EP1100937A1 - Vegetaux synthetisant un amidon modifie, leurs procedes de preparation, leur utilisation, ainsi que l'amidon modifie - Google Patents

Vegetaux synthetisant un amidon modifie, leurs procedes de preparation, leur utilisation, ainsi que l'amidon modifie

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Publication number
EP1100937A1
EP1100937A1 EP99936576A EP99936576A EP1100937A1 EP 1100937 A1 EP1100937 A1 EP 1100937A1 EP 99936576 A EP99936576 A EP 99936576A EP 99936576 A EP99936576 A EP 99936576A EP 1100937 A1 EP1100937 A1 EP 1100937A1
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EP
European Patent Office
Prior art keywords
starch
plant
nucleic acid
enzymes
cells
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
EP99936576A
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German (de)
English (en)
Inventor
Volker Landschütze
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Bayer CropScience AG
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Aventis CropScience GmbH
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Filing date
Publication date
Application filed by Aventis CropScience GmbH filed Critical Aventis CropScience GmbH
Publication of EP1100937A1 publication Critical patent/EP1100937A1/fr
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1051—Hexosyltransferases (2.4.1)
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B30/00—Preparation of starch, degraded or non-chemically modified starch, amylose, or amylopectin
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B30/00—Preparation of starch, degraded or non-chemically modified starch, amylose, or amylopectin
    • C08B30/04—Extraction or purification
    • C08B30/048—Extraction or purification from potatoes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C12N15/8245—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving modified carbohydrate or sugar alcohol metabolism, e.g. starch biosynthesis

Definitions

  • the present invention relates to recombinant nucleic acid molecules which contain two or more nucleotide sequences which encode enzymes involved in starch metabolism, processes for producing transgenic plant cells and plants which synthesize a starch which has been modified with regard to its phosphate content and its side chain structure. Furthermore, the present invention relates to vectors and host cells which contain the nucleic acid molecules according to the invention, the plant cells and plants resulting from the processes according to the invention, the starch synthesized by the plant cells and plants according to the invention and processes for producing this starch.
  • polysaccharides are important, renewable raw materials from plants.
  • starch which is one of the most important storage substances in higher plants, plays a central role in the polysaccharides.
  • the potato plays an important role in starch production.
  • the polysaccharide starch is a polymer made up of chemically uniform building blocks, the glucose molecules. However, it is a very complex mixture from different molecular forms that differ in terms of their degree of polymerization and the occurrence of branches of the glucose chains. Starch is therefore not a uniform raw material.
  • amylose starch an essentially unbranched polymer composed of ⁇ -1,4-glycosidically linked glucose molecules, and amylopectin starch, which in turn is a complex mixture of differently branched glucose chains.
  • the branches come about through the occurrence of additional ⁇ -1,6-glycosidic linkages.
  • the synthesized starch consists of approximately 25% amylose starch and approximately 75% amylopectin starch.
  • the molecular structure of the starch which is largely determined by the degree of branching, the amylose / amylopectin ratio, the average length and distribution of the side chains and the presence of phosphate groups, is crucial for important functional properties of the starch or its aqueous solutions .
  • important functional properties are solubility, retrograding behavior, film-forming properties, viscosity, color stability, gelatinization properties, and binding and adhesive properties.
  • the starch grain size can also be important for various applications.
  • the production of starches containing high amylose is of particular interest for certain applications.
  • a modified starch contained in plant cells can advantageously change the behavior of the plant cell under certain conditions.
  • the biochemical pathways that lead to starch building are essentially known.
  • the starch synthesis in plant cells takes place in the plastids.
  • Important enzymes involved in starch synthesis are, for example, branching enzymes, ADP-glucose pyrophosphorylases, starch-bound Starch synthases, soluble starch synthases, debranching enzymes, disproportionation enzymes, plastid starch phosphorylases and the R1 enzymes (R1 proteins).
  • the object of the present invention is to develop further or alternative genetic engineering approaches for modifying the starch metabolism in starch-forming plants (for example rye, barley, oats, maize, wheat, millet, sago, rice, pea, marker pea, cassava, potato, tomato, rape, To provide soybean, hemp, flax, sunflower, cow pea, mung bean, bean, banana or arrowroot) or suitable nucleic acid molecules by means of which plant cells can be transformed, so that the synthesis of modified, advantageous starch varieties is made possible.
