CZ20004155A3 - Nucleic acid molecules encoding wheat enzymes involved in starch synthesis - Google Patents

Abstract

The solution relates to nucleic acid molecules encoding enzymes involved in starch synthesis in plants. These enzymes include wheat isoamylases. The solution further relates to vectors and host cells containing said nucleic acid molecules, in particular transformed plant cells and plants that can be regenerated from said cells and that exhibit increased or decreased isoamylase activity.

Description

NUCLEIC ACID MOLECULES CODING ENZYMES FROM WHEAT INVOLVED IN STARCH SYNTHESIS

Technical field

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

Furthermore, the present invention relates to vectors, host cells, as well as plant cells and plants, which contain the nucleic acid molecules of the present invention.

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

State of the art

Recently, the importance of plant-based ingredients as renewable sources of raw materials has been constantly increasing, and therefore one of the tasks of biotechnological research is to strive to adapt these plant-based raw materials to the requirements of the processing industry. In order to make the area of applicability of these renewable raw materials as wide as possible, it is necessary to achieve their maximum diversity.

Polysaccharides are an important renewable plant raw material, alongside oils, fats and proteins. The most important9 · • · · · « · « polysaccharides, besides cellulose, is starch, which is one of the main storage substances in higher plants. One of the most important cultivated plants is wheat, which accounts for 20% of the total starch production in the European Community.

The polysaccharide starch is a polymer consisting of chemically uniform basic units, namely glucose molecules. It 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 can also be derivatized, for example phosphorylated. This means that starch is not a uniform raw material. Amylose, which is formed by an essentially unbranched polymer consisting of 1,4-glycosidic glucose molecules, differs from amylopectin, which is formed by a complex mixture of differently branched glucose chains. 1,6-glycosidic bonds also participate in this branching. Starch synthesized in wheat contains approximately 11 to 37% amylose.

In order to ensure the usability of suitable starches for a wide range of industrial purposes, it is necessary to have plants that are capable of synthesizing modified starches that are particularly suitable for various uses. One option for obtaining such plants is breeding measures. However, breeding measures are very difficult to apply due to the polyploid nature of wheat (tetra- and hexaploid). Only recently has it been possible to obtain waxy (amylose-free) wheat by crossing natural wheat mutants (Nakamura et al., Mol. Gen. Genet. 248 (1995), 253-259).

An alternative to breeding measures is targeted ge• « • · · · «·· *·· • · · · · · · <> ♦ V · · * · · · • · · · · * · ······ ·

The biochemical synthesis pathways leading to the construction of starch are essentially known. Starch synthesis in plant cells takes place in plastids. In photosynthetically active tissues these are chloroplasts, in photosynthetically inactive starch storage tissues these are amyloplasts.

For further targeted genetic engineering transformation of the branching of starch synthesized in plants, it is still necessary to identify DNA sequences encoding enzymes that participate in starch metabolism, especially in chain branching or its degradation.

In addition to the so-called Q-enzymes, which introduce branching into the starch molecule, plants also contain enzymes that can break down the branching. These enzymes are called debranching enzymes and are divided into three groups according to their substrate specificity:

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

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

(b) Isoamylases, which do not require 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 require border dextrins as a substrate.

Li et al. (Plant Physiol. 98 (1992), 1277 - 1284) demonstrated in sugar beet, in addition to five endo- and two exoamylases, only one debranching enzyme of the pullulanase type. This enzyme, with a size of about 100 kD and an optimum pH of 5.5, is localized in chloroplasts. A debranching enzyme was also described in spinach, which used pullulan as a substrate.

Both the debranching enzyme from spinach and sugar beet have a fivefold lower activity when reacting 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 - potatoes - the activity of the Debranching enzyme was monitored by Hobson et al. (J.Chem.Soc, (1951), 1451). These authors demonstrated that the relevant enzyme, unlike the Q-enzyme, does not show any activity in chain elongation, but only hydrolyzes alpha-1,6-glycosidic bonds. However, this enzyme has not been characterized in more detail so far.

In potatoes, a procedure for purifying the debranching enzyme and also partial peptide sequences of the purified protein has already been proposed (VO 95/04826). In spinach, purification of the debranching enzyme and also isolation of the corresponding cDNA have been described (Renz et al., Plant Physiol. 108 (1995), 1342).

In maize, only one debranching enzyme has been described in the literature. This enzyme was classified in the isoamylas group according to its substrate specificity (see e.g. Hannah et al., Scientia Horticulturae 55 (1993), 177-197 or Garwood (1984) in the publication Starch Chemistry and Technology, Whistler, R.L., BeMiller, J.N., Puschall, E.F. (eds.), Academy Press San Diego, New York, Boston, 25-86). The corresponding mutants were designated as sugary. This sugar locus gene was recently 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 has been demonstrated in maize. There is also no evidence to date that other forms of debranching enzymes occur in maize. To obtain a transgenic maize plant that does not have any debranching enzyme activity, for example to achieve a higher degree of amylopectin branching, it is necessary to identify all forms of debranching enzymes that occur in maize and isolate the relevant genes or cDNA sequences.

To obtain further possibilities for performing transformations of any starchy plants, preferably cereals, and especially wheat, so that these plants synthesize modified starch, it is necessary to identify the relevant DNA sequences that encode other isoforms of enzymes affecting chain branching.

The essence of the invention

The aim of the present invention is the preparation of nucleic acid molecules that encode enzymes involved in starch biosynthesis and their use to obtain genetically modified plants that enable the production of starches with altered chemical and/or physical properties.

The present invention therefore relates to nucleic acid molecules encoding a protein with wheat isoamylase function, in particular a protein which is defined in particular by the amino acid sequence set out in Seq ID No. 3 or 7. The present invention relates in particular to a nucleic acid molecule which comprises the nucleotide sequence set out in Seq ID No. 1, 2 or 6 or a part thereof, preferably a molecule comprising the coding region set out in Seq ID No. 1, 2 or 6 and also the corresponding ribonucleotide sequences. Particularly preferred is a nucleic acid molecule comprising further regulatory elements which effect transcription and also optionally translation of said nucleic acid molecules.

