr Va Lys Leu His Leu Tyr Gin Ser Phe Ser WO 00/66745 WO 0066745PCT/AUOO/00385 32 tgg T rp acc Thr gta Val1 705 gct Ala tgc Cys tat Tyr aat Asn gat Asp 785 cat His gga Gly gtc ValI agg Arg gtc Val 865 eag Lys ctt Leu gcc Ala ggt ggt Gly Gly 675 gtt tac Val Tyr 690 tat gga Tyr Gly cta gag Leu Glu cat gat His Asp tcc caa Ser Gin 755 ctt gaa Leu Glu 770 aaa gcc Lys Ala ggc gcc Gly Ala att gat Ile Asp cct tat Pro Tyr 835 gcc ttg Ala Leu 850 gga atc Gly Ilie cac gca His Ala ggt tca Gly Ser gat gct Asp Ala 915 eaa gta Lys Vai 680 cct caa Pro Gin 695 gec cgc Asp Arg cag aat Gin Asn gct ccg Ala Pro gca agc Ala Ser 760 cat tat His Tyr 775 tct cct Ser Pro cat cgt His Arg tgg gat Trp Asp aat gtt Asn Val 840 ttt gga Phe Giy 855 ctg ace Leu Thr acc ctc Thr Leu cat cge His Arg gtt tac Val Tyr 920 670 geaa Glu gtt Val1 tgt Cys a ta Ile aag Lys 750 acc Th r aca Thr gtg Val1 att Ile ttt Phe 830 gct Ala gtc Vel cac His g tg Val1 tgc Cys 910 ctt Le u gac ctg Asp Leu gga tgt Gly Cys cat tct His Ser 720 ata cat Ile His 735 gaa cac Glu His etc cac Ile His tac tgt Tyr Cys gce ggc Ala Gly 800 ctc aat Leu Asn 815 atc ccg Ile Pro gca aaa Ala Lys cct att Pro Ile ctc etc Leu Ile 880 gtt ttg Val Leu 895 age ttg Arg Leu gtt cta Val Leu 2064 2112 2160 2208 2256 2304 2352 2400 2448 2496 2544 2592 2640 2688 2736 2784 WO 00/66745 WO 0066745PCT/AUOO/00385 -33acc tac Thr Tyr 930 att att Ile Ile 945 gcc atg Ala Met tac gac Tyr Asp ett ggt Leu Gly ggc gtg Gly Val 1010 egt gat Arg Asp 1025 ect ctt tct cae Pro Leu Ser His 935 tca atc ttt gaa.

Ser Ile Phe Glu 950 gga tcg atc cet Gly Ser Ile Pro 965 tte gae gta gac Phe Asp Val Asp eca aat ggg tte Pro Asn Gly Phe 1000 gce etc aac aga Ala Leu Asn Arg 1015 cac tee ctg tgt His Ser Leu Cys 1030 ect gca ctg gac Pro Ala Leu Asp 1045 ata tac gct ggc Ile Tyr Ala Gly 940 tgt ggc tta aea Cys Gly Leu Thr 955 gtt cgg aaa ace Val Arg Lys Thr 970 gat aag gac cgg Asp Lys Asp Arg ttc gac gga gee Phe Asp Gly Ala 1005 ate ggc gct tgg Ile Gly Ala Trp 1020 agg gtc atg gag Arg Val Met Glu 1035 att gaa ttg tac gac ttc Asp Phe ctt gtt Leu Val 960 gga ett Gly Leu 975 egg tet Arg Ser age aat Ser Asn gat gee Asp Ala gae tgg Asp Trp 1040 gee get 2832 2880 2928 2976 3024 3072 3120 3168 3217 3277 3337 3397 3457 3517 3577 3621 teg tgg aae egg Ser Trp Asn Arg Tyr Ile Glu Leu Tyr His Ala Ala 1050 1055 ega aaa tte tgacaeeeaa etgaaeeaat ggeaagaaea agegeattgt Arg Lys Phe gggategaet aeagteatae agggetgtge agategtett getteagtta gtgeeetett eagttagttc eaagegeaet aeagtegtae atagetgagg ateetettge eteeteeaee aggggaaaea aageagaaat geataagtge attgggaaga ettttatgta. tattgttaaa tttttccttt tetttteett eeetgeaeet ggaaatggtt aagegeateg eegagataag aaeeaeagta aeattetgtg agtagetttg tatattetet eatettgtga aaaetaatgt geatgttagg etctctgatc atgtggaage tttgttata- gttacttatg gttatatggt ataeateaat gatatttaea tttgtggaaa aaaaaaaFiaa aaaa <210> <211> 1059 <212> PRT <213> Tritieum aestivui <400> Asp Ala Leu Tyr Val Asn Gly Leu Glu Ala Lys Glu Gly Asp His Thr 1 5 10 Ser Glu Lys Thr Asp Giu Asp Ala Leu His Val Lys Phe Asn Val Asp 25 Asn Val Leu Arg Lys His Gin Ala Asp Arg Thr Gin Ala Val Glu Lys 40 WO 00/66745 PCT/AUOO/00385 -34- Lys Gin Ser Leu Glu Va1 Ala 145 Arg Ile Asp Thr Lys 225 Asn Gly Lys Arg Ala 305 Lys Asn Arg Ile Va1 Thr Lys Ile Ser Asp Glu 130 Leu Asn Asp Va1 Glu 210 Leu Gly Ile Glu Arg 290 Asp Lys Leu Leu Trp 370 Glu Trp Arg Thr Glu Asp 115 Gin Lys Lys Leu Val 195 Arg Tyr Arg Glu Lys 275 Thr Glu Lys Trp Tyr 355 M4et Ser Lys Ala Glu Glu 100 Gly Asp Val Leu Tyr 180 Ile Leu Ile Thr Gly 260 Gin Gin Ala Leo Tyr 340 Tyr His Phe Lys Ala Ile Glu Gin Ile Met Phe 165 Leo Lys His Pro Val 245 Thr Arg Thr Val Gin 325 Ile Asn Gly Val Va1 Glu 70 Gly Leu Tyr Gin Leu 150 Va1 Asn Gly Lys Lys 230 Tyr Met Glu Glu Arg 310 Ser Glu Arg Gly Lys Asp 55 Gly Met Ser Glu Gly 135 Gin Phe Arg Ala Ser 215 Glu Glu Asn Leu I Glu 295 Ala Met Ala Asn Tyr 375 Cys I Glu Gin Gly Trp Val 120 Ser Glu Pro Asp Phe 200 Asp Ala Asn Glu Glu 280 Gin Gln Leu Ser Ser 360 ksn ksn Glu Met Arg Ser 105 Asp Pro Leo Glu Leo 185 Asn Leu Tyr Asn Asp 265 Lys Arg Ala Ser Thr 345 Arg Asn Asp I His Val Gly 90 Glu Glu Gln Ala Val 170 Thr Gly Gly Arg Gly 250 Leu Leu Arg Lys Leo 330 Asp Pro rrp Lys Leu Va1 75 Asp Asp Thr Asp Glu 155 Val Ala Trp Gly Leu 235 Asn Phe Ala Arg Ala 315 Ala Thr Leu Ser Asp Tyr Asn Lys Glu Ser Va1 140 Lys Lys Leu Lys Val 220 Asp Asn Glu Met Lys 300 Glu Arg Arg Ala I Asp 380 Gly I Met Glu Ile Val Val 125 Va1 Asn Ala Ala Trp 205 Trp Phe Asp Asp Glu 285 Glu Ile [hr iis 365 ksp Thr Asp Gin Gin 110 Ser Asp Tyr Asp Asn 190 Arg Trp Val Phe Phe 270 Glu Ala Glu Cys Asp 350 Ser Leu Trp Glu Glu His Leu Val Pro Ser Ser 175 Glu Leu Ser Phe Cys 255 Leu Ala Arg Ile Val 335 Thr Thr Ser Trp His Leu Va1 Ile Asn Gin Met 160 Va1 Pro Phe Cys Phe 240 Ile Val Glu Ala Lys 320 Asp Ile Glu Ile Tyr WO 00/66745 PCT/AUOO/00385 385 390 395 400 Ala Asp Val Ile Pro Pro Giu Lys Ala Leu Val Leu Asp Trp Val Phe 405 410 415 Ala Asp Gly Pro Ala Gly Asn Ala Arg Asn Tyr Asp Asn Asn Ala Arg 420 425 430 Gin Asp Phe His Ala Ile Leu Pro Asn Asn Asn Val Thr Giu Giu Giy 435 440 445 Phe Trp Ala Gin Glu Giu Gin Asn Ile Tyr Thr Arg Leu Leu Gin Giu 450 455 460 Arg Arg Giu Lys Giu Giu Thr Met Lys Arg Lys Ala Giu Arg Ser Ala 465 470 475 480 Asn Ile Lys Ala Giu Met Lys Ala Lys Thr Met Arg Arg Phe Leu Leu 485 490 495 Ser Gin Lys His Ile Val Tyr Thr Arg Thr Xaa Leu Lys Tyr Val Pro 500 505 510 Gly Thr Thr Val Asp Val Leu Tyr Asn Pro Ser Asn Thr Val Leu Asn 515 520 525 Gly Lys Ser Glu Giy Trp Phe Arg Cys Ser Phe Asn Leu Trp Met His 530 535 540 Ser Ser Gly Ala Leu Pro Pro Gin Lys Met Val Lys Ser Giy Asp Gly 545 550 555 560 Pro .Leu Leu Lys Ala Thr Val Asp Val Pro Pro Asp Ala Tyr Met Met 565 570 575 Asp Phe Val Phe Ser Glu Trp Giu Giu Asp Gly Ile Tyr Asp Asn Arg 580 585 590 Asn Gly Met Asp Tyr His Ile Pro Val Ser Asp Ser Ile Giu Thr Giu 595 600 605 Asn Tyr Met Arg Ile Ile His Ile Ala Vai Giu Met Ala Pro Val Ala 610 615 620 Lys Val Gly Giy Leu Gly Asp Val Vai Thr Ser Leu Ser Arg Ala Ile 625 630 635 640 Gin Asp Leu Gly His Thr Val Giu Val Ile Leu Pro Lys Tyr Asp Cys 645 650 655 Leu Asn Gin Ser Ser Val Lys Asp Leu His Leu Tyr Gin Ser Phe Ser 660 665 670 Trp Giy Gly Thr Glu Ile Lys Val Trp Val Gly Arg Val Giu Asp Leu 675 680 685 Thr Val Tyr Phe Leu Giu Pro Gin Asn Gly Met Phe Gly Val Gly Cys 690 695 700 Vai Tyr Gly Arg Asn Asp Asp Arg Arg Phe Gly Phe Phe Cys His Ser 705 710 715 720 Ala Leu Giu Phe Ile Leu Gin Asn Giu Phe Ser Pro His Ile Ile His 725 730 735 WO 00/66745 WO 0066745PCT/AUOO/00385 -36- Cys His Asp Trp Ser 740 Ser Ala Pro Val Ala Trp Leu Tyr Tyr Ser Asn Leu 770 Asp Lys 785 His Gly Gly Ile Val Pro Arg Ala 850 Val Gly 865 Lys His Leu Gly Ala Asp Thr Tyr 930 Ile Ile 945 Ala Met Tyr Asp Leu Gly Gly Val 1010 Arg Asp 1025 Gin 755 Giu Ala Ala Asp Tyr 835 Leu Ile Al a Se r Ala 915 Asp Val Arg Th r Leu 995 Asp T rp Se r Phe Th r Ile Pro 820 Thr Gin Ile Ile Ala 900 Leu Giu Pro Tyr Val 980 Giu Tyr Phe Arg Gly Th r Ala 805 Asp Cys Gin Thr His 885 Pro His Pro Se r Gly 965 Phe Pro Ala His Met Ala Ser 760 Ala His Tyr 775 Vai Ser Pro 790 Pro His Arg Ile Trp Asp Giu Asn Val 840 Lys Phe Gly 855 Arg Leu Thr 870 Arg Thr Leu Asp His Arg Gly Val Tyr 920 Leu Ser His 935 Ile Phe Glu 950 Ser Ile Pro Asp Val Asp Asn Gly Phe 1000 Leu Asn Arg 1015 Ser Leu Cys 745 Th r Ile Thr Giu Pro 825 Val Leu Ala Giu Ile 905 His Leu Pro Ile Asn 985 Ser Ala Lys Arg Gly Tyr Lys 810 Tyr Glu Gin Gin Se r 890 Gin Giy Ile Cys ValI 970 Asp Phe Ile Arg Vai Val Phe 765 Lys Ala Met 780 Ser Arg Asp 795 Phe Tyr Giy Thr Asp Asn Gly Lys Arg 845 Gin Thr Asp 860 Lys Giy Ile 875 Asn Giy Gin Gly Asp Phe Arg Val Lys 925 Tyr Ala Gly 940 Gly Leu Thr 955 Arg Lys Thr Lys Asp Arg Asp Giy Ala 1005 Giy Ala Trp 1020 Val Met Giu Lys Giu His 750 Thr Ile His Thr Tyr Cys Vai Ala Gly 800 Ile Leu Asn 815 Phe Ile Pro 830 Ala Ala Lys Val Pro Ile His Leu Ile 880 Val Vai Leu 895 Cys Arg Leu 910 Leu Val Leu Ser Asp Phe Gin Leu Val 960 Gly Gly Leu 975 Ala Arg Ser 990 Asp Ser Asn Phe Asp Ala Gin Asp Trp 1040 1030 1035 Ser Trp Asn Arg Pro Ala Leu Asp Tyr Ile Giu Leu Tyr His Ala Ala 1045 1050 1055 Arg Lys Phe (210> 11 <211> 728 WO 00/66745 WO 0066745PCT/AUOO/00385 -37- <212> DNA <213> Triticum sp.

