MXPA00010988A - Nucleic acid molecules which code for enzymes derived from wheat and which are involved in the synthesis of starch - Google Patents

Abstract

The invention relates to nucleic acid molecules which code for enzymes and which are involved in the synthesis of starch in plants. These enzymes concern isoamylases derived from wheat. The invention also relates to vectors and host cells which contain the described nucleic acid molecules, especially transformed plant cells and plants which can be regenerated therefrom, which exhibit an increased or reduced activity of the inventive isoamylases.

Description

NUCLEIC ACID MOLECULES THAT CODIFY FOR WHEAT ENZYMES INVOLVED IN THE STARCH SYNTHESIS DESCRIPTIVE MEMORY The present invention relates to nucleic acid molecules that encode a wheat enzyme involved in the synthesis of starch in plants. This enzyme is an isoamylase. The invention further relates to vectors, host cells, plant cells and plants comprising the nucleic acid molecules according to the invention. In addition, methods for the generation of transgenic plants are described which, thanks 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 vegetable constituents as a renewable raw material, one of the objectives of biotechnological research refers to the adaptation of this vegetable raw material to the needs of the processing industries. In addition, to allow the use of renewable raw material in as many fields as possible, a wide variety of materials must be generated. Apart from oils, fats and proteins, polysaccharides are the most important renewable raw material in plants. Apart from cellulose, starch - which is one of the most important storage substances in higher plants - takes a central position among 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 types of molecules that are different with respect to their degree of polymerization, the occurrence of branching of the glucose chains and their chain lengths, which, in addition, can be derived, for example phosphorylated The starch is therefore not a uniform raw material. In particular, a distinction is made between amylose starch, an essentially unbranched polymer of glucose molecules linked alpha-1, 4-glycosidically and amylopectin starch, which, in turn, constitutes a complex mixture of glucose chains with several ramifications. Branches occur through the occurrence of additional alpha-1, C-glycosidic bonds. In wheat, amylose starch constitutes approximately 11 to 37% of the synthesized starch. To allow the use of suitable starches in the broadest possible manner for the widest possible scale of industrial needs, it is desirable to provide plants that are capable of synthesizing modified starches that are particularly well suited for various purposes. One possibility of providing such plants is to employ measures of plant breeding. However, since wheat is polyploid in character (tetra-and hexaploid), the effect of influence by plant breeding proves to be very difficult. A "waxy" wheat (free of amylose) was generated only recently by crossing naturally occurring mutants (Nakamura et al., Mol.Gen. Genet, 248 (1995), 253-259). An alternative for plant breeding methods is the specific modification of starch-producing plants by recombinant methods. However, the prerequisites are the identification and characterization of the enzymes that are involved in the synthesis of starch and / or starch modification, and the isolation of the nucleic acid molecules that code for these enzymes. The biochemical pathways leading to the synthesis of starch are essentially known. The synthesis of starch in plant cells takes place in the plastids. In photosynthetically active tissue, these plastids are chloroplasts, and in starch storage tissue and photosynthetically inactive are amyloplasts. An additional specific alteration of the degree of branching of the starch synthesized in plants with the aid of recombinant methods still requires the identification of DNA sequences, which code for enzymes involved in the metabolism of starch, in particular in the introduction or degradation of the branching inside the starch molecules.

Apart from the so-called Q enzymes, which introduce branching into starch molecules, enzymes occur in plants that are capable of breaking the branches. These enzymes are called debranching enzymes and, according to their specificity in the substrate, they are divided into three groups: a) pullulanases, which, in addition to pullulan, also use amylopectin as a substrate, are found in microorganisms, for example Klebsiella and in plants. In plants, these enzymes are also called enzymes R. b) soamilases, which do not use pullulan, but in fact use glycogen and amylopectin as a substrate, are also found in microorganisms and plants. For example, soamilases have been described in corn (Manners &Carbohydr, Res. 9 (1969), 107) and potato (Ishizaki et al., Agrie. Biol. Chem. 47 (1983), 771-779). c) amyl-1, 6-glucosidases are described in mammals and levators, and use grenzdextrins as a substrate. In beet, Li et al. (Plant Physiol. 98 (1992), 1277-1284) could only find a pullulanase-like debranching enzyme, in addition to five endoamylases and two exoamylases. This enzyme, which has a size of approximately 100 kD and an optimum pH of 5.5, is located in the chloroplasts. In spinach, too, a debranching enzyme was described that uses pullulan as the substrate. The activity of both the spinach debranching enzyme and the beet debranching enzyme after the reaction with amylopectin as a substrate is five times lower compared to pullulan as a substrate (Ludwig et al., Plant Physiol. 74 (1984) , 856-861; Li et al., Plant Physiol. 98 (1992), 1277-1284). In the agriculturally important starch storage culture plant called potato, the activity of a debranching enzyme was studied by Hobson et al. (J. Chem. Soc, (1951), 1451). It was successfully proved that, in contrast to enzyme Q, this enzyme does not have chain extension activity, but simply hydrolyzes alpha-1, 6-glucosidic bonds. However, it has been impossible to characterize the enzyme in greater detail so far. 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 interim (Renz et al., Plant Physiol, 108 (1995), 1342). In corn, only the existence of a debranching enzyme in the literature has been described so far. Thanks to its substrate specificity, this enzyme is classified as belonging to the group of isoamylases (see, for example, Hannah et al., Scientia Horticulturae 55 (1993), 177-197 or Garwood (1994) in Starch Chemistry and Technology, Whistler RL, BeMiller, JN, Puschall, EF (eds), San Diego Academic Press, New York, Boston, 25-86). The corresponding mutant is called "sugary". The sugar locus gene has recently been cloned (see James et al., Plant Cell 7 (1995), 417-429). Apart from the sugar locus, no other gene locus encoding a protein with debranching enzyme activity in corn is known to date. Likewise, there have been no indications to date of the occurrence of other forms of debranching enzyme in corn. If transgenic maize plants are to be generated that no longer have any debranching enzyme activity, for example to extend the degree of branching of amylopectin starch, it is necessary to identify all forms of debranching enzymes that occur in corn and isolate the corresponding genes or cDNA sequences. To provide additional possibilities to alter any starch storage plant, preferably cereals, in particular wheat, in such a way as to synthesize a modified starch, it is necessary to identify in each case DNA sequences that code for additional isoforms of debranching enzymes. The aim of the present invention is therefore to provide nucleic acid molecules that code for enzymes involved in the synthesis of starch, which allows the generation of genetically modified plants that make possible the production of plant starches whose chemical and / or physical characteristics are altered . This objective is achieved by providing the forms of use designated in the patent claims.

Therefore, the present invention relates to a nucleic acid molecule that encodes a protein with the function of a wheat isoamylase, preferably a protein that is essentially defined by the indicated amino acid sequence Seq ID No. 3 or 7 In particular, the invention relates to a nucleic acid molecule comprising the nucleotide sequence indicated Seq ID No. 1, 2 or 6, or a portion thereof, preferably a molecule comprising the coding region indicated in Seq ID No. 1, 2 or 6, and corresponding ribonucleotide sequences. A nucleic acid molecule which further comprises regulatory elements that ensure transcription and, if appropriate, translation of said nucleic acid molecules is very particularly preferred. The present invention is also a nucleic acid molecule that hybridizes with one of the nucleic acid molecules according to the invention. The present invention is also a nucleic acid molecule encoding wheat isoamylase whose sequence deviates from the nucleotide sequences of the molecules described above due to the degeneracy of the genetic code. The invention also relates to a nucleic acid molecule with a sequence that is complementary to all or part of one of the sequences mentioned above. The term "hybridization" is used in the context of the present invention to denote hybridization under conventional hybridization conditions, preferably under stringent conditions as described, for example, by Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Ed. (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY). The "hybridization" takes place especially preferably under the following conditions: Hybridization pH regulator: 2 x SSC; 10 x solution Denhardt (Fikoll 400+ PEG + BSA, ratio 1: 1: 1); 0.1% SDS; 5 mM of EDTA; 50 nM Na2HPO4; 250 μg / ml Herring sperm DNA; 50 μg / ml tRNA or 0.25 M phosphate pH regulator, pH 7.2; 1 mM EDTA; 7 out of SDS Hybridization temperature: T = 65 to 70 ° C Wash pH regulator: 0.2 x SSC; 0.1% SDS Washing temperature: T = 40 to 75 ° C. The nucleic acid molecules that hybridize with the nucleic acid molecules according to the invention are capable, in principle, of coding isoamylases of any wheat plant expressing said proteins. Nucleic acid molecules that hybridize with the molecules according to the invention can be isolated, for example, from genomic libraries or libraries of wheat cDNA or wheat plant tissue. Alternatively, they can be generated by recombinant methods or chemically synthesized.

The identification and isolation of said nucleic acid molecules can be carried out using the molecules according to the invention or parts of these molecules or the inverse 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 that can be used are, for example, nucleic acid molecules having exactly or essentially the nucleotide sequence indicated Seq ID No. 1, 2 or 6 or parts of these sequences. The fragments used as a hybridization probe can also be synthetic fragments that have been prepared with the aid of normal synthetic techniques whose sequence essentially matches that of a nucleic acid molecule according to the invention. The molecules that hybridize with the nucleic acid molecules according to the invention also include fragments, derivatives and allelic variants of the nucleic acid molecules described above which code for a wheat isoamylase according to the invention. The term "fragments" should be understood as meaning parts of the nucleic acid molecules of sufficient length to encode one of the described proteins. The term derivative means in this context that the sequences of these molecules are different from the sequences of the nucleic acid molecules described above in 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 at least 60%, preferably more than 80%, particularly preferably more than 90%. The deviations relative to the nucleic acid molecules described above may have been generated by deletion, substitution, insertion or recombination. Homology further means that there is functional and / or structural equivalence between the nucleic acid molecules in question or the proteins encoded by them. Nucleic acid molecules that are homologous to the molecules described above and constitute derivatives of these molecules are, as a rule, variations of these molecules that constitute modifications that exert the same biological function. They may be variations that occur naturally, for example, sequences from other organisms, or mutations that have occurred naturally or have been introduced by site-directed mutagenesis. In addition, the variations can be synthetically generated sequences. Allelic variants can be either variants that occur naturally or synthetically generated variants, or variants produced by recombinant DNA techniques. The soamilases encoded by the different 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 any physical properties such as, for example, the migratory behavior in gel electrophoresis, the chromatographic behavior, sedimentation coefficients, solubility, characteristics spectroscopic, load characteristics, stability; Optimum pH, optimum temperature and the like. The protein encoded by the nucleic acid molecules according to the invention is a wheat soamilase. These proteins show certain homology indices with isoamylases of other plant species that are already known. The nucleic acid molecules according to the invention can be DNA molecules, in particular cDNA molecules or genomic molecules. In addition, the nucleic acid molecules according to the invention can be RNA molecules that can be the result of, for example, 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 may have been generated by recombinant or synthesized techniques. The present invention is also oligonucleotides that hybridize specifically with a nucleic acid molecule according to the invention. Said oligonucleotides preferably have a length of at least 10, in particular at least 15 and especially preferably at least 50 nucleotides. The oligonucleotides according to the invention hybridize specifically with the nucleic acid molecules according to the invention, that is, not or only to a very low degree with nucleic acid sequences that code for 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 a hybridization probe for the isolation of the related genes. Likewise, they can be constituents of antisense constructs or of DNA molecules that code for suitable ribozymes. The invention further relates to vectors, in particular plasmids, cosmids, phagemids, viruses, bacteriophages and other vectors conventionally used in genetic engineering comprising the nucleic acid molecules described above according to the invention. Said vectors are suitable for the transformation of pro- or eukaryotic cells, preferably plant cells. The vectors especially preferably allow the 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 that can be used in agrobacterial-mediated gene transfer. Preferably, the integration of a nucleic acid molecule according to the invention in sense or antisense orientation ensures that a translatable or, if appropriate, non-translatable RNA is synthesized in the transformed pro-or eukaryotic cells. The term "vector" generally indicates a suitable auxiliary known to the person skilled in the art that allows the targeted transfer of a single or double stranded nucleic acid molecule into a host cell, for example a DNA or RNA virus, a fragment of virus, a plasmid 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 any metal particles that may be employed, for example, in the particle gun method, but may also encompass a nucleic acid molecule that can be introduced directly into a cell by chemical or physical methods. In a preferred embodiment, the nucleic acid molecules within the vectors are linked to regulatory elements that ensure the transcription and synthesis of a translatable RNA in pro-or eukaryotic cells or which, if desired, ensure the synthesis of a non-specific RNA. translatable The 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 the synthesis of starch in the plant cell. This allows conclusions to be drawn regardless of the function that the protein in question exerts during the synthesis of starch 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 common molecular biology techniques (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 can be altered. In the present it is possible, on the one hand, the generation of deletion mutants in which the nucleic acid molecules are generated by successive deletions from the 5 'or 3' end of the coding DNA sequence leading to the synthesis of correspondingly truncated proteins. Said deletions at the 5 'end of the nucleotide sequences allow, for example, to identify amino acid sequences that are responsible for the translocation of the enzyme in the plastids (transit peptides). This allows the directed generation of enzymes that, thanks to the removal of the sequences in question, are no longer located in the plastids, but in the cytosol, or which, thanks to the addition of other signal sequences, are located in other compartments. On the other hand, it is also possible to introduce point mutations in positions in which the altered amino acid sequence affects, for example, the activity of the enzyme or the regulation of the enzyme. In this way, it is possible to generate, for example, mutants that have an altered Km value or which are no longer subject to the regulatory mechanisms by means of allosteric regulation or covalent modification that are normally present in the cell. In addition, it is possible to generate mutants of the protein according to the invention having a substrate or altered product specificity.

