WO2005021587A1

WO2005021587A1 - Novel storage protein - encoding gene and uses therefor

Info

Publication number: WO2005021587A1
Application number: PCT/AU2003/001129
Authority: WO
Inventors: Mohammad Hassani; Cristina Gianibelli; Peter John Sharp; William George Rathmell
Original assignee: Value Added Wheat Crc Limited
Priority date: 2003-09-02
Filing date: 2003-09-02
Publication date: 2005-03-10
Also published as: AU2003257245A1

Abstract

The present invention provides a novel isolated nucleic acid (SEQ. ID. No 1) encoding a y-type high molecular weight (HMW) glutenin polypeptide (SEQ. ID. No 2) of about (43-48) kDa as determined by SDS/PAGE. This sequence is characterized by a truncated central repetitive domain (delection of 215 amino acids at position 274 and deletion of 6 amino acids at position 540).Also provided are recombinant proteins, plant parts, food products and non-food products produced therewith.

Description

NOVEL STORAGE PROTEIN - ENCODING GENE AND USES THEREFOR

FIELD OF THE INVENTION The present invention relates to novel, isolated plant genes and proteins and novel plants produced therefor. More particularly, the present invention relates to isolated polynucleotides encoding y-type high molecular weight (HMW) glutenin polypeptides, the deduced amino acid sequences encoded by the isolated polynucleotides, and the use of the polynucleotides and proteins to produce transformed plants having modified seed composition and/or bread-making quality and/or noodle-making quality.

BACKGROUND OF THE INVENTION

1. General 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. Unless the context requires otherwise or specifically stated to the contrary, integers, steps, or elements of the invention recited herein as singular integers, steps or elements clearly encompass both singular and plural forms of the recited integers, steps or elements. The embodiments of the invention described herein with respect to any single embodiment shall be taken to apply mutatis mutandis to any other embodiment of the invention described herein. 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 elements or integers. 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 examples described herein. Functionally equivalent products, compositions and methods are clearly within the scope of the invention, as described herein. The present invention is performed without undue experimentation using, unless otherwise indicated, conventional techniques of molecular biology, microbiology, virology, recombining DNA technology, peptide synthesis in solution, solid phase peptide synthesis, and immunology. Such procedures are described, for example, in the following texts that are incorporated herein by reference: 1. Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, New York, Second Edition (1989), whole of Vols I, II, and III; 2. DNA Cloning: A Practical Approach, Vols. I and II (D. N. Glover, ed., 1985), IRL Press, Oxford, whole of text;

3. Oligonucleotide Synthesis: A Practical Approach (M. J. Gait, ed., 1984) IRL Press, Oxford, whole of text, and particularly the papers therein by Gait, ppl- 22; Atkinson et al, pp35-81; Sproat et al, pp 83-115; and Wu et al, pp 135- 151;

4. Nucleic Acid Hybridization: A Practical Approach (B. D. Hames & S. J. Higgins, eds., 1985) IRL Press, Oxford, whole of text;

5. Perbal, B., A Practical Guide to Molecular Cloning (1984);

6. Wϋnsch, E., ed. (1974) Synthese von Peptiden in Houben-Weyls Metoden der Organischen Chemie (Mϋler, E., ed.), vol. 15, 4th edn., Parts 1 and 2, Thieme, Stuttgart.

7. Handbook of Experimental Immunology, Vols. I-IV (D. M. Weir and C. C. Blackwell, eds., 1986, Blackwell Scientific Publications). This specification contains nucleotide and amino acid sequence information prepared using Patentln Version 3.1, presented herein after the claims. Each nucleotide sequence is identified in the sequence listing by the numeric indicator <210> followed by the sequence identifier (e.g. <210>1, <210>2, <210>3, etc). The length and type of sequence (DNA, protein (PRT), etc), and source organism for each nucleotide sequence, are indicated by information provided in the numeric indicator fields <211>, <212> and <213>, respectively. Nucleotide sequences referred to in the specification are defined by the term "SEQ ID NO:", followed by the sequence identifier (eg. SEQ ID NO: 1 refers to the sequence in the sequence listing designated as <400>1). 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. For the purposes of nomenclature, the nucleotide sequences set forth in the Sequence Listing are as follows: SEQ ID NO: 1 relates to a novel polynucleotide sequence encoding the novel high molecular weight glutenin subunit polypeptide set forth in SEQ ID NO: 2; SEQ ID NO: 3 relates to a high molecular weight glutenin subunit DylO from

Triticum tauschii var. Cheyenne (Anderson et al. 1989); SEQ ID NO: 4 relates to a high molecular weight glutenin subunit Dyl2 from Triticum tauschii var. Chinese Spring (Thompson et al. 1985); SEQ ID NO: 5 relates to a high molecular weight glutenin subunit Dyl2^c from Triticum tauschii (Accession 18964); SEQ ID NO: 6 relates to a 5' primer located 36 bp upstream of the encoding region that corresponds with SEQ ID NO: 1; SEQ ID NO: 7 relates to a 3' primer located 32 bp downstream of the encoding region that corresponds with SEQ ID NO: 1; SEQ ID NO: 8 relates to a 5' primer for amplification of the central repetitive domain; and SEQ ID NO: 9 relates to a 3' primer for amplification of the central repetitive domain.

2. Description of the related art It is well-established that the elasticity and extensibility of endosperm proteins, named high _r(HMW) glutenin are important determinants of bread-making quality in wheat. In particular, two structural features, viz. the central repetitive domain and the two non-repetitive terminal domains that contain the majority of the cysteine residues present in the high M_τ glutenin subunits, are considered the most important characteristics affecting the quality of dough elasticity. The quality of wheat cultivars and the noodle-making and bread-making quality of the flour derived therefrom depends on the number and composition of the HMW glutenin subunits present. Prolamins are a novel group of storage proteins found in the endosperm of cereal grains which are divided into two groups, gliadins and glutenins. Gliadins are monomeric proteins while the glutenins are polymeric and obtained as monomers when treated with reducing agents. Glutenin subunits are divided in two major groups according to their electrophoretic mobility in SDS-PAGE, viz. high molecular weight (HMW) and low molecular weight (LMW) glutenin subunits (Bietz et al. 1975, Gupta and Shepherd 1990). The HMW glutenin subunits are of two types (x- and y-) and are encoded by two closely linked genes at the Glu-1 loci, Glu-1-1 and Glu-1-2 respectively, that are located on the long arms of group 1 homoeologous chromosomes (Payne et al. 1987, reviewed by Shewry et al. 1992). The primary structure of HMW glutenin subunits was deduced by characterizing the encoding genes at the Glu-1 loci (Thompson et al. 1985, Anderson et al. 1989, Reddy and Appels 1993). The subunits comprise a large central repetitive domain flanked by non-repetitive N- and C-terminal domains. The two non- repetitive terminal domains contain most of the cysteine residues present in the HMW glutenin subunits. The number and position of cysteine residues are important features of the structure of the HMW glutenin subunits (Shewry et al. 1992, 1997, MacRitchie and Lafiandra 1997). The N-terminal region has a non-repetitive sequence ranging from 81 to 104 residues and comprising three or five cysteine residues (present in x- and y-type, respectively). The C-terminal domains of both x- and y-type subunits with 42 amino acid residues comprise only one cysteine. One cysteine residue is also present near the end of the central repetitive domain of the y-type subunits. Disulfide bonds between HMW and LMW glutenin subunits are responsible for the formation of the glutenin polymers, with a range of different sizes that can reach up to tens of millions of Daltons affecting dough properties (Shewry et al. 1992, MacRitchie 1992, Wrigley 1996). Although intermolecular disulfide bonds are clearly important in the formation of the glutenin polymeric structures, recent studies based on Nuclear Magnetic Resonance (NMR) have indicated that hydrogen bonds between HMW glutenin subunits could have an important role in stabilizing the polymeric structure of glutenin (Belton et al, 1994, 1995). Recently, Belton (1999) has proposed a loop and train model to explain the role of hydrogen bonds in conferring gluten elasticity. More recently, Tilley et al. (2001) have pointed out the possible contribution of tyrosine cross-links as part of the molecular basis of the gluten structure. According to these authors, the double tyrosine residues that occur within the repetitive motifs could also contribute to the formation of gluten structure, a continuous proteinaceous network, formed during the mixing of wheat flour with water to make dough. One of the progenitors of hexaploid wheat is T. tauschii (Coss.) Schmal, which contributed the D genome (Kihara 1944, McFadden and Sears 1946). A recent study of HMW glutenin subunits of 173 accessions of T. tauschii has revealed significant genetic variability in the D genome (Gianibelli et al. 2001b). T tauschii has been used as a gene pool source to improve disease, nematode and insect resistance, tolerance to salinity as well as bread making quality of common wheat (Kihara et al. 1965, Kerber and Dyck 1969, Appels and Lagudah 1990, Eastwood et al. 1991, Schachrman et al. 1991, Mackie et α/. 1996). Low (weak) gluten elasticity is responsible for the poor bread-making and/or noodle-making qualities of wheat cultivars which otherwise have desirable agronomic properties. Faced with sub-optimal gluten flours, mixing of flours from different cultivars is required to produce a suitable product for bread-making and/or noodle- making applications. Accordingly, there exists a continuing need for enhanced wheat varieties that express novel glutenin subunits with enhanced end-use product quality. One approach to providing such enhanced varieties is to isolate novel glutenin-encoding genes for introduction into plants by traditional breeding approaches (eg., introgression or other standard breeding) or by recombinant means. It is therefore desirable to obtain novel genes encoding HMW glutenin polypeptides, which are capable of being expressed in planta thereby enhancing end-use product quality of the flour.