<400> 11 gatcttgaac ggcacgtgaa tcatcgatca tgacagagca gagatctcca cgccagagcg gctaggcggg cgaggcagag tggtgggtgg tgggttttgc ccggccacgg agccctctgt cggccgcggc ggcggcctcg acggaatgtt gtttgatttg aaaaaaaaag gtttatagtg gagtcaaagg gcattggttt tgagctggaa ttcatactgc agacttgtaa tagtattatg ttgtattcca gggatagggc gggcggggtt gccctcggag cgcaggtacg gttctgtccc attttgattt tgatttgcat ttaaaacgaccaacatcccc gcatagaatg attttagttc agtcgccgct tagtaggttc cagtcagccg ggtgattatg gggtcaggtt ctttcatctc gcggaacata gtgattttaa gagacctcca aaaaaaaggc tttccccg tg gcgtggtgga ccggaaatgg ctcgtcgtcg gttcttgatt catagtgatt gggaacattt ttggttattt ttgctggaag acctatgaga ataaggtgat aggaggggag ctgactggtg agatggctct tccggccggc cggtcggttc ttattccgca ttatatctgg attaatgtgg aggtaaagaa 120 180 240 300 360 420 480 540 600 660 catgaattct atcatgga tgttatattt gttaaaaaaa atcccctgtt ctagcgtttc aatctgcatg <210> 12 <211> 2446 <212> DNA <213> Triticum sp.

<400> 12 gtgggtctat aaaagacagg gatgtgggat gcaattgatg cgggaaatgc ttcaagctgc gatcagaaga ggaaacittt atatggcagt ggatcatgtg atccagtacc gtcttcattc tgaatcctga gctgcgactt aaaaagacct gtcaattgat aacaagataa atcaattgct aacaacaccg gtcaattgtt agcaaaagca gtccatagtt aacaaaacgt accaattgtt ttgatagaca ggatgcgttg ccgagaaaac cgatgaggat tttgagcgga aaaccgtggc gcgacataca gcgatggatt ggtgaagcta tctatgtggg gtcagggttg gatttaccag gatgttgcgg gctttcccca ggattccgta ggtacgtcga tatgtgaatg gtgcttcatg ttcgtcagga ttgatcaaga gagaagtgga tgtttgcaag ccgatgaaga acaaggctat aagaacaagg gacaaaacca gaccgaccca aacaaaacca gtcaagatct gagagggtca gactggaagc taaaatttaa aatgtttcaa cgcagttgag tgatgtggtg tgaatcaggc agagacttac tgctaaaaca caaagtaaat atcgatcatt atcaattttt gtcaattgtt ttcggctgtt aacaaagcaa taaggaggga tgttgacaat caagtgcgac gcggatttgt gatgaaacta catgagaaac caacagcaat ggtgtaagtt tttagtgata ggttcctata ggttctagta agtgtcactg agtctcccta gttcctgttg gatcacacat gtgttgcgga WO 00/66745 WO 0066745PCT/AUOO/00385 -38agcatcaggc aacatcttta aggatgagct tttctgagga aatatgaagt cacaggatgt attat tcgat ttgatcttta aaggagcatt ttggaggggt ttgtgttctt gaatagaagg gggagcttga ggcgaagtaa tagagatcaa atttgtggta acagaaactc attggtcaga attggtggta attttttttg atgaatttcc aagctcaaca tgtaatgtaa tgttggactg ctcgacaaga tgcaagagga ccatgaaaag agatagaacc catgactgaa ttctataact agagct t tca tgatgagacc tgtggatccg gaggaacaag tttcaatcgt caatggttgg ttggtggtct caacggtcgc cactatgaat gaaacttgcc ggaagcaagg gaacaaaaaa catagaggct gaggccactt tggactctct tgcagatggt ttgaggaaac ttgttttatt tttaccatag tacctgtctt ggtttttgct tttccatgct gcaaaacatc aaaggtgagt caagcagtgg catcagatag gaaattggaa tggtctgaag tctgtgtccg caagcactaa ctgtttgttt gacctaacag aaatggaggc tgcaaactgt acggtctatg gaagatctgt atggaagaag gctgcagatg ttgcagagta agcacagata gcgcatagta attgttgaaa acgacacctc atttgttttg gatgtcatga acagacgctt ttctctatat gatgggccag attcttccaa tatacaaggc tgcaacaaaa aaacgataac gtgctgccga tggggagagg atgaagtgca ttaacgttga aggtgatgct ttccagaggt ctttggcgaa ttttcactga acatacccaa agaacaatgg ttgaggattt ctgaaaggag aagctgtcag tgttgagttt caagcggaga ctgagatttg gctttgtcaa aacctttgta attctagcat gaaagtattt aaagatcatt gtacagttat ctgggaatgc acaacaatgt ttctgcaaga tctttgcata ttggaagaaa aggacagatg tgataaaatt gttaattgag acaagatatc gcaagaactc agtgaaagct tgaacccgat aagattgcat ggaggcctac caacaatgat cttggttaaa gacacagact ggcacaagcg ggccagaaca tactatcagg gatgcatggt gtgcaatgac cataaggcaa aatgctccta tattaactcg tgtattccgt tccacctgaa aaggaactat aaccgaggaa.

aaggagagaa tagatc gttgatgagg 900 gtagttaacg 960 cagcatgtgc 1020 gatgatggac 1080 caggggtcac 1140 gctgagaaaa 1200 gattcagtta 1260 gttgtcatca 1320 aagagtgacc 1380 agattagact 1440 ttctgtatag 1500 gaaaagcaaa 1560 gaagaacagc 1620 aaggccgaga 1680 tgtgttgata 1740 ttatactata 1800 ggttacaaca 1860 agagacggcg 1920 cattgttttg 1980 caaatatggc 2040 aaggccatgg 2100 ggatcatata 2160 aaagcacttg 2220 gacaacaatg 2280 ggcttctggg 2340 aaggaagaaa 2400 2446 <210> 13 <211> 1032 <212> DNA <213> Triticum sp.

<400> 13 gatctctata attttggcag ttaacccctg agtgatggca aatatattcc ctttcgtcta ttttccaaat tcaaaatgca tggttccatg caagcttatc caaaatcact tgataatata 120 WO 00/66745 WO 0066745PCT/AUOO/00385 -39ccaatcacaa ctccctttcg ctgatgtttg cctacacagg aggtttctgc accacagtgg tggtttagat atggtgaaat actgttatta gatgcacaga gcatagcaca cttagaccat gtcacaacgt gggttggcac cccactcggt cgagaattcc cataactttg aggctggttg gaacaaaaca ctgagagaag tttcccagaa atgtgctata gctcttttaa caggggatgg gtatatatat taggatcaca agatcotgta gagattgtgc aactgggtga gaatcgagac aatgcatcat cg tttaccataa gcttgatgag tatgatcagg tgcaaatatc acacattgtt caatccctct cctttggatg gccgctctta aaccattttt tcattaggag gttcgttcgc aactcccgga ctataaaggt tgggatttgt cataatgagc gaacattcct taactggcaa gttgtttggg aaagctgaga tataccgaac aacacagtgc catccaagtg aaagccacag atgcaatcaa aatgatgtga tagagctttt tatcgtagga gcactacagg cactccgtat tcaatgtgac acttaaaatt ttaacaaaga ttgactcatg tgaaggcaaa cgcttgaaat taaatggaaa gagcattgcc gtttattgcg tagagtcaag tggacaagac ctaatgtcaa gtgctttggg tatctccgaa gacggagagg taaggagtta tgcaaggtaa aaagatatat ttccttttta aactatgcga acgtgccgga gccggaggtt accccagaag ttattacatc tgcaactaat ccaatcctaa gtatcatttc tgtatcaaat agtttctgtt tatctttggg gccacgggat 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1032 <210> 14 <211> 892 <212> DNA <213> Triticum sp.

<400> 14 aatatttctt gttctattat gatatgcaac tactccctcc caaagttata ctaaagctgt tagctgtttt gagaccgagt caggcacctc ttctcta .,aa tggtcacctt tcttcttaaa tagctatttt ttcggagcgt ttgatatata ttgtgaagtc ttggccaaat ccctgttaaa ccggggggta ataaagcttc attactgagt acaatacaga ctgtgttcct tgtctagtta cgagtgggaa gaagatggga tggtaataat aatccatatt gacaagtaat gtctgctcgg aaaagtgctt aattatggca gaatgggagc acttaagcct ttggtttact cattttctac gcaccgaaaa atgttccacc tctatgacaa tagctagttt acttgtcgca atggaccgga gtggctagct gcagcccccc ccaagggaaa gtctttctgt tgttaaaacg gtgtactaga aatgaagtta gctgtatcct ggatgcctat caggaatggg aatgccataa actttggtac gggagtacta ggagcgggct cgccccctcc ttctcggctg ataaggccta tagaaactta tgcatcgatg attatcctac tcctacttcc atgatggact atggactatc gcccataaca aactttagta tataagcttg gaagtgcttg atagggtgag gtcgagcttg taggcttact gttccgcaac gcgcagagtc ttgccttgta ttatgtttat ttgttttctc atattcctgt WO 00/66745 WO 0066745PCT/AUOO/00385 ttctgattca attgaaacag agaattacat gcgtattatc cacattgccg ttgagatggc 840 ccccgttgca aaggtaatat aattctaagg ctagtttctt tgatgcgagg cg 892 <210> <211> 871 <212> DNA <213> Triti <400> aggttatcct tgactttttc atcttgtatt gtttcctttc atccccttcc acattgccat atccagaatg ttatattggt ttgtaatttt gttcttgtgt tattcaaggg attctcaatg tctcgaggtc taatacgtga .cum sp.

ccagaatgaa tttgtgagac cagcgcgtta ctactctact atttctcagt gattggtcaa gcaagcactc aaagcaatga taatctttct tttgtacgta acgtggcagg gaattgatcc tttacattgc aaacattaat ttttttccag tacactttct ctttcagttt atctgaatgc taaaaaaact gtgctccggt gggttgtatt catactgtga gtttggcgca ttcaactatt ccatggtgcc agatatctgg tggtgctctt tcttttctca tacgtattat.

tgtttaccat ctttactact tacttgtgtt tgcatctgt~a cgcctggcta taccatccac taaagccaca cagaaaatct tccacccaaa at tgctcctc gatcctgatt taccccgact acaagggacg ttagaatact tccagtgcac agcttatttg ttcgcaacag ttcacgtgac tataaggaac aatcttgaat actgtgagtg tccacatttt cttttcaggt atcgtgagaa gccaacatgc ttctggcgtg gacaaacgcg agcggtatat catgttcaaa gtgcattggt ttgcttcttt agca tat aa t actattccca ttggagcaca ccttactgtc acagaatcat ttctcctaca attctacggc tgtttggtcg aatgatggag cgagat tgcc tcctacctgg cttcggaact gaaagaactg g <210> 16 <211> 1592 <212> DNA <213> Triticum sp.

