CZ20004155A3 - Molecules of nucleic acids encoding enzymes from wheat and which take part in starch synthesis - Google Patents

Abstract

The present invention relates to nucleic acid molecules encoding enzymes involved in starch synthesis in plants. These enzymes include wheat isoamylases. Next, the solution relates vectors and host cells containing said molecules nucleic acids, especially transformed plant ones cells and plants that can be regenerated from these cells and which exhibit increased or decreased isoamylase activity.

Description

NUCLEIC ACID MOLECULES CODING WHEAT ENZYMES, WHICH PARTICIPATE IN STARCH SYNTHESIS

Technical field

The present invention relates to nucleic acid molecules encoding an enzyme from wheat which is involved in the synthesis of starch in plants. This enzyme is isoamylase.

Further, the present invention relates to vectors, host cells as well as plant cells and plants comprising the nucleic acid molecules of the invention.

Further described is a process for obtaining transgenic plants which, upon introduction of the nucleic acid molecules of the invention, synthesize starch with some altered property.

BACKGROUND OF THE INVENTION

Recently, the importance of plant-based substances as a renewable resource of raw materials has been steadily increasing and therefore one of the tasks of biotechnology research is to strive to adapt these plant-derived materials to the requirements of the processing industry. In order to maximize the usability of these renewable raw materials, it is essential to maximize their diversity.

In addition to oils, fats and proteins, polysaccharides are an important renewable vegetable raw material. The most important position among polysaccharides is in addition to cellulose starch, which is one of the main storage substances in higher plants. One of the most important crop plants is wheat, from which 20% of the total starch production is obtained in the European Community.

The starch polysaccharide is a polymer consisting of chemically uniform base units, namely glucose molecules. This is a very complex mixture of different molecular forms, which differ in their degree of polymerization, branching of glucose chains and their length, and which, moreover, can be derivatized, for example phosphorylated. This means that starch is not a uniform raw material. Amylose, which consists of a substantially unbranched polymer consisting of 1,4-glycosidically linked glucose molecules, differs from amylopectin, which is a complex mixture of differently branched glucose chains. 1,6-glycosidic linkages also contribute to this branching. Starch synthesized in wheat contains about 11 to 37% amylose.

In order to ensure the applicability of suitable starches for a wide variety of industrial purposes, there is a need for plants that are capable of synthesizing modified starches that are particularly suitable for a variety of uses. One of the possibilities for obtaining such plants is breeding measures. However, breeding measures are very difficult to apply due to the polyploid nature of wheat (tetraa hexaploid). Only recently, by crossing natural wheat mutants, waxy (amylose-free) wheat was obtained (Nakamura et al., Mol. Gen. Genet. 248 (1995), 253-259).

An alternative to the breeding measures is the targeted geography. A new-technological modification of starch-producing plants. However, the prerequisite for this is the identification and characterization of enzymes involved in the synthesis or modification of starch, as well as the isolation of nucleic acid molecules that encode these enzymes.

The routes of biochemical synthesis leading to starch formation are essentially known. The synthesis of starch in plant cells takes place in plastids. They are chloroplasts in photosynthetically active tissues, and amyloplasts in photosynthetically inactive starch storage tissues.

For further targeted genetic engineering of branching of starch synthesized in plants, it is still necessary to identify DNA sequences coding for enzymes involved in starch metabolism, in particular chain branching or degradation.

In addition to the so-called Q-enzymes which introduce branching into the starch molecule, there are enzymes in the plants which can break down the branching. These enzymes are referred to as Debranching enzymes and are classified into three groups according to their substrate specificity:

(a) Pullulanases, which in addition to pullulan need amylopectin as a substrate, are found in microorganisms such as Klebsiella and in plants.

In plants these enzymes are also referred to as R-enzymes.

(b) Isoamylases which do not need pullulan as a substrate but glycogen and amylopectin also occur in microorganisms and in plants. Isoamylases have been described, for example, in maize (Manners & Carbohydr. Res. 9 (1969), 107) and potatoes (Ishizaki et al., Agric.

Biol. Chem. 47 (1983) 771-779).

(c) Amylo-1,6-glucosidases are described in mammals and yeast and need borderline dextrins as substrate.

Li et al. (Plant Physiol. 98 (1992), 1277 - 1284), in addition to five endo- and two exoamylases, only one Debranching-enzyme of the pullulanase type was found in sugar beet. This enzyme of about 100 kD with an optimum pH of 5.5 is located in chloroplasts. Also, in spinach, one Debranching enzyme has been described using pullulan as a substrate.

Both spinach and sugar beet debranching enzymes have five times less activity in reaction with amylopectin as a substrate 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 an economically important starch-storing crop plant - in potatoes - they monitored the activity of Debranching-enzyme Hobson et al. (J. Chem. Soc., (1951), 1451). These authors have shown that the enzyme in question, unlike the Q-enzyme, exhibits no chain-extending activity but merely hydrolyzes alpha-1,6-glycosidic bonds. However, this enzyme has not yet been characterized in more detail.

In potatoes, a procedure for purifying the Debranching enzyme as well as partial peptide sequences of the purified protein has already been proposed (WO 95/04826). Meanwhile, spinach has been described to purify the Debranching enzyme as well as to isolate the appropriate cDNA (Renz et al., Plant Physiol. 108 (1995), 1342).

In the case of maize, only the existence of one Debranching enzyme has so far been described in the literature. This enzyme has been classified into the isoamylase family according to its substrate specificity (see, e.g., Hannah et al., Scientia Horticulturae 55 (1993), 177-197 or Garwood (1984) in Starch Chemistry and Technology, Vhistler, RL, BeMiller, JN, Puschall, EF (eds.), Academic Press of San Diego, New York, Boston, 25-86). The respective mutants were designated as sugars. This sugar center locus gene was briefly cloned (see James et al., Plant Cell 7 (1995), 417-429). In addition to the sugary-locus center, no other gene center (Genlocus) that encodes a protein with Debranching enzyme activity has been shown in maize. So far, there is no evidence that other forms of Debranching enzymes are present in maize. In order to obtain a transgenic maize plant having no Debranching enzyme activity, for example to achieve a higher degree of branching of amylopectin, it is necessary to identify all forms of Debranching enzyme that are present in the maize and to isolate the appropriate genes or cDNA sequences.

In order to provide further possibilities for transforming any starchy plants, preferably cereals, and in particular wheat, so that these plants synthesize the modified starch, it is necessary to identify the appropriate DNA sequences that encode other isoforms of the chain-branching enzymes.

SUMMARY OF THE INVENTION

It is an object of the present invention to prepare nucleic acid molecules that encode enzymes involved in starch biosynthesis and thereby obtaining a genetically modified plant that allows the production of grids of linear starches with altered chemical and / or physical properties.

Accordingly, the present invention relates to nucleic acid molecules encoding a protein having the function of wheat isoamylase, in particular a protein which is defined in particular by the amino acid sequence set forth in Seq ID No.3 or 7. The present invention particularly relates to a nucleic acid molecule comprising the nucleotide sequence set forth in Seq ID No. 1, 2 or 6, or a portion thereof, preferably a molecule comprising the coding region set forth in Seq ID No. 1, 2 or 6, as well as the corresponding ribonucleotide sequences. Particularly preferred is a nucleic acid molecule comprising additional regulatory elements which effect transcription as well as optionally translation of said nucleic acid molecules.

Further, the invention relates to nucleic acid molecules that hybridize to some of the nucleic acid molecules of the invention.

The present invention also provides a nucleic acid molecule which encodes wheat isoamylase and whose sequence differs from the nucleotide sequence of the aforementioned molecules due to the degeneracy of the genetic code.

The present invention also relates to a nucleic acid molecule comprising a sequence complementary to all or part of the above sequences.

The term hybridization in the context of the present invention means hybridization under conventional hybridization conditions, preferably under the strictly defined conditions described, for example, in Sambrook et al., Molecular Cloning.

Ί

A Laboratory Manual, 2nd Ed. (1989) Cold Spring Harbor

Laboratory Press, Cold Spring Harbor, NY).