SUMMARY OF THE INVENTION In work leading up to the present invention, the inventors sought to identify novel y-type HMW glutenin-encoding genes for bread-making and/or noodle-making applications. The inventors identified and characterised a novel glutenin-encoding gene capable of being expressed in planta, and showed that the encoded protein has an unusually low molecular mass (M_r). Surprisingly, the inventors found that the encoded glutenin polypeptide comprises an unusually-small central repetitive domain and, remarkably, has significantly enhanced bread-making and/or noodle-making characteristics. In particular, dough comprising the glutenin of the invention exhibits significantly enhanced elasticity and extensibility when compared to dough prepared from flour comprising known glutenins having larger central repetitive domains. Accordingly, the present invention relates to novel polynucleotides encoding novel wheat glutenin, such as, for example, a wheat glutenin of Triticum spp., more particularly to a novel glutenin-encoding gene derived from T. tauschii designated "12.4'". The size of the novel glutenin is significantly smaller than that seen for known high- ,- glutenin, in particular about 43 kDa to about 48 kDa, due to the presence of a truncated central repetitive domain (i.e. the 14.2* gene product has a deletion of a considerable number of amino acids in the central repetitive domain relative to other known glutenin polypeptides). The polynucleotide encoding the glutenin of present interest is used to modify glutenin in transgenic plants or plants provided by methods such as introgression or recombinant DNA approaches. The polynucleotides, polypeptides and plants of the invention are useful in providing the novel glutenin and end-products comprising the glutenin of present interest. The invention further provides a flour having enhanced noodle-making and/or bread-making qualities, such as an enhanced noodle-making quality while maintaining good bread-making quality relative to that obtained using flour containing known glutenin proteins. According to a first aspect of the invention there is provided an isolated polynucleotide comprising a nucleotide sequence selected from the group consisting of: a) the nucleotide sequence set out in SEQ ID NO: 1; b) a nucleotide sequence which is degenerate as a result of the genetic code to the nucleotide sequence set out in SEQ ID NO: 1; c) a sequence that selectively hybridises to a sequence that is complementary to (a) or (b) wherein said sequence encodes a glutenin subunit having a molecular weight of about 43 - 48 kDa; d) a sequence that is at least about 70% identical to (a) or (b) and encodes a glutenin subunit having a molecular weight of about 43 - 48 kDa; e) a sequence that hybridizes to at least about 15 contiguous nucleotides of (a) or (b) under at least low stringency hybridization conditions and encodes a glutenin subunit having a molecular weight of about 43 - 48 kDa. Preferably, the percentage identity to SEQ ID NO: 1 or a complementary sequence thereto is at least about 80%, more preferably at least about 90%, even more preferably at least about 95% and still more preferably, at least about 98%. In a particularly preferred embodiment of the invention, there is provided an isolated polynucleotide encoding a glutenin polypeptide and comprising a nucleotide sequence selected from the group consisting of: a) that set forth in SEQ ID NO: 1 ; b) a sequence encoding the amino acid sequence set forth in SEQ ID NO: 2; and c) a sequence that is complementary to (a) or (b). According to a second aspect of the invention there is provided an isolated or recombinant glutenin polypeptide having a molecular weight of about 43 - 48 kDa and comprising an amino acid sequence selected from the group consisting of: a) the amino acid sequence set out in SEQ ID NO: 2; and b) an amino acid sequence having at least 70 % sequence identity to the amino acid sequence set out in SEQ ID NO: 2. Preferably, the percentage identity to SEQ ID NO: 2 or a complementary sequence thereto is at least about 80%, more preferably at least about 90%, even more preferably at least about 95% and still more preferably, at least about 98%. It will be understood from the disclosure provided in International Patent Application No. PCT/AU99/00563, incorporated herein by reference, that it is possible to produce novel wheat storage proteins by fusing or swapping one or more domains of one protein with the protein domains from another storage protein, wherein the fusion protein products have novel ligand-binding characteristics. Accordingly, a third aspect of the invention provides a method for producing a modified glutenin subunit polypeptide, the method comprising adding a glutenin polypeptide or a domain thereof encoded by a polynucleotide having nucleotide sequence according to the first aspect of the invention to an exogenous amino acid domain which confers upon the modified glutenin subunit polypeptide an ability to bind a ligand or other macromolecule, and wherein the modified glutenin subunit polypeptide has an ability to incorporate into gluten. It will be understood that the modified protein is generally produced by producing an in-frame fusion of the open reading frames of each storage protein domain and expressing the fusion protein in a suitable host cell such as, for example, a bacterial cell, yeast cell or plant cell. According to a fourth aspect of the invention there is provided a modified glutenin subunit polypeptide provided according to the method of the third aspect of the invention. According to a fifth aspect of the invention there is provided an isolated polynucleotide encoding a modified glutenin subunit according to the fourth aspect of the invention. According to a sixth aspect of the invention there is provided a vector comprising the polynucleotide according to either the first or the fifth aspects of the invention. According to a seventh aspect of the invention there is provided a host cell comprising the vector according to the sixth aspect of the invention. According to an eighth aspect of the invention there is provided a cell culture comprising the host cell according to the seventh aspect of the invention. According to a ninth aspect of the invention there is provided a plant comprising the polynucleotide according to either the first or the fifth aspects of the invention. According to the tenth aspect of the invention there is provided propagating material or a part of the plant according to the ninth aspect of the invention wherein said propagating material or plant part comprises the polynucleotide according to either the first or the fifth aspects of the invention. According to an eleventh aspect of the invention there is provided a seed of the plant according to the ninth aspect of the invention wherein said seed comprises the polynucleotide according to either the first or the fifth aspects of the invention. According to a twelfth aspect of the invention there is provided a crossed fertile plant prepared by a process comprising: a) obtaining a fertile plant comprising the polynucleotide according to the first or fifth aspects of the invention; b) crossing the fertile plant with a plant thereby producing a seed; c) selecting seed that comprises the polynucleotide according to either the first or the fifth aspects of the invention; and d) germinating the seed to obtain the crossed fertile plant. According to a thirteenth aspect of the invention there is provided a method for detecting a polynucleotide according to either the first or the fifth aspects of the invention, said method comprising contacting a nucleic acid sample, such as, for example, plant genomic DNA, plant mRNA or cDNA derived therefrom, with the isolated polynucleotide according to either the first or the fifth aspects of the invention or a probe that comprises at least about 15 contiguous nucleotides of the isolated polynucleotide according to either the first or the fifth aspects of the invention for a time and under conditions sufficient for hybridization to occur and then detecting the hybridization. The presence or absence of a hybridization signal indicates the presence or absence, respectively, of the polynucleotide according to either the first or the fifth aspects of the invention in said sample. In a preferred embodiment, the method further comprises obtaining a nucleic acid-containing sample from a cell, organ or whole organism (eg. , a plant) . According to a fourteenth aspect of the invention there is provided a use of the polypeptide according to either the second or fourth aspects of the invention in the preparation of a food product, in particular flour or a by-product (eg., gluten) or end- product (eg., bread or noodle) thereof. According to a fifteenth aspect of the invention there is provided a use of the polypeptide according to either the second or fourth aspects of the invention in the preparation of a non- food product.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows a one-dimensional one-step SDS-PAGE separation of reduced and alkylated polymeric proteins from several accessions of T. tauschii:

(A) HMW glutenin subunits,

(B) LMW glutenin subunits;

(C) LMW glutenin subunits; The 12.4' glutenin subunit (SEQ ID NO: 2) is arrowed (accession: AUS24092) and (h) hexaploid wheats. Figure 2 shows PCR products of y-type HMW glutenin gene. Lanes 1 and 8: pUC19/HpaII, 2 and 7: 1 Kb ladder, 3: central repetitive domain of the gene from T. tauschii accession AUS24092, 4: Australian cultivar Baxter, 5: encoding region of the gene from T. tauschii accession AUS24092, 6: Australian cultivar Baxter. Figure 3 provides the nucleotide sequences of the Glu-Dl-2 allele from T. tauschii accession AUS24092 (SEQ ID NO: 1). Figure 4 shows a comparison of the derived amino acid sequence of HMW glutenin subunit Dyl2.4^l from AUS24092 (SEQ ID NO: 2) with DylO from the cultivar Cheyenne (Anderson et al. 1989)(SEQ ID NO: 3), Dyl2 from the cultivar Chinese Spring (Thompson et al. 1985)(SEQ ID NO: 4) and Dyl2* from T. tauschii accession AUS 18964 (Mackie et al. 1996)(SEQ ID NO: 5). *= conserved residues, bold residues = differences among subunits. Figure 5 shows (a) SDS-PAGE and (b) RP-HPLC separation of reduced and alkylated polymeric proteins of two T. tauschii accessions (1=CPI 110750 and 2-

CPI110745), x- and y-type-high M_r glutenin subunits; B- and C-type = low M_r glutenin subunits. (c) Polymerase chain reaction amplification on 1.5% agarose gel of (*in lane 2) complete encoding region and (- in lane 3) central repetitive region of y-type high M_r glutenin gene (12.4¹) present in accession CPU 10750. Lanes 1 and 4 are DNA molecular size markers (kb).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Glutenin polynucleotides and polypeptides, modified glutenin subunits, and methods for producing same The HMW glutenins are composed primarily of a central domain of 45-90 repeating two or three simple motifs, comprising chiefly glutamine and proline. This unusual primary structure results in a rod-like secondary structure as assessed by circular dichroism and SEM. Cysteines in the C- and N-terminal domains of the molecules are known to be critical for the formation of intermolecular disulfide bonds. The quaternary interactions of these proteins are of interest because the disulfide crosslinkages among HMW and LMW subunits is thought to be integral to the elastic properties of dough. According to one aspect of the invention there is provided an isolated polynucleotide comprising a nucleotide sequence selected from the group consisting of: a) the nucleotide sequence set out in SEQ ID NO: 1 ; b) a nucleotide sequence which is degenerate as a result of the genetic code to the nucleotide sequence set out in SEQ ID NO: 1 ; c) a sequence that selectively hybridises to a sequence that is complementary to (a) or (b) wherein said sequence encodes a glutenin subunit having a molecular weight of about 43 - 48 kDa; d) a sequence that is at least about 70% identical to (a) or (b) and encodes a glutenin subunit having a molecular weight of about 43 - 48 kDa; e) a sequence that hybridizes to at least about 15 contiguous nucleotides of (a) or (b) under at least low stringency hybridization conditions and encodes a glutenin subunit having a molecular weight of about 43 - 48 kDa. In preferrd embodiments, the percentage identity to SEQ ID NO: 1 or a complementary sequence thereto is at least about 80%, more preferably at least about 90%, even more preferably at least about 95% and still more preferably, at least about 98%. In a particularly preferred embodiment of the invention, there is provided an isolated polynucleotide encoding a glutenin polypeptide of wheat (Triticum spp.) and comprising a nucleotide sequence selected from the group consisting of: a) the set forth in SEQ ID NO: 1 ; b) a sequence encoding the amino acid sequence set forth in SEQ ID NO: 2; and c) a sequence that is complementary to (a) or (b). The term "selectively hybridises" means that a target polynucleotide of the invention is found to hybridize to a further polynucleotide at a level significantly above background. The background hybridization may occur because of other polynucleotides present, for example, in the cDNA or genomic DNA library being screening. In this event, background implies a level of signal generated by interaction between the polynucleotide of interest and a non-specific DNA member of the library which is less than 10 fold, preferably less than 100 fold as intense as the specific interaction observed with the target polynucleotide. The intensity of interaction may be measured, for example, by radiolabelling the probe, e.g. with ³²P. Polynucleotides of the invention comprise DNA or RNA. They are single- stranded or double-stranded. They are also polynucleotides which include within them synthetic or modified nucleotides. A number of different types of modification to oligonucleotides are known in the art. These include methylphosphonate and phosphorothioate backbones, addition of acridine or polylysine chains at the 3' and/or 5' ends of the molecule. For the purposes of the present invention, it is to be understood that the polynucleotides described herein are capable of modification by any method available in the art. Such modifications are carried out in order to enhance the in vivo activity or life span of polynucleotides of the invention. Hybridization conditions are based on the melting temperature (Tm) of the nucleic acid binding complex, as taught in Berger and Kimmel (1987, Guide to Molecular Cloning Techniques, Methods in Enzymology, Vol 152, Academic Press, San Diego CA), and confer a defined "stringency" as explained herein. Maximum stringency typically occurs at about Tm-5°C (5°C below the Tm of the probe); high stringency at about 5°C to 10°C below Tm; intermediate stringency at about 10°C to 20°C below Tm; and low stringency at about 20°C to 25°C below Tm. As will be understood by those of skill in the art, a maximum stringency hybridization can be used to identify or detect identical polynucleotide sequences while an intermediate (or low) stringency hybridization can be used to identify or detect similar or related polynucleotide sequences. Where the polynucleotide of the invention is double-stranded, both strands of the duplex, either individually or in combination, are encompassed by the present invention. Where the polynucleotide is single-stranded, it is to be understood that the complementary sequence of that polynucleotide is also included within the scope of the present invention. In one embodiment of the invention, variants and strain/species homologues are obtained using degenerate PCR which will use primers designed to target sequences within the variants and homologues encoding conserved amino acid sequences within the sequences of the present invention. Conserved sequences can be predicted, for example, by aligning the amino acid sequences from several variants/homologues. Sequence alignments can be performed using computer software known in the art. For example the GCG Wisconsin PileUp program is widely used. The primers used in degenerate PCR will contain one or more degenerate positions and will be used at stringency conditions lower than those used for cloning sequences with single sequence primers against known sequences. Alternatively, in a further embodiment of the present invention, such polynucleotides are obtained by site-directed mutagenesis of characterised sequences, such as the sequence referred to in SEQ ID NO: 1. This is useful where for example silent codon changes are required to sequences to optimise codon preferences for a particular host cell in which the polynucleotide sequences are being expressed. Other sequence changes are desired in order to introduce restriction enzyme recognition sites, or to alter the property or function of the polypeptides encoded by the polynucleotides. In yet another embodiment of the present invention, polynucleotides of the invention are used to produce primers, e.g. PCR primers (e.g. SEQ ID NO: 6; SEQ ID NO: 7; SEQ ID NO: 8; and SEQ ID NO: 9), a primer for an alternative amplification reaction, a probe e.g. labelled with a revealing label by conventional means using radioactive or non-radioactive labels, or where the polynucleotides are cloned into vectors. Such primers and/or probes will be at least 15, preferably at least 20, for example at least 25, 30 or 40 nucleotides in length, and are also encompassed by the term polynucleotides of the invention as used herein. Polynucleotides such as DNA polynucleotides and probes according to the invention are capable of being produced recombinantly, synthetically, or by any means available to those of skill in the art. They are also capable of being cloned by standard techniques. In general, primers will be produced by synthetic means, involving a step wise manufacture of the desired nucleic acid sequence one nucleotide at a time. Techniques for accomplishing this using automated techniques are readily available in the art. Longer polynucleotides will generally be produced using recombinant means, for example using PCR (polymerase chain reaction) cloning techniques. This will involve making a pair of primers (e.g. of about 15 to 30 nucleotides) flanking a region of the sequence which it is desired to clone, bringing the primers into contact with mRNA or cDNA obtained from a sample, performing a polymerase chain reaction under conditions which bring about amplification of the desired region, isolating the amplified fragment (e.g. by purifying the reaction mixture on an agarose gel) and recovering the amplified DNA. In preferred embodiments, the primers are designed to contain suitable restriction enzyme recognition sites so that the amplified DNA can be cloned into a suitable cloning vector. In preferred embodiments, polynucleotides or primers of the invention carry a revealing label. Suitable labels include proteins, enzymes, radionuclides, fluorophores, luminophores, enzyme inhibitors, coenzymes, luciferins, paramagnetic metals and spin labels. Such labels are detected using by techniques known in the art. According to another aspect of the invention there is provided an isolated or recombinant glutenin polypeptide (e.g. Triticum spp.) having a molecular weight of about 43 - 48 kDa and comprising an amino acid sequence selected from the group consisting of: a) the amino acid sequence set out in SEQ ID NO: 2; and b) an amino acid sequence having at least 70 % sequence identity to the amino acid sequence set out in SEQ ID NO: 2. In preferred embodiments, the percentage identity to SEQ ID NO: 2 or a complementary sequence thereto is at least about 80%, more preferably at least about 90%, even more preferably at least about 95% and still more preferably, at least about 98%. It will be understood from the disclosure provided in International Patent