  • starch-forming plants for example rye, barley, oats, maize, wheat, millet, sago, rice, pea, marker pea, cassava, potato, tomato, rape,
  • suitable nucleic acid molecules by means of which plant cells can be transformed, so that the synthesis of modified, advantageous starch varieties is made possible.
  • Such changed starch varieties e.g. Modifications related to their degree of branching, the amylose / amylopectin ratio, the phosphate content, the starch grain size and / or the average length and distribution of the side chains (i.e. side chain structure).
  • Another object of the invention is to provide methods which enable the production of transgenic plants which synthesize a modified (modified) starch variety.
  • transgenic plants or plant cells which have been transformed with the nucleic acid molecules according to the invention or to be used according to the invention synthesize a starch which has been changed in its particular physicochemical properties and / or in its side chain structure in the particular manner according to the invention.
  • known starches which are synthesized by transgenic plants do not show the changes according to the invention.
  • the invention therefore relates to a recombinant nucleic acid molecule (nucleotide sequence) comprising a) at least one nucleotide sequence (polynucleotide or nucleic acid molecule) coding for a protein with the function of a soluble starch synthase III or fragments of said nucleotide sequence and b) one or more nucleotide sequences which are suitable for one Encode protein, selected from group A, consisting of proteins with the function of branching enzymes (BE I, Ha and 11b), ADP-glucose pyrophosphorylases, starch-bound starch synthases, soluble starch synthases I, II or others, debranching enzymes, disproportionation enzymes, plastidary Starch phosphorylases, R1 enzymes, amylases and glucosidases or their fragments - preferably soluble starch synth
  • group A consisting of proteins with the function of branching enzymes (BE
  • Nucleotide sequences which are suitable according to the invention and which code for a protein with the function of a soluble starch synthase III are known, for example, from EP-A-0779363.
  • the term “nucleotide sequence coding for a protein with the function of a soluble starch synthase IN” should be understood in particular to mean those sequences whose coding region has a length of 3000-4500 bp, preferably 3200-4250 bp, particularly preferably 3400- 4000 bp and their homology to the entire coding region of a nucleic acid coding for a protein with the function of a starch synthase is at least 70%, preferably at least 80%, particularly preferably at least 90% and very particularly preferably at least 95%.
  • Nucleotide sequences which are suitable according to the invention and which code for a group A protein are, for example, soluble starch synthases (type I, II or other) or starch grain-bound starch synthase isoforms (for example Hergersberg, 1988, dissertation, University of Cologne; Abel, 1995, dissertation FU Berlin ; Abel et al., 1996, Plant Journal 10 (6): 981-991; Visser et al., 1989, Plant Sei. 64: 185-192; van der Leij et al., 1991, Mol. Gen. Genet.
  • soluble starch synthases type I, II or other
  • starch grain-bound starch synthase isoforms for example Hergersberg, 1988, dissertation, University of Cologne; Abel, 1995, dissertation FU Berlin ; Abel et al., 1996, Plant Journal 10 (6): 981-991; Visser et al., 1989, Plant Sei. 64: 185-192; van der Lei
  • Branching enzyme isoforms (branching enzyme I, IIa, Mb), debranching enzyme isoforms (debranching enzymes, isoamylases, pullulanases), • or disproportionation enzyme isoforms described for example in WO 92/14827; WO 95/07335; WO 95/09922; WO 96/19581; WO 97/22703; WO 97/32985; WO 97/42328; Takaha et al., 1993, J. Biol.
  • Botany 41 (Suppl.) 5-7; Lin et al., 1991, Plant Physiol. 95: 1250-1253; Sonnewald et al., 1995, Plant Mol. Biol. 27: 567-576; DDBJ No. D23280; Lorberth et al., 1998, Nature Biotechnology 16: 473-477 and for amylases and glucosidases.
  • nucleotide sequences to be used according to the invention are of pro- or eukaryotic origin, preferably of bacterial, fungal or vegetable origin.
  • fragment means parts of the Nucleic acid molecules according to the invention or to be used according to the invention which are at least 15 bp, preferably at least 150 bp, particularly preferably at least 500 bp long, but generally do not exceed a length of 5000 bp, preferably 2500 bp.
  • hybridization means hybridization under conventional hybridization conditions, preferably under stringent conditions, as described, for example, in Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd ed. (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).
  • fragment encompasses biologically active molecules.