It is also possible to generate mutants of the protein according to the invention having an altered activity-temperature profile. 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 that allow the mutagenesis to take place or that a sequence be altered by recombinant DNA sequences. Base exchanges can be carried out or natural or synthetic sequences can be added with the help of standard methods (see, Sambrook et al., 1989, Molecular Cloning, A laboratory manual, 2nd Ed., Cold Spring Harbor Laboratory Press, NY, USA). ). To link the DNA fragments with one another, adapters or linkers can be attached to the fragments. In addition, manipulations can be employed that provide suitable restriction cleavage sites or which eliminate superfluous DNA or restriction cleavage sites. When insertions, deletions or substitutions are suitable, in vitro mutagenesis, primer repair, restriction or ligation can be employed. The analytical methods that are generally used 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 a nucleic acid molecule described above according to the invention, or with a vector according to the invention, and to cells that are derived from cells transformed in this way and that comprise a nucleic acid molecule according to the invention or a vector. They are preferably pro-or eukaryotic cells, in particular plant cells. The present invention also relates to 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 to processes for their preparation, wherein a host cell according to the invention invention is cultivated under suitable conditions that are known to the person skilled in the art and which allow the synthesis of the protein according to the invention and which is subsequently isolated from the host cells and / or the culture medium. By providing the nucleic acid molecules according to the invention, and with the aid of recombinant methods, it is now possible to intervene in the metabolism of starch in plants in a targeted manner, and to alter it in such a way that the resulting synthesis is of modified 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 gel or film formation properties, the granule size of starch and / or the shape of the starch granule are altered in comparison with those of the known starch. In this way, it is possible to express the nucleic acid molecules according to the invention in plant cells to increase the activity of the isoamylase in question, or to introduce them into cells that do not naturally express this enzyme. The expression of the nucleic acid molecules according to the invention also makes it possible to decrease the level of natural activity of the isoamylase according to the invention in plant cells. Furthermore, it is possible to modify the nucleic acid molecules according to the invention by methods known to the person skilled in the art to obtain isoamylases according to the invention which are no longer subject to the intrinsic regulatory mechanism of the cell or which have profiles of temperature-activity or altered substrate or product specificities. When the nucleic acid molecules according to the invention are expressed in plants, it is possible, in principle, for the synthesized protein to be located in any desired compartment of the plant cell. To achieve localization in a particular compartment, the sequence that ensures plastid localization must be eliminated, and the remaining coding region must, if necessary, be linked to DNA sequences that 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. Natil.k, Acad. Sci. USA 85 (1988), 846-850; Sonnewald et al., Plant J. 1 (1991), 95-106). The present invention also relates to a method for generating transgenic plant cells that have been transformed with a nucleic acid molecule or a vector according to the invention, wherein a nucleic acid molecule according to the invention or a vector according to the invention. with the invention it is integrated into the genome of a plant cell, to the transgenic plant cells that have been transformed by means of a vector or nucleic acid molecule according to the invention, and to transgenic plant cells derived from cells transformed in this way. The cells according to the invention comprise one or more nucleic acid molecules or vectors according to the invention, these being preferably linked to regulatory DNA elements that ensure transcription in plant cells, in particular to a suitable promoter. Said 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 that does not occur naturally in these cells, or by the fact that said molecule exists integrated in a place in the genome of the cell where it does not occur otherwise, that is, in a different genomic environment. In addition, said 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 said molecule that occur naturally in the cells. If the nucleic acid molecules introduced into the cells are additional copies of molecules that 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, are, or are, located in places in the genome where they do not occur naturally, or do not occur naturally. This can be verified, for example, with the help of a Southern blot analysis. If the nucleic acid molecule according to the invention that has been introduced into the genome of the plant is heterologous to the plant cell, the transgenic plant cells exhibit transcripts of the nucleic acid molecules according to the invention that can be detected in a simple by methods known to the person skilled in the art, for example, by Northern blot analysis. If the nucleic acid molecule according to the invention that has been introduced is homologous to the plant cell, the cells according to the invention can be distinguished from naturally occurring cells, for example, based on the further expression of acid molecules. nucleic acid according to the invention. The transgenic plant cells preferably comprise more transcripts than the nucleic acid molecules according to the invention. This can be detected, for example, by Northern 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 the corresponding non-transformed cells. The cells preferably also exhibit a corresponding increase or decrease in the activity of the protein according to the invention (at least 10%, 20% or 50%). The transgenic plant cells can be regenerated in intact plants by techniques known to the person skilled in the art. Another important aspect of the present invention is a method for generating transgenic plants, wherein 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 of said plant is regenerated. plant cell. Another embodiment of the invention are plants comprising the transgenic plant cells described above. At first, the transgenic plants can be plants of any species, that is, not only monocotyledonous but also dicotyledonous plants. Useful plants are preferably plants, preferably starch-synthesizing plants or starch storages, in especially preferably rye, oats, wheat, sorghum and millet, sago, corn, rice, peas, squash, potatoes, tomatoes, rapeseed oil, soybeans , hemp, flax, sunflowers, chickpeas or arrowroot, in particular, wheat, corn, rice and potatoes. The invention also relates to propagation material of plants according to the invention, for example, fruits, seeds, tubers, roots, seedlings, barbs, calluses, protoplasts, cell cultures, and the like. The present invention further relates to a process for the preparation of a modified starch comprising the step of extracting the starch from a plant described above according to the invention and / or starch storage portions of said plant.

Methods for extracting starch from plants or storage parts of starch from plants, in particular from wheat, are known to the person skilled in the art, see, 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: production of corn starch and sorghum, by Watson, chapter XIII, pages 469-479, production of tapioca starches, arrowroot and sago, by Corbishley and Miller, chapter XIV, pages 479-490: production and uses of potato starch, by Mitch, chapter XV, pages 491 to 506: production, modification and uses of wheat starch, by Knight and Oson, and chapter XVI, pages 507 to 528: production and uses of rice starch, by Rohmer and Klem). The devices normally used in the processes for extracting starch from plant materials are separators, decanters, hydrocyclones, spray dryers and fluidized milk dryers. Thanks to the expression of a nucleic acid molecule according to the invention, the plant cells and transgenic plants according to the invention synthesize a starch whose physicochemical properties, for example the amylose / amylopectin ratio, the degree of branching, the length of The average chain, the phosphate content, the gelatinization behavior, the size of the starch granule and / or the shape of the starch granule are altered, compared to the starch synthesized in wild type plants. In particular, said starch can be altered with respect to the viscosity and / or film-forming properties of gels made from this starch in comparison with known starches. The present invention further relates to a starch which can be obtained from the plant and plant cells according to the invention and its propagation material, and to starch which can be obtained by the process according to the invention described above. It is also possible to generate, with the aid of the nucleic acid molecules according to the present 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, comparatively with the starch of wild-type plant cells. Another aspect of the invention is also in this way a transgenic plant cell comprising a nucleic acid molecule according to the invention, in which the activity of an isoapylase is reduced comparatively with an isoamylase of non-transformed cells. Plant cells with reduced activity of an isoamylase can be obtained, for example, by expressing a suitable antisense RNA, a sense RNA to achieve a cosuppression effect, or by expressing a properly constructed ribozyme which specifically cuts transcripts coding for an isoamylase, making use of of the nucleic acid molecules according to the invention by methods known to the person skilled in the art; see, for example, Jorgensen (Trends Biotechnol., 8 (1990), 340-344), Niebel et al. (Curr. Top, Microbiol, Immunol., 197 (1995), 91-103), Flavell et al. (Top Curr., 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), Borne et al. (Mol., Gen. Genet, 243 (1994), 613-621). To reduce the activity of the soamilase according to the invention, it is preferred to reduce, in plant cells, the number of transcripts that encode it, for example, by expressing an antisense RNA. Here, it is possible to make use, on the one hand, of a DNA molecule that encompasses the entire sequence coding for a protein according to the invention, including any flanking sequence that may be present., or also of DNA molecules that only span parts of the coding sequence, it being necessary that these parts be sufficiently long to cause an antisense effect in the cells. In general, sequences with a minimum length of up to 15 bp, preferably with a length of 100 to 500 bp, can be used for efficient antisense inhibition, in particular sequences with a length of more than 500 bp. As a rule, DNA molecules are used that are less than 5,000 bp, preferably sequences that are less than 2,500 bp. Also possible is the use of DNA sequences that show a high degree of homology, but are not completely identical, with the sequences of the DNA molecules according to the invention. The minimum homology should exceed approximately 65%. The use of sequences with homologies between 95 and 100% is preferred. Another aspect of the invention is a process for producing a modified starch comprising the step of extracting the starch from a cell or plant according to the invention, and / or parts of said plant that store starch. Yet another aspect of the invention is starch which can be obtained from the cells or plants according to the invention and propagation material, or parts thereof, 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 those skilled in the art and are suitable, in their modified or unmodified form, for a variety of uses in the food or non-food sector. In principle, the possible uses of the starches according to the invention can be divided into two important sectors. One sector includes starch hydrolysates, mainly glucose and glucan units, which can be obtained by enzymatic or chemical methods. They are used as starting material for other chemical modifications and processes such as fermentation. What can be significant to reduce costs is the simplicity and economic design of a hydrolytic method. It is currently carried out enzymatically using amyloglucosidase. What would be feasible is financial savings through less use of enzymes. This could be done by altering the structure of the starch, for example, by increasing the surface area of the granule, or by facilitating the possibility of digestion, for example, by allowing a lower degree of branching or a steric structure that limits accessibility for the enzymes. used. The other sector in which the starches according to the invention can be used as the so-called native starch, due to its polymer structure, can be divided into two other fields of application: 1. The food industry Starch is a traditional additive for a large number of food products in which its function is essentially to bind aqueous additives or increase viscosity or gelation. Important characteristics are rheology, absorption characteristics, swelling temperature, gelatinization temperature, viscosity, thickener capacity, starch solubility, transparency and gel structure, thermal stability, shear stability, acid stability, the tendency to undergo retrogradation, film forming capacity, freeze-thaw stability, visco-stability in saline solutions, the ability to digest and the ability to form complexes with, for example, organic or inorganic 2. The non-food industry In this important sector, starch can be used as an auxiliary for various preparation procedures or as an additive in industrial products. When starch is used as an auxiliary, mention should be made, in particular, of the paper and cardboard industry. The starch acts mainly for delaying purposes (retention of solids), for joining particles and filler fines, as stiffener and for dehydration. In addition, the advantageous properties of the starch are used with respect to stiffness, hardness, sound, touch, luster, uniformity, bond strength and surfaces. 2. 1 Paper and cardboard industry Within the papermaking process, four fields of application must be distinguished, namely, surface, coating, supply material and aspersion. The demands of the starch with respect to the surface treatment are essentially high whiteness, an adapted viscosity, high visco-stability, good film formation and low dusting. When used for coating, the solids content, a suitable viscosity, a high binding capacity and a high affinity for pigments, play an important function. Of importance when used as an additive for supply material, they are fast, uniform and loss-free distribution, high mechanical strength and complete retention in the paper reel. If the starch is used in the spraying sector, again, an adapted content of solids, high viscosity and high binding capacity are of importance. 2. 2 The adhesive industry An important field of application for starches is the adhesive industry, where the potential uses are divided into 4 subsections: the use as a paste of pure starch, the use in starch pastes which have been treated with specialty chemicals, the use of starch as an additive for synthetic resins and polymer dispersions, and the use of starches as thinners for synthetic adhesives. 90% of the starch-based adhesives are used in the sectors of corrugated cardboard production, production of bags and paper bags, production of mixed materials for paper and aluminum, production of cardboard for boxes and adhesives of gumming for wrappings, prints , and similar. 2. 3 Textile industry and textile care products industry An important field of application for starches as auxiliaries and additives is the textile production and textile care products sector. The four following fields of application within the textile industry should be distinguished: the use of starch as a sizing agent, that is, as an aid to standardize and reinforce the roughing behavior as protection against the tension forces applied during the weaving, and for increase the abrasion resistance during weaving, starch as a textile finishing agent, in particular after previous treatments that reduce the quality, such as bleaching, dyeing and the like, starch as a thickener in the preparation of dye pastes to prevent bleeding , and starch as an additive for luster agents for sewing threads. 2. 4 Construction materials industry The fourth field of application is the use of starches as additives in building materials. An example is the production of gypsum boards, where the starch which is mixed with the gypsum suspension, gelatinizes with water, diffuses to the surface of the gypsum core and joins the cardboard to the core. Other fields of application are as a mixture to make mineral fibers. In the case of ready-mix concrete, starch products are used to delay agglutination. 2. 5 Soil stabilization Another area of use of starch is the production of soil stabilizers, which are used for the temporary protection of soil particles from water when the soil is artificially disturbed.

