BRPI0017574B1 - Method for Production of a Transgenic Plant Cell with Increased Activity of an Amylosacarase Protein and a Branching Enzyme - Google Patents

Description

Report of the Invention Patent for "METHOD FOR PRODUCTION OF A TRANSGENIC PLANT CELL WITH INCREASED ACTIVITY OF AN AMYLOSACARASE PROTEIN AND A RAMIFICATION ENZYME".

Divided from PI 0010989-4, filed May 26, 2000. The present invention relates to methods for producing a transgenic plant cell with increased activity of an amylosaccharide protein and increased activity of a branching enzyme. Such plant cells synthesize a modified starch and / or synthesize branched alpha-1,4-glucans with a modified branch degree at the 0-6 position and / or give higher yield with genetically wild type plants. corresponding unmodified (plant cells).

In the area of agriculture and sivilculture it was a permanent effort to produce high yielding plants, in particular to secure food for the continually growing population of the world and to ensure the supply of regenerating raw materials. Traditionally, attempts have been made to obtain reproductive productive plants. For each plant species of interest a corresponding breeding program has to be carried out. This is, however, time consuming and labor intensive. Progress has been made, partly by genetic manipulation of plants, that is, by introducing and intentionally expressing recombinant amino acid molecules in plants. Such approaches have the advantage that in general they are not being limited to one plant species but can be transferred to other plant species. Therefore, it seems desirable to provide plants and plant cells that give increased yields as well as to offer methods for producing such plant and plant cells.

With reference to the growing importance that was attached to plant substances as a source of raw material regeneration recently, one of the tasks in biotechnology research is to strive to adjust these plant raw materials to the needs of the production industry. In order to facilitate the use of regenerating raw materials in as many application areas as possible, it is essential to achieve a wide variety of substances. Moreover, it is necessary to increase the yield of these plant substances in order to increase the efficiency of the production of regenerating raw material sources. Despite oils, fats and proteins, polysaccharides are the most important regenerating plant raw materials. Despite cellulose, starch plays a vital role with polysaccharides as it is one of the most important reserve substances in higher plants.

Despite its use in food, polysaccharide starch is also widely used as a regenerating raw material for the production of industrial products. Polysaccharide starch consists of chemically uniform basic components, glucose molecules, but forms a complex mixture of several molecules that have different degrees of branching and polymerization and thus differ substantially in their physical and chemical properties.

A differentiation is made between amylose starch, a basically unbranched polymer of alpha-1,4-glycosidically bonded glucose units, and amylopectin starch, a branched polymer in which branching is caused by alpha- Additional 1,6-glycosides. According to the literature Voet and Voet, Biochemistry, John Wiley & Sons, 1990) alpha-1,6-glycosidic bonds occur on average every 24 ° every 30 ° glucose residue. This corresponds to a degree of branching of about 3% - 4%. Details of the degree of branching are variable and depend on the source (eg plant species, plant variety, etc.) of the individual starch. Plants typically used for industrial starch production vary in their amylose content from the total starch content between 10 and 25%. In order to facilitate a very wide use of polysaccharides such as, for example, starch it seems desirable to provide plants that are modified in their polysaccharide composition and, for example, are capable of synthesizing modified and / or alpha-1,6-alpha starch. Highly branched -1,4-glucans which are particularly suitable for various uses. One possibility for producing such plants is - despite the form of breeding methods - intentional modification of starch metabolism in starch producing plants by genetic engineering methods. A prerequisite to date, however, is the identification and characterization of enzymes playing a role in starch modification and / or synthesis as well as the isolation of the corresponding DNA molecules encoding these enzymes.

The paths of chemical synthesis leading to starch formation are essentially known. Starch synthesis in plant cells occurs in plastids. In photosynthetically active tissues these are chloroplasts, in photosynthetically inactive starch storage tissues or amyoplasts.

The most important enzymes involved in starch synthesis are starch synthases (see, for example, WO 96/15248), R1 enzyme (see, for example, WO 97/11188) as well as branching enzymes (cf. WO 92/14827). The exact role of other enzymes such as, for example, starch phosphorylases (cf., for example, WO 98/40503) during starch biosynthesis is not known. In order to provide further possibilities for modifying any plants such that they synthesize modified starch, it is also possible to introduce foreign, such as bacterial or fungal, nucleic acid molecules, which are not present in wild type plants and which encode. proteins involved in polysaccharide synthesis. For example, the synthesis of so-called "Amylofructan" could be shown to be possible by amyloplast expression of bacterial fructosyltransferases in amyloplasts (Smeekens, Trends in Plant Science vol. 2 No. 8 (1997), 286-288). Heterologous expression of a bacterial glycogen synthase in potato plants leads to a slight decrease in amylose content, an increase in branching and a change in the branching pattern of amylopectin compared to wild type plants (Shewmaker et al. Plant Physiol. 104 (1994), 1159-1166).

Moreover, the expression of a bacterial branching enzyme in potato plants in amylose-free (amf) potato mutants (Jacobsen et al., Euphytica, 44 (1989), 43-48) leads to amylose molecules. pectin having 25% more branch points (Kortstee et al., The Plant Journal, 10 (1), (1996), 83-90) than control molecules (amf). The increase in branching points was due to a modification of the chain length distribution of longer side chains in favor of shorter side chains. Reduction in mean chain length and reduction in λιτίθχ after iodine staining are also an indication for a higher branched amylopectin structure in transformed plants compared to unprocessed plants (Kortstee et al, see above). The degree of glycogen branching of about 10% could, however, not be achieved via this approach. Glycogen, a polysaccharide, which is found primarily in animals and bacteria, contains highly branched alpha-1,6-alpha-1,4-glucans. Glycogen differs from starch also in the average length of the side chains and the degree of polymerization. According to the literature (Voet and Voet, Biochemistry, John Wiley & Sons, 1990) contains one alpha-1,6-branch point on each 8th or 12th glucose residue on average. This corresponds to a degree of branching from about 8% to 12%. There are several figures, for glycogen molecular weight ranging from 1 million to more than 1000 million (DJ Manners in: Advances in Carbohydrate Chemistry, Ed. ML Wolfrom, Academic Press, New York (1957), 261-298; Geddes et al., Carbohydr Res., 261 (1994), 78-89). These figures, too, depend to a great extent on the organism of the corresponding source, its nutritional status as well as the kind of glycogen isolation. It is usually obtained by costly and time-consuming methods from mussels (eg, Mytillus edulis), from mammalian liver muscles (eg, rabbits, rats) (Bell et al., Biochem. J. 28 ( Bueding and Orrel, J. Biol. Chem. 236 (1961), 2854). In addition, in plants there is, for example, the southern corn mutant the so-called phytoglycogen which has a branching degree of about 10% and which shows, in comparison with amylopectin, a branched side chain distribution. (Yun and Matheson, Carbohydrate Research 243, (1993), 307-321) and a different solubility behavior. Such phytoglycogen accumulation plants, however, show a reduction in starch content of up to 90% (Zeeman et al., Plant Cell 10, (1998), 1699-1711).

Furthermore, an in vitro method using amylosaccharase and a branching enzyme for the synthesis of alpha-1,6-branched alpha-1,4-glucans are described which among others allows the production of highly branched glucans (similar to glycogen) (German patent application DE 19846635.8). The production of such glucans in plants, however, is not described there.

It therefore appears desirable to provide alternative medium which allows for the reasonably estimated production of modified starches and / or alpha-1,6-alpha-1,4-glycans with a modified branch degree at the 0-6 position compared to wild type plants. in plants.

Thus, the underlying technical problem of the present invention is to provide plant and plant cells which, in comparison to unmodified wild-type plant cells and corresponding wild-type plants, contain a modified composition of the polysaccharides contained in plant and plant cells. and, if possible, also show a higher yield.

This problem has been solved by providing the embodiments characterized in the claims.

Therefore, the present invention relates to transgenic plant cells that are genetically modified wherein the genetic modification is the introduction of a foreign nucleic acid molecule or several foreign nucleic acid molecules in the presence or absence of which leads to an activity. of an amylosacarase protein and an increased activity of a branching enzyme protein compared to genetically unmodified plant cells of wild-type plants. Genetic modification can be any modification that leads to an increase in amylosacarase activity and an increase in branching enzyme activity.

In a preferred embodiment the genetic modification consists of introducing a foreign nucleic acid molecule encoding an amylosaccharide protein and a branching enzyme into the genome of a plant cell.

This foreign nucleic acid molecule may, for example, be called a "double construct" which is a simple vector for plant transformation that contains the genetic information encoding both an amylosaccharide protein and a branching enzyme.

Nucleic acid molecules encoding the amylosaccharide enzyme and the branching enzyme which are both contained within the "foreign nucleic acid molecule" may or, independently of each other, be under the control of a promoter each or they may, after fusion as traditional unit, be under the control of the same promoter.

In another preferred embodiment several foreign nucleic acid molecules are introduced into the plant cell genome wherein a foreign nucleic acid molecule encodes an amylosaccharide protein and another foreign nucleic acid molecule encodes a branching enzyme.

Hereby, foreign nucleic acid molecules may be introduced into the plant cell genome at the same time or consecutively. In the former case it is called a "co-transformation", in the latter a "supertransformation". The term "transgenic" therefore means that the plant cell of the invention contains at least one preferably two foreign nucleic acid molecules stably integrated into the genome, preferably one or two foreign nucleic acid molecules encoding an amylosaccharide protein in an enzyme. branching The term "foreign nucleic acid molecule" preferably means a nucleic acid molecule that encodes a protein with amylosaccharide activity and a protein with branching enzyme activity that either does not occur in the corresponding naturally occurring or non-naturally occurring plants. in the actual spatial order in plants or that is located in a place in the plant genome where it does not occur naturally. Preferably, the foreign nucleic acid molecule is a re-combining molecule consisting of several elements the combination or specific spatial order of which does not occur naturally in plants. The plants of the invention contain at least one nucleic acid molecule encoding an amylosaccharide activity protein and a branching enzyme activity protein preferably linked with regulatory DNA elements that ensure transcription in plants, in particular with a promoter. The term "various foreign nucleic acid molecules" preferably means two nucleic acid molecules wherein one foreign nucleic acid molecule encodes an amylosaccharide protein and the second foreign nucleic acid molecule encodes a branching enzyme.

In principle, the foreign nucleic acid molecule (s) may be any nucleic acid molecule (s) encoding an amylosaccharide protein and a branching enzyme. .

Within the present invention an amylosacarase protein (sucrose; 1,4-alpha-D-glucan 4-alpha glycotransferase, EC2.4.1.4) refers to an enzyme that preferably catalyzes the conversion of sucrose into fructose and water insoluble alpha-1,4-glucans. The following reaction scheme is suggested for this enzyme Sucrose + (alpha-1,4-D-glucan) n -> D-fructose + (alpha-1,4-glucan) n + i This is a transglycosylation reaction. The products of this in vitro reaction are water insoluble fructose and alpha-1,4-glucans.

Nucleotide activated cofactors or sugars are not required for this reaction. The enzyme, however, is stimulated in vitro by the presence of glycosyl group acceptors (or initiators) such as, for example, maltooligo, dextrin or glycogen saccharide onto which sucrose glycosyl residue is transferred accordingly. with the above reaction scheme with concomitant alpha-1,4-glucan chain extension (Remaud-Simeon et al., In SB Peterson, B. Svenson and S. Pedersen (Eds.), Carbohydrate bioengineering, 313-320 ( 1995); Elsevier Science BV, Amsterdam, The Netherlands).

Within the present invention, in principle, all suitable amylosacases are suitable for catalyzing the synthesis of linear alpha-1,4-glucans from sucrose.

Amylosaccharides have so far only been known from bacterial species, in particular mainly from the Neisseria species (MacKenzie et al., Can. J. Microbiol. 24 (1978) 357-362).

Therefore, an amylosaccharose of prokaryotic origin is preferably used. Amylosaccharases are known, for example, from Neisseria perflava (Okada and Hehre, J. Biol. Chem. 249 (1974), 126-135; Mackenzie et al., Can. J. Microbiol. 23 (1977), 1303-1307) or Neisseria canis, Neisseria cinerea, Neisseria denitrificans, Neisseria sicca and Neisseria subflava (MacKenzie et al., Can. J. Microbiol. 24 (1978), 357-362). Furthermore, WO 95/31553 and PCT / EP 98/05573 describe an amylosaccharide from Neisseria polysaccharea.

In a preferred embodiment of the invention the nucleic acid molecule encodes an amylosacarase from a bacterium of the genus Neisseria.

In a particularly preferred embodiment of the invention the foreign nucleic acid molecule encodes an amylosaccharide from Neisseria polysaccharea, more preferably an amylosaccharide having the amino acid or nucleic acid sequence as disclosed in international patent application PCT / EP 98/05573. The enzyme which is expressed in Neisseria polysaccharea is extremely stable, is firmly bound to polymerization products and is competitively inhibited by reaction product fructose (MacKenzie et al., Can. J. Microbiol. 23 (1977) 1303-1307). . With the species of Neisseria Neisseria polysaccharea amylosaccharase is secreted (Riou et al., Can. J. Microbiol. 32 (1986), 909-911), while with another species of Neisseria it remains in the cell.