Furthermore, the present invention relates to nucleic acid molecules that hybridize with certain nucleic acid molecules of the present invention.

The subject of this invention is also a nucleic acid molecule that encodes wheat isoamylase and whose sequence differs from the nucleotide sequence of the above-mentioned molecules due to the degeneracy of the genetic code.

The present invention also relates to a nucleic acid molecule which contains a sequence complementary to the complete above-mentioned sequences or to parts thereof.

The term hybridization means, within the scope of the present invention, hybridization under conventional hybridization conditions, preferably under strictly defined conditions described, for example, in the publication 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 with nucleic acid molecules of the present invention can in principle encode isoamylases from any wheat plant that expresses these proteins.

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

Identification and isolation of these nucleic acid molecules can be performed using molecules according to this

Φ Φ φ · ·· · ·· φφφφ · φ · · · ♦ · • φ φ φ φ φ · φ •φφφφφφ φφφ ·· φφφ of the invention or parts of these molecules or using reverse complements of these molecules, for example by hybridization according to standard procedures (see, for example, Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).

As a sample for hybridization, it is possible to use, for example, nucleic acid molecules containing exactly the same or substantially the same nucleotide sequences or parts thereof, as set forth under Seq ID No. 1, 2 or 6. These fragments used as a sample for hybridization may also be synthetic fragments prepared using conventional synthetic techniques, the sequence of which substantially corresponds to the nucleic acid molecules of the field of this invention.

The molecules hybridized with the nucleic acid molecules of the present invention also include fragments, derivatives and allelic variants of the above-mentioned nucleic acid molecules which encode the wheat isoamylases of the present invention. Fragments are understood to mean parts of nucleic acid molecules which are long enough to be able to encode the proteins mentioned. The term derivative in this context means that the sequence of these molecules differs from the sequences of the above-mentioned nucleic acid molecules at one or more points and that they show a high degree of homology to these sequences. Homology means a sequence identity of at least 40%, in particular an identity of at least 60%, in particular above 80% and particularly preferably above 90%. Deviations from the previously described nucleic acid molecules can arise by cleavage, substitution, insertion or recombination.

Homology further means that there is a functional and/or

structural equivalence between the relevant nucleic acid molecules or the proteins encoded by them. Nucleic acid molecules that are homologous to the above-mentioned molecules and represent derivatives of these molecules are usually variations of these molecules that represent modifications with the same biological functions. This can be both naturally occurring variations, for example sequences from other organisms, and mutants, whereby these mutants can occur naturally or can be introduced by targeted mutagenesis. Furthermore, these are variations of synthetically prepared sequences. Allelic variants can be both naturally occurring variants and variants prepared synthetically or obtained by recombinant DNA technology.

Isoamylases encoded by different variants of nucleic acid molecules according to the invention exhibit certain common properties. These include, for example, enzymatic activity, molecular weight, immunological reactivity, conformation, etc., as well as physical properties, such as gel electrophoresis mobility, chromatography behavior, sedimentation coefficients, solubility, spectroscopic properties, electrical charge, stability; pH-optimum, temperature optimum, etc.

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

The nucleic acid molecules according to the invention may be DNA molecules, in particular cDNA or genomic molecules. Furthermore, the nucleic acids according to the invention may be RNA molecules, which may be generated, for example,

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

The invention also provides oligonucleotides specifically hybridized with a nucleic acid molecule according to the invention. These oligonucleotides are preferably at least 10, in particular at least 15 and particularly preferably at least 50 nucleotides in length. The oligonucleotides according to the invention are characterized in that they hybridize specifically with the nucleic acid molecules according to the invention, i.e. they do not hybridize or hybridize only to a small extent with nucleic acid sequences encoding other proteins, in particular other isoamylases. The oligonucleotides according to the invention can be used, for example, as a primer for a PCR reaction or as a hybridization template for the isolation of related genes. They can also form part of antisense constructs or DNA molecules encoding a suitable ribozyme.

The invention further includes vectors, in particular plasmids, cosmids, phagemids, viruses, bacteriophages and other vectors common in genetic engineering, which contain the above-mentioned nucleic acid molecules according to the invention. These vectors are suitable for the transformation of prokaryotic or eukaryotic, preferably plant, cells.

Particularly preferred are vectors that allow the integration of nucleic acid molecules according to 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 transfer mediated by • ·

Agrobacteria. The synthesis of transferable or optionally non-transferable nucleic acid molecules in transformed pro- or eukaryotic cells by integration of the nucleic acid molecule in the sense- or anti-sense-orientation according to the invention is preferred.

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

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

The expression of nucleic acid molecules according to the invention in prokaryotic cells, for example in Escherichia coli, is of importance for a more precise characterization of the enzymatic activity of the enzymes encoding these molecules. In particular, it is possible to characterize the product synthesized by the respective enzymes in the absence of other enzymes involved in starch synthesis in plant cells. This allows conclusions to be drawn about the function of the respective protein in starch synthesis in plant cells.

In addition, it is possible to introduce various mutations into the nucleic acid molecules of the invention using conventional 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), whereby proteins with possibly altered biological properties are obtained during this synthesis. This allows the preparation of deletion mutants in which, by successive cleavage from the 5'- or 3'-end, the coding DNA sequence of the nucleic acid molecules can be obtained, which lead to the synthesis of the corresponding truncated proteins. This deletion at the 5'-end of the nucleotide sequence makes it possible, for example, to identify amino acid sequences that cause the translocation of enzymes in plastids (transit peptides). This allows the targeted preparation of enzymes that, after removal of the corresponding sequences, are no longer localized in the plastids, but in the cytosol, or that, after addition of other signal sequences, are localized in other parts of the cell.

Furthermore, it is possible to introduce local mutations at positions where the change in the amino acid sequence affects, for example, enzyme activity or enzyme regulation. In this way, it is possible to obtain, for example, mutants with an altered K _m value or mutants that are no longer subject to normal regulatory mechanisms in the cell by the action of allosteric regulation or covalent modification.