Application No. PCT/AU99/00563, incorporated herein by reference, that it is possible to produce novel wheat storage proteins by fusing or swapping one or more domains of one protein with the protein domains from another storage protein, wherein the fusion protein products have novel ligand-binding characteristics. According to yet another aspect of the invention there is provided a method for producing a modified glutenin subunit polypeptide, the method comprising adding a glutenin polypeptide or a domain thereof encoded by a polynucleotide having nucleotide sequence according to the first aspect of the invention to an exogenous amino acid domain which confers upon the modified glutenin subunit polypeptide an ability to bind a ligand or other macromolecule, and wherein the modified glutenin subunit polypeptide has an ability to incorporate into gluten. It will be understood that the modified protein is generally produced by producing an in-frame fusion of the open reading frames of each storage protein domain and expressing the fusion protein in a suitable host cell such as, for example, a bacterial cell, yeast cell or plant cell. According to a further aspect of the invention there is provided a modified glutenin subunit provided according to the afore-mentioned methods of the invention. According to still another aspect of the invention there is provided an isolated polynucleotide encoding a modified glutenin subunit polypeptide according to the invention. The term "adding" as used herein refers to the joining together of at least two proteins/polypeptides, notably, in this aspect of the invention, a polypeptide of the invention and an exogenous amino acid domain according to methods known in the art. In a preferred embodiment of this aspect, the exogenous amino acid domain is fused or joined to a further protein/polypeptide. In further preferred embodiments of the present aspect of the invention, examples of exogenous amino acid domains include enzymes, polypeptides having ligand binding domains, polypeptides having macromolecule binding domains etc. In a particularly preferred embodiment, the fusion protein may comprise an enzymatic or chemical cleavage site upstream and preferably adjacent the N-terminus of the second protein/polypeptide and/or an enzymatic or chemical cleavage site downstream and preferably adjacent the C-terminus of the second protein/polypeptide thereby providing a means for recovering the second protein from the modified glutenin subunit polypeptide through use of a suitable cleaving agent. In a preferred embodiment of this aspect of the invention, the ligand or other macromolecule is selected from the group consisting of starch and lipid. Preferably, the domain capable of binding lipid is derived from barley oleosin protein or the lipid- binding regions of wheat CM 16 protein. The isolation of polynucleotides of the present invention may be accomplished by a number of techniques. In one embodiment, oligonucleotide probes based on the sequences disclosed in the prior art can be used to isolate the desired polynucleotide from a cDNA or genomic DNA library. To construct genomic libraries, large segments of genomic DNA are generated by random fragmentation, e.g. using restriction endonucleases, and are ligated with vector DNA to form concatemers that can be packaged into the appropriate vector. To prepare a cDNA library, mRNA is isolated from endosperm and a cDNA library which contains the glutenin gene transcript is prepared from the mRNA according to standard methods. In an alternative embodiment, the polynucleotides of interest can be amplified from nucleic acid samples using amplification techniques, for instance, polymerase chain reaction (PCR) technology to amplify the sequences of the glutenin and related genes directly from genomic DNA, from cDNA, from genomic libraries or cDNA libraries. PCR and other in vitro amplification methods may also be useful, for example, to clone nucleic acid sequences that code for proteins to be expressed, to make nucleic acids to use as probes for detecting the presence of the desired mRNA in samples, for nucleic acid sequencing, or for other purposes. For a general overview of PCR see PCR Protocols: A Guide to Methods and Applications. (Innis, M, Gelfand, D., Sninsky, J. and White, T., eds., Academic Press, San Diego (1990)). In yet another embodiment, the polynucleotides may be synthesized by well- known techniques as described in the technical literature. See, e.g., Carruthers et al, Cold Spring Harbor Symp. Quant. Biol. 47:411-418 (1982), and Adams et al, J. Am. Chem. Soc. 105:661 (1983). Double stranded DNA fragments may then be obtained either by synthesizing the complementary strand and annealing the strands together under appropriate conditions, or by adding the complementary strand using DNA polymerase with an appropriate primer sequence. Isolated polynucleotides prepared as described herein can then be used to modify glutenin gene expression and therefore glutenin content in plants. One of skill will recognize that the nucleic acid encoding a functional glutenin protein need not have a sequence identical to the exemplified genes disclosed here. Thus, genes encoding chimeric glutenin polypeptides can be used in the present invention. According to a further aspect of the invention there is provided a vector comprising a polynucleotide according to the invention. Preferably, the vector comprises regulatory elements which ensure transcription in bacterial, yeast or plant cells. In a further preferred embodiment, the vector is used to replicate the polynucleotides of the invention in a compatible host cell. According to yet another aspect of the invention there is provided a host cell comprising a vector according to the invention. In a preferred embodiment, the invention provides a method of making polynucleotides of the invention by introducing a polynucleotide of the invention into a replicable vector, introducing the vector into a compatible host cell, and growing the host cell under conditions which bring about replication of the vector. Preferably, the host cell is a plant cell. Preferably, the plant cell is selected from the group consisting of Triticum spp. (e.g. T tauschii, T. aestivum), maize, rice, oats, barley and rye and/or wild varieties and/or hybrids or derivatives and/or ancestral progenitors of same. In a further preferred embodiment, the vector is recovered from the host cell. Suitable host cells include bacteria such as E. coli, yeast, plant cell lines and other eukaryotic cell lines, for example insect Sf9 cells. In a preferred embodiment, a regulatory region is "operably linked" to a the polynucleotides of the invention in such a way that expression of the polynucleotides of the invention is achieved under conditions compatible with the regulatory region. Another aspect of the invention provides a cell culture comprising the host cell according to the invention, prepared and cultured according to methods well-known to those skilled in the art. In preferred embodiments, the vectors are plasmid or virus vectors provided with an origin of replication, optionally a promoter for the- expression of the polynucleotide of present interest and optionally a regulator of the promoter. Preferably, the vectors contain one or more selectable marker genes, for example an ampicillin resistance gene in the case of a bacterial plasmid or a neomycin resistance gene for a mammalian vector. In a further preferred embodiment, the vectors of the invention includes a reporter gene such as chloramphenicol acetyltransferase, β-galactosidase, β- glucuronidase, luciferase, green fluorescent protein, red fluorescent protein, placental alkaline phosphatase, or secreted embryonic alkaline phosphatase. In another preferred embodiment of the invention, a DNA sequence coding for the desired polypeptide of interest or the modified glutenin subunit, for example a cDNA or a genomic sequence encoding a full length protein, is conveniently used to construct a recombinant expression cassette which is introduced into the desired plant. The expression cassette will typically comprise the polynucleotide sequence of present interest operably linked to a promoter sequence and other transcriptional and translational initiation regulatory sequences which will direct the transcription of the sequence from the glutenin gene in the intended tissues (e.g., endosperm) of the transformed plant. In a further preferred embodiment, a constitutive plant promoter fragment is employed which will direct expression of the glutenin in all tissues of a plant. Such promoters are active under most environmental conditions and states of development or cell differentiation. Examples of constitutive promoters useful in this embodiment include the cauliflower mosaic virus (CaMV) 35S transcription initiation region, the 1'- or 2'-promoter derived from T-DNA of Agrobacterium tumafaciens, and other transcription initiation regions from various plant genes known to those of skill. Alternatively, the plant promoter is under environmental control. Such promoters are referred to here as "inducible" promoters. Examples of environmental conditions that may effect transcription by inducible promoters include pathogen attack, anaerobic conditions, or the presence of light. Typically, the promoters used in the constructs of the invention will be "tissue- specific" and are under developmental control such that the desired gene is expressed only in certain tissues, such as leaves, roots, fruit, seeds, or flowers. Promoters that direct expression in seeds, particularly the endosperm are particularly preferred. Examples of such promoters include the preferred promoter from genes encoding seed storage proteins, such as napin, cruciferin, phaseolin, and the like (see, U.S. Pat. No. 5,420,034). Other promoters suitable for expressing glutenin genes in cereals include promoters from genes encoding gliadins, cereal prolamines (e.g., zein, hordein, secalin, and avenin) and starch biosynthetic enzymes. The endogenous promoters from glutenin genes are particularly useful for directing expression of the polynucleotides of present interest in the seed, particularly the endosperm. These seed-specific promoters can also be used to direct expression of heterologous structural genes. Thus, the promoters can be used in recombinant expression cassettes to drive expression of the gene of present interest in seeds. In a preferred embodiment of the invention, the glutenin promoters are typically at least about 400 base pairs in length, and often at least about 800 or about 1000 base pairs. The length of the promoters is typically less than about 3500 base pairs, usually less than about 2800 base pairs and often less than about 2000 base pairs in length. The length of the promoters is counted upstream from the translation start codon of the native gene. One of skill will recognize that use of the "about" to refer to lengths of nucleic acid fragments is meant to include fragments of various lengths that do not vary significantly from the lengths recited here and still maintain the functions of the claimed promoters (i.e., seed-specific gene expression). To identify glutenin promoters, the 5¹ portions of a genomic glutenin gene clone can be analyzed for sequences characteristic of promoter sequences. For instance, promoter sequence elements include the TATA box consensus sequence (TATAAT), which is usually 20 to 30 base pairs upstream of the transcription start site. In plants, further upstream from the TATA box, at positions -80 to -100, there is typically a promoter element with a series of adenines surrounding the trinucleotide G (or T) (Messing et al, in Genetic Engineering in Plants, pp. 221-227 (Kosage, Meredith and Hollaender, eds. 1983). In preparing expression vectors of the invention, sequences other than the promoter and the gene of present interest are also preferably used. If proper polypeptide expression is desired, a polyadenylation region at the 3 '-end of the glutenin coding region should be included. The polyadenylation region can be derived from the natural gene, from a variety of other plant genes, or from T-DNA. The vector comprising the sequences of a glutenin gene will typically comprise a marker gene which confers a selectable phenotype on plant cells. For example, the marker may encode biocide resistance, particularly antibiotic resistance, such as resistance to kanamycin, G418, bleomycin, hygromycin, or herbicide resistance, such as resistance to chlorosulfuron, or phosphinothricin (the active ingredient in bialaphos and Basta). With a working knowledge of conventional techniques for cloning, DNA isolation, amplification and purification, for enzymatic reactions involving DNA ligase, DNA polymerase, restriction endonucleases and the like, and various separation techniques known and commonly employed by those skilled in the art (See, for example, R. Wu, ed. (1979) Meth. Enzymol. 68; R. Wu et al, eds. (1983) Meth. Enzymol. 100, 101: L. Grossman and K. Moldave, eds. (1980) Meth. Enzymol. 65: J. H. Miller (1972) Experiments in Molecular Genetics; R. Davis et al. (1980) Advanced Bacterial Genetics; R. F. Schleif and P. C. Wensink (1982) Practical Methods in Molecular Biology; and T. Maniatis et al. (1982) Molecular Cloning.), one of ordinary skill can employ a suitable gene construct containing the genes coding for the polypeptides of the invention. All parts of the relevant DNA constructs (promoters, regulatory-, stabilizing-, targeting- or termination sequences) of the present invention may be modified, if desired, to affect their control characteristics using methods known to those skilled in the art. In a preferred embodiment, in constructing the modified glutenin subunit expression vector, the nucleic acid encoding the polynucleotides encoding the glutenin subunit of the invention will be linked or joined to the nucleic acid encoding the exogenous amino acid domain such that the open reading frame of the glutenin subunit and the exogenous amino acid domain is intact, allowing translation of the modified glutenin subunit to occur. The modified glutenin subunit encoding sequence of this preferred embodiment of the invention is designed and constructed to comprise the codon(s) necessary to achieve cleavage by the desired cleaving agent at desired positions, i.e. upstream, preferably adjacent the N-terminus of the exogenous amino acid domain encoding region of the modified glutenin subunit encoding sequence or downstream, and preferably adjacent the C-terminus of the exogenous amino acid domain encoding region or both if the exogenous amino acid domain encoding region of the modified glutenin subunit is linked to a further protein/polypeptide. The present invention clearly excludes any gliadin proteins comprising the amino acid sequences set forth in any one of SEQ ID NOs 3 - 5, or polynucleotides encoding said gliadin proteins. Preferred embodiments of the glutenin subunits and modified glutenin subunits of the invention are particularly useful in both noodle-making and bread-making as they exhibit significantly enhanced characteristics, in particular with respect to the elasticity and extensibility of dough. Sequence comparisons between two (or more) polynucleotides or polypeptides are typically performed by comparing sub-sequences of the two sequences over a segment or "comparison window" to identify and compare local regions of sequence similarity. Optimal alignment of sequences for comparison may be conducted by eye, or more usually, with the aid of readily available sequence comparison programs such as the local homology algorithm of Smith and Waterman Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman and Wunsch J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson and Lipman Proc. Natl. Acad. Sci. (U.S.A.) 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis.), the BLAST package (see Ausubel et al, 1999 ibid - Chapter 18), FASTA (Atschul et al, 1990, J. Mol. Biol., 403-410) or by inspection. Percentage homology may be calculated over contiguous sequences, i.e. one sequence is aligned with the other sequence and each nucleotide in one sequence directly compared with the corresponding nucleotide in the other sequence, one base at a time. This is called an "ungapped" alignment. Typically, such ungapped alignments are performed only over a relatively short number of bases (for example less than 50 contiguous nucleotides). Although this is a very simple and consistent method, it fails to take into consideration that, for example, in an otherwise identical pair of sequences, one insertion or deletion will cause the following nucleotides to be put out of alignment, thus potentially resulting in a large reduction in percentage homology when a global alignment is performed. Consequently, most sequence comparison methods are designed to produce optimal alignments that take into consideration possible insertions and deletions without penalising unduly the overall homology score. This is achieved by inserting "gaps" in the sequence alignment to try to maximise local homology. However, these more complex methods assign "gap penalties" to each gap that occurs in the alignment so that, for the same number of identical amino acids, a sequence alignment with as few gaps as possible - reflecting higher relatedness between the two compared sequences - will achieve a higher score than one with many gaps. "Gap costs" are typically used that charge a relatively high cost for the existence of a gap and a smaller penalty for each subsequent residue in the gap. This is the most commonly used gap scoring system. High gap penalties will of course produce optimized alignments with fewer gaps. Most alignment programs allow the gap penalties to be modified. However, it is preferred to use the default values when using such software for sequence comparisons. Calculation of maximum percentage homology therefore firstly requires the production of an optimal alignment, taking into consideration gap penalties. A suitable computer program for carrying out such an alignment is the above-mentioned GCG Wisconsin BESTFIT package (University of Wisconsin, U.S.A.; Devereux et al, 1984, Nucleic Acids Research 12:387). The default scoring matrix has a match value of 10 for each identical nucleotide and -9 for each mismatch. The default gap creation penalty is -50 and the default gap extension penalty is -3 for each nucleotide. Both BLAST and FASTA are available for offline and online searching