Particularly preferably, the hybridization is carried out under the following conditions:

Hybridization Buffer: 2 x SSC; 10 x Denhardt solution (Fikoll

400+ PEG + BSA; ratio 1: 1: 1); 0.1% SDS; 5 mM EDTA; 50 mM Na ₂ HPO ₄ ; 250 pg / ml herring sperm DNA (Heringssperma-DNA);

pg / ml tRNA; or 0.25 M sodium phosphate buffer pH 7.2; 1 mM EDTA; 7% SDS;

Hybridization temperature: T = 65 to 70 ° C

Wash Buffer: 0.2 x SSC; 0.1% SDS;

Washing temperature: T = 40 to 75 ° C

Nucleic acid molecules hybridized to the nucleic acid molecules of the invention can, in principle, encode isoamylases from any wheat plant that expresses these proteins.

For example, nucleic acid molecules hybridized to the nucleic acid molecules of the invention can be isolated from genomic wheat or wheat tissues or from cDNA libraries of wheat or wheat tissues. Alternatively, they may be prepared by genetic engineering methods or by chemical synthesis.

The identification and isolation of these nucleic acid molecules can be performed using the molecules of this invention

The invention or parts of these molecules, respectively, using reverse complements of these molecules, for example by hybridization according to standard methods. (See, e.g., Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).

For example, nucleic acid molecules containing exactly the same or substantially the same nucleotide sequences or portions thereof set forth in Seq ID No. 1, 2 or 6 can be used as the sample for hybridization. fragments, prepared using conventional synthetic techniques, whose sequence substantially corresponds to the nucleic acid molecules of the field of the invention.

The molecules hybridized to the nucleic acid molecules of the invention also include fragments, derivatives and allelic variants of the above-mentioned nucleic acid molecules that encode the wheat isoamylases of the invention. Fragments are understood to mean portions of nucleic acid molecules that are long enough to encode said proteins. The term derivative in this context means that the sequences of these molecules differ from the sequences of the above-mentioned nucleic acid molecules at one or more sites and that they exhibit a high degree of homology with these sequences. Homology means a sequence identity of at least 40%, in particular an identity of at least 60%, especially above 80% and particularly preferably above 90%. Deviations from the previously described nucleic acid molecules may result from cleavage, substitution, insertion, or recombination.

Homology further means that there is a functional and / or

structural equivalence between the respective nucleic acid molecules or proteins encoded by them. Nucleic acid molecules which are homologous to the above-mentioned molecules and represent derivatives of these molecules are generally variations of these molecules which represent modifications with the same biological functions. These can be both naturally occurring variations, for example sequences from other organisms, as well as mutants, which mutants can be naturally occurring or can be introduced by targeted mutagenesis. Further, there are variations of synthetically prepared sequences. Allelic variants may be both naturally occurring variants and variants prepared synthetically or obtained by recombinant DNA techniques.

The isoamylases encoded by the various variants of the nucleic acid molecules of the invention exhibit certain common properties. These include, for example, enzyme activity, molecular weight, immunological reactivity, conformation, and the like, as well as physical properties such as gel electrophoresis mobility, chromatography behavior, sedimentation coefficients, solubility, spectroscopic properties, electrical charge, stability; pH optimum, temperature optimum and the like.

The protein encoded by the nucleic acid molecules of the invention is wheat isoamylase. These proteins show certain homologous regions with known isoamylases from other plant species.

The nucleic acid molecules of the invention may be DNA molecules, in particular cDNA or genomic molecules. Further, the nucleic acids of the invention may be RNA molecules which may be produced, for example

by transcription of a nucleic acid molecule of the invention. For example, the nucleic acids of the invention may be obtained from natural sources or by recombinant techniques or synthetically.

The present invention also relates to oligonucleotides specifically hybridized to a nucleic acid molecule of the invention. These oligonucleotides preferably have a length of at least 10, particularly at least 15, and particularly preferably a length of at least 50 nucleotides. The oligonucleotides of the invention are characterized in that they hybridize specifically to the nucleic acid molecules of the invention, that is, the nucleic acid sequences which encode other proteins, in particular other isoamylases, are not subject to hybridization or only to a limited extent. The oligonucleotides of the invention can be used, for example, as a primer for a PCR reaction or as a hybridization sample for the isolation of related genes. They may also form part of antisense constructs or DNA molecules encoding a suitable ribozyme.

The present invention further encompasses vectors, particularly plasmids, cosmids, phagemids, viruses, bacteriophages, and other vectors known in the art, comprising the aforementioned nucleic acid molecules of the invention. These vectors are suitable for the transformation of prokaryotic or eukaryotic, preferably plant cells.

Particularly preferred are vectors allowing the integration of the nucleic acid molecules of the invention into the genome of a plant cell, optionally together with shielding regulatory regions. Examples include binary vectors that can be used in gene-mediated gene transfer.

agrobacteria. Preferred is the synthesis of transferable or optionally non-transferable nucleic acid molecules in transformed pro- or eukaryotic cells by integrating the nucleic acid molecule in sense or anti-sense orientation of the invention.

The term vector refers to a generally suitable, art-recognized adjuvant enabling the targeted transfer of a single or double stranded nucleic acid molecule to a host cell, such as a DNA or RNA virus, a fragment of a virus, a plasmid construct which in the presence or absence of regulatory elements it may be suitable for transferring nucleic acids into cells, carrier materials such as glass fibers or even metal particles, used for example in the particle gun process, but may also be a nucleic acid molecule that can be inserted directly into the cell by a suitable chemical or physical process.

In a preferred embodiment, the nucleic acid molecules contained in the vectors are linked to regulatory elements which allow the transcription and synthesis of the transferable RNA into pro- or eukaryotic cells or, if desired, allow the synthesis of the non-transferable RNA.

Expression of the nucleic acid molecules of the invention in prokaryotic cells, for example in Escherichia coli, is important for more precise characterization of the enzymatic activity of the enzymes that encode these molecules. In particular, it is possible to characterize the product synthesized by the respective enzymes in the absence of other enzymes involved in the synthesis of starch in plant cells. This makes it possible to draw conclusions about the function of the respective protein in the synthesis of starch in plant cells.

In addition, various mutations can be introduced into nucleic acid molecules according to conventional molecular biology techniques (see, for example, Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, NY). of the invention, whereby proteins with optionally converted biological properties are obtained in this synthesis. This allows the preparation of deletion mutants in which, by sequential cleavage from the 5 'or 3' ends, the coding DNA sequence of the nucleic acid molecules that results in the synthesis of the respective truncated proteins can be obtained. This deletion at the 5 'end of the nucleotide sequence makes it possible, for example, to identify amino acid sequences that cause translocation of enzymes in plastids (transit peptides). This makes it possible to carry out a targeted preparation of enzymes which, after removal of the respective sequences, are no longer localized in the cytosol but in the cytosol, or which are located in other parts of the cell after the addition of other signal sequences.

Further, it is possible to introduce local mutations at positions where the amino acid sequence change affects, for example, enzyme activity or enzyme regulation. For example, mutants with altered K _m or mutants that are no longer subject to normal regulatory mechanisms in the cell by allosteric regulation or covalent modification can be obtained in this way.

Further, mutants of the proteins of the invention having altered substrate or product specificity can be obtained. In addition, protein mutants that have an altered activity-temperature profile can be prepared.

• · · · · · · · · · · · ·

In carrying out a genetic-engineering modification of prokaryotic cells, the nucleic acid molecules of the invention or parts thereof can be inserted into plasmids which also allow mutagenesis or sequence alteration by recombination of the DNA sequences. Standard exchanges (see Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY) can be used to base exchanges or to attach natural or synthetic sequences. For linking DNA fragments to each other, it is possible to attach linking or binding groups to these fragments (Adaptoren o. Linker). Manipulations that release the appropriate cleavage restriction sites or that remove excess DNA or restriction sites can also be used. Where insertion, deletion or substitution is contemplated, primer repair, restriction, or ligation may be used for in vitro mutagenesis. Sequence analysis, restriction analysis or other biochemical or molecular biology methods were generally used as analytical methods.

In other embodiments, the invention relates to host cells, in particular pro- or eukaryotic cells, which are transformed with one of the above-mentioned nucleic acid molecules of the invention or one of the vectors of the invention, as well as cells resulting from the transformed cells and comprising a nucleic acid molecule of the invention or a vector. These are preferably pro- or eukaryotic cells, in particular plant cells.

The present invention further provides proteins which can be produced by recombinant means and which have isoamylase activity encoded by nucleic acid molecules

according to the invention, as well as a process for their preparation, wherein the host cell of the invention is cultured under suitable conditions known to those skilled in the art to synthesize the protein of the invention and ultimately isolate the protein from the host cells and / or the culture medium.