A branching enzyme (alpha-1,4-glucan; alpha-1,4-glucan 6-glycosyltransferase, EC2.41.18) is a protein that catalyzes a transglycolysis reaction in which a donor's alpha-1,4 bonds. alpha-1,4-glucan chains are hydrolysed and the free established alpha-1,4-glucan chains in this process are transferred over an alpha-1,4-glucan acceptor chain and thereby transformed into alpha-1 bonds, 6

In connection with the present invention, in principle all branching enzymes of any bacterial origin (bacterial, fungal, plant, animal) are suitable, for example, branching enzymes derived from maize (see, for example, Baba et al. Biochem, Biophys, Res. Comm. 181 (1991), 8794; Genbank Acc. No. AF072724, AF072725), from potatoes (Kossmann et al., Mol. Gen. Genet. 203 (1991), 237- 244; Jobling et al., Genbank Acc. No. AJ011885), from rice (Mizuno et al., J. Biochem. 112 (1992), 643-651; Kawasaki et al., Mol Gen. Genet. 237 (1993)). 10-16; Mizuno et al., J. Biol. Chem. 268 (1993), 190844-19091; Nakamura and Yamanouchi, Plant Physiol. 99 (1992), 1265-1266) from wheat (Baga et al. Plant Mol. Biol. 40 (1999), 1019-1030; Rahman et al., Theor. Appi. Genet. 98 (1999), 156-163 and Genbank Acc. No. Y12320), derived from barley (Genbank Acc. No. AF064561), from Synechocystis (Genbank Acc. No. D63999), from E.coli (Baecker et al., J. Biol Chem 261 (1986), 8738-8743; Genbank Acc. No. M13751), from Bacillus stearo-thermophilus (Genbank Acc. No. M35089), Streptomyces aureofaciens (Genbank Acc. No. L11647), Bacillus caldolyticus (Genbank Acc. No. Z14057), Syn-echococcus PCC6301 (Genbank Acc. (M151544), Synechococcus sp. PCC7942 (Kiel et al., Gene 78 (1989), 9-17) and from Agrobacterium tumefaciens (Genbank Acc. No. AF033856). Isolation of the corresponding genes is possible for the person skilled in the art by standard molecular biological procedures, as described, that is, by Sambrook et al. (Sambrook et al., Molecular cloning: A laboratory manual, 2nd edition, Cold Spring Harbor Laboratory Press, NY, USA (1989)).

In a preferred embodiment of the invention the foreign nucleic acid molecule encodes a branching enzyme from a prokaryote, preferably from a bacterium of the genus Neisseria, with particular preference from Neisseria denitrificans and even more preferably from a branching enzyme. with a nucleotide sequence shown in SEQ ID No. 1 or with the amino acid sequence shown in SEQ ID No. 2.

In another preferred embodiment the foreign nucleic acid molecule encodes a plant branching enzyme. There are a variety of techniques for introducing DNA into a plant host cell. These techniques include transformation of plant cells with T-DNA using Agrobacterium tumefaciens or Agrobacterium rhizogenes as a transformation agent, fusion of protoplasts, injection, DNA electroporation, DNA introduction with the biolistic approach as well as other methods. possibilities. The use of Agrobacterium-measured transformation of plant cells has been intensively examined and sufficiently described in EP 0 120516; Hoekema, IN: The Binary Plant Vector System Offsetdrukkerij Kanters B.V., Alblasserdam (1985), Chapter V; Fraley et al., Crit. Rev. Plant I know. 4, 1-46 and An et al. EMBO J. 4, (1985), 277-287. For potato processing, see, for example, Rocha-Sosa et al., EMBO J. 8, (1989), 2933). Transformation of monocotyledonous plants by Agrobacterium-based vectors has been described (Chan et al., Plant Mol. Biol. 22, (1993), 491-506; Hiei et al., Plant J. 6, (1994) 271 -282; Deng et al., Science in China 33, (1990), 28-34; Wilmink et al., Plant Cell Reports 11, (1992), 76-80; May et al., Bio / Technology 13, ( 1995), 486-492; Connor and Domisse, Int. J. Plant Sci. 153 (1992), 550-555; Ritchie et al., Trangenic Res. 2 (1993), 252-265). An alternative system for the transformation of monocotyledonous plants is transformation with the biolistic approach (Wan and Lemaux, Plant Physiol. 104, (1994), 37-48; Vasil et al., Bio / Technology 11 (1993), 1553-1558). ; Ritala et al., Plant Mol. Biol. 24, (1994), 317-325; Spencer et al., Theor. Appl. Genet. 79, (1990), 625-631), protoplast transformation, electroporation partially permeabilized cells, the introduction of DNA via glass fibers. Corn transformation, in particular, has been described in the literature repeatedly (cf. for example, WO 95/06128, EP 0513849, EP 0465875, EP 0 292435; Fromm et al Biotechnology 8, (1990), 833-844; Gordon Kamm et al., Plant Cell 2, (1990), 603-618; Koziel et al., Biotechnology 11 (1993), 194-200; Moroc et al., Thear. Appl. Genet. 80 (1990), 721-726). Successful transformation of too many other species of grain has already been described, for example, for barley (Wan and Lernaux, see above; Ritala et al., See above; Krens et al., Nature 296, (1982), 72 -74) and for wheat (Nehra et al., Plant J. 5, (1994), 285-297).

In general any promoter active in plant cells can be used for expression of the foreign nucleic acid molecule (foreign nucleic acid molecules). The promoter may be chosen in such a way that expression in the plants of the invention occurs either constitutively or only in a certain tissue at a certain point in time of plant development or at a time determined by factors that exceed external influence. With reference to the plant the promoter may be homologous or heterologous.

Suitable promoters are, for example, the Cauliflower Mosaic Virus RNA 35S promoter and the maize ubiquitin promoter for constitutive expression, the B33 patatin gene promoter (Ro-cha-Sosa et al., EMBO). J. 8 (1989), 23-29) for a tuber-specific expression in potatoes or a promoter which guarantees expression in photosynthetically active tissue only, for example, the ST-LS1 promoter promoter (Stockhaus et al., Proc. Natl. Acad. Sci. USA 84 (1987), 7943-7947; Stockhaus et al., EMBO J. 8 (1989), 2445-2451), the Ca / b promoter (see, for example, US Patent No. 5656496, US Patent No. 5639952, Bansai et al., Proc. Natl. Acad. Sci. USA 89, (1992), 3654-3658) and the Rubisco SSU promoter (see, for example, US Patent No. 5034322, US Patent No. 4962028) or the glutelin promoter for an endosperm specific expression (Leisy et al., Plant Mol. Biol. 14, (1990), 41-50; Zheng et al., Plant J 4, (1993), 357-366; Yoshihara et al. ., FEBS Lett. 383, (1996), 213-218), the shrunken-1 promoter (Werr et al., EMBO J. 4, (1985), 1373-1380), the wheat HMG promoter, the USP promoter, the phaseolin promoter or maize zein gene promoters (Pedersen et al., Cell 29, (1982), 1015-1026; Quatroccio et al., Plant Mol. Biol. 15 (1990), 81-93). Expression of the foreign nucleic acid molecule (the foreign nucleic acid molecules) is particularly advantageous in those plant organs that have an increased sucrose content or store sucrose. Such organs are, for example, the beet turnip or the trunk of sugar cane or millet sugar. Preferably, therefore, promoters used are those that mediate expression in these organs. Other promoters, however, may also be used, that is, those that are only active at a point in time determined by factors that exert outside influence (cf., for example, WO 9307279). Here, heat shock protein promoters may be of special interest as they allow for simple induction. In addition, seed specific promoters such as, for example, the Vicia faba USP promoter, which guarantees seed specific expression in Vicia faba and other plants, may be used (Fiedler et al., Plant Mol. Biol 22, (1993), 669-679; Baumlein et al., Mol. Gen. Genet. 225 (1991), 459-467). In addition, fruit-specific promoters may be used as described, for example, in WO 91/01373, WO 99/16879, van Haaren and Houck (Plant Mol. Biol. 21 (1993), 625-640). .

In addition, a determination sequence may be present which is useful for the correct transcription termination as well as for the addition of poly-A end to the transcript which is assigned a function in transcript stabilization. Such elements have been described in the literature (cf., for example, Gielen et al., EMBO J. 8 (1989), 23-29 and are arbitrarily interchangeable.

The plant cells of the invention may be differentiated from naturally occurring plant cells among others in that they contain one or more foreign nucleic acid molecule (s) that do not naturally occur in these cells or that such a molecule (s) is found integrated into such a place in the genome of plants where it does not normally occur, that is, in another genomic adjacency. Moreover, such transgenic plant cells of the invention may be differentiated from naturally occurring plant cells when they contain at least one copy of the foreign nucleic acid molecule (foreign nucleic acid molecules) stably integrated into their genome, possibly beyond those of the genome. copies of such a naturally occurring molecule in plant cells. If the nucleic acid molecule (s) that is introduced into the cell is an additional copy (s) of naturally occurring molecules in plants then the plant cells of the invention may be be differentiated from naturally occurring plant cells in particular by the fact that this additional copy (s) is located in the places in the genome where it does not occur naturally. This can be tested, for example, by Southern Blot analysis.

Furthermore, the plant cells of the invention may be differentiated from naturally occurring plant cells preferably by one of the following characteristics: if the introduced nucleic acid molecule (s) is heterologous with reference to to the plant, transgenic plant cells show transcripts of introduced nucleic acid molecules. These can be detected, for example, by Northern Blot analysis. Preferably, the plant cells of the invention contain proteins that are encoded by the foreign nucleic acid molecule (s). This can be tested, for example, by immunological methods, in particular by Western blot analysis.

If the introduced molecule is homologous with reference to the plant, the transgenic plant cells of the invention may be differentiated from naturally occurring plant cells, for example due to the additional expression of the introduced foreign nucleic acid molecules. Transgenic plant cells preferably contain more transcripts of foreign nucleic acid molecules. This can be tested, for example, by Northern Blot analysis. The term "genetically modified" means that the plant cell is modified in its genetic information by introducing a foreign nucleic acid molecule or several foreign nucleic acid molecules and that the presence or expression of foreign nucleic acid molecule (s) (s) leads to a phenotypic change. Thus, hay-typical change preferably means a measurable change of one or more plant functions (plant cells). Plant cells of the invention, for example, show increased activity of an ami-lossacarase protein and a protein with branching enzyme activity due to the presence or expression of the introduced nucleic acid molecule.

In the context of the present invention the term "increased activity" means increased expression of the nucleic acid molecule (various nucleic acid molecules) that encodes a protein with amylosacarase activity and a protein with branching enzyme activity or an increase in the activity of nucleic acid. a protein with amylosacarase activity and a protein with branching enzyme activity in plants.

Increased expression can be determined, for example, by measuring the amount of transcripts encoding such proteins, for example by Northern Blot analysis. There, an increase in preference means an increase in the amount of transcripts compared to corresponding genetically unmodified plant cells by at least 10%, preferably by at least 20%, particularly preferably by at least 50% and especially by preference. at least 75%. The increase in the amount of protein with amylosaccharide activity or with branching enzyme activity can be determined, for example, by Western blot analysis. An increase in preference means an increase in the amount of protein with amylosaccharide activity or with branching enzyme activity and / or an increase in amylosaccharide activity or branching enzyme activity compared with corresponding genetically unmodified cells for at least 10 %, preferably at least 20%, particularly preferably at least 50% and especially preferably at least 75%. The activity of the amylosacarase protein and branching enzyme may, for example, be tested as described in the examples. Moreover, the activity of a branching enzyme can be determined as described in Lloydeta; (Biochem. J. 338 (1999), 515-521). Amylosacarase activity can also be determined as described below in the "Materials and Methods ..." section 3.

Surprisingly, it has been found that plants containing such plant cells with increased activity of an amylosacarase and a branched alpha-1,4-glucan branching enzyme with a branching degree modified at the 0- 6 which are not synthesized by corresponding genetically modified wild-type plant cells.

In one embodiment of the invention plant cells of the invention contain alpha-1,6-branched alpha-1,4-glucans with a degree of branching at the 0-6 position of at least 2%, preferably at least 4%. In another embodiment the degree of branching is at least 6%, preferably at least 8%, particularly preferably at least 10% and especially preferably at least 12%.

Within the framework of the present invention "branching degree" means the average number of branches at the 0-6 position compared to all differently linked glucose units. The degree of branching can be determined via a methylation analysis as, for example, described below. General information on this method can also be found, for example, in "Analysis of Carbohydrates by GLC and MS" (Biermann, CJ and McGinnis, G. D (eds) CRC Press (1989) Chapter 9 by Carpita, NC and Shea, MS, 157-216) or Biordal H. et al. (Angew. Chem., 82, (1970), 643-652; Int. Ed. Engl. 9, (1970), 610-619).

In another embodiment of the invention the plant cells of the invention synthesize modified starches that differ from corresponding wild-type plant cell starches in their physicochemical properties, in particular the amylose / amylopectin ratio, the degree of ramification. medium chain length, phosphate content, pulping properties, size and / or shape of the starch granule. In particular, such starch may be modified with reference to the viscosity and / or gel forming capacity of starch pastes compared to wild type starch.