  • a “specific hybridization” is particularly preferably carried out under the following • highly stringent conditions:
  • Hybridization buffer 2 x SSC; 10 x Denhardt's solution (Fikoll 400 + PEG + BSA; ratio 1: 1: 1); 0.1% SDS; 5mM EDTA; 50 mM Na 2 HPO 4 ; 250 ⁇ g / ml herring sperm DNA; 50 ⁇ g / ml tRNA; or 0.25 M sodium phosphate buffer pH 7.2; 1mM EDTA; 7% SDS in one
  • the molecules hybridizing with the nucleic acid molecules according to the invention or to be used according to the invention also include fragments, derivatives and allelic variants of the nucleic acid molecules according to the invention or to be used according to the invention.
  • “Fragments” are not only understood to mean parts of the nucleic acid molecules that are long enough to encode a functionally active part of the proteins described.
  • the term “derivative” in the context of the present invention means that the sequences of these molecules differ from the Sequences of the invention or according to the invention distinguish using nucleic acid molecules at one or more positions and have a high degree of homology to these sequences.
  • Homology means a sequence identity of at least 60%, preferably over 70% and particularly preferably over 85%, in particular over 90% and very particularly preferably over 95%.
  • the deviations from the nucleic acid molecules according to the invention or to be used according to the invention may have arisen from one or more deletions, substitutions, insertions (addition) or recombinations.
  • nucleic acid molecules which are homologous to the molecules according to the invention or to be used according to the invention and which are derivatives of these molecules are generally variations of these molecules which represent modifications which have the same, an almost identical or similar biological function.
  • These can be both naturally occurring variations, for example sequences from other plant species, or mutations, wherein these mutations can have occurred naturally or have been introduced by targeted mutagenesis.
  • the variations can be synthetically produced sequences.
  • allelic variants can be both naturally occurring variants and also synthetically produced variants or those produced by recombinant DNA techniques.
  • the nucleic acid molecules according to the invention or to be used according to the invention can be DNA molecules, in particular cDNA or possibly the combination of genomic molecules. Furthermore, the nucleic acid molecules according to the invention or to be used according to the invention can be RNA molecules.
  • the nucleic acid molecules according to the invention or to be used according to the invention or fragments thereof can e.g. B. obtained from natural sources, be produced by recombinant techniques or synthetically.
  • nucleic acid molecules according to the invention or to be used according to the invention in sense or antisense orientation in plant cells are linked to regulatory DNA elements which ensure transcription in plant cells.
  • regulatory DNA elements which ensure transcription in plant cells.
  • These include promoters in particular.
  • any promoter active in plant cells can be used for the expression.
  • the promoter can be selected so that the expression takes place constitutively or only in a certain tissue, at a certain time in plant development or at a time determined by external influences, which can be chemically or biologically inducible, for example.
  • the promoter - like the nucleotide sequence - can be homologous or heterologous.
  • Suitable • promoters are, for example, the 35S RNA promoter of the Cauliflower Mosaic Virus and the ubiquitin promoter from maize for a constitutive expression, the patatin promoter B33 (Rocha-Sosa et al, 1989, EMBO J. 8:. 23-29) for a bulb-specific expression in potatoes or a promoter which ensures expression only in photosynthetically active tissues, for example the ST-LS1 promoter (Stockhaus et al., 1987, Proc. Natl. Acad. Sci. USA 84: 7943-7947; Stockhaus et al., 1989, EMBO J. 8: 2445-2451) the Ca / b promoter (e.g.
  • a termination sequence terminating the nucleic acid molecule according to the invention can serve for the correct completion of the transcription, as well as the addition of a poly-A tail to the transcript, which is believed to have a function in stabilizing the transcripts.
  • Such elements are described in the literature (cf. Gielen et al., 1989, EMBO J. 8: 23-29) and are generally interchangeable.
  • the nucleic acid molecules according to the invention or to be used according to the invention can be used for the production of transgenic plant cells and plants which are each increased and / or decreased in the activity of the soluble starch synthase III and at least one further enzyme, the starch metabolism.
  • the nucleic acid molecules according to the invention or to be used according to the invention are introduced into suitable vectors, provided with the necessary regulatory nucleic acid sequences for efficient transcription in plant cells and introduced into plant cells. It consists • a possibility to use the invention or used according to nucleic acid molecules to inhibit the synthesis of endogenous soluble starch synthase III and / or at least one further protein of the group A in the cells.
  • nucleic acid molecules according to the invention or to be used according to the invention can be used to express the soluble starch synthase III and / or at least one other protein of group A in cells of transgenic plants and thus lead to an increase in the activity of the respectively expressed enzymes in the cells.
  • nucleic acid molecules according to the invention or to be used according to the invention for inhibiting the synthesis of the endogenous soluble starch synthase III and the overexpression of at least one further protein of group A in the cells.