The nucleic acid molecules obtained according to the present invention now make it possible, by means of genetic engineering methods, to carry out targeted interventions in the starch metabolism in plants and to alter this metabolism to the extent that such modified starches are synthesized that have other physical starches compared to known starches. chemical properties such as amylose / amylopectin ratio, chain branching degree, average chain length, phosphate content, lubricating properties, gel or film forming properties, starch grain size and / or shape.

It is also possible to express the nucleic acid molecules of the invention in plant cells to increase the activity of the respective isoamylase, or to introduce the nucleic acid molecules of the invention into cells that do not normally express the enzyme. Expression of the nucleic acid molecules of the invention also provides the ability to reduce the natural activity of the isoamylase of the invention in plant cells. Further, the nucleic acid molecules of the present invention can be modified by methods known to those skilled in the art to obtain isoamylases of the present invention that are no longer subject to the natural regulatory mechanisms of the cell itself, or which have an altered temperature / activity profile or altered specific substrate or product properties.

• · ·

When expressing the nucleic acid molecules of the invention in plants, there is in principle the possibility of localizing the synthesized protein to any part of the plant cell.

In order to localize to a particular part of the plant cell, the plasmids providing the localization in the plastids must be cleaved and the remaining coding region optionally linked to the DNA sequences which provide the localization to the respective site. Such sequences are known (see, eg, Braun et al., EMBO J. 11 (1992), 3219-3227; Volter et al., Proc. Natl. Acad. Sci. USA 85 (1988), 846-850; Sonnenwald et al. et al., Plant J. 1 (1991), 95-106).

The invention also relates to a process for the preparation of transgenic plant cells which are transformed with a nucleic acid molecule or a vector according to the invention, wherein the nucleic acid molecule according to the invention or a vector according to the invention integrates into the genome of the plant cell. plant cells transformed with a nucleic acid molecule or vector of the invention, and also includes transgenic plant cells derived from such transformed cells. The cells of the invention comprise one or more nucleic acid molecules or vectors of the invention, the molecules or vectors being preferably linked to regulatory DNA elements that provide transcription in plant cells, in particular a suitable promoter. These cells can be distinguished from natural plant cells by, inter alia, the fact that they contain the nucleic acid molecules of the invention that do not normally occur in these cells, or that such a molecule is integrated at a particular location in the cell genome where does not occur, ie in another genomic neighborhood. Further, these transgenic plant cells according to the invention may be used

of the present invention to distinguish it from natural plant cells by containing at least one copy of the nucleic acid molecule of the invention stably integrated in their genome, optionally in addition to the naturally occurring copies thereof. If the nucleic acid molecule (s) introduced into the cells are copies in addition to the naturally occurring molecules in the cell, the plant cells according to the invention can be distinguished from the natural cells in particular by the extra copy (s) being / are located in the genome in the place (s) where it does not occur in nature. This can be readily demonstrated, for example, by Southern blot analysis according to procedures known to those skilled in the art.

When the nucleic acid molecule of the invention introduced into the plant genome is heterologous to the plant cell, the transgenic plant cells contain transcripts of the nucleic acid molecules of the invention, as can be readily demonstrated, for example, by Northern blotanalysis according to procedures known to those skilled in the art.

If the nucleic acid molecule of the invention is homologous to a plant cell, the cells of the invention can be distinguished from naturally occurring cells, for example, by further expressing the nucleic acid molecules of the invention. The transgenic plant cells preferably contain multiple transcripts of the nucleic acid molecules of the invention. This can be demonstrated, for example, by Northern blot analysis. More preferably at least 10% more, in particular 20% more and in particular at least 50% more transcripts than the corresponding untransformed cells. Preferably, these cells also exhibit a corresponding (at least 10%, 20%, or 50%) increase.

activity, or optionally reducing the activity of the proteins of the invention. Transgenic plant cells can be regenerated into whole plants by methods known to those skilled in the art.

The present invention also provides a process for obtaining transgenic plants, wherein one or more of the nucleic acid or vector molecules of the invention are integrated into the genome of a plant cell and the whole plant is regenerated therefrom. It is a further object of the present invention to provide plants comprising the above transgenic plant cells. These transgenic plants can in principle be plants of any plant species, i.e. both monocotyl and dicotyl plants. They are preferably useful plants, in particular plants which synthesize or store starch, particularly preferably rye, barley, oats, wheat, millet, sago, corn, rice, peas, peas, cassava, potatoes, tomatoes, rape, soybeans , hemp, flax, sunflowers, fodder peas or maranta, and in particular wheat, maize, rice and potatoes.

The invention also relates to plant propagation material of the invention, such as fruits, seeds, tubers, root parts, seedlings, seedlings, tissues, protoplasts, cell cultures and the like.

The present invention further relates to a method for producing a modified starch comprising the operation of extracting starch from the aforementioned plants according to the invention and / or from the starchy portions thereof.

Processes for extracting starch from plants or from their starchy portions, in particular from wheat, are known to those skilled in the art, and are known to those skilled in the art. See, for example, Eckhoff et al. (Cereal Chem. 73 (1996) 54-57 Starch - Chemistry and Technology (edited by Vhistler, BeMiller and Paschall (1994), 2nd edition, Academic Press Inc. London Ltd; ISBN 0-12-746270-8; see, for example) Chapter XII, pp. 412 - 468: Starches from maize and sorghum: Manufacture; Vatson; Chapter XIII, pp. 469 - 479: Tapioca, marzipan and sago starch: Manufacture; Corbishlev and Miller; Chapter XIV, pp. 479 - 490: Potato starch: Manufacture and use; Mitch; Chapter XV, pp. 491-506: Wheat starch: Manufacture, modification and use; Knight and Oson; and Chapter XVI, pp. 507 to 528: Rice starch: Manufacture and use; Devices which are generally used to extract starch from plant material include separators, decanters, hydrocyclones, spray dryers and fluid bed dryers.

The transgenic plant cells and plants of the invention synthesize starches based on the expression of the nucleic acid molecules of the invention which, with their physicochemical properties, such as amylose / amylopectin ratio, chain branching degree, average chain length, phosphate content, lubricating properties , the size of the starch grains and / or their shape differs from the starches synthesized by natural plants. In particular, their viscosity and / or their film-forming properties and the properties of the resulting starch sebum may be varied in comparison with those of known starches.

The present invention further provides starch obtainable from plant cells, plants and their propagation material according to the invention and starch obtainable by the above processes of the invention.

Further, plant cells and plants in which the activity of the protein of the invention is reduced can be produced using the nucleic acid molecules of the invention. This also leads to the synthesis of starch with altered chemical and / or physical properties compared to starches from native plant cells.

It is a further object of the present invention to provide transgenic plant cells comprising a nucleic acid molecule of the present invention wherein the isoamylase activity is reduced compared to untransformed cells.

The preparation of plant cells with reduced isoamylase activity can be carried out, for example, by expressing an appropriate anti-sense RNA, a sense RNA for a co-suppression effect, or by expressing a correspondingly constructed ribozyme that specifically cleaves a transcript encoding isoamylase using the nucleic acid molecules of the invention a procedure known to those skilled in the art, see Jorgensen (Trends Biotechnol. 8 (1990), 340-34), Niebel et al., Immunol. 197 (1995), 91-103),

Top. Microbiol. Immunol. 197 (1995),

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

(Curr. Top. Microbiol Flavell et al. (Curr

Preferably, a reduction in the number of coding transcripts in plant cells, for example by expression of antisense-RNA, is used to reduce the isoamylase activity of the invention.

It is possible to use a DNA molecule which comprises the complete sequence coding for the protein according to the invention, including any shielding sequences present, as well as DNA molecules containing only parts of the coding sequence, these parts being long enough to produce an antisense effect in the cells . In general, sequences of at least 15 bp in length, preferably 100 to 500 bp in length, may be used for effective antisense inhibition, in particular, sequences over 500 bp in length. As a rule, DNA molecules that are shorter than 5000 bp are used, preferably sequences that are shorter than 2500 bp.

DNA sequences which exhibit a high degree of homology to the DNA molecule sequences of the invention, but which are not entirely identical, may also be used. The minimum homology should be greater than about 65%. Preferably, sequences with a degree of homology between 95 and 100% are used.

The present invention also provides a process for preparing modified starch, comprising a process for extracting starch from a cell or plant of the present invention and / or from starchy portions of such plants.