In accordance with the present knowledge, combinations of starch product and polymer emulsions will be on a par with previously used products with respect to their erosion and bark reducing effect, but they are markedly less expensive. 2. 6 Use in products for the protection of crops and fertilizers A field of application of starch is in products for the protection of crops to alter the specific properties of the products. In this way, the starch can be used to improve the wetting of fertilizers and crop protection products, for the controlled release of the active ingredients, to convert liquid, volatile and / or malodorous active ingredients into stable, configurable microcrystalline substances , to mix incompatible products, and to prolong the duration of action by reducing decomposition. 2J Pharmaceutical, medicinal and cosmetic industry Another field of application is the pharmaceutical, medical and cosmetic industry. In the pharmaceutical industry, the starch can be used as a binder for tablets, or to dilute the binder in capsules. In addition, starch can be used as a tablet disintegrant, since it absorbs fluid after swallowing, and swells after a short time to such an extent that the active ingredient is released. The medicinal lubricating powders and the powder for wounds are based on starch for qualitative reasons. In the cosmetics sector, starches are used, for example, as vehicles for powder additives such as fragrances and salicylic acid. A relatively large field of application for starch are toothpastes. 2. 8 Addition of coal starch and briquettes A field of application of starch is as an additive for charcoal and briquettes. With the addition of starch, the coal can be agglomerated or briquetted, in terms of high amounts, thus preventing premature decomposition of the briquettes. In the case of charcoal for grilling, the addition of starch is between 4 and 6%, and in the case of charcoal, it is between 0.1 and 0.5%. In addition, starches are gaining importance as binders since the emission of harmful substances can be markedly reduced when charcoal starches and briquettes are added. 2. 9 Processing of mineral and coal suspensions In addition, starch can be used as a flocculant in the coal ash and mineral oil layer processing sector. 2. 10 Cast iron auxiliary Another field of application of starch is as an additive for foundry auxiliaries. Several molding processes require cores made of sand treated with binders. Today, the binder that is used predominantly is bentonite, which is treated with modified starches, in most cases inflatable starches. The purpose of adding starch is to increase the fluidity and improve the agglutination capacity. In addition, the inflatable starches can satisfy other demands of engineering production, such as being dispersible in cold water, rehydratable, easily miscible with sand and have high agglutination capacity in water. 2. 11 Use in the rubber industry In the rubber industry, starch can be used to improve technical and visual quality. The reasons are the improvement of the surface luster and the improvement of the handling and appearance, and for this purpose, the starch is dispersed on the rubbery sticky surface of rubber materials before the cold cure, as well as the improvement of the rubber printing capacity. 2. 12 Production of leather substitutes Modified starches can also be marketed 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 incorporation of starch degradation products in the processing process (the starch acts only as filler, and there is no direct link between the synthetic polymer and starch) or, alternatively, the incorporation of starch degradation products in the production of polymers (the starch and the polymer form a stable bond). The use of starch as pure filler is not competitive, comparatively with other substances such as talc. However, this is different when the specific properties of the starch have an impact and in this way significantly alter the spectrum of characteristics of the final products. An example of this is the use of starch products in the processing of thermoplastics such as polyethylene. In this case, the starch and the synthetic polymer are combined by coexpression at a ratio of 1: 1 to give a master batch, from which various granulated polyethylene products are obtained, using conventional processing techniques. By incorporating starch into polyethylene films, a permeability can be achieved, by increasing the amount of substances in the case of hollow bodies, improved permeability for water vapor, improved antistatic performance, improved antiblock behavior and ease of improved printing with aqueous inks. Another possibility is the use of starch in polyurethane foams. By adapting the starch derivatives and optimizing by engineering and processing, it is possible to control the reaction between the synthetic polymers and the hydroxyl groups of the starches in a targeted manner. This results in polyurethane films that acquire the following spectrum of properties, due to the use of starch: a reduced coefficient of thermal expansion, a reduced shrinkage behavior, an improved pressure-tension behavior, an increase in vapor permeability of water without altering water absorption, reduced flammability and reduced ultimate tensile strength, no droplet formation with combustible parts, halogen freedom, or reduced aging. The disadvantages that still exist are the reduced compressive strength and the reduced impact resistance. Currently, product development is no longer restricted to movies. Solid polymer products such as molds, plates and saucers comprising a starch content of more than 50% can also be made. In addition, starch / polymer mixtures are considered advantageous because their biodegradable capacity is much higher. Starch graft polymers have become extremely important due to their extremely high water binding capacity. They are products with a starch base structure and a side chain of a synthetic monomer, grafted following the principle of the chain mechanism of free radicals. The starch graft polymers that are currently available are distinguished by a better binding capacity and retention of up to 1000 g of water per gram of starch in combination with high viscosity. The fields of application of these superabsorbents have extended widely in recent years and are, in the hygiene sector, products such as diapers and mattress covers and, in the agricultural sector, for example in seed coatings. Decisive for the application of novel genetically modified starches are, on the one hand, the structure, the water content, the protein content, the lipid content, the fiber content, the ash / phosphate content, the ratio of amylose / amylopectin, the distribution of molecular mass, the degree of branching, the size and shape of the granule and the crystallinity, and, on the other, also the characteristics that effect the following aspects: flow and sorption behavior, gelatinization temperature, viscosity, Viscostability in salt solutions, thickening powder, solubility, gel structure and gel transparency, thermal stability, shear stability, acid stability, tendency to undergo retrogradation, gel formation, freeze-thaw stability, complex formation, Iodine binding, film formation, adhesive powder, enzyme stability, digestion capacity and reactivity idad. 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 chemical or physical methods do not seem to be necessary anymore. On the other hand, starches that have been altered by recombinant methods can undergo additional chemical modifications, which leads to other improvements in quality for some of the fields of application described above. These chemical modifications are known in principle. These are, in particular, modifications by thermal treatment, treatment with organic or inorganic acids, oxidation and esterification leading, for example, to the formation of phosphate starches, nitrate starches, sulfate starches, xanthate starches, acetate starches and citrate starches. In addition, the mono- or polyhydric alcohols in the presence of strong acids can be used to produce starch ethers, resulting in alkyl starch ethers, O-allyl ethers, hydroxyalkyl ethers, O-carboxymethyl ethers, N-containing starch ethers. , starch ethers containing P, S-containing starch ethers, cross-linked starches or starch graft polymers. A preferred use of the starches according to the invention is the production of packaged materials and disposable articles, on the one hand, and as food products or precursors of food products on the other. In order 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 that ensure transcription in plant cells. These include, in particular, promoters, enhancers and terminators. In general, any promoter that is active in plant cells is suitable for expression.

The promoter can be selected in such a way that the expression forms part or takes place only in a particular tissue, at a particular point in the time of development of the plant or at a point in time determined by external factors. In relation to the plant, the promoter can be homologous or heterologous. Examples of such suitable promoters are the 35S RNA promoter of the cauliflower mosaic virus and the corn ubiquitin promoter for constitutive expression, the patatine B33 promoter (Rocha-Sosa et al., EMBO J. 8 (1989), 23-29) for tuber-specific expression, or a promoter that ensures expression only in photosynthetically active tissues, for example the ST-LS1 promoter (Stockhaus et al., Proc. Nati. Acad. Sci. USA 84 (1987) , 7943-7947, Stockhaus et al., EMBO J. 8 (1989), 2445-2451) or, for specific expression of the endosperm, the AMG promoter of wheat, the USP promoter, the phaseolin promoter or zein gene promoters. of corn. A terminator sequence can also be present which serves to correctly terry the transcript and to add a poly-A tail to the transcript, which is thought to have a function to stabilize the transcript. Such elements have been described in the literature (see Gielen et al., EMBO J. 8 (1989), 23-29) and can be exchanged as desired. The present invention provides nucleic acid molecules that code for a protein with a wheat isoamylase function. The nucleic acid molecules according to the invention allow the production of this enzyme whose functional identification in the biosynthesis of starch, the generation of plants that have been altered by recombinant technology where the activity of this enzyme is altered and therefore it allows a starch to be synthesized whose structure is altered and whose physicochemical properties are altered. In principle, the nucleic acid molecules according to the invention can also be used to generate plants in which the activity of the amylasease according to the invention is increased or reduced simultaneously than the activities of other enzymes involved in the synthesis of starch are altered. The alteration of activities of a soamilasa in plants results in the synthesis of a starch with altered structure. In addition, the nucleic acid molecules encoding an isoamylase, or suitable antisense constructs can be introduced into plant cells wherein the synthesis of endogenous starch synthetases 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 inhibition of the synthesis of several enzymes involved in starch biosynthesis in transformed plants, the transformation may involve DNA molecules that simultaneously comprise several regions encoding the enzymes in question in antisense orientation under the control of a promoter. suitable. Here, it is possible as an alternative for each sequence to be under the control of its own promoter, or the sequences can be transcribed as fusion from a linker promoter or be under the control of a linker promoter. The last mentioned alternative will generally be preferred, since in this case the synthesis of the proteins in question should be inhibited to the same degree. As regards the length of the individual coding regions used in said construction, what has been mentioned above for the generation of antisense constructions also applies here. In principle, there is no upper limit for the number of antisense fragments transcribed in said DNA molecule starting from a promoter. However, the preferably formed transcript will not exceed a length of 10 kb, in particular a length of 5 kb. The coding regions located in said DNA molecules in combination with other coding regions in antisense orientation behind a suitable promotercan be derived from DNA sequences that code for the following proteins: starch synthetases bound to starch granules (GBSS I and II) and soluble starch synthetases (SSS I and II), branching enzymes (isoamylases, pullulanases, R enzymes, branching enzymes, debranching enzymes), starch phosphorylases and disproportionation enzymes. This list is only by way of example. It is also possible to use other DNA sequences for the purposes of said combination.