<400> 16 cgggaattct cgatcccgtg ttaccgagtg ggcccaaaga tcgtgccaac ccaacagaaa ttacgttgtg acatttgata tcatggtcta aggaaatgat cttgtgctat gcttaggatt atacactgac aattttatcc caaggccaca gatagtttta cttgtgagaa tgttgtcgaa gctaactcct tacctctccg ctttcggaga cacccaaagc acttgacatt gggtcttgtc cggtaccaga tgcttaacta ggcaagagag tagtcacatt tcatacggag tacctgtagt actcctacga agaaaagctc catcacatca ttttttccca tgtgtttcat ctgcaaaaag gagctcatta tgacaaatcc gcacctttat tatccgggag tagcgaacga ttctcctaat gagtgcaagt actacttcag ggccttgcag tgatgatgca cagtctcgat agtcacccag ttgcacaatc actacacgat gatgtgatcc agatatatac gtcccttata cagaagtttg WO 00/66745 WO 0066745PCT/AUOO/00385 -41gattacagca aactgatgtc cctattgtcg gaatcatcac ccgtctgaca gcccagaagg 600 gaatccacct atcccttgtg ttttgtttac ttggttcagc acggtgttta tggtgagctc tactttataa ttattgtccc gatcgatccc actgatttgc gactttacga ttgaaccaaa acaggcaagt ttgcatgttc tccactccct actacattga.

catcaagcac aacgaataaa ttcaaaacaa tccagatcat ccacggtagg caatatccta tttaggttga ttcaatcttc tatagttcgg atatgtcgag cactgtcttc tgggttcagt atcgttcctc catacctcat gtgtaagagg attgtaccat gcaattcacc catcaaacgt aagtctgaaa cgaatacaag gtgaagcttg cacaccatct tgatcgatca gaaccctgtg aaaaccggag ctgttggaca gacgtagaca ttcgacggag aattagccct ttcagagcaa gtcatggaac gccgctcgaa gaaccctcga aagcaacgga tttgtttata atgaagtgtt gcgatttttg ttctaaccta agccagccct tgctgcagat gcttaacaca gtgtgtgact tgaaatggaa atgataagga ccgacagcaa gaattcagca tcggcgcttg aagactggtc aattctgaca aaaagttgct tggttcctag cagattggcc cgatgagcct tcattatggg atacgctggc acttgttgcc atttctctcc actatccttt ccgggctcgg cggcgtggat gtagtgctag gttcgatgcc atggaaccgg cccaactgaa caggttcatc 660 tactatttgt 720 gtggttttgc 780 gatgctcttc 840 ctttctcacc 900 agctggagac 960 tccgacttca 1020 atgcgttatg 1080 attatgctgc 1140 ggtatcgcag 1200 tctcttggtc 1260 tatgccctca 1320 gttatttacc 1380 cgtgattggt 1440 cccgcactgg 1500 ccaatggcaa 1560 gaacaagcgc attgtgggat cgagaattcc cg <210> 17 <211> <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:PEPTIDE MOTIF <400> 17 Asp Val Gin Leu Val Met Leu Gly Thr Gly 1 5 <210> 18 <211> <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:PEPTIDE MOTIF <400> 18 Ala Ala Gly Lys Lys Asp Ala Gly Ile Asp 1 5 1592 <210> 19 WO 00/66745 PCT/AU00/00385 -42- <211> <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:PEPTIDE MOTIF <400> 19 Ala Thr Gly Lys Lys Asp Ala Gly Ile Asp 1 5 <210> <211> <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:PEPTIDE MOTIF <400> Ala Leu Gly Lys Lys Asp Ala Gly Ile Asp 1 5 <210> 21 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:PEPTIDE MOTIF <400> 21 Ala Thr Gly Lys Lys Asp Ala Leu 1 <210> 22 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:PEPTIDE MOTIF <400> 22 Ala Leu Gly Lys Lys Asp Ala Leu 1 <210> 23 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:PEPTIDE MOTIF <400> 23 Ala Ala Gly Lys Lys Asp Ala Arg Val Asp Asp Asp Ala Ala 1 5 WO 00/66745 PCT/AU00/00385 -43- <210> 24 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:PEPTIDE MOTIF <400> 24 Ala Leu Gly Lys Lys Asp Ala Gly lie Val Asp Gly Ala 1 5 <210> <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:PRIMER <400> tgttgaggtt ccatggcacg ttc 23 <210> 26 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:PRIMER <400> 26 agtcgttctg ccgtatgatg tcg 23 <210> 27 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:PRIMER <400> 27 ccaagtacca gtggtgaacg c 21 <210> 28 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:PRIMER <400> 28 cggtgggatc caacggccc 19 <210> 29 <211> <212> DNA <213> Artificial Sequence WO 00/66745 PCT/AU00/00385 -44- <220> <223> Description of Artificial Sequence:PRIMER <400> 29 ggaggtcttg gtgatgttgt <210> <211> <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:PRIMER <400> cttgaccaat catggcaatg <210> 31 <211> <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:PRIMER <400> 31 cattgccatg attggtcaag <210> 32 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:PRIMER <400> 32 accacctgtc cgttccgttg c 21 <210> 33 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:PRIMER <400> 33 gcacggtcta tgagaacaat ggc 23 <210> 34 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:PRIMER <400> 34 WO 00/66745 WO 0066745PCT/AUOO/00385 tctgcatacc accaatcgcc g <210> <211> <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:PEPTIDE MOTIF <400> Lys Val Gly Gly Leu Gly Asp Val Val Thr Ser Leu Ser Arg Ala Val 1 5 10 Gin Asp Leu Gly His Asn Val Glu Val <210> 36 <211> <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:PEPTIDE MOTIF <400> 36 Lys Val Gly Gly Leu Gly Asp Val Val Thr Ser Leu Ser Arg Ala Ile 1 5 10 Gln Asp Leu Gly His Thr Val Glu Val <210> 37 <211> 9024 <212> DNA <213> Triticun sp.