In another embodiment of the invention plants containing the plant cells of the invention have a higher yield compared to corresponding genetically unmodified wild type plants.

Within the present invention, the term "wild-type plant" means that the plants served as starting materials for the production of the plants of the invention, ie whose genetic information, in addition to the genetic modification introduced, corresponds to that of a plant of the invention. .

Here the term "increased yield" means an increase in yield by at least 5%, preferably by at least 10%, particularly preferably by at least 20% and especially preferably by at least 30%. The term "increased yield" preferably means an increase in the production of substances and / or biomass, particularly when measured based on fresh weight per plant.

Such increase in yield preferably refers to plant parts that can be collected such as seeds, fruit, storage roots, roots, tubers, flower, bud, bud, logs or wood.

According to the invention the yield increase is at least 3% with reference to biomass substances and / or content compared to corresponding unprocessed plants of the same genome type if grown under the same conditions, preferably at least 10%, particularly preferably at least 20% and especially preferably at least 30% or even 40% compared to wild type plants.

In another embodiment of the present invention the plant cells of the invention have an increased caloric value compared to corresponding non-genetically modified wild-type plant cells. The term "caloric value" is defined as the amount of energy (given in calories or joule) the body gets through food digestion and that is used to cover energy needs. The term "increased caloric value" means an increase in caloric value by at least 5%, preferably by at least 10%, particularly preferably by at least 20% and especially preferably by at least 30%.

High calorie plants are of interest to the food industries, in particular to the diet of people with high energy needs, such as, for example, sick or elderly people, children or competitive athletes.

In a preferred embodiment the nucleotide sequence encoding an amylosaccharide enzyme and a branching enzyme comprise a protein targeting signal sequence that assures localization in a specific cell compartment, such as vacuole or plastids. In a particularly preferred embodiment the nucleotide sequences encoding the two enzymes comprise a protein targeting signal sequence which ensures that both enzymes are located in the same cell compartment. In this context, the foreign nucleic acid molecule may comprise one or more protein targeting signal sequence (s) that ensure the location of the amylosaccharide enzyme and the branching enzyme in the same cell compartment. It is in particular possible that each coding region encoding the amylosaccharide or branching enzyme comprises more than one combination sequence or a combination of different signal sequences.

In another embodiment of the invention the foreign nucleic acid molecule has one or more protein targeting signal sequence (s) that mediate a vacuolar location of the amylosaccharide protein and branching enzyme.

Nucleic acid molecules encoding the amylosaccharide enzyme and the branching enzyme that are both contained within the "foreign nucleic acid molecule" may either be under the control of one or more of the targeting signal sequence (s). independently of each other or they may be under the control of one or more protein targeting signal sequence (s) together after fusion as a translational unit.

In one embodiment of the invention foreign nucleic acid molecules have one, each or several protein targeting signal sequence (s) each mediating a vacuolar location of the amylosaccharide protein and branching enzyme.

In this embodiment several foreign nucleic acid molecules are introduced into the plant cell genome wherein a foreign nucleic acid molecule encodes an amylosaccharide protein and another nucleic acid molecule encodes a branching enzyme. As mentioned earlier, foreign nucleic acid molecules can be introduced into the plant cell genome simultaneously or consecutively.

Each of the foreign nucleic acid molecules contains one or more protein targeting signal sequence (s) that measures a vacuolar location of each amylosaccharide protein and the branching enzyme in which the targeting signal sequences of protein may be identical or may differ from each other. The N-terminal sequence (146 amino acids) of patathin protein, for example, can be used as a vacuolar targeting sequence (Sonnewald et al., Plant J. 1, (1998), 95-106). In a preferred embodiment the signal sequence described in SEQ ID NO: 7 is used. In addition, the following signal sequences may be used as vacuolar targeting sequences: the tomato acid (Genbank Acc. No. LMS1081) or potato acid (Genbank Acc. No. L29099) invertase signal sequence , the N-terminal signal sequence of sweet potato sporamine (Koide et al., Plant Physiol. 114 (1997), 863-870), the N-terminal signal sequence of barley aleurain (Vitale and Raikhel, Trends in Plant Science 4 (1999), 149-155), the N-terminal signal sequence of the potato proteinase inhibitor (Genbank Acc. No. X04118) in combination with the barley lectin C-terminal vacuolar targeting signal peptide (Vitale and Raikhel, loc. Cit.), Other vacuolar signal sequences are described, for example, Matsuoka and Neuhaus, Journal of Experimental Botany 50, (1999), 165-174; Chrispeel and Raikel, Cell 68 (1992), 613-616; Matsuoka and Nakamu-ra, Proc. Natl. Acad. Know. USA 88, (1991), 834-838; Bednarek and Raikhel, Plant Cell 3, (1991), 1195-1206; Nakamura and Matsuoka, Plant Phys. 101, (1993), 1-5. In general, a combination may be used comprising an N-terminal signal sequence, which assures the transport of the respective protein into the endoplasmic reticulum, and the C-terminal vacuolar targeting sequence. An overview of vacuolar targeting sequences can be found in Chrispeels and Raikhel (Cell 68 (1992), 613-616).

Since vacuole can usually store large amounts of sucrose that serves as a substrate for amylosaccharide, this compartment is suitable for producing plant cells which, due to increased activity of an amylosaccharide protein and increased activity of a branching enzyme synthesize alpha-1,4-glucans alpha-1,6-branched in the vacuole. In one embodiment of the invention these glucans at position 0-6 have a branching degree of at least 1%, preferably at least 4%, particularly preferably at least 7% and especially preferably at least 10%.

In another embodiment of the invention the degree of branching at the 0-6 position may be controlled by selection of transgenic plants showing different ratios of branching enzyme activity to amylosaccharide activity.

In a particularly preferred embodiment plant cells according to the invention in which both amylosaccharide and branching enzyme are located in the vacuole show an increased caloric value. For the definition of this term, see above.

In another embodiment of the invention the foreign nucleic acid molecule has one or more protein targeting signal sequence (s) that mediate a plastidic location of the amylosaccharase protein and the branching enzyme protein.

The nucleic acid molecules for the amylosaccharide enzyme and the branching enzyme are both contained within the "foreign nucleic acid molecule" or may independently be under the control of one or more signal sequence (s). Each of them may, after fusion as a translational unit, be under the control of one or more protein targeting signal sequence (s).

In another embodiment of the invention the foreign nucleic acid molecules have one or more protein targeting signal sequence (s) each that mediates a plastidic location of the amylosacase protein and the branching enzyme protein.

In this embodiment several foreign nucleic acid molecules are introduced into the plant cell genome wherein a foreign nucleic acid molecule encodes an amylosaccharide protein and another foreign nucleic acid molecule encodes a branching enzyme. As mentioned earlier, foreign nucleic acid molecules can be introduced into the plant cell genome simultaneously or consecutively.

Each of the introduced foreign nucleic acid molecules contains one or more protein targeting signal sequence (s) that mediate a plastidic location of each of the amylosaccharide protein and the branching enzyme protein in which the sequences. Protein targeting signals are identical or different from each other. The signal sequence of spinach ferradine: NADP + oxidoreductase (FNR), for example, can be used as the signal sequence. The sequence contains the 5 'untranslated region as well as the cDNA flanking transit peptide sequence of the iron-doxin plastid protein: NADP + spinach oxidoreductase (nucleotide -171 to +165; Janens et al., Current Genetics 13, (1988), 517-522).

In addition, for example, the cornflower protein transit peptide plus the first 34 amino acids of the mature waxy protein (Klösgen et al., Mol. Gen. Get. 217, (1989), 155-161) may be used. as the signal sequence.

Other plastid targeting sequences that can be used are: Rubisco's small subunit signal sequence (Wolter et al., Proc. Natl. Acad. Sci. USA 85 (1988), 846-850; Nawrath et al., Proc Natl. Acad. Sci. USA 91 (1994), 12760-12764), the NADP-maleate dehydrogenase signal sequence (Gallardo et al., Plant 197 (1995), 324-332) and the reductase signal sequence de glutation (Creissen et al., Plant J. 8 (1995), 167-175).

In a preferred embodiment of the invention the maize waxy protein transit peptide (see above) is used (see example 1) without the first 34 amino acids of the mature waxy protein.

In a particularly preferred embodiment the plastid signal sequence of potato-derived R1 protein is used (Lorberth et al., Nature Biotechnology 16 (1998), 437-477).

With the amyloplast expression of fructosyltransferase bacteria-na it could be shown that plastids also contain sucrose which can be transformed into "amylofrutane" by fructosyltransferase into amylplasts (Smeekens, Trends in Plant Science Vol. 2 No. 8 (1997), 286 -288). Therefore the compartment is also suitable for the combined expression of an amylosaccharide gene and a branching enzyme gene and allows the synthesis of modified starch which is modified, for example, in its physicochemical properties, particularly the amylose / amylopec ratio. tina, the degree of branching, the average chain length, the phosphate content, the pulping properties, the size and / or shape of the starch granule compared to starch synthesized in wild type plants.

Therefore, in another embodiment of the invention the transgenic plant cells of the invention synthesize modified starches.

In a preferred embodiment of the invention the gel stability of these starches is changed compared to starches extracted from wild type plants. In a particularly preferred embodiment the maximum gel stability is increased by at least 20%, more preferably by at least 50%, even more preferably by at least 100% and especially preferably by at least 200% compared to extracted starches. of wild type plants. Gel stability can be determined as described in example 9.

Starches isolated from the plant cells of the invention may also be modified according to methods known to the person skilled in the art, suitable in both modified and unmodified form for various food and non-food applications.

In principle, the starch application area can be subdivided into two large areas. One area comprises starch hydrolysis products, mainly glucan and glucose components which are obtained via enzymatic or chemical methods. They serve as starting materials for other chemical modifications and processes, such as fermentation. With reference to cost savings the simplicity and cost efficient driving of a hydrolysis method may be of importance. At the moment, it is mainly enzymatic when amyloglycosidase is used. It would be conceivable to protect costs by reducing the amount of enzymes used. A modification of starch structure, for example grain surface extension, easier digestibility through lower branching or a steric structure that limits accessibility to the enzymes used could achieve that. The other area in which starch is used due to its polymer structure with so-called native starch can be divided into two other application areas: 1. Food Industry Starch is a classic additive of various foods, where essentially it serves the purpose of bind aqueous additives or cause increased viscosity or increased gel formation. Important characteristics sorption and flow behavior, swelling and slurry temperature, viscosity and thickening performance, starch solubility, transparency and slurry structure, film forming capacity, freeze / thaw resistance, digestibility as well as the ability to complex with, for example, organic or inorganic ions. 2. Non-food Industry In this vast area starch can be used as an aid in various production processes or as an additive in technical products. The main field where starch is used as an aid is the paper and cardboard industry. In this field where starch is mainly used for retention (retention solids) for the bonding of filler and fine particles as solidifying substance and for dehydration. In addition, the advantageous properties of starch with reference to stiffness, hardness, sound, toughness, gloss, smoothness, tear resistance as well as surfaces are used. 2.1 Paper and cardboard industry Within the paper production process, a differentiation can be made between four fields of application, namely surface, coating, mass and spraying.