Furthermore, it is possible to obtain mutants of the proteins of the present invention which have altered substrate or product specificity. In addition, it is possible to prepare mutants of the proteins which exhibit altered activity-temperature profiles.

• · · · · · * ······ ·· · · · · · · ·

When carrying out genetic engineering modification of prokaryotic cells, it is possible to insert nucleic acid molecules according to the invention or parts thereof into plasmids, which also allow mutagenesis or sequence change by recombination of DNA sequences. Using standard procedures (see Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA) it is possible to carry out base substitutions or to attach natural or synthetic sequences. To connect DNA fragments to each other, it is possible to attach connecting or binding groups (Adaptoren o. Linker) to these fragments. It is also possible to use manipulations that release the relevant restriction cleavage sites or that remove excess DNA or restriction sites. In cases where insertions, deletions or substitutions are considered, primer repair, restriction or ligation can be used in in vitro mutagenesis. Sequence analysis, restriction analysis or other biochemical or molecular biological methods have generally been used as analytical methods.

In further 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 according to the invention or with one of the vectors according to the invention, as well as cells which are generated from such transformed cells and contain a nucleic acid molecule according to the invention or a vector. These are preferably pro- or eukaryotic cells, in particular plant cells.

The present invention also provides recombinantly produced proteins having isoamylase activity encoded by nucleic acid molecules.

according to the present invention and also a process for their preparation, wherein the host cell according to the present invention is cultured under suitable conditions known to those skilled in the art, allowing the synthesis of the protein according to the present invention and the final isolation of this protein from the host cells and/or from the culture medium.

The nucleic acid molecules obtained according to the present invention now allow, using genetic engineering methods, to carry out targeted interventions in starch metabolism in plants and to change this metabolism to the extent that such modified starches are synthesized which, compared to known starches, will have different physicochemical properties, such as the ratio of amylose to amylopectin, the degree of chain branching, the average chain length, the phosphate content, the lubricating properties, the gel or film forming properties, the size of the starch grains and/or their 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 this enzyme. The expression of the nucleic acid molecules of the invention also provides the possibility of reducing the natural activity of the isoamylase of the invention in plant cells. Furthermore, the nucleic acid molecules of the invention can be modified by methods known to those skilled in the art to obtain isoamylases of the invention that are no longer subject to the natural regulatory mechanisms of the cell itself, or that have an altered temperature-activity profile or altered specific properties of the substrate or product.

• · ·

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 specific part of the plant cell, the plastid-localizing sequence must be excised and the remaining coding region optionally linked to DNA sequences that localize to the appropriate site. Such sequences are known (see, for example, Braun et al., EMBO J. 11 (1992), 3219-3227; Volter et al., Proc. Natl. Acad. Sci. USA 85 (1988), 846-850; Sonnenwald et al., Plant J. 1 (1991), 95-106).

The present invention also relates to a process for preparing 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 the vector according to the invention is integrated into the genome of the plant cell, and further relates to transgenic plant cells transformed with a nucleic acid molecule or a vector according to the invention, and also includes transgenic plant cells which are derived from such transformed cells. The cells according to the present invention contain one or more nucleic acid molecules or vectors according to the invention, wherein these molecules or vectors are preferably linked to regulatory DNA elements which ensure transcription in plant cells, in particular to a suitable promoter. These cells can be distinguished from natural plant cells, inter alia, by the fact that they contain nucleic acid molecules according to the invention which are not normally present in these cells, or by the fact that such a molecule is integrated at a specific location in the genome of the cell where it does not otherwise occur, i.e. in a different genomic environment. Furthermore, these transgenic plant cells can be

of the invention can be distinguished from natural plant cells by the fact that these cells contain at least one copy of the nucleic acid molecule of the invention stably integrated into their genome, optionally in addition to the copies of this molecule naturally occurring in the cells. If the nucleic acid molecule(s) introduced into the cells are copies in addition to the molecules naturally occurring in the cell, the plant cells of the invention can be distinguished from natural cells in particular by the fact that this/these additional copies are/are located in the genome at a location(s) where they do not occur in nature. This can be easily demonstrated, for example, by Southern blot analysis according to methods known to those skilled in the art.

If the nucleic acid molecule of the invention introduced into the plant genome is heterologous with respect to the plant cell, the transgenic plant cells contain transcripts of the nucleic acid molecules of the invention, which can be easily demonstrated, for example, by Northern blot analysis according to methods known to those skilled in the art.

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

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

The present invention also provides a method for obtaining 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 the entire plant is regenerated from this cell. The present invention also provides plants comprising the above transgenic plant cells. These transgenic plants can in principle be plants of any plant species, i.e. both monocotyledonous and dicotyledonous plants. They are preferably useful plants, in particular plants which synthesize or store starch, particularly preferably rye, barley, oats, wheat, millet, sago, maize, rice, peas, string beans, cassava, potatoes, tomatoes, rapeseed, soybeans, hemp, flax, sunflower, field peas or arrowroot and in particular wheat, maize, rice and potatoes.

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

The present invention further relates to a process for producing modified starch, comprising the operation of extracting starch from the above-mentioned plants according to the invention and/or from starchy parts of these plants.

Processes for extracting starch from plants or their starchy parts, especially from wheat, are known to those skilled in the art, see for example Eckhoff et al. (Cereal Chem. 73 (1996) 54 - 57 Starch - Chemistry and Technology (published by Whistler, BeMiller and Paschall (1994), 2nd ed., Academie Press lne. London Ltd; ISBN 0-12-746270-8; see for example Chapter XII, pp. 412 - 468: Maize and sorghum starches: Production; Vatson; Chapter XIII, pp. 469 - 479: Tapioca, arrowroot and sago starches: Production; Corbishlev and Miller; Chapter XIV, pp. 479 - 490: Potato starch: Production and uses; Mitch; Chapter XV, pp. 491 - 506: Wheat starch: Production, modification and uses; Knight and Oson; and Chapter XVI, pp. 507 to 528: Rice starch: Production and uses; Rohmer and Klem). The equipment typically used to extract starch from plant material includes separators, decanters, hydrocyclones, spray dryers and fluidized bed dryers.