Said constructions allow the synthesis of a plurality of enzymes to be inhibited simultaneously in plant cells transformed with said constructions. In addition, the constructs can be introduced into plant mutants that 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 can be related to the following proteins: starch synthetases linked to starch granules (GBSS I and II) and soluble starch synthetases (SSS I and II), branching enzymes (BE I and II), debranching enzymes (enzymes) R), disproportionation enzymes and starch phosphorylases. This list is given only as an example. Said process also allows the synthesis of a plurality of enzymes to be inhibited simultaneously in plant cells transformed with them. To prepare for the introduction of strangers in higher plants, a large number of cloning vectors containing a replication signal for E. coli and a marker gene are available to select the transformed bacterial cells. Examples of such vectors are pBR322, pUC series, M13mp series, pACYC184, and the like. The desired sequence can be introduced into the vector at a suitable restriction cleavage site. The obtained plasmid is used to transform E. coli cells. Transformed E. coli cells are grown in a suitable medium and subsequently harvested and lysed. The plasmid is recovered. The analytical methods to characterize plasmid DNA obtained that are generally used are restriction analysis, gel electrophoresis and other methods of biochemistry and molecular biology. After each manipulation, the plasmid DNA can be cut and the resulting DNA fragments linked to other DNA sequences. Each plasmid DNA sequence can be cloned into the same or different plasmids. A large number of techniques are available to introduce DNA into a host plant cell. These techniques comprise the transformation of plant cells with T-DNA using Agrobacterium tumefaciens or Agrobacterium rhizogenes as transformation agents, protoplast fusion, injection, DNA electroporation, introduction of DNA by the biolistic method, and other possibilities. The injection and electroporation of DNA in plant cells does not require anything in particular on the plasmids used. Simple plasmids such as, for example, pUC derivatives can be used. However, if the intact plants will be regenerated from cells transformed in this manner, the presence of a selectable marker gene is required. Depending on the method for introducing desired genes into the plant cell, additional DNA sequences may be required. If, for example, the Ti or Ri plasmid is used to transform the plant cell, at least the right edge, but frequently the right and left border, of the T-DNA of the Ti and Ri plasmid should be linked to the genes to be introduced as a flanking region. If agrobacteria are used for transformation, the DNA that will be introduced must be cloned into specific plasmids, either in an intermediate vector or in a binary vector. Intermediary vectors can be integrated into the Ti or Ri plasmid of agrobacteria by homologous recombination due to sequences that are homologous to the sequences in the T-DNA. Said plasmid also comprises the vir region, which is required for the transfer of T-DNA. Intermediary vectors can not replicate in agrobacteria. The intermediate vector can be transferred to Agrobacterium tumefaciens by means of an auxiliary plasmid (conjugation). The binary vectors are capable of replicating in E. coli and in agrobacteria. They contain a selection marker gene and a linker or polylinker, which are bordered by the left and right T-DNA border region. They can be transformed directly into agrobacteria (Holsters et al., Mol. Gen. Genet. 163 (1978), 181-187). The agrobacteria that acts as the host cell should comprise a plasmid carrying a vir region. The vir region is required to transfer the T-DNA into the plant cell. Additional T-DNA can be presented. Agrobacteria transformed in this way can be used to transform plant cells. The use of T-DNA to transform plant cells has been extensively investigated and described sufficiently in EP 120 516; Hoekema, in: The Binary Plant Vector System Offsetdrukkerij Kanters B.V., Alblasserdam (1985), Chapter V; Fraley et al., Crit Rev. Plant. Sci., 4, 1-46 and An et al. EMBO J. 4 (1985), 277-28J. To transfer the DNA into the plant cell, plant explants can be co-cultivated in a timely manner with Agrobacterium tumefaciens or Agrobacterium rhizogenes. Intact plants can be regenerated from the infected plant material (eg, leaf sections, stem sections, roots, but also protoplasts, or plant cells grown in suspension culture) in a suitable medium which may comprise, inter alia, certain sugars, amino acids, antibiotics or biocides to select transformed cells. The resulting plants can be examined for the presence of the DNA that has been introduced. Other possibilities of introducing foreign DNA using the biolistic method or by protoplast transformation are known (see, for example, Willmitzer, L., 1993 Transgenic Plants, In: Biotechnology, A Multi-Volume Comprehensive Treatise (HJ Rehm, G. Reed, A. Pühier, P. Stadler, eds.), Vol. 2, 627-659, VCH Weinheim-New York-Basel-Cambridge). Although the transformation of dicotyledonous plants through vector systems of Ti plasmid with the help of Agrobacterium tumefaciens is well established, more recent research suggests that even monocotyledonous plants are accessible to transformation by vectors based on agrobacteria (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 through a biolistic approach, the transformation of protoplasts, or the absorption of physically or chemically induced DNA into protoplasts, for example by electroporation of partially permeabilized cells, transfer of DNA by means of glass fibers, macroinjection of DNA in inflorescences, microinjection of DNA into microspores or proembryos, absorption of DNA by germination of pollen and absorption of DNA in embryos by swelling (see Potrykus, Physiol. Plant (1990), 269-273). Three of the aforementioned transformation systems have been established in the past for several cereals: tissue electroporation, protoplast transformation and DNA transfer by bombardment of particles in regenerable tissues and cells (see Jáhne et al., Euphytica 85 (1995), 35-44). Different methods for transforming wheat have been described in the literature (see Maheshwari et al., Critical Reviews in Plant Science 14 (2) (1995), 149 to 178): Hess et al. (Plant Sci 72 (1990), 233) where the macroinjection method is used to carry pollen and agrobacteria in immediate proximity. The mobilization of the plasmid containing the nptll gene as a 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 that was regenerated after bombardment with DNA bound to microprojectiles was described by Vasil et al. (Biop "echnology 10 (1992), 667-674) The target tissue for bombardment was an embryogenic callus culture (callus type C.) The selection marker used was the bar gene coding for a phosphinothricin acetyltransferase and therefore Both average resistance to the herbicide phosphinothricin Another 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 DNA transformation is the scutellum of immature embryos that were stimulated in a preliminary in vitro phase to induce somatic embryos.The transformation efficiency in the system developed by Becker et al. (Loe cit.) It is 1 transgenic plant for 83 embryos of the variety "Florida" and therefore significantly higher than the system established by Weeks et al., which produces 1 to 2 transgenic plants per 1000 embryos of the variety "Bohwhite". developed by Becker et al. (loe cit.) form the basis of the transformation experiments described in the examples. Once the introduced DNA is integrated into the genome of the plant cell, it is, as a rule, stable there and is also retained in the progeny of the originally transformed cell. It usually contains one of the selection markers mentioned above that mediates resistance to a biocide such as phosphinothricin or an antibiotic such as kanamycin, G 418, bleomycin or hrythromycin, to transformed plant cells or that allows selection through the presence or absence of certain sugars or amino acids. The individually selected marker should allow the selection of transformed cells on cells lacking introduced DNA. Within the plant, the transformed cells grow in a traditional manner (see also McCormick et al., Plant Cell Reports 5 (1986), 81-84). The resulting plants can be grown normally and hybridized with plants having the same transformed germ plasm or other germ plasmids. The resulting hybrid individuals have the corresponding phenotype properties. The seeds can be obtained from plant cells. Two or more generations should be cultivated in order to ensure that the characteristic phenotype is retained and inherited stably. In addition, the seeds must be harvested to ensure that the phenotype in question or other properties have been retained. The following examples are intended to illustrate the invention and do not constitute a restriction thereof. 1. Cloning methods The vector pBluescript II SK (Stratagene) was used to clone in E. coli. 2. Bacterial Strains The E. coli strain DH5a (Bethesda Research Laboratories, Gaithersburg, E.U.A.) was used for the Bluescript vector and for the antisense constructs. Strain XL1-Blue from E. coli was used for excision in vivo. 3. Transformation of immature wheat embryos Media MS: 100 ml / l macrosal (D. Becker and H. Lórz, 1 ml / l of microsal Plant Tissue Culture 2 ml / l Fe / NaEDTA Manual (1996), B 12: 1-20) 30 g / l of sucrose # 30: MS + 2,4-D (2 mg / l) # 31: MS + 2,4-D (2 mg / l) + phosphinothricin (PPT active ingredient of herbicide BASTA (2 mg / l) ) # 32: MS + 2,4-D (0.1 mg / l) + PPT (2 mg / l) # 39: MS + 2,4-D (2 mg / l) + every 0.5 N mannitol / sorbitol established media were brought to a pH of 5.6 using KOH and solidified using 0.3% Gelrite. The method for transforming immature wheat embryos was developed and optimized by Becker and Lórz (D. Becker and H. Lorz, Plant Tissue Culture Manual (1996), B12: 1 to 20).

In the experiments described hereinafter, the procedure developed by Becker and Lórz (loe. Cit) was adhered to. For the transformation, ears were harvested and sterilized on the surface with caryopses in the development stage 12 to 14 days after anthesis. Isolated scutes were placed on plates in induction medium # 30 with the axis of the embryo oriented towards the middle. After preculturing for 2 to 4 days (26 ° C, in the dark), the explants were transferred to medium # 39 for the osmotic preculture (2 to 4 hours, 26 ° C, in the dark). For the biolistic transformation, approximately 29 μg of gold particles were used for each shot in which some μg of the target DNA had been precipitated previously. Because the experiments performed are co-transformations, the target DNA added to the precipitation batch is composed of a target gene and a resistance marker gene (gene bar) in the ratio 1: 1. 4. DIG labeling of DNA fragments The DNA fragments used as selection probes were labeled by a specific PCR with the incorporation of DIG labeled with DIG (Boehringer Mannheim, Germany). The media solutions used in the examples: 20 x SSC 175.3 g of NaCI 88.2 g of sodium citrate H20 twice distilled to 1000 ml 10 N NaOH at pH 7.0 Plasmid pTaSU 8A was deposited at the DSMZ in Braunschweig, Federal Republic of Germany, as specified in the Budapest Treaty under the number DSM 12795, and plasmid pTaSU 19 under the number DSM 12796.

EXAMPLE 1 Identification, isolation and characterization of a cDNA encoding an isoamylase ("sugary" homologue) of wheat (Triticum aestivum, vc Florida) To identify a cDNA coding for a isoform of wheat soamilasa (sugary), a heterologous selection strategy was followed. For this purpose, a wheat cDNA library was selected with a corn sweetener probe. The probe (sugar probe) was isolated from a corn cDNA library by specific primers using PCR amplification. The corn cDNA library was cloned from poly (A) + RNA from a mixture of equal amounts of 13-, 17-, 19-, 20-, 22-, 25- and 28-day cariopses. (DAP) in a Lambda Zap II vector following the manufacturer's instructions (Lambda ZAP ll-cDNA Synthesis Kit Stratagene GmbH, Heidelberg, Germany). In all the cariopses used, with the exception of the 13-day grains, the embryo had been removed before isolating the RNA. The DNA fragment used as a probe to select the wheat cDNA library was amplified with the following primers: sul p-1: 57 \ AAGGCCCAATATTATCCTTTAGG 3 '(SEQ ID No. 4) su1 p-2: 5'GCCATTTCAACCGTTCTGAAGTCGGGAAGTC 3' (SEQ ID No. 5) The template used for the PCR was 2 μl of the amplified corn cDNA library. In addition, the PCR contained 1.5-3 mM MgCl2, 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 0.8 mM mixed dNTP, 1 μM su1 p-1a primer, 1 μM primer su1 p- 2 and 2.5 units of Taq polymerase (recombinant, Life Technologies). The amplification was carried out using a Biometra Trioblock following the scheme: 4 min / 95 ° C; 1 min / 95 ° C, 45 sec / 58 ° C; 1 min 15 sec / 72 ° C; 30 cycles 5 min / 72 ° C. The amplified DNA band of approximately 990 bp was separated on an agarose gel and cut. A second amplification is [lagoon] of this fragment following the scheme as described above. The 990 bp fragment obtained from this second amplification was cut with the restriction enzyme BAM Hl in a fragment of 220 bp and 770 bp. After the sugar fragment had separated again on an agarose gel, the band cut and the fragment isolated, the probe was labeled with DIG. 500 ng of sugary fragment were used for the labeling of random primer with digoxigenin. 10 μl of random primer was added to the fragment that would be labeled and the reaction heated for 5 minutes at 95-100 ° C. After heating, 0.1 mM of dATP, 0.1 mM of dGTP, 0.1 mM of dCTP and 0.065 mM of dTTP and 0.035 mM of digoxigenin-11-dUTP (Boehringer Mannheim) and Klenow regulator (standard) and 1 unit of Klenow polymerase were added. . The reaction was allowed to continue at room temperature overnight. To verify the marking, a dot test was performed 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) + caryopsis RNA of approximately 21 days ("starched" endosperm) in a Lamda Zap II vector following the manufacturer's instructions (Lambda ZAP ll-cDNA Synthesis Kit, Stratagene GmbH, Heidelberg). After determining the title of the cDNA library, a primary titer of 1.26 x 106 pfu / ml was determined. To select the wheat cDNA gene, approximately 350,000 phages were plated. The phages were plated and the plates blotted following standard protocols. The filters were prehybridized and hybridized in 5x SSC, 3% Blocking (Boehringer Mannheim), 0.2% (SDS), 0.1% sodium lauryl sarcosine and 50 μg / ml herring sperm DNA at 55 ° C. 1 ng / ml of the labeled sugar 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 room temperature; 2 x 10 min in 1 X SSC, 0.5% SDS at 55 ° C; 2 x 10 min in 0.5 X SSC, 0.2% SDS at 55 ° C. The positive clones were separated by additional selection cycles. Individual clones were obtained through in vivo excision as phagemid pBluescript SK (analogous procedure to manufacturer's instructions, Stratagene, Heidelberg, Germany). After the clones had been analyzed through mini-preparations and after the plasmid DNA had been restricted, the clone pTaSU-19 was deposited in the DSMZ Deutsche Sammiung für Mikroorganismen und Zellkulturen GmbH under the number DSM 12796 and analyzed in more detail.

EXAMPLE 2 Sequence analysis of the cDNA inserts of the plasmid pTaSU19 The plasmid DNA was isolated from the clone pTASU19 and the sequence of the cDNA inserts were determined by the dideoxynucleotide method (Sanger et al., Proc.Nat.Acid.Sci.U.A. 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 in SEQ ID No. 1. A comparison that already published sequences revealed that the sequence under the number SEQ ID No. 1 comprises a coding region having homologies to isoamylases of other organisms.

Sequence analysis also revealed that two introns are located in the cDNA sequence at positions 297-396 (intern 1) and 1618-2144 (intern 2). If these introns are removed, a protein sequence that exhibits homologies to the protein sequences of soamilases from other organisms can be derived. The amino acid sequence corresponding to the coding regions of SEQ ID No. 1 is shown under SEQ ID No. 3.

EXAMPLE 3 Generation of the transformation vector pTa-alpha-SU19 in plants To express an antisense RNA corresponding to TaSU19-cDNA, pTa-alpha-SU19 transformation vectors in plants were constructed based on the basic plasmid pUC19 by binding the pTa-alpha-SU19 plasmid cDNA insert in antisense orientation to the 3 'end of the ubiquitin promoter. This promoter is composed of the first non-translated exon and the first intron of the maize ubiquitin 1 gene (Christensen A.H. et al., Plant Molecular Biology 18 (1992), 675-689). Parts of the NOS polylinker and terminator are derived from the pACTLca plasmid (CHANGE, TG 0063; Change, GPO Box 3200, Canberra ACT 2601, Australia). The vector constructs with this terminator and the constructions based on pActl. Cas are described by McEIroy et al. (Molecular Breeding 1 (1995), 27-37). The resulting vector is called pUbi.cas.

The vector was cloned by restricting a 2kb fragment of the Ta-SU19 clone with the restriction enzyme Xba I. The fragment was filled at the ends by a Klenow reaction and the fragment was subsequently ligated into the Sma I cloning site of the vector. expression pUbi.cas. The resulting expression vector was designated TA-alpha-SU 19 and was used as described above to transform wheat.

