AU2004200536A1 - Nucleic acid molecules - Google Patents

Description

P001 Section 29 Regulation 3.2(2)

AUSTRALIA

Patents Act 1990 COMPLETE SPECIFICATION STANDARD PATENT Application Number: Lodged: Invention Title: Nucleic acid molecules The following statement is a full description of this invention, including the best method of performing it known to us: Nucleic acid molecules encoding wheat enzymes Involved In starch synthesis The present invention relates to nucleic acid molecules which encode a wheat enzyme involved in starch synthesis in plants. This enzyme is an isoamylase.

The invention furthermore relates to vectors, host cells, plant cells and plants comprising the nucleic acid molecules according to the invention.

Furthermore, there are described methods for the generation of transgenic plants which, owing to the introduction of nucleic acid molecules according to the invention, synthesize starch with altered characteristics.

In view of the increasing Importance attributed lately to plant constituents as renewable raw materials, one of the objects of biotechnology research addresses the adaptation of these plant raw materials to the needs of the processing industries. Moreover, to allow renewable raw materials to be used in as many fields as possible, a wide diversity of materials must be generated.

Apart from oils, fats and proteins, polysaccharides constitute the important renewable raw materials from plants. Apart from cellulose, starch which is one of the most important storage substances in higher plants takes a central position amongst the polysaccharides. In this context, wheat is one of the most important crop plants since it provides approximately 20% of the total starch production in the European Community. The polysaccharide starch Is a polymer of chemically uniform units, the glucose molecules. However, it is a highly complex mixture of different molecule types which differ with regard to their degree of polymerization, the occurrence of branching of the glucose chains and their chain lengths, which, in addition, may be derivatized, for example phosphorylated. Starch therefore does not constitute a uniform raw material. In particular, a distinction is made between amylose starch, an essentially unbranched polymer of alpha-1,4-glycosidically linked glucose molecules, and amylopectin starch, which, in turn, constitutes a complex mixture of glucose chains with various branchings. The branchings occur by the occurrence of additional alpha-1,6-glycosidic linkages. In wheat, amylose starch makes up approximately 11 to 37% of the starch synthesized.

To allow suitable starches to be used in the widest possible manner for the widest possible range of industrial needs, it is desirable to provide plants which are capable of synthesizing modified starches which are particularly well suited to various purposes. One possibility of providing such plants is to employ plant-breeding measures. However, since wheat is polyploid in character (tetra- and hexaploid), the exertion of influence by plant breeding proves to be very difficult. A "waxy" (amylose-free) wheat was generated only recently by crossing naturally occurring mutants (Nakamura et al., Mol.

Gen. Genet. 248 (1995), 253-259).

An alternative to plant-breeding methods is the specific modification of starch-producing plants by recombinant methods. However, prerequisites are the identification and characterization of the enzymes which are involved in starch synthesis and/or starch modification and the isolation of the nucleic acid molecules encoding these enzymes.

The biochemical pathways which lead to the synthesis of starch are essentially known. Starch synthesis in plant cells takes place in the plastids. In photosynthetically active tissue, these plastids are the chloroplasts and In photosynthetically inactive, starch-storing tissue are amyloplasts.

A further specific alteration of the degree of branching of starch synthesized in plants with the aid of recombinant methods still requires identification of DNA sequences, which encode enzymes involved in starch metabolism, in particular in the introduction or degradation of branching within the starch molecules.

Besides the so-called Q enzymes, which introduce branchings into starch molecules, enzymes occur in plants which are capable of breaking down branchings. These enzymes are called debranching enzymes and, according to their substrate specificity, they are divided into three groups: The pullulanases, which, in addition to pullulan, also utilize amylopectin as substrate, are found in microorganisms, for example Klebsiella and in plants. In plants, these enzymes are also termed R enzymes.

The isoamylases, which do not utilize pullulan, but indeed glycogen and amylopectin as substrate, are also found in microorganisms and plants. For example, isoamylases have been described in maize (Manners Carbohydr. Res. 9 (1969), 107) and potato (Ishizaki et al., Agric. Biol. Chem. 47 (1983), 771-779).

The amylo-1,6-glucosidases are described in mammals and yeasts and utilize grenzdextrins as substrate.

In sugar beet, Li et al. (Plant Physiol. 98 (1992), 1277-1284) were only able to find one debranching enzyme of the pullulanase type, in addition to five endoamylases and two exoamylases. This enzyme, which has a size of approx. 100 kD and a pH optimum of 5.5, is localized in the chloroplasts. In spinach, too, a debranching enzyme was described which utilizes pullulan as substrate. The activity both of the spinach debranching enzyme and of the sugar beet debranching enzyme upon reaction with amylopectin as substrate is five times lower in comparison with pullulan as substrate (Ludwig et al., Plant Physiol. 74 (1984), 856-861; U et al., Plant Physiol. 98 (1992), 1277-1284).

In the agronomically important starch-storing crop plant potato, the activity of a debranching enzyme was studied by Hobson et al. Chem. Soc., (1951), 1451). It was proved successfully that, in contrast to the Q enzyme, this enzyme has no chain-extending activity, but merely hydrolyzes alpha- 1,6-glycosidic bonds. However, it has been impossible as yet to characterize the enzyme in greater detail. In the case of potatoes, processes for purifying the debranching enzyme and partial peptide sequences of the purified protein have already been proposed (WO 95/04826). In the case of spinach, the purification of a debranching enzyme and the isolation of suitable cDNA have been described in the meantime (Renz et al., Plant Physiol. 108 (1995), 1342).

In maize, only the existence of one debranching enzyme has been described as yet in the literature. Owing to its substrate specificity, this enzyme is classified as belonging to the group of the isoamylases (see, for example, Hannah et al., Scientia Horticulturae 55 (1993), 177-197 or Garwood (1994) in Starch Chemistry and Technology, Whistler, R.L, BeMiller, Puschall, E.F. Academic Press San Diego, New York, Boston, 25-86). The corresponding mutant is termed "sugary". The gene of the sugary locus has been cloned recently (see James et al., Plant Cell 7 (1995), 417-429). Apart from the sugary locus, no other gene locus which encodes a protein with debranching enzyme activity is as yet known in maize. Also, there have been no indications to date that other debranching enzyme forms occur in maize. If transgenic maize plants are to be generated which no longer have any debranching enzyme activities whatsoever, for example in order to extend the degree of branching of the amylopectin starch, it is necessary to identify all debranching enzymes forms which occur in maize and to isolate the corresponding genes or cDNA sequences.

To provide further possibilities of altering any starch-storing plant, preferably cereals, in particular wheat, so that it synthesizes a modified starch, it is necessary to identify in each case DNA sequences which encode further isoforms of branching enzymes.

The object of the present invention is therefore to provide nucleic acid molecules encoding enzymes involved in starch synthesis, which allow genetically modified plants to be generated which make possible the production of plant starches whose chemical and/or physical characteristics are altered.

This object is achieved by providing the use forms designated in the patent claims.

The present invention therefore relates to a nucleic acid molecule which encodes a protein with the function of a wheat isoamylase, preferably a protein which is essentially defined by the amino acid sequence stated under Seq ID No. 3 or 7. In particular, the invention relates to a nucleic acid molecule comprising the nucleotide sequence stated under Seq ID No. 1, 2 or 6, or a part thereof, preferably a molecule comprising the coding region stated in Seq ID No. 1, 2 or 6, and corresponding ribonucleotide sequences. Very especially preferred is a nucleic acid molecule furthermore comprising regulatory elements which ensure transcription and, if appropriate, translation of said nucleic acid molecules. The subject matter of the invention is furthermore a nucleic acid molecule which hybridizes with one of the nucleic acid molecules according to the invention.

The subject matter of the invention is also a nucleic acid molecule encoding a wheat isoamylase whose sequence deviates from the nucleotide sequences of the above-described molecules owing to the degeneracy of the genetic code.

The invention also relates to a nucleic acid molecule with a sequence which is complementary to all or part of one of the abovementioned sequences.

The term "hybridization" as used in the context of the present invention denotes hybridization under conventional hybridization conditions, preferably under stringent conditions, as they are described, for example, by Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Ed.

(1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).

"Hybridization" especially preferably takes place under the following conditions: Hybridization buffer 2 x SSC; 10 x Denhardt solution (Fikoll 400 PEG BSA; ratio 0.1% SDS; 5 mM EDTA; 50 mM Na2HPO 4 250 pg/ml Herring sperm DNA; 50 tg/ml tRNA; or 0.25 M sodium phosphate buffer pH 7.2; 1 mM EDTA; 7% SDS Hybridization temperature T 65 to Wash buffer: 0.2 x SSC; 0.1% SDS Wash temperature T 40 to Nucleic acid molecules which hybridize with the nucleic acid molecules according to the invention are capable, in principle, of encoding isoamylases from any wheat plant which expresses such proteins.

Nucleic acid molecules which hybridize with the molecules according to the invention can be isolated for example from genomic libraries or cDNA libraries of wheat or wheat plant tissue. Alternatively, they can be generated by recombinant methods or synthesized chemically.

Identification and isolation of such nucleic acid molecules can be effected using the molecules according to the invention or parts of these molecules or the reverse complements of these molecules, for example by means of hybridization by standard methods (see, for example, Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).

Hybridization probes which can be used are, for example, nucleic acid molecules which have exactly or essentially the nucleotide sequence stated under Seq ID No. 1, 2 or 6 or parts of these sequences. The fragments used as hybridization probe may also be synthetic fragments which have been prepared with the aid of the customary synthetic techniques whose sequence essentially agrees with that of a nucleic acid molecule according to the invention.

The molecules hybridizing with the nucleic acid molecules according to the invention also encompass fragments, derivatives and allelic variants of the above-described nucleic acid molecules which encode a wheat isoamylase according to the invention. Fragments are to be understood as meaning parts of the nucleic acid molecules of sufficient length so as to encode one of the proteins described. The term derivative means in this context that the sequences of these molecules differ from the sequences of the abovedescribed nucleic acid molecules at one or more positions and have a high degree of homology with these sequences. Homology means a sequence identity of at least 40%, in particular of at least 60%, preferably over especially preferably over 90%. The deviations relative to the abovedescribed nucleic acid molecules may have been generated by deletion, substitution, insertion or recombination.

Homology furthermore means that functional and/or structural equivalence exists between the nucleic acid molecules in question or the proteins encoded by them. The nucleic acid molecules which are homologous to the above-described molecules and constitute derivatives of these molecules are, as a rule, variations of these molecules which constitute modifications exerting the same biological function. They may be naturally occurring variations, for example, sequences from other organisms, or mutations which may have occurred naturally or been introduced by directed mutagenesis. Furthermore, the variations may be synthetically generated sequences. The allelic variants may be both naturally occurring variants and synthetically generated variants or variants produced by recombinant DNA techniques.

The isoamylases encoded by the various variants of the nucleic acid molecules according to the invention share certain characteristics. These may include, for example, enzyme activity, molecular weight, immunological reactivity, conformation and the like, or else physical properties such as, for example, the migration behavior in gelelectrophoresis, the chromatographic behavior, sedimentation coefficients, solubility, spectroscopic characteristics, charge characteristics, stability; pH optimum, temperature optimum and the like.

The protein encoded by the nucleic acid molecules according to the invention is a wheat isoamylase. These proteins show certain homology ranges with isoamylases from other plant species which are already known.

The nucleic acid molecules according to the invention may be DNA molecules, in particular cDNA or genomic molecules. Furthermore, the nucleic acid molecules according to the invention may be RNA molecules which may result, for example, from the transcription of a nucleic acid molecule according to the invention. The nucleic acid molecules according to the invention may have been obtained, for example, from natural sources or they may have been generated by recombinant techniques or synthesized.

Subject matter of the invention are also oligonucleotides which hybridize specifically with a nucleic acid molecule according to the invention. Such oligonucleotides preferably have a length of at least 10, in particular of at least 15 and especially preferably of at least 50 nucleotides. The oligonucleotides according to the invention hybridize specifically with nucleic acid molecules according to the invention, i.e. not or only to a very low degree with nucleic acid sequences which encode other proteins, in particular other isoamylases. The oligonucleotides according to the invention can be used, for example, as primers for a PCR reaction or as hybridization probe for the isolation of the related genes. Equally, they may be constituents of antisense constructs or of DNA molecules encoding suitable ribozymes.

The invention furthermore relates to vectors, in particular plasmids, cosmids, phagemids, viruses, bacteriophages and other vectors conventionally used in genetic engineering comprising the above-described nucleic acid molecules according to the invention. Such vectors are suitable for the transformation of pro- or eukaryotic cells, preferably plant cells.

The vectors especially preferably permit integration of the nucleic acid molecules according to the invention, if appropriate together with flanking regulatory regions, into the genome of the plant cell. Examples are binary vectors which can be employed in agrobacterial-mediated gene transfer.

Preferably, integration of a nucleic acid molecule according to the invention in sense or antisense orientation ensures that a translatable or, if appropriate, nontranslatable RNA is synthesized in the transformed pro- or eukaryotic cells.

The term "vector" generally denotes a suitable auxiliary known to the skilled worker which allows the directed transfer of a single- or double-stranded nucleic acid molecule into a host cell, for example a DNA or RNA virus, a virus fragment, a plasmrid construct which, in the absence or presence of regulatory elements, may be suitable for transferring nucleic acid into cells, or support materials such as glass fibers or else metal particles as can be employed, for example, in the particle gun method, but it may also encompass a nucleic acid molecule which can be introduced directly into a cell by means of chemical or physical methods.

In a preferred embodiment, the nucleic acid molecules within the vectors are linked to regulatory elements which ensure transcription and synthesis of a translatable RNA in pro- or eukaryotic cells or which if desired ensure synthesis of a nontranslatable RNA.