<400> 37 aaatatgaaa ccaaaaaaaa taatggataa aaaataaaac gttcccttat tatctcccgt acccaacgtc acaagctcac aaaggaaacg ggctgagagc ccctcgacaa aatatgaatc ccaaaagaat tctcagctga agattttttt tttgcaaaaa catctcgaac attataaaca ccgcgaaaag aaaaggattc ttggtctcat tgcgcgctaa ctccgcagca agaagacaag aatagaaaaa atcaaagaaa gcaccccggt acatgcccag cgaaacacat gcgacgtgat atgagtttta taatccaaat ttacactgag ggaagacaga acaacctgga tcgtcacatg ggaaaggtaa aacgaaatgc agcgtaggac cgagagaaaa gggctgcgct tggatcctat gcaagactga ctattagcaa attccaggac aattatccaa cagcagaaga tcagacgccg aatagaaagt agaagaaaaa aaaaagaaaa gaaaaatggt ttgttcgcta ggtggaaaaa tcattatatc agttcagtag caccaaacaa accacgt tcg agcaaagcag tcactcaagc taaataggaa aacgtcactt aatagaacgg gcgacaaaaa cgaagctctc gtgaatgaga caacataaat aagtacaaag cccactactg tccttggttg tgtgcttccg aagcaaactg WO 00/66745 WO 0066745PCT/AUOO/00385 -46 caatgcttct cgcaacgcat atatacgcaa cacgtggatt actggccccg gttcgcttct gctggtgagg ggctgccgcc agcgtgctcg tacccgcccc tcggcctgac tgccaccgcg ggaccaacec gccgcatcct gtgagcgcgc cagcgcacgg atcgacgacg aaggtagt ta cgctcacgaa gggatcccgt cagcgaggca acaaatctac cgccccatcc cctcgccgcc tcagcgacaa caaatttcga aactgaagaa ctgcagcccc agcccgtgga accaccagaa tggctgctga ttcctgttgt cgttcggttt gcatgcacaa tcaacgcggg ttgcaacttc tgggcggaac ctagcgggca agcctgccgc ctcgtgtgct cccgcaaacc tcgtgtaaag cccgt tcgtt ctactctcca gcgaaccgta tcctcgcgct agccacccca ctcgcgacgg ccgccgcgtc gttatgacca ttgtttattt caagacgctc ggacgccgcc cggcggcggc gtcgacccag gacaagcata ggcgccggag gtcctcggcc gggtgCggtc cgctgtacaa ggccaaggat tcacgactcc gtgttctccc tcataaaact atcccctagc accaacaaac cctcctcctt cttccagcag ggggcctccg ccgacagaag tcgttgtgcc gctcgcgtgg gcagacccaa ccgccgccgt tacccccaca ctcccactgc ccatctcccg cgcgtcagcc cgccggggcc agct gtggcg cgtgaggcag agttatgacg atacaaaacg gaccgcgacg cgtccgccga ggcgcgacta aacagagcac gccgaggccg tccgttgtcc tctgctcccg gttgtcgaag gaagaccttt gatggccggg ggacctttgg tggtgcaaaa ttgctcgaat acgcacgaac gaaacagtgc cgccgcgagc cttgtcacca ctcgcccctt gggccggcgc gcagctggag cacgccgcaa cacgccagcc cgtcgccgtc cagagcacac caccacctcc ccccgatcca tcccccggga ggcaggttgc gcgctcgccg ccccgcgca c cgtgcgcgcg cacgcccgcg ccgcggaagg gtatgaacgg aagacagcgg cggtgaacgg eggcttcgga cagctgagaa ggtctgacac aagctccaaa gggatttcaa ctgtcgcaga caggggagaa caggcatgga tactcataag gcatgtgccg agttgcagtg cccgttccgt cggacgcttc gcgaaaccca agggtaggac accgagcggg cggcaccggg gccagggggc ccccgctcgc tccagtccag gcctgcgccg tgtcgtcggc gatcacgcag actggccgcc ccgggaagaa tccgcggtgg cctcgagatc tgtgcaggtc cggcgggccg catgccggtg gctgcccacg tgaaaacaaa ttccgcagct gacgccgccg tgtcagcgac gccaaaggct gaaatacatt tgatgcgggc tgtcatgaac cattacctct aacaaacatt caccgcgtca ctctatctac cctcggtctt ctctctgaca cggctcgtcc caggctgtca gcaacggaac cccgctttcc cgttcgtacg ggccatttct tccagcccac cgctctgggc ggtcgcgtcc gcgggcgagg gtggccgccg ggacgcgggg cgccgccacc atcgtcgtct gcggagcgaa tccccgccgg aacggcgaga cccgcacgcg gctaacgtcg accatttcca tcgtccggct gtggaacaag ctttcgccgc ggtttcgagg tcctttgaac gtggtcgtcg tcagtctctc gtgttgcata 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 2640 WO 00/66745 WO 0066745PCT/AUOO/00385 -47ggtggtctgg gttatggtac tgagtagtat gtaaattaat tttacagacg tgggtggggg ggctgataac ccaacgtcgg accaaatatc tttccggtta aatgtagatg gagctagttt gtgagagagg ccagtaaaga gaaaatagtt gctagacttt tcttaggttg aaatactaca tattgcttat aagtgaatta tcttccgaca ttggtgtttg ttacacattt gttgaggtat atgtatttca acct tacatg tgaacaatta gcgaattgat tcatccaatt catttttatt atggggatgg atctgaaagc tacataacat gagatgttgc tacaagcttt aatgttatta ataagagcgt gagtagagta agggggtttg cacaccatca gtttacccgc gccagcgccc atggtttcta atggataaat cgcggt tcgg gttttgggga gtaaactatt atggtatcat gataatctga tggtaccaag aggctgctgg tgtcataata tttccatgct ccgtcaggaa attgcactga tattttttca ctctccaact acagatacat cacatttggt tcttgatgat ttggaaatag tagatatttt cctctataat aaatctggtg atattacagg cgctcaccag gggtgctctg catttaactc agtgcaagac ttagattact tttcatagcc aaacaagtgg gtgaaggaat cctatagatc gtgtgtgtat tcatattcac gtatgttgtc t tagagccat gttaactttc ttctgcaggc ataaaccata aattttaaat gtatggggac acaggtaagc aatcaatttt tatatcgatg gacatttatg taaactgaga ggaaattatg caattgacaa aatctcttgt caagcgttat ccttgttact catttccacc cqtacttggc ttgcaggttc tttattgcaa gaccatggtt gttccttttc cccaaggctt tgttgggtcc atgaaagtgt actttagtga aaccctggag tggttagcag gaatgtcggg cgaataagta ttatactact tgtaattgtt gagctttcat caaaacccca gggattcagt atcccaatta tattattcaa ttgatgataa tatgaagaag aaaaatgcaa taagtgtttt gagttgattt ggggcagcag acaagccaag aagcgcatga cctattacca gaagtgcata gatt taactt tcatcgttat tgccacaaac atatcatccc catggcacgt atgattggca tgatgcagta tcctaatctt tggcaaagag atatgttcga tcttctgtca tctaaacgct gttaggttgc ccagatttca tacccgatcg gttcctatct ggatgatcaa agtaaacagt ttcaatgcaa gaatttttgg tagagacgct ttctgtagaa aatctagaat ttgagaaatg cctacgatgt tcgaagggga ttttgtcctg tgtgttcatt acaggttaat gcctactgac ttttgttctg ctatacaatt tatactaata ctgataatct gtttccatgt aataatatac attaaatatt tccatgcggc cacggcactc cactcggtcc gatttttctc aggacatcgt 2700 ataatatcag 2760 tactccctcc 2820 cttatagtag 2880 tgaggcctac 2940 caaagaagga 3000 accgttttgc 3060 tcaattaggt 3120 tttatcaaca 3180 agatgtttgt 3240 ttttgattgg 3300 gagttggctt 3360 cttactagtt 3420 attagaagtg 3480 catggacttg 3540 atcctttcta 3600 cggagtccga 3660 gctgaaattt 3720 caggatatgg 3780 gacgctcctc 3840 cttctatatg 3900 tggcatatga 3960 caaggccgct 4020 atgtgtatgc 4080 acatttcaat 4140 attgcactga 4200 tctcttcacc 4260 actcctactt 4320 attggtccat 4380 ggtgtccctt 4440 ctgcctgtct 4500 attatggtga 4560 tagtctctac 4620 WO 00/66745 WO 0066745PCT/AUOO/00385 48 tatttactcc acattgtttg aggaaactaa acgggttgca aaattatgat ggcttatgaa 4680 agttatagtc tttggtgctt tacggtgcat ctatagctgt gggccccact tggggtcttg tcccctatgc atgctgtttt ggccagcgcc tgtcatgacc gcccctcttg agttgtgacc cctacttggg atcgcaaacc gcaatacatg ctctaagcct cctacgtcgg tcggcgacat catccatgtg cactgttcct atcccttggg cacacgccac tacttagcca tcttaacggc tcatactgca gcgagcatca ggtgcggtgg cggtgccgaa ggatgaggga gcgaagcgct atttctcagg ttatagaggt acagttgtag tttccgtatg tagaccgtgc tgtcaaccta aaaatgggac acttcatgtc tctttggttc ttcatgatgg atccaccaac tcctttcccc atcgcctgcg agcccatacc aacttctcct ccgagttggc agcacctttt ctgcagttgc ctcctcgacc aaccgaatca ctattccccc tcatcggttc actaagccct gagagagaac t aca aacaa c tgcgagagcc aagtgtacat gtggctcaaa Ccatattgaa cataaaggat tcatgatttc tcgcttctcg aaatgcacca actatgaaaa taggagtcaa ctacgtcgcc tgacataaat ctcgcaggta ttgtgtatgt aaggctacca cccaagtgct ccaacacaca tcgtacccaa tcgcctcata tccctgcaca ctccttctcc catggcccta cccctcacca ctgccgcctc tcccattcca tcatagaact caagccgtgt aatggctatt ttctttctcc atgagcttgt ggatatggtg agagccaggt atgccgaacc gacaccccaa gtggtgaggt ctcataaata catctcccct tctaaatccg gtggtgcttg acgggtgcaa acgttgccta attgggccca aaatggaaat tgctggtagc gtqtgtctgt tgctcaacaa ccgcaccatc aaatcctcaa acatgtcttg gagcccgacc tcctcctctt cactctggcc ttccccaatt attgcaagtt aactctgatc cacgtggcct aacaccggag cgcgtcataa tctttctcct tacccgttga tggtgccacg tcacgtgagc ttttgcacca aaagtgaaca tagaagctat cagttgcttg ttgcaatgtt agatcagaga catatgtagc aacttgtcac aaacttgctg tgtgggcatt cacactctct ttataataaa cagcacgccc gtggggaggg gcccacctcc gctcaaagcg catccgcaaa ataacccttg tagccggacc tccccataga gtttcccccg gctcgcacta ggggagcccc cagacctcgt ggcgaggatc aggtcatccc tataagacgg gtctactgat taagtgggac tcggcaagcc gtttacgaat gttttctgta tggtgagtcc tgcctccgac tgctatgact cattcaaatt cacttggtcg tcacttcaat gcgtgttcac 4740 ttgtgtgcca 4800 gtattcccgt 4860 atttacatgt 4920 aatgatggcc 4980 taaacattaa 5040 gggggtatgc 5100 gcttcaacac 5160 gcaacgtcgt 5220 tagtgagcat 5280 gagctgcaca 5340 gttatagaag 5400 tcgtgccgcc 5460 ccgcgaagct 5520 ggaggtcctc 5580 tcgcgagctc 5640 tcccgtggcc 5700 accgcatgtt 5760 gacggcgtcg 5820 acttatttgt 5880 aagtgggacc 5940 ccacacacag 6000 atgtcagcag 6060 ggaaagtgca 6120 ttttacaact 6180 attcttttct 6240 attgccaatt 6300 actaggtatt 6360 cttaacattt 6420 tgtacactga 6480 gacttgcctt 6540 WO 00/66745 WO 0066745PCT/AUOO/00385 -49 tggtccagct atttggatca tgatccgctc tgtctctcca aggaacaggg ttgatttgga tattagacgg gtcactattt tgctcaattt aagtagaaaa aaaaatgaga tatacaacac cgttgggcta ccgttcaccg ggtgaacacg agccccgggt atacggcaga aaccccgagg ctggactccg cgcgccgacg atcatcgcgg accgggcgcc gtgcgcgggt gcgctcctca tacggcaccg gaccccttca atcgaggcgc ctccaggagc gacgtcctcg gcatgcgtgc ggcat ccgcg cccgtagcag gttgtgtgca aacgccattt cgggcagacg ttcccgaaga tggtcgcccc tgccaccttc aggaacacaa atcaagtgtg ggctaggtcg gtattttgtt aaattctcct acatccgtgg tgacatgccg attctttctc agttgcctga ccaactactt acctgtggga acgactggaa tggacgccca gcaagcggca tgccgctgct acgccatgcc acgacctgga gggtggggtt tgccctcccg tccccgtcgt accactccgg tcgggcactg gcggcatgtc tcaaggccaa atgacaggat aagtacagtg agtagagcgg ttattacaat gcgtagcaaa cgctagacaa cacttgtgat agccatagat ggacaagaag caacagtctt atgaatccta cttgccatcc gttatgtgtt ccataatgat caagt ttaag aattacatgc ttcatgttgc gcactacctg cgccgccggc gctgaagacg gacccgcggc cctcaagtcg gtgcaaggag cggcttcatc ctggatcgtg gagcatgctg ctccgtgcgc gttcgagccg gcacgccgt t gctcgggtgg cctccgcacc gcaggacttc gtaccagtgg ggaactgcat acatgaggtg aggtatatgg gttgttactt tttttcatat ggtcttccgc cttattacga gccttgttct ttgcgtagtt tgaaggcaaa ctggtacaat cgctctgtgc tttagtaatg cacatacagt caccaccggt tttggtcagt atgcagggcc gaacacttca ctgaagatgg gtggagggcg atcgtcaacg .gacggctaca gccctgcagc ggccgcctgg agccaggacg cggcacttcg ctggcgcacc tgcgggctga ggcggcctca acgttcgacc taccgagact agctgggagc tgaacgctag tgcgcacgca tgtgtggttg gaatcttaac attcttgtta ggctcgctct acaatgaaca gttgtgccat ctgaatggtg tggtcgtctt gctaattcct gccgttgcta taagcgcttg taacctgaac tctcctgcat gcatttttac tattccattc gtggccctgt gactgtacga cggaccaggt gctgggggct gcatcgacaa ccaacttctc gcgagctggg acgggcagaa tgcagctggt agcgggagca ggatcacggc accagctcta gggacaccgt gcgccgaggc tcaaggagag acgccgccaa ctgctagccg ggaaagtgcc agacgctgat ttggtattgt agtcggaggc gcgcaagagg 6600 ttgagttttt 6660 ttcaaacatc 6720 ggtttcagct 6780 aactgcttgg 6840 tcgatcaagt 6900 gttgcttgga 6960 gggtcgcttt 7020 tttctggact 7080 ggttcgaaaa 7140 ctcaaagtta 7200 ttcggtactc 7260 agatgaattc 7320 ccccgtgggt 7380 tgtcgtggtg 7440 tcacgacatc 7500 catggagtgg 7560 cctgaggacg 7620 cctgcaggtc 7680 gggcgtggag 7740 gatgctgggc 7800 ccacgacaag 7860 gggggcggac 7920 cgccatggcc 7980 gccgccgttc 8040 gcacaagctg 8100 ctggagggcc 8160 gctctacgag 8220 ctccagcccc 8280 atggagcgcc 8340 tccaatccgg 8400 aatttgttat 8460 caagggcgaa 8520 WO 00/66745 WO 0066745PCT/AUOO/00385 agctagctca cggcaagaat cagttaaaac tggactcata ccatcaacat ccagatacat caagtgaagg gaagagttga acacgtcacc catgtctgat gggaagtgaa aatagcactt gcatgttacc ttgaacctat aggtgccaaa caacaagcat agttgtgatt gtccaatcca ggatgcacgt ttcctccctg cgagtggaag aaaaaatgcc acaaactaga gggctacaac cactacggag tgacgaaacc aaga gccatggttg cttgaattag tgaacaagaa tttcgccccg acacaccact cacaacacac cctccggcgt gtgcgctcca gtttggtagc cactttcagt aaccaacatc ctgtatatat caaaacccac cgaaagactc ccttccgatg aaacggtgcc gcagtgcaaa aataatcagt acacccggta aaagcaacga acactcaggg acatagacta aagaaatcat ttcaggaagg 8580 8640 8700 8760 8820 8880 8940 9000 9024 <210> 38 <211> 11611 <212> DNA <213> Triticum aestivum taatccgttt cacccccccc acaattttgg aaatatcagg ctgtttgggt ttttcacaaa caagttcttt tccaggagct acaatctcag tcagaagtgg tcctgcacga agaaagcaag ttccatgtgt tcgaagctgc tccacgccag cgggcgaggc gtggtgggtt acggagccct cggcggcggc t gt tgt t tga gtctaatgaa ctatatgagt gatactaaac aaacgtattc ggatcatagg acttcacatg cgaattttcc ctggtgtatt aaaaaactgg ccctcagccc acattcgcgt tacaaaaaac gcgcacacgg tctcggacaa agcgttgtat agaggggata ttgcgggcgg ctgtgccctc ctcgcgcagg tttggttctg atatatgtga attaaattca ctggatgctc tcagtaaaaa tcagactata agagtagatt tagtattttt tttcaatata acggtttatt ctcactcttc tgaagttttt accagccatc agaagcagct aatggttgaa tccaatttta gggcagtcgc ggtttagtag ggagcagtca tacgggtgat tcccgggtca tgggagagga aaaaacaaac agtctactcc cagacaaaaa ttttcttcca tgggcatcca tgaaattaat tgtatggtta ctagctgatt ttcctcctac tcaaaagaaa caccaccgtc cgaacaaaaa ggacgaagga gttctttccc cgctgcgtgg gttcccggaa gccgctcgtc tatggttctt ggttcatagt tttggagcat cgaggatatt catgtgaagt attcttatgc tggatacatg agtttgatat attcgtatag tttaaaaaaa ttgtgtgcag ct tctgctct acaatatact cgttactggt aaccaaacga gcctttttgg cgtgaggagg tggactgact atggagatgg gtcgtccggc gattcggtcg gattttattc tggggtgctc caaaaagtct ttcatgaaaa acagaaaaaa tcatggtatt ccccatattc gggtggagca aactcgtaca tttcccataa gtcttccgct tgctggaaaa ccacctgcat aaataaagga tgcgcagatc ggaggctagg ggtgtggtgg ctctccggcc cggccggccg gttcacggaa cgcaaaaaaa 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 WO 00/66745 WO 0066745PCT/AUOO/00385 51 aaaggtttat aagggcattg ggaattcata ttctgttata aaatgttaat caagaaacag tctgcacctt tccgtatgta gagtgtatca atataaattg ggtcagaaaa gaggtcagaa aaatgacctt tatctataag cttttgtgtt atgcatggta gtttggaagc tgacaatgaa gtatgtttgg gttgcgtttc actacagttt tcaaagtatt atactttgca tcattcagat ttcttctaga caataatcgg agaatggaca aagcagttct gacagtgaat agttaaagaa ggatgtgatg gatggatgat agtgattttg gt tttgatt t ctgcttaaaa tttgttaaaa gttaatgctg aaattcattg gtatgtttgt aggcgagcat gtagttcgaa ga tt acat cc tgagatacag ggcgatttca gataattctg aaggtt tcct ttccagcctt gcaagttcag aatgtcttat tactgtagtt caatcaaaag tcagtttttt tgtggatact taaaaccata acgaaacaca cctcctaata ggatatacga gatgaagaaa gatactagag ataatcgggg tcattaagca gacgtatttg gatcataatg gcggcggaca atttctttca gcatgcggaa cgacgtgatt aaaatcccct gttaatttgg cgaaaaaatg gatgaagtta tgccatcttt ttgcgctaat tttctgttga tggggacctt gtagaattta ttccgattgt ttttacgctc ttttgatgaa gtttgaggaa tcaaacctca tatgaaacca tatacagcgt aaaaagaggt gtagtttata gtttttagaa gcccagatgt ggaaatcaag caagactcat ctcttgatac aagccgagac gagtggatgt gtataacgaa agctggattt ggactgtaca aagctagagt tctcgggaac catattggtt ttaattgctg gttctagcgt agtgaagatt gtggagcgaa tttccatata ctataagctg gttttttgac acggagaaat ccctactgta tatgagaggc tcgcaaatac aaacatgttg tggcagattt taatctgtca gtattttgat acagtctttc tgcaataggc cccaactact atactacaat aaactgtagt tctgttaact aaagatggta tgttgaacca atacaatgcg tgctaaagcg ggcggatgaa