EXAMPLE 4 Isolation and characterization of an additional cDNA encoding an isoamylase (sugary counterpart 1) of wheat (Triticum aestivum I., vc Florida) A wheat cDNA library was selected with a sugar probe representing a part of clone pTaSU19, positions 489-1041 of SEQ ID No. 1. The wheat-specific digoxigenin-labeled sugary probe used to select the cDNA library was prepared by PCR amplification. The primers used in this reaction were: SUS01: 5'-GCT TTA CGG GTA CAG GTT CG-3 '(SEQ ID No. 8), and SUS02: 5-AAT TCC CCG TTT GTG AGC3- '(SEQ ID No. 9) 1 ng of plasmid pTaSU19 was used in the reaction as a template. In addition, the PCR contained in each case 300 nM of the initiators SUS01 and SUS02, in each case 100 μM of the nucleotides dATP, dGTP, dCTP, 65 μM of dTTP, 35 μM of digoxigenin-11-dUTP (Boehringher Mannheim), 1.5 mM MgCl 2, and 2.5 U (units) of Taq Polymerase and 10 μl of 10 times concentrated reaction buffer of Taq polymerase (both from Life Technologies). The final volume of the reaction was 100 μl. The amplification was performed in a PCR apparatus (TRIO® Thermoblock, Biometra) with the following temperature regime: 3 min at 95 ° C (one time); 45 sec at 95 ° C - 45 sec at 55 ° C - 2 min at 72 ° C (30 cycles); 5 min at 72 ° C (one time). A 553 bp DNA fragment resulted. The incorporation of digoxigenin-11-dUTP into the PCR product was revealed due to reduced mobility in the agarose gel compared to the product of a controlled reaction without digoxigenin-11-dUTP. The caryopsis specific wheat cDNA library of Example 1 was selected with the resulting digoxigenin labeled probe. The hybridization step was performed overnight in 5x SSC, 0.2% SDS, 0.1% lauryl sarcosine sodium and 50 μg / ml herring sperm DNA at 68 ° C in the presence of 1 ng / ml of the labeled probe with digoxigenin. After annealing, 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 0.5x SSC, 0.2% SDS at 68 ° C. The positive clones were separated by at least two additional selection cycles. The plasmids were obtained from pBluescript SK phage clones through in vivo excision (protocols according to the manufacturer's instructions, Stratagene, Heidelberg, Germany). After the restriction analysis, the clone obtained pTaSU8A was deposited in the Deutsche Sammiung für Mikroorganismen und Zellkulturen under the number DSM 12795 and studied in greater detail.

EXAMPLE 5 Sequence analysis of the cDNA insert in the pTaSU8A plasmid The nucleotide sequence of the cDNA insert in the pTaSUdA plasmid was determined by the dideoxynucleotide method (SEQ ID No. 6). The insertion of the pTaSUdA clone is 2437 bp in length and constitutes a partial cDNA. A comparison that already published sequences revealed that the sequence under SEQ ID No. 6 comprises a coding region that has homologies to the soamilases of other organisms. Similarly, the sequence of proteins derived from the coding region of the clone pTaSUdA shown in SEQ ID No. 7 exhibits homologies to the protein sequences of soamilases from other organisms. After comparing the sequences of clones pTaSU19 (SEQ ID No. 1) and pTaSUdA (SEQ ID No. 6), a similarity of 96.8% results. Most of the differences in relation to the sequences are in the 3 'untranslated region of the cDNAs. The remaining differences with respect to the sequences in the coding region lead to different amino acids in a total of 12 positions of the derived protein sequences (SEQ ID No. 3 and 7). The cDNAs contained in pTaSU19 and pTaSUdA are not identical and encode isoforms of wheat isoamylase.

EXAMPLE 6 Generation of the transformation vector pTa-alpha-SU8A in plants To express an antisense RNA corresponding to the TaSUdA cDNA, the transformation vector pTa-alpha-SUdA in plants was constructed based on the basic plasmid pUC19 by binding a portion of the cDNA of TASUE generated by PCR amplification in antisense orientation at 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 Molecular Biology 1d (1992), 675-669). Parts of the NOS polylinker and terminator are derived from the plasmid pACT1.cas (CHANGE, TG 0063, Change, GPO Box 3200, Canberra ACT 2601, Australia).

The vector constructs with this terminator and the constructions based on pActl. Cas are described by McEIroy et al. (Molecular Breeding 1 (1995), 27-37). The vector containing ubiquitin promoter, polylinker and NOS terminator and based on pUC19 was named pUbi.cas.