Expression of the nucleic acid molecules according to the invention in prokaryotic cells, for example, in Escherichia coli, is of importance for a more detailed characterization of the enzymatic activities of the enzymes encoded by these molecules. In particular, it is possible to characterize the product synthesized by the enzymes in question in the absence of other enzymes involved in starch synthesis in the plant cell. This permits conclusions to be drawn regarding the function which the protein in question exerts during starch synthesis in the plant cell.

In addition, various types of mutations can be introduced into the nucleic acid molecules according to the invention by means of customary techniques of molecular biology (see, for example, Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY), resulting in the synthesis of proteins whose biological properties may be altered. Possible here is, on the one hand, the generation of deletion mutants in which nucleic acid molecules are generated by successive deletions from the 5' or the 3' end of the coding DNA sequence which lead to the synthesis of correspondingly truncated proteins. Such deletions at the 5' end of the nucleotide sequence allow, for example, amino acid sequences to be identified which are responsible for translocation of the enzyme into the plastids (transit peptides). This allows the directed generation of enzymes which, owing to the removal of the sequences in question, are no longer localized in the plastids, but in the cytosol, or which, owing to the addition of other signal sequences, are localized in other compartments.

On the other hand, it is also possible to introduce point mutations at positions where an altered amino acid sequence affects, for example, enzyme activity or enzyme regulation. In this manner, It is possible to generate, for example, mutants which have an altered Km value or which are no longer subject to the regulatory mechanisms via allosteric regulation or covalent modification which are normally present in the cell.

Furthermore, it is possible to generate mutants which have an altered substrate or product specificity of the protein according to the invention.

Furthermore, it is possible to generate mutants which have an altered activity-temperature profile of the protein according to the invention.

To carry out the recombinant modification of prokaryotic cells, the nucleic acid molecules according to the invention or parts of these molecules can be introduced into plasmids which allow mutagenesis to take place or a sequence to be altered by recombining DNA sequences. Base exchanges can be carried out or natural or synthetic sequences added with the aid of standard methods (cf. Sambrook et al., 1989, Molecular Cloning: A laboratory manual, 2nd Ed., Cold Spring Harbor Laboratory Press, NY, USA). To link the DNA fragments to each other, adapters or linkers may be attached to the fragments. Furthermore, manipulations may be employed which provide suitable restriction cleavage sites or which eliminate superfluous DNA or restriction cleavage sites. Where insertions, deletions or substitutions are suitable, in vitro mutagenesis, primer repair, restriction or ligation may be employed. Analytical methods which are generally employed are sequence analysis, restriction analysis or other methods of biochemistry and molecular biology.

In a further embodiment, the invention relates to host cells, in particular pro- or eukaryotic cells, which have been transformed with an abovedescribed nucleic acid molecule according to the invention or a vector according to the invention, and to cells which are derived from cells transformed thus and comprise a nucleic acid molecule according to the invention or a vector. They are preferably pro- or eukaryotic cells, in particular plant cells.

Subject matter of the invention are furthermore proteins with isoamylase activity which are encoded by the nucleic acid molecules according to the invention and which can be prepared by recombinant technology, and processes for their preparation, where a host cell according to the invention is cultured under suitable conditions which are known to the skilled worker and which permit synthesis of the protein according to the invention and it is subsequently isolated from the host cells and/or the culture medium.

Providing the nucleic acid molecules according to the invention now makes it possible to intervene, with the aid of recombinant methods, in a directed fashion in the starch metabolism of plants and to alter it so that the resultant synthesis is of modified starch whose physicochemical properties, for example the amyloselamylopectin ratio, the degree of branching, the average chain length, the phosphate content, the gelatinization behavior, the gel- or film-forming properties, the starch granule size and/or the starch granule shape is altered in comparison to known starch.

Thus, it is possible to express the nucleic acid molecules according to the invention in plant cells in order to increase the activity of the isoamylase in question, or to introduce them into cells which do not naturally express this enzyme. Expressing the nucleic acid molecules according to the invention also makes it possible to lower the natural activity level of the isoamylase according to the invention in the plant cells. Furthermore, it is possible to modify the nucleic acid molecules according to the invention by methods known to the skilled worker in order to obtain isoamylases according to the invention which are no longer subject to the cell's intrinsic regulatory mechanism or which have altered temperature-activity profiles or substrate or product specificities.

When expressing the nucleic acid molecules according to the Invention in plants, it is possible, in principle, for the protein synthesized to be localized in any desired compartment of the plant cell. To achieve localization in a particular compartment, the sequence ensuring localization in plastids must be deleted and the remaining encoding region must, if necessary, be linked to DNA sequences which ensure localization in the compartment in question. Such sequences are known (see, for example, Braun et al., EMBO J. 11 (1992), 3219-3227; Wolter et al., Proc. Natl., Acad. Sci. USA 85 (1988), 846-850; Sonnewald et al., Plant J. 1 (1991), 95-106).

The present invention thus also relates to a method for generating transgenic plant cells which have been transformed with a nucleic acid molecule or a vector according to the invention, where a nucleic acid molecule according to the invention or a vector according to the Invention is integrated into the genome of a plant cell, the transgenic plant cells which have been transformed by means of a vector or nucleic acid molecule according to the invention, and transgenic plant cells derived from cells transformed thus. The cells according to the invention comprise one or more nucleic acid molecules or vectors according to the invention, these preferably being linked to regulatory DNA elements which ensure transcription in plant cells, in particular to a suitable promoter. Such cells can be distinguished from naturally occurring plant cells inter alia by the fact that they comprise a nucleic acid molecule according to the invention which does not occur naturally in these cells, or by the fact that such a molecule exists integrated at a location in the cell's genome where it does not occur otherwise, i.e. in a different genomic environment. Furthermore, such transgenic plant cells according to the invention can be distinguished from naturally occurring plant cells by the fact that they comprise at least one copy of a nucleic acid molecule according to the invention stably integrated into the genome, if appropriate in addition to the copies of such a molecule which occur naturally in the cells. If the nucleic acid molecule(s) introduce into the cells is(are) additional copies to molecules which already occur naturally in the cells, then the plant cells according to the invention can be distinguished from naturally occurring plant cells in particular by the fact that this additional copy, or these additional copies, is, or are, localized at locations in the genome where it does not occur naturally, or they do not occur naturally. This can be checked, for example, with the aid of a Southern blot analysis.

If the nucleic acid molecule according to the invention which has been introduced into the plant genome is heterologous to the plant cell, the transgenic plant cells exhibit transcripts of the nucleic acid molecules according to the invention which can be detected in a simple manner by methods known to the skilled worker, for example by Northern blot analysis.

If the nucleic acid molecule according to the invention which has been introduced is homologous to the plant cell, the cells according to the invention can be distinguished from naturally occurring cells, for example, on the basis of the additional expression of nucleic acid molecules according to the invention. The transgenic plant cells preferably comprise more transcripts of the nucleic acid molecules according to the invention.

This can be detected, for example, by Northem blot analysis. "More" in this context means preferably at least 10% more, preferably at least 20% more, especially preferably at least 50% more transcripts than corresponding untransformed cells. The cells furthermore preferably exhibit a corresponding increase or decrease in the activity of the protein according to the invention (at least 10%, 20% or The transgenic plant cells can be regenerated into intact plants by techniques known to the skilled worker.

Another subject matter of the present invention is a method for the generation of transgenic plants, where one or more nucleic acid molecules or vectors according to the invention are integrated into the genome of a plant cell and a complete plant is regenerated from said plant cell. Subject matter-of the invention are furthermore plants which comprise the abovedescribed transgenic plant cells. In principle, the transgenic plants can be plants of any species, i.e. not only monocotyledonous but also dicotyledonous plants. They are preferably useful plants, by preference starch-synthesizing or starch-storing plants, especially preferably rye, barley oats, wheat, sorghum and millet, sago, maize, rice, peas, marrowfat peas, cassava, potatoes, tomatoes, oilseed rape, soybeans, hemp, flax, sunflowers, cowpeas or arrowroot, in particular wheat, maize, rice and potatoes.

The invention also relates to propagation material of the plants according to the invention, for example fruits, seeds, tubers, rootstocks, seedlings, cuttings, calli, protoplasts, cell cultures and the like.

The present invention furthermore relates to a process for the preparation of a modified starch comprising the step of extracting the starch from an above-described plant according to the invention and/or starch-storing parts of such a plant.

Processes for extracting the starch from plants or starch-storing parts of plants, In particular from wheat, are known to the skilled worker, cf., for example, Eckhoff et al. (Cereal Chem. 73 (1996) 54-57) "Starch: Chemistry and Technology (Eds.: Whistler, BeMiller and Paschall (1994), 2nd Edition, Academic Press Inc. London Ltd; ISBN 0-12-746270-8; see, for example, Chapter XII, pages 412-468: Corn and sorghum starches: production; by Watson; Chapter XIII, pages 469-479; Tapioca, arrowroot and sago starches: production; by Corbishley and Miller; Chapter XIV, pages 479- 490: Potato starch: production and uses; by Mitch; Chapter XV, pages 491 to 506: Wheat starch: production, modification and uses; by Knight and Oson; and Chapter XVI, pages 507 to 528: Rice starch: production and uses; by Rohmer and Klem). Devices normally used in processes for extracting starch from plant material are separators, decanters, hydrocyclones, spray dryers and fluidized-bed dryers.

Owing to the expression of a nucleic acid molecule according to the invention, the transgenic plant cells and plants according to the invention synthesize a starch whose physicochemical properties, for example the amylose/amylopectin ratio, the degree of branching, the average chain length, the phosphate content, the gelatinization behavior, the starch granule size-and/or starch granule shape is altered compared with starch synthesized in wild-type plants. In particular, such a starch may be altered with regard to viscosity and/or the film- or gel-forming properties of gels made from this starch in comparison with known starches.

Subject matter of the present invention is furthermore a starch which is obtainable from the plant cells and plants according to the invention and their propagation material and starch which is obtainable by the abovedescribed process according to the invention.

It is furthermore possible to generate, with the aid of the nucleic acid molecules according to the invention, plant cells and plants in which the activity of a protein according to the invention is reduced. This also leads to the synthesis of a starch with altered chemical and/or physical characteristics compared with starch from wild-type plant cells.

A further subject matter of the invention is thus also a transgenic plant cell comprising a nucleic acid molecule according to the invention in which the activity of an isoamylase is reduced in comparison with untransformed cells.

Plant cells with a reduced activity of an isoamylase can be obtained, for example, by expressing a suitable antisense RNA, a sense RNA for achieving a cosuppression effect or by expressing a suitably constructed ribozyme which specifically cleaves transcripts which encode an isoamylase, making use of the nucleic acid molecules according to the invention by methods known to the skilled worker, cf. Jorgensen (Trends Biotechnol. 8 (1990), 340-344), Niebel et al., (Curr. Top. Microbiol.

Immunol. 197 (1995), 91-103), Flavell et al. (Curr. Top. Microbiol. Immunol.

197 (1995), 43-46), Palaqui and Vaucheret (Plant. Mol. Biol. 29 (1995), 149-159), Vaucheret et al., (Mol. Gen. Genet. 248 (1995), 311-317), de Borne et al. (Mol. Gen. Genet. 243 (1994), 613-621).

To reduce the activity of an isoamylase according to the invention, it is preferred to reduce, in the plant cells, the number of transcripts encoding it, for example by expressing an antisense RNA.

Here, it is possible to make use, on the one hand, of a DNA molecule which encompasses all of the sequence encoding a protein according to the invention, inclusive of any flanking sequences which may be present, or else of DNA molecules which only encompass parts of the coding sequence, it being necessary for these parts to be sufficiently long so as to cause an antisense effect in the cells. In general, sequences up to a minimum length of 15 bp, preferably with a length of 100-500 bp, may be used for efficient antisense inhibition in particular sequences with a length of over 500 bp. As a rule, DNA molecules are used which are shorter than 5000 bp, preferably sequences which are shorter than 2500 bp.

Also possible is the use of DNA sequences which show a high degree of homology with the sequences of the DNA molecules according to the invention, but are not completely identical. The minimum homology should exceed approx. 65%. The use of sequences with homologies between and 100% is to be preferred.

Subject matter of the invention is also a process for producing a modified starch encompassing the step of extracting the starch from a cell or plant according to the invention and/or from starch-storing parts of such a plant.

Subject matter of the invention is furthermore starch which can be obtained from the cells or plants according to the invention and their propagation material or parts, and also starch which can be obtained by a process according to the invention.

The starches according to the invention can be modified by methods known to the skilled worker and are suitable, in their unmodified or modified form, for a variety of applications in the food or non-food sectors.

In principle, the possible uses of the starches according to the invention can be divided into two important sectors. One sector encompasses the hydrolyzates of the starch, mainly glucose and glucan units, which are obtained by enzymatic or chemical methods. They are used as starting material for further chemical modifications and processes such as fermentation. What would be feasible for reducing the costs is the simplicity and economic design of a hydrolytic method. It currently proceeds essentially enzymatically using amyloglucosidase. What would be feasible is a financial saving by using less enzyme. This could be caused by altering the structure of the starch, for example by increasing the surface area of the granule, better digestibility, for example owing to a lower degree of branching or a sterical structure which limits the accessibility for the enzymes employed.

The other sector in which the starch according to the invention can be used as so-called native starch, owing to its polymeric structure, can be divided into two further fields of application: 1. The food industry Starch is a traditional additive to a large number of foodstuffs in which its function is essentially to bind aqueous additives or to cause increased viscosity or else increased gelling. Important characteristics are the rheology, the sorptive characteristics, the swelling temperature, the gelatinization temperature, the viscosity, the thickening power, the starch solubility, the transparency and gel structure, the thermal stability, the shear stability, the stability to acids, the tendency to undergo retrogradation, the film-forming capacity, the freeze-thaw stability, the viscosity stability in salt solutions, the digestibility and the ability to form complexes with, for example, inorganic or organic ions.