Starch requirements with respect to surface treatment are essentially a high degree of gloss, corresponding viscosity, high viscosity stability, good film formation as well as low dust formation. When used in coating the solid content, a corresponding viscosity, a high bindability as well as a high pigment affinity play an important role. As an additive for uniform, quick mass, oil free dispersion, high mechanical stability and complete paper pulp retention are of importance. When spraying starch, corresponding solids content, high viscosity as well as high binding capacity are also significant. 2.2 Adhesive Industry A major field of application is, for example, in the adhesive industry, where starch is used in four areas: use as a pure starch glue, use in starch glues prepared with special chemicals, use of starch as an additive for synthetic resins and polymer dispersions as well as the use of starches as diluents for synthetic adhesives. 90% of all starch-based adhesives are used in the production of corrugated cardboard, paper bags and pouches, aluminum and paper composite materials, boxes and moisten glue for envelopes, stamps, etc. 2.3 Textile and Textile Care Industry Another possible use of starch as an auxiliary and additive is in the production of textile and textile care products. Within the textile industry, a differentiation can be made between the following four fields of application: the use of starch as a sizing agent, i.e. an aid for smoothing and strengthening of tuning behavior for protection against stressors as well as for increased resistance to use during weaving, as an agent for textile improvement especially after quality deteriorating pretreatment such as bleaching, dyeing, etc., as a thickener in the production of dye pastes. the prevention of dye diffusion and as an additive for sewing thread warping agents. 2.4 Construction Industry The fourth area of starch application is its use as an additive in building materials. An example is the production of plasterboard, in which starch is mixed in the thin plaster pastifiers with water, diffuses on the surface of the plasterboard and thus connects the cardboard to the board. Other fields of application are mixed with plaster and mineral fibers. In ready-to-use concrete, starch can be used to slow down the bonding process. 2.5 Soil stabilization In addition, starch is advantageous for the production of soil stabilization medium used for the temporary protection of soil particles against water on artificial earth displacement. According to the prior art, combination products consisting of polymer and starch emulsions can be considered to have the same erosion reduction and scaling effects as the products used thus far; however, they are considerably less expensive. 2.6 Use of Starch in Fertilizers and Plant Protectors Another field of amplification is the use of starch in plant protectors to modify the specific properties of these preparations. For example, starches are used to improve the wetting of protectors and fertilizers, for the metered release of active ingredients, for the conversion of liquid, aromatic and / or volatile active ingredients into stable deformable, microcrystalline substances by mixing incompatible compositions. and for prolonging the effect duration due to slower decomposition. 2.1 Drug, medicine and cosmetics industry Starch can also be used in the drug, medicine and home appliance fields. In the pharmaceutical industry, starch may be used as a tablet binder or for diluting the binder in capsules. In addition, starch is suitable as a tablet disintegrant since on swallowing it absorbs fluid and after a short while it swells so much that the active ingredient is released. For qualitative reasons, medical lubricating powders and medical wound powders are starch based. In the field of cosmetics, starch is for example used as a carrier for powder additives such as perfume and salicylic acid. A relatively wide field of application for starch is toothpaste. 2.8 Starch as a charcoal and briquettes additive Starch can also be used as a charcoal and briquettes additive. By adding starch, charcoal can be quantitatively agglomerated and / or briquetted in high quality, thus preventing premature disintegration of briquettes. Barbecue charcoal contains between 4 and 6% added starch, "hot" charcoal between 0.1 and 0.5%. What's more, the toxic substances starch. 2.9 Coal and Ore Paste Processing Furthermore, starch can be used as a flocculating agent in the processing of coal and ore paste. 2.10 Starch as a Molding Additive Another field of application is the use of starch as an additive for molding process materials. For various molding processes cores produced from sands mixed with bonding agents are required. Today, the most commonly used binding agent is bentonite mixed with modified starches, most often swelling starches. The purpose of starch addition is increased flow resistance as well as increased bond strength. In addition, swelling starches can fulfill other prerequisites for the production process, such as cold water dispersibility, rehydratability, good area mixability and high water binding capacity. 2.11 Use of starch in the rubber industry In the rubber industry starch can be used to improve technical and optical quality. Reasons for this are improved surface gloss, toughness and appearance. For this purpose, starch is dispersed on sticky starched surfaces and rubber substance before cold vulcanization. It can also be used for rubber stamping enhancement. 2.12 Production of Leather Substitutes Another field of application for modified starch is the production of leather substitutes. 2.13 Starch in Synthetic Polymers In the plastics market the following fields of application are appearing: the integration of starch products in the processing process (starch is just a filler, there is no direct link between synthetic polymer and starch) or, alternatively , the integration of starch products into polymer production (starch and polymer form a stable bond). The use of starch as a pure filler cannot compete with other substances such as talc. Those changes when specific starch properties become effective and the property profile of the end products are thus clearly changed. An example is the use of starch products in the processing of thermoplastic materials such as polyethylene. Thus, starch and synthetic polymer are combined at a ratio of 1: 1 by co-expression to form a master batch from which various products are produced by common techniques using granulated polyethylene. Integration of starch in polyethylene films can cause increased substance permeability in hollow bodies, improved water vapor permeability, improved antistatic behavior, improved antiblock behavior as well as improved stamping with aqueous dyes.

Another possibility is the use of starch in polyurethane foams. Due to the adaptation of starch derivatives as well as the optimization of processing techniques, it is possible to specifically control the reaction between synthetic polymers and starch hydroxy groups. The results are polyurethane films that achieve the following property profiles due to the use of starch: reduced coefficient of thermal expansion, decreased shrinkage behavior, improved stress / pressure behavior, increased water vapor permeability without a change in acceptability. water, cracking density and reduced flammability, no falling flammable parts, no halogen aging or reduced aging. Disadvantages that still exist today are impact resistance and reduced pressure.

Film product development is no longer the only option. Also solid plastic products such as pots, plates and bowls can be produced with starch content of more than 50%. Moreover, starch / polymer blends offer the advantage that they are biodegradable to a greater extent.

Moreover, due to their extreme ability to bind water, starch graft polymers have gained greater importance. These are products having a starch backbone and a grafted synthetic monomer side network according to the radical chain mechanism principle. Starch graft polymers available today are characterized by improved bonding and capacity retention of up to 1000 g water per g starch at a high viscosity. In recent years these superabsorbers have been more widely used - mainly in the field of hygiene, for example in products such as diapers and sheets, as well as in the agriculture sector, for example in seed pellets.

Decisive factors for the use of new starch modified by recombinant DNA techniques are, on the one hand, structure, water content, protein content, lipid content, fiber content, ash / phosphate content, ami-lose ratio / amylopectin, relative molar mass distribution, branching degree, granule size and shape as well as crystallization, and on the other hand, properties that result in the following characteristics: flow and sorption behavior, slurry temperature, viscosity, saline viscosity, thickening performance, solubility, transparency and paste structure, acid, shear and heat resistance, tendency to retrograde, gel forming capacity, freeze / thaw resistance, complex forming capacity, bonding of iodine, film formation, adhesive resistance, enzyme stability, digestibility and reactivity. Genetically modified starch production with a transgenic plant can modify the starch properties obtained from the plant in such a way as to make further modifications by superfluous physical or chemical methods. On the other hand, starches modified by recombinant DNA techniques could undergo further chemical modifications, which will result in further quality improvement for certain of the above described fields of application. These chemical modifications are primarily known to the person skilled in the art. These are particularly modifications by - heat treatment - acid treatment - oxidation and - esterification leading to the formation of phosphate, nitrate, sulfate, xanthate, acetate and citrate starches. Other organic acids may also be used for esterification: - formation of starch ethers starch alkyl ether, O-allyl ether, hydroxyalkyl ether, O-carboxymethyl ether, N-containing starch ethers, P-containing starch ethers and starch ethers containing S. - branched starch formation - starch graft polymer formation.

In another embodiment of the invention a foreign nu-cyclic acid molecule has one or more protein targeting signal sequence (s) that mediates a specific cell wall location of the amylosaccharide protein and branching enzyme.

Nucleic acid molecules encoding the amylosaccharide enzyme and branching enzyme that are both contained within the "foreign nucleic acid molecule" may either be under the control of one or more protein targeting signal sequence (s) independently. each other or they may be under the control of one or more protein targeting signal sequence (s) together after fusion as a translational unit.

In another embodiment of the invention foreign nucleic acid molecules have one or more protein targeting signal sequence (s) each mediating a specific cell wall location of the amylosaccharide protein and the branching enzyme protein. In this embodiment several foreign nucleic acid molecules are introduced into the plant cell genome wherein a foreign nucleic acid molecule encodes an amylosaccharide protein and another foreign nucleic acid molecule encodes a branching enzyme. As mentioned earlier, foreign nucleic acid molecules can be introduced into the plant cell genome simultaneously or consecutively. In the first case it is called "co-transformation" in the last "supertransformation".

Each of the introduced foreign nucleic acid molecules contains one or more protein targeting signal sequence (s) that mediate a specific cell wall location of the amylosaccharide protein and the branching enzyme protein each in which they are present. Protein targeting signal sequences are the same or different from each other.

When signal sequence that of the potato proteinase II inhibitor may be used (von Schaewen et al., EMBO J. 9, (1990), 3033-3044; Keil et al., Nucleic Acid Research 14, (1986), 5641- 5650).

In another embodiment of the invention the foreign nucleic acid molecule (s) mediate a cytosolic location of the amylosaccharide protein and branching enzyme.

Moreover, the present invention relates to transgenic plants containing such plant cells with increased activity of an α-kilosacase and a branching enzyme.

The plants of the invention may belong to any plant species, that is, they may be monocotyledonous or dicotile-donea plants. Preferably, they are plants derived from agriculturally useful plants, that is, from plants that are grown by man for use as food or for technical, particularly industrial use. The invention preferably relates to fiber forming plants (e.g. flax, cannabis, cotton), oil storage plants (e.g. rapeseed, sunflower, soybeans), starch storage plants (e.g. wheat barley, oats, rice, potatoes, maize, rice, peas, cassava), sugar storage plants (eg sugar beet, sugar cane, millet) and protein storage plants (eg plants legumes).

In another preferred embodiment the invention relates to food plants (for example, forage harvest and pasture plants (alfalfa, clover etc.)), vegetable plants (for example tomatoes, salad, chicory). With particular preference are beet, sugar cane, corn, wheat and rice. The present invention also relates to a method for producing transgenic plants giving increased yield with wild type plants wherein a) a plant cell is genetically modified by introducing one (several) nucleic acid molecule (s) strange (s) the presence or expression of which leads to increased activity of a protein with amylosaccharide activity and an increase in activity of a protein with branching enzyme activity; b) a plant is regenerated from the cell produced according to a); and c) other plants are optionally produced from the plant produced according to step b).

Furthermore, the present invention relates to a method for producing a transgenic plant which synthesizes alpha-1,4-branched alpha-1,4-glucans with a modified branching degree at the 0-6 position compared to genetically unmodified wild-type plants in which a) a plant cell is genetically modified by introducing one or more foreign nucleic acid molecule (s) the presence or expression of which leads to increased activity of a protein with activity of an amylosacarase and an increased activity of a protein with activity of a branching enzyme; b) a plant is regenerated from the cell produced according to a); and c) other plants are optionally produced from the plant produced according to step b).

Another subject matter of the present invention is a method for producing a transgenic plant which synthesizes a modified starch compared to corresponding genetically unmodified wild type plants wherein a) a plant cell is genetically modified by introducing one or more nucleic acid molecule (s) strange to the presence or expression leading to increased activity of a protein with amylosaccharide activity and increased activity of a protein with activity of a branching enzyme; b) a plant is regenerated from the cell produced according to a); and c) other plants are optionally produced from the plant produced according to step b). The same as described above in another context with reference to the plants of the invention applies to the genetic modification introduced according to step a). Plant regeneration according to step b) may be performed according to methods known to the person skilled in the art. The generation of other plants according to step c) of the method of the invention is achieved for example by vegetative propagation (eg via seedlings, tubers or callus culture and regeneration of whole plants) or through reproduction. sexual. Sexual reproduction is preferably performed under control, that is, selected plants with certain properties are crossed and propagated. The present invention relates to plants obtainable by the methods of the invention. The person skilled in the art knows that he can obtain the plants of the invention not only by the above-mentioned methods of the invention but also by crossing, for example, a genetically modified plant that has an increased activity of a protein with amylosaccharide activity due to the introduction of a foreign nucleic acid molecule with a transgenic plant that has increased activity of a protein with branching enzyme activity due to the introduction of a foreign nucleic acid molecule. Furthermore, it is known to the person skilled in the art that overtransformation described above should certainly not be performed with primary transformants but preferably with stable transgenic plants that have been selected before and have been favorably tested in corresponding experiments with reference to, for example. , fertility, stable expression of foreign gene, hemi- and homozygosity etc. Therefore, also transgenic plants and plant cells are the subject matter of the present invention which are obtainable by the above methods and which show the phenotype described in the above embodiments. The present invention also relates to plant propagation material of the invention as well as transgenic plants produced according to the methods of the invention. The term "propagating material" includes those plant components that are suitable for the production of offspring in a vegetative and generative manner. For vegetative propagation, for example, seedlings, callus crops, rhizomes or tubers are suitable. Other propagation material comprises, for example, fruit, seeds, seedlings, protoplasts, cell cultures etc. Preferably, the propagating material is tubers and seeds.

Furthermore, the present invention relates to the use of a foreign nucleic acid molecule (s) encoding a protein with the enzymatic activity of an amylosaccharide and a protein with enzymatic activity of a branching enzyme to production of plants that give increased yield compared to wild-type plants and / or synthesize starch which is modified compared to starch from wild-type plants and / or synthesizes alpha-1,4-glucans alpha-1,6 -ramified with a degree of branching modified at position 0-6 compared to corresponding genetically unmodified wild-type plants.

Description of the Figures: Figure 1: Modified "Multiple Cloning Site" (MCS) pBinAr Figure 2: Plasmid Map pAmsu-wxy-Hyg Figure 3: Plasmid Map pAmsu-pat-Hyg Figure 4: Plasmid Map pBE-fnr -Km Figure 5: Plasmid map pBE-pat-Km Figure 6: Plasmid map pAmsu-cyt-Km Figure 7: Gel amylosaccharide activity Figure 8: Gel branch enzyme activity Figure 11: Gel activity of a protein extract from transgenic tobacco plants (Samsung NN variety) described in Example 7. In this experiment the FNR signal peptide (Example 6) was used. 25 - 32, 33 - 37, 39 and 40: protein extracts (75 pg total protein) from different independent transgenic tobacco lines. K = control; purified recombinant amylosaccharide produced in E. coli as described in patent application WO 99/67412: 50 ng Wt protein: protein extract (75 pg total protein) from wild-type tobacco plant (Samsung NN).

Figure 12: Gel activity of a protein extract from transgenic tobacco plants (Samsung NN variety) described in Example 7. In this experiment the signal peptide R1 (Example 6) was used. 9 - 16 and 41 - 48: protein extracts (75 pg total protein) from different independent transgenic tobacco lines. K = control: purified recombinant amylosaccharase produced in E. coli as described in patent application WO 99/67412; 50 ng Wt protein: protein extract (75 pg total protein) from wild-type tobacco plant (Samsung NN).