gaaggaagtg gccagcaact agagacattg tgaagaagac atttttatat atttattaat gaagaggtaa ttcagtctgc tccacggcaa ttcggagagt ttttttgcga gtatttgtct gaaacgaaa c ttatccttgc ttatgctaaa ataaataatt cttcggattt agctgcacaa actcgaagca aatggcctat actacggttt taagtatttc caccagtaga ttttttaata ttttattaca atccttgaaa tcatgtcttt tcacctcagg agcaccgaga ctattaagta gactcgtcgc gatatacttg gatgcagtgg acattgagaa agaagtgtga gtatttgagc ctgggagtca gtggtgagct agaacatgaa atgatcatgg gagtttcgaa atttacattg taaagttact gccagatagc tatgaagacg ttagaagtga aagaagaagt tggtaggatt tctcaagcat cttattttcc ggacccttcg cattctatct tctatagcga ggcaacagtg caaggccttt ctgcaaaaac gccaaacacc tactttgaga ccaaattgca ttaaagtcat atatagaaca ccgagacagc aaaatgcttt cggctgatct acaaagctag gtgtgatagt tagtagatgt tggatttgtc 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 2640 2700 2760 2820 2880 2940 3000 3060 3120 WO 00/66745 WO 0066745PCT/AUOO/00385 -52aggaaatatt tcaagcagtg cgacgaccgt ggaactagat tgttcaagac ggaagtggat aggaaatgtt tataaaagac ggatgcaatt tgcttcaagc agaggaaaca agtggatcat accgtcttca tgagctgcga cctgtcaatt taaatcaatt ccggtcaatt gcagtccata cgtaccaatt acaggatgcg aaccgatgag ggcagataga ttacatgact gctttctata ggaagagctt agttgatgag tgttgtggat gatgaggaac ttat ttcaat attcaatggt ggtttggtgg cttcaacggt aggcactatg tgagaaactt taaggaagca caagaacaaa acatttgagg acgagtgctg ttttcaagca aggtttgaga gatgaaaccg tgcgcgacat tttgcgatgg gtgggtgaag ttctctatgt cttgtcaggg gatgatttac gctgatgttg gttgctttcc gttggattcc gttggtacgt ttgtatgtga gatgtgcttc acccaagcag gaaca tcaga actgaaattg tcatggtctg acctctgtgt ccgcaagcac aagctgtttg cgtgacctaa tggaaatgga tcttgcaaac cgcacggtct aatgaagatc gccatggaag agggctgcag aaattgcaga cgaactcgtc aagctgggaa gtacaacagt cggattcgtc tggctgatca acagagaagt atttgtttgc ctaccgatga gggacaaggc ttgaagaaca caggacaaaa cgggaccgac ccaaacaaaa gtagtcaaga cgagagaggg atggactgga atgtaaaatt tggaaacgat taggtgctgc gaatggggag aagatgaagt ccgttaacgt taaaggtgat tttttccaga cagctttggc ggcttttcac tgtacatacc atgagaacaa tgtttgagga aagctgaaag atgaagctgt gtatgt tgag aggaaatgtt tgatcaaggc ggaagtgggt aggaaatgtt agacgcagtt ggatgatgtg aagtgaatca agaagagact tattgctaaa aggcaaagta ccaatcgatc ccaatcaatt ccagtcaatt tctttcggct tcaaacaaag agctaaggag taatgttgac aacttggaag cgaaggacag aggtgataaa gcagttaatt tgaacaagat gctgcaagaa ggtagtgaaa g aat gaa ccc tgaaagattg caaggaggcc tggcaacaat tttcttggtt gaggacacag cagggcacaa tttggccaga gcggttgacg tcaaacagtg atatttagag gcagtggatg tcaacaagtg gaggcggatt gtggatgaaa ggccatgaga taccaacagc acaggtgtaa aattttagtg attggttcct tttggttcta gttagtgtca gttagtctcc caagttcctg ggagatcaca aatgtgttgc aaagttgatg atggtagtta attcagcatg gaggatgatg atccaggggt ctcgctgaga gctgattcag gatgttgtca cataagagtg tacagattag gatttctgta aaagaaaagc actgaagaac gcgaaggccg acatgtgttg aagtcgggcc 3180 caacggtacg 3240 cagatttgtc 3300 aagctgggtc 3360 cgacgatgtg 3420 tgtcgggaaa 3480 ctagatcaga 3540 aacatatggc 3600 aatatccagt 3660 gtttgaatcc 3720 ataaaaaaga 3780 ataaacaaga 3840 gtaaacaaca 3900 ctgagcaaaa 3960 ctaaacaaaa 4020 ttgttgatag 4080 catccgagaa 4140 ggaagcatca 4200 aggaacatct 4260 acgaggatga 4320 tgctttctga 4380 gacaatatga 4440 caccacagga 4500 aaaattattc 4560 ttattgatct 4620 tcaaaggagc 4680 accttggagg 4740 actttgtgtt 4800 taggaataga 4860 aaagggagct 4920 agcggcgaag 4980 agatagagat 5040 ataatttgtg 5100 WO 00/66745 WO 0066745PCTAUOOIOO385 53 gtacatagag ctcgaggcca agatggactc gtatgcagat ttgttgagga tccttgtttt acatttacca taatacctgt dtgggttttt agatttccat ggagcaaaac aagaaaggtg cctgagtgat catgcaagct ataagaacat tgagtaactg cagggttgtt tatcaaagct tgtttatacc ctctaacaca gatgcatcca cttaaaagcc ttttatgcaa ttgttctatt actactccct tactaaagct ttgagaccga tcttctctaa tttcttctta ttttcggagc tattgtgaag atccctgtta gctagcacag cttgcgcata tctattgttg ggtacgacac aacatttgtt attgatgtca tagacagacg cttttctcta gctgatgggc gctattcttc atctatacaa agttgcaaca ggcaaatata tatccaaaat tcctacttaa gcaattaaca tgggttgact gagatgaagg gaaccgcttg gtgctaaatg agtggagcat acaggtttat tcaatagagt attggtaata ccaatccata gtgacaagta gtgtctgctc aaaaaagtgc aaaattatgg gtgaatggga t cact taagc aattggttta atacaagcgg gtactgagat aaagctttgt ctcaaccttt t tgattctag tgagaaagta cttaaagatc tatgtacagt cagctgggaa caaacaacaa ggcttctgca aaatctttgc ttccctttcg cacttgataa aatttgcaag aagaaaagat catgttcctt caaaaactat aaatacgtgc gaaagccgga tgccacccca tgcgttatta caagtgcaac attagctagt ttacttgtcg atatggaccg gggtggctag ttgcagcccc caccaaggga gcgtctt tct cttgttaaaa ctgtgtacta agatactatc ttgga tgcat caagtgcaat gtacataagg cataatgctc ttttattaac atttgtattc tattccacct tgcaaggaac tgtaaccgag agaaaggaga atatgatctc tctattttcc tataccaatc gtaactccct atatctgatg tttacctaca gcgaaggttt cggaaccaca ggtttggttt gaagatggtg catcactgtt taatgatgca ttaatgccat caactttggt gagggagtac ctggagcggg cccgccccct aattctcggc gtataaggcc cgtagaaact gatgcatcga aggttatact ggtggttaca gacagagacg caacattgtt ctacaaatat tcgaaggcca cgtggatcat gaaaaagcac tatgacaaca gaaggcttct gaaaaggaag tataattttg aaattcaaaa acaacataac t tcgaggctg tttggaacaa caggctgaga ctgctttccc gtggatgtgc agatgctctt aaatcagggg attagtatat cagataggat aagc 2cataa acaactttag tatataagct ctgaagtgct ccatagggtg tggtcgagct tataggctta tagttccgca tggcgcagag ataacagaaa acaattggtc gcgattggtg tt gat tttt t ggcatgaatt tggaagctca atatgtaatg ttgtgttgga atgctcgaca gggtgcaaga aaaccatgaa gcagttaacc tgcatggttc tttgtttacc gttggcttga aacatatgat gaagtgcaaa agaaacacat tatacaatcc ttaacctttg atgggccgct atataaccat ccaatatttc cagatatgca ta caaag tt a tgtagctgtt tgcaggcacc agtggtcacc tgtagctatt ctttgatata acttggccaa tccggggggt 5160 5220 5280 5340 5400 5460 5520 5580 5640 5700 5760 5820 5880 5940 6000 6060 6120 6180 6240 6300 6360 6420 6480 65d0 6600 6660 6720 6780 6840 6900 6960 7020 WO 00/66745 WO 0066745PCT/AUOO/00385 54 aataaagctt tacaatacag ttgtctagtt agaagatggg aattgaaaca aaaggtaata gccttttttt tggaggtctc tgtcgaggtt tgtactacat caactgaagc taaaccaata tgttttatat tttacattta agtcgaagac atagtttttt aaaattatta aactacttat caggctgaga agctaaagga caccctagga aaaggttgtg ttacagtttt gtagcaaact aagatgttca tagattgcag catttcaatt aaggcacaag tttttggttc cttcacaacc atggaaggaa tccagaatga tctttgtgag ccattttcta agcaccgaaa aatgttccac atctatgaca gagaattaca taattctaag cattctatgc ggggatgttg attctcccga aatctattca taaagaattc tatgaccaat ccattataca tatcaaagtt ctgaccgttt ttgtgggaaa ggctagagcc cgtagactat gctagttata atttgctgct cacgcagcct cattaaggct tattctgata attgccatct acctttattt acacacttgc aagagaaata caatcagcac tcttcctgta tattcatttt tgatgaccgc attttctcca actacacttt caatgaagtt agctgtatcc cggatgccta acaggaatgg tgcgtattat gctagtttct cataatacta ttacaagtct agtacgactg cttagtcttt tgagctgcga aagggggtgc ttattaatca tttcttgggg acttcctgga tgtatagttg ttgtactggt agaagccaat acaatgatat agatgagcca gcacaaccaa aggccctggt tctcttgcgc gtcactcaat atgcttgagg acgtgctgtg tgaaagaaag catttatcaa agtggatgcc gacatttcct agatttgggt gtacgtatta cttgtttacc aattatccta ttcctacttc tatgatggac gatggactat ccacat tgcc ttgatgcgag tg ctct gt ca ttcacgtgcc tttgaaccaa aaaatttcaa tggagcgcag caagatcagt agttacatct tggtacagaa acctcaaaat agtgatataa taataatgtg tagtaacaca gtgagattag atcaatccaa gaacacagct cagtggctgt ccatatgacg gataaggtag atgcgtttga tacaccttcc aaatgttatc aaataaatga aatatctcaa tttctaggat tcttctgtca tttagaatac attccagtgc cttgccttgt cttatgttta tttgttttct cat at tcc tg gttgagatgg gcgagatctc tgatcgatga gttcaagatc agcagtgtaa ctcaaaatgc tagggtggca aggcactaat atttcaatgc ataaaagtat gggtatgaat aacatattac tacctttttc atacattggc tggctctata ctaattttaa aagatcat cg caaggactcc ctaccaaacg acaatctttc ttgtcaaatt attatctggc acctgaatgt tttacttgtc gaactctcct gtttggcgtc ttctgctctt tagctgctat accatgttca aattactgag tctgtgttcc ccgagtggga tttctgattc cccccgttgc atcaccttat tctcataggt tagggcatac gttgaagtac cacgaagctt cagatcccaa gaatttcctt aggtcaagga gggttggacg cagctaatgt ttcttttcac tcattcatat cttggcattc accacttttg attccatgat cgtgggcaca atggggctcc ct tgtaacct ctttcccttt tcagtttctc at gggat t tg tagagcttaa tagttgtCtc gaggattttt ggatgtgtat gagtttatcc attgactttt aaatcttgta 7080 7140 7200 7260 7320 7380 7440 7500 7560 7620 7680 7740 7800 7860 7920 7980 8040 8100 8160 8220 8280 8340 8400 8460 8520 8580 8640 8700 8760 8820 8880 8940 9000 WO 00/66745 WO 0066745PCT/AUOO/00385 55 ttcagcgcgt tcctactcta ccatttctca atgattggtc tggcaagcac gtaaagcaat tttaatcttt gttttgtacg ggacgtggca tggaattgat tttcccagag gtttcatact caaaaagggc tcatcacccg ccctcgaaag gtttataaaa aagtgtttgg atttttgcag taacctacga cagcccttca tgcagatata taacacaact tgtgactatt aatggaaact ataaggaccg acagcaacgg attcagcagt ggcgcttggt gactggtcat ttctgacacc tacagggctg gctgaggatc tactttcagt ctatctgaat gttaaaaaaa aagtgctccg tcgggttgta gacatactgt ctgtttggcg tattcaacta ggccatggtg ccagatatct tgcaagtaga acttcaggtc cttgcagcag tctgacagcc caacggacag agttgcttac ttcctaggtg attggccgat tgagcctctt ttatgggagc cgctggctcc tgttgccatg tctctccatt atcctttggt ggctcggtct cgtggattat agtgctaggt tcgatgcccg ggaaccggcc caactgaacc tgcagatcgt ctcttgcctc ttctttacta gctacttgtg cttgcatctg gtcgcctggc tttaccatcc gataaagcca cacagaaaat tttccaccca ccattgctcc gggatccata tatataccaa ccttatactt aagtttggat cagaagggaa gttcatcatc tatttgtttt gttttgcttg gctcttcacg tctcacctgg tggagactac gacttcatta cgttatggat atgctgcact atcgcaggac cttggtcttg gccctcaaca tatttacctt tgattggttc cgcactggac aatggcaaga cttgcttcag ctccaccagg ctagcttatt ttttcgcaac tattcacgtg tatataagga acaatcttga caactgtgag cttccacatt aacttttcag tcatcgtgag cactgacaat ggccacagat gtgagaatgt tacagcaaac tccacctcat ccttgtgaac tgtttacttc gttcagctcc gtgtttacca tgagctccaa tttataattt ttgtcccttc cgatccctat gatttgcata tttacgacac aaccaaatgg gagcaagtat gcatgttcca cactccctgt tacattgaat acaagcgcat ttagttccaa gggaacaaag tggtgcattg agttgcttct acagcatata acactattcc atttggagca tgccttactg ttacagaatc gtttctccta aaattctacg tttatcccgg agttttatgc tgtcgaaggc tgatgtccct caagcacgca gaataaacat aaaacaaaag agatcatcga cggtagggtg tatcctacac aggttgatga aatcttcgaa agttcggaaa tgtcgagctg tgtcttcgac gttcagtttc cgttcctcaa tacctcattt gtaagagggt tgtaccatgc tgtgggatcg gcgcactgca cagaaatgca gtgtttcctt ttatcccctt atacattgcc caatccagaa cattatattg tcttgtaatt atgttcttgt catattcaag gcattctcaa taccagattt ttaactatgt aagagagctg attgtcggaa attcaccgaa caaacgtttt tctgaaaatg atacaaggcg aagcttgttc accatctagc tcgatcatgc ccctgtggct accggaggtg ttggacatga gtagacaatg gacggagccg ttagccctga cagagcaatc catggaacaa cgctcgaaaa actacagtca gtcgtacata tgagtgcatt 9060 9120 9180 9240 9300 9360 9420 9480 9540 9600 9660 9720 9780 9840 9900 9960 10020 10080 10140 10200 10260 10320 10380 10440 10500 10560 10620 10680 10740 10800 10860 10920 WO 00/66745 WO 0066745PCT/AUOO/00385 56 gggaagactt aatggttaag attctctcat gttatatgtt ttggtttctg ctgaaacaga tctattagta ttgtagacat ttttttttat ttaggaagcg cagttcttgc ttatgtatat cgcatcggca cttgtgcaaa acttatggta ctatctgttt cacgctcctc taaactaagg tcactagaag gtgtgtgtgg ctcaagcaat gctcctgctg tgttaagatt atataagaac cttatgtgca tacatcaatg tgtgaaatgg taagctgtga tacatcaacg tataagcgcg atatttcact tggccaatgg ggatatcatc ttccttttct cgcagtgaca tgctaggctc atatttacat cagggccatg ctgtgagctc gtgaagattt cttttctgcg atgacctgtg gaaggtgccg aagggagatc tttccttccc ttttgtgagt tctgatcatg ttgtggatga attatgcaga tgaaaacagc acgagctaaa ccgcctaggc ggcaaaaggc gccctgatgg gagaattccc tgcacctgga agctttgtat tggaagcttt gctactgcac ttcactggtt attgttaacactccgtttgg tgcaatgatt tggccgagat tttcacggcc gggatccgcg 10980 11040 11100 11160 11220 11280 11340 11400 11460 11520 11580 11611 gccgcgagct tccctatagt gagtcgtatt a <210> 39 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:PEPTIDE <400> 39 Lys Val Gly Gly Leu Gly Asp Val Val Thr Ser 1 5 <210> <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:PEPTIDE <400> Gly His Thr Val Glu Val Ile Leu Pro Lys Tyr 1 5 <210> 41 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:PEPTIDE <400> 41 His Asp Trp Ser Ser Ala Pro Val Ala Trp Leu Tyr Lys Glu His Tyr 1 5 10 <210> 42 WO 00/66745 PCT/AU00/00385 -57- <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:PEPTIDE <400> 42 Gly lie Leu Asn Gly Ile Asp Pro Asp Ile Trp Asp Pro Tyr Thr Asp 1 5 10 <210> 43 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:PEPTIDE <400> 43 Asp Val Pro lie Val Gly Ile Ile Thr Arg Leu Thr Ala Gln Lys Gly 1 5 10 <210> 44 <211> <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:PEPTIDE <400> 44 Asn Gly Gin Val Val Leu Leu Gly Ser Ala 1 5 <210> <211> 27 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:PEPTIDE <400> Ala Gly Ser Asp Phe Ile Ile Val Pro Ser Ile Phe Glu Pro Cys Gly 1 5 10 Leu Thr Gin Leu Val Ala Met Arg Tyr Gly Ser <210> 46 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:PEPTIDE <400> 46 Thr Gly Gly Leu Val Asp Thr Val 1 WO 00/66745 PCT/AU00/00385 -58- <210> 47 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:PEPTIDE <400> 47 Lys Thr Gly Gly Leu Gly Asp Val Ala Gly Ala 1 5 <210> 48 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:PEPTIDE <400> 48 Gly His Arg Val Met Val Val Val Pro Arg Tyr 1 5 <210> 49 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:PEPTIDE <400> 49 Asn Asp Trp His Thr Ala Leu Leu Pro Val Tyr Leu Lys Ala Tyr Tyr 1 5 10 <210> <211> 16 <212> PRT <213> Artificial Sequence <220> -223> Description of Artificial Sequence:PEPTIDE <400> Gly Ile Val Asn Gly Ile Asp Asn Met Glu Trp Asn Pro Glu Val Asp 1 5 10 <210> 51 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:PEPTIDE <400> 51 Asp Val Pro Leu Leu Gly Phe Ile Gly Arg Leu Asp Gly Gln Lys Gly 1 5 10 WO 00/66745 PCT/AU00/00385 -59- <210> 52 <211> <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:PEPTIDE <400> 52 Asp Val Gin Leu Val Met Leu Gly Thr Gly 1 5 <210> 53 <211> 27 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:PEPTIDE <400> 53 Ala Gly Ala Asp Ala Leu Leu Met Pro Ser Arg Phe Xaa Pro Cys Gly 1 5 10 Leu Asn Gln Leu Tyr Ala Met Ala Tyr Gly Thr <210> 54 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:PEPTIDE <400> 54 Val Gly Gly Xaa Arg Asp Thr Val 1