2. The non-food industry In this large sector, starch can be employed as auxiliary for various preparation processes or as an additive in industrial products. When using starch as an auxiliary, mention must be made, in particular, of the paper and board industry. Starch acts mainly for retardation purposes (retaining solids), for binding filler particles and fines, as stiffener and for dehydration. Moreover, the advantageous properties of starch regarding stiffness, strength, sound, touch, luster, smoothness, bonding strength and the surfaces is utilized.

2.1 Paper and board industry Within the papermaking process, four fields of application must be distinguished, i.e. surface, coating, stock and spraying. The demands on starch with regard to surface treatment are essentially high whiteness, an adapted viscosity, high viscosity stability, good film formation and low dust formation. When used for coating, the solids content, a suitable viscosity, a high binding capacity and a high pigment affinity play an important role. Of importance when used as additive to the stock is-rapid, uniform, loss-free distribution, high mechanical strength and complete retention in the paper web. If the starch is used in the spraying sector, again, an adapted solids content, high viscosity and high binding capacity are of importance.

2.2 The adhesives industry An important field of application for starches is the adhesives industry, where the potential uses can be divided into four subsections: the use as a pure starch paste, the use in starch pastes which have been treated with specialty chemicals, the use of starch as additive to synthetic resins and polymer dispersions, and the use of starches as extenders for synthetic adhesives. 90% of the starchbased adhesives are employed in the sectors production of corrugated board, production of paper sacks and bags, production of composite materials for paper and aluminum, production of boxes and gumming adhesives for envelopes, stamps and the like.

2.3 Textile industry and textile care products industry An important field of application for starches as auxiliaries and additives is the sector production of textiles and textile care products. The following four fields of application must be distinguished within the textile industry: the use of starch as sizing agent, i.e. as auxiliary for smoothing and strengthening the smoothing behavior as protection from the tensile forces applied during weaving, and for increasing abrasion resistance during weaving, starch as a textile finishing agent, in particular after qualityreducing pretreatments such as bleaching, dyeing and the like, starch as thickener in the preparation of dye pastes for preventing bleeding, and starch as additive to glazing agents for sewing threads.

2.4 Construction materials industry The fourth field of application is the use of starches as additives in construction materials. An example is the production of gypsum plasterboards, where the starch which is admixed to the gypsum slurry gelatinizes with the water, diffuses to the surface of the plaster core and there binds the board to the core. Other fields of application are the admixture to rendering and mineral fibers. In the case of ready-mixed concrete, starch products are employed for delaying binding.

Soil stabilization Another market for starch products is the production of soil stabilizers, which are employed for the temporary protection of the soil particles from water when the soil is disturbed artificially.

According to present knowledge, product combinations of starch and polymer emulsions equal the previously employed products with regard to their erosion- and crust-reducing effect, but are markedly less expensive.

2.6 Use in crop protection products and fertilizers One field of application for using starch is in crop protection products for altering the specific properties of the products. Thus, starch can be employed for improving the wettability of crop protection products and fertilizers, for the controlled release of the active ingredients, for converting liquid, volatile and/or malodorous active ingredients into microcrystalline, stable, shapeable substances, for mixing incompatible compounds and for extending the duration of action by reducing decomposition.

2.7 Pharmaceuticals, medicine and cosmetics industry Another field of application is the sector of the pharmaceuticals, medicine and cosmetics industry. In the pharmaceuticals industry, starch can be employed as binder for tablets or for diluting the binder in capsules. Moreover, starch can be employed as tablet disintegrant since it absorbs fluid after swallowing and swells within a short time to such an extent that the active ingredient is liberated.

Medicinal lubricating powders and wound powders are starch-based for reasons of quality. In the cosmetics sector, starches are employed, for example, as carriers of powder additives such as fragrances and salicylic acid. A relatively large field of application for starch is toothpaste.

2.8 Addition of starch to coal and briquettes A field of application for starch is as additive to coal and briquettes.

With an addition of starch, coal can be agglomerated, or briquetted, in terms of high quantity, thus preventing premature decomposition of the briquettes. In case of barbecue coal, the starch addition amounts to between 4 and in the case of calorized coal to between 0.1 and Moreover, starches are gaining importance as binders since the emission of noxious substances can be markedly reduced when starches are added to coal and briquettes.

2.9 Ore slick and coal silt processing Furthermore, starch can be employed as flocculant in the ore slick and coal silt processing sector.

2.10 Foundry auxiliary A further field of application is as additive to foundry auxiliaries.

Various casting processes require cores made with sands treated with binders. The binder which is predominantly employed nowadays is bentonite, which is treated with modified starches, in most cases swellable starches.

The purpose of adding starch is to increase flowability and to improve the binding power. In addition, the swellable starches can meet other demands of production engineering, such as being coldwater-dispersible, rehydratable, readily miscible with sand and having high water-binding capacity.

2.11 Use in the rubber industry In the rubber industry, starch is employed for improving the technical and visual quality. The reasons are the improvement of the surface luster, the improvement of handle and of appearance, and to this end starch is scattered on to the tacky gummed surface of rubber materials prior to cold curing, and also the improvement of the rubber's printability.

2.12 Production of leather substitutes Modified starches may furthermore also be sold for the production of leather substitutes.

2.13 Starch in synthetic polymers In the polymer sector, the following fields of application can be envisaged: the use of starch degradation products in the processing process (starch only acts as filler, there is no direct bond between the synthetic polymer and the starch) or, alternatively, the use of starch degradation products in the production of polymers (starch and polymer form a stable bond).

The use of starch as a pure filler is not competitive in comparison with other substances such as talc. However, this is different when the specific properties of starch make an impact and thus markedly alter the spectrum of characteristics of the end products. An example of this is the use of starch products in the processing of thermoplasts, such as polyethylene.

Here, the starch and the synthetic polymer are combined by coexpression In a ratio of 1:1 to give a master batch, from which various products are produced with granulated polyethylene, using conventional process techniques. By using starch in polyethylene films, an increased substance permeability in the case of hollow bodies, an improved permeability for water vapor, an improved antistatic behavior, an improved antiblock behavior and an improved printability with aqueous inks can be achieved.

Another possibility is the use of starch in polyurethane foams. By adapting the starch derivatives and by process-engineering optimization, it is possible to control the reaction between synthetic polymers and the starches' hydroxyl groups in a directed manner. This results in polyurethane films which have the following spectrum of properties, owing to the use of starch: a reduced heat expansion coefficient, a reduced shrinking behavior, an improved pressure-tension behavior, an increase in permeability for water vapor without altering the uptake of water, a reduced flammability and a reduced ultimate tensile strength, no drop formation of combustible parts, freedom from halogens, or else reduced aging.

Disadvantages which still exist are reduced printability and reduced impact strength.

Product development is currently no longer restricted to films. Solid polymer products such as pots, slabs and dishes which have a starch content of over 50% may also be produced. Moreover, starch/polymer mixtures are considered advantageous since their biodegradability is much higher.

Starch graft polymers have become exceedingly important owing to their extremely high water binding capacity. They are products with a starch backbone and a side lattice of a synthetic monomer, grafted on following the principle of the free-radical chain mechanism. The starch graft polymers which are currently available are distinguished by a better binding and retention capacity of up to 1000 g of water per g of starch combined with high viscosity. The fields of application of these superabsorbers have extended greatly in recent years and are, in the hygiene sector, products such as diapers and pads and, in the agricultural sector, for example seed coatings.

What is decisive for the application of novel, genetically modified starches are, on the one hand, structure, water content, protein content, lipid content, fiber content, ash/phosphate content, amylose/amylopectin ratio, molecular mass distribution, degree of branching, granule size and granule shape and crystallinity, and, on the other hand, also the characteristics which effect the following features: flow and sorption behavior, gelatinization temperature, viscosity, viscosity stability in salt solutions, thickening power, solubility power, gel structure and gel transparency, thermal stability, shear stability, stability to acids, tendency to undergo retrogradation, gel formation, freeze-thaw stability, complex formation, iodine binding, film formation, adhesive power, enzyme stability, digestibility and reactivity.

The production of modified starches by recombinant methods can, on the one hand, alter the properties of the starch derived from the plant in such a way that other modifications by means of chemical or physical processes are no longer required. On the other hand, starches which have been altered by recombinant methods may be subjected to further chemical modification, which leads to further improvements in quality for some of the above-described fields of application. These chemical modifications are known In principle. They are, in particular, modifications by thermal treatment, treatment with organic or inorganic acids, oxidation and esterifications, which lead, for example, to the formation of phosphate starches, nitrate starches, sulfate starches, xanthate starches, acetate starches and citrate starches. Moreover, mono- or polyhydric alcohols in the presence of strong acids may be employed for producing starch ethers, resulting in starch alkyl ethers, 0-allyl ethers, hydroxyalkyl ethers, 0-carboxy methyl ethers, N-containing starch ethers, P-containing starch ethers), S-containing starch ethers, crosslinked starches or starch graft polymers.

A preferred use of the starches according to the invention is the production of packaging materials and disposable articles, on the one hand, and as foodstuff or foodstuff precursor on the other hand, To express the nucleic acid molecules according to the invention in sense or antisense orientation in plant cells, they are linked to regulatory DNA elements which ensure transcription in plant cells. These include, in particular, promoters, enhancers and terminators. In general, any promoter which is active in plant cells is suitable for expression.

The promoter may be chosen in such a way that expression is constitutive or takes place only in a particular tissue, at a particular point in time of plant development or at a point in time determined by external factors. Relative to the plant, the promoter can be homologous or heterologous. Examples of suitable promoters are the cauliflower mosaic virus 35S RNA promoter and the maize ubiquitin promoter for constitutive expression, the patatin promoter B33 (Rocha-Sosa et al., EMBO J. 8 (1989), 23-29) for tuberspecific expression, or a promoter which ensures expression only in photosynthetically active tissue, for example the ST-LS1 promoter (Stockhaus et al., Proc. Natl. Acad. Sci. USA 84 (1987), 7943-7947; Stockhaus et al., EMBO J. 8 (1989), 2445-2451) or, for endosperm-specific expression, the wheat AMG promoter, the USP promoter, the phaseolin promoter or promoters from maize zein genes.

A termination sequence which serves to correctly terminate transcription and to add a poly-A tail to the transcript, which is considered to have a function in stabilizing the transcripts, may also be present. Such elements have been described in the literature (cf. Gielen et al., EMBO J. 8 (1989), 23-29) and are exchangeable as desired.

The present invention provides nucleic acid molecules which encode a protein with a wheat isoamylase function. The nucleic acid molecules according to the invention permit the production of this enzyme whose functional identification in starch biosynthesis, the generation of plants which have been altered by recombinant technology in which the activity of this enzyme is altered and thus allows a starch to be synthesized in plants modified thus whose structure is altered and whose physicochemical properties are altered.

In principle, the nucleic acid molecules according to the invention may also be used for generating plants in which the activity of the isoamylase according to the invention is increased or reduced while simultaneously the activities of other enzymes which participate in starch synthesis are altered.

Altering the activities of an isoamylase in plants results in the synthesis of a starch with altered structure. Furthermore, nucleic acid molecules which encode an isoamylase or suitable antisense constructs can be introduced into plant cells in which the synthesis of endogenous starch synthases or branching enzymes is already inhibited (as, for example, in WO 92/14827 or Shannon and Garwood, 1984, in Whistler, BeMiller and Paschall, Starch: Chemistry and Technology, Academic Press, London, 2nd Edition: 25-86).

If it is intended to achieve the inhibition of the synthesis of several enzymes involved in starch biosynthesis in transformed plants, the transformation may involve DNA molecules which simultaneously comprise several regions encoding the enzymes in question in antisense orientation under the control of a suitable promoter. Here, it is possible for each sequence to be under the control of its own promoter, or for the sequences to be transcribed by a joint promoter as a fusion or to be under the control of a joint promoter. The last-mentioned alternative will generally be preferred, since in this case the synthesis of the proteins in question should be inhibited roughly to the same extent. As regards the length of the individual coding regions used in such a construct, what has been mentioned above for the generation of antisense constructs also applies here. In principle, there is no upper limit for the number of antisense fragments to transcribed in such a DNA molecule starting from one promoter. However, the transcript formed should preferably not exceed a length of 10 kb, in particular a length of 5 kb.

Coding regions localized in such DNA molecules in combination with other coding regions in antisense orientation behind a suitable promoter may be derived from DNA sequences which encode the following proteins: starchgranule-bound starch synthases (GBSS I and II) and soluble starch synthases (SSS I and II), branching enzymes (isoamylases, pullulanases, R enzymes, branching enzymes, debranching enzymes), starch phosphorylases and disproportioning enzymes. This enumeration is only by way of example. The use of other DNA sequences for the purposes of such a combination is also feasible.

Such constructs allow the synthesis of a plurality of enzymes to be inhibited simultaneously in plant cells transformed with them.

Furthermore, the constructs can be introduced into plant mutants which are deficient for one or more starch biosynthesis genes (Shannon and Garwood, 1984, in Whistler, BeMiller and Paschall, Starch: Chemistry and Technology, Academic Press, London, 2nd Edition: 25-86). These defects may relate to the following proteins: starch-granule-bound starch synthases (GBSS I and II) and soluble starch synthases (SSS I and II), branching enzymes (BE I and II), debranching enzymes (R enzymes), disproportioning enzymes and starch phosphorylases. This enumeration is only by way of example.

Such a procedure furthermore allows the synthesis of a plurality of enzymes to be inhibited simultaneously in plant cells transformed with them.