To clone pTa-alpha-SU3A, a portion of approximately 2.2 kb of the TaSUdA cDNA, positions 140-2304 of SEQ ID No. 6, was amplified by PCR. The primers used 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 TAC AAC ATC AGG GCG CAA TAC-3' (SEQ ID No . eleven ). 1 ng of plasmid pTaSUdA was used in the reaction as a template. In addition, the PCR contained: in each case 300 nM of the primers SUEX3 and SUEX4, in each case 200 μM of the nucleotides dATP, dGTP, dCTP and dTTP, 1.6 mM of MgCl2, 60 mM of Tris-S04 (pH 9.1), 1d mM of (NH4) 2SP and 1 μl of Elongase® enzyme mixture (mixture of Taq polymerase and DNA polymerase, Life Technologies). The final volume of the reaction was 50 μl. The amplification was carried out in a PCR apparatus (TRIO® Thermoblock, Biometra) with the following temperature regime: 1 min at 94 ° C (one time); 30 sec at 95 ° C- 30 sec at 55 ° C- 2 min 30 sec at 6d ° C (30 cycles); 10 min at 6d ° C (one time). The reaction gave a DNA fragment of 2205 bp in length. The 2.2 kb product was restricted with Kpnl and Sali and ligated into the pUbi.cas expression vector that had previously been cut with Kpn / and Sali. The resulting transformation vector in plants was designated pTa-alpha-SUAd and was used as described above to transform wheat. 53 LIST OF SEQUENCES < 110 > Hoechst Schering AgrEvo GmbH < 120 > Nucleic acid molecules encoding wheat enzymes involved in the synthesis of starch < 130 > 199d / M206 < 140 > PCT / EP99 / 03141 < 141 > 1999-05-07 < 150 > DE 19320606.9 < 151 > 1998-05-08 < 160 > 11 < 170 > PatentlnVer.2.1 < 210 > 1 < 211 > 2997 < 212 > DNA < 213 > Triticum aestivum L. cv. Florida < 220 > < 221 > CDS < 222 > (3) ... (296) < 220 > < 221 > CDS < 222 > (397) ... (1617) < 220 > < 221 > CDS < 222 > (2145) ... (2960) < 400 > 1 gg teg ggg eeg geg eeg ege atg cga SSGG tgg Oga oco aat Gog aeg 47 S * Gly Pro Ala Pro Arg Leu Arg Arg Trp Arg Pro Asn Ala Thr 1 10 May 15 'gcg ggg aag ggg gtc ggc gag gtg tgc gcc gcg gtt gtc gag gcg gcg Ala Gly Lys Gly 95 Gly Glu Val Cys a.1 Ala Val Ala Val Ala Glu Ala 20 25 30 acg aag gta gag gac ggg gag gag gag gag gac gac gag gcg gtg ceg 143 Thr Val Glu Asp Glu Lya Gly Glu Glu Asp Glu Pro Val Ala Glu Asp 35 40 45 agg tac gcg ctc ggc ggc gcg tgc agg gtg ctc gcc gga atg ccc gcg 191 Arg Tyr Ala Leu Gly Gly Ala Cys Arg Val Leu Ala Gly Met Pro Ala 50 55 60 ceg ctg ggc gcc acc gcg ctc gcc ggc ggg gtc aat ttc gcc gtc tat 239 HSRP Leu Gly Ala Thr Ala Leu Ala Gly Gly Val Assn Pha Ala Val Tyr 65 70 75 aka ggc gga gcc acc gcc gcg gcg ctc tgc ctc ttc acg cea gaa gat 237 Ser Gly Gly Ala Thr Ala Ala Ala Leu Cya Leu Phe Thr Pro Glu Asp TO 35 90 95 ctc aag gcg gtggggttgc ctccsgagta gagttcatca. gctttgcgtg 336 Leu Lya Ala cgccgcgsgc cccttttttg ggcctgcaat ttaagttttg tactggggea aatgctgcag 396 gat agg gtg acc gag gag gtt acc att gao coc otg atg aat cgg acc 444 Asp Arg Val Thr Glu Glu Val Pro Leu Aap Pro Lau Mat Asn Arg Thr 100 105 110 ggg aac gtg tgg cat gtc gtc cat atc gaa ggc gag ctg cac aac atg ett 492 Gly? an Val Trp Hia Val Phe lie Glu Gly Glu Leu Hia Asn Met Leu 115 120 125 130 tac ggg tac agg ttc gac ggc acc ttt gct cct cac tgc. ggg falls tac 540 Tyr Gly Tyr Arg Phe Aap Gly Thr Phe Wing Pro His Cys Gly His Tyr 135 140 145 ett gat gtt tcc aat gtc gtg gtg gat cct tat get aag gca gtg ata 588 Leu Aap Val Ser Aan Val Val Val Asp Pro Tyr Ala Lya Ala Val He 150 155 160 age cga ggg gag tat ggt gtt cea gcg cgt ggt aac aat tgc tgg cct 636 Ser Arg Gly Glu Tyr Gly Val Pro Wing Arg Gly Asn Aßn Cya Trp Pro 165 170 175 cag atg gct ggc atg ate cet ett oca tat age acg ttt gat tgg gaa 684 Gln Met Wing Gly Met lie Pro Leu Pro Tyr Ser Thr Phe Aap Trp Glu 180 185 190 ggc gac ata cct eta aga tat cct caa aag gac ctg gta ata tat gag 732 Gly Aap Leu Pro Leu Arg Tyr Pro Gln Lyc Aap Leu Val Ilc Tyr Glu 195 200 205 210 atg falls ttg cst ssa tte acs aag cat gat tea age aat gta gaa cat 780 Met His Leu Arg Gly Phe Thr Lya Hia Aßp Ser Ser Aan Val Glu Hia 215 220 225 ceg ggt act tte att gga gctg teg aag ett gac tat ttg aag gag 323 Pro Gly Thr Phe lie Gly Ala Val Ser Lys Leu Aap Tyr Leu Lya Glu 230 235 240 ett gga gtt aat tgt att gaa t ta atg cce tgc eat gag ttc aac gag 976 Leu Gly Val Aan Cya He Glu Leu Met Pro Cya Hia Glu Phe Asn Glu 245 250 255 ctg gag tac tea acc tet tet tcc aag atg aac ttt tgg gga tat tet 924 Leu Glu Tyr 3er Thr 3ßr Ser Ser Lya Met Aan Phe Trp Gly Tyr 3er 260 26S 270 ace ata aac ttc ttt ate cea at g aga aga ta g a te g gc ggg ata 972 Thr lie Aan Phe Phe Ser Pro Met Thr Arg Tyr Thr Ser Gly Gly He 275 280 285 290 aaa aac tgt ggg cgt gat gcc ata aat gag tte aaa act ttt gta aga 1020 Lya Aan Cys Qly Arg Asp Ala lie Asn Glu Phe Lys Thr Phe al Arg 295 300 305 gag get falls aaa cgg gga att gag gtg atc ctg gat gtt gtc ttc aac 1068 Glu Wing His Lys Arg Gly Ha Glu Val Ilß Leu Asp Val Val Phe Asn 310 315 320 cat here get gag ggt aat gag aat ggt cea ata tta tea ttt aag ggg 1116 His Thr Ala Glu Gly Aan Glu Aan Gly Pro He Leu Ser Phe Lya Gly 325 330 335 ga gat aat aat ta ta tat atg ett goa ccc aag gga gag ttt tat 1164 Val Aap Aan Thr Thr Tyr Tyr Met Leu Wing Pro Lys Gly Glu Phe Tyr 340 345 350 aac tat tet ggc tgt ggg aat acc ttc aac tgt aat eat cet gtg gtt 1212 Asn Tyr Ser Glv Cys Gly Asn Thr Phe Asn Cys Aan Hia Pro Val Val 355 360 365 370 cgt cata ttc att gta gat tgt tta aga tac tgg gtg acg gaa atg cat 1260 Arg Glp Phß Il «Val Aap Cya Leu Axg Tyr Txp Val Thr Glu Met His 375 330 385 gtt gat ggt ttt cgt ttt gat ett gca tcc ata atg acc aga ggt tee 1308 Val Asp Gly Phe Arg Phe Asp Leu Wing Being Met Thr Arg Gly Ser 390 395 400 agt ctg tgg gat cea gtt aac gtg tat gga gct cea ata gaa ggt gac 1356 n Ser Leu Trp Asp Pro Val Asn Val Tyr Gly Ala Pro He Glu Gly Asp u 40S 410 415 ata ate ac here sct cct ett ott act et cea ett att gae atg 1404 Met He Thr Thr Gly Thr Pro Leu Val Thr Pro Pro Leu lie Asp Met 420 425 430 atc age aat gac cea att ett gga ggc gtc aag ctc att gct gaa gca 1452 He Ser Aßn Aap Pro lie Leu Gly Gly Val Lya Leu Ilß Ala Glu Wing 435 4-40 44S 450 tgg gat gca gga ggc ctc tat ca gta ggt ca ttc cct cac tgg aat 1500 Trp Asp Wing Gly Gly Leu Tyr Gln Val Gly Gln Phe Pro His Trp Aan 455 460 465 gtt tgg tet gag t? g aat ggg aag tac cgg gac att gtg cgt cate tte 1548 Val Trp Ser Glu Trp Aan Gly Lys Tyr Arg Asp He Val Arg Gln Phe 470 475 490 att aaa ggc aet gat gga ttt get ggt ggt ttt gcc gag tgt ett tgt 1596 He Lys Gly Thr Aap Gly Phe Wing Gly Gly Phe Wing Glu Cya Leu Cya 485490495 GGA AGT cea eac cta tae cag gtaagttgtg goaataettg taaatgagtt 1647 Gly Ser Pro His Leu Tyr Gln 500 505 gagtgaatgt oaootggatt ttttatatat aeeaeatgat taaatatata gataeaeate 1707 acaatcatag tstatgcata tgeatttggc taagaagtat tagtgtatac actagtgeta 1767 tatataggtt ttaaeaccca aettgceaat gaaggaacat agggctttct agttatctta 1827 tttatttgtc cggtgaataa tccactgaaa aattecagee atgtcatttt ttaggggggg 1887 agaagaaact atattgattt gcccccctaa aagaagceat etcagaatte atagg aagt 1947 tgcttttctg taaagaaagg aaaaegaatt catactttct atcggtgcta acttagctcg 2007 atgtatattt gtaagatgaa tgecaaattt aatttgtcgg ataatttgat otgttattc a 2067 caaatttcta tttggtttct ctagaaatca aaecagtaac ttgttattgg cactgcaact 2127 tcttattgat taatcag gca gga gga agg aaa cet tgg cac agt atc aac 217 wing Gly Gly Arg Lys Pro Trp His Ser He Aan 510 515 ttt ata tgt be cat gat ssa ttt here ctg gct gat ttg gta aea tat 2225 Phe Val Cys Wing Hia Asp Gly Phe Thr Leu Wing Ápp Leu Val Thr Tyr 520 525 530 aat aag aat tata aat tta cea aat ggg gag aac aga aga gat gga gaa 2273 Aan Lys Lys Tyr Aan Leu Pro Aan Gly Glu Asn Asn Arg Asp Gly Glu 535 540 545 aat cac aat ett age tgg aat tgt ggg gag gaga gga gaa tte gca aga 2321 Aan His Asn Leu be Trp Asn cys Gly Slu Glu Gly Glu Phe Ala Arg 550 555 560 ttg tet gte aaa aga ttg agg aag agg eag atg cgs aat tta ttt gtt 2369 Leu 3rd val Lys Arg Leu Arg Lys Arg Gln Met Arg Asn Phe Phß val 565 570 575 580 tgt ote atg gtt tet ca * gga gtt co »atg ttc tac atg ggt gat gaa 2417 Cya Leu Met val Ser Gln Gly Val Pro Met Phe Tyr Met Sly Aap Glu 585 590 595 tat gga cac ac aaa ggg ggc aae aat aat aat tae ge eat gat tet 2465 Tyr G and Hia Thr Lys Gly Gly Asn Asn Asn Thr Tyr Cys His Asp Ser 600 605 610 tat gte aat tat ttt cgc tgg gat aaa aaa gaa ca tac tet gag ttg 2513 Tyr Val Asn Tyr Phe Arg Trp Asp Lys Lya Glu Gln Tyr Ser Glu Lau 615 620 625 cac cga ttc tgc tgc cte atg aec aaa ttc cgc aag gag tgc gag ggt 2S61 His Arg Ph "Cya Cya Leu Met Thr Lys Phe Arg Lys Glu Cya Glu Gly 630 635 640 ett ggc ett gag gac ttt cea acg gee aaa cgg etg cag tgg cat ggt 2609 Leu Gly Leu Glu Asp Phe Pro Thr Wing Lys Arg Leu Gln Trp His Gly 645 6S0 655 660 cat cag eet ggg aag cet gat tgg tst gag aat age ega tte gtt gee 2657 His Gln Pro Gly Lya Pro Aßp Trp Ser Glu Asn Ser Arg Phe Val Wing 665 670 675 ttt tcc atg aaa gat gaa aga cag ggc gag atc tat gtg gcc ttc aac 2705 Phe Ser Met Lyß Aßp Glu Arg Gln Gly Glu llß Tyx Val Wing Phe Asn 680 635 690 accc cac tta cec gcc gtt gtt gag ctc cea gag cgc gca ggg cgc 2753 Thr Ser Hia Leu Pro Wing Val Val Glu Leu Pro Glu Arg Wing Gly Arg 695 700 705 cgg tgg gag ceg gtg gtg gac here ggc aag cea gca cea tac gac ttc 2801 Arg Trp Glu Pro Val Val Asp Thr Gly Lys Pro Wing Pro Tyr Asp Phe 710 715 720 ctc acc gac gac tta cct gat cgc get ctc ace ata cac cag ttc teg 2849 Leu Thr Asp Asp Leu Pro Aap Arg Ala Leu Thr He Hia Gln Phe Ser 725 730 735 740 ßat ttc ctc tao tco aac ctc tac coo atg ctc ago tac tea teg gtc 2997 His Phe Leu Tyr Ser Asn Leu Tyr Pro Met Lau Ser Tyr Ser Ser Val 745 750 755 atc cta gta ttg cgc cct gat gtt tga gag ace aat ata tac agt aaa 2945 He Leu Val L «u Arg Pro Asp Val * Glu Thr Aan Il« a Tyr S * r Lya 760 765 770 taa tat gtc tat atg aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa Val Tyr Met 775 < 210 > 2 < 211 > 2997 < 212 > DNA < 213 > Triticum aestivum L. cv. Florida < 400 > 2 ggtcggggcc ggcgcegcgc ctgcgacggt ggcgacacaa tgcgacggcg gggaaggggg 60 tcggcgaggt gtgcgccgcg gttgtcgagg eggcgacgaa ggtagaggae gagggggagg 120 aggacgagec ggtggcggag gacaggtacg egeteggegg egegtgeagg gtgetegaeg 180 gaatgcccgc gccgctgggc gccaccgcgc tcgccggcgg ggtcaatttc gccgtctatt 240 ccggcggagc caccgccgcg gcgctctgcc tcttcacgcc agaagatctc aaggcggtgg 300 ggttgcctcc cgagtagagt tcatcagctt tgcgtgegcc gcgcgcccct tttttgggcc 360 tgcaatttaa gttttgtact ggggeaaatg etgeaggata gggtgaeega ggaggttccc 420 cttgaccccc tgatgaatcg gaccgggaac gtgtggcatg tcttcotcga aggcgagctg 480 caeaacatgo ttasgggta caggttegae ggcaectttg oteotoaotg cgggcaetac 540 ettsatsttt ccaatgtcgt ggtggatcct tatgetaagg cagtgataag ccgaggggag 600 tatggtgttc cagegegtgg taacaattga tggeotcaga tggetggeat gateeetett 660 ccatatagca cgtttgattg ggaaggcgac ctacctctaa gatatcctca aaaggacctg 720 g aatatatg agatgcactt gcgtggattc acgaagcatg attcaagcaa tgtagaacat 780 ccgggtaett tcattggagc tgtgtcgaag cttgactatt tgaaggaget tggagttaat 340 tgtattgaat taatgceet g ccatgagttc aacgagctgg agtaoteaae etcttettßc 900 tttggggata aagatgaact ttctaccata aacttctttt aagatacaca caccaatgac 960 tcaggcggga tsaaaaactg tgggegtgat gccataaatg agttcaaaac ttttgtaaga 1020 gaggeteaca aacggggaat tgaggtgatc etggatgttg tettcaaaca taaagctgag 1080 ggtaatgaga atggtccaat attateattt aagggggtcg ataatactac atactatatg 1140 ettgeaccca agggagagtt ttataactat tetggetgtg ggaatacctt caactgtaat 1200 catcctgtgg ttcgtcaatt oattgtagat tgtttaagat aetgggtgae ggaaatgcat 1260 ttcgttttga gttgatggtt tcttgcatee ataatgacca gaggttccag tctgtgggat 1320 ccagttaacg tgtatggage tccaatagaa ggtgacatga tcacaacagg gacacctctt 1380 cacttattga gttactcaaa oatgatoagc aatgacocaa ttottggagg cgtoaagotß 1440 attscteraaa catsggatgc aggaagcctc tatcaagtas steaatteec tcactggaat 1500 gtttggtctg agtggaatgg gaagtaecgg gacattgtga gteaatteat taaaggeact 1560 gatggatttg ctggtggttt tgccgaatgt ctttgtggaa gtccacacct ataccaggta 1620 atacttgtaa agttgtggca atgagttgag tgaatgtcac ctggattttt tatatatacc 1680 aoatgatgat acacatctaa ata tataaca atcatagtgt atgcatatgc atttggctaa 1740 gaagtattag tgtatacact agtgctatat ataggtttta acacccaact tgccaatgaa 1800 ggaacatagg getttctagt tatettattt atttgtsegg tgaataatcc actgaaaaat 1860 tccsgccatg tcatttttta gg gg aga agaaactata ttgatttgcc cccctaaaag 1920 aageeatc and agaattoata ggtaagttge ttttetgtaa agaaaggaaa aegaetteat 1980 actttctatc ggtgctaact tagctcgatg tatatttgta agatgaatgc caaatttaat 2040 ttgtcggata atttgatetg ttatteacaa atttetattt ggtttctata gaaateaaae 2100 cagtaacttg ttattggcac tgcaacttct tattgattaa tcaggcagga ggaaggaaac 2160 cttggcacag tatcaacttt gtatgtgcac atgatggatt tacactggct gatttggtaa 2220 catataataa gaagtacaat ttaccaaatg gggagaacaa gaaaatcaca cagagatgga 2280 atcttagctg gaattgtggg gaggaaggag aattcgcaag attgtatgtc aaaagattga 2340 ggaagaggea gatgcgcaat ttctttsttt stcteatggt ttctcaaaga gttccaatgt 2400 tctocatggg tgatgaatat ggccacacaa aagggggeaa ßaacaataoa tactgocatg 2460 attcttatgt caattatttt cgctgggata aaaaagaaea atactctgag ttseaccsat 2520 tctgctgcct catgaccaaa ttccgeaag g agtgcgaggg tcttggcctt gaggactttc 2580 eaaeggccaa acggctgcag tggeatggta atcagcctgg gaagcotgat tggtctgaga 2640 atagccgatt cgttgccttt tccatgaaag atgaaagaca gggcgagatc tatgtggcct 2700 tcaacaccag ccacttaccg gcegttgttg agetcccaga gcgcgcaggg cgceggtggg 2760 aaccggtggt ggacacaggc aagccagcac catacgactt cctcaccgac gacttaeetg 2820 ategegetct eaccatacae cagttetcge atttectcta ctccaacctc taccccatgc 2880 teagctactc atcggtcatc ctagtattgc gccctgatgt ttgagagacc aatatataca 2940", taaataata tgtetatatg taaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaa 2997 < 210 > 3 < 211 > 764 < 212 > PRT < 213 > Triticum aestivum L. cv. Florida < 400 > 3 Ser Oly Pro Wing Pro Arg Leu Arg Arg Trp Arg Pro Asn Wing Thr Wing 1 5 10 15 Gly Lys Gly Val Gly Glu Val Cya Ala Ala Val Val Glu Ala Wing Thr 20 25 30 Lys val Glu Asp Glu Glu Glu Glu Asp Glu Pro Val Wing Glu Asp Arg 35 40 45 T r Ala Leu ßly Gly Ala Cys Arg Val Leu Ala Gly Met Pro Pro Ala 30 55 so Leu Gly Ala Thr Ala Leu Ala ßly aly val Aan Phe Ala val Tyr be 65 70 75 80 Gly Gly Ala Thr Ala Ala Ala Leu Cys Leu Phe Thr Pro Glu Asp Leu 85 90 95 Lya Wing Asp Arg val Thr Glu Glu val Pro Leu ASD Pro Leu Met Asn 100 105 110 Arg Thr Gly Asn Val Trp Hiß Val Phe He Glu Gly Glu Leu Hia Asn H5 120 125 Met Leu Tyr aly Tyr Arg Phe Asp sly Thr Pha Ala Pro His cys ßlv 130 135 140 His Tyr Leu Asp val be Aßn val val val Aßp pro Tyr Ala Lys Ala 145 150 155 160 val lie be Arg aiy aiu Tyr Gly val pro Ala Arg Gly Asn Asn cys 165 170 175 Trp Pro Gln Met