Figure 13: Gel activity of a protein extract from transgenic potato plants (Desirée variety) described in example 8. 8 to 12, 14, 16, 32 and 35 - 40: protein extracts (75 pg total protein) from different independent transgenic potato lines. Κ = control: purified recombinant amylosacarase produced in E. coli as described in patent application WO 99/67412; 50 ng Wt protein: protein extract (75 pg total protein) from wild-type potato (Desirée) plant. BE: protein extract from a transgenic potato plant expressing the branching enzyme derived from Neisseria denitrificans on plastids (example 5).

Figure 14: Texture analyzer profiles of transgenic plants (see example 9), control (transgenic plants expressing the branching enzyme from Neisseria denitrificans as described in example 5) and wild type plants.

Comments on Figures 11 - 13 All plant-derived protein gels that were analyzed by the amylosaccharide activity test described in example 4 show a brownish strip just above the amylosaccharase-specific strip. This brownish strip is particularly visible when using green plant material for the production of protein extracts (see, for example, Fig. 12, lines 9, 42 and 43). This brownish strip also exists in wild type plants. It is already visible as a green strip after electrophoresis. During gel incubation in a sucrose-containing buffer (example 4) the green to brown changes before staining with Lugol's solution. From these observations it can be concluded that this strip does not appear due to amylosaccharide activity.

In plant-derived gels having a high expression of amylosaccharide the nonspecific brownish strip is superimposed on the strip arising due to amylosacase activity.

Materials and methods which are important in combination with the description and which are used in the examples: 1. Determination of the degree of branching by methylation analysis The degree of branching of the obtained glucans can be determined by methylation analysis.

In general - methylation of all free OH groups from the gluten samples, double determinations each - hydrolysis of the permethylated polymers, followed by reduction in C-1 and acetylation of the monomer mixture - gas chromatographic analysis and quantification of reaction products. Examination of the degree of branching of the glucan samples was performed via a methylation analysis. The free OH groups of the polymers are labeled by conversion to methyl ether. The degradation in monomers is performed by acid hydrolysis and leads to partially methylated glucose molecules that are present in the form of pyranoside / furanoside as well as alpha- and beta-glycosides. These variants are focused on the corresponding partially methylated sorbitol derivative by reduction with NaBH4 or NaBD4. Final acetylation of free OH groups allows reaction products to be analyzed via gas chromatography.

Experimental part a) Production of DMSO solutions

1% of solutions (w / v) is produced in DMSO b) Methylation 2 ml of DMSO solution (ie 20 mg of polymer) is transferred into a 50 ml nitrogen flask, added to the N2 atmosphere with 5 equivalents / OH (eq / OH) of fresh dimsila solution and are stirred for 30 minutes. The contents of the flask are frozen in an ice bath, 10 eq / OH methyliodide is added and then frozen and shaken for at least two hours. Excess methyliodide is removed in vacuo prior to the second deprotonization and methylation step. Subsequently, excess methyliodide was removed by the addition of 50 ml of water and one extraction with each 10 ml of dichloromethane (5 times). To remove traces of DMSO from the organic phase were extracted by water three times. First using a sample, it is tested as many methylation steps are required for the permethylation of hydroxyl groups. After the first methylation half of the preparation is further processed, the other half is again methylated. After the decay of both samples the results of the GC analyzes are compared. A second methylation always follows in order to check for possible C-3 branching that can be simulated by subjecting at this position. c) Hydrolysis 2 mg of the methylated sample is weighed into a 1 ml pressure glass, 0.9 ml of 2 M trifluoracetic acid is added and stirred for 2.5 hours at 120 ° C. After the glass has cooled concentration follows in the atmosphere of N2. For the removal of traces of toluolene acid is added three times and is extracted. d) Reduction 0.5 ml of 0.5 M alkaline ammonium NaBD4 solution is added to the residue of the reaction step above and is stirred for 1 hour at 60 ° C. The reagent is carefully disintegrated with a few drops of glacial acetic acid, the borate is removed with boric acid trimethyl ester by five additions of 15% methanol containing acetic acid and extracted. e) Acetylation 50 μΙ pyridine and 250 μΙ acetic acid anhydride are added to the residue of the reaction step before and are stirred for 2 hours at 95 ° C. After cooling the reaction mixture is dripped into 10 ml saturated NaHCO3 solution and extracted with dichloromethane five times. The reaction products in the organic phase are analyzed via gas chromatography. fl Gas Chromatography Gas chromatographic analyzes are performed with a Cario Erba GC 6000 Vega Series 2 instrument with column inlet and FID detector. Separations are performed with a Supelco SPB5 fused silica capillary column (internal diameter 0.2 mm, length 30 m) with hydrogen as the carrier gas and with a pressure of 80 kPa. The following temperature program is used: 60 ° C (1 min) -25 ° C / min 130 ° C-4 ° C / min 280 ° C.

Results The evaluation of gas chromatograms is performed by identifying peaks, integrating peak areas and correcting data using the Sweet and others RCT concept (Sweet and others, Carbohydr. Res. 40 (1975), 217). 2. Purification of an amylosaccharide from Neisseria polvsaccharea For the production of an amylosacase used E. coli cells that have been transformed with an amylosacase from Neisseria polysaccharea. DNA trunks come from an N. polysaccharea genomic library and have a nucleotide sequence given in International Patent Application PCT / EP 98/05573.

An overnight culture of these E. coli cells expressing the gene encoding amylosaccharide from Neisseria polysaccharea was centrifuged and resuspended in a volume of about 1/20 50mM sodium citrate buffer ( pH 6.5), 10 mM DTT (lithium threitol), 1 mM PMSF (phenylmethyl sulfonyl fluoride). Then the cells were twice disintegrated with a French press at 112 kg / cm2 (16,000 psi). After 1 mM MgCfe was added to the cell extract as well as benzonase (from Merck; 100,000 units; 250 μΓ1 units) at a final concentration of 12.5 units ml'1. Then the preparation was incubated at 37 ° C for at least 30 minutes while shaking gently. The extract was allowed to stand on ice for at least 1.5 hours. Then it was centrifuged at 4 ° C at about 40,000 g for 30 minutes until the supernatant was relatively clear.

Pre-filtration was performed with PVDF membrane (Millipo-re "Durapore" or similar) which had a pore diameter of 0.45 pm. The extract was allowed to stand overnight at 4 ° C. Before hydrophobic interaction chromatography of H1 was performed, solid NaCl was added to the extract and adjusted to give a 2 M NaCl concentration. Then again, it was concentrated at 4 ° C and about 40,000 g for 30 min. After the extract was released from the last E. coli residue by filtration using a PVDF membrane (Millipore "Durapore" or similar) that had a pore diameter of 0.22 µm. The filtrate extract was separated by passing it over a 4B butylsepharose (Pharmacia) column column (column volume: 93 ml, length: 17.5 cm). About 50 ml of extract with an amylosacarase activity of 1 to 5 μΓ1 Units were placed on the column. Then non-binding proteins were removed by washing the column with 150 ml buffer B (buffer B: 50 mM sodium citrate, pH 6.5, 2% NaCl). Amylosacarase was finally eluted by a linear, decreasing gradient of NaCl (from 2 M to 0 M NaCl in 50 mM sodium citrate in a volume of 433 ml at a flow rate of 1.5 ml min -1). ) which was generated by an automatic pump system (FPLC, Pharmacia). Amylosaccharide elution occurs between 0.7 M and 0.1 M NaCl. Fractions were collected, desalted on a PD10 Sephadex column (Pharmacia), stabilized with 8.7% glycerol, tested for amylosaccharide activity. and finally frozen in storage buffer (8.7% glycerol, 50 mM citrate). 3. Determination of amylosacarase activity Purified protein or crude protein extract at various dilutions is introduced into 1 ml preparations containing 5% sacarose, 0.1% glycogen and 100 mM citrate, pH 6.5. and were incubated at 37 ° C. After 5 minutes, 10 min., 15 min., 20 min., 25 min. and 30 min. 10 μΙ every hour is taken from this preparation and the enzymatic activity of amylosaccharase is stopped by immediate heating to 95 ° C. In the coupled photometric test the ratio of the free fructose pool to the amylosaccharide is then determined. Therefore, 1 μΙ to 10 μΙ of the inactivated sample is placed in 1 ml of 50 mM imidazole buffer, pH 6.9, 2 mM MgCh, 1 mM ATP, 0.4 mM NAD, and 0.5 U / ml hexokinase. Following the sequential addition of glucose-6-phosphate dehydrogenase (from Leuconostoc mesenteroids) and phosphoglycosis isomerase the change in absorption is measured at 340 nm. Then the set amount of free fructose is calculated according to Lambert's Law.

When the value obtained is related to the time taken to sample, the number of Units (1U = pmol fructose / min) (per μΙ protein extract or μ9 purified protein), respectively, can be determined.

Vectors used in examples 1. pBinAR-N

In plasmid pBinAR-N (Hofgen and Willmitzer, Plant Science 66, (1990), 221-230) the polylinker between the 35S promoter and the OCS terminator was changed (Fig. 1) using nucleic acid oligonucleotides via methods. molecular biological standards (see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, NY, USA (1989)). This is how plasmid pBinAR-N was obtained.

2. pBinAR-Hva-N The EcoRI / HinDIlI fragment from pBinAR-N containing the 35S promoter, the following polylinker and the OCS terminator was cloned into the same restriction sites of pBIB-Hyg plasmid (Becker, Nucleic A- cids Research 18, (1990), 203) by use of standard molecular biological methods (see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, NY, USA (1989) ). The resulting plasmid is called pBinAR-Hyg-N. 3. pBinAR-wxv-Hva For the cloning of sequences encoding the Zea mays waxy protein signal peptide (see, for example, Klösgen et al., Mol. Gen. Genet. 217 (1989), 155-161) The corresponding sequences were amplified by PCR using the oligonucleotides (see SEQ ID NOS: 3 and 4), starting from the genomic DNA from Zea mays (Stra-tagene) as a template. The DNA fragments thus obtained were incubated with restriction endonucleases Xbal and Sall and cloned into the pBinAR-Hyg-N vector and cleaved with Spel and Sall. The resulting plasmid was called pBinAR-wxy-Hyg.

PCR Conditions

Gibco BRL Buffer and Polymerase (High Fidelity Platinum Taq DNA Polymerase No. 11304-011) DNA 0.2 pg 10x Buffer 5 μΙ MgS04 (50 mM) 2.0 μΙ dNTPs (10 nM each) 1 μΙ Sp Primer -wxy-5 '100 nM

Initiator Sp-wxy-3 '100 nM

Taq Platinum Hifi Polymerase 1.5 units Water Dest. ad 50 μΙ Conditions for the reaction Step 1 95 ° C 2:30 min Step 2 95 ° C 0:30 min Step 3 60 ° C 0:30 min Step 4 68 ° C 0:25 min (plus 1 sec per cycle ) Step 5 68 ° C 3:00 min Steps 2-4 were repeated in cycles 35 times 4. pBinAR-pat-Hyq and pBinAR-pat The sequences encoding the potato patatin gene signal peptide (Rosahl and Others, Mol. Gen. Genet. 203, (1986), 214-220; Sonnewald et al., Plant J. 1 (1998), 95-106) were amplified from plasmid pgT5 using the Sp-pat oligonucleotides. -5 'and Sp-pat-3' (see SEQ ID NO: 5 and SEQ ID NO: 6). The obtained fragments were digested with restriction endonucleases Xbal and Sall and were cloned into plasmids pBinAR-N and pBinAR-Hyg, respectively, were cleaved with Spel and Sall. The resulting plasmids were called pBinAR-pat and pBinAR-pat-Hyg, respectively. The sequence of nucleic acids contained in these plasmids encoding the patatin protein used peptide signal is illustrated in SEQ ID NO: 7 as it deviates from the published signal sequence (amino acid exchange of the third amino acid).

PCR Conditions: Boehringer Mannheim Buffer and Polymerase (PWO Polymerase No. 1644947) DNA 0.2 pg 10χ Buffer + MgS04 5 pl dNTPs (10 nM each) 1 μΙ Primer Sp-pat-5 '120 nM

Initiator Sp-pat-3 '120 nM

Pwo polymerase 1.0 units Water dest. ad 50 μΙ Conditions for the reaction Step 1 95 ° C 2:30 min Step 2 95 ° C 0:30 min Step 3 64 ° C 0:30 min Step 4 72 ° C 0:30 min (plus 1 sec per cycle ) Step 5 72 ° C 5:00 min Steps 2 through 4 were repeated in 35-fold cycles 5. Spinach-derived FNR signal peptide cloning Spinach-derived sequences encoding the FNR signal peptide were amplified using the primers Sp-fnr-5 'and Sp-fnr-3' (see SEQ ID NO: 8 and SEQ ID NO: 9) and plasmid p6SocFNR-15 as a template (Jansen et al., Current Genetics 13, (1988), 517-522). After digestion of fragments obtained with restriction endonucleases Xbal and SalI they were cloned into plasmid pBinAR-N cleaved with Spel and SalI. The resulting plasmid was called pBinAR-fnr-N.