Transgenic plant cells and plants according to the invention synthesize starches based on the expression of nucleic acid molecules according to the invention, which differ from starches synthesized by natural plants in their physicochemical properties, such as the ratio of amylose to amylopectin, the degree of chain branching, the average chain length, the phosphate content, the lubricating properties, the size of the starch grains and/or their shape, based on the expression of the nucleic acid molecules according to the invention. In particular, the viscosity and/or the film-forming properties and the properties of the starch greases formed may be altered in these starches compared to the properties of known starches.

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

Furthermore, it is possible to produce plant cells and plants using the nucleic acid molecules of the invention in which the activity of the protein of the invention is reduced. This also leads to the synthesis of starch with altered chemical and/or physical properties compared to starches from the original plant cells.

Another object of the present invention are transgenic plant cells containing a nucleic acid molecule according to the present invention, in which 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 the appropriate anti-sense RNA, sense RNA to achieve a co-suppressive effect, or by expressing a correspondingly constructed ribozyme that specifically cleaves the transcript encoding isoamylase using the nucleic acid molecules of the invention according to a procedure known to those skilled in the art, see Jorgensen (Trends Biotechnol. 8 (1990), 340-344), 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, the reduction of the number of coding transcripts in plant cells, for example by expression of antisense RNA, is used to reduce the isoamylase activity of the present invention.

It is possible to use a DNA molecule which contains the complete sequence encoding 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 having to be long enough to induce an antisense effect in cells. In general, sequences of at least 15 bp in length can be used, preferably 100 to 500 bp in length, for effective antisense inhibition in particular sequences of over 500 bp in length. As a rule, DNA molecules which are shorter than 5000 bp, preferably sequences which are shorter than 2500 bp, are used.

It is also possible to use DNA sequences which show a high degree of homology to the sequences of the DNA molecules according to the invention, but which are not completely identical. The minimum homology should be higher than about 65%. Sequences with a degree of homology between 95 and 100% are preferably used.

The subject of the present invention is also a process for preparing modified starch, comprising a process for extracting starch from a cell or from a plant according to the present invention and/or from starchy parts of these plants.

The subject of the present invention is further starch obtainable from cells, plants and also propagation material or parts thereof according to the present invention and further starch obtainable by the process according to the present 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 uses in the food or non-food sector.

The possibilities of using starches according to this invention are discussed in

is essentially divided into two large areas. One area includes the products of starch hydrolysis, especially glucose and glucan building blocks, which can be obtained by enzymatic or chemical processes. These substances serve as starting materials for further chemical modifications and processes, such as fermentation. In this case, the simplicity and inexpensive implementation of the hydrolysis process can be of considerable importance for reducing costs. At present, this hydrolysis is mainly carried out enzymatically using amyloglucosidase.

It can be assumed that cost savings could be achieved by reducing the amount of enzymes used. Structural transformation of starch, such as reducing the degree of branching or changing the spherical structure, which determines the accessibility of the molecule for the enzymes used, could also have some influence.

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

1. Food industry

Starch is a classic ingredient in many foods, where it usually acts as a binder for aqueous components or is used to increase viscosity or cause increased gel formation. Important properties include fluidity and sorption, swelling and greasing temperature, viscosity and thickening ability, starch solubility, transparency and structure of starch grease, stability to heat, shear forces and acids, tendency to retrogradation, film-forming ability, freeze/thaw stability, viscosity stability in salt solutions, digestibility and also the ability to form complexes with, for example, inorganic or organic ions.

2. Non-food industry

In this broad area, starch can be used as an auxiliary in various production processes or as an additive in technical products. Starch as an auxiliary is used mainly in the paper and cardboard industries. In this application, starch is used mainly for retardation (binding of solids), binding of fillers and fine particles, as a stiffening agent and as a dewatering agent. In addition, the advantageous properties of starch are used to achieve stiffness, hardness, acoustic properties, the desired feel, gloss, smoothness, resistance to splitting and also to achieve the desired surface properties.

2.1 Paper and cardboard manufacturing industry

In papermaking, four areas of application are distinguished: surface treatment, coating, stock treatment and spraying. The requirements for starch for surface treatment are in particular a high degree of whiteness, appropriate viscosity, high viscosity stability, good film-forming ability and minimal dust formation. When starch is used for coatings, solids content, appropriate viscosity, high binding and also a high affinity for pigments are important. When starch is used as an additive in stock, its rapid, uniform and loss-free dispersion, high mechanical stability and

• · and complete retention of the starch in the stock stream. When using starch for spraying, adequate solids content, high viscosity and high binding are also important properties.

2.2. Adhesives industry

The adhesives industry represents a broad area of application for starches, which can be divided into four sub-areas: use as pure starch adhesive, use in starch adhesives with the addition of special chemicals, use of starch as an additive to synthetic resins and polymer dispersions, and finally use of starches as a setting agent in synthetic adhesives. 90% of starch-based adhesives are used in the production of corrugated cardboard, paper bags, sacks and cones, for the production of bonding materials for paper and aluminum, for the production of cardboard and as a wetting adhesive for envelopes, stamps and the like.

2.3 Textile industry and textile auxiliary products industry

A major consumer of starches is the textile and textile preparation industry, where starches are used as an auxiliary agent 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 auxiliary agent for smoothing, strengthening and increasing the cohesion of textile raw materials to protect against tensile forces during spinning and weaving and also to increase abrasion resistance during weaving, the use of starch as an agent for refining textiles, • · especially after quality-impairing treatments such as bleaching, dyeing and the like, starch is also used as a thickening agent in the production of dye pastes to suppress the diffusion of dyes and finally starch is used as an additive to agents for joining threads.

2.4 Construction

The fourth area of application is the use of starch as an additive to building materials. An example is plasterboard panels, where starch added to the gypsum slurry, under the influence of lubricating water, diffuses to the surface of the gypsum board and binds the cardboard to this board. Another area of application is the addition of starch to plasters and mineral fibers. In the transportation of concrete, starch products are used to slow down its hardening.