Claims (31)

1. An isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide having starch synthase activity or a nucleotide sequence complementary thereto, wherein said nucleotide sequence is selected from the group consisting of: a nucleotide sequence having at least 97% identity to the nucleotide sequence set forth in SEQ ID NO: 1 or the protein-encoding region thereof or a degenerate nucleotide sequence thereto; (ii) a nucleotide sequence having at least 97% identity to the nucleotide sequence set forth in SEQ ID NO: 3 or the protein-encoding region thereof or a degenerate nucleotide sequence thereto; (iii) the nucleotide sequence set forth in SEQ ID NO: 5 or the protein-encoding region thereof or a degenerate nucleotide sequence thereto; (iv) a nucleotide sequence having at least 85% identity to the nucleotide sequence set forth in SEQ ID NO: 7 or the protein-encoding region thereof or a degenerate nucleotide sequence thereto; Oo* ••go °ooo oo o o oo *oe oooo ooQ• oo**o* a nucleotide sequence having at least 85% identity to the forth in SEQ ID NO: 9 or the protein-encoding region thereof or sequence thereto; (vi) a nucleotide sequence having at least 85% identity to the forth in SEQ ID NO: 11 or the protein-encoding region thereof or sequence thereto; (vii) a nucleotide sequence having at least 85% identity to the forth in SEQ ID NO: 12 or the protein-encoding region thereof or sequence thereto; (viii) a nucleotide sequence having at least 85% identity to the forth in SEQ ID NO: 13 or the protein-encoding region thereof or sequence thereto; nucleotide sequence set a degenerate nucleotide nucleotide sequence set a degenerate nucleotide nucleotide sequence set a degenerate nucleotide nucleotide sequence set a degenerate nucleotide Q:\OPERUMS\40924.00 claims doc 12/10/04 (ix) a nucleotide sequence having at least 85% identity to the forth in SEQ ID NO: 14 or the protein-encoding region thereof or sequence thereto; a nucleotide sequence having at least 85% identity to the forth in SEQ ID NO: 15 or the protein-encoding region thereof or sequence thereto; (xi) a nucleotide sequence having at least 85% identity to the forth in SEQ ID NO: 16 or the protein-encoding region thereof or sequence thereto; (xii) a nucleotide sequence having at least 85% identity to the forth in SEQ ID NO: 37 or the protein-encoding region thereof or sequence thereto; (xiii) a nucleotide sequence having at least 85% identity to the forth in SEQ ID NO: 38 or the protein-encoding region thereof or sequence thereto; nucleotide sequence set a degenerate nucleotide nucleotide sequence set a degenerate nucleotide nucleotide sequence set a degenerate nucleotide nucleotide sequence set a degenerate nucleotide nucleotide sequence set a degenerate nucleotide (xiv) a nucleotide sequence encoding a polypeptide having at least 97% identity to the amino acid sequence set forth in SEQ ID NO: 2; (xv) a nucleotide sequence encoding a polypeptide having at least 97% identity to the amino acid sequence set forth in SEQ ID NO: 4; (xvi) a nucleotide sequence encoding a polypeptide having the amino acid sequence set forth in SEQ ID NO: 6; (xvii) a nucleotide sequence encoding a polypeptide having at least 85% identity to the amino acid sequence set forth in SEQ ID NO: 8; (xviii) a nucleotide sequence encoding a polypeptide having at least 85% identity to the amino acid sequence set forth in SEQ ID NO: 10; and(xix) a nucleotide sequence which is complementary to any one of to (xviii). The isolated nucleic acid molecule according to claim 1 wherein the starch synthase polypeptide comprises one or more amino acid sequences selected from the group consisting Q:\OPER\MS\40924-00 claims doc 12/10/04 of: KVGGLGDVVTS; GHTVEVILPKY; HDWSSAPVAWLYKEHY; GILNGIDPDIWDPYTD; DVPIVGIITRLTAQKG; NGQVVLLGSA; AGSDFIIVPSIFEPCGLTQLVAMRYGS; TGGLVDTV; KTGGLGDVAGA; GHRVMVVVPRY; NDWHTALLPVYLKAYY; GIVNGIDNMEWNPEVD; DVPLLGFIGRLDGQKG; DVQLVMLGTG; AGADALLMPSRF(EN)PCGLNQLYAMAYGT; and VGG(V/L)RDTV.