PCR Conditions

Gibco BRL Buffer and Polymerase (High Fidelity Platinum Taq DNA Polymerase No. 11304-011) DNA 0.2 pg 10x Buffer 5 μΙ MgS04 2.0 μΙ dNTPs (10 nM each) 1 μΙ Sp-fnr-5 Primer '150 nM

Primer Sp-fnr-3 '150 nM

Taq Platinum Hifi Polymerase 1.5 units Water Dest. ad 50 μΙ Conditions for the reaction Step 1 95 ° C 2:00 min Step 2 95 ° C 0:30 min Step 3 58 ° C 0:30 min Step 4 68 ° C 0:20 min (plus 1 sec per cycle ) Step 5 68 ° C 3:00 min Steps 2 to 4 were repeated in cycles 35 times 6. pBinAR-R1-Hyg In order to clone the R1 protein signal peptide coding sequence for Solanum tuberosum (Lorbeth and others, Nature Biotechnology 16 (1998), 473-477), the corresponding sequences were PCR amplified using the cDNA clone RL2 as a template (Lorber-th, PhDthesis, "Charakterisierung von RL1: ein neues des Stärkemetabolismus" Freie Universitat Berlin (1996) and oligonucleotides SEQ ID NOs: 14 and 15 as primers The resulting DNA fragments were digested with restriction endonucleases Xbal and Sall and were then cloned into the pBinAr-Hyg-N vector cleaved with Spel and Sall. The resulting plasmid was called pBinAR-R1-Hyg.

PCR Reaction Conditions: Boehringer Mannheim Buffer and Polymerase (PWO DNA Polymerase No. 1644955) DNA 0.05 pg 10x Buffer 5 μΙ dNTPs (10 nM each) 1 μΙ Primer Sp-R1 -5 '100 nM

Primer Sp-R1 -3 '100 nM

Pwo polymerase 1.0 units Water dest. ad 50 μΙ Conditions for the reaction Step 1 95 ° C 2:00 min Step 2 95 ° C 0:30 min Step 3 60 ° C 0:30 min Step 4 72 ° C 0:25 min (plus 1 sec per cycle ) Step 5 72 ° C 3:00 min Steps 2-4 were repeated in 30-fold cycles 7. pBinAR-fnr-Hyg The sequences encoding the spinach-derived FNR signal peptide were amplified using plasmid p6SocFNR-15 (Jan-sen et al., Current Genetics 13 (1988), 517-522) as a model and the initiators Sp-fnr-5 'and Sp-fnr-3' (SEQ ID NO: 8 and SEQ ID NO: 9 ). The resulting DNA fragments were digested with restriction endonucleases Xbal and Sall and were cloned into the pBinAr-Flyg-N vector and cleaved with Spel and Sall. The resulting plasmid was called pBinAR-fnr-Flyg.

Conditions for PCR Reaction

Gibco BRL Buffer and Polymerase (High fidelity Platinum Taq DNA Polymerase # 11304-011) DNA 0.2 pg 10x Buffer 5 pl dNTPs (10 nM each) 1 pl Primer Sp-fnr-5 '100 nM

Primer Sp-fnr-3 '100 nM

Taq Platinum Hifl Polymerase 1.0 units Water Dest. ad 50 μΙ Conditions for reaction Step 1 95 ° C 2:30 min Step 2 95 ° C 0:30 min Step 3 58 ° C 0:30 min Step 4 72 ° C 0:30 min (plus 1 sec per cycle ) Step 5 72 ° C 5:00 min Steps 2-4 were repeated in cycles 35 times Example 1 Production of expression cassettes for plant transformation: vacuolar and plastidic expression, respectively, of an amylosaccharase from Neisseria polysaccharea Using AS-5 'and AS-3' oligonucleotides (SEQ ID NO: 10 and SEQ ID NO: 11) the amylosaccharide coding sequences were amplified by PCR using plasmid pNB2 as a template (see International Patent WO 95/31553, filed with the "Deutsche Sammlung für Mikrooganismen und Zellkulturen" (DSMZ) in Braunschweig, Germany, under accession number DSM 9196). Their obtained amplifications were digested with restriction endonucleases Xhol and Pstl and were cloned into plasmids pBinAR-wxy-Hyg and pBinAR-pat-Hyg, respectively, were cleaved with Sall and Sdal. The resulting plasmids were called pAm-su-wxy-Hyg (Fig. 2) and pAmsu-pat-Hyg (Fig. 3), respectively.

PCR Conditions: Boehringer Mannheim Buffer and Polymerase (PWO Polymerase No. 1644947) DNA 0.2 pg 10x Buffer + MgSo4 5 μΙ dNTPs (10 nM each) 1 μΙ Primer Sp-AS-5 '100 nM

Primer Sp-AS-3 '100 nM

Pwo polymerase 1.0 units Water dest. ad 50 μΙ Conditions for the reaction Step 1 95 ° C 2:00 min Step 2 95 ° C 0:30 min Step 3 58 ° C 0:30 min Step 4 68 ° C 2:00 min (plus 1 sec per cycle ) Step 5 68 ° C 5:00 min Steps 2 to 4 were repeated in 40-fold cycles The pAmsu-wxy-Hyg and pAmsu-pat-Hyg plasmids, respectively, can be used for plant transformation according to the methods. default (see above).

Example 2 Production of Expression Cassettes for Plant Transformation: Vacuolar and Plastid Expression, respectively, of a branching enzyme from Neisseria denitrificans Using oligonucleotides BE-5 'and BE-3' (SEQ ID NO: 12 and SEQ ID NO: 13) The sequence encoding the Neisseria denitrificans branching enzyme was amplified by PCR using plasmid pBB48 as a template (deposited in the Deutsche Sammlung für Mikroo-ganismen und Zellkulturen (DSMZ)). in Braunschweig, Germany under access number DSM 12425). Their obtained magnifications were digested with restriction endonucleases Sall and Sdal and were cloned into pBinAR-fnr and pBinAR-pat plasmids, respectively, were cleaved with Sall and Sdal. The resulting plasmids were called pBE-fnr-Km (Fig. 4) and pBE-pat-Km (Fig. 5), respectively.

PCR Conditions: Boehringer Mannheim Buffer and Polymerase (PWO Polymerase No. 1644947) D NA 0.2 pg 10x Buffer + MgSo4 5 μΙ dNTPs (10 nM each) 1 μΙ Primer Sp-BE-5 '120 nM

Initiator Sp-BE-3 '120 nM

Pwo polymerase 1.0 units Water dest. ad 50 μ Conditions for reaction Step 1 95 ° C 2:00 min Step 2 95 ° C 0:30 min Step 3 58 ° C 0:30 min Step 4 72 ° C 2:00 min (plus 1 sec per cycle ) Step 5 72 ° C 8:00 min Steps 2-4 were repeated in cycles 40 times Plasmids pBE-fnr-Km and pBE-pat-Km, respectively, can be used for plant transformation according to the methods. default (see above).

Example 3 Production of Expression Cassettes for Plant Transformation: Cytosolic Expression of a Neisseria polysaccharea Amylosaccharide A fragment encoding a Neisseria polysaccharea amylosaccharase was isolated with restriction endonucleases Xmnl and Eagl from plasmid pNB2 (see above). and the ends of the fragment were filled with Klenow DNA polymerase. Then cloning of the fragment into the Smal cleaved plasmid pBinAR is followed (Hofgen and Willmitzer, Plant Science 66, (1990), 221-230). The resulting plasmid was called pAmsu-cyt-Km (Fig. 6) and can be used for plant transformation.

Example 4 Identification and testing of transgenic potato plants with amylosaccharide activity Via Northern Blot analysis transgenic potato plants could be identified that have the mRNA of an amylosaccharide from Neisseria polysaccharea. Then it could be shown that amylosaccharide in such plants is active.

For detection of amylosaccharide activity in stably transformed plants leaf material of the plants to be tested was frozen in liquid nitrogen and then ground in a mortar that had been pre-cooled with liquid nitrogen. Before the milled material thawed, extraction buffer (50 mM sodium citrate, pH 6.5, 4 mM DTT, 2 mM calcium chloride) was added. About 500 μΙ of extraction buffer was added to about 100 mg of plant material (fresh weight). Solid components of the disintegrated plant material suspension and extraction buffer were separated by centrifugation (10,000 x g). An aliquot of the obtained clear supernatant was mixed with a quarter volume volume buffer extract (40% glycerin, 250 mM Tris pH 8.8, 0.02% bromophenol blue) and separated on polyacrylamide gel (see below) at constant current intensity of 20 mA per gel. (Before protein extracts were applied to the gel, an electrophoresis of the gels was performed for 20 minutes under the conditions described above). After the bromophenol blue staining agent had left the gel the electrophoresis was stopped. The gel was then equilibrated 5 times in wash buffer (100 mM sodium citrate, pH 6.5) with 5 times the volume of gel each for 20 min. each while rotating at room temperature. Then the gel was incubated in incubation buffer (100 mM sodium citrate, pH 6.5, 5% sucrose) with 5 times the amount of gel volume at 37 ° C for 16 h. After decantation of the incubation buffer the glucan produced by the amylosaccharide was detectable as a brownish bluish strip when Lugol's solution (diluted 1: 5) was added (Fig. 7). Polyacrylamide gel composition a) 375 mM Tris gel separation, pH 8.8 7.5% polyacrylamide (Biorad No. EC-890) For polymerization: 1/2000 volume TEMED 1/100 volume persulphate b) Collection of 125 mM Tris gel, pH 6.8 4% polyacrylamide (Biorad No. EC-890) For polymerization: 1/2000 TEMED volume 1/100 ammonium persulphate volume c) Buffer 375 mM Tris electrophoresis, pH 8.8 200 mM glycine

Example 5 Identification and testing of transgenic potato plants with branching enzyme activity Via Northern Blot analysis transgenic potato plants could be identified that had the mRNA of a Neisseria denitrificans branching enzyme. For detection of branching enzyme activity in stably transformed plants plant material from the plants to be tested was frozen in liquid nitrogen and then ground in a mortar that had been cooled with liquid nitrogen. Before the milled material thawed, extraction buffer (50 mM sodium citrate, pH 6.5, 4 mM DTT, 2 mM calcium chloride) was added. About 200 μΙ of extraction buffer was added to about 100 mg of plant material (fresh weight). Solid components of the disintegrated plant material suspension and extraction buffer were separated by centrifugation (10,000 x g). An aliquot of the obtained clear supernatant was mixed with a quarter of the tidal buffer extract (40% glycerin, 250 mM Tris, pH 8.8, 0.02% bromofenol blue) and gel separated of polyacrylamide (see below) at constant current intensity of 20 mA per gel. (Before protein extracts were applied to the gel, an electrophoresis of the gels was performed for 20 minutes under the conditions described above). After the bromophenol blue staining agent present in the standard buffer had left the gel, the electrophoresis was stopped. The gel was then equilibrated 5 times in wash buffer (100 mM sodium citrate, pH 6.5) with 5 times the volume of gel each for 20 min. each while rotating at room temperature. Then the gel was incubated in incubation buffer (100mM sodium citrate, pH 6.5, 5% sacarose, 0.625 units of purified Neisseria polysaccharea amylosaccharide (enzyme purification and activity determination see above)) with 5 times the volume amount of gel at 30 ° C for 16 hs. After decantation of the incubation buffer the glucan produced by the amylosaccharide in combination with the branching enzyme was detectable as a brownish bluish strip when Lugol's solution (diluted 1: 5) was added (Fig. 8). All remaining polyacrylamide gel turns blue due to the amylosaccharide activity present in the incubation buffer.

Polyacrylamide gel composition a) 375 mM Tris gel separation, pH 8.8 7.5% polyacrylamide (Biorad No. EC-890) For polymerization: 1/2000 volume TEMED 1/100 volume persulphate d) Collection of 125 mM Tris gel pH 6.8 4% polyacrylamide (Biorad No. EC-890) For polymerization: 1/2000 TEMED volume 1/100 ammonium persulphate volume e) Buffer 375 mM Tris electrophoresis, pH 8.8 200 mM glycine

Example 6 Construction of a Plant Expression Cassette: Plastid Expression of an Amylosaccharide from Neisseria polysaccharea By Use of Plasmid pNB2 (Deutsche Sammlung für Mikrooga-nismen and Zellkulturen (DSMZ) Braunschweig, Germany, DMS 9196) as a model and oligonucleotides AS-5 'and AS-3' (SEQ ID NOs: 10 and 11) as PCR primers the amylosaccharide coding region from Neisseria polysaccharea was expanded. The PCR product was then digested by restriction enzymes XhoI and Pstl. The resulting fragment containing the coding region was cloned into Sall and Sdal-digested plasmids pBinAR-R1-Hyg and pBinAR-fnr-Hyg. The resulting plasmids were called pAmsu-R1-Hyg (Fig. 9) and pAmsu-fnr-Hyg (Fig. 10), respectively.