2.5 Soil stabilization

Another area of starch consumption is the production of soil stabilizers, which are used during soil movements to temporarily protect soil particles from water. According to current knowledge, combined products consisting of starch and polymer emulsions are comparable in their effects on reducing erosion and crust formation to those used to date, but are considerably cheaper.

2.6 Use in plant protection products and fertilizers

In this area, starch is used in plant protection products to modify their specific properties. Starches are used for improved wetting of plant protection products and fertilizers, for the gradual release of active ingredients, for the conversion of liquid, volatile and/or odorous substances into microcrystalline, stable and moldable substances, for the mixing of incompatible compounds and for the extension of the period of effectiveness by slowing down decomposition.

2.7 Pharmaceutical, healthcare and cosmetic industries

Another area of application of 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. In addition, starch is used as a means for rapid disintegration of tablets, where the tablets absorb liquid after swallowing and swell after a short time, releasing the active substance. Medical powders and powders for covering wounds contain starch as a basic substance. In the cosmetics sector, starches are used, for example, in powders as carriers for ingredients such as fragrances and salicylic acid. Relatively significant amounts of starches are used in toothpastes.

2.8 Adding starches to coal and briquettes

Starches are also added to coal and briquettes. Coal agglomerates or briquettes better after the addition of starch, which prevents premature disintegration of briquettes. 4 to 6% starch is added to barbecue coal, 0.1 to 0.5% starch to fuel coal. Starches are becoming increasingly important as a binder for coal, because the addition of starch to coal and briquettes can significantly reduce emissions of harmful substances.

2.9 Treatment of ore and coal sludges

Starches can also be used for the treatment of ore and coal sludges as a flocculating agent.

2.10 Foundry aids

Another area is the use of starch as an additive in foundry auxiliaries. Various casting processes require cores that are made from sands with a binder. The binder currently used is mainly bentonite, to which modified starches, especially swelling starches, are added. The addition of starch is used to increase the flow strength and to improve the binding capacity. In addition, swelling starches can meet other technical requirements, such as dispersibility in cold water, easy rehydration, good miscibility with sand 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 surface gloss, feel and appearance by dusting sticky rubberized surfaces with starch before cold vulcanization. Starch is also used to improve the printability of rubber products.

2.12 Production of leather substitutes

Another area of application of modified starches is the production of leather substitutes.

2.13 Starches in synthetic polymers

In the plastics sector, starches are used for the following purposes: addition of starch products during processing (starch acts only as a filler, no direct bond is formed between the synthetic polymer and starch) or alternatively for binding starch products during polymer production (starch and polymer form a strong bond).

The use of starch as a mere filler is not promising compared to other substances such as talc. However, the situation changes if the specific properties of starch are used and the properties of the final product are significantly changed. An example is the use of starch products in the processing of thermoplastics such as polyethylene. In this case, starch and synthetic polymer are processed by co-expression in a ratio of 1:1 to a so-called master batch, from which various products are produced with granulated polyethylene using conventional technological processes. The binding of starch in polyethylene films can increase the material permeability of the film hollow bodies, improve the water vapor permeability, improve the antistatic properties and also improve the printability with water-soluble inks.

Another option is the use of starch in polyurethane foams. By modifying starch derivatives and also optimizing technological parameters, it is possible to specifically control the reaction between synthetic polymers and hydroxyl groups of starch. The result is polyurethane films to which the use of starch gives the following properties: reduction of the coefficient of thermal expansion, reduction of shrinkage, improvement of properties under pressure/tension, increase of water vapor permeability without changing the ability to absorb water, reduction of flammability and possibility of tearing, no dripping of burning parts, absence of halogens and delayed aging. The disadvantages that are currently apparent are reduced compressive strength and reduced impact resistance.

However, product development was not limited to foils.

Nowadays, even solid products, such as cups, plates and bowls, containing more than 50% starch are produced. Starch and polymer mixtures are also considered very favorable from an ecological point of view, since they have significantly higher biodegradability.

Grafted starch polymers have gained special importance due to their extreme water-binding capacity. These are products whose main chain is starch and to this chain are grafted side chains of a synthetic monomer according to the principle of the radical mechanism. Grafted starch polymers that are currently available are characterized by an improved ability to bind and retain water, up to 1000 g of water per 1 g of starch at high viscosity. The areas of application of these superabsorbent substances have expanded greatly in recent years and these substances are used in the field of hygiene such as diapers and pads and also in agriculture, such as for seed coating.

The use of new genetically modified starches is determined by their 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, and by the properties that result in the following characteristics: flow behavior and sorption properties, greasing temperature, viscosity, viscosity stability in salt solutions, thickening ability, solubility, structure and transparency of starch grease, resistance to heat, shear forces and acids, tendency to retrogradation, gel formation, freeze/thaw stability, complex formation ability, iodine binding, film formation, adhesive bond strength, enzyme stability, digestibility and reactivity.

Modified starches obtained from the respective plants by means of genetic engineering processes can have their properties changed to such an extent that further modification by means of chemical or physical processes is no longer necessary. Alternatively, starches modified by genetic engineering processes can be further modified by chemical processes, so that further quality improvements are achieved for the above-mentioned purposes of use. These chemical treatments are known in principle. In particular, treatments using heat, organic or inorganic acids, oxidation and reesterification are used, whereby, for example, phosphates, nitrates, sulfates, xanthates, acetates or citrates of starch are formed. Furthermore, monofunctional or polyfunctional alcohols can be used in the presence of strong acids for the preparation of starch ethers, whereby alkyl ethers, O-allyl ethers, hydroxyalkyl ethers, O-carboxymethyl starch ethers, nitrogen-containing starch ethers, phosphorus-containing starch ethers, sulfur-containing starch ethers, cross-linked starches or grafted starch polymers are formed.

A preferred use of the starches according to the present invention is both the production of packaging materials and disposable products and the use of these starches as food and feed additives.