The present invention further provides starch obtainable from cells, plants as well as propagation material or parts thereof according to the invention and furthermore starch obtainable by the process according to the invention.

The starches of the present invention can be modified by methods known to those skilled in the art, and these starches in modified or unmodified form are suitable for various applications in the food or non-food field.

Possibilities of use of the starches according to the invention are described in U.S. Pat

basically divided into two large areas. One field includes the products of starch hydrolysis, in particular glucose and glucan building units, obtainable by enzymatic or chemical processes. These substances serve as starting materials for other chemical modifications and processes such as fermentation. In this case, simplicity and not costly performance of the hydrolysis process can be of great importance for reducing costs. Currently, this hydrolysis is carried out mainly enzymatically using amyloglucosidase.

It can be assumed that cost savings could be achieved by reducing the amount of enzymes used. A structural conversion of starch, such as a reduction in the degree of branching or a change in the spherical structure that determines the accessibility of the molecule to the enzymes used, could also have some effect.

The second area in which the starches of the present invention could be used as so-called native starches due to their polymeric structure is divided into two main fields of application:

1. Food industry

Starch is a classic additive to a variety of foods in which it generally serves as a binder for aqueous ingredients or is used to increase viscosity or cause increased gel formation. Important properties are fluidity and sorption, swelling and lubrication temperature, viscosity and thickening ability, starch solubility, starch sebum transparency and structure, stability to heat, shear and acidity, tendency to retrogradation, film formation, freeze / thaw stability , viscosity stability in salt solutions, digestibility, as well as the ability to form complexes with, for example, inorganic or organic ions.

2. Non-food industry

In this broad field, starch can be used as an auxiliary in various manufacturing processes or as an additive to technical products. Starch as an excipient is mainly used in the paper and board industries. Starch in this application is mainly used for retardation (solids binding), binding of fillers and fine particles, as a reinforcing agent and as a dewatering agent. In addition, the advantageous properties of starch are utilized to achieve stiffness, hardness, acoustic properties, desirable feel, gloss, smoothness, resistance to splitting, as well as to achieve desirable surface properties.

2.1 Paper and board industry

There are four areas of application for paper manufacture: surface treatment, coating, paper processing and spraying. The requirements for starch for surface treatment are in particular a high degree of whiteness, adequate viscosity, high viscosity stability, good film-forming capacity and minimal dust formation. When using starch for coatings, the solids content, the corresponding viscosity, the high binding power as well as the high affinity for pigments are important. When starch is used as an additive in paper pulp, its fast, uniform and lossless dispersion, high mechanical stability is required

And the complete retention of starch in the pulp stream. When using starch for spraying, the corresponding solids content, high viscosity and high binding properties are also important properties.

2.2. Adhesive industry

The adhesive industry is a broad field of application of starches, which can be divided into four sub-areas: use as pure starch adhesive, use in starch adhesives with special chemical additives, use of starch as an additive to synthetic resins and polymer dispersions, and finally use of starches as extender into synthetic adhesives. 90% of starch based adhesives are used in the manufacture of corrugated cardboard, paper bags, sacks and cones, for the production of fasteners for paper and aluminum, for the manufacture of cardboard and as a moisturizing adhesive for letter envelopes, stamps and the like.

2.3 Textile industry and textile auxiliary industry

A major consumer of starches is the textile and textile preparations industry, where starches are used as an adjuvant and additive. Within the textile industry, four areas of application can be distinguished: the use of starch as a sizing agent, i.e. as an aid for smoothing, consolidating and enhancing the cohesiveness of textile raw materials to protect against the tensile forces of spinning and weaving, the use of starch as a textile refining agent, especially after adjustments deteriorating quality such as bleaching, dyeing and the like, starch is further used as a thickening agent in the manufacture of dye pastes for inhibiting color diffusion, and finally the starch is used as an additive .

2.4 Construction

A fourth field of application is the use of starch as an additive to building materials. By way of example, gypsum panels are added where the starch added to the gypsum slurry by water lubrication, diffuses to the surface of the gypsum board and binds the cardboard to the gypsum board. Another field of application is the addition of starch to plasters and mineral fibers. When transporting concrete, starch products are used to retard curing.

Soil stabilization

Another area of starch consumption is the production of soil stabilizers, which are used in soil movements to temporarily protect soil particles from water. Combination products consisting of starch and polymer emulsions are, to the present knowledge, comparable in their erosion and crust-reducing effects to those used to date, but they are substantially cheaper.

2.6. Use in plant protection products and fertilizers

In this field, starch is used in plant protection products to alter their specific properties. Starches are used for improved wetting of plant protection products and fertilizers, for the gradual release of active substances, for the conversion of liquid, volatile and / or odorous substances to microcrystalline, stable and formable substances, for mixing incompatible compounds and for prolonging the efficiency by retarding decomposition.

2.7 Pharmaceutical, medical and cosmetic industries

Another field of use for starch is the pharmaceutical, medical and cosmetic industries. In the pharmaceutical industry, starch is used as a binder for tablets or for diluting binders in capsules. Furthermore, starch is used as a rapid disintegration tablet, where the tablets absorb swallowed liquid and swell after a short time, releasing the active ingredient. Medical powders and powders for wound filling contain starch as the basic substance. In the field of cosmetics, starches are used, for example, in powders as excipients such as fragrances and salicylic acid. Relatively large amounts of starches are used in toothpastes.

2.8 Addition of starches to coal and briquettes

Starches are also added to coal and briquettes. The coal, after the addition of starch, agglomerates or briquetts better, preventing premature disintegration of the briquettes. 4 to 6% starch is added to the charcoal, 0.1 to 0.5% starch to the fuel charcoal. Starches as a binder for coal are becoming increasingly important as the addition of starch to coal and briquettes can greatly reduce emissions of harmful substances.

2.9 Treatment of ore and coal sludge

Starches can also be used for the treatment of ore and coal sludge as flocculating agents.

2.10 Foundry Aids

Another field is the use of starch as an additive to foundry aids. Various casting processes require cores which are made of sands with a binder additive. Currently, mainly bentonite is used as binder, to which modified starches, in particular swelling starches, are added. The addition of starch is used to increase flow strength and improve binding. In addition, swellable starches can meet other technical requirements such as cold water dispersibility, easy rehydration, good sand miscibility and high water binding capacity.

2.11 Use in the rubber industry

In the rubber industry, starch can be used to improve the technical and optical quality of products. It is used to improve the gloss of the surface, to improve its feel and appearance, while starch-coated gummed surfaces are dusted with starch before cold vulcanization. Starch is also used to improve the printability of rubber products.

2.12 Manufacture of leather substitutes

Another field of application of modified starches is the manufacture of leather substitutes.

2.13 Starches in synthetic polymers

In the field of plastics, starches are used for the following purposes: addition of starch products in processing (starch acts only as a filler, there is no direct bond between the synthetic polymer and starch) or alternatively to bind starch products in the production of polymers (starch and polymer form a strong bond).

The use of starch as a mere filler is not perspective compared to other substances such as talc. However, the situation changes when the specific properties of starch are applied and the properties of the final product are substantially altered. An example is the use of starch products in the processing of thermoplastics such as polyethylene. The starch and the synthetic polymer are coexpressed in a 1: 1 ratio to a so-called master batch, from which various products are produced with granulated polyethylene using conventional techniques. The binding of starch in polyethylene films can increase the fabric permeability of the film hollow bodies, improve the water vapor permeability, improve the antistatic properties, and also improve the printability of the water-dilutable paints.

Another possibility is to use starch in polyurethane foams. By adjusting the starch derivatives and also optimizing the process parameters, the reaction between the synthetic polymers and the hydroxyl groups of the starch can be targeted. The result is a polyurethane film which gives the following properties to the use of starch: a reduction in the thermal coefficient φ · «φ • φ • φ φ φ φ • φ φ φ φ φ φ φ φ φ φ extensibility, reduced shrinkage, improved pressure / pull properties, increased water vapor permeability without altering water-absorption capacity, reduced flammability and tear potential, no dripping of flammable components, absence of halogens and retarded aging. The disadvantages presently present are the reduced compressive strength and the reduced impact resistance.

However, product development was not limited to foils.

Massive products such as mugs, plates and bowls, containing more than 50% starch, are currently being produced. The starch / polymer blends are also evaluated ecologically because they have a significantly higher biodegradability.