(Ausubel et al., 1999 ibid, pages 7-58 to 7-60). However it is preferred to use the GCG Bestfit program. Once the software has produced an optimal alignment, it is possible to calculate percentage homology, preferably percentage sequence identity. The software typically does this as part of the sequence comparison and generates a numerical result. The term "polypeptide" as used herein is used in its broadest sense, i.e., any polymer of amino acids (dipeptide or greater) linked through peptide bonds. Thus, the term "polypeptide" includes proteins, oligopeptides, protein fragments, analogues, fusion proteins and the like. The term also encompasses amino acid polymers as described above that include additional non-amino acid moieties. Thus, the term "polypeptide" includes glycoproteins, lipoproteins, phosphoproteins, metalloproteins, nucleoproteins, as well as other conjugated proteins. The term "polypeptide" contemplates polypeptides as defined above that are recombinantly produced, isolated from an appropriate source, or synthesized. In another preferred embodiment of the invention, vectors and polynucleotides are introduced into host cells for the purpose of replicating the vectors/polynucleotides and/or expressing the polypeptides encoded by the polynucleotides of the invention. Although the polypeptides may be produced using prokaryotic cells as host cells, it is preferred to use eukaryotic cells, for example yeast or plant cells, in particular plant cells. In a further preferred embodiment, vectors/polynucleotides are introduced into suitable host cells using a variety of techniques known in the art, such as transfection, transformation and electroporation. Where vectors/polynucleotides of the invention are to be administered to propagation material, several techniques are known in the art, for example, direct injection of polynucleotides/vectors and biolistic transformation. Preferably, the vectors will comprise regulatory elements which ensure transcription in bacterial, yeast or plant cells.