9 9 9 9 · · ·»····· ·

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 effect transcription in the plant cells.

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

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

Furthermore, a termination sequence may be present, which ensures the correct termination of transcription and also ensures the addition of a poly-A-terminus to the transcript, which is attributed to a function in stabilizing transcripts. These elements are described in the literature (see e.g. Gielen et al., EMBO J. 8 (1989) and are arbitrarily interchangeable.

• · ··· · · ·

The present invention provides nucleic acid molecules that encode a protein with the function of wheat isoamylase. The nucleic acid molecules of the present invention allow the preparation of this enzyme, which is functionally involved in starch biosynthesis, and which allow the production of genetically modified plants in which the activity of this enzyme is altered, thus allowing the synthesis of starch with an altered structure and with altered physicochemical properties in such modified plants.

The nucleic acid molecules of the invention can in principle also be used to obtain plants in which the activity of the isoamylase of the invention is increased or decreased and at the same time in which the activity of other enzymes involved in starch synthesis is altered. This change in the activity of the isoamylase in plants leads to the synthesis of starch with an altered structure. Furthermore, nucleic acid molecules encoding the isoamylase or corresponding anti-sense constructs can be introduced into plant cells in which the synthesis of endogenous starch synthases or enzymes affecting chain branching has already been 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 ed.: 25-86).

If the inhibition of several enzymes involved in biosynthesis is to be achieved in transformed plants, DNA molecules can be used for transformation which simultaneously contain several regions coding for the corresponding enzymes in antisense orientation under the control of a suitable promoter. Alternatively, either each sequence can be controlled by its own promoter, or the sequences can be transcribed as a fusion by a common promoter, or all sequences can be controlled by a common promoter. As a rule, the latter alternative is preferred, since in this case the synthesis of the relevant proteins is inhibited to approximately the same extent. The length of the individual coding regions used in the resulting construct is the same as that previously mentioned for the preparation of antisense constructs. There is no upper limit to 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 exceeding 5 kb in length.

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

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

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

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

Using this procedure, it is also possible to simultaneously inhibit the synthesis of several enzymes in plant cells transformed in this way.

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

The desired sequence can be introduced into the vector at the appropriate restriction site. The obtained plasmid is used to transform E.coli cells. The transformed E.coli cells are cultured in a suitable medium, and finally their isolation and lysis are performed. 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 obtained DNA fragments can be bound to other DNA sequences. Each plasmid DNA sequence can be cloned in the same plasmid or in other plasmids.

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

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

Depending on the method of introducing the desired genes into the plant cell, additional DNA sequences may need to be used. 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 Ri plasmid T-DNA must be linked as a border region to the gene to be introduced.

If Agrobacteria are used for transformation, the DNA to be introduced 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 sequences in the T-DNA. This plasmid also contains the viral region (vir-Region) required for the transfer of the T-DNA. Intermediate vectors cannot be replicated in Agrobacteria. Using a 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 selection marker gene and a binding member (linker or polylinker) that frame the T-DNA border region on the right and left. These vectors can be transformed directly in Agrobacteria (Holsters et al.

·

• 9

Mol. Gen. Genet. 163 (1978), 181 - 187). The Agrobacterium host cell should contain a plasmid carrying the vir-region. This vir-region is necessary for the transfer of the T-DNA into the plant cell. Other T-DNAs may also be present. Agrobacterium cells transformed in this way can be used for the transformation of plant cells.

The use of T-DNA for the transformation of plant cells has been the subject of intensive study and is described in detail 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,

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

For the transfer of DNA into a plant cell, it is possible to co-cultivate plant explants with Agrobacterium tumefaciens or Agrobacterium rhizogenes. From infected plant material (for example, pieces of leaves, parts of stems, roots, but also protoplasts or suspension-cultured plant cells), it is then possible to regenerate whole plants again in a suitable medium containing, among other things, 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 obtained in this way. Other possibilities for the introduction of foreign DNA using a biolistic procedure or by means of protoplast transformation are also known (see e.g. Villmitzer, L., 1933 Transgenic plants, in the publication Biotechnology, A MultiVolume Comprehensive Treatise (edit. H.J. Rehm, G.Reed, A.Puhler, P.Stadler), vol. 2, 627 - 659, VCH Veinheim-New York-Basel-Cambridge).

While the transformation of dicotyledonous plants by means of Ti-plasmid vector systems using Agrobacterium is well known, recent work shows that transformation using vectors derived from Agrobacterium can also be performed in monocotyledonous plants (Chán et al., Plant Mol. Biol. 22 (1993), 491-506; Hiei et al., Plant J. 6 (1994), 271-282).

Alternative methods for transforming monocot plants include the use of biolistic methods, protoplast transformation, or physically or chemically induced DNA insertion into protoplasts, for example by electroporation of partially permeabilized cells, DNA transfer using glass fibers, macroinjection of DNA into inflorescences, microinjection of DNA into microspores or pro-embryos, DNA insertion using germinating pollen, and DNA insertion into embryos by swelling (Review: Potrykus, Physiol. Plant (1990), 269-273).

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

Transformation of wheat has been described in a number of papers (Review: Maheswari et al., Critical Reviews in Plant Science 14 (2) (1995), 149-178). Hess et al. (Plant Sci. 72 (1990), 233) used a macroinjection procedure to bring pollen and Agrobacterium into close proximity. Mobilization of a plasmid containing the nptll gene as a selectable marker was demonstrated by Southern blot analysis and the NPTII assay. These transformants showed a normal phenotype and were fertile. Resistance to kanamycin could be demonstrated in two successive generations.

The first transgenic fertile wheat plant that could be regenerated after injection of DNA bound to microprojectiles was described by Vasil et al. (Bio/Technology 10 (1992), 667-674). The target tissue for injection was an embryogenic culture of potassium (callus type C). A pure gene (bar Gen) encoding phosphinothricin-acetyltransferase, which provided resistance to the herbicide Phosphinothricin, was used as a selection marker. Another system was 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 scutellum of immature embryos that were stimulated in the initial in vitro phase to induce somatic embryos. The efficiency of this transformation was significantly higher in the system developed by Becker et al. (see previous citation), with 1 transgenic plant per 83 embryos of the Florida species, than in the system developed by Veeks et al. with 1 to 2 transgenic plants per 1000 embryos of the Bohwhite species.