To prepare the introduction of foreign genes into higher plants, a large number of cloning vectors containing a replication signal for E.coll and a marker gene for selecting transformed bacterial cells is available. Examples of such vectors are pBR322, pUC series, M13mp series, pACYC184 and the like. The desired sequence may be introduced into the vector at a suitable restriction cleavage site. The plasmid obtained is used to transform E.coli cells. Transformed E.coli cells are grown in a suitable medium and subsequently harvested and lyzed. The plasmid is recovered. Analytical methods for characterizing the plasmid DNA obtained which are generally used are restriction analyses, gel electrophoresis and further methods of biochemistry and molecular biology. After each manipulation, the plasmid DNA can be cleaved and resulting DNA fragments linked to other DNA sequences. Each plasmid DNA sequence can be cloned in identical or different plasmids.

A large number of techniques is available for introducing DNA into a plant host cell. These techniques encompass transformation of plant cells with t- DNA using Agrobacterium tumefaciens or Agrobacterium rhizogenes as transformation agents, protoplast fusion, injection, the electroporation of DNA, the introduction of DNA by means of the biolistic method, and other possibilities.

The injection and electroporation of DNA into plant cells per se require no particular aspect of the plasmids used. Simple plasmids such as, for example, pUC derivatives may be used. However, if intact plants are to be regenerated from such transformed cells, the presence of a selectable marker gene is generally required.

Depending on the method of introducing desired genes into the plant cell, further DNA sequences may be required. If, for example, the Ti or Ri plasmid is used for transforming the plant cell, at least the right border, but frequently the right and left borders, of the Ti and Ri plasmid T-DNA must be linked to the genes to be introduced as flanking region.

If agrobacteria are used for the transformation, the DNA to be introduced must be cloned into specific plasmids, either into an intermediate vector or into a binary vector. The intermediate vectors can be integrated into the agrobacterial Ti or Ri plasmid by homologous recombination owing to sequences which are homologous to sequences in the T-DNA. The former also contains the vir region, which Is required for the T-DNA transfer.

Intermediate vectors cannot replicate in agrobacteria. The intermediate vector can be transferred to Agrobacterium tumefaciens (conjugation) by means of a helper plasmid. Binary vectors are capable of replication in E.coli and in agrobacteria. They contain a selection marker gene and a linker or polylinker, which are framed by the left and right T-DNA border regions. They can be transformed directly into the agrobacteria (Holsters et al. Mol. Gen. Genet. 163 (1978), 181-187). The agrobacterium which acts as the host cell should contain a plasmid carrying a vir region. The vir region is required for transferring the T-DNA into the plant cell. Additional T-DNA may be present. The agrobacterium thus transformed can be used for transforming plant cells.

The use of T-DNA for transforming plant cells has been researched intensively and been described in EP 120 516; Hoekema, In: The Binary Plant Vector System Offsetdrukkerij Kanters Alblasserdam (1985), Chapter V; Fraley et al., Crit. Rev. Plant. Sci., 4, 1-46 and An et al. EMBO J. 4 (1985), 277-287.

To transfer the DNA into the plant cell, plant explants can expediently be cocultured with Agrobacterium tumefaciens or Agrobacterium rhizogenes.

Intact plants can then be regenerated again from the infected plant material (for example leaf sections, stalk sections, roots, but also protoplasts, or plant cells grown in suspension culture) in a suitable medium which can contain, inter alia, certain sugars, amino acids, antibiotics or biocides for selecting transformed cells. The resulting plants can then be examined for the presence of the DNA which has been introduced. Other possibilities of introducing foreign DNA using the biolistic method or by protoplast transformation are known for example, Willmitzer, L, 1993 Transgenic plants. In: Biotechnology, A Multi-Volume Comprehensive Treatise Rehm, G. Reed, A. POhler, P. Stadler, eds.), Vol. 2, 627-659, VCH Weinheim-New York-Basel-Cambridge).

While the transformation of the dicotyledonous plants via Ti-plasmid vector systems with the aid of Agrobacterium tumefaciens is well established, more recent work suggests that even monocotyledonous plants are indeed accessible to transformation by means of agrobacterium-based vectors (Chan et al., Plant Mol. Biol. 22 (1993), 491-506; Hiei et al., Plant J. 6 (1994), 271-282).

Alternative methods for the transformation of monocotyledonous plants are the transformation by means of the biolistic approach, protoplast transformation, or the physically- or chemically-induced DNA uptake into protoplasts, for example by electroporation of partially permeabilized cells, transfer of DNA by means of glass fibers, macroinjection of DNA into inflorescences, the microinjection of DNA into microspores or proembryos, DNA uptake by germinating pollen and DNA uptake in embryos by swelling (review: Potrykus, Physiol. Plant (1990), 269-273).

Three of the abovementioned transformation systems have been established in the past for various cereals: the electroporation of tissue, the transformation of protoplasts and the DNA transfer by particle bombardment into regenerable tissue and cells (review: JAhne et al., Euphytica 85 (1995), 35-44).

Different methods of transforming wheat have been described in the literature (review: Maheshwari et al., Critical Reviews in Plant Science 14 (1995), 149 to 178): Hess et al. (Plant Sci. 72 (1990), 233) employ the macroinjection method to bring pollen and agrobacteria into the immediate -vicinity. The mobilization of the plasmid which contained the nptll gene as selectable marker was detected by Southern blot analysis and NPTII test.

The transformants showed a normal phenotype and were fertile.

Kanamycin resistance was detected in two consecutive generations.

The first transgenic fertile wheat plant which was regenerated after bombardment with DNA bound to microprojectiles was described by Vasil et al. (Bio/Technology 10 (1992), 667-674). The target tissue for the bombardment was an embryogenic callus culture (type C callus). The selection marker employed was the bar gene which encodes a phosphinothricin acetyl transferase and thus mediates resistance to the herbicide phosphinothricin. A further system was described.by Weeks et al.

(Plant Physiol. 102 (1993), 1077-1084), and Becker et al. (Plant J. 5(2) (1994), 299-307). Here, the target tissue for the DNA transformation is the scutellum of immature embryos which was stimulated in a preliminary in-vitro phase to induce somatic embryos. The transformation efficacy in the system developed by Becker et al. (loc cit.) is 1 transgenic plant per 83 embryos of the variety "Florida" and thus markedly higher than the system established by Weeks et al., which yields 1 to 2 transgenic plants per 1000 embryos of the variety "Bohwhite".

The system developed by Becker et al. (loc Cit) forms the basis for the transformation experiments described in the examples.

Once the DNA introduced is integrated into the genome of the plant cell, it is, as a rule, stable and is also retained in the progeny of the originally transformed cell. It normally contains one of the above-mentioned selection markers which mediates, for example, resistance to a blocide such as phosphinothricin or an antibiotic such as kanamycin, G 418, bleomycin or hygromycin, to the transformed plant cells or which permits selection via the presence or absence of certain sugars or amino acids. The marker chosen individually should therefore allow the selection of transformed cells over cells which lack the DNA introduced.

Within the plant, the transformed cells grow in the customary manner (see also McCormick et al., Plant Cell Reports 5 (1986), 81-84). The resulting plants can be grown normally and hybridized with plants which have the same transformed germ plasm or other germ plasm. The resulting hybrid individuals have the corresponding phenotype properties. Seeds may be obtained from the plant cells. Two or more generations should be grown in order to ensure that the phenotype characteristic is stably retained and inherited. Also, seeds should be harvested in order to ensure that the phenotype in question or other characteristics have been retained.

The examples which follow are intended to illustrate the invention and do not constitute any restriction whatsoever.

1. Cloning methods The vector pBluescript II SK (Stratagene) was used for cloning in E.coli.

2. Bacterial strains The E. coli strain DH5a (Bethesda Research Laboratories, Gaithersburg, USA) was used for the Bluescript vector and for the antisense constructs. The E. coli strain XL1-Blue was used for the in vivo excision.

3. Transformation of immature wheat embryos Media MS: 100 mVlI macrosalt Becker and H. L6rz, 1 ml/I microsalt Plant Tissue Culture 2 ml/I Fe/NaEDTA Manual (1996), B 12:1-20) g/I sucrose MS 2,4-D (2 mg/I) #31: MS 2,4-D (2 mg/I) phosphinothricin (PPT, active component of herbicide BASTA (2 mg/I)) #32: MS 2,4-D (0.1 mg/I) PPT (2 mg/I) #39: MS 2,4-D (2 mg/ml) of each 0.5 N mannitol/sorbitol The media stated were brought to pH 5.6 using KOH and solidified using 0.3% Gelrite.

The method for transforming immature wheat embryos was developed and optimized by Becker and L8rz Becker and H. L6rz, Plant Tissue Culture Manual (1996), B12:1 to In the experiments described hereinbelow, the procedure developed by Becker and L6rz (loc. Cit) was adhered to.

For the transformation, ears with caryopses of developmental stage 12 to 14 days after anthesis were harvested and surface-sterilized. The isolated scutella were plated onto induction medium #30 with the embryo axis orientated toward the medium.

After preculture for 2 to 4 days (26 0 C, in the dark), the explants are transferred to medium #39 for the osmotic preculture (2 to 4 h, 26°C, in the dark).

For the biolistic transformation, approx. 29 Ig of gold particles on which a few pg of the target DNA had previously been precipitated were employed per shot. Since the experiments carried out are cotransformants, the target DNA composed of the target gene and a resistance marker gene (bar gene) in the ratio 1:1 is added to the precipitation batch.

4. DIG labeling of DNA fragments DNA fragments employed as screening probes were labeled via a specific PCR with the incorporation of DIG-labeled dUTP (Boehringer Mannheim, Germany).

Media and solutions used in the examples: x SSC 175.3 g NaCI 88.2 g sodium citrate twice-distilled H20 to 1000 ml N NaOH to pH Plasmid pTaSU 8A was deposited at the DSMZ in Braunschweig, Federal Republic of Germany, as specified in the Budapest Treaty under the No. DSM 12795, and plasmid pTaSU 19 under the No. DSM 12796.

Example 1: Identification, isolation and characterization of a cDNA encoding an isoamylase ("sugary" homolog) from wheat (Triticum aestivum cv Florida) To identify a cDNA which encodes a wheat isoamylase isoform (sugary), a heterologous screening strategy was followed. To this end, a wheat cDNA library was screened with a maize sugary probe.

The probe (sugary probe) was isolated from a maize cDNA library by means of specific primers using PCR amplification. The maize cDNA library was cloned from poly(A) RNA from a mixture of equal amounts of 13-, 17-, 19-, 20-, 22-, 25- and 28- day (DAP) old caryopses in a Lambda Zap II vector following the manufacturer's instructions (Lambda ZAP II-cDNA Synthesis Kit Stratagene GmbH, Heidelberg, Germany). In all the caryopses used, with the exception of the 13-day-old kernels, the embryo had been removed prior to isolating the RNA.

The DNA fragment employed as probe for screening the wheat cDNA library was amplified with the following primers: sulp-1: 5'AAAGGCCCAATATTATCCTTTAGG 3' (Seq ID No. 4) sulp-2: 5' GCCATTTCAACCGTTCTGAAGTCGGGAAGTC 3' (Seq. ID No. The template employed for the PCR reaction was 2 0l of the amplified maize cDNA library. Furthermore, the PCR reaction contained 1.5-3 mM MgCI 2 20 mM Tris-HCI (pH 50 mM KCI, 0.8 mM dNTP mix, 1 pM primer sulp-la, 1 pM primer sulp-2 and 2.5 units Taq polymerase (recombinant, Life Technologies).

The amplification was carried out using a Trioblock from Biometra following the scheme: 4 min/95 0 C; 1 min/95°C, 45 sec/58°C; 1 min 15 sec/72 0 C; cycles 5 min/72°C. The amplified DNA band of approx. 990 bp was separated in an agarose gel and excised. A second amplification was [lacuna] from this fragment following the scheme as described above. The 990 bp fragment obtained from this second amplification was cleaved with the restriction enzyme BAM HI into a 220 bp and a 770 bp fragment. After the sugary fragment had again been separated in an agarose gel, the band excised and the fragment isolated, the probe was DIG-labeled. 500 ng of sugary fragment were employed for the random-prime labeling with digoxygenin. 10 p of random primer were added to the fragment to be labeled and the reaction was heated for 5 min at 95-100°C. After heating, 0.1 mM dATP, 0.1 mM dGTP, 0.1 mM dCTP and 0.065 mM dTTP and 0.035 mM digoxygenin-11-dUTP (Boehringer Mannheim) and Klenow buffer (standard) and 1 unit of Klenow polymerase were added. The reaction was allowed to proceed at RT (room temperature) overnight. To check the labeling, a dot test was carried out following the manufacturer's instructions ("The DIG System User's Guide for Filter Hybridization" by Boehringer, Mannheim, Germany).

The wheat cDNA library was synthesized from poly(A) RNA of approx.

21-day ("starchy" endosperm) old caryopses in a Lamda Zap II vector following the manufacturer's instructions (Lambda ZAP II-cDNA Synthesis Kit, Stratagene GmbH, Heidelberg). After determination of the titer of the cDNA library, a primary titer of 1.26 x 10 pfu/ml was determined.

To screen the wheat cDNA library, approx. 350,000 phages were plated out. The phages were plated out and the plates blotted by standard protocols. The filters were prehybridized and hybridized in 5X SSC, 3% blocking (Boehringer, Mannheim), 0.2% SDS, 0.1% sodium laurylsarcosin and 50 gg/ml herring sperm DNA at 550C. 1 ng/ml of the labeled sugary probe was added to the hybridization solution and the hybridization was incubated overnight. The filters were washed 2 x 5 mins in 2 X SSC, 1% SDS at RT; 2 x 10 min in 1 X SSC, 0.5% SDS at 55 0 C; 2 x 10 min in X SSC, 0.2% SDS at 550C. Positive clones were singled out by further screening cycles. Single clones were obtained via in vivo excision as pBluescript SK phagemids (procedure analogous to the manufacturer's instructions; Stratagene, Heidelberg, Germany).