Graft starch polymers have gained particular importance due to their extreme water-binding capacity. These are products whose starch is the main chain and the side chains of the synthetic monomer are grafted onto this chain according to the principle of the free radical mechanism. The currently available graft starch polymers are characterized by improved water binding and retention capacity, up to 1000 g water per g starch at high viscosity. The fields of application of these superabsorbent substances have been very widespread in recent years and are used in the field of hygiene as diapers and mats, as well as in agriculture, such as seed coating.

The structure, moisture content, protein content, lipid content, fiber content, ash / phosphate content, amylose / amylopectin ratio, molecular weight distribution, degree of branching, grain size and their shape and crystallinity are decisive for the use of the novel genetically converted starches. and properties resulting in the following characteristics: flow behavior and sorption properties, lubricating temperature, viscosity, stability of viscosity in salt solutions, thickening ability, solubility, structure and transparency of starch sebum, resistance to heat, shear and acids, tendency for retrogradation, gel formation, freeze / thaw stability, complexing ability, iodine binding, film formation, adhesive bond strength, enzyme stability, digestibility and reactivity.

Modified starches obtained from the respective plants by genetic engineering may change their properties such that further modification by chemical or physical methods is no longer necessary. Alternatively, the starches modified by genetic engineering can be further modified by chemical methods so that further quality improvement is achieved for the above-mentioned uses. These chemical treatments are known in principle. In particular, heat treatment, organic or inorganic acids, oxidation and re-esterification are used, for example, phosphates, nitrates, sulfates, xanthates, acetates or starch citrates are formed. Further, monohydric or multifunctional alcohols in the presence of strong acids may be used to prepare starch ethers to form alkyl ethers, O-allyl ethers, hydroxyalkyl ethers, starch O-carboxymethyl ethers, nitrogen-containing starch ethers, phosphorus-containing starch ethers, sulfur-containing starch ethers, crosslinked starches or graft starch polymers.

A preferred use of the starches of the present invention is to produce both packaging materials and disposable products and to use these starches as food and to digest them. 9 9 9 9 9 9 9 9

9 9 9 9 · Semi-finished products.

To express the nucleic acid molecules of the invention in sense or antisense orientation in plant cells, these molecules are linked to regulatory DNA elements which then transcribe in plant cells.

In particular, promoters, enhancers and terminators are also associated with this. In general, any active promoter in a plant cell can participate in expression.

The promoter can be selected such that expression takes place constitutively, or only in a particular tissue, at a certain time of plant development, or at a point in time determined by external influences. In relation to a plant, this promoter may be homologous or heterologous. Suitable promoters for constitutive expression are, for example, the cauliflower mosaic virus 35S RNA promoter and the maize ubiquitin promoter, and the B33 patatin promoter is suitable for tuber-specific expression (Rocha-Sosa et al., EMBO J. 8 (1989), 23-29). for example, the ST-LS1 promoter (Stockhaus et al., Proc. Natl. Acad. Sci. USA 84 (1987), 7943-7947; Stockhaus et al., EMBO J. 8 (suitable) as a promoter providing expression only in photosynthetically active tissues; 1989), 2445-2451), the HMG promoter from wheat, the USP promoter, the faseolin promoter or the promoters from maize zein genes may be used for endosperm-specific expression.

In addition, a termination sequence may be provided to ensure proper transcription termination and also to add poly-A-terminus to the transcript to which the transcript stabilization function is attributed. These elements are described in the literature (see, eg, Gielen et al., EMBO J. 8 (1989)) and are arbitrarily interchangeable.

• · ··· · · ·

The present invention provides nucleic acid molecules that encode a protein having the function of wheat isoamylase. Nucleic acid molecules according to the invention allow the preparation of this enzyme, which is involved in starch biosynthesis and which enables the production of genetically modified plants in which the activity of the enzyme is altered, thus allowing synthesis of starch with altered structure and altered physico-chemical properties.

The nucleic acid molecules according to the invention can in principle also be used for obtaining plants in which the activity of the isoamylase according to the invention is increased or decreased and at the same time the activity of other enzymes involved in starch synthesis is altered. This alteration of isoamylase activity in plants leads to the synthesis of starch with altered structure. In addition, nucleic acid molecules that encode isoamylase or corresponding anti-sense constructs may be introduced into plant cells in which the synthesis of endogenous starch synthases or chain branching enzymes has been inhibited (such as in WO 92/14827 or Shannon and Garwood, 1984, Vhistler, BeMiller and Paschall, Starch: Chemistry and Technology, Academic Press, London, 2nd edition: 25-86).

If inhibition of multiple enzymes involved in biosynthesis is to be achieved in transformed plants, DNA molecules which simultaneously contain multiple regions encoding the corresponding enzymes in antisense orientation can be used for transformation under the control of a suitable promoter. Alternatively, each sequence may be controlled by a single promoter of its own, or the sequences may be transcribed. Tf tf tf tf tf tf tf tf tf tf tf tf tf tf tf tf tf Tftf tf as fusions to a single common promoter or all sequences can be controlled by a single common promoter. As a rule, this latter alternative is preferred, since in this case the synthesis of the respective proteins is inhibited to approximately the same extent. For the length of each coding region used in the obtained construct, what was previously stated in the preparation of antisense constructs. In principle, there is no upper limit on the number of antisense-transcribed fragments in the promoter of a single DNA molecule. However, the resulting transcript should not be longer than 10 kb, preferably not longer than 5 kb.

The coding regions that are located in these DNA molecules in combination with the other coding regions in the antisense orientation downstream of the respective promoter may originate from DNA sequences that encode the following proteins: starch-grain synthases (GBSS I and II) and soluble synthases (SSS I and II), branching enzymes (isoamylases, pullulanases, R-enzymes, Branching-enzymes, Debranching-enzymes), starch phosphorylases and disproportionation enzymes. This list is for reference only. Other DNA sequences may be used in such combinations.

These constructs allow simultaneous inhibition of the synthesis of multiple enzymes in plant cells transformed by their action.

Furthermore, these constructs can be introduced into plant mutants that are defective for one or more of the starch biosynthesis genes (Shannon and Garwood, 1984, Vhistler, BeMiller and Paschall, Starch: Chemistry and Technology, Academic Press, London, 2nd ed. : 25-86). These defects may relate to the following enzymes: bound synthases »· f ·

for starch grain (GBSS I and II) and soluble synthases (SSS I and II), branching enzymes (BE I and II), Debranching enzymes (R-enzymes), disproportionation enzymes and starch phosphorylases. This list is for reference only.

Furthermore, the synthesis of several enzymes can be inhibited simultaneously in plant cells transformed by this method.

For the introduction of foreign genes into higher plants, a large number of cloning vectors are available which contain a replication signal for E. coli and a marker gene for the selection of transformed bacterial cells. Examples of such vectors include pBR322, pUC-series, M13mp-series, pACYC184 and others.

The desired sequence can be introduced into the vector at the appropriate restriction cleavage site. The obtained plasmid was used to transform E. coli cells. The transformed E. coli cells are cultured in a suitable medium, and finally isolated and lysed. The plasmid is regenerated. Restriction analysis, gel electrophoresis and other biochemical and molecular-biological methods are generally used as analytical methods for characterizing the obtained plasmid-DNA. After each manipulation, the plasmid DNA can be cleaved and the DNA fragments obtained can be ligated to other DNA sequences. Each plasmid-DNA sequence can be cloned in the same plasmid or in other plasmids.

There are a number of procedures for introducing DNA into a plant host cell. These procedures include transforming plant cells with T-DNA using Agrobacterium tumefaciens or Agrobacterium rhizogenes as a transforming agent, protoplast fusion, injection, DNA electroporation, DNA insertion using the biolistic method, and other options.

There are no special requirements for the plasmids used when injecting and electroporating DNA into plant cells. Simple plasmids such as pUC derivatives can be used. However, if the entire plant is to be regenerated from such transformed cells, the presence of the appropriate marker gene is usually necessary.

Depending on the method of introducing the genes of interest into the plant cell, additional DNA sequences may be required. For example, if a Ti- or Ri-plasmid is used to transform a plant cell, at least the right border, but often both the right and left borders of the Ti- and Riplasmid T-DNA, must be joined as a border region to the introduced gene.