The system developed by Becker et al. (see citation above) forms the basis of the transformation experiments described in the examples.

Once the inserted DNA has integrated into the genome, it usually remains stably in it and is also retained in the progeny of the original transformed cells. It usually contains one of the above-mentioned selection markers which provides the transformed plant cells with, for example, resistance to biocides 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. The individually chosen marker should therefore allow selection of transformed cells • · over cells lacking the inserted DNA.

The transformed cells grow normally in the plant (see, e.g., McCormick et al., Plant Cell Reports 5 (1986), 81-84). The resulting plants can be grown normally and can be crossed with plants that have the same or different heritable characteristics. The hybrid individuals thus formed have the corresponding phenotypic characteristics. Seeds can be obtained from the plant cells. To ensure that the phenotypic characteristic is maintained and is heritable, it is necessary to grow for two or more generations. The seeds must also be harvested to ensure that the relevant phenotype or other characteristic characteristics are maintained.

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

Examples of embodiments of the invention

Example 1

Cloning procedure

For cloning in E.coli, the pBluescript II SK vector (Stratagene) was used.

Example 2

Bacteria strains

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

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

Example 3

Transformation of immature wheat embryos

Media:

MS: 100 ml/1 macrosalt (D.Becker and H. Lorz, 1 ml/1 microsalt Plant Tissue Culture #30: 2 mL/1 Fe/NaEDTA 30 g/l sucrose MS + 2,4-D (2 mg/1) Manual (1996), B 12 : 1 - 20) #31: MS + 2,4-D (2 mg/l) + Phosphinothricin (PPI, active

component of the herbicide BASTA

(2mg/1) #32: World Cup + 2,4-D (0.1mg/1) + PPT (2 mg/1) #39: World Cup + 2,4-D (2mg/1) + after 0.5 N mannitol/sorbitol

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

The method of transformation of immature wheat embryos was developed and optimized by Becker and Lórz (D.Becker and H.Lórz,

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

In the experiments described below, the protocol developed by Becker and Lórz (see citations earlier) was followed.

« · φ ·

For transformation, ears with caryopsis at the stage of development 12 to 14 days after anthesis were harvested and surface sterilized. The isolated scutella were plated with the ebrya axis attached to the medium onto induction medium #30.

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

For biolistic transformation, approximately 29 pg of gold particles were used per shot, on which several pg of target DNA had previously been precipitated. Since the experiments involved co-transformants, the target DNA for precipitation was added with the target gene and the resistance marker gene (bar-Gen) in a 1:1 ratio.

Example 4

DIG-labeling of DNA fragments

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

Media and solutions used in the examples:

x SSC 175.3 g NaCl

88.2 g sodium citrate, make up to 1000 ml with redist. water, adjust to pH 7.0 with 10 N NaOH solution

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

Example A

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

A heterologous screening strategy was used to identify the cDNA encoding the isoform of isoamylase (saccharide - sugar) from wheat. A wheat cDNA library was screened using a maize carbohydrate probe.

Isolation of this carbohydrate probe from the maize cDNA library was performed using a specific primer by PCR-amplification. Cloning of the maize cDNA library was performed from the poly(A) + RNA from a mixture of equal proportions of 13, 17, 19, 20, 22, 25 and 28 day (DAP) old caryopsis in the Lambda Zap II vector according to the manufacturer's instructions (Lambda ZAP II-cDNA synthesis kit, Stratagene GmbH, Heidelberg, Germany). For all caryopsis used, except for the 13-day-old grains, the embryo was removed before RNA isolation.

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

sulp-1a: 5'AAAGGCCCAATATTATCCTTTAGG 3' (Seq.ID No.4) sulp-2 : 5'GCCATTTCAACCGTTCTGAAGTCGGGAAGTC 3' (Seq.ID No.5)

As templates for the PCR reaction, 2 μΐ ·· *« ·· · *· · ··· · · · · * • · · · · · « ···« »··· • · · · · · · · · ···»· ·· ··· *· amplified corn cDNA library was used. This PCR reaction mixture also contained 1.5 - 3 mM MgC^, 20 mM Tris-HCl (pH 8.4), mM KC1, 0.8 mM dNTP Mix, 1 μΜ primer sulp-1a, 1 μΜ primer sulp-2 and 2.5 units Taq polymerase (recombinant, Life Technologies). The amplification was performed in a Trioblock device from Biometra according to the scheme: 4’(min)/95 °C; 1’/95 °C; 45” (sec.)/58 °C; 1’15”/72 °C; 30 cycles of 5’/72°C. The amplified DNA bands of approximately 990 bp were separated 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 sugary fragment on an agarose gel, band excision and isolation of this fragment, DIG-labeling of the probe was performed. 500 ng of the sugary fragment was used for labeling the random prime with digoxygenin. 10 μί of Random Primer was added to this labeled fragment and the reaction mixture was heated for 5’ at a temperature of 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-ll-dUTP (Boehringer Mannheim) and Klenow buffer (standard) and 1 unit of Klenow polymerase were added. The reaction was carried out at RT (laboratory temperature) overnight. Dot-test was used to check the labeling according to the manufacturer's instructions (Dig System User's Guide for Filter Hybridization from Boehringer, Mannheim,

Germany).

The synthesis of the wheat cDNA library was performed from poly(A)+RNA from 21-day-old caryopsis (starch-endosperm) in the Lambda ZAP II vector according to the manufacturer's instructions (Kit for Lambda ZAP II-cDNA synthesis, Stratagene GmbH, • · • »

Heidelberg, Germany). After titration of the cDNA library, the primary titer was determined to be 1.26 x 10 pfu/ml.