PCR Conditions: Boehringer Mannheim Polymerase Buffer (PWO Polymerase No. 1644947) DNA 0.2 pg 10x Buffer + MgSo4 5 pl dNTPs (10 nM each) 1 μΙ Sp-BE-5 'Primer 100 nM

Initiator Sp-BE-3 '100 nM

Pwo polymerase 1.0 units Water dest. at 50 μ Reaction conditions: Step 1 95 ° C 2:00 min Step 2 95 ° C 0:30 min Step 3 56 ° C 0:30 min Step 4 72 ° C 2:00 min (1 sec more per cycle) Step 5 72 ° C 5:00 min Steps 2 to 4 were cyclically repeated.

Plasmids pAmsu-R1-Hyg and pAmsu-fnr-Hyg can be used for plant transformation according to standard methods. Example 7 Identification of transgenic tobacco plants showing the activity of an amylosacarase The pAmsu-R1-Hyg (fig. 9) and pAmsu-fnr-Hyg (Fig. 10) constructs described in example 6 were used to transform tobacco plants from according to Rosahl et al. (EMBO J. 6 (1987), 1155-1159). By performing a Northern Blot analysis transgenic tobacco plants were identified having the mRNA of an amylosacarase. Furthermore, those plants expressing the amylosaccharide gene in plastids also show the enzymatic activity of an amylosaccharase (Figs. 11 and 12). Enzymatic activity was tested as described in example 4.

Example 8 Production and identification of transgenic potato plants expressing a gene encoding a branching enzyme derived from Neisseria deni-trificans and a gene encoding amylosaccharide derived from Neisseria polv-saccharea Three transgenic potato plant lines that were previously transformed with plasmids pBE-fnr-Km (example 2) and showing the enzymatic activity of a plastid branching enzyme (enzymatic activity test was performed as described in example 5, fig. 8), were selected .

Then, leaf explants from these plants were transformed again via agrobacteria with the plasmid pAmsu-R1-Hyg (Example 6). By using the activity tests described in examples 4 and 5 plants were identified which show in parallel the activity of an amylosaccharide protein and a branching enzyme (fig. 13) with both enzymes located in the plastids.

Example 9 Determination of starch gel stability by use of a texture analyzer 2g of starch (dw) extracted from transgenic plants as described in example 8 was added to an appropriate volume of distilled water to produce a suspension containing final concentration at 8 ° C. % starch (w / v). This suspension was then heated on a Visco Rapid Analyzer (Newport Scientific Pty Ltd., Investiment Support Group, Warriewood NSW 2102, Australia) using the following temperature profile: First, the suspension was heated from 50 ° C to 95 ° C. C with a temperature increase rate of 12 ° C per minute. Then the temperature was maintained for 2.5 minutes at 95 ° C. Finally, the suspension was cooled to 50 ° C at a rate of 12 ° C per minute. The resulting probe was stored airtight for 24 h at 25 ° C. The probes are then fixed in a texture analyzer, model TA-XT2, produced by Stable Micro Systems (Haslemere, England). A round seal was used. Gel stability was determined by adjusting the parameters as follows: - test speed 0.5 mm / s - distance 7 mm - contact area 113 mm2 - trigger force 2 g The resulting profiles of transgenic lines 006 and 035 in Comparison with wild-type plants and control plants (transgenic plants expressing the branching enzyme from Neisseria denitrificans as described in example 5) are shown in Figure 14.

The texture analyzer profiles (see Figure 14) of starches from transgenic plants show significant differences for the starch profile of wild type and control plants.

In case the "distance" is set at 7.0 mm the profile of the transgenic plants can be described as "crown type".

SEQUENCE LISTING <110> Bayer Cropscience AG

<120> METHOD FOR PRODUCTION OF A TRANSGENIC PLANT CELL WITH INCREASED ACTIVITY OF AN AMYLOSACARASE PROTEIN AND A RAMIFICATION <130> D1703 BR / 1S3 <140> PI0017574-9 <141> 26/05/2000 <150> DE19924342.5 <151> 27/05/1999 <160> 17 <170> Patentln version 3.5 <210> 1 <211> 2475 <212> DNA <213> Neisseria denitrificans <220>