Wing Sly Mefe Pro Pro Leu Pro Tyr Ser Thr Phe Asp iaa isa 190 Trp Glu Gly Aßp Leu Pro Leu Arg Tyr Pro Gln Lya Aßp Leu val He 195 200 205 'Tyr Glu Met His Leu Arg Gly Phe Thr Lys His Asp Ser Ser Aan Val 210 215 220 slu Hie Pro ßly Thr Phe lie Gly Ala val be Lya Leu Aßp Tyr Leu 225 330 235 240 Lyß Glu Leu aly Val Asn Cya He Glu Leu Met Pro Cyß Hia Glu Phe 245 250 255 Aan Olu Lau Glu Tyr be Thr ser be Lys Mac Aßn Phß Trp Gly 260 265 a ™ Tyr Ser Thr He Asn he Phe Ser Pro Met Thr Arg Tyr Thr ser 3ly 27S 2S0 285 _ Gly lie Lya Aßn Cyß Gly Arg Asp Ala He Asn ßlu Phe Lyß Thr Phe ° a90 293 300 Val Arg Glu Ala l? Io Lya Arg Gly Xle ßlu Val Zle Leu Asp val Val 305 310 315 320 Phe Aßn His Thx Ala? Lu ßly Aßn ßlu Asn Gly Pro He Leu Ser Phe 32S 330 335 Lya ßly Val Asp Asn Thr Thr Tyr Tyr Met Leu Wing Pro Lys Gly Olu 340 345 350 Phe Tyr Asn Tyr be Gly cys Gly Asn Thr Phe Asn Cys Asn His Pro 355 360 365 Val Val Arg Gln Phe He Val Asp Cys Leu Arg Tyr Trp Val Thr Glu 370 375 380 Mee His val Asp Gly he Arg Phe Asp Leu Ala be He Mee Thr Arg 385 390 395 400 Gly be Leu Trp Aßp Pro val Asn val Tyr Gly Wing Pro He ßlu 40S 410 41S Gly Asp Mee ie xnr Thr Gly Thr Pro Leu val Tur pro Pro Leu pe 420 425 430 Asp M? T He Ser As Asp Pro He Leu Gly Gly Val Lys Leu He Wing 435 440 445 Glu Wing Trp Aßp Wing Gly Gly Leu Tyr Gln val Gly Gln Phe pro Hia 450 455 460 Trp Aan val trp be Glu Trp Aßn Gly Lyß Tyr Arg Asp He val Arg 465 470 475 480 Gn Phe He Lys ßly Thr Asp ßly Phe Ala ßly ßly Phe Ala ßlu cys 485 490 495 Leu Cys Gly Ser Pro Hiß Leu Tyr Gln Wing Gly Gly Arg Lys Pro Trp 500 505 510 Hia Ser He Asn Phe Val Cys Wing His Aßp ßly Phe Thr Leu Ala Aßp S15 520 525 Leu val Thr Tyr Aan Lys Lys Tyr Asn Leu pro Asn ßly ßlu Asn Asn 530 535 540 Arg Asp Gly Glu Aan His Asn Leu be Trp Asn cys ßly ßlu ßlu ßly 545 S50 555 560 Sl- »he Ai» rg Leu S »r Val Lyß Arg Leu Arg Ly» Arg olp. Mßt Arg SS5 S70 S75 Aan Phe Phß Val Cya Leu Met Val Ser 31n Sly Val Pro Mßt Phe Tyr S80 S85 590 Met Qly Aap Slu Tyr Cly Ria Thr Lya aly Oly Aßan Aan Aan Thr Tyr S9S COO SOS Cya Lyß Lyß Glu wing Tyr Ser Olu Leu wing Arg Phe Cya Cya Leu Het Thr Lya Phe Arg Lyo 625 £ 30 635 640 Olu Cya aiu Sly Leu Oly Leu Slu ap Phe Pro Thr Ala Lya Arg Leu S4S £ 50 £ 55 Gln Trp Hia Qly Hia Ola Pro aly Lya Pro Aßp Trp Ser Olu Aßn Ser 660 665 670 Arg Phe Val Ala? Ha Ser Met Lya Aap Slu Arg Ola aly aiu He Tyr 67S 680 SßS Val Wing Phe Aßn Thr Ser Sio Leu Pro Wing Val Val Qlu Leu Pro ßlu 690 595 700 Arg Wing Arg Arg Trp ßlu Pro Val Val? ßp Thr ßly Lya Pro Wing 70S 710 71S 720 Pro Tyr Asp Phe Leu Thr Asp Aßp Leu Pro Asp Arg Ala Leu Thr lio 725 730 735 Hia Glp. Phß Ser Hia Phe Lau Tyr Ser Aan Leu Tyr ro Mßt Leu Ser 740 74S 750 Tyr Ser Ser V l Ilc Leu Val Lou Arg Pro Aßp Val 755 760 < 210 > 4 < 211 > 24 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the artificial sequence: initiator < 400 > 4 aaaggcccaa tattateett tagg 2 «< 210 > 5 < 211 > 31 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the artificial sequence: initiator < 400 > 5 gccatttsaa ccgttctgaa gtcgggaagt c < 210 > 6 < 211 > 2437 < 212 > DNA < 213 > Triticum aestivum L. cv. Florida < 220 > < 222 > CDS < 223 > (16) ... (2304) < 400 > 6"" ""., ".,« «-" g, gj JOJ g. j; JW? JJJ «" «« - «« H ^ 5 i? S3 S! SS 5! I !? ? S? S S! S! S K H! S Yes 3H VI n fi 35 ! is f "t gae agg tac gcg ctc ggc ggc gcg tgc agg gtg ctc gee gga atg ecc 195 acg ceg ctg gge gee aoc gcg ctc gcc ggc gss ste aat ttc gcc gte 243 Thr Pro Leu Gly Ala Thr Ala Leu Ala Gly Gly Val Aan Phe Wing Val 65 70 75 tac tce ggc gga gcc here gcc geg gcg etc tge ctc ttc acg cea gaa 291 ivr Ser G1V Gly Ala Thr Ala Ala Ala Leu Cya Leu Phe Thr Pro Glu 30 95 90 gat ete aag gcg gat agg gtg acg gag gag gtt eee ett gac ecc ctg 339 Asp Leu Lys Wing Asp Arg Val Thr Glu Val Val Leu Asp Pro Leu 95 100 Ios atg aat cgg act ggg aac gta tgg cat * c ttc ate gaa ggc gag ctg 387 Met Asn Arg Thr Gly Aan to Trp Hls val Phe He Glu Gly Glu Leu 110 115 120 oag sa atg ett tae ggg tac agg ttc gae gge ace ttt gct cct drops 435 gaac ßin AAsspp Met Leu Tyr Gly Tyr Arg Phe Asp Gly Thr Phe Ala Pro His 125 0 135 140 tac ggg falls tac ett gat gtt toe aat gta gtg gtg gat cct tat gct 483 Cvs Glv His Tyr Leu Asp Val 3er Asn al al al Aap Pro Tyr Ala * 143 150 155 aag gca gtg ata age cga ggg gag tat ggt gtt acg gcg egt ggt aae 531 Lya Ala val He Ser Arg Gly siu Tyr Gly val Pro Ala Arg Sly Aan 160 165 170 aat tge tgg oot aag atg got gga atg ato ect ett coa tat age acg 579 Asn Cya Trp Pro Gln Met Wing Gly Met He Pro Pro Leu Pro Tyr Ser Thr 175 180 1S5 ttt gat tgg gaa gga gac cta act ota aga tat act ca aag gac ctg 627 Phß Aap Trp Glu Gly Aßp Leu Pro Leu Axg Tyr Pro Gln Lya Aap Leu 190 195 200 gta ata tat gag atg cac ttg cgt g? A tte acg aag eat gat tea age € 75 Val He Tyr Glu Met His Leu Arg Gly Phe Thr Lys Hia Aap Ser Ser 205 210 215 220 aat gta gaa cat ccc ggt act ttc att ggg gct gtg teg aag ett gac 723 Aan Val Glu His Pro Gly Thr? He He Gly Wing Val Ser Lya Leu Asp 225 230 235 tat ttg aag gag ett gga gtt aat tgt att gag tta atg ccc tgc cat 771 Tyr Leu Lya Glu Leu Gly Val Asn Cya He Glu Leu Met Pro Cys His 240 245 250 gag ttc aac gag ctg gag tac tea acc tet tet tcc aag atg aac ttt T19 Glu Phe Aan Glu Leu Glu Tyr Ser Thr Ser Ser Lyß Met Asn Phe 2S5 260 265 t ra rat tet acc ata a tc t t t t t ate t a t t a t t a t t t t t t t t t t t t t t t t t t t t Tyr Thr 270 275 280 tea? R e agg ata aaa aae tgt ggg cgt gat gcc ata aat gag ttc aaa 915 Ser Gly 31y He Lys Asn Cys Gly Arg Aap Wing He Aon Glu Phe Lya 285 290 295 300 act ttt gta aga gag gct cac aaa cgg gga att gag gtg atc ctg gat 963 Thr Phe Val Arg Glu Wing Hi3 Lya Arg Gly He Glu Val He Leu Asp 30S 310 315 gtt gte tte aae cat gg sa ggt aat sg aat ggt eea atatta 1011 Val Val Phe Aan Hia Thr Ala Glu Giy Asn Glu Asn Gly Pro He Leu 320 325 330 tea ttt agg ggg gtc gat aat act tac tat atg ett gca CCC aag 1059 Ser Phe Arg Gly Val Aap Asn. Thr Thr Tyr Tyr Met Leu Wing Pro Lya 335 340 345 gga gag ttt tat aac tat tet ggc tgt ggg aat ace ttc aac tgt aat 1107 Gly Glu Phe Tyr Asn Tyr Ser Gly Cys Gly Aan Thr Phe Aan Cya Asn 350 355 360 cat cct gtg gtt cgt cat ttc att gta gat tgt tta aga tac tgg gtg 1155 H13 Pro val val Arg Gln Píie He val Aap cys Leu Arg Tyr Trp val 365 370 375 380 aeg gaa atg cat gtt gat ggt ttt egt ttt gat ett gea tee ata atg 1203 Thr Glu Met His Val Asp Gly Phe Arg Phe Aap Leu Ala Ser He Met 385 390 395 acc aga ggt toe agt ctg gat eea gtt aae gtg tat gga get oca 1251 Thr Arg siy ser ser Leu Trp Aßp pro < val Asn val Tyr Gly Wing Pro 400 403 410 ata gaa ggt gac atg atc here ggg ac ect ett gtt aet cea cea 1299 He Glu Gly Asp Met He Thr Thr Gly Thr Pro Leu Val Thr Pro Pro 415 420 425 ett att gac atg atc age aat gac cea att ett gga ggc gtc aag cte 1347 Leu He Aep Met He Ser Aan Aap Pro llß Leu Gly Gly Val Lys Leu 430 435 440 gtt gct gca tgg gat gca gga ggc ctc tat ca gta gta gta ca ttc 1395 Val Ala Glu Ala Trp Aap Ala Sly Gly Leu Tyr Gln Val Gly Gln Phe 445 450 455 460 cct cag tgg aat gtt tgg tet gag tgg aat ggg aag tac cgg gac att 1443 Pro His Trp Afin Val Trp Ser Glu Trp Aan Gly Lys Tyr Arg Asp Ha 465 470 475 gtg cgt cat ttc att aaa ggc act gat gga ttt gct ggt gtt ttt gcc 1491 Val Axg Gln Pile He Lys Gly Thr Asp Gly Phe Wing Gly Gly Phe Wing 480 4S5 490 gaa tgt ett tgt gga agt cea cac cta tac cag gca gga gga agg aaa 1539 Glu Cye Leu Cya Gly Ser Pro Hia Leu Tyr Gln Wing Gly Gly Arg Lys 495 500 505 cet tgg eac agt atc aaa ttt gta tgt gca cac gat gga ttt here ctg 1537 Pro Trp His Ser He Aan Phe Val Cya Wing His Asp Gly Phe Thr Leu 510 515 520 get gat ttg gta ac tat aat aat aat tata aat tta oca aat ggg gag 1635 Wing Asp Leu Val Thr Tyr Aan Aan Lya Tyr Aan Leu Pro Aan Gly Glu 525 530 535 540 aa aa aga aga gat gga gaa aat cao aat ott age tgg aat tgt ggg gag 1693 Asn Aan Arg Asp Gly Glu Aan Hia Aan Leu be Trp Asn Cys Gly Glu 545 550 555 gaa gga gaa ttc gca aga ttg tet gtc aaa aga ttg agg aag agg cag 1731 Giu Giy aiu Fiia Wing Arg Leu be val Lys Arg Leu Arg Lya Arg Gin 560 565 S70 atg ege aat tet ttt gtt tgt etc atg gtt tet ea gga gtt cea atg 1779 Met Arg Asn Phe Phe Val Cys Leu Met Val Ser Gln Gly Val Pro Met 575 530 585 tte tae atg ggt gat gaa tat gge eae aea aaa ggg ggc aae aat aat 1827 Phß Tyr Met Gly Asp Glu Tyr Gly His Thr Lys Gly Gly Aan Aan Asn 590 595,600 ac tac tgc cat gat tet tat gte aat tat ttt ege tgg gat aaa aaa 1975 Thr Tyr Cy? Hia Aßp Ser Tyr Val Asn Tyr Phe Arg Trp As Lya Lya 605 610 615 620 gaa eaa tae tet gac ttg eae tte tgt tgc tgc etc atg aaa tta 1923 Glu Sin Tyr Ser As Lau Hia Arg Phe Cys Cya Leu Met Thr Lys Phe 625 630 635 cgc aag gag tge gag ggt ett gge ett gag gat ttt eea aeg gec gaa 1971 Arg Lys Glu Cys Glu Gly L «u Gly Leu Glu Aap Phe Pro Thr Wing Glu fi d 645 645 cgg cg tg cag tgg cat ggt eat cag ect ggg aag cct gat tgg tet gag 2019 Arg Leu Gln Trp His Gly His Gln Pro Gly Lys Pro Asp Trp Ser Glu 655 660 665 aat age ega ttc gtt gee ttt tcs atg aaa gat gaa aga cag ggc gag 2067 A3n Ser Arg Phe Val Ala Phe Ser Met Lya Asp Glu Arg Gln Gly Glu 670 675 680 ate tat gtg gee ttc aac age eae tta eeg gee gtt gtt gag eto 2115 He Tyr Val Ala Phe Asn Thr Ser His Leu Pro Ala Val Val Glu Leu 685 690 695 700 ceg gag cgc here ggg cgc cgg tgg gaa ecg gtg gtg gac here ggc aag 2163 Pro Glu Arg Thr Gly Arg Arg Trp Glu Pro Val Val Asp Thr Gly Lys 705 710 715 eea gea cea tac gac tte ctc act gac gac tta ect g at ege gct ete 2211 Pro Wing Pro Tyr Asp Ph * L «u Thr Asp Asp Leu Pro Aap Arg Wing Leu 720 725 730 ace ata cac tg tt tet cat ttc ctc aac tcc aac ctc tac cec atg 2259 Thr He His Gln Phß Ser His Phe Leu Asn Ser Asn Leu Tyr Pro Met 735 740 745 ctc age tac tea teg gtc atc cta gta ttg cgc cct gat gtt tga 2304 Leu Ser Tyr Ser Val Val Leu Val Leu Arg Pro Asp Val 750 755 760 gaggsggata tacagtaaat aatatgtata tatgtagtcc tttggegtat tatcagtgtg 2-64 cacaattget etattgecaa tgatetatte gatecacaga tacatgtgca aaaaaaaaaa 2424 aaaaaaactc gag 2437 < 210 > 7 < 211 > 762 < 212 > PRT < 213 > Triticum aestivum L. cv. Florida < 400 > 7 Pro Ala Pro Arg Leu Arg Arg Trp Arg Pro Asn Ala Thr Ala Gly Lys 1 5 10 15 Gly Val Gly Glu Val Cys Aia Wing Val Val Glu Val Wing Thr Lys Wing 20 25 30 Glu Asp Glu Glu Glu Glu Asp Glu Pro Val Wing Glu Asp Arg Tyr Wing 35 40 45 'Leu Gly Gly Aia Cya Arg Val Leu Wing Gly Met Pro Thr Pro Leu Gly 50 55 60 Ala Thr Ala Leu Ala Gly Gly Val Aan Phe Ala Val Tyr Ser Gly Gly 65 70 75 60 Ala Thr Ala Ala Ala Leu Cys Leu Phe Tnr Pro Glu Aap Leu Lys Ala T3 90 95 Asp Arg Val Thr Glu Glu Val Pro Leu Aap Pro Leu Met Aan Arg Thr 100 105 110 Gly Aan Val Trp His Val Phe He Glu Gly Glu Leu Gln Asp Met Leu 115 120 125 Tyr Gly Tyr Arg Phe Asp Gly Thr Phe Wing Pro Hia Cys Gly His Tyr 130 135 140 Leu Asp Val Ser Aßn Val Val Val Aap Pro Tyr Ala Lyß Ala Val He 145 150 155 160 Being Arg ßly Glu Tyr Gly Val Pro Wing Arg Gly Asn Aan Cya Trp Pro 165 170 175 Gln Met Wing Gly Met He pro Pro Leu pro Tyr Ser Thr Phß Asp Trp Glu 180 135 190 Gly Asp Leu Pro Leu Axg Tyr Pro Gln Lys Asp Leu Val Ha Tyr Glu 195 200 203 Met Hls Leu Arg Gly Phe Thr Lys His Aep Ser As As Val Glu Hia 210 215 220 Pro Gly Thr Phe He Gly Ala Ser Lys Leu Asp Tyr Leu Lys Glu 225 230 235 240 Leu Gly Val Asn Cys XI »Glu Leu Met Pro Cys His Glu Phe Asn Glu 245 250 253 Leu Glu Tyr Being Thr Being Being Lys Met Aßn Phe Trp Gly Tyr Ser 260 265 270 Thr He Asn Phe Phe Ser Pro Met Thr Arg Tyr Thr Ser Gly Gly He 275 2T0 295 Lys Asn Cys Gly Arg Asp Wing He Asn Glu Phe Lys Thr Phe Val Arg 290 295 300 Glu Wing His Lys Arg Gly He Glu Val He Leu Asp Val Val Phe Asn 305 310 315 320 His Thx Wing Glu Gly Asn Glu Aan Gly Pro He Leu Ser Phe Axg Gly 325 330 335 Asp Asn Thr Thr Tyr Tyr Met Leu Wing Pro Lys Gly Glu Phe Tyr 340 345 350 Aßn Tyr Ser Gly Cys Gly Asn Thr Ph9 Asn Cys Asn His Pro Val Val 355 360 365 Arg Gln Phe Xle Val Asp Cya Leu Arg Tyr Trp Val Thr Glu Met Hia 370 375 380 Val Aap Gly Pha Arg Phe Aap Leu Wing Being He Met Thr Arg Gly Ser 385 390 395 400 Ser Leu Trp Asp Pro Val Asn Val Tyr Gly Ala Pro He Glu Gly Asp 405 410 415 Met He Th »Thr Gly Thr Pro Leu Val Thr Pro Pro Leu He Aβp Met 420 425 430 He Ser Asn Aßp Pro He Leu Gly Gly Val Lys Leu Val Wing Ala Glu Wing 435 440 445 Trp Asp Wing Gly Gly Leu Tyr Gln Val ßly Gln Phe Pro His Trp Asn 450 455 460 Val Trp Ser Glu Trp Asn Gly Lyc Tyr Arg Asp He Val Arg Gln Pha 465 470 475 4T0 He Lys Gly Thr Asp Gly Phe Wing Gly Gly Phe Wing Glu Cys Leu Cys 4T5 490 495 Gly Ser Pro His Leu Tyr Gln Wing Gly Gly Arg Lys Pro Trp His Ser 500 505 510 He Asn Phe Val Cys Wing His Asp Gly Phe Thr Leu Wing Aßp Leu Val 515 520 525 Thr Tyr Asn Asn Lys Tyr Asn Leu Pro Asn Gly Glu Asn Asn Arg Asp 530 535 540 Gly Glu Asn His Aan Leu Ser Trp Asn Cys Gly Glu Glu Gly Glu Phe 545 550 555 560 Wing Arg Leu S9X Val Lyß Arg Leu Arg Lys Arg ßln Met Arg Asn Phe 565 570 575 Phe Val Cys Leu Met Val Ser Gln Gly Val Pro Met Phe Tyr Met Gly 530 595 590 Asp Glu Tyr Gly His Thr Lys Gly Gly Asn Asn Asn Thr Tyr Cys His 393 600 605 Asp Ser Tyr Val Aan Tyr Phe Arg Trp Asp Lys Lys Glu Gln Tyr Ser 610 615 620 Asp Leu His Arg Phe Cys Cys Leu Met Thr Lys Phe Arg Lya Glu Cys 625 630 635 640 Glu Gly Leu Gly Leu Glu Asp Phe Pro Thr Glu Wing Arg Leu Gln Trp 645 650 655 His ßly His Gln Pro Gly Lys Pro Asp Trp Ser Glu Asn Ser Arg Phe 660 665 670 Val Wing Phe Ser Met Lys Asp G¿u Arg Gln Gly Glu He Tyr Val Wing 675 690 685 Phe Asn Thr Ser His Leu Pro Wing Val Val Glu Leu Pro Glu Arg Thr 690 695 700 Gly Arg Arp Trp Glu Pro val val Asp Thr Gly Lys Pro Wing Pro Tyr 705 710 715 720 Asp Phe Leu Thr Asp Asp Leu Pro Asp Arg Ala Leu Thr Zle His Gln 725 730 735 Phe Ser His Phe Leu Asn Ser Asn Leu Tyr Pro Mßt Leu Ser Tyr Ser 740 743 750 Ser val Ha Leu val Leu Arg Pro Asp val < 210 > 8 < 211 > 20 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the artificial sequence: initiator < 400 > 8 gctttacggg tacaggttcg 20 < 210 > 9 < 211 > 18 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the artificial sequence: initiator < 400 > 9 18 aattccccgt ttgtgagc < 210 > 10 < 211 > 51 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the artificial initiator sequence > 400 > 10 51 goggtac oto tagaaggaga tataeatatg gaggagg »" ggtaegeget < 21O > 11 < 211 > 33 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the artificial sequence: initiator < 400 > 11 33 gctcgagtcg aeteaaaeat eagggcgcaa tac