3. The isolated nucleic acid molecule according to claim 2 wherein the starch synthase polypeptide comprises at least three of said amino acid sequences selected from the group consisting of to

4. The isolated nucleic acid molecule according to claim 2 wherein the starch synthase polypeptide comprises at least six of said amino acid sequences selected from the group consisting of to Q:\OPER\IMS\4092400 claims doc 12/10/04 The isolated nucleic acid molecule according to claim 1 encoding a wheat starch synthase II polypeptide.

6. The isolated nucleic acid molecule according to claim 1 encoding a wheat starch synthase III polypeptide.

7. The isolated nucleic acid molecule according to claim 1 wherein the a starch synthase polypeptide comprises one or more amino acid sequences selected from the group consisting of: GHTVEVILPKY; HDWSSAPVAWLYKEHY; DVPIVGIITRLTAQKG; NGQVVLLGSA; AGSDFIIVPSIFEPCGLTQLVAMRYGS; 0 o*.0 TGGLVDTV; GIVNGIDNMEWNPEVD; and 9 AGADALLMPSRF(EN)PCGLNQLYAMAYGT.

8. The isolated nucleic acid molecule of claim 5 encoding a wheat starch synthase II polypeptide which comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 2; (ii) SEQ ID NO: 4; (iii) SEQ ID NO: 6; and (iv) a homologue of or (ii) having at least 97% identity thereto. Q:\OPER\JMS\40924-OO claimsidoc- 12/10/04 86

9. The isolated nucleic acid molecule of claim 6 encoding a wheat starch synthase III polypeptide which comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 8; (ii) SEQ ID NO: 10; and (iii) a homologue of or (ii) having at least 85% identity thereto. A probe or primer comprising at least 15 contiguous nucleotides in length obtained from an isolated nucleotide sequence according to any one of claims 1 to 9.

11. The probe or primer according to claim 10 comprising a nucleotide sequence selected from the group consisting of: the nucleotide sequence set forth in SEQ ID NO: (ii) the nucleotide sequence set forth in SEQ ID NO: 26; (ii) the nucleotide sequence set forth in SEQ ID NO: 27; (iii) the nucleotide sequence set forth in SEQ ID NO: 27; v) the nucleotide sequence set forth in SEQ ID NO: 2 (iv) the nucleotide sequence set forth in SEQ ID NO: 28; the nucleotide sequence set forth in SEQ ID NO: 29; (vii) the nucleotide sequence set forth in SEQ ID NO: 31; (viii) the nucleotide sequence set forth in SEQ ID NO: 32; (ix) the nucleotide sequence set forth in SEQ ID NO: 33; (vii) the nucleotide sequence set forth in SEQ ID NO: 34; (ix) the nucleotide sequence set forth in SEQ ID NO: 33; the nucleotide sequence set forth in SEQ ID NO: 34; (xi) a nucleotide sequence which encodes an amino acid sequence selected from the group consisting of: KVGGLGDVVTS; GHTVEVILPKY; HDWSSAPVAWLYKEHY; Q \OPER\JMS\40924-00 claims doc 12/10/04 87 GILNGIDPDIWDPYTD; DVPIVGIITRLTAQKG; NGQVVLLGSA; AGSDFIIVPSIFEPCGLTQLVAMRYGS; TGGLVDTV; KTGGLGDVAGA; GHRVMVVVPRY; NDWHTALLPVYLKAYY; GIVNGIDNMEWNPEVD; DVPLLGFIGRLDGQKG; 9* DVQLVMLGTG; AGADALLMPSRF(EN)PCGLNQLYAMAYGT; and VGG(V/L)RDTV; (xii) a nucleotide sequence comprising at least 15 contiguous nucleotides of an intron region of SEQ ID NO: 37; (xiii) a nucleotide sequence comprising at least 15 contiguous nucleotides of an intron region of SEQ ID NO: 38; and oe9*o (xiv) a nucleotide sequence which is complementary to any one of to (xiii).

12. An isolated or recombinant polypeptide having starch synthase activity selected from the group consisting of: a polypeptide having at least 97% identity to the amino acid sequence set forth in SEQ ID NO: 2 or the mature protein region thereof; (ii) a polypeptide having at least 97% identity to the amino acid sequence set forth in SEQ ID NO: 4 or the mature protein region thereof; Q:\OPERUMS\40924-00 clirsdoc 12110/04 (iii) the amino acid sequence set forth in SEQ ID NO: 6 or the mature protein region thereof; (iv) a polypeptide having at least 85% identity to the amino acid sequence set forth in SEQ ID NO: 8 or the mature protein region thereof; and a polypeptide having at least 85% identity to the amino acid sequence set forth in SEQ ID NO: 10 or the mature protein region thereof.

13. The isolated or recombinant polypeptide according to claim 12 comprising one or more amino acid sequences selected from the group consisting of: KVGGLGDVVTS; 0*o* GHTVEVILPKY; HDWSSAPVAWLYKEHY; GILNGIDPDIWDPYTD; DVPIVGIITRLTAQKG; NGQVVLLGSA; AGSDFIIVPSIFEPCGLTQLVAMRYGS; TGGLVDTV; KTGGLGDVAGA; GHRVMVVVPRY; NDWHTALLPVYLKAYY; GIVNGIDNMEWNPEVD; DVPLLGFIGRLDGQKG; DVQLVMLGTG; AGADALLMPSRF(EN)PCGLNQLYAMAYGT; and VGG(V/L)RDTV. Q \QPERUMS\40924-OO clzims.doc- 12110/04 89

14. The isolated or recombinant polypeptide according to claim 13 wherein the starch synthase polypeptide comprises at least three of said amino acid sequences selected from the group consisting of to The isolated or recombinant polypeptide according to claim 13 wherein the starch synthase polypeptide comprises at least six of said amino acid sequences selected from the group consisting of to

16. The isolated or recombinant polypeptide according to claim 12 encoding a wheat starch synthase II polypeptide.

17. The isolated or recombinant polypeptide according to claim 12 encoding a wheat starch synthase III polypeptide.

18. The isolated or recombinant polypeptide according to claim 12 which comprises one or more amino acid sequences selected from the group consisting of: GHTVEVILPKY; HDWSSAPVAWLYKEHY; .:.oo DVPIVGIITRLTAQKG; NGQVVLLGSA; AGSDFIIVPSIFEPCGLTQLVAMRYGS; TGGLVDTV; GIVNGIDNMEWNPEVD; and AGADALLMPSRF(EN)PCGLNQLYAMAYGT. Q \OPERUMS\40924-DO clnims.doc- 12/I0/04

19. The isolated or recombinant polypeptide according to claim 16 consisting of a wheat starch synthase II polypeptide which comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 2; (ii) SEQ ID NO: 4; (iii) SEQ ID NO: 6; and (iv) a homologue of or (ii) having at least 97% identity thereto. The isolated or recombinant polypeptide according to claim 17 consisting of a wheat starch synthase III polypeptide which comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 8; (ii) SEQ ID NO: 10; and (iii) a homologue of or (ii) having at least 85% identity thereto.