  • nucleic acid molecules according to the invention or to be used according to the invention can also be used to express soluble starch synthase III and to inhibit at least one other Group A protein in cells of transgenic plants. The latter two embodiments of the invention thus lead to a simultaneous inhibition and increase in the activities of the respectively inhibited or expressed enzymes in the cells.
  • Another object of the invention is a vector containing a nucleic acid molecule according to the invention.
  • vector encompasses plasmids, cosmids, viruses, bacteriophages and other vectors which are common in genetic engineering and which contain the nucleic acid molecules according to the invention and are suitable for transforming cells.
  • Such vectors are preferably suitable for transforming plant cells. They particularly preferably allow the nucleic acid molecules according to the invention, optionally together with flanking regulatory regions, to be integrated into the genome of the plant cell. Examples of this are binary vectors, such as pBinAR or pBinB33, which can be used in the gene transfer mediated by agrobacteria.
  • the vector according to the invention is characterized in that the nucleotide sequence which codes for a protein with the function of a soluble starch synthase III or whose fragments are present in the sense or anti-sense direction.
  • the vector according to the invention is characterized in that the nucleotide sequence which codes for one or more proteins selected from group A or fragments thereof is present in the sense or antisense direction.
  • the vector according to the invention is characterized in that the nucleotide sequence which is for several Proteins selected from group A are encoded or fragments thereof, partly in the sense direction and partly in the anti-sense direction.
  • the vector according to the invention very particularly preferably contains one or more regulatory elements which ensure the transcription and synthesis of an RNA in a pro- or eukaryotic cell.
  • deletions at the 5 'end of the DNA sequence make it possible, for example, to specifically produce enzymes which, by removing the corresponding transit or signal sequences, no longer in their original (homologous) compartment, but in the cytosol, or due to the addition of other signal sequences in one or more other (heterologous) compartments (eg plastid, vacuole, mitochondrium, apoplast) are localized.
  • heterologous compartments eg plastid, vacuole, mitochondrium, apoplast
  • the introduction of point mutations is also conceivable at positions in which a change in the amino acid sequence has an influence, for example on the enzyme activity or the regulation of the enzyme.
  • the DNA sequences according to the invention or to be used according to the invention or fragments of these sequences can be introduced into plasmids which permit mutagenesis or a sequence change by recombination of DNA sequences. With the help of standard molecular biological methods (cf.
  • Base exchanges can be carried out or natural or synthetic sequences can be added.
  • adapters or links can be attached to the parts.
  • Manipulations that provide suitable restriction sites or that remove excess DNA or restriction sites that are no longer required can also be used. Where insertions, deletions or substitutions are possible, w ' fra mutagenesis, "primer repair", restriction or ligation can be used. Sequence analysis, restriction analysis and possibly other biochemical-molecular biological methods are generally carried out as the analysis method.
  • Another object of the invention is a transgenic host cell with altered starch metabolism, in particular prokaryotic or eukaryotic cells, preferably bacterial or plant (monocotyledon or dicotyledonous) cells (for example from E. coli, Agrobacterium, Solananceae, Poideae, rye, barley, oats, maize, Wheat, millet, sago, rice, pea, marker pea, cassava, potato, tomato, rapeseed, soybean, hemp, flax, sunflower, cow pea, mung bean, bean, banana or arrowroot), the one or more nucleic acid molecules according to the invention or one or more according to the invention Contains vectors, or which is derived from such a cell.
  • prokaryotic or eukaryotic cells preferably bacterial or plant (monocotyledon or dicotyledonous) cells (for example from E. coli, Agrobacterium, Solananceae, Poideae, rye
  • Yet another object of the invention is a transgenic host cell with an altered starch metabolism, in particular prokaryotic or eukaryotic cells, preferably bacterial or plant cells (for example from E. coli, Agrobacterium, Solananceae, Poideae, Rye, Barley, Oats, Corn, Wheat, Millet, Sago, Rice, Pea, Pea, Cassava, Potato, Tomato, Rapeseed, Soybean, Hemp, Flax, Sunflower, Cow Pea, Mung Bean, Bean, Banana or Arrowroot) which contains a) at least one nucleotide sequence coding for a protein with the function of a soluble starch synthase III or its fragments and b) one or more nucleotide sequences which code for a protein selected from group A, or whose fragments or nucleotide sequences hybridizing with these nucleic acid molecules , or which is derived from such a cell.