<221> CDS <222> (170) .. (2458) <400> 1 actgtatgcc gtgcagctgg aaaacctgct gggcgtacgc gacaacctca atattcccgg 60 cgtggccgaa ggctatccga actgggcgcg caaaatgccg 120 cagcctctgg aagcctttgc ccgccacccg caaatgggca agcagcttgc catgatggga gacatccgc atg aac cga 178 Met Arg Asn 1 cat aac cgc ate cga cgc ggc tac cac ccg gaa gcc gga gaa cgc caa 226 Asn Arg His Ile Arg Arg Gly Tyr His Pro Glu Ala Gly Glu Arg Gin 5 10 15 up to gac age ctg ttt gcc gcc acc cac age gat ccg ttt gcc tat 274 Ile Ile Asp Ser Leu Phe Ala Wing Thr His Ser Asp Pro Phe Ala Tyr 20 25 30 35 ctt ggg cgg cat cgt gtc aac gac gaa gcc gtg cgc gtg ctg 322 Leu Gly Arg His Arg Go Asn Asp Glu Arg Glu Ala Go Arg Vai Leu 40 45 50 cgt ccc gac gcg cac cac to gac until gac cgc cac aca ggc gea 370 Arg Pro Asp Ala His His Ile Asp Ile Ile Asp Arg His Thr Gly Ala 55 60 65 gtc up to atg ccg tet gaa aaa to gac gag cgc ggc ctg ttt gcc gcc 418 Go Ile Met Pro Be Glu Lys Ile Asp Glu Arg Gly Leu Phe Wing Ala 70 75 80 gta ttg ccc gaa cac gcg ccc ga c tac gcc ctg ctg gtg aca tac cac 466 Go Leu Pro Glu His Ala Pro Asp Tyr Ala Leu Leu Go Thr Tyr His 85 90 95 gag ggc gaa gcc gcc gta cgc gaa gaa gat gac tac cgc ttc ggc age 514 Glu Gly Glu Ala Wing Go Arg Glu Glu Asp Asp Tyr Arg Phe Gly Ser 100 105 110 115 gcg ctg caa cat acc gat gcc tgg ctg ctg ggc gag acg cac ctg 562 Ala Leu Gin His Thr Asp Ala Trp Leu Gly Glu Gly Thr His Leu 120 125 130 cgc cct tat gaa acg ctg ggc gea cat ttc gcc gaa atg gac ggc gta 610 Arg Pro Tyr Glu Thr Leu Gly Ala His Phe Ala Glu Met Asp Gly Go 135 140 145 tcc ggc gtg cgc ttt gcc gcg ccc aac cgg cgg gta tcg 658 Ser Gly Go Arg Phe Ala Go Trp Ala Pro Asn Wing Arg Arg Will Be 150 155 160 gtc up to ggc gaa ttc aac ggc tgg gac age cgc cgc cat gcc atg cgt 706 Go Ile Gly Glu Phe Asn Gly Trp Asp Ser Arg Arg His Ala Met Arg 165 170 175 ccg cac aca ggc aac ggc ctg tgg gac until ttt till ccc ggc gtc ggc 754 Pro His Thr Gly Asn Gly Leu Trp Asp Ile Phe Ile Pro Gly Go Gly 180 185 190 195 ctc aac gcg ctg t at aaa ttc tcc gta ctc gat gcc aac ggc aac up to 802 Leu Asn Ala Leu Tyr Lys Phe Will Go Leu Asp Ala Asn Gly Asn Ile 200 205 210 cgc gaa aaa gcc gac cct tac ggg gg cg cc ccg acc 850 Glu Lys Ala Asp Pro Tyr Ala Phe Gly Ala Glu Leu Arg Pro Thr 215 220 225 acc gca tcc gtg gtg cgc ttg ccg gcc aaa gcc gcg ccc gct 898 Thr Ala Will Go Go Arg Gly Leu Pro Ala Lys Ala Glu Ala Pro Ala 230 235 240 ttc cgc cgc cgc aac tcc gtg gaa gcg ccc until age att tac gaa 946 Phe Arg Arg Arg Wing Asn Will Go Glu Ala Pro Ile Ser Ile Tyr Glu 245 250 255 gtc cat ctc ggc tgg cgc aat cc gaa aac aac tac tgg ctc 994 Go His Leu Gly Ser Trp Arg Arg Asn Pro Glu Asn Tyr Trp Leu 260 265 270 275 acc tac acg cag ctg gcc gac gaa ttg gtg aac tat gta aaa gac atg 1042 Thr Tyr Thr Gin Leu Ala Asp Glu Leu Goes Asn Tyr Goes Lys Asp Met 280 285 290 ggc ttc acc cac up gag ctg ctg cct tg tcc gaa tat ccg ttc gac 1090 Gly Phe Thr His Ile Glu Leu Pro Leu Ser Glu Tyr Pro Phe Asp 295 300 305 ggc tea tgg ggc tac caa gcc acc ggc ctg tat gca ccg acc age cgc 1138 Gly Ser Trp Gly Tyr Gin Ala Thr Gly Leu Tyr Ala Pro Thr Ser Arg 310 315 320 ttc ggc tcg ccc gat gag ctg aaa gcc ctg att gac gcc gcc cac gcc 1186 Phe Gly Ser Pro Asp Glu Leu Lys Wing Leu Ile Asp Wing His Wing Wing 325 330 335 gcc ggc till age gtg att ctc gac ggg ggg cac ttc ccc acc 1234 Wing Gly Ile Will Go Ile Leu Asp Trp Go Ala Gly His Phe Pro Thr 340 345 350 355 gac gac cac ggc ctc aac acc ttc gac ggc acg gcg ctt tac gaa cac 1282 Asp Asp His Gly Leu Asn Thr Phe Asp Gly Thr Ala Leu Tyr Glu His 360 365 370 gcc gac ccg cgc gaa ggc tac cat cag gat tgg aac acg ctg att tac 1330 Ala Asp Pro Arg Glu Gly Tyr His Gin Asp Trp Asn Thr Leu Ile Tyr 375 380 385 aac ttc ggc cgc aac gaa gtc cag ggc aac gcg ctc 1378 Asn Pn Arg Asn Glu Go Lys Asn Phe Leu Gin Gly Asn Ala Leu 390 395 400 tac tgg att gag cgt ttc ggc ttc gac ggc up to cgc gtg gac gcc gtg 1426 Tyr Trp Ile Glu Arg Phe Gly Phe Asp Gly Ile Arg Go Asp Ala Go 405 410 415 gcc tcg atg att tac cgc aac tac tcg cgc aaa gac ggc gag tgg att 1474 Ala Ser Met Ile Tyr Arg Asn Tyr Ser Arg Lys Asp Gly Glu Trp Ile 420 425 430 435 ccc aac cgc tac ggc ggc age gaa aat ctg gaa gcc up to gcc ttt ttg 1522 Pro Asn Arg Tyr Gly Gly Ser Glu Asn Leu Glu Ala Ile Ala Phe Leu 440 445 450 cgc caa acc aat gcc gtc tta aaa age aca ccc ggc ggc tcg 1570 Arg Gin Thr Asa Ala Vai Leu Lys Ser Glu Thr Pro Gly Ala Gly Ser 455 460 465 ttt gcc gaa gaa tcg act tcc gcc gac gta acc cgc gaa gcc ggc 1618 Phe Ala Glu Glu Be Thr Phe Ala Gly 470 475 480 ctg aac ttc gat ttc aaa tgg aat atg ggc tgg atg aac gac acc ctg 1666 Leu Asn Phe Asp Phe Lys Trp Asn Met Gly Trp Met Asn Thr Leu 485 490 495 cgc tat atg cag gaa gac ccc gtc cac cac aaa 1714 Arg Tyr Met Gin Glu Asp Pro Goes His Arg Lys Tyr His His Gly Lys 500 505 510 515 atg aca ttc ggc atg tac at tac tac hunting gaa aac ttc gtt ctg ccc 1762 Met Thr Phe Gly Met Met Tyr Gin Tyr S er Glu Asn Phe Vai Leu Pro 520 525 530 ctg tcg cac gac gaa gtg gta cac ggc aaa cgc tcg ctg ctg ggc aaa 1810 Leu Ser His Asp Glu Goes His Gly Lys Arg Be Leu Gly Lys 535 540 545 atg ccg ggc gac tgc tgg cag cag ttt gcc aac cg cgc gcc tat tac 1858 Met Pro Gly Asp Cys Trp Gin Gin Phe Ala Asn Leu Arg Ala Tyr Tyr 550 555 560 ggc ttt atg tac ggc ttc ccc gta aaa aaa ctc cta ttt atg ggly a06 Phe Met Tyr Gly Phe Pro Gly Lys Lys Leu Read Phe Met Gly Asn 565 570 575 gaa ttt gcc caa ggc cgc gag tgg aat tat cag gaa gga ctg gat tgg 1954 Glu Phe Ala Gin Gly Arg Glu Trp Asn Tyr Gin Glu Gly Leu Trp 580 585 590 595 cat ctg ctc gac ggg ggc ggc tgg cac aaa ggc gtg cag gat tat 2002 His Leu Leu Asp Glu Ala Gly Trp His Lys Gly Go Gin Asp Tyr 600 605 610 gta cgc gac ctg aac cac acc gcc cac gcc ccg etc tac cag 2050 Going Arg Asp Leu Asn His Ile Tyr Thr Ala His Wing Pro Leu Tyr Gin 615 620 625 etc gac cag cag ccc gag ggc ttt gaa tgg ctg gcc gac gac age 2098 Leu Asp Gin Gin Pr the Glu Gly Phe Glu Trp Leu Go Ala Asp Asp Ser 630 635 640 gac aat tcg gta ttc gta cgc gg cgc gea ggc aac cgc 2146 Asp Asn Will Go Phe Go Phe Glu Arg Arg Asp Arg Ala Gly Asn Arg 645 650 655 up to gtc till age aac ttt acc ccg gtg gtg cgc gaa cac tac cgc 2194 Ile Ile Go Ile Be Asn Phe Thr Pro Go Go Arg Glu His Tyr Arg 660 665 670 675 ttc ggc gtc aac gcg ccc ggc tat acc gaa ate ctg aat tcc gac 2242 Phe Gly Go Asn Ala Pro Gly Arg Tyr Thr Glu Ile Leu Asn Ser Asp 680 685 690 cgc acg cag tat caa ggc age ggc up gea aac ggc gcg gac up acg 2290 Arg Thr Gin Tyr Gin Gly Ser Gly Ile Wing Asn Gly Wing Asp Ile Thr 695 700 705 gcg gaa aac gtg cct tcg cac ggc aaa gcg cag tcg ctg acc 2338 Wing Glu Asn Going to Be His Gly Lys Ala Gin Being Read To Be Leu Thr 710 715 720 ctg ccg ctg gcc acg gtc tat ctg tat cag aaa gcc gcg ccc gea 2386 Leu Pro Pro Leu Thr Wing Go Tyr Leu Tyr Gin Lys Wing Pro Wing 725 730 735 acg gaa att cag acg gcc tcc gcc ag cag gcgta aaa 2434 Thr Glu Ile Gin Thr Wing Leu Arg Wing Asp Lys Gin Pro Wing Go Lys 740 745 750 755 gat aag cag gea aaa gcc aaa taa agcggcacca tactgcc 2475 Asp Lys Gin Ala Lys Wing Alys 760 <210> 2 <211> 762 <212> PRT <213> Neisseria denitrificans <400> 2 Met Asn Arg Asn Arg His Ile Arg Gly Tyr His Pro Glu Wing Gly 15 10 15 Glu Arg Gin Ile Ile Asp Be Read Phe Wing Wing Thr His Be Asp Pro 20 25 30 Phe Wing Tyr Leu Gly Arg His Arg Go Asn Asp Glu Arg Glu Wing Go 35 40 45 Arg Go Read Arg Pro Asp Wing His His Ile Asp Ile Ile Asp Arg His 50 55 60 Thr Gly Wing Go Ile Met Pro Be Glu Lys Ile Asp Glu Arg Gly Leu 65 70 75 80 Phe Wing Ala Goes Leu Pro Glu His Wing Pro Asp Tyr Wing Leu Leu Goes 85 90 95 Thr Tyr His Glu Gly Glu Wing Ala Goes Arg Glu Glu Asp Asp Tyr Arg 100 105 110 Phe Gly Ser Ala Leu Gin His Thr Asp Wing Trp Read Leu Gly Glu Gly 115 120 125 Thr His Read Leu Arg Pro Tyr Glu Thr Leu Gly Wing His Phe Ala Glu Met 130 135 140 Asp Gly Will Be Gly Go Arg Phe Ala Go Trp Ala Pro Wing 145 145 150 155 160 Arg Will Be Go Ile Gly Glu Phe Asn Gly Trp Asp Be Arg Arg His 165 170 175 Ala Met Arg Pro His Thr Gly Asn Gly Leu Trp Asp Ile Phe Ile Pro 180 185 190 Gly Go Gly Leu Asn Wing Tyr Lys Phe Be Go Read Asp Ala Asn 195 200 205 Gly Asn Ile Arg Glu Lys Wing Asp Pro Tyr Phe Wing Gly Wing Glu Leu 210 215 220 Arg Pro Thr Thr Wing Will Go Go Arg Gly Leu Pro Wing Lys Wing Glu 225 230 235 240 Wing Pro Wing Phe Arg Arg Arg Wing Asn Will Go Glu Wing Pro Ile Be 245 250 255 Ile Tyr Glu Go His Leu Gly Ser Trp Arg Arg Asn Pro Glu Asn 260 265 270 Tyr Trp Leu Thr Tyr Thr Gin Leu Wing Asp Glu Leu Go Asn Tyr Go 275 280 285 Lys Asp Met Gly Phe Thr His Ile Glu Read Leu Pro Leu Be Glu Tyr 290 295 300 Pro Phe Asp Gly Be Trp Gly Tyr Gin Wing Thr Gly Leu Tyr Wing Pro 305 310 315 320 Thr Be Arg Phe Gly Ser Pro Asp Glu Leu Lys Wing Leu Ile Asp Wing 325 330 335 Wing His Wing Wing Gly Ile Will Go Ile Leu Asp Trp Go Wing Gly His 340 345 350 Phe Pro Thr Asp His Gly Leu Asn Thr Phe Asp Gly Thr Wing Leu 355 360 365 Tyr Glu His Wing AspPro Arg Glu Gly Tyr His Gin Asp Trp Asn Thr 370 375 380 Leu Ile Tyr Asn Phe Gly Arg Asn Glu Go Lys Asn Phe Leu Gin Gly 385 390 395 400 Asn Ala Leu Tyr Trp Ile Glu Arg Phe Gly Phe Asp Gly Ile Arg Go 405 410 415 Asp Wing Go Go Wing Be Met Ile Tyr Arg Asn Tyr Be Arg Lys Asp Gly 420 425 430 Glu Trp Ile Pro Asn Arg Tyr Gly Gly Be Glu Asn Leu Glu Ala Ile 435 440 445 Ala Phe Leu Arg Gin Thr Asn Ala Go Read Lys Be Glu Thr Pro Gly 450 455 460 Wing Gly Be Phe Wing Glu Glu Be Thr Be Phe Wing Asp Go Thr Arg 465 470 475 480 Glu Wing Gly Leu Asn Phe Asp Phe Lys Trp Asn Met Gly Trp Met Asn 485 490 495 Asp Thr Read Leu Tyr Met Gin Glu Asp Pro Goes His Arg Lys Tyr His 500 505 510 His Gly Lys Met Thr Phe Gly Met Met Tyr Gin Tyr Be Glu Asn Phe 515 520 525 Will Read Leu Pro Read His Be Asp Glu Goes His Gly Lys Arg Ser Leu 530 535 540 Read Gly Lys Met Pro Gly Asp Cys Trp Gin Gin Phe Wing Asn Leu Arg 545 550 555 560 Wing Tyr Tyr Gly Phe Met Tyr Gly Phe Pro Gly Lys Liu Leu Phe 565 570 575 Met Gly Asn Glu PheAla Gin Gly Arg Glu Trp Asn Tyr Gin Glu Gly 580 585 590 Leu Asp Trp His Leu Read Asp Glu Ala Gly Gly Trp His Lys Gly Go 595 600 605 Gin Asp Tyr Go Arg Asp Leu Asn His Ile Tyr Thr Ala His Ala Pro 610 615 620 Leu Tyr Gin Leu Asp Gin Gin Pro Glu Gly Phe Glu Trp Leu Go Ala 625 630 635 640 Asp Asp Be Asp Asn Will Go Phe Go Phe Glu Arg Arg Asp Arg Wing 645 650 655 Gly Asn Arg Ile Ile Will Ile Be Asn Phe Thr Pro Go Go Arg Glu 660 665 670 His Tyr Arg Phe Gly Go Asn Wing Pro Gly Arg Tyr Thr Glu Ile Leu 675 680 685 Asn Be Asp Arg Thr Gin Tyr Gin Gly Ile Wing Asn Gly Ala 690 695 700 Asp Ile Thr Wing Glu Asn Goes To Be His Gly Lys Wing Gin Gin Read Le 705 710 715 720 Be Read Thr Read Leu Pro Pro Leu Wing Thr Go Tyr Leu Tyr Gin Lys Wing 725 730 735 Wing Pro Ala Thr Glu Ile Gin Wing Ala Thr Wing Ala Asp Lys Gin Pro 740 745 750 Wing Go Lys Asp Lys Gin Wing Lys Wing Lys 755 760 <210> 3 <211> 27 <212> DNA <213> Artificial <220> <223> Artificial <400> 3 tctagaggaa ttagcggc 27 < 210> 4 <211> 25 <21 2> DNA <213> Artificial <220> <223> Artificial <400> 4 gtcgacgctg gcgcacacga cgagc 25 <210> 5 <211> 26 <212> DNA <213> Artificial <220> <223> Artificial <400> 5 tctagactgc aaaatggcaa ctacta 26 <210> 6 <211> 26 <212> DNA <213> Artificial <220> <223> Artificial <400> 6 gtcgacggtt tcatttggag tagtta 26 <210> 7 <211> 92 <212> PRT <213> Solanum tuberosum <400> 7 Met Thr Thr Wing Lys Be Phe Leu Ile Leu Phe Phe Met Ile Leu Wing 15 10 15 Thr Thr Be Ser Cys Wing Lys Leu Glu Glu Met Will Thr Go Leu 20 25 30 Ser Ile Asp Gly Gly Gly Ile Lys Gly Ile Ile Pro Wing Ile Ile Leu 35 40 45 Glu Phe Leu Glu Gly Gin Leu Gin Glu Go Asp Asn Asn Lys Asp Wing 50 55 60 Arg Leu Wing Asp Tyr Phe Asp Go Ile Gly Gly Thr Be Thr Gly Gly 65 70 75 80 Leu Leu Thr Thr Ala Met Ile Thr Thr Asn Glu Thr 85 90 <210> 8 <211> 25 <212> DNA <213> Artificial <220> <223> Artificial <400> 8 tctagacgta ctccgccatg accac 25 <210> 9 <211> 24 <212> DNA <213> Artificial <220> <223> Artificial <400> 9 gtcgacgatc tgggccctga tggg 24 <210> 10 <211> 26 <212> DNA <213> Artificial <220> <223> Artificial <400> 10 ctcgagatgt tgacccccac gcagca 26 <210> 11 <211> 23 <212> DNA <213> Artificial <220> <223> Artificial <400> 11 ctgcagacgg catttgggaa gcg 23 <210> 12 <211> 27 <212> DNA <213> Artificial <220> <223> Artificial <400> 12 gtcgacatga accgaaacc ccatatc 27 <210> 13 <211> 29 <212> DNA <213> Artificial <220> <223> Artificial <400> 13 cctgcaggta tggtgccgct ttatttggc 29 <210> 14 <211> 35 <212> DNA <213> Artificial <220> <223> Artificial <400> 14 ggcgcgtcta gatgaggga tccttg ataac 35 <210> 15 <211> 36 <212> DNA <213> Artificial <220> <223> Artificial <400> 15 gcgccggtcg acagcatgag gagaactaga aaaagc 36 <210> 16 <211> 4173 <212> DNA <213> Neisseria polysaccharea <220>

Claims (8)

Method for producing a transgenic plant cell that synthesizes modified starch, characterized in that the transgenic plant cell is genetically modified by introducing an exogenous nucleic acid molecule or exogenous nucleic acid molecules, the (s) ) nucleic acid molecule (s) comprising the sequence of SEQ ID NO: 16 encoding an amylosacarase protein and the sequence of SEQ ID NO: 1 encoding a branching enzyme, the expression of which leads ( m) increased activity of an amylosaccharide protein and increased activity of a branching enzyme in comparison with corresponding genetically unmodified wild-type plant cells. Method for producing a transgenic plant cell according to claim 1, characterized in that it synthesizes alpha-1,4-branched alpha-1,4-glucans with a modified branch degree at position 0-6 which are not synthesized by corresponding genetically unmodified wild-type plant cells or synthesizing a modified starch that is not synthesized by corresponding genetically unmodified wild-type plant cells. Method for producing a transgenic plant cell according to claim 1 or 2, characterized in that the amylosaccharide is derived from a bacterium of the genus Neisseria. Method for producing a transgenic plant cell according to any one of claims 1 to 3, characterized in that the branching enzyme is derived from a bacterium of the genus Neisseria. Method for producing a transgenic plant cell according to claim 3 or 4, characterized in that the branching enzyme is derived from Neisseria denitrificans. Method for producing a transgenic plant cell according to any one of claims 1 to 5, characterized in that the exogenous nucleic acid molecule (s) has one or more protein targeting signal sequence (s) that mediates a vacuolar location of the amylosacarase protein and the branching enzyme protein. Method for producing a transgenic plant cell according to any one of claims 1 to 5, characterized in that the exogenous nucleic acid molecule (s) has one or more protein targeting signal sequence (s) that mediates a plastidic localization of the amylosacarase protein and the branching enzyme protein. Method for producing a transgenic plant cell according to any one of claims 1 to 5, characterized in that the exogenous nucleic acid molecule (s) has one or more protein targeting signal sequence (s) mediating (m) a specific cell wall location of the amylosacarase protein and the branching enzyme protein.