Production of Plants and Breeding Methods Recent scientific progress shows that in principle monocots are amenable to transformation and that fertile transgenic plants can be regenerated from transformed cells. The development of reproducible tissue culture systems for these crops, together with the powerful methods for introduction of genetic material into plant cells has facilitated transformation. Presently the methods of choice for transformation of monocots are microprojectile bombardment of explants or suspension cells, and direct DNA uptake or electroporation of protoplasts. For example, transgenic rice plants have been successfully obtained using the bacterial hph gene, encoding hygromycin resistance, as a selection marker. The gene was introduced by electroporation (Shimamoto et al, 1989). Transgenic maize plants have been obtained by introducing the bar gene from Streptomyces hygroscopicus, which encodes phosphinothricin acetyltransferase (an enzyme which inactivates the herbicide phosphinothricin), into embryogenic cells of a maize suspension culture by microparticle bombardment (Gordon-Kamm et al., 1990). The introduction of genetic material into aleurone protoplasts of other monocot crops such as wheat and barley has been reported (Lee et al, 1989). The stable transformation of wheat cell suspension cultures via microprojectile bombardment has recently been described (Vasil et al, 1991). Wheat plants have been regenerated from embryogenic suspension culture by selecting only the aged compact and nodular embryogenic callus tissues for the establishment of the embryogenic suspension cultures (Vasil et al, 1990). The combination of regeneration techniques with transformation systems for these crops enables the application of the present invention to monocots. These methods may also be applied for the transformation and regeneration of dicots. In another aspect of the invention, the vectors of the invention may be used to transform plants as desired, to provide plants according to the invention as discussed herein. In another aspect of the invention there is provided propagating material or a part of the plant according to the ninth aspect of the invention wherein said propagating material or plant part comprises a polynucleotide according to the invention. In a preferred embodiment, the vector is introduced into a plant cell using any convenient technique, including, for example, chemically induced uptake, electroporation, liposomes, retroviruses, electrophoresis, microparticle bombardment, and microinjection into cells from any plant species, including monocotyledonous or dicotyledonous plants, in cell or tissue culture or in whole plants where applicable, to provide transformed plant cells or plants containing as foreign DNA at least one copy of the DNA sequence of the plant expression cassette. Using known techniques, protoplasts can be regenerated and cell or tissue culture can be regenerated to form whole fertile plants which carry and express the desired gene for the selected protein. See generally Chapters 6, 7 and 9 in Glick and Thompson, Methods in Plant Molecular Biology and Biotechnology, CRC Press, Boca Raton, 1993. Preferably, plants are transformed using the Biolistic PDS-1000 He (Bio-Rad laboratories, USA) device or the like via microprojectile bombardment. In a preferred embodiment, the plant is selected from the group consisting of Triticum spp. (e.g. T. tauschii, T. aestivum), maize, rice, oats, barley and rye and/or wild varieties and/or hybrids or derivatives and/or ancestral progenitors of same. In another aspect of the invention there is provided a seed of a plant according to the invention wherein said seed comprises a polynucleotide according to the invention. In preferred embodiments of the invention transgenic wheat plants are obtained from bombarded immature embryos by the methods described by Weeks et al. (1993) and Vasil et al. (1993) using bialaphos (Meiji Seika Kaisha Ltd, Japan) selection. The resistant calli may be induced to produce shoots and roots following which putative transgenic plantlets can be transferred to the greenhouse and allowed to self-fertilize. A number of further methods for transforming cereals have been described in the literature and are used in this embodiment of the invention, for instance, transformation of rice is described by Toriyama et al Bio/Technology 6:1072-1074 (1988), Zhang et al. Theor. Appl. Gen. 76:835-840 (1988), and Shimamoto et al. Nature 338:274-276 (1989). Transgenic maize regenerants have been described by Fromm et al, Bio/Technology 8:833-839 (1990) and Gordon-Kamm et al, Plant Cell 2:603-618 (1990)). Similarly, oats (Somers et al, Bio/Technology 10:1589-1594 (1992)), sorghum (Casas et al, Proc. Natl. Acad. Sci. USA 90:11212-11216 (1993)), rice (Li et al, Plant Cell Rep. 12:250-255 (1993)), barley (Wan and Lemaux, Plant Physiol. 104:37-48 (1994)), and rye (Castillo et al, Bio/Technology 12:1366-1371 (1994)) have been transformed via bombardment. Transformed plant cells which are derived by any of the above transformation techniques are cultured to regenerate a whole plant which possesses the transformed genotype and thus the desired phenotype. Such regeneration techniques rely on manipulation of certain phytohormones in a tissue culture growth medium and identification of transformants, typically relying on a biocide and/or herbicide marker which has been introduced together with the glutenin polynucleotide sequences. Plant regeneration from cultured protoplasts is described in Evans et al, Protoplasts Isolation and Culture, Handbook of Plant Cell Culture, pp. 124-176, MacMillan Publishing Company, New York, 1983; and Binding, Regeneration of Plants, Plant Protoplasts, pp. 21-73, CRC Press, Boca Raton, 1985. Regeneration is also obtained from plant callus, explants, organs, or parts thereof. Such regeneration techniques are described generally in Klee et al. Ann. Rev. of Plant Phys. 38:467-486 (1987). The methods of the present invention are particularly useful for incorporating the polynucleotides of present interest into plants in ways and under circumstances which are not found naturally. In particular, the glutenin subunit is expressed at times or in quantities which are not characteristic of natural plants and in plants which do not naturally express glutenin. One of skill will recognize that after an expression cassette is stably incorporated in transgenic plants and confirmed to be operable, it can be introduced into other plants by sexual crossing. Any of a number of standard breeding techniques can be used, depending upon the species to be crossed. The invention has use over a broad range of types of plants. In a preferred embodiment, the glutenin polypeptides or modified glutenin are expressed in a cereal species commonly used for production of flour, e.g., wheat, rye, oats, barley, rice, corn, millets and the like. The invention is particularly useful for improvement of wheat cultivars. Any wheat cultivar is improvable by the methods of the invention. Exemplary lines include hexaploid lines such as Anza, Shasta, Yecoro Rojo, Siouxiand, Freedom, Taml07, Tam200, Karl 92, Hartog, Kukri, and Janz. The effect of the modification of glutenin gene expression can be measured by detection of increases or decreases in desired glutenin protein levels using, for instance, gel electrophoresis. In a further embodiment, quantification of HMW glutenin content can be carried out by SDS-PAGE densitometry methods as known in the art. Other methods for quantifying glutenin content include sonication of flour dispersions in SDS buffer to solubilize glutenin and other relatively insoluble proteins (Singh and MacRitchie in Wheat End-Use Properties: Wheat and Flour Characterization for Specific End-Uses H. Salovaara, ed., pp 321-326 (University of Helsinki, Helsinki, 1989). This method is useful in solubilizing at least 95% of total flour protein. Quantification of the glutenin fraction can then be performed using size exclusion high performance liquid chromatography as described by Batey et al, Cereal Chem. 68:207-209 (1991). Other suitable methods include reverse phase HPLC and capillary electrophoresis. Another measure of increased glutenin content in transgenic plants of the invention is glutenin content as a percent of total protein of a mature seed or flour derived from the mature seed. Different wheat cultivars have different glutenin contents as measured using standard methods. Thus, the final glutenin content of the plants of the invention will depend upon the parental line. In the case where HMW glutenin genes are used, the wheat plants of the invention will typically have HMW glutenin contents of at least about 15%, usually at least about 18%, and preferably at least about 20% as measured using standard methods such as those described herein. Introgression oftransgenes into elite inbreds and hybrids Backcrossing can be used to improve a starting plant. Backcrossing transfers a specific desirable trait from one source to an inbred or other plant that lacks that trait. This can be accomplished, for example, by first crossing a superior inbred (A) (recurrent parent) to a donor inbred (non-recurrent parent), which carries the appropriate gene(s) for the trait in question, for example, a construct prepared in accordance with the current invention. The progeny of this cross first are selected in the resultant progeny for the desired trait to be transferred from the non-recurrent parent, then the selected progeny are mated back to the superior recurrent parent (A). After five or more backcross generations with selection for the desired trait, the progeny are hemizygous for loci controlling the characteristic being transferred, but are like the superior parent for most or almost all other genes. The last backcross generation would be crossed to self to give progeny which are pure breeding for the gene(s) being transferred. Therefore, through a series of breeding manipulations, a selected transgene may be moved from one line into an entirely different line without the need for further recombinant manipulation. Transgenes are valuable in that they typically behave genetically as any other gene and can be manipulated by breeding techniques in a manner identical to any other gene. Therefore, one may produce inbred plants which are true breeding for one or more transgenes. By crossing different inbred plants, one may produce a large number of different hybrids with different combinations of transgenes. In this way, plants may be produced which have the desirable agronomic properties frequently associated with hybrids ^hybrid vigor"), as well as the desirable characteristics imparted by one or more transgene(s). In yet another aspect of the invention there is provided a crossed fertile plant prepared by a process comprising: a) obtaining a fertile plant comprising a polynucleotide according to the invention; b) crossing the fertile plant with a plant thereby producing a seed; c) selecting seed that comprises the polynucleotide according to the invention; and germinating the seed to obtain the crossed fertile plant. In a preferred embodiments of this aspect of the invention, the polynucleotide of the invention is introduced into the plant by introgression. Preferably, the plant is selected from the group consisting of Triticum spp. (e.g. T tauschii, T. aestivum), maize, rice, oats, barley and rye and or wild varieties and/or hybrids or derivatives and/or ancestral progenitors of same. In a further preferred embodiment, the polynucleotide according to the invention is inherited through a female parent. In another preferred embodiment, the polynucleotide according to the invention is inherited through a male parent. In another aspect of the invention there is provided a seed of the plant comprising a polynucleotide of the invention wherein the seed also comprises the polynucleotide of the invention.

Marker assisted selection and polynucleotide probes Genetic markers may be used to assist in the introgression of one or more transgenes of the invention from one genetic background into another. Marker assisted selection offers advantages relative to conventional breeding in that it can be used to avoid errors caused by phenotypic variations. Further, genetic markers may provide data regarding the relative degree of elite germplasm in the individual progeny of a particular cross. For example, when a plant with a desired trait which otherwise has a non-agronomically desirable genetic background is crossed to an elite parent, genetic markers may be used to select progeny which not only possess the trait of interest, but also have a relatively large proportion of the desired germplasm (i.e. genotype). In this way, the number of generations required to introgress one or more traits into a particular genetic background is minimized. In the process of marker assisted breeding, DNA sequences are used to follow desirable agronomic traits (Tanksley et al, 1989) in the process of plant breeding. Marker assisted breeding may be undertaken as follows. Seed of plants with the desired trait are planted in soil in the greenhouse or in the field. Leaf tissue is harvested from the plant for preparation of DNA at any point in growth at which approximately one gram of leaf tissue can be removed from the plant without compromising the viability of the plant. Genomic DNA is isolated using a procedure modified from Shure et al. (1983). Approximately one gram of leaf tissue from a seedling is lyophilized overnight in 15 ml polypropylene tubes. Freeze-dried tissue is ground to a powder in the tube using a glass rod. Powdered tissue is mixed thoroughly with 3 ml extraction buffer (7.0 urea, 0.35 M NaCl, 0.05 M Tris-HCl pH 8.0, 0.01 M EDTA, 1% sarcosine). Tissue/buffer homogenate is extracted with 3 ml phenol/chloroform. The aqueous phase is separated by centrifugation, and precipitated twice using 1/10 volume of 4.4 M ammonium acetate pH 5.2, and an equal volume of isopropanol. The precipitate is washed with 75% ethanol and resuspended in 100-500 μl TE (0.01 M Tris-HCl, 0.001 M EDTA, pH 8.0). Genomic DNA is then digested with a 3-fold excess of restriction enzymes, electrophoresed through 0.8%) agarose (FMC), and transferred (Southern, 1975) to Nytran (Schleicher and Schuell) using 10 x SCP (20 SCP: 2M NaCl, 0.6 M disodium phosphate, 0.02 M disodium EDTA). The filters are prehybridized in 6 x SCP, 10% dextran sulfate, 2% sarcosine, and 500 μg/ml denatured salmon sperm DNA and ³²P- labeled probe generated by random priming (Feinberg & Vogelstein, 1983). Hybridized filters are washed in 2 x SCP, 1% SDS at 65 °C for 30 minutes and visualized by autoradiography using Kodak XAR5 film. Genetic polymorphisms which are genetically linked to traits of interest are thereby used to predict the presence or absence of the traits of interest. Those of skill in the art will recognize that there are many different ways to isolate DNA from plant tissues and that there are many different protocols for Southern hybridization that will produce identical results. Those of skill in the art will also recognize that a Southern blot can be stripped of radioactive probe following autoradiography and re-probed with a different probe. In this manner one may identify each of the various transgenes that are present in the plant. Further, one of skill in the art will recognize that any type of genetic marker which is polymorphic at the region(s) of interest may be used for the purpose of identifying the relative presence or absence of a trait, and that such information may be used for marker assisted breeding. Each lane of a Southern blot represents DNA isolated from one plant. Through the use of multiplicity of gene integration events as probes on the same genomic DNA blot, the integration event composition of each plant may be determined. Correlations may be established between the contributions of particular integration events to the phenotype of the plant. Only those plants that contain a desired combination of integration events may be advanced to maturity and used for pollination. DNA probes corresponding to particular transgene integration events are useful markers during the course of plant breeding to identify and combine particular integration events without having to grow the plants and assay the plants for agronomic performance. It is expected that one or more restriction enzymes will be used to digest genomic DNA, either singly or in combinations. One of skill in the art will recognize that many different restriction enzymes will be useful and the choice of restriction enzyme will depend on the DNA sequence of the transgene integration event that is used as a probe and the DNA sequences in the genome surrounding the transgene. For a probe, one will want to use DNA or RNA sequences which will hybridize to the DNA used for transformation. One will select a restriction enzyme that produces a DNA fragment following hybridization that is identifiable as the transgene integration event. Thus, particularly useful restriction enzymes will be those which reveal polymorphisms that are genetically linked to specific transgenes or traits of interest. According to another aspect of the invention there is provided a method for detecting a polynucleotide according to the invention, said method comprising contacting nucleic acid, such as, for example, plant genomic DNA, plant mRNA or cDNA derived therefrom, with the isolated polynucleotide according to the invention or a probe that comprises at least about 15 contiguous nucleotides of the isolated polynucleotide according to the invention for a time and under conditions sufficient for hybridization to occur and then detecting the hybridization. The presence or absence of a hybridization signal indicates the presence or absence, respectively, of the polynucleotide according to the invention in said sample. In a preferred embodiment, the method further comprises obtaining a nucleic acid-containing sample from a cell, organ or whole organism (eg., a plant). In a preferred embodiment, the polynucleotide probe is labelled with a detectable marker. Preferably the detectable marker is selected from the group consisting of proteins, enzymes, radionuclides, fluorophores, luminophores, enzyme inhibitors, coenzymes, luciferins, paramagnetic metals and spin labels. In a preferred embodiment, the protein is selected from the group consisting of biotin, a biotin analog and a single-stranded histone binding protein. Preferably, the detection of the polynucleotide is performed by a method selected from Southern Blotting and Northern Blotting. In another preferred embodiment of the invention there is provided a method for the detection of the polypeptide according to the invention or the modified glutenin polypeptide according to the invention comprising an immunodetection assay. Preferably, the immunodetection assay is Western Blotting. Preferably, the increased production of the protein is detected by a detectable antibody specific for a polypeptide according to the invention and/or the modified glutenin subunit polypeptide according to the invention. Preferably, the antibody is labeled with a detectable marker. Preferably, the marker is selected from the group consisting of proteins, enzymes, radionuclides, fluorophores, luminophores, enzyme inhibitors, coenzymes, luciferins, paramagnetic metals and spin labels.