After the clones had been analyzed via minipreps and restriction of the plasmid DNA, clone pTaSU-19 was deposited at the DSMZ Deutsche Sammlung for Mikroorganismen und Zellkulturen GmbH under the number DSM 12796 and analyzed in greater detail.

Example 2: Sequence analysis of cDNA insertions of plasmids pTaSU19 The plasmid DNA was isolated from clone pTaSU19 and the sequence of the cDNA insertions determined by means of the dideoxynucleotide method (Sanger et al., Proc. Natl. Acad. Sci. USA 74 (1977), 5463-5467).

The insertion of clone TaSU-19 is 2997 bp In length and constitutes a partial cDNA. The nucleotide sequence is shown under Seq ID No. 1.

A comparison with already published sequences revealed that the sequence shown under Seq ID No. 1 encompasses a coding region which has homologies to isoamylases from other organisms.

Sequence analysis also reveals that two introns are located in the cDNA sequence in positions 297-396 (intron 1) and 1618-2144 (intron If these introns are removed, a protein sequence may be derived which exhibits homologies to the protein sequences of isoamylases of other organisms.

The amino acid sequence which corresponds to the coding regions of Seq ID No. 1 is shown under Seq ID No. 3.

Example 3 Generation of the plant transformation vector pTa-alpha- SU19 To express an antisense RNA corresponding to the TaSU19-cDNA, the plant transformation vectors pTa-alpha-SU19 were constructed on the basis of the basic plasmid pUC19 by linking the cDNA insertion of plasmid pTa-alpha-SU19 in antisense orientation to the 3' end of the ubiquitin promoter. This promoter is composed of the first untranslated exon and the first intron of the maize ubiquitin 1 gene (Christensen A.H. et al., Plant Molecular Biology 19 (1992), 675-689). Parts of the polylinker and the NOS terminator are derived from plasmid pAct .cas (CAMBIA, TG 0063; Cambia, GPO Box 3200, Canberra ACT 2601, Australia). Vector constructs with this terminator and constructs based on pActl.cas are described by MCEIroy et al. (Molecular Breeding 1 (1995), 27-37). The vector thus formed was termed pUbi.cas.

The vector was cloned by restricting a 2kb fragment from clone Ta-SU19 with the restriction enzyme Xba I. The fragment was filled up at the ends by means of Klenow reaction and subsequently ligated into the Sma I cloning site of the expression vector pUbi.cas.

The resulting expression vector is termed Ta-alpha-SU 19 and is used as described above for transforming wheat.

Example 4: Isolation and characterization of a further cDNA encoding an isoamylase (sugary 1 homolog) from wheat (Triticum aestivum cv Florida) A wheatcDNA library was screened with a sugary probe which represents a part of .clone pTaSU19, viz. positions 489-1041 of Seq ID No. 1.

The wheat-specific digoxygenin-labeled sugary probe employed for screening the cDNA library was prepared by means of PCR amplification.

The primers employed in this reaction were: SUSO1: 5'-GCT TTA CGG GTA CAG GTT CG-3' (Seq ID No. and SUSO2: 5'-AAT TCC CCG TTT GTG AGC-3' (Seq ID No. 9) 1 ng of plasmid pTaSU19 was employed in the reaction as template. In addition, the PCR reaction contained in each case 300 nM of the primers SUSO1 and SUSO2, in each case 100 1gM of the nucleotides dATP, dGTP, dCTP, 65 1M dTTP, 35 iM digoxygenin-11-dUTP (Boehringer Mannheim), mM MgCI2, and 2.5 U (units) Taq polymerase and 10 pl of concentrated Taq polymerase reaction buffer (both Life Technologies). The final volume of the reaction was 100 1l. The amplification was performed in a PCR apparatus (TRIO® Thermoblock, Biometra) with the following temperature regime: 3 min at 95°C (once); 45 sec at 95°C 45 sec at -2 min at 72°C (30 cycles); 5 min at 72°C (once). A 553 bp DNA fragment resulted. The incorporation of dogoxygenin-11-dUTP into the PCR product was revealed owing to the reduced mobility in the agarose gel in comparison with the product of a controlled reaction without digoxygenin- 11-dutp.

The caryopses-specific wheat cDNA library of Example 1 was screened with the resulting digoxygenin-labeled probe.

The hybridization step was performed overnight in 5x SSC, 0.2% SDS, 0.1% sodium laurylsarcosin and 50 pg/ml herring sperm DNA at 680C in the presence of 1 ng/ml of the digoxygenin-labeled probe. After the hybridization, the filters were washed as follows: 2 x 5 min in 2x SSC, 1% SDS at RT; 2 x 10 min in 1x SSC, 0.5% SDS at 68°C; 2 x 10 min in SSC, 0.2% SDS at 68°C. Positive clones were singled out by at least two further screening cycles. Plasmids were obtained from the phage clones pBluescript SK via in vivo excision (protocols in accordance with the manufacturers instructions; Stratagene, Heidelberg, Germany). After restriction analysis it clones obtained, clone pTaSU8A was deposited at the Deutsche Sammlung for Mikroorganismen und Zellkulturen under the number DSM 12795 and studied in greater detail.

Example 5: Sequence analysis of the cDNA insert in plasmid pTaSU8A The nucleotide sequence of the cDNA insert in plasmid pTaSU8A was determined by means of the dideoxynucleotide method (Seq ID No. 6).

The insertion of clone pTaSU8A is 2437 bp in length and constitutes a partial cDNA. A comparison with already published sequences reveals that the sequence shown under Seq ID No. 6 comprises a coding region which has homologies to isoamylases from other organisms. Equally, the protein sequence derived from the coding region of clone pTaSU8A and shown in Seq ID No. 7 exhibits homologies to the protein sequences of isoamylases of other organisms. Upon comparison of the sequences of clones pTaSU19 (Seq ID No. 1) and pTaSU8A (Seq ID No. a similarity of 96.8% results.

Most of the differences regarding the sequences are in the 3'-untranslated region of the cDNAs. The remaining differences regarding the sequences in the coding region lead to different amine acids at a total of 12 positions of the derived protein sequences (Seq ID No. 3 and The cDNAs contained in pTaSU19 and pTaSU8A are not identical and encode isoforms of the wheat isoamylase.

Example 6: Generation of the plant transformation vector pTa-alpha- SU8A To express an antisense RNA corresponding to the TaSU8A cDNA, the plant transformation vector pTa-alpha-SU8A was constructed on the basis of the basic plasmid pUC19 by linking a part of the TaSU8A cDNA generated by PCR amplification in antisense orientation to the 3' end of the ubiquitin promoter. This promoter is composed of the first untranslated exon and the first intron of the maize ubiquitin I gene (Christensen A.H. et al., Plant Mol. Biol 1 (1992), 675-689). Parts of the polylinker and the NOS terminator are derived from plasmid pActl.cas (CAMBIA, TG 0063; Cambia, GPO Box 3200, Canberra ACT 2601, Australia). Vector constructs with this terminator and constructs based on pActl.cas are described by McElroy et al. (Molecular Breeding 1 (1995), 27-37). The vector containing ubiquitin promoter, polylinker and NOS terminator and based on pUC19 was termed pUbi.cas.

To clone pTa-alpha-SUBA, an approx. 2.2 kb portion of the TaSU8A cDNA, viz. positions 140-2304 of Seq ID No. 6 was amplified by means of PCR.

The primers employed in this reaction were: SUEX3: 5'-GCG GTA CCT CTA GAA GGA GAT ATA CAT ATG GCG GAG GAC AGG TAC GCG CTC-3' (Seq ID No. 10), and SUEX4: 5'-GCT CGA GTC GAC TCA AAC ATC AGG GCG CAA TAC-3' (Seq ID No. 11).

1 ng of plasmid pTaSU8A was employed in the reaction as template. In addition, the PCR reaction contained: in each case 300 nM of the primers SUEX3 and SUEX4, in each case 200 M of the nucleotides dATP, dGTP, dCTP and dTTP, 1.6 mM MgCI 2 60 mM Tris-S0 4 (pH 18 mM (NH 4 2

SP

4 and 1 pl of Elongase® enzyme mix (mixture of Taq polymerase and DNA polymerase, Life Technologies). The final volume of the reaction was 50 pl. Amplification was performed in a PCR apparatus (TRIO@ Thermoblock, Biometra) with the following temperature regime: 1 min at 94°C (once); 30 sec at 950°C 30 sec at 55°C 2 min 30 sec at 68°C (30 cycles); 10 min at 680°C (once). The reaction gave rise to a DNA fragment 2205 bp in length.

The 2.2 kb product was restricted with Kpnl and Salland ligated into the expression vector pUbi.cas which had previously been cleaved with KpnI and Sail. The resulting plant transformation vector was termed pTa-alpha-SU8A and used as described above for transforming wheat.

Comprises/comprising and grammatical variations thereof when used in this specification are to be taken to specify the presence of stated features, integers, steps or components or groups thereof, but do not preclude the presence or addition of one or more other features, integers, steps, components or groups thereof.

Claims (21)

1. A nucleic acid molecule encoding a protein with the function of a wheat isoamylase, selected from the group consisting of a nucleic acid molecule encoding a protein comprising the amino acid sequence shown under Seq ID No. 3, a nucleic acid molecule comprising the nucleotide sequence shown under Seq ID No. 2 or a part thereof or a ribonucleotide sequence corresponding hereto; a nucleic acid molecule which hybridizes with a nucleic acid molecule mentioned under or or is complementary thereto, and a nucleic acid molecule whose nucleotide sequence deviates from the sequence of a nucleic acid molecule mentioned under or owing to the degeneracy of the genetic code, the nucleic acid molecule mentioned under and having a homology of over 90% with Seq ID No. 2.

2. A nucleic acid molecule as claimed in claim 1, which is a DNA molecule.

3. A DNA molecule as claimed in claim 2, which is a cDNA molecule.

4. A nucleic acid molecule as claimed in one or more of claims 1 to 3 containing regulatory elements. A nucleic acid molecule as claimed in claim 1, which is an RNA molecule.

6. A nucleic acid molecule which specifically hybridizes with a nucleic acid molecule as claimed in any of claims 1 to 5 and has a homology of over 90% with Seq ID No. 2.

7. A nucleic acid molecule as claimed in claim 6 which, is an oligonucleotide with a length of at least 15 nucleotides.

8. A vector containing a DNA molecule as claimed in any of claims 1 to

9. A vector as claimed in claim 8, wherein said nucleic acid molecule is linked in sense orientation to regulatory elements which ensure transcription and synthesis of a translatable RNA in pro- or eukaryotic cells. A vector as claimed in claim 8, wherein said nucleic acid molecule is linked in sense orientation to regulatory elements which ensure the synthesis of an untranslatable RNA in pro- or eukaryotic cells.

11. A vector as claimed in claim 8, wherein said nucleic acid molecule is linked in antisense orientation to regulatory elements which ensure the synthesis of an untranslatable RNA in pro- or eukaryotic cells.

12. A host cell which is transformed with a nucleic acid molecule as claimed in one or more of claims 1 to 5 or a vector as claimed in one or more of claims 8 to 11 or which is derived from such a cell.

14. A process for the preparation of a protein as claimed in claim 13, wherein a host cell as claimed in claim 12 is cultured under conditions which permit said protein to be synthesized and said protein is isolated from the cultured cells and/or the culture medium. A process for generating a transgenic plant cell, wherein a) a nucleic acid molecule as claimed in one or more of claims 1 to or b) a vector as claimed in one or more of claims 8 to 11 is integrated into the genome of a plant cell.

16. A transgenic plant cell which has been transformed with a. nucleic acid molecule as claimed in one or more of claims 1 to 4 or one or more vector as claimed in claim 8 to 11 or which is derived from such a cell.

17. A process for generating a transgenic plant cell, wherein 38 al) a nucleic acid molecule as claimed in one or more of claims 1 to or a2) a vector as claimed in one or more of claims'8 to 11 is integrated into the genome of a plant cell and b) an intact plant is regenerated from said plant cell.

18. A plant containing a plant cell as claimed in claim 16.

19. A plant as claimed in claim 18, which is a crop plant. A plant as claimed in claim 19, which is a starch-storing plant.