21. The isolated or recombinant polypeptide according to claim 12 substantially free of conspecific or non-specific proteins.

22. A method comprising: hybridising single-stranded or double-stranded mRNA, cDNA or genomic DNA with a nucleotide sequence selected from the group consisting of: an isolated nucleotide sequence according to any one of claims 1 to 9; a probe or primer obtained from a nucleotide sequence according to sub- paragraph and comprising at least 15 contiguous nucleotides of said nucleotide sequence in length; and (ii) detecting the hybridised mRNA, cDNA or genomic DNA using a detecting means. Q:\OPER\MS\40924-00 claims doc 12/10/04 91

23. The method according to claim 22 wherein the detecting means consists of a reporter molecule covalently attached to the probe or primer molecule.

24. The method according to claim 22 wherein the detecting means consists of a polymerase chain reaction. The method according to claim 22 wherein the probe or primer comprises a nucleotide sequence selected from the group consisting of: the nucleotide sequence set forth in SEQ ID NO: (ii) the nucleotide sequence set forth in SEQ ID NO: 26; (iii) the nucleotide sequence set forth in SEQ ID NO: 27; (iv) the nucleotide sequence set forth in SEQ ID NO: 28; the nucleotide sequence set forth in SEQ ID NO: 29; (vi) the nucleotide sequence set forth in SEQ ID NO: (vii) the nucleotide sequence set forth in SEQ ID NO: 31; (viii) the nucleotide sequence set forth in SEQ ID NO: 32; (ix) the nucleotide sequence set forth in SEQ ID NO: 33; the nucleotide sequence set forth in SEQ ID NO: 34; (xi) a nucleotide sequence which encodes an amino acid sequence selected from the group consisting of: KVGGLGDVVTS; GHTVEVILPKY; HDWSSAPVAWLYKEHY; GILNGIDPDIWDPYTD; DVPIVGIITRLTAQKG; NGQVVLLGSA; Q:\OPER\JMS\40924-00 claims doc 12/10/04 AGSDFIIVPSIFEPCGLTQLVAMRYGS; TGGLVDTV; KTGGLGDVAGA; GHRVMVVVPRY; NDWHTALLPVYLKAYY; GIVNGIDNMEWNPEVD; DVPLLGFIGRLDGQKG; DVQLVMLGTG; AGADALLMPSRF(EN)PCGLNQLYAMAYGT; and VGG(V/L)RDTV; (xii) a nucleotide sequence comprising at least 15 contiguous nucleotides of an intron region of SEQ ID NO: 37; (xiii) a nucleotide sequence comprising at least 15 contiguous nucleotides of an intron region of SEQ ID NO: 38; and (xiv) a nucleotide sequence which is complementary to any one of to (xiii).

26. A method of assaying for the presence or absence of a nucleotide sequence encoding a starch synthase polypeptide in a plant or a plant extract or in an isolated nucleic acid sample from a plant, said method comprising the steps of: hybridising single-stranded or double-stranded mRNA, cDNA or genomic DNA from said plant, plant extract or nucleic acid sample with a nucleotide sequence selected from the group consisting of: a nucleotide sequence having at least 85% identity to the nucleotide sequence set forth in SEQ ID NO: 1 or the protein-encoding region thereof or a degenerate nucleotide sequence thereto; (ii) a nucleotide sequence having at least 85% identity to the nucleotide sequence set forth in SEQ ID NO: 3 or the protein-encoding region thereof or a degenerate Q:\OPER\JMS\40924- clhims .doc 12/10/04 nucleotide sequence thereto; (iii) a nucleotide sequence having at least 85% identity to the nucleotide sequence set forth in SEQ ID NO: 5 or the protein-encoding region thereof or a degenerate nucleotide sequence thereto; (iv) a nucleotide sequence having at least 85% identity to the nucleotide sequence set forth in SEQ ID NO: 7 or the protein-encoding region thereof or a degenerate nucleotide sequence thereto; a nucleotide sequence having at least 85% identity to the nucleotide sequence set forth in SEQ ID NO: 9 or the protein-encoding region thereof or a degenerate nucleotide sequence thereto; (vi) a nucleotide sequence having at least 85% identity to the nucleotide sequence set forth in SEQ ID NO: 11 or the protein-encoding region thereof or a degenerate nucleotide sequence thereto; (vii) a nucleotide sequence having at least 85% identity to the nucleotide sequence set forth in SEQ ID NO: 12 or the protein-encoding region thereof or a degenerate nucleotide sequence thereto; (viii) a nucleotide sequence having at least 85% identity to the set forth in SEQ ID NO: 13 or the protein-encoding degenerate nucleotide sequence thereto; (ix) a nucleotide sequence having at least 85% identity to the set forth in SEQ ID NO: 14 or the protein-encoding degenerate nucleotide sequence thereto; a nucleotide sequence having at least 85% identity to the set forth in SEQ ID NO: 15 or the protein-encoding degenerate nucleotide sequence thereto; nucleotide sequence region thereof or a nucleotide sequence region thereof or a nucleotide sequence region thereof or a (xi) a nucleotide sequence having at least 85% identity to the nucleotide sequence set forth in SEQ ID NO: 16 or the protein-encoding region thereof or a degenerate nucleotide sequence thereto; (xii) a nucleotide sequence having at least 85% identity to the nucleotide sequence Q \OPER\JMS\40924-00 claimsdoc 12/10/04 set forth in SEQ ID NO: 37 or the protein-encoding region thereof or a degenerate nucleotide sequence thereto; (xiii) a nucleotide sequence having at least 85% identity to the nucleotide sequence set forth in SEQ ID NO: 38 or the protein-encoding degenerate nucleotide sequence thereto; (xiv) a nucleotide sequence encoding a polypeptide having at the amino acid sequence set forth in SEQ ID NO: 2; (xv) a nucleotide sequence encoding a polypeptide having at the amino acid sequence set forth in SEQ ID NO: 4 (xvi) a nucleotide sequence encoding a polypeptide having at the amino acid sequence set forth in SEQ ID NO: 6; (xvii) a nucleotide sequence encoding a polypeptide having at the amino acid sequence set forth in SEQ ID NO: 8; (xviii) a nucleotide sequence encoding a polypeptide having at the amino acid sequence set forth in SEQ ID NO: region thereof or a least 85% identity to least 85% identity to least 85% identity to least 85% identity to least 85% identity to (xix) a nucleotide sequence which is complementary to any one of to (xviii); and (xx) a probe or primer obtained from a nucleotide sequence according to any one of to (xix) and comprising at least 15 contiguous nucleotides of said nucleotide sequence in length; and detecting the hybridised mRNA, cDNA or genomic DNA using a detecting means. The method according to claim 26 wherein the detecting means consists of a reporter molecule covalently attached to the probe or primer molecule. The method according to claim 26 wherein the detecting means consists of a polymerase chain reaction. The method according to claim 26 further comprising preparing the plant extract or Q:\OPER\JMS\40924-00 claims doc 12/10/04 nucleic acid sample. A method of marker-assisted breeding and/or selection of a plant comprising performing the method comprising the steps and according to claim 26.

31. The method according to claim 30 further comprising selecting a plant which expresses a desirable starch synthase characteristic.

32. The method according to claim 30 further comprising crossing a plant which expresses a desirable starch synthase characteristic to another plant.

33. The method according to claim 32 further comprising selecting a progeny plant of the cross which expresses a desirable starch synthase characteristic.

34. A plant produced by the method according to any one of claims 30 to 33 wherein said plant expresses a starch synthase polypeptide at a desired level detectable using the method according to any one of claims 22 to a

35. A method of modifying the starch content and/or starch composition of one or more tissues or organs of a plant, comprising expressing in said plant a nucleic acid molecule for a time and under conditions sufficient for the enzyme activity of one or more starch synthase isoenzymes to be modified, wherein said nucleic acid molecule is selected from the group consisting of: an isolated nucleic acid molecule according to any one of claims 1 to 9; (ii) a nucleic acid molecule comprising a fragment of which is capable of being expressed to down-regulate the expression of an endogenous starch synthase isoenzyme of said plant; and Q:\OPER\JMS\40924-00 claims doc 12/10/04 96 (iii) a nucleic acid molecule comprising a fragment of which encodes a functional starch synthase isoenzyme of said plant.

36. The method according to claim 35 wherein the nucleic acid molecule at sub-paragraph (ii) is an antisense molecule, ribozyme molecule, co-suppression molecule, or gene-targeting molecule.

37. The method according to claim 35 further comprising introducing the nucleic acid molecule to an isolated plant cell, tissue ororgan.

38. The method according to claim 37 further comprising regenerating an intact plant from the isolated plant cell, tissue ororgancomprising the introduced nucleic acid molecule. a

39. The method according to claim 37 wherein the nucleic acid molecule is introduced to the plant cell, tissue or organ by transformation.

40. The method according to any one of claims 35 to 39 wherein the plant is a wheat plant. a

41. An isolated promoter sequence comprising a nucleotide sequence selected from the group consisting of: nucleotides 1 to about 287 of SEQ ID NO: 11; (ii) nucleotides 1 to about 1416 of SEQ ID NO: 37; (iii) nucleotides 1 to about 973 of SEQ ID NO: 38; (iv) a fragment of any one of to (iii) capable of conferring expression on a heterologous gene in a m plant cell, tissue or organ; and a complementary nucleotide sequence to any one of to (iv). Q:\0PER\JMS\4O924-OO claims doc- 12/I0/04 0 0*0t *00 00*0 0 0 0 *0 0. 0 *0*00 97 The isolated promoter sequence according to claim 41 that is operable in the endosperm. A plant comprising an isolated nucleic acid molecule according to any one of claims 1 to 9 as an exogenous complement to its genome. A progeny plant of the plant according to claim 43 wherein said progeny plant comprises said introduced nucleic acid molecule. A propagule of the plant according to claim 43 or 44 wherein said propagule comprises said introduced nucleic acid molecule. A gene construct or vector which comprises an isolated nucleic acid molecule according to any one of claims 1 to 9 and one or more origins of replication. The gene construct or vector according to claim 46 further comprising a promoter sequence in operable connection with said isolated nucleic acid molecule. A gene construct or vector which comprises a probe or primer according to claim 10 or 11 and one or more origins of replication. A modified starch derived from the plant according to claim 34 or 43 wherein said starch is modified by virtue of the use of an isolated nucleic acid according to any one of claims 1 to 9 to produce said plant. A modified starch derived from the progeny plant according to claim 44 wherein said Q.\OPER\[MS\40924-00 claims doc 1210/04 98 starch is modified by virtue of the use of an isolated nucleic acid according to any one of claims 1 to 9 to produce said progeny plant. A modified starch derived from the propagule according to claim 45 wherein said starch is modified by virtue of the use of an isolated nucleic acid according to any one of claims 1 to 9 to produce said propagule. A food product comprising the modified starch according to any one of claims 49 to 51. a S 4 *5.5 S.. S 4955 a c. S 4. S S The food product according to claim 52 consisting of flour or a flour-based food product. The food product according to claim 52 or 53 selected from the group consisting of: flour-based sauce; leavened bread; unleavened bread; pasta, noodle; cereal; snack food; cake; and pastry. Use of the modified starch according to any one of claims 49 to 51 in the preparation of a food product for consumption by an animal or human. Dated this 12 t day of October 2004. Commonwealth Scientific and Industrial Research Organisation AND Goodman Fielder Limited AND Groupe Limagrain Pacific Pty. Ltd. By their Patent Attorneys Davies Collison Cave Q.\OPERJMS\40924-00 claims doc 1210/04