End-use products and goods manufactured with or from same, In another aspect of the invention, there is provided a method for enhancing and increasing the bread-making and noodle-making characteristics of plant products, preferably wheat. In one preferred embodiment of this aspect of the invention, the method includes the transformation of the plants with genes encoding the glutenin subunit and/or modified glutenin of the invention. In accordance with another preferred embodiment, at least one copy of the polynucleotide encoding the glutenin subunit and/or modified glutenin of the invention is integrated into the genome and expressed in the plant cell. In another preferred embodiment of this aspect, the genes encoding the polypeptide of the invention may be introduced into the plant by the methods of plant breeding described herein, particularly introgression. The present invention also encompasses bread, noodles and the like prepared from plant products and seeds of the present invention. In another aspect of the invention there is provided a use of a polypeptide according to the invention in the preparation of a food product, in particular flour or a by-product (eg., gluten) or end-product (eg., bread or noodle) thereof. Preferably, the food product is selected from the group consisting of leavened or unleavened breads, pasta, noodles, breakfast cereals, snack foods, cakes, pastries, and food containing flour-based sauces. In another aspect there is provided a use of a polypeptide according the invention in the preparation of a non-food product. Preferably, the non-food product is selected from the group consisting of films, coatings, adhesives, building materials, and packaging materials. It is therefore clear that, according to these teachings, the person skilled in the art is enabled to increase the total number of the genes of the glutenin subunit and/or modified glutenin of the invention, and thus the amount of HMW glutenin accumulated, resulting in enhanced dual bread-making and noodle-making quality. Further, the present invention provides the opportunity to routinely manipulate the composition of glutenin, and thus its effect on bread-making and noodle-making quality, by the introduction into wheat and other plants/cereals of genes mutated by means well known in the art to cause alterations in the structure of HMW glutenin, particularly the glutenin subunit and/or modified glutenin of present interest. In a preferred embodiment of this aspect, there is provided a method in terms of which a polynucleotide of present interest is stably integrated, expressed, and inherited as a single dominant locus in the wheat genome following Mendelian inheritance thus facilitating the production of flour associated with good bread-making and noodle- making quality. Similarly, the composition of maize, rice, and other cereals can be altered as taught herein and according to known transformation and selection techniques. The ultimate goal in plant transformation or breeding is to produce plants which are useful to man. In this respect, plants created in accordance with the current invention are useful for the production of end-products of both bread and noodle nature. For example, one may wish to harvest seed from the plants. This seed is in turn useful for a wide variety of purposes. The seed can be sold to farmers for planting in the field or can be directly used as food, either for animals or humans. Alternatively, products can be made from the seed itself. Examples of products which can be made from the seed include, oil, gluten, starch, animal or human food, pharmaceuticals, and various industrial products. The food uses of wheat, in addition to human consumption of wheat kernels, include both products of dry- and wet-milling industries. The principal products of wheat dry milling are meal and flour. The wheat wet-milling industry can provide wheat starch, wheat syrups, and dextrose for food use. Wheat, including both grain and non-grain portions of the plant, also is used extensively as livestock feed, primarily for beef cattle, dairy cattle, hogs, and poultry. The industrial applications of wheat starch and flour are based on functional properties, such as viscosity, film formation, adhesive properties, and ability to suspend particles. Specific methods for crop utilization may be found in, for example, Sprague and Dudley (1988), and Watson and Ramstad (1987). Analysis of the rheological properties of flour derived from the transgenic plants of the invention can be carried out according to standard physical dough-testing instruments widely used to measure flour and dough quality (MacRitchie, Advances in Food and Nutrition Research 36:1-87 (1992)). Such methods include, for instance, use of extensographs to measure tensile strength. Two of the main parameters measured are maximum resistance (R_ma ) and extensibility (Ext). Other methods include mixographs and bake-test loaf volume (MacRitchie and Gras Cereal Chem. 50:292-302 (1973) as well as SDS-sedimentation tests, amylographs, and cookie spread methods.

The present invention is further described with reference to the following non-limiting Examples and the accompanying drawings.

EXAMPLE 1 CHARACTERIZATION OF THE NUCLEOTIDE- AND DERIVED AMINO ACID SEQUENCE OF THE GLUTENIN SUBUNIT OF THE INVENTION Polymerase chain reaction (PCR) is a fast and reliable alternative to conventional methods for the study of the genes controlling wheat proteins. It allows the specific amplification of a target DNA segment using a pair of flanked oligonucleotide primers (D'Ovidio et al. 1990, 1994, 1995). PCR was used to characterize HMW glutenin genes encoding x- and y-type subunits from bread wheat (Tahir et al. 1996, Lafiandra et al. 1997). The aims of this study were to characterize a novel y-type HMW glutenin gene, with unusually low molecular weight, deduce its amino acid sequence, compare the deduced sequence with previously published sequences of y-type subunits associated to the D genome to describe the degree of similarity among them.

Materials and methods Seeds of T tauschii accession AUS 24092 were supplied by the Australian

Winter Cereals Collection (AWCC), Tamworth NSW. Seeds were planted under quarantine conditions and at the three to four leaf stage, fresh material was collected for DNA extraction.

Glutenin protein extraction and electrophoresis Wholemeal flour of T. tauschii accession AUS 24092 was used for protein extraction and electrophoretic analysis. The monomeric proteins (gliadins, albumins and globulins) were first extracted with dimethyl sulphoxide (DMSO) and then with 70% ethanol, 10 mg of wholemeal sample being mixed with 1.3 mL of each extractant solution (w/v). Polymeric proteins were solubilized from the remaining pellet by a further extraction with 70% ethanol and reduced with 5% β-mercaptoethanol, and alkylated with 10% 4-vinylpyridine as described previously by Gupta and MacRitchie (1991). Reduced and alkylated glutenin subunits were fractionated by one-step one- dimensional SDS-PAGE to determine the high and low M_t glutenin subunit compositions of this accession. Gels were run at constant voltage (200 V) during 4 h and then the gels were stained with Coomassie Brilliant Blue G-250 overnight following the method of Neuhoff et al. (1988). The gels were destained with distilled water overnight and then stored in 20% (w/v) ammonium sulphate solution at 4°C. Primers and PCR conditions The oligonucleotide primers used to amplify the y-type HMW glutenin gene were those of Gianibelli and Solomon (2001). The primers (PI, P2) (SEQ ID NO: 6 and SEQ ID NO: 7, respectively) were located respectively at 36 bp upstream (5') and 32 bp downstream (3') of the encoding region. A second pair of oligonucleotide primers (P3, P4) (SEQ ID NO: 8 and SEQ ID NO: 9, respectively) was designed from the published sequences of the y-type glutenin gene reported by D'Ovidio et al. (1995) to amplify the central repetitive domain.