If agrobacteria are used for transformation, the introduced DNA must be cloned in a special plasmid, either in an intermediate vector or in a binary vector. Intermediate vectors can be integrated into the Ti- or Ri-plasmid of agrobacteria by homologous recombination based on sequences homologous to those in T-DNA. This plasmid also contains the vir-region required for T-DNA transfer. Intermediate vectors cannot be replicated in agrobacteria. Using the helper plasmid, the intermediate vector can be transferred to Agrobacterium tumefaciens (conjugation). Binary vectors can be replicated in both E. coli and agrobacteria. These vectors contain a selectable marker gene and a linker (linker or polylinker) that frame the right and left T-DNA border regions. These vectors can be transformed directly into agrobacteria (Holsters et al.

·

• 9

Mol. Gene. Genet. 163 (1978), 181-187). Agrobacterium as the host cell should contain a plasmid carrying the vir-region. This vir-region is required for the transfer of T-DNA into the plant cell. Additional T-DNA may be present. Agrobacterium cells thus transformed can be used to transform plant cells.

The use of T-DNA for the transformation of plant cells is the subject of intensive study and is described in detail in EP 120 516; Hoekema, The Binary Plant Vector System, Offsetdrukkerij Kanters B.V, Alblasserdam (1985), Chapter V; Fraley et al., Crit Rev. Plant. Sci., 4,

46 and An et al., EMBO J. 4 (1985), 277-287.

Conveniently, plant explants with Agrobacterium tumefaciens or Agrobacterium rhizogenes can be co-cultured for transferring DNA into a plant cell. Whole plants can then be regenerated again from the infected plant material (e.g. leaf pieces, stem parts, roots, but also protoplasts or suspension-cultivated plant cells) in a suitable medium containing, inter alia, certain sugars, amino acids, antibiotics or biocides for the selection of transformed cells. . The presence of the introduced DNA can then be monitored in the plants thus obtained. Other possibilities for introducing foreign DNA using a biolistic procedure or by protoplast transformation are known (see, eg, Villmitzer, L., 1933 Transgenic plants, Biotechnology, A MultiVolume Comprehensive Treatise (edited by HJ Rehm, G.Reed, A.). Puhler, P. Stadler), Vol. 2, 627-659, VCH Veinheim-New York-Basel-Cambridge).

While the transformation of dicotyl plants by Ti-plasmid vector systems using Agrobacterium is well known, more recent work suggests that transformation with vectors derived from Agrobacterium can also be performed in monocotyl plants (Khan et al., Plant Mol. Biol. 22 ( 1993), 491-506, Hiei et al., Plant J. 6 (1994), 271-282).

Alternative methods to transform monocotyl plants are the use of biolistic techniques, protoplast transformation or physically or chemically induced DNA insertion into protoplasts, for example by electroporation of partially permeabilized cells, glass fiber DNA transfer, DNA macroinjection to inflorescence, DNA microinjection to microspores or pro-embryons. insertion of DNA by germinating pollen and insertion of DNA into embryos by swelling (Review: Potrykus, Physiol. Plant (1990), 269-273).

Three of the above transformation systems have previously been used for various grain types: tissue electroporation, protoplast transformation, and DNA-transfer by firing particles into regenerable tissue and cells (Review: Jáhne et al., Euphytica 85 (1995), 25-44).

Transformation of wheat has been described in a number of studies (Review: Maheswari et al., Critical Research in Plant Science 14 (2) (1995), 149-178). Hess et al. (Plant Sci. 72 (1990), 233) used a macroinjection procedure to bring pollen and agrobacteria in close proximity to each other. Mobilization of the plasmid containing the nptII gene as a selectable marker was demonstrated by Southern blot analysis and NPTII assay. These transformants showed a normal phenotype and were fertile. Kanamycin resistance was demonstrated in two consecutive generations.

The first transgenic fertile wheat plant that could be regenerated after the injection of DNA bound to the microprojectiles was described by Vasil et al. (Bio / Technology 10 (1992), 667-674). The target tissue for the firing was an embryogenic culture of Kali (Kallus type C). A pure gene (bar Gen) encoding phosphinothricin acetyltransferase was used as a selectable marker to confer resistance to the Phosphinothricin herbicide. Another system has been described by Veeks et al. (Plant Physiol. 102 (1993), 1077-1084) and Becker et al. (Plant J. 5 (2) (1994), 299-307). In this case, the target tissue for DNA transformation was the skutellum of immature embryos that were stimulated in the early in vitro phase to induce somatic embryons. The efficiency of this transformation was in the system developed by Becker et al. (cited earlier), with 1 transgenic plant per 83 Florida embryos substantially higher than the system developed by Veeks et al. with 1 to 2 transgenic plants per 1000 Bohwhite embryos.

The system developed by Becker et al. (cited above) form the basis of the transformation experiments described in the Examples.

Once the inserted DNA has been integrated into the genome, it usually remains stable in the genome and remains in the progeny of the original transformed cells. As a rule, it contains one of the aforementioned selection markers which provides transformed plant cells with, for example, resistance to biocidal agents such as Phosphinothricin or to antibiotics such as kanamycin, G418, Bleomycin or Hygromycin or allows selection in the presence or absence of certain sugars or amino acids. An individually selected marker should therefore allow the selection of transformed cells • for cells lacking the inserted DNA.

Transformed cells grow in the plant in a normal manner (see, eg, McCormick et al., Plant Cell Reports 5 (1986), 81-84). The resulting plants can be grown normally and can be crossed with plants having the same or different hereditary properties. The hybrid individuals thus formed have corresponding phenotypic properties. Seeds can be obtained from plant cells. In order to ensure that the phenotypic property has been retained and is inherited, two or more generations must be grown. Seeds should also be harvested to ensure that the phenotype or other characteristic is retained.

The following examples illustrate the invention but do not limit it in any way.

DETAILED DESCRIPTION OF THE INVENTION

Example 1

Cloning procedure

PBluescript II SK (Stratagene) was used for cloning in E. coli.

Example 2

Strains of bacteria

For the Bluescript vector and for the antisense constructs, it was • · · * · ·

E. coli strain DH5a (Bethesda Laboratories, Gaithersburg, USA) was used. E.coli strain XL1-Blue was used for in vivo excision.

Example 3

Transformation of immature wheat embryons

Media:

MS: 100 ml / 1 macro salt (D. Becker and H. Lórz, 1 ml / 1 microsalt Plant Tissue Culture # 30: 2 ml / 1 Fe / NaEDTA 30 g / l sucrose MS + 2,4-D (2mg / L) Manual B 12: 1-20) # 31: MS + 2,4-D (2mg / L) + Phosphinothricin (PPI, Active

component of BASTA herbicide

(1 mg / 1) # 32: MS + 2,4-D (0.1 mg / l) + PPT mg / 1) # 39: MS + 2,4-D (2 mg / L) + 0.5 N each mannit / sorbit

In these media, the pH was adjusted to 5.6 with KOH and the media was solidified with 0.3% Gelrite.

Becker and Lórz have developed and optimized the method for transforming immature wheat embryons (D.Becker and H.Lórz,

Plant Tissue Culture Manual (1996), B 12: 1-20).

In the experiments described below, the protocol prepared by Becker and Lórz was adhered to (cited above).

«· Φ ·

For transformation, caryopsis ears were harvested and surface sterilized 12 to 14 days after the anesthetic. The isolated skutella was plated with the ebrya axis assigned to the medium on # 30 induction medium.

After a two to four day preculture (26 ° C, dark), explants were transferred to medium # 39 for osmotic pre-culture (osmotische Vorkultur) (2-4 hours, 26 ° C, dark).

For biolistic transformation, about 29 pg of gold particles were used per shot and previously precipitated several pg of target DNA. Since the experiments were co-transformants, the target DNA for clotting was added with the target gene and the resistant marker gene (bar-Gen) at a ratio of 1: 1.

Example 4

DIG-labeling of DNA fragments

Labeling of DNA fragments used as screening probes was performed by specific PCR with the insertion of DIG-labeled dUTP (Boehringer Mannheim, Germany).

Media and solutions used in the examples:

SSC 175.3 g NaCl

Make up 88,2 g of sodium citrate to 1000 ml redest. water adjustment to pH 7.0 with 10 N NaOH solution

At the DSMZ in Braunschweig, Germany, plasmid pTaSU 8A was deposited under the Budapest Treaty under number DSM 12795 and plasmid pTaSU 19 under number DSM 12796.

Example A

Identification, isolation and characterization of cDNA encoding isoamylase (saccharide homolog / sugary H. /) from wheat (Triticum aestivum L., variety Florida)

A heterologous screening strategy was used to identify the cDNA encoding the isamylase (sugar) isoform of wheat. The wheat cDNA bank was examined using a maize saccharide probe.