21. A plant as claimed in claim 20, which is a monocotyledonous plant or maize

22. A plant as claimed in claim 21, which is a barley, rye or wheat plant.

23. A propagation material of a plant as claimed in one or more of claims 18 to 22.

24. The use of a plant cell as claimed in claim 16, a plant as claimed in one or more of claims 18 to 22 or of propagation material as claimed in claim 23 for the production of starch. DATED this 15th day of February 2004 BAYER CROPSCIENCE GMBH WATERMARK PATENT TRADE MARK ATTORNEYS 290 Burwood Road Hawthorn, Victoria 3122 Australia SEQUENCE LISTING <110> Hoechst Schering AgrEvo GmbH <120> Nucleic acid molecules which code for enzymes derived from wheat and which are involved in the synthesis of starch <130> 1998/M 206 <140> PCT/EP99/03141 <141> 1999-05-07 <150> DE 198 20 608.9 <151> 1998-05-08 <160> 11 <170> PatentIn Ver. 2.1 <210> <211> <212> <213> <220> <221> <222> <220> <221> <222> <220> <221> <222> 1 2997 DNA Triticum aestivum L. cv. Florida CDS CDS (397)..(1617) CDS (2145)..(2960) <400> .1 gg tcg ggg ccg gcg ccg cgc Ser Gly Pro Ala Pro Arg ctg cga cgg tgg cga ccc aat gcg acg Leu Arg Arg Trp Arg Pro Asn Ala Thr gcg ggg aag ggg Ala Gly Lys Gly gtc ggc Val Gly gag gtg tgc Glu Val Cys gcg gtt gtc gag Ala Val Val Glu gcg gcg Ala Ala acg aag gta Thr Lys Val agg tac gcg Arg Tyr Ala gac gag ggg Asp Glu Gly gag gag Glu Glu tgc agg Cys Arg 55 gac gag ccg gtg Asp Glu Pro Val gcg gag gac Ala Glu Asp atg ccc gcg Met Pro Ala ctc ggc ggc gcg Leu Gly Gly Ala gtg ctc gcc Val Leu Ala gga Gly ccg ctg Pro Leu ggc gcc acc gcg Gly Ala Thr Ala gcc ggc ggg gtc Ala Gly Gly Val aat Asn ttc gcc gtc tat Phe Ala Val Tyr tcc Ser ggc gga gcc acc Gly Gly Ala Thr gcc Ala 85 gcg gcg ctc tgc ctc ttc acg cca gaa Ala Ala Leu Cys Leu Phe Thr Pro Glu ctc aag gcg gtggggttgc ctcccgagta gagttcatca gctttgcgtg 336 Leu Lys Ala cgccgcgcgc cccttttttg ggcctgcaat ttaagttttg tactggggca aatgctgcag 396 gat agg Asp Arg 100 gtg acc gag gag Val Thr Glu Giu ccc ctt gac ccc Pro Leu Asp Pro atg.aat cgg acc Met Asn Arg Thr ggg Gly 115 aac gtg tgg cat Asn Val Trp His ttc atc gaa ggc Phe Ile Giu Gly ctg cac aac atg Leu His Asn Met ctt Leu 130 tac ggg tac agg Tyr Gly Tyr Arg ttc Phe 135 gac ggc acc ttt Asp Gly Thr Phe gct Ala 140 cct cac tgc ggg Pro His Cys Gly cac tac His Tyr 145 ctt gat gtt Leu Asp Val agc cga ggg Ser Arg Gly 1165 aat gtc gtg gtg Asn Val Val Val gat Asp 155 cct tat gct aag Pro Tyr Ala Lys gca gtg ata Ala Val Ile 160 tgc tgg cct Cys Trp Pro gag tat ggt gtt Giu Tyr Gly Val cca Pro 170 gcg cgt ggt aac Ala Arg Gly Asn aat Asn 175 cag atg Gin Met 180 gct ggc atg atc Ala Gly Met Ile ctt cca tat agc Leu Pro Tyr Ser ttt gat tgg gaa Phe Asp Trp Giu ggc Gly 195 gac cta cct cta Asp Leu Pro Leu aga Arg 200 tat cct caa aag Tyr Pro Gin Lys gac Asp 205 ctg gta ata tat Leu Val Ile Tyr gag Giu 210 atg cac ttg cgt Met His Leu Arg gga Giy 215 ttc acg aag cat Phe Thr Lys His gat Asp 220 tca agc aat gta Ser Ser Asn Val gaa cat Giu His 225 ccg ggt act Pro Giy Thr ctt gga gtt Leu Gly Val 245 ttc Phe 230 att gga gct gtg Ile Gly Ala Val tcg Ser 235 aag ctt gac. tat Lys Leu Asp Tyr ttg aag gag Leu Lys Giu 240 .tt c aac gag Phe Asn-Glu 828 8'76 aat tgt att gaa Asn Cys Ile Giu atg ccc tgc cat Met Pro cys His gag Giu' 255 ctg gag Leu Glu 260 tac tca acc tct Tyr Ser Thr Ser tct Ser 265 tcc aag atg aac Ser Lys Met Asn ttt tgg gga tat tct Phe Trp Gly Tyr Ser 270 aca tca, ggc ggg ata Thr Ser Gly Gly Ile acc Thr 275 ata aac ttc ttt Ile Asn Phe'Phe tca Ser 280 cca atg aca aga Pro Met Thr Arg tac Tyr 285 924 972 1020 aaa aac tgt ggg Lys Asn Cys Gly gat gcc ata aat Asp Ala Ile Asn ttc aaa act ttt Phe Lys Thr Phe gta aga Val Arg 305 gag gct cac Glu Ala His .cat aca gct His Thr Ala 325 aaa Lys 310 cgg gga att gag Arg Gly Ile Giu gtg Vai 315 atc ctg gat gtt Ile Leu Asp Val gtc ttc aac. Val Phe Asn 320 ttt aag ggg Phe Lys' Gly 1068 1116 gag ggt aat gag Glu Gly Asn Giu aat Asn 330 ggt cca ata tta Gly Pro Ile Leu tca Ser 335 gtc gat aat act aca tac tat atg ctt Val Asp Asn Thr Thr Tyr Tyr Met Leu 340 345 gca ccc aag. Ala Pro Lys 350 gga gag ttt tat Gly Giu Phe Tyr aao Asn 355 cgt Arg tat tot ggc tgt Tyr Ser Gly Cys ggg Gly 360 acc tto aa Thr Phe As: tgt Cys 365 tgg r Trp 0 aat oat cot gtg Asn His Pro Val gtt Val 370 oaa tto att Gin Phe Ile gat tgt tta aga Asp Cys Leu Arg ta TY: 38 gtg aog gaa Vai Thr Giu atg oat Met His 385 gtt. gat ggt Val Asp Giy agt otg tgg Ser Leu Trp .405 atg ato aoa Met Ile Thr ttt Phe 390 gat Asp aca Thr ttt gat ott Phe Asp Leu too ata atg aco Ser Ile Met Thr ooa gtt aac Pro Val Asn ggg aoa oct Giy.Thr Pro 425 ooa att ott Pro Ile Leu gga got ooa Gly Ala Pro ata Ile 415 ot t Leu aga ggt too Arg Gly Ser 400 gaa ggt gao Giu Giy Asp att gao atg Ile Asp Met gtt aot oca Vai Thr Pro 420 ato ago Ile Ser oca Pro 430 oto Leu