PI 5ΑCA AAA TAG AGA TCA ATT CAC 3' (SEQ ID NO: 6);

P2 5 ' CCC AAG CAC CAT GCA AG 3 ' (SEQ ID NO: 7);

P3 5 ' GGG AAC ATC TTC ACA AAA CAG TAC AA 3 ' (SEQ ID NO: 8); and

P4 5' CTG TGT TAA CAT GGT ATG GGT TGT C 3' (SEQ ID NO: 9) Genomic DNA (50-75ng) was added to a 20μL reaction, containing lx PCR buffer (Advanced Biotechnologies), 1.5mM MgCl₂, 0.5μM of each primer, 200μM of each dNTP and 1.0 unit Taq DNA polymerase. Thermal cycling consisted of pre- denaturation at 95 °C for 4 min, five cycles of denaturation at 94°C for 1 min, annealing at 60°C for 1.5 min and extension at 72°C for 1.5 min, 25 cycles of denaturation at 94°C for 30 sec, annealing at 60°C 1.5 min and extension at 72°C for 1.5 min, followed by a final cycle of extension at 72°C for 7 min. PCR was carried out in a thermal cycler with a 36 tube holder block (Corbett Research, Australia). Amplified products were analyzed on 1.2% agarose gels. DNA fragments were cut from gel and purified by BRESA-clean DNA Purification Kit (Gene Works Pty Ltd Australia). Molecular weight standards were used to estimate the size of the amplified products and the two different PCR products were cloned into pGEM-T vector (Promega) and subsequently sequenced. The Genetics Computer Group package was used in the Australian National Genomic Information Service to analyze the sequence data. Results and discussion SDS-PAGE analysis of accession AUS 24092 , with the unusually small molecular weight subunit 12.4¹ of 45000 Da, is shown in Figure 1. The PCR reactions selectively amplified a HMW glutenin (y-type) allele. The primers used in this study (P1+P2 and P3+P4) (SEQ ID NO: 6 and SEQ ID NO: 7; SEQ ID NO: 8 and SEQ ID NO: 9, respectively, respectively) amplified two fragments of 1.45 and 0.85 Kb corresponding to the complete gene and its central repetitive domain, respectively (Figure 2). Nucleotide sequences from both fragments were analyzed. The 1.4 kb fragment corresponded to an open reading frame of 1317 base pair nucleotide sequence of the entire encoding region of the Glu-D'l y-type 12.4* gene (Figure 3). The deduced y-type 12.4¹ protein sequence of the Glu-D^ll gene corresponds to a small HMW glutenin subunit of 439 amino acids (including the signal peptide). As in published HMW glutenin subunits, it contains the 21 amino acid residues corresponding to the signal peptide, an N-terminal sequence of 104 amino acid residues followed by a central repetitive domain of 272 amino acid residues and the C-terminal domain of 42 amino acid residues. In agreement with previous observations in other gluten protein genes, no introns were observed. A remarkable reduction in the size of the central repetitive domain was responsible for the small size of the Dyl2.4^l subunit. In this region, a large and a small deletion of 215 and 6 amino acid residues, was observed at position 274 and 540 of subunit Dyl2, respectively. The latter was located close to the cysteine present at the end of the central repetitive domain (Figure 4). The Dyl2.4^l subunit also differed from the other three y-type subunits in six amino acid residues. These residues were located at positions 2 (the signal peptide), 239, 252 (the central repetitive domain), and 612, 647, 649 (the C-terminal region) of subunit Dyl2. Four other changes were observed between subunits Dyl2.4^l and Dyl2. They were located at positions 186, 550, 561 and 616 in subunit Dyl2. A total of five and six changes were observed compared with subunits Dyl2^l and DylO, respectively (Figure 4). Two deletions of six amino acid residues were observed in subunits DylO and Dyl2^l compared with the deduced sequence of subunit Dyl2.4^l. Thus, the highest similarity was observed between subunits Dyl2.4⁴ and Dyl2, with ten different amino acid residue changes (plus the two deletions at position 274 and 540, mentioned above). No differences were observed in the N-terminal region of subunits Dyl2.4^l, DylO and Dyl2, but at position 81, isoleucine was observed in the subunit Dyl2^l of Triticum tauschii. In the C-terminal region subunit Oyl2A^t presented three different amino acid residues, compared with the other y-type HMW glutenin subunits. Thus, leucine, serine and alanine were observed at positions 612, 647 and 649, respectively, while serine, proline and valine were observed in the other three subunits. The largest variation among the Dy-type subunits was observed in the central repetitive domain (Figure 4). The number and position of the cysteine residues among the four y-type subunits, were compared. In Dyl2.4^l subunit, five cysteines were observed in the N- terminal region and one in the C-terminal region, plus one cysteine close to the end of the central repetitive region. These seven cysteines were observed at the same position in all y-type subunits considered in this study, suggesting that number and position of cysteine residues are very conserved among y-type HMW glutenin subunits. The repetitive motifs PGQGQQ and GYYPTSPQQ form more than 90% of the repetitive domain of the y-type HMW glutenin subunits (Anderson and Greene 1989, Shewry et al. 1992, 1997). A comparison of repetitive motifs between different HMW glutenin subunits and the Dyl2.4⁴ subunit is presented in Table 1.

Table 1: Number of repeat motifs present in the central repetitive domain of HMW glutenin subunits and Dyl2.4^l (in bold).

A large decrease in the number of repetitive motifs was observed in the Dyl2.4^l subunit, with 29 hexapeptide and 10 nonapeptide sequences, resulting in a very small y- type HMW glutenin subunit. Also a decrease in the number of tyrosine residues was observed in the Dyl2.4^l subunit compared to the other y-type HMW glutenin subunits. The lack of 12 tyrosine amino acid residues located at the large deletion of the central repetitive domain in the Dyl2.4^l subunit, could affect to some extent the formation of the glutenin polymeric structure and elasticity of dough. The estimated molecular weight of the Dyl2.4^l subunit, calculated on the basis of the deduced amino acid sequence, was 45228 Daltons. This value is slightly smaller than the molecular weight (47900 Daltons) estimated by Gianibelli and Solomon (2001) by the length of gene (PCR reaction). The deduced amino acid composition (expressed as mol %) among subunits (Table 2), indicates a clear decrease in the % of glutamine and glycine, which are present in the repetitive motifs of the central domain was observed. On the other hand, as a result of the smaller size of this protein, an increase in the proportion of other amino acids, such as cysteine, asparagine, glutamic acid, arginine, serine and valine, arises relative to known HMW glutenin subunits.

Table 2: Deduced amino acid composition (expressed as mol %) of Dyl2.4^l, Dyl2^l from T. tauschii and Dyl2 and DylO from hexaploid wheat sequence data.

In contrast to previous reports where a large insertion within the repetitive domain was reported (Tahir et al. 1996, D'Ovidio et al. 1996), this is the first example of a large deletion detected in the repetitive domain of an active HMW glutenin subunit gene. The close similarity observed between subunit Dyl2 and Dyl2.4' strongly indicates their close relationship, suggesting that the T tauschii variant could have originated from the Glu-l-2a locus (Dyl2), and differentiated through point mutations and deletions in the central repetitive domain. Several mechanisms such as gene conversion or unequal crossing over could be responsible for the large deletion observed in the gene reported here, resulting in the change in size of this particular HMW glutenin subunit gene. These mechanisms are also suggested as responsible for variation in size of the different HMW glutenin subunit alleles (Shewry et al. 1989, D'Ovidio et al. 1996). Moreover, characterization of genes at Glu-1 loci in bread wheat and wild relatives has indicated that the N-terminal and C-terminal domains of the y- type HMW glutenin subunits encoded at the Glu-Dl locus, are fairly conserved regions. Most of the variability observed between subunits occurrs as a result of changes within the central repetitive region. The elasticity of gluten could be partly explained by the formation of intermolecular hydrogen bonding between adjacent HMW glutenin subunits (Belton et al. 1994; Belton, 1999, for details). The number of amino acid residues that contribute to the formation of hydrogen bonds plays an important role in defining the elastic properties of this type of proteins. The repetitive domain contains a very high level of glutamine, which has a very high capacity to form intra- and intermolecular hydrogen bonds (Belton 1999). Changes in the length of the central repetitive domain directly affect the number of hydrogen bonds possible, and therefore, the functional properties. The fact that in this subunit other critical features such as number and distribution of cysteine residues, are similar to those observed in other HMW glutenin subunits, makes this protein of unique value. The effect of individual proteins (HMW and LMW glutenin subunits, hordeins, gliadins) on dough properties is evaluated by means of studying the mixing behavior of a base flour, modified either by incorporation or addition of the specific proteins (Bekes et al. 1994a, 1994b). Recent advances in micro-scale mixing and protein engineering systems have proved to be valuable in elucidating the structure/function relationship in gluten proteins (Bekes et al. 1998).

EXAMPLE 2 CHARACTERISATION OF THE GLUTENIN SUBUNIT OF THE INVENTION The high and low M_r glutenin subunits of accession AUS 24092 were identified by SDS-PAGE (Gianibelli et al. 2002). This accession contains the x-type high M_t glutenin subunit 2.1¹ [Figure 5] with slower elecfrophoretic mobility than subunit 2 in hexaploid wheat (Gianibelli et al. 2001). The estimation of the M_r of the 12.4¹ subunit was carried out by comparison with molecular size standards using SDS-PAGE. High M_r glutenin subunits were isolated by RP-HPLC (Gianibelli et al. 2001) and fractions were collected, freeze-dried and analysed by SDS-PAGE. N-terminal sequencing analysis was performed on aliquots of the sample collected from the RP-HPLC. DNA was extracted from leaved of T.tauschii using published methods (Lagudah et al. 1991). The following primers (a) 5'ACA AAA TAG AGA TCA ATT CAC 3' (SEQ ID NO: 6), (b) 5'CCC AAG CAC CAT GCA AG 3' (SEQ ID NO: 7) were used for amplification of the complete region of y-type high M_r glutenin genes using the conditions described by D'Ovidio et al. (1994).

Results and Discussion The 12.4* subunit, although having an unusually low molecular weight, has been considered a y-type high M_r glutenin subunit (Gianibelli et al. 2001). However, the peculiar molecular weight of the novel subunit (M_r 45.228 according to SDS-PAGE analysis) made it difficult to determine whether it is high or low M_r glutenin subunit. In this respect, RP-HPLC has helped to classify the 12.4¹ subunit as a high M_r glutenin subunit. Figure 5(b) allows the RP-HPLC separation of reduced and alkylated polymeric proteins of the accession AUS 24092 and the peaks corresponding to the x- and y-type high M_r glutenin subunit. The two types of high M_r glutenin subunits (x- and y-type) encoded by the Glu-Dl locus have clearly different elution times in RP-HPLC, allowing their easy separation in the conditions used in the present study. There were only small differences in terms of elution time when x-type or y-type subunits were compared with similar glutenin subunits from the Glu-Dl locus in hexaploid wheat or other T tauschii accessions [Figure 5(b)]. Considering that the central repetitive domains of high M_r glutenin subunits are both highly repetitive and relatively hydrophilic, it is reasonable to expect that changes in their size will not strongly modify the hydrophobic behaviour of these subunits. However, high M_r glutenin subunits with a smaller central repetitive domain are expected to elute later than those with a larger repetitive domain [Figure 5]. This was the case for subunit 12.4¹, where the increase of approximately 1 min