Isolation of this saccharide probe from the corn cDNA flask was performed using a specific primer by PCR-amplification. The cloning of the corn cDNA flask was performed from (A) + RNA from a mixture of equal proportions of 13, 17, 19, 20, 22, 25 and 28 day (DAP) old karyops in the Lambda Zap II vector according to the manufacturer's data. synthesis of Lambda ZAP II-cDNA, Stratagene GmbH, Heidelberg, Germany). The embryo was removed in all karyops used except the 13-day-old grains.

Amplification of the DNA fragment used as a probe for the wheat cDNA library was performed with the following primers:

sulp-la: 5'AAAGGCCCAATATTATCCTTTAGG 3 '(Seq.ID No.4) sulp-2: 5`GCCATTTCAACCGTTCTGAAGTCGGGAAGTC 3' (Seq.ID No.5)

2 μΐ were used as templates for the PCR reaction. Ϊ́ 2 2 2 2 2 2 2 PCR 2 PCR 2 2 2 2 2 2 2 Amplified maize cDNA banks. This PCR reaction mixture further contained 1.5-3 mM MgCl2, 20 mM Tris-HCl (pH 8.4), mM KCl, 0.8 mM dNTP Mix, 1 μΜ sulp-la primer, 1 μΜ sulp- primer. 2 and 2.5 units of Taq polymerase (recombinant, Life Technologies). Amplification was performed in a Trioblock from Biometra according to the scheme: 4 '(min) / 95 ° C; l '/ 95 ° C; 45 ”(sec) / 58 ° C; l'15 ”/ 72 ° C; 30 cycles of 5 '/ 72 ° C. Amplified DNA bands of about 990 bp were resolved in an agarose gel and excised. A second amplification was performed with this fragment according to the above scheme. The 990 bp fragment obtained from this second amplification was digested with the restriction enzyme BAM HI into a 220 bp fragment and a 770 bp fragment.

After repeated separation of the saccharide (sugary) fragment on an agarose gel, excision of the band and isolation of this fragment, DIG-labeling of the probe was performed. A 500 ng saccharide fragment was used to label random prime with digoxygenin. To this labeled fragment was added 10 μί of Random Primer and the reaction mixture was heated for 5 na to 95-100 ° C. After heating, 0.1 mM dATP, 0.1 mM dGTP, 0.1 mM dCPT and 0.065 mM dTTP and 0.035 mM Digoxygenin-11-dUTP (Boehringer Mannheim) and Klenow buffer (standard) and 1 unit of Klenow polymerase were added. The reaction was carried out at RT (room temperature) overnight. The dot-test according to the manufacturer's data was used to check the tagging (Dig System User’s Guide for Filter Hybridization from Boehringer, Mannheim,

Germany).

Wheat cDNA-bank synthesis was performed from poly (A) + RNA from 21 days old karyops (starchy-endosperm) in the Lambda ZAP II vector Vector according to the manufacturer's data (Kit / kit / for Lambda ZAP II-cDNA synthesis, Stratagene GmbH) · • »

Heidelberg, Germany). After determining the titer of the cDNA flask, a primary titer value of 1.26 x 10 ° pfu / ml was determined.

Approximately 350,000 phages were plated on the plate to examine the wheat cDNA bank. Phage loading and plate evaluation (Abziehen) were performed according to standard protocol. Prehybridization and filter hybridization was performed in 5X SSC, 3% Blocking (Boehringer, Mannheim),

0.2% SDS, 0.1% sodium lauryl sarcosine and 50 µg / ml herring sperm DNA (Heringssperma) at 55 ° C. 1 ng / ml of labeled saccharide probe was added to the hybridization solution and the hybridization was incubated overnight. The filters were washed 2X5 'in 2XSSC, 1% SDS at room temperature; 2X10 ’in 1XSSC, 0.5% SDS at 55 ° C; 2X10 ’in 0.5XSSC, 0.2% SDS at 55 ° C. Positive clones were pooled in other screening procedures. Pooled clones such as pBluescript SK Phagemide (performed according to manufacturer's data; Stratagene, Heidelberg, Germany) were obtained by in vivo excision.

After analysis of this clone by miniprepation and restriction of the obtained plasmid-DNA, clone pTaSU-19 was deposited in the German Collection of Microorganisms and Cell Cultures DSMZ - Deutsche Sammlung für Mikroorganismen und Zellkulturen GmbH under the number DSM 12796 and further analyzed.

Example B

Sequence analysis of cDNA insertions of plasmid pTaSU-19

Plasmid DNA was isolated from the clone pTaSU-19 and the sequence of the cDNA insert was determined by the didesoxynucleotide method (Sanger et al., Proc. Nat. Acad. Sci. USA 74 (1977), 5463-5467).

The insertion of clone pTaSU-19 is 2997 bp long and represents a partial cDNA. The nucleotide sequence is designated Seq ID No. 1. Comparison with the previously published sequences showed that the Seq ID No. 1 sequence comprises a complete coding region that exhibits homology to isoamylases from other organisms. Analysis of this sequence also showed that the cDNA sequence contained two introns at positions 297-396 (intron 1) and 1618-2144 (intron 2). After removal of these introns, it is possible to derive a protein sequence which exhibits homology to the isoamylase protein sequences from other organisms. The amino acid sequence corresponding to the coding regions of Seq ID No. 1 is designated Seq ID No.3.

Example C

Preparation of the plant transformation vector pTa-alpha-SU19

To express the antisense-RNA corresponding to TaSU19-cDNA, a plant transformation vector pTa-alpha-SU19 was constructed based on the base plasmid pUC19, in which the cDNA insertion of the plasmid pTa-alpha-SU19 was linked in antisense orientation to the 3´-end of the ubiquitin promoter. This promoter consists of a first untranslatierter Exon and a first intron of the ubiquitin 7-gene from maize (Christensen A.H. et al., Plant Molecular Biology 18 (1992), 675-689). The polylinker portions and the NOS terminator were derived from plasmid pACT1.cas (CAMBIA, TG 0063; Cambia, GPO Box 3200, Canberra ACT 2601, Australia). Vector constructs with this terminator and constructs based on pACTl.cas are described in McElroy et al. (Molecular Breeding 1 (1995))

- 37). The resulting vector was named PUbi.cas.

• ·

The cloning of this vector was accomplished by restriction of the 2 kb fragment from the Ta-SU19 clone with the restriction enzyme Xba 1. This fragment was complemented at the ends by the Klenow reaction and then ligated to the Sma I cloning site of the pUbi.cas expression vector.

The resulting expression vector was designated Ta-alpha-SU19 and was used for the transformation of wheat as described above.

Example D

Isolation and characterization of another cDNA encoding isoamylase (Sugaryl-homolog) from wheat (Triticum aestiviim L., variety Florida).

The wheat cDNA bank was probed with a Sugary-Probe probe that represented part of the pTaSU19 clone at position 489-1041 of Seq.ID No. 1.

The preparation of wheat-specific digoxygenin-labeled Sugary-Probe used for screening the cDNA bank was performed by PCR amplification.

The primers used in this reaction were as follows:

SUSO1: 5'-GCT TTA CGG GTA CAG GTT CG-3 '(Seq.ID No.8), and

SUSO2: 5 '-AAT TCC CCG TTT GTG AGC-3' (Seq.ID No.9).

1 ng of plasmid pTaSU19 was used as template. In addition, the PCR reaction mixture contained 300 nM of SUSO1 and SUSO2 primers, each having 100 μΜ of dATP, dGTP, dCTP, 65 μΜ of dTTP, 35 μΜ of Digoxygenin-11-dUTP (Boehringer Mannheim), 1.5 mM MgCl ₂ and 2, 5 U (unit)

• Taq Polymerase and 10 μΐ 10-fold concentrated Taq Polymerase reaction buffer (both preparations from Life Technologies). The final volume of the reaction mixture was 100 μΐ. Amplification was performed in a PCR-Gerate (TRIO ^ Thermoblock, Biometra) at the following temperature program:

3 ´ (min) at 95 ° C (once); 45 '(sec) at 95 ° C - 45' at 55 ° C - 2 'at 72 ° C (30 cycles); 5 ’at 72 ° C (once). The resulting DNA fragment was 553 bp long. The insertion of Digoxygenin-11-dUTP into the PCR product was shown by slight agarose mobility compared to the control reaction product without Digoxygenin-11-dUTP.