1164. 1212 1260 1308 1356 1404 1452 1500 1548 1596 1647 aat gao Asn Asp gga ggo gto Giy Gly Vai att got gaa Ile Ala Glu 435 tgg Trp goa Ala 450 gat goa gga Asp Ala Gly ggo Giy 455 tgg Trp tat oaa gta Tyr Gin Val ggt Giy 460 ttc cot oao Phe Pro His tgg aat Trp Asn 465 gtt tgg tot Val Trp Ser att aaa ggc Ile Lys Giy 485 gga agt oca Giy Ser Pro 500 gag Glu 470 act Thr aat ggg aag Asn Gly Lys tao Tyr 475 ggt Gly cgg. gao att gtg Arg Asp le. Val ggt ttt goc gaa Gly Phe Ala Giu ogt caa tto Arg Gin Phe 480 tgt ott tgt Cys Leu Cys gat gga ttt got Asp Gly Phe Ala cac ota tao cag gtaagttgtg goaataottg taaatgagtt His Leu Tyr Gin 505 gagtgaatgt aoaatcatag tatataggtt tttatttgtc agaagaaaot tgottttctg atgtatattt caaatttcta oaootggatt tgtatgoata ttaaoaoooa oggtgaataa atattgattt taaagaaagg gtaagatgaa tttggtttct ttttatatat tgcatttggc aottgooaat tooaotgaaa gooooootaa aaaaogaott tgooaaattt ctagaaatca aooaoatgat taagaagtat gaaggaacat aattooagoo aagaagooat oataotttct aatttgtogg aaccagtaac gatacacato tagtgtatao agggotttot atgtoatttt. otoagaatto atoggtgota ataatttgat ttgttattgg taaatatata actagtgcta agttatotta ttaggggggg ataggtaagt aottagotog otgttattoa cactgoaact 1707 1767 1827 1887 1947 2007 2067 2127 tcttattgat taatcag gca gga gga agg aaa cct tgg cac agt atc aac Ala Gly Gly Arg Lys Pro Trp His Ser Ile Asn 2177 ttt gta tgt Phe Val Cys cat gat gga ttt His Asp Gly Phe aca Thr 525 ctg gct gat ttg Leu Ala Asp Leu gta aca tat Val Thr Tyr 530 2225 aat. aag Asn Lys aat cac Asn His 550 aag tac Lys Tyr 535. aat tta cca Asn Leu Pro aat Asn 540 ggg gag aac aac aga gat gga gaa Gly Glu Asn Asn Arg Asp Gly Giu 545 2273 aat ctt. agc tgg Asn Leu Ser Trp tgt ggg gag gaa Cys Gly Giu Giu gga Giy 560 gaa ttc gca-aga Giu Phe Ala Arg 2321 ttg tct gtc aaa aga ttg agg aag agg cag atg cgc Leu Ser Val Lys Arg Leu Arg Lys Arg Gin Met Arg 565 570 *575. aat ttc ttt Asn Phe Phe gtt. Val 580 2369 2417 tgt ctc atg gtt Cys Leu Met Val caa gga gtt cca Gin Gly Val Pro ttc tac atg ggt Phe Tyr Met Gly gat gaa Asp Giu 595 tat ggc cac Tyr Gly His tat gtc aat Tyr Val Asn 615 aca Thr 600 aaa ggg ggc aac Lys Gly Gly Asn aac Asn 605 aat aca tac tgc Asn Thr Tyr Cys cat gat tct, His Asp Ser 610 tct gag ttg Ser Giu Leu 2465 2513 tat ttt cgc-tgg Tyr Phe Arg Trp aaa aaa gaa caa Lys Lys Giu Gin cac cga His Arg 630 ttc tgc tgc ctc Phe Cys Cys Leu atg Met 635 acc aaa ttc cgc Thr Lys Phe Arg aag Lys 640 gag tgc gag ggt Giu Cys Glu Gly ctt Leu 645 ggc ctt gag gac Gly Leu Glu Asp cca acg gcc aaa Pro Thr Aia Lys cgg Arg 655 ctg cag tgg cat Leu Gin Trp, His 2561 2609 2657 cat cag cct ggg His Gin Pro Gly aag Lys 665 cct gat tgg tct Pro Asp Trp Ser aat agc cga ttc Asn Ser Arg Phe gtt. gcc Val Ala 675 ttt tcc atg Phe Ser Met acc agc cac Thr Ser His 695 aaa Lys 680 gat gaa aga cag Asp Giu Arg Gin ggc Gly 685 gag atc tat gtg Giu Ile Tyr Vai gcc ttc aac Aia Phe Asn 690 gca ggg cgc Ala Gly Arg 2705 2753 tta ccg gcc gtt Leu Pro Ala Val gtt Val1 700 gag. ctc cca gag Glu Leu Pro Giu cgg tgg Arg Trp 710 gaa ccg gtg gtg Giu Pro Val Val aca ggc aag cca Thr Giy.Lys Pro gca. Aia .720 dca tac gac ttc Pro T yr Asp Phe 2801 ctc acc gac gac tta cct gat cgc Leu Thr Asp Asp Leu Pro Asp Arg gct ctc acc ata cac cag ttc Ala Leu Thr Ile His Gin Phe tcg Ser 740 2849 725 25730 735 cat ttc ctc tac tcc aac ctc tac ccc atg ctc agc tac. tca tcg gtc His Phe Leu Tyr Ser Asn Leu Tyr Pro Met Leu Ser.Tyr Ser Ser Val 745 750 755 atc cta gta ttg cgc cct gat gtt tga gag acc aat ata tac, agt aaa Ile Leu Val Leu Arg Pro Asp Val Glu Thr Asn Ile Tyr Ser Lys 760 765 770 taa tat gtc tat atg taaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaa *Tyr Val Tyr Met 775 <210>. 2 <211> 2997 <212> DNA <213> Triticum aestivun L. cv. Florida 2897 2945 2997 <400> 2 ggtcggggcc tcggcgaggt aggacgagcc gaatgcccgc ccggcggagc ggttgcctcc tgcaatttaa cttgaccccc cacaacatgc cttgatgttt tatggtgttc ccatatagca gtaatatatg ccgggtactt tgtattgaat aagatgaact tcaggcggga gaggctcaca ggtaatgaga cttgcaccca catcctgtgg gttgatggtt ggcgccgcgc gtgcgccgcg ggtggcggag gccgctgggC caccgccgcg cgagtagagt gttttgtact tgatgaatcg tttacgggta ccaatgtcgt cagcgcgtgg cgtttgattg agatgcactt tcattggagc taatgccctg tttggggata taaaaaactg aacggggaat atggtccaat agggagagtt ttcgtcaatt ttcgttttga c tgcgacggt gttgtcgagg gacaggtacg gccaccgcgc gcgctctgCC tcatcagctt ggggcaaatg gaccgggaac caggttcgac ggtggatcct taacaattgc ggaaggcgac gcgtggattc tgtgtcgaag ccatgagttc ttctaccata tgggcgtgat tgaggtgatc attatcattt ttataactat cattgtagat tcttgcatcc ggcgacccaa cggcgacgaa cgctcggcgg tcgccggcgg tcttcacgcc tgcgtgcgcc ctgcaggata gtgtggcatg ggcacctttg tatgctaagg tggcctcaga ctacctctaa acgaagcatg Ct tgac tat t aacgagctgg aacttctttt gccataaatg ctggatgttg aagggggtcg tctggctgtg tgtttaagat ataatgacca tgcgacggcg ggtagaggac cgcgtgcagg ggtcaatttc agaagatctc gcgcgcccct gggtgaccga tcttcatcga ctcctcactg cagtgata ag tggctggcat gatatcctca attcaagcaa tgaaggagc t agtac tcaac caccaatgac agttcaaaac tcttcaacca ataatactac ggaatacctt actgggtgac gaggttccag gggaaggggg gagggggagg 120 gtgctcgccg 180 gccgtctatt 240 aaggcggtgg 300 tttttgggcc 360 g gaggttccc 420 aggcgagctg 480 cgggcactac 540 ccgaggggag 600 gatccctctt 660 aaaggacctg 720 tgtagaacat 780 tggagttaat 840 ctcttcttcc 900 aagatacaca 960 ttttgtaaga 1020 tacagctgag 1080 atactatatg 1140 caactgtaat 1200 ggaaatgcat. 1260 tctgtgggat 1320 ccagttaacg gttactccac attgctgaag tgtatggagc tccaatagaa cacttattga catgatcagc cat'gggatgc aggaggcctc gtttggtctg agtggaatgg gaagtaccgg gatggatttg agttgtggca acatgatgat gaagtat tag ggaacatagg tccagccatg, aagccatctc actttctatc ttgtcggata cagtaacttg cttggcacag catataataa atcttagctg ggaagaggca tctacatggg attcttatgt tctgctgcct caacggccaa atagccgatt tcaacaccag aaccggtggt atcgcgctct tcagctactc ctggtggttt atacttgtaa acacatctaa tgtatacact gctttctagt tcatttttta agaattcata ggtgctaact atttgatctg ttattggcac tatcaacttt gaagtacaat gaattgtggg gatgcgcaat tgatgaatat caattatttt catgaccaaa acggctgcag cgttgccttt ccacttaccg ggacacaggc caccatacac atcggtcatc tgccgaatgt atgagttgag atatataaca agtgctatat tatcttattt gggggggaga ggtaagttgc tagctcgatg ttattcacaa tgcaacttct gtatgtgcac ttaccaaatg gaggaaggag ttctttgttt ggccacacaa cgctgggata ttccgcaagg tggcatggtc tccatgaaag gccgttgttg aagccagcac cagttctcgc ctagtattgc ggtgacatga aatgacccaa tatcaagtag gacattgtgc ctttgtggaa tgaatgtcac atcatagtgt ataggtttta atttgtccgg agaaactata ttttctgtaa tatatttgta atttctattt tattgattaa atgatggatt gggagaacaa aattcgcaag gtctcatggt aaggggg~caa aaaaagaaca agtgcgaggg atcagcctgg atgaaagaca agctcccaga catacgactt atttcctcta gccctgatgt tcacaacagg ttcttggagg gtcaattccc gtcaattcat gtccacacct ctggattttt atgcatatgc acacccaact tgaataatcc ttgatttgcc agaaaggaaa agatgaatgc ggtttctcta tcaggcagga tacactggct cagagatgga attgtctgtc ttctcaagga caacaataca atactctgag tcttggcctt gaagcctgat gggcgagatc gcgcgcaggg cctcaccgac ctccaacctc ttgagagacc gacacctctt cgtcaagctc tcactggaat taaaggcact ataccaggta tatatatacc atttggctaa tgccaatgaa actgaaaaat cccctaaaag acgacttcat caaatttaat gaaatcaaac ggaaggaaac gatttggtaa gaaaatcaca -aaaagattga gttccaatgt tactgc catg ttgcaccgat gaggactttc. tggtctgaga tatgtggcct cgccggtggg gacttacctg taccccatgc aatatataca 13 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2220 2280 2340 2400 2460 2520 2580 2640 2700 2760 2820 2880 2940 2997 gtaaataata tgtctatatg taaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaa <210> 3 <211> 764 <212> PRT <213> Triticum aestivum L. cv. Florida <400> 3 Ser Gly Pro Ala Pro Arg Leu Arg Arg Trp Arg Pro Asn Ala Thr Ala 1 5 10 Gly Lys Gly Val Gly Glu Val Cys Ala Ala Val Val Glu Ala Ala Thr 25 Lys Val Glu Asp Glu Gly Glu Glu Asp Glu Pro Val Ala Glu Asp Arg 40 Tyr Ala Leu Gly Gly Ala Cys Arg Val Leu Ala Gly Met Pro Ala Pro 55 Leu Gly Ala Thr Ala Leu Ala Gly Gly Val Asn Phe Ala Val Tyr Ser 70 75. Gly Gly Ala Thr Ala Ala Ala Leu Cys Leu Phe Thr Pro Glu Asp Leu 90 Lys Ala Asp Arg Val Thr Glu Glu Val Pro Leu Asp Pro Leu Met Asn 100 105 110 Arg Thr Gly Asn Val-Trp His Val Phe Ile Glu Gly Glu Leu His Asn 115 120 125 Met Leu Tyr Gly Tyr Arg Phe Asp Gly Thr Phe Ala Pro His Cys Gly 130 135 140 His Tyr Leu Asp Val Ser Asn Val Val Val Asp Pro Tyr Ala Lys Ala 145 150 155 160 Val Ile Ser Arg Gly Glu Tyr Gly Val Pro Ala Arg Gly Asn Asn Cys 165 170 175 Trp Pro Gln Met Ala Gly Met Ile Pro Leu Pro Tyr Ser Thr Phe Asp 180 185 190 Trp Glu Gly Asp Leu Pro Leu Arg Tyr Pro Gln Lys Asp Leu Val Ile 195 200 205 Tyr Glu Met His Leu Arg Gly Phe Thr Lys His Asp Ser Ser Asn Val 210 215. 220 Glu His Pro Gly Thr Phe Ile Gly Ala Val Ser Lys Leu Asp Tyr Leu 225 230 235 240 Lys Glu Leu Gly Val Asn Cys Ile Glu Leu Met Pro Cys His Glu Phe 245 250 255 Asn Glu Leu Glu Tyr Ser Thr Ser Ser Ser Lys Met Asn Phe Trp Gly 260 265 270 Tyr Ser Thr Ile Asn Phe Phe Ser Pro Met Thr Arg Tyr Thr Ser Gly 275 280 285 Gly Ile Lys Asn Cys Gly.Arg Asp Ala Ile Asn Glu Phe Lys Thr Phe 290 295 300 Val Arg Glu Ala His Lys Arg Gly Ile Glu Val Ile Leu Asp Val Val 305 310 315 320 Phe Asn His Thr Ala Glu Gly Asn Glu Asn Gly Pro Ile Leu Ser Phe 325 330 335 Lys Gly Val Asp Asn Thr Thr Tyr Tyr Met Leu Ala Pro Lys Gly Glu Phe Val Met 385 Gly Gly Asp Glu Trp 465 Gin Leu His Leu Arg 545 Giu Asn Met Cys Tyr 625 Glu Gin Arg Val Iz Tyr Val1 370 His Ser Asp Met Ala 450 Asn Phe Cys Ser Val 530 Asp Phe Phe Gly H~is 610 SerC 2ys C rrp F- ?he 1 la P As~ Ar Va S6 Met IiE 435 Trp Val Ile Gly Ile 515 Thr Gly Ala Phe ksp 595 k.sp 1lu ;iu is ~al 1 he 34' n Ty: 5 g Gli L Asi -Let 420 Ser Asp *Trp Lys *Ser 500 Asn Tyr Giu Arg Val1 580 Giu Ser Leu Gly Gly 660 Ala Asn 0 r Ser Gly Cys i Phe Ile Val 375 SGly Phe Arg 390 Trp Asp Pro 405 Thr Thr Giy Asn Asp Pro Ala Gly Gly 455 Ser Giu Trp 470 Gly Thr Asp 485 Pro His Leu Phe Val Cys Asn Lys Lys 535 Asn His Asn 550 Leu Ser.Vai 565 Cys Leu Met Tyr Gly His Tyr Val Asn 615 His Arg Phe 630 Leu Gly Leu 645 His Gin Pro Phe Ser Met Thr Ser HisI 695 Gli, 360 Asp Phe Vai Thr Ile 440 Leu Asn Giy Tyr Ala 520 Tyr Leu Lys Val Thr 600 T'yr -ys 3lu .71y -,ys 680 .'eu 345 Asn Cys Asp Asn Pro 425 Leu Tyr Giy Phe Gin 505 His Asn Ser Arg Ser 585 Lys Phe Cys Asp Lys 665 Asp Pro 350 Thr Leu Leu Val 410 Leu Gly Gin Lys Ala 490 Ala Asp Leu Trp Leu 570 Gin Giy Arg Leu Phe 650 Pro 31u klia Phe Asn Cys Asn His Pro 365. *Arg Ala 395 Tyr Vai Gly Val Tyr 475 Gly Gly Gly Pro Asn 555 Arg Gly Gly Trp Met 635 ,Pro Asp Arg Val Tyr 380 *Ser *Giy Thr *Val Gly 460 Arg Gly Gly Phe Asn. .540 Cys *Lys. Val Asn Asp *620 Thr Thr Trp Gin Val 700 Trp Ile Ala Pro Lys 445 Gin Asp Phe Arg Thr 525 Gly Gly Arg Pro Asn 605 Lys Lys Aia Ser Gly 685 Glu Val Thr Met Thr Pro Ile 415 Pro Leu 430 Leu Ile Phe Pro Ile Vai Ala Giu 495 Lys Pro 510 Leu Ala Giu Asn Giu Giu Gin Met 575 Met Phe 590 Asn Thr Lys Giu Phe Arg Lys Arg 655 Giu Asn 670 Giu Ile Leu Pro Giu Arg 40.0 Giu 'le Ala His Arg 480 Cys Trp Asp Asn Giy 560 Arg Tyr Tyr Gin Lys 640 Leu Ser Tyr Glu *Arg Ala 705 Giy Arg Arg Trp 710 Giu Pro Val Val Thr Giy Lys Pro Aia 720 Pro Tyr Asp Phe Leu Thr Asp Asp Leu Pro Asp Arg Ala Leu Thr Ile 725 730 735 His Gln Phe Ser His Phe Leu Tyr Ser Asn Leu Tyr Pro Met Leu Ser 740 745 750 Tyr Ser Ser Val Ile Leu Val Leu Arg Pro Asp Val 755 760 <210> 4 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 4 aaaggcccaa tattatcctt tagg <210> <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> gccatttcaa ccgttctgaa gtcgggaagt c 31 <210> 6 <211> 2437 <212> DNA <213> Triticum aestivum L. cv. Florida <220> <221> CDS <222> (16)..(2304) <400> 6 gaattcggca cgagg ccg gcg ccg cgc ctg cga cgg tgg cgg ccc aat gcg 51 Pro Ala Pro Arg Leu Arg Arg Trp Arg Pro Asn Ala 1 5 acg gcg ggg aag ggg gtc ggc gag gtc tgc gcc gcg gtt gtc gag gtg 99 Thr Ala Gly Lys Gly Val Gly Glu Val Cys Ala Ala Val Val Glu Val 20 gcg acg aag gcc gag gat gag ggg gag gag gac gag ccg gtg.gcg gag 147 Ala Thr Lys Ala Glu Asp Glu Gly Glu Glu Asp Glu Pro Val Ala Glu 35 gac agg tac gcg ctc ggc ggc gcg tgc agg gtg ctc gcc gga atg ccc 195 Asp Arg Tyr Ala Leu Gly Gly Ala Cys Arg Val Leu Ala Gly Met Pro 50 55 acg ccg ctg ggc gcc acc gcg ctc gcc ggc ggg gtc aat ttc gcc gtc 243 Thr Pro Leu Gly Ala Thr Ala Leu Ala Gly Gly Val Asn Phe Ala Val 70 tac tcc ggc gga gcc aca gcc gcg gcg ctc tgc ctc ttc acg cca gaa 291 Tyr Ser Gly Gly Ala Thr Ala Ala Ala Leu Cys Leu Phe Thr Pro Glu 85 gat ctc aag gcg gat agg gtg acg gag gag gtt ccc ctt gac ccc ctg 339 Asp Leu Lys Ala Asp Arg Val Thr Glu Glu Val Pro Leu Asp Pro Leu 100 105 atg aat cgg act ggg aac gta tgg cat gtc ttc atec gaa ggc gag ctg 387 Met Asn Arg Thr Gly Asn Val Trp His Val Phe Ile Glu Gly Glu Leu 110 115 120 cag gac atg ctt tac ggg .tac agg ttc gac ggc acc ttt gct cct cac 435 Gln Asp Met Leu Tyr Gly Tyr Arg Phe Asp Gly Thr Phe Ala Pro His 125 130 135 140 tgc ggg cac tac ctt gat gtt tcc aat gtc gtg gtg gat cct tat gct 483 Cys Gly His Tyr Leu Asp Val Ser Asn Val Val Val Asp Pro Tyr Ala 145 150 155 aag gca gtg ata agc cga ggg gag tat ggt gtt ccg gcg cgt ggt aac 531 Lys Ala Val aat tgc tgg Asn Cys Trp 175 ttt gat tgg Phe Asp Trp 190 Ser Arg Gly Giu Tyr 165 Gly Val Pro Ala Arg Gly Asn 170 tat agc acg Tyr Ser Thr cct cag atg gct Pro Gin Met Ala atg atc cct ctt Met Ile Pro Leu cca Pro 185 gaa ggc gac Glu Gly Asp cta Leu 195 cct cta aga tat Pro Leu Arg Tyr cc t Pro 200 caa aag gac ctg Gln Lys Asp Leu gta Val 205 ata tat gag atg Ile Tyr Giu Met ttg cgt gga ttc Leu Arg Giy Phe aag cat gat tca Lys His Asp Ser agc Ser 220 aat gta gaa cat Asn Val Giu His ggt act ttc att Gly Thr Phe Ile ggg Gly 230 gct gtg tcg aag Aia -Val Ser Lys ctt gac Leu Asp 23.5 tat ttg aag Tyr Leu Lys gag ttc aac Glu Phe Asn 255 gag Giu 240 ctt gga gtt aat Leu Gly Val Asn att gag tta atg Ile Glu Leu Met ccc tgc cat Pro Cys Hit 250 atg aac ttt Met Asn Phe 771 819 gag ctg gag tac Glu Leu Giu Tyr acc tct tct tcc Thr Ser Ser Ser aag Lys 265 tgg gga Trp Gly 270 tat tct acc ata Tyr Ser Thr Ile aac Asn 275 ttc ttt tca cca Phe Phe Ser Pro atg Met 280 acg aga tac aca Thr Arg Tyr Thr tca Ser 285 ggc ggg ata aaa Gly Gly Ile Lys tgt ggg cgt gat Cys Gly Arg Asp ata* aat gag ttc Ile Asn Glu Phe act ttt gta aga Thr Phe Val Arg gag Glu 305 gct cac aaa cgg Ala His Lys Arg gga Gly 310 att gag gtg atc Ile Giu Val Ile ctg gat Leu Asp 315 gtt gtc ttc Val Val Phe tca ttt agg Ser Phe Arg 335 aac Asn 320 cat aca gct gag His Thr Ala Giu ggt Gly 325 aat gag aat ggt Asn Glu Asn Gly cca at a tta Pro Ile Leu 330 gca ccc aag Ala Pro Lys 101l 1059 ggg gtc gat aat Gly Val Asp Asn act Thr 340 aca tac tat atg Thr Tyr'Tyr Met gga gag Gly Glu 350 ttt tat aac tat Phe Tyr Asn Tyr tct Ser 355 ggc tgt ggg aat Gly Cys Gly Asn acc Thr 360 ttc aac tgt aat Phe Asn Cys Asn cat His 365 cct gtg gtt cgt Pro Val Val Arg caa Gin 370 ttc att gta gat Phe Ile Vai Asp tta aga tac tgg Leu Arg Tyr Trp gtg Val 380 1107 1203 acg gaa atg cat Thr Glu Met His gtt Val 385 gat ggt ttt cgt Asp Gly Phe Arg ttt Phe 390 gat ctt gca tcc Asp Leu Ala Ser ata atg Ile Met 395 acc aga ggt Thr Arg Gly tcc Ser 400 agt ctg tgg gat Ser Leu Trp Asp cca Pro 405 gtt aac gtg tat Val Asn Val Tyr gga gct cca Giy Ala Pro 410 1251 ata gaa ggt gac Ile Glu Giy Asp 415 atg atc aca Met Ile Thr ggg aca cct ctt Gly Thr Pro Leu gtt Val 425 act cca cca 19 Thr Pro Pro ctt att Leu Ile 430 gac atg atc agc Asp Met Ile Ser aa t Asn 435 gac cca att ctt gga ggc gtc aag ctc Asp Pro Ile Leu Gly'Gly Val Lys Leu 440 1347 gtt Vai 445 gct gaa gca tgg Aia Gi~u Aia Trp gca gga ggc ctc Aia GiyGiy Leu tat Tyr 455 caa gta ggt caa Gin Val Giy Gin tt 'c Phe' 460 1395 1443 cct cac Pro His tgg aat gtt Trp Asn Val 465 tgg tct gag tgg Trp Ser Glu Trp ggg aag tac cgg Giy Lys Tyr Arg gac att Asp Ile 475 gtg cgt caa Vai Arg Gin gaa tgt ctt Giu Cys.Leu .495 ttc Phe 480 att aaa ggc act Ile Lys Giy Thr gat Asp 485 gga ttt gct ggt Giy Phe Aia Gly ggt ttt gcc, Gly Phe Ala 490 gga agg aaa Giy Arg Lys 1491 1539 tgt gga agt cca Cys Giy Ser Pro cta tac cag gca Leu Tyr Gin Aia cct tgg Pro Trp 510 cac agt atc aac His Ser Ile Asn ttt Phe 515 gta tgt gca cac Vai Cys Ala His gat Asp 520 gga ttt aca ctg Gly Phe Thr Leu gct Aia 525 aac Asn gat ttg gta aca Asp Leu Val Thr aac aga gat gga Asn Arg Asp Gly 545 tat aat Tyr Asn 530 aac aag tac Asn Lys Tyr tta cca aat ggg Leu Pro Asn Gly gag Giu 540 1587 1635 1683 gaa aat cac aat Glu Asn His Asn ctt Leu 550 agc *tgg aat tgt Ser Trp Asn Cys ggg gag Gly Glu 555 gaa gga gaa Glu Gly Glu atg cgc aat Met Arg Asn 575 ttc Phe 560 gca aga ttg tct Aia Arg Leu Ser gtc Val 565 aaa aga ttg agg Lys Arg Leu Arg aag agg cag Lys Arg Gin 570 gtt cca atg Val Pro Met 1731 1779 ttc ttt gtt tgt Phe Phe Val Cys atg gtt tct caa Met Val Ser Gin gga Gly 585 ttc tac Phe Tyr 590 atg ggt gat gaa Met Gly Asp Giu tat Tyr 595 ggc cac aca aaa Gly His Thr Lys ggg ggc aac aac aat Gly Gly Asn Asn Asn 600 cgc tgg gat aaa aaa Arg Trp Asp Lys Lys aca Thr 605 tac tgc cat gat Tyr Cys His Asp tct Ser 610 tat gtc aat tat Tyr Val Asn Tyr ttt Phe 615 1827