30s in the elution time has been attributed to a decrease in the length of the central domain of the subunit (the hydophilic part of the protein). Nevertheless, this consideration is only valid among the same type of glutenin subunits (for instance y- type or x-type), since the two types of high M_r glutenin subunits do not elute in order of decreasing size (Margiotta et al. 1993). The N-terminal sequence could be extended up to the first 20 amino acid residues, though the amino acid residue in position 10 was not determined. The sequence of the 12.4¹ subunit, obtained from the fraction collected from the RP-HPLC, was identical to the other y-type glutenin polypeptides 10, 12, 8, 9, the y-type subunits of the different Glu-1 loci have greater similarity in N-terminal sequences than that found between x- and y-type subunit encoded by the same locus (Margiotta et al. 1993). To further confirm that the novel glutenin subunit (12.4¹) detected in T. tauschii AUS 24092 is a typical y-type glutenin subunit encoded at the Glu-Dl locus, PCR analysis was carried out. Since, in T. tauschii, there is only one y-type high M_r glutenin gene, the y-type specific primers used were able to amplify only a single PCR product corresponding to the y-type glutenin gene of the ID chromosome. The primers (SEQ ID NO: 6 and SEQ ID NO: 7) were located outside the encoding region of the gene, more specifically at 36 bp upstream (5') and 32 bp downstream (3') of the encoding region. Accession AUS 24092 showed an amplified product of about 1.45kb [Figure 5(c)], corresponding to an encoding region of 1.38 kb (the primers amplified 70 bp outside of the encoding region of the gene) which in turn was about 600 and 560 bp smaller than the genes corresponding to subunits 12 and 10 of the hexaploid wheats 'Chinese Spring' and 'Cheyenne', respectively. The Dy-type glutenin genes in common wheat are approximately 1.94 or 1.98 kb, although an unusually large y-type gene (named 12j) has been reported in landrace wheat (D'Ovidio et al. 1994). A clear correlation between the size of high M_r glutenin subunits separated in SDS-PAGE and the size of PCR products of high M_r glutenin genes was reported in hexaploid wheats (D'Ovidio et al. 1994). Also a strong correlation was established between the molecular weight of the glutenin subunit deduced from the gene length measurement of the glutenin gene (for instance, 1DX2, 1DX5, IDylO and lDyl2) and with those calculated from the deduced amino acid sequences (D'Ovidio et al. 1994). Thus the molecular weight of the 12.4¹ glutenin subunit, estimated on the basis of gene length would be around a _r of 47 900 (or 1.38kb). For a more detailed analysis of the high M_r glutenin genes encoded at the Glu- Dl locus, D'Ovidio et al. (1995) designed specific primers for the repetitive domain of the y-type genes. Using these primers, an amplified product of about 950 bp was clearly identified, corresponding to a central repetitive region of the glutenin gene containing 900 bp. Thus, it was possible to demonstrate that the internal repetitive domain of the 12.4¹ gene was responsible for the size variation of the whole glutenin gene and its encoded subunit. Therefore, the glutenin subunit encoded by the 12.4¹ gene is formed by about 435 amino acid residues forming the central repetitive domain of the subunit. Two different results herein support the hypothesis that a deletion within the central repetitive domain has been responsible for the reduction in the size of the 12.4 glutenin subunit. Firstly, the size of the fragment amplified with primers specific for the central repetitive domain of the y-type glutenin gene is much smaller than seen for related high M_r glutenin polypeptides. Secondly, the calculation of the gene length differences between the central repetitive domain and the complete encoding region in the 12.4¹ glutenin gene is about 480 bp (or 160 amino acids), a value very close to the 167 amino acids that represent the sum of both N- and C-terminal regions, including signal peptide, of other y-type glutenin genes. These results are in agreement with those reported by Shewry et al. (1989) in relation to the very conservative nature of the non- repetitive regions of y-type M_r glutenin subunits. As has been pointed out by other authors (Payne et al. 1983; D'Ovidio et al. 1996), the most likely mechanism giving rise to this unusual glutenin subunit is an unequal crossing-over event. The unequal crossing-over could also produce very long genes, as a result of insertion of several blocks of repetitive motifs (D'Ovidio et al. 1996). In this case, the small gene of the 12.4' glutenin subunit might be the result of deletion of several blocks of repetitive motifs. The results presented herein are consistent with the theory postulated by Shewry et al. (1989) about the origin and evolution of the high M_r glutenin genes. These could be summarised as follows: (a) minor changes in the N-terminal and C-terminal domains have evolved by combination of single amino acid substitution and small insertions and/or deletions; (b) major changes in the repetitive block structure of the central domain have provided the basis for a more rapid evolution and divergence by duplication and/or deletion of whole blocks, or several blocks of repetitive motifs. According to this, the deletion of about 200 amino acids in the repetitive domain has produced the subunit 12.4¹ in T tauschii.

Claims

WE CLAIM:

1. An isolated polynucleotide comprising a nucleotide sequence selected from the group consisting of: a) the nucleotide sequence set out in SEQ ID NO: 1 ; b) a nucleotide sequence which is degenerate as a result of the genetic code to the nucleotide sequence set out in SEQ ID NO: 1 ; c) a sequence that selectively hybridises to a sequence that is complementary to (a) or (b) wherein said sequence encodes a glutenin subunit having a molecular weight of about 43 - 48 kDa; d) a sequence that is at least about 70% identical to (a) or (b) and encodes a glutenin subunit having a molecular weight of about 43 - 48 kDa; e) a sequence that hybridizes to at least about 15 contiguous nucleotides of (a) or (b) under at least low stringency hybridization conditions and encodes a glutenin subunit having a molecular weight of about 43 - 48 kDa.

2. An isolated polynucleotide encoding a glutenin polypeptide having a molecular weight of about 43 - 48 kDa as determined by SDS/PAGE wherein said polynucleotide comrpises a nucleotide sequence selected from the group consisting of: a) that set forth in SEQ ID NO: 1; and b) a sequence encoding the amino acid sequence set forth in SEQ ID NO: 2.

3. An isolated or recombinant glutenin polypeptide having a molecular weight of about 43 - 48 kDa and comprising an amino acid sequence selected from the group consisting of: a) the amino acid sequence set out in SEQ ID NO: 2; and b) an amino acid sequence having at least 70% sequence identity to the amino acid sequence set out in SEQ ID NO: 2.

4. An isolated or recombinant glutenin polypeptide having a molecular weight of about 43 - 48 kDa as determined by SDS/PAGE and comprising the amino acid sequence set forth in SEQ ID NO: 2.

5. A method for producing a modified glutenin subunit polypeptide, the method comprising adding a glutenin polypeptide encoded by a polynucleotide having nucleotide sequence according to claim 1 or 2 to an exogenous amino acid domain which confers upon the modified glutenin subunit an ability to bind a ligand or other macromolecule, and wherein the modified glutenin subunit has an ability to incorporate into gluten.

6. A modified glutenin subunit polypeptide produced according to the method of claim 3.

7. The modified glutenin subunit polypeptide according to claim 6, wherein the ligand or other macromolecule is selected from the group consisting of starch and lipid.

8. The modified glutenin subunit polypeptide according to claim 6 or claim 7, wherein the domain capable of binding lipid is derived from barley oleosin protein or the lipid-binding regions of wheat CM 16 protein.

9. An isolated polynucleotide encoding a modified glutenin subunit polypeptide according to any one of claims 6 to 8.

10. A vector comprising the polynucleotide according to any one of claims 1, 2 or 9.

11. The vector according to claim 10 wherein the isolated polynucleotide is operably connected to a regulatory element that is operable in a bacterial cell, yeast cell or plant cell.

12. A host cell comprising the vector according to claim 10 or 11.

13. The host cell according to claim 12, wherein the host cell is a plant cell.

14. The host cell according to claim 13, wherein the plant cell is selected from the group consisting of Triticum spp., maize, rice, oats, barley and rye and/or wild varieties and/or hybrids or derivatives and/or ancestral progenitors of same.

15. A cell culture comprising the host cell according to any one of claims 12 to 14.

16. A plant comprising the polynucleotide according to any one of claims 1, 2 or 9 introduced into its genome.

17. The plant according to claim 16, wherein the plant is selected from the group consisting of Triticum spp., maize, rice, oats, barley and rye and/or wild varieties and/or hybrids or derivatives and/or ancestral progenitors of same.

18. The plant according to claim 16 or 17 wherein the polynucleotide is introduced into the genome of the plant by a process comprising introgression of the nucleic acid.

19. The plant according to claim 16 or 17 wherein the polynucleotide is introduced into the genome of the plant by a process comprising tranforming a plant cell with the nucleic acid and regenerating a whole plant or plant part comprising the polynucleotide.

20. Propagating material or a part of the plant according to any one of claims 16 to 19 wherein said propagating material or part comprises the polynucleotide according to any one of claims 1 , 2 or 9 inserted into its genome.

21. A seed of the plant according to any one of claims 16 to 19 wherein said seed comprises the polynucleotide according to any one of claims 1 , 2 or 9 inserted into its genome.

22. A crossed fertile plant prepared by a process comprising: a) obtaining a fertile plant comprising the polynucleotide according to any one of claims 1 , 2 or 9 inserted into its genome; b) crossing the fertile plant with a plant thereby producing a seed; c) selecting seed that comprises the polynucleotide according to any one of claims 1 , 2 or 9 inserted into its genome; and d) germinating the seed to obtain the crossed fertile plant.

23. The crossed fertile plant according to claim 22, wherein the crossed fertile plant is selected from the group consisting of Triticum spp., maize, rice, oats, barley and rye and/or wild varieties and/or hybrids or derivatives and/or ancestral progenitors of same.

24. The crossed fertile plant according to claim 22 or 23 wherein the fertile plant is produced by a process comprising introgression of the nucleic acid.

25. The crossed fertile plant according to any one of claims 22 to 24 wherein the fertile plant is produced by a process comprising tranforming a plant cell with the nucleic acid and regenerating a whole plant or plant part comprising the polynucleotide.

26. The crossed fertile plant according to any one of claims 22 to 25 wherein the polynucleotide is inherited through a female parent.

27. The crossed fertile plant according to any one of claims 22 to 26 wherein the polynucleotide is inherited through a male parent.

28. Propagating material or a part of the crossed fertile plant according to any one of claims 22 to 27 wherein said propagating material or part comprises the polynucleotide according to any one of claims 1 , 2 or 9 inserted into its genome.

29. A seed of the crossed fertile plant according to any one of claims 22 to 27 wherein said seed comprises the polynucleotide according to any one of claims 1 , 2 or 9 inserted into its genome.

30. A method for detecting a polynucleotide encoding a glutenin polypeptide having a molecular weight of about 43 - 48 kDa as determined by SDS/PAGE, said method comprising contacting nucleic acid with the isolated polynucleotide according to any one of claims 1, 2 or 9 or a probe that comprises at least about 15 contiguous nucleotides of said isolated polynucleotide for a time and under conditions sufficient for hybridization to occur and then detecting the hybridization.

31. The method of claim 31 further comprising selecting a polynucleotide encoding a glutenin polypeptide having a molecular weight of about 43 - 48 kDa as determined by SDS/PAGE.

32. The method of claim 30 or 31 wherein the nucleic acid consists of genomic DNA.

33. The method of claim 30 or 31 wherein the nucleic acid consists of mRNA or cDNA.

34. The method according to any one of claims 30 to 33 wherein the nucleic acid is derived from a plant.

35. The method of claim 34 wherein the plant is selected from the group consisting of Triticum spp., maize, rice, oats, barley and rye and/or wild varieties and/or hybrids or derivatives and/or ancestral progenitors of same.

36. Use of the isolated or recombinant glutenin polypeptide of claim 3 or 4 or the modified glutenin subunit polypeptide according to any one of claims 6 to 8 in the preparation of a food product.

37. The use according to claim 36 wherein the food product is selected from the group consisting of leavened bread, unleavened bread, pasta, noodle, breakfast cereal, snack food, cake, pastry, flour, gluten and a food comprising a flour-based sauce.

38. Use of the the isolated or recombinant glutenin polypeptide of claim 3 or 4 or the modified glutenin subunit polypeptide according to any one of claims 6 to 8 in the preparation of a non-food product.

39. The use according to claim 38 wherein the non-food product is selected from the group consisting of a film, a coating, an adhesive, a building material, and a packaging material.