Claims (24)

NOVELTY OF THE INVENTION CLAIMS

1. - A nucleic acid molecule that codes for a protein with the function of a wheat isoamylase, selected from the group consisting of a) a nucleic acid molecule that codes for a protein comprising the amino acid sequence shown under SEQ ID No. 3, b) a nucleic acid molecule comprising the nucleotide sequence shown under SEQ ID No. 2 or a part thereof, or a sequence of ribonucleotides corresponding thereto; c) a nucleic acid molecule that hybridizes with one of the nucleic acid molecules mentioned under a) or b) or is complementary thereto, and d) a nucleic acid molecule whose nucleotide sequence deviates from the sequence of a molecule of nucleic acid mentioned under a), b) or c) due to the degeneracy of the genetic code, the nucleic acid molecule mentioned under a), c) and d) having a homology of more than 90% with SEQ ID No.

2. .- A nucleic acid molecule according to claim 1, further characterized in that it is a DNA molecule.

3. A DNA molecule according to claim 2, further characterized in that it is a cDNA molecule.

4. A nucleic acid molecule according to any of claims 1 to 3, further characterized in that it contains regulatory elements.

5. - A nucleic acid molecule according to claim 1, further characterized in that it is an RNA molecule.

6. A nucleic acid molecule that hybridizes specifically with a nucleic acid molecule according to any of claims 1 to 5 and has a homology of more than 90% with SEQ ID No. 2.

7.- A molecule of nucleic acid according to claim 6, further characterized in that it is an oligonucleotide with a length of at least 15 nucleotides.

8. A vector containing a DNA molecule according to any of claims 1 to 5.

9. A vector according to claim 8, further characterized in that said nucleic acid molecule is linked in sense direction to regulatory elements. that ensure the transcription and synthesis of a translatable RNA in pro- or eukaryotic cells.

10. A vector according to claim 8, further characterized in that said nucleic acid molecule binds in sense orientation to regulatory elements that ensure the synthesis of a nontranslatable RNA in pro- or eukaryotic cells.

11. A vector according to claim 8, further characterized in that said nucleic acid molecule binds in antisense orientation to regulatory elements that ensure the synthesis of a nontranslatable RNA in pro- or eukaryotic cells.

12. - A host cell that is transformed with a nucleic acid molecule according to any of claims 1 to 5, or with a vector according to any of claims 8 to 11, or derived from said cell.

13. A protein encoded by a nucleic acid molecule according to any of claims 1 to 4.

14. A process for the preparation of a protein according to claim 13, wherein a host cell in accordance with the Claim 12 is cultured under conditions that allow said protein to be synthesized, and said protein is isolated from the cultured cells and / or the culture medium.

15. A method for generating a transgenic plant cell, wherein a) a nucleic acid molecule according to any of claims 1 to 5 or b) a vector according to any of claims 8 to 11 is integrated into the genome of a plant cell.

16. A transgenic plant cell that has been transformed with a nucleic acid molecule according to any of claims 1 to 4 or with one or more vectors according to any of claims 8 to 11 or that is derived from said cell .

17.- A procedure to generate a transgenic plant cell, wherein a1) a nucleic acid molecule according to any of claims 1 to 5 or a2) a vector according to any 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 that contains a plant cell according to claim 16.

19. A plant according to claim 18, further characterized in that it is a crop plant.

20. A plant according to claim 19, further characterized in that it is a plant that stores starch.

21. A plant according to claim 20, further characterized in that it is a monocotyledonous plant or corn.

22. A plant according to claim 21, further characterized in that it is a plant of barley, rye or wheat.

23. A propagation material of a plant according to any of claims 18 to 22. 24.- The use of a plant cell as claimed in claim 16, of a plant according to any of the claims 18 to 22, or of a propagation material according to claim 23 for the production of starch.