  • prokaryotic or eukaryotic cells
  • host cells according to the invention can also be produced by successive transformation (for example by so-called “supertransformation"), in which several parts of the nucleotide sequence according to the invention or several vectors containing parts of the nucleotide sequence according to the invention which code for a protein with the function of a soluble starch synthase III or their fragments and also one or more proteins selected from group A consisting of branching enzymes, ADP-glucose pyrophosphorylases, starch granule-bound starch synthases, soluble starch synthases I, II or others, debranching enzymes, disproportionation enzymes, plastid starch phosphorylases, amylases, R1 - Enzymes, their fragments - preferably soluble starch synthases II, soluble starch synthases I and / or branching enzymes and their fragments - and nucleic acid molecules, which with one of the hybridize said
  • the cell is transformed with a) at least one nucleic acid molecule which encodes a protein with the function of a soluble starch synthase III, its fragment or with a vector containing said nucleic acid molecule, and b) with one or more nucleic acid molecules which encode a protein, which is selected from group A, consisting of branching enzymes, ADP-glucose pyrophosphorylases, starch-bound starch synthases, soluble starch synthases I, II or others, Branching enzymes, disproportionation enzymes, amylases, glucosidases, plastid starch phosphorylases and R1 enzymes, or their fragments - preferably soluble starch synthases II, soluble starch synthases I and / or branching enzymes and their fragments - and nucleic acid molecules which hybridize with one of said or their nucleotide sequences or their nucleotide sequences one or more vectors containing one or more of said
  • Another object of the invention is a method for producing a transgenic host cell, bacterial cell, plant cell or plant which synthesizes a modified starch, characterized in that a) at least one nucleic acid molecule which encodes a protein with the function of a soluble starch synthase III or its fragments and b) one or more nucleic acid molecules which encode a protein which is selected from group A, consisting of branching enzymes, ADP-glucose pyrophosphorylases, starch-bound starch synthases, soluble starch synthases I, II or others, debranching enzymes, disproportionation enzymes, amylases, Glucosidases, plastidic starch phosphorylases and R1 enzymes or their fragments - preferably soluble starch synthases II, soluble starch synthases I and / or branching enzymes and their fragments - and nucleic acid molecules which are associated with one of said nucleotide sequences or their fragment
  • a further embodiment of the present invention relates to a method for producing a transgenic host cell, bacterial cell, plant cell or plant which synthesizes a modified starch, characterized in that one or more nucleic acid molecules according to the invention or one or more vectors according to the invention are integrated into the genome of a cell and if necessary, an entire plant is regenerated from the transgenic plant cell.
  • nucleic acid molecules according to the invention it is possible, using genetic engineering methods, to intervene in the starch metabolism of plants and to change it in such a way that a modified starch is synthesized which, for example, in terms of structure, water content, protein content, lipid content, fiber content, Ash / phosphate content, amylose / amylopectin ratio, molecular weight distribution, degree of branching, grain size and shape as well as crystallization or also in their physico-chemical properties such as flow and sorption behavior, gelatinization temperature, viscosity, thickening performance, solubility, paste structure, transparency, heat, shear and Acid stability, tendency to retrogradation, gel formation, freeze / thaw stability, complex formation, iodine binding, film formation, adhesive strength, enzyme stability, digestibility or reactivity is changed compared to starch synthesized in wild-type plants.
  • a modified starch which, for example, in terms of structure, water content, protein content, lipid content, fiber content, Ash
  • the synthesized protein can be located in any compartment of the plant cell.
  • the transit or signal sequence ensuring the localization may have to be deleted (removed) and the remaining coding region may have to be linked to DNA sequences, which ensure localization in the respective compartment.
  • sequences are known (see, for example, Braun et al., EMBO J. 11 (1992), 3219-3227; Wolter et al., Proc. Natl. Acad. Sci.
  • the production of plant cells with a reduced activity of a protein involved in starch metabolism can be achieved, for example, by the expression of a corresponding antisense-RNA, a sense-RNA to achieve a cosuppression effect, in vivo mutagenesis or the expression of a correspondingly constructed ribozyme that specifically transcripts cleaves which encode one of the proteins involved in starch metabolism using a nucleic acid molecule according to the invention, preferably by expression of an antisense transcript.
  • a DNA molecule can be used which comprises the entire sequence coding for a protein involved in starch metabolism, including any flanking sequences which may be present, and also DNA molecules which only comprise parts of the coding sequence, these parts having a minimum length of 15 bp, preferably of at least 100-500 bp, and in particular of over 500 bp.