For the investigation of the wheat cDNA library, approximately 350,000 phages were plated on the plate. The phage plating and the evaluation (Abziehen) of the plates were carried out according to a standard protocol. Prehybridization and filter hybridization were carried out in 5X SSC, 3% Blocking (Boehringer, Mannheim),

0.2% SDS, 0.1% sodium lauryl sarcosine and 50 pg/ml herring sperm DNA (Heringssperma) at 55 °C. 1 ng/ml of labeled carbohydrate probe was added to the hybridization solution and hybridization was incubated overnight. 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 for further screening procedures. Pooled clones were obtained by in vivo excision as pBluescript SK Phagemide (performed according to manufacturer’s instructions; Stratagene, Heidelberg, Germany).

After analysis of this clone by minipreparation and restriction of the obtained plasmid DNA, the 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 was further analyzed.

Example B

Sequence analysis of cDNA-insertion plasmid pTaSU-19

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

The insert of the clone pTaSU-19 has a length of 2997 bp and represents a partial cDNA. The nucleotide sequence is given under the designation Seq ID No.l. Comparison with previously published sequences showed that the sequence given under the designation Seq ID No.l includes the complete coding region, which shows homology to isoamylases from other organisms. Analysis of this sequence also showed that the cDNA sequence contains 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 shows homology to protein sequences of isoamylases from other organisms. The amino acid sequence corresponding to the coding regions of Seq ID No.l is given under the designation Seq ID No.3.

Example C

Preparation of the plant transformation vector pTa-alpha-SU19

For the expression of antisense RNA corresponding to TaSU19 cDNA, the plant transformation vector pTa-alpha-SU19 was constructed based on the basic plasmid pUC19, in which the cDNA insert of the plasmid pTa-alpha-SU19 was linked in the antisense orientation to the 3'-end of the ubiquitin promoter. This promoter consists of the first untranslated exon and the first intron of the ubiquitin 7 gene from maize (Christensen A.H. et al., Plant Molecular Biology 18 (1992), 675-689). The polylinker parts and the NOS terminator come from the plasmid pACTl.cas (CAMBIA, TG 0063; Cambia, GPO Box 3200, Canberra ACT 2601, Australia). Vector constructs with this terminator and constructs based on pACTl.cas are described in the work of McElroy et al. (Molecular Breeding 1 (1995),

- 37). The vector thus created was named PUbi.cas.

• ·

Cloning of this vector was performed by restriction of a 2 kb fragment from clone Ta-SU19 using the restriction enzyme Xba 1. This fragment was filled in at the ends using the Klenow reaction and then ligated into the Sma I cloning site of the expression vector pUbi.cas.

The resulting expression vector was designated Ta-alpha-SU19 and was used for wheat transformation using the above procedure.

Example D

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

The wheat cDNA library was probed with the Sugary-Probe, which represented a part of the pTaSU19 clone, namely positions 489-1041 of Seq.ID No.1.

The preparation of the wheat-specific digoxygenin-labeled Sugary-Probe used for screening the cDNA library was carried out 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 a template in the reaction. The PCR reaction mixture also contained 300 nM primer SUSO1 and SUSO2, 100 μΜ each of nucleotides dATP, dGTP, dCTP, 65 μΜ dTTP, 35 μΜ of Digoxygenin-ll-dUTP preparation (Boehringer Mannheim), 1.5 mM MgCl ₂ and 2.5 U (units)

• · of Taq Polymerase preparation and 10 μΐ of tenfold 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-Gerát instrument (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 incorporation of Digoxygenin-ll-dUTP into the PCR product was demonstrated by the slight mobility in an agarose gel compared to the product of a control reaction without Digoxygenin-ll-dUTP.

The karyotype-specific wheat cDNA library from Example 1 was probed using the obtained digoxygenin-labeled probe.

The hybridization procedure was performed overnight in 5X SSC, 0.2% SDS, 0.1% sodium lauryl sarcosine and 50 pg/ml herring sperm DNA (Heringssperma) at 68°C with the addition of 1 ng/ml digoxygenin-labeled probe. After hybridization, the filters were washed 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 the phage clones by in vivo excision (protocols according to the manufacturer’s instructions; 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 E

Sequence analysis of the cDNA insert in plasmid pTaSU8A

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

This insertion of the clone pTaSU8A is 2437 bp long and represents a partial cDNA. When compared with previously published sequences, it was shown that the sequence designated as Seq. ID No.6 includes a coding region showing homology to isoamylases from other organisms. Likewise, the protein sequence derived from the coding region of the clone pTaSU8A, shown as Seq. ID No.7, showed homology to protein sequences of isoamylases from other organisms.

A comparison of the sequences of clones pTaSU19 (Seq.ID No.1) and pTaSU8A (Seq.ID No.6) shows a 96.8% similarity. Most of the differences between the two sequences lie in the 3’-untranslated region of the cDNA. Other sequence differences in the coding region are caused by different amino acids in a total of twelve positions of the derived protein sequences (Seq.ID Nos.3 and 7). The cDNAs contained in pTaSU19 and pTaSU8A are not identical and encode isoforms of isoamylase from wheat.

Example F

Preparation of the plant transformation vector pTa-alpha-SU8A

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

For cloning pTa-alpha-SU8A, a portion of TaSU8A-cDNA of approximately 2.2 kb in size, namely positions 140 - 2304 of Seq. ID No.6, was amplified by PCR.

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 a template in the reaction. The PCR reaction mixture also contained: 300 nM each of primers SUEX3 and SUEX4, 200 μΜ each of nucleotides dATP, dGTP, dCTP and dTTP, 1.6 mM MgCl ₂ , 60 mM Tris-S04 (pH 9.1), 18 mM (NH^^SO^ and also 1 μΐ of Elongase^ Enzym Mix (mixture • · · · · * · · · • · · · · · · · · · · · ·

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Taq Polymerase and DNA Polymerase, Life Technologies). The final volume of the reaction mixture was 50 μί. Amplification was performed in a PCR-Gerát device (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 DNA fragment with a length of 2205 bp.;

This 2.2 kb product was restricted with KpI and SaZ1 and ligated into the expression vector pUbi.cas modified with KpI and SaZ1. 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.