The caroyptic-specific wheat cDNA bank of Example 1 was examined using a digoxygenin-labeled probe.

The hybridization procedure was performed overnight in 5X SSC, 0.2% SDS, 0.1% sodium lauryl sarcosine and 50 µg / ml herring sperm DNA (Heringssperma) at 68 ° C with the addition of 1 ng / ml digoxygenin labeled probe. After hybridization, the filters were washed with 2X5 'in 2XSSC, 1% SDS at room temperature; 2X10 ’in 1XSSC, 0.5% SDS at 68 ° C; 2X10'in 0.5XSSC, 0.2% SDS at 68 ° C. Positive clones were obtained by at least two additional screening procedures. PBluescript SK plasmids were obtained from phage clones (protocols according to manufacturer's specifications; Stratagene, Heidelberg, Germany).

After restriction analysis, the obtained clone pTaSU8A was deposited in the German Collection of Microorganisms and Cell Cultures DSMZ - Deutsche Sammlung für Mikroorganismen und Zellkulturen GmbH under the number DSM 12795 and was further analyzed.

Example Ε

Sequence analysis of cDNA insert in plasmids pTaSU8A

The nucleotide sequence of the cDNA insert in plasmids pTaSU8A was determined using the didesoxynucleotide method (Seq.ID No.6).

This insertion of clone pTaSU8A is 2437 bp in length and represents partial cDNA. In comparison with previously published sequences, Seq. ID No.6 includes the coding region showing homology to isoamylases from other organisms. Also, a protein sequence derived from the coding region of pTaSU8A clone, shown as Seq. ID No.7, showed homology to the protein sequences of isoamylases from other organisms.

Comparison of the sequences of the clones pTaSU19 (Seq.ID No. 1) and pTaSU8A (Seq.ID No. 6) shows 96.8% similarity. Most of the differences between the two sequences lie in the 3'-non-cDNA region. Other differences in the sequences in the coding region cause different amino acids at a total of twelve positions of derived protein sequences (Seq.ID No.3 and 7). The cDNAs contained in pTaSU19 and pTaSU8A are not identical and encode isoforms of wheat isoamylases.

Example F

Preparation of plant transformation vector pTa-alpha-SU8A

To express the antisense-RNA corresponding to TaSU8A-cDNA, a plant transformation vector pTa-alpha-SU8A was constructed based on the base plasmids pUC19, in which a portion of the TaSU8A-cDNA was joined in antisense orientation with the 3´-end of the ubiquitin promoter. This promoter consists of the first non-transferable exon and the first intron of the ubiquitin I maize gene (Christensen A.H. et al., Plant Molecular Biology 18 (1992), 675-689). The polylinker portions and the NOS terminator were derived from plasmid pACT1.cas (CAMBIA, TG 0063; Cambia, GPO Box 3200, Canberra ACT 2601, Australia). Vector constructs with this terminator and constructs based on pACTl.cas are described in McElroy et al. (Molecular Breeding 1 (1995), 27-37). The ubiquitin promoter, polylinker, and NOS terminator based on pUC19 was named pUbi.cas.

For cloning of pTa-alpha-SU8A, approximately 2.2 kb TaSU8A-cDNA was amplified by PCR using position 140-2304 of Seq. ID No.6.

The primers used in this reaction were as follows:

SUEX3:

5 '-GCG GTA CCT CTA GAA GGA GAT ATA CAT ATG GCG GAG GAC AGG TAC GCG CTC-3' (Seq.ID No.10), and

SUEX4:

5 '-GCT CGA GTC GAC TCA AAC ATC AGG GCG CAA TAC-3' (Seq.ID No.11).

1 ng of plasmid pTaSU8A was used as template in the reaction. The PCR reaction mixture additionally contained: 300 nM primers SUEX3 and SUEX4 each, 200 μΜ nucleotides dATP, dGTP, dCTP and dTTP, 1.6 mM MgCl ₂ , 60 mM Tris-SO 4 (pH 9.1), 18 mM ( NH ^^ SO ^ as well as 1 μΐ of Elongase ^ Enzyme Mix (mixture)

- 49 · · · · ··· * ··········

Taq Polymerase and DNA Polymerase, Life Technologies). The final volume of the reaction mixture was 50 μί. Amplification was performed in a PCR-Gerate (TRIO ^ Thermoblock, Biometra) at the following temperature program: 1 '(min) at 94 ° C (once); 30 ´ ´ (sec.) At 95 ° C - 30 ”at 55 ° C - 2´30 ´’ at 68 ° C (30 cycles); 10 ’at 68 ° C (once). This reaction produced a 2205 bp DNA fragment;

This 2.2 kb product was restricted with Kp I and SaZ1 and ligated into the expression vector pUbi.cas engineered with Kp I and S Z1. The resulting plant transformation vector was designated pTa-alpha-SU8A and was used to transform wheat according to the above procedure.

Claims (20)

PATENT CLAIMS A nucleic acid molecule encoding a protein having the function of wheat isoamylase selected from the group consisting of (a) a nucleic acid molecule encoding a protein comprising the amino acid sequence set forth in Seq ID NO. (B) a nucleic acid molecule comprising the nucleotide sequence set forth in Seq ID No. 3; 2 or a portion thereof or a corresponding ribonucleotide sequence; (c) a nucleic acid molecule hybridized to or complementary to any of the nucleic acid molecules referred to in (a) or (b); the acid of (a), (b) or (c), wherein the nucleic acid molecule of (a), (c) and (d) exhibits a homology of over 90% with Seq ID No. 2. 2. The nucleic acid molecule of claim 1 which is a DNA molecule. DNA molecule according to claim 2, characterized in that it is a cDNA molecule. • · · · ··· ······· · · ··· · · ··· The nucleic acid molecule of one or more of claims 1 to 3, which comprises regulatory elements. 5. The nucleic acid molecule of claim 1 which is an RNA molecule. A nucleic acid molecule that is specifically hybridized to the nucleic acid molecule of any one of claims 1 to 5 and which exhibits homology to Seq ID No. 5. 2 over 90%. The nucleic acid molecule of claim 6, which is an oligonucleotide of at least 15 nucleotides in length. A vector comprising a DNA molecule according to any one of claims 1 to 5. The vector of claim 8, wherein said nucleic acid molecule is linked to regulatory elements in sense orientation, which provides for transcription and synthesis of transfer RNA into pro- or eukaryotic cells. The vector of claim 8, wherein said nucleic acid molecule is linked to regulatory elements in a sense orientation that provides for transcription and synthesis of non-transfer RNA into pro- or eukaryotic cells. The vector of claim 8, wherein said nucleic acid molecule is linked to regulatory elements in an antisense orientation that provides for transcription and synthesis of non-transfer RNA into pro- or eukaryotic cells. 11 11 1112 1 A host cell which is transformed with a nucleic acid molecule according to one or more of claims 1 to 12 5 or a vector according to one or more of claims 8 to 11, or a cell derived from such a cell. A protein encoded by a nucleic acid molecule according to one or more of claims 1 to 4. A method for producing a protein according to claim 13, wherein the host cell according to claim 12 is cultured under conditions permitting the synthesis of said protein and said protein is isolated from the cultured cells and / or the culture medium. A method of preparing a transgenic plant cell comprising: (a) a nucleic acid molecule according to one or more of claims 1 to 5; or b) a vector according to one or more of claims 8 to 11 integrating plant cells into the genome. A transgenic plant cell transformed with a nucleic acid molecule according to one or more of claims 1 to 4 or one or more vectors according to claims 8 to 11, or a cell derived from such a cell. 17. A method for producing a transgenic plant, wherein a1) a nucleic acid molecule according to one or more of claims 1 to 5 or a process according to one or more of Claims 1 to 5, or (A2) a vector according to one or more of claims 8 to 11 integrates into the plant cell genome and b) regenerating the entire plant from said plant cell. A plant comprising a plant cell according to claim 16 19 Dec nou. Plant according to claim 19, which Yippee utility rostli- 20 May .mu.Ci Plant starch. according to claim 19, which Yippee plant imposes- 21. linou Plant by or plant Claim 20 maize. which Yippee monocotyl grate 22nd Plant according to claim 21, which Yippee plant rye, barley or warbler. Plant propagation material according to one or more of claims 18 to 22. Use of a portion of a plant according to claim 16, a plant according to one or more of claims 18 to 22 or propagation material according to claim 23 for the manufacture of starch.