1875- 1923 gaa caa tac tct Glu Gin Tyr Ser ttg cac cga ttc Leu His Arg Phe tgc ctc atg acc Cys Leti Met Thr aaa ttc Lys Phe 635 cgc aag gag Arg Lys Giu gag ggt ctt ggc Giu Giy Leu Gly ctt Leu 645 gag gat ttt cca Glu Asp Phe Pro acg gcc gaa. Thr Ala Glu 650 1971 cgg ctg cag tgg cat ggt cat Arg Leu Gin Trp'His Giy His 655 aa t Asn atc Ile 685 ccg Pro agc Ser 670 tat Tyr gag Giu cga Arg gtg Val cgc Arg cca Pro cac His 735 tac Tyr ttc Phe gcc Ala aca Thr tac Tyr 720 cag Gin tca Ser gtt Val1 ttc Phe ggg Giy 705 gac Asp ttc Phe tcg Ser gcc Ala aac Asn 690 cgc Arg ttc Phe tc t Ser gtc Val 13 cag cct ggg Gin Pro Gly 660 tcc atg aaa Ser Met Lys agc cac tta Ser His Leu tgg gaa ccg Trp Giu Pro 710 act gac gac Thr Asp Asp 725 ttc ctc aac Phe Leu Asn 740 cta gta ttg Leu Val Leu aag Lys gat Asp ccg Pro 695 gtg Val tta Leu tcc Ser cgc Arg cct Pro gaa Glu 680 gcc Ala gtg Vai cct Pro aac Asn cct Pro 760 gat Asp 665 aga Arg gtt *Vai gac Asp gat Asp c tc Leu 745 gat Asp tgg tct gag Trp Ser Glu cag ggc gag Gin Gly Giu gtt gag ctc Val Giu Leu 700 aca ggc aag Thr Giy Lys 715 cgc gct ctc Arg Ala Leu 730 tac ccc Atg Tyr Pro Met gtt tga Val cca gca Pro Ala acc ata Thr Ile ctc agc Leu Ser 750 2019 2067 2115 2163 2211 2259 2304 2364 2424 2437 Ile gaggcggata tacagtaaat aatatgtata tatgtagtcc cacaattgct ctattgccaa tgatctattc gatccacaga aaaaaaactc gag <210> 7 <211> 762 <212> *PRT <213> Triticum aestivum L. cv. Florida <400> 7 Pro Aia Pro Arg Leu Arg Arg Trp Arg Pro Asn 1 5 10 Gly Val Gly Glu Val Cys Ala Ala Val Val Glu 25 Glu Asp Giu Gly Giu Giu Asp Glu Pro Vai Ala 40 Leu Gly Gly Ala Cys Arg Val Leu Ala Gly. Met 55 Ala Thr Ala Leu Ala Gly Gly Val Asn Phe Ala 70 75 Ala Thr Ala Ala Ala Leu Cys Leu Phe Thr Pro 90 Asp Arg Val Thr Giu Giu Val Pro Leu Asp Pro 100 105 tttggcgtat tatcagtgtg tacatgtgca aaaaaaaaaa Ala Val Glu Pro Val Glu Leu Thr Ala Asp Thr Tyr Asp Met Ala, Thr Arg Pro Ser Leu Asn 110 Gly Lys Tyr Leu Gly Lys Arg Lys Ala Ala Gly Gly Ala Thr '14 Gly Asn Val Trp, His .Val Phe Ile Glu Gly Glu Leu Gin Asp Met Leu 115 120 125' Tyr Gly Tyr Arg Phe Asp Gly Thr Phe Ala Pro 'His Cys Gly His Tyr 130 135 140 Leu Asp Val Ser Asn Val Val Val Asp Pro Tyr Ala Lys Ala Val Ile 145 150 155 160 Ser Arg Gly Glu Tyr Gly Val Pro.Ala Arg Gly Asn Asn Cys Trp, Pro* 165 170 175 Gin Met Ala Gly Met Ile Pro Leu Pro Tyr Ser Thr Phe Asp Trp Glu 180 185 190 Gly Asp Leu Pro Leu Arg Tyr Pro Gin Lys Asp Leu Val Ile Tyr Gil 195 200 205 Met His Leu Arg Gly Phe Thr Lys His Asp Ser. Ser Asn Val Glu His 210 215 220 Pro Giy-Thr Phe Ile Gly Ala Vai Ser Lys Leu Asp Tyr Leu Lys Glu 225 230 235 240 Leu Gly Val Asn Cys Ile Giu Leu Met Pro Cys His Glu Phie Asn Glu 245 250 255 Leu Giu Tyr Ser Thr Ser Ser Ser Lys Met Asn Phe Trp Gly.Tyr Ser 260 265 270 Thr Ile Asn Phe Phe Ser Pro'Met Thr Arg Tyr Thr Ser Gly. Gly Ile 275 280. 28 Lys Asn Cys Gly Arg Asp Ala Ile Asn Glu Phe Lys Thr Phe Val Arg 290 295 300 Giu Ala His Lys Arg GlyIle Glu Val Ile Leu Asp Val Val Phe Asn 305 310 315 '320 His Thr Ala Giu Gly Asn Glu Asn Gly Pro Ile Leu Ser Phe Arg Gly 325 .330 335 Val Asp Asn Thr Thr Tyr Tyr Met Leu Ala Pro Lys Gly Glu Phe 'Pyr 340 345 350 Asn Tyr Ser Gly Cys Gly Asn Thr Phe Asn Cys Asn His Pro Val Val 355 360 365 Arg Gin Phe Ile Val Asp Cys Leu Arg Tyr Trp Val Thr Glu Met His 370 375 380 Val Asp Gly Phe Arg Phe Asp Leu Ala Ser Ile Met Thr Arg Gly Ser 385 390 395 400 Ser Leu Trp Asp Pro Val Asn Val Tyr Gly Ala Pro Ile Giu Gly Asp 405 410 415 Met Ile Thr Thr Gly Thr Pro Leu Val Thr Pro Pro Leu Ile Asp Met 420 425 430 Ile Ser Asn Asp Pro Ile Leu Gly Gly Val Lys Leu Val Ala Giu Ala 435 4Afl AAIr Trp Asp Ala Gly Giy Leu Tyr Gin Val 450 455 Val 465 Ile Gly Ile Thr Giy 545 Ala Phe Asp Asp Asp 625 Giu His Val Phe Giy 705 Asp Phe Trp Lys Ser Asn Tyr 530 Giu Arg Val Glu Ser 610 Leu Giy Gly Ala Asn 690 Arg Phe Ser Ser Gly Pro Phe 515 Asn Asn Leu Cys Tyr 59.5 Tyr His Leu His Phe 675 Thr Arg Leu His Glu Thr His 500 Vai Asn His Ser Leu 580 Gly Val Arg Gly Gin 660 Ser Ser Trp Thr Phe 740 Trp Asp 485 Leu Cys Lys Asn Vai 565 Met His Asn Phe Leu 645 Pro Met His Giu Asp 725 Leu Asn 470 Gly Tyr Ala Tyr Leu 550 Lys Vai Thr Tyr Cys 630 Giu Gly Lys Leu Pro 710 Asp Asn Gly Phe Gin His Asn 535 Ser Arg Ser Lys Phe 615 Cys Asp Lys Asp Pro 695 Val Leu Ser Lys Ala Ala Asp 520 Leu Trp Leu Gin Gly 600 Arg Leu Phe Pro Giu 680 Ala Val1 Pro Asn Tyr Giy Gly 505 Gly Pro Asn Arg Gly 585 Gly Tr~p Met Pro Asp 665 Arg Val Asp Asp Leu 745 Gly Arg Gly 49.0 Gly Phe Asn Cys Lys 570 Val Asn Asp Thr Thr 650 Trp Gin Val Thr Arg 730 Tyr Gin Asp 475 Phe Arg Thr Gly Gly 555 Arg Pro Asn Lys Lys 635 Ala Ser Gly Giu Gly 715 Ala Pro Phe 460. Ile Ala Lys Leu Giu 540 Giu Gin Met Asn Lys 620 Phe Giu Glu Glu Leu 700 Lys Leu Met Pro Val Giu Pro Ala 525 Asn Giu Met Phe Thr 605 Giu Arg Arg Asn Ile 685 Pro Pro Thr. Leu His Arg Cys Trp 510* Asp Asn Gly Arg Tyr 590 Tyr Gin Lys Leu Ser 670 Tyr Giu Ala Ile Ser 750 Trp, Gi:n Leu 495 His Leu Arg Glu Asn 575 Met Cys Tyr Giu Gin 655 Arg Val Arg Pro His 735 Tyr Asn Phe 480 Cys Ser .Val Asp Phe 560 Phe Gly His Ser Cys 640 Trp Phe Ala Thr Tyr 720 Gin Ser Ser Val Ile Leu Val Leu Arg Pro Asp Val 16 <210> 8 <211> <212> DNA <213> A tificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 8 gctttacgggtacaggttcg <210> 9 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 9 aattccccgt ttgtgagc 18 <210> <211> 51 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> gcggtacctc tagaaggaga tatacatatg gcggaggaca ggtacgcgct c 51 <210> 11 <211> 33 <212> .DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 11 gctcgagtcg actcaaacat cagggcgcaa tac 33