  • DNA molecules are used which are shorter than 5000 bp, preferably shorter than 2500 bp.
  • DNA sequences that have a high degree of Have homology to the sequences of the DNA molecules according to the invention, but are not completely identical.
  • the minimum homology should be greater than approximately 65%.
  • sequences with a homology of about 75 to 85% and in particular of about 85 to 95% is preferred.
  • ribozymes to reduce the activity of certain proteins in cells is known to the person skilled in the art and is described, for example, in EP-B1-0 321 201.
  • the expression of ribozymes in plant cells has been described e.g. described in Feyter et al. (Mol. Gen. Genet. 250 (1996), 329-338).
  • the reduction of the proteins involved in starch metabolism in the plant cells according to the invention can also be achieved by the so-called "in vivo mutagenesis", in which a hybrid RNA-DNA oligonucleotide ("chimeroplast”) is introduced into cells by transforming cells (Kipp PB et al, Poster session at.
  • in vivo mutagenesis in which a hybrid RNA-DNA oligonucleotide (“chimeroplast”) is introduced into cells by transforming cells (Kipp PB et al, Poster session at.
  • Part of the DNA component of the RNA-DNA oligonucleotide used here is homologous to a nucleic acid sequence of an endogenous protein, but has a mutation in comparison to the nucleic acid sequence of the endogenous protein or contains a heterologous region which is enclosed by the homologous regions.
  • the mutation or heterologous region contained in the DNA component of the RNA-DNA oligonucleotide can be transferred into the genome of a plant cell. This leads to a decrease in the activity of the protein involved in starch metabolism.
  • the enzyme activities involved in starch metabolism in the plant cells can be reduced by a cosuppression effect.
  • This method is described, for example, in Jorgensen (Trends Biotechnol. 8 (1990), 340-344), Niebel et al., (Curr. Top. Microbiol. Immunol. 197 (1995), 91-103), Flavell et al. (Curr. Top. Microbiol. Immunol. 197 (1995), 43-46), Palaqui and Vaucheret (Plant. Mol. Biol. 29 (1995), 149-159), Vaucheret et al., (Mol. Gen. Genet 248 (1995), 311-317), de Borne et al. (Mol. Gen. Genet. 243 (1994), 613-621).
  • DNA molecules can be used for the transformation, which simultaneously contain several regions coding for the corresponding enzymes in antisense orientation under the control of a suitable promoter.
  • Each sequence can alternatively be under the control of its own promoter, or the sequences can be transcribed as a fusion from a common promoter, so that the synthesis of the proteins in question is inhibited to approximately the same or different extent.
  • the length of the individual coding regions which are used in such a construct what has already been stated above for the production of antisense constructs applies. In principle, there is no upper limit for the number of antisense fragments transcribed from a promoter in such a DNA molecule. However, the resulting transcript should generally not exceed a length of 25 kb, preferably 15 kb.
  • nucleic acid molecules according to the invention With the aid of the nucleic acid molecules according to the invention or to be used according to the invention, it is therefore possible to transform plant cells and to inhibit the synthesis of several enzymes simultaneously.
  • Nucleic acid molecules are introduced into classic mutants that are defective or defective in relation to one or more genes of starch biosynthesis (Shannon and Garwood, 1984, in Whistler, BeMiller and Paschall, Starch: Chemistry and Technology, Academic Press, London, 2nd Edition: 25 -86).
  • defects can relate, for example, to the following proteins: starch-grain-bound (GBSS I) and soluble starch synthases (SS I, II, III and others), branching enzymes (BE I, Ha and IIb), "debranching" enzymes (R-enzymes , Isoamylases, pullulanases), disproportionation enzymes and plastid starch phosphorylases.
  • GBSS I starch-grain-bound
  • SS I, II, III and others branching enzymes
  • BE I, Ha and IIb branching enzymes
  • R-enzymes "debranching" enzymes
  • Isoamylases Isoamylases, pullulanases
  • disproportionation enzymes plastid starch phosphorylases.
  • the present invention relates thus also to transgenic plant cells obtainable by a method according to the invention which are derived with an inventive or have been transformed according to the invention to be used in nucleic acid molecule or vector, as well as transgenic plant cells or plants transformed by such ⁇ cells.
  • the cells according to the invention contain one or more nucleic acid molecules according to the invention or to be used according to the invention, these preferably being linked to one or more regulatory DNA elements (for example promoter, enhancer, terminator) which ensure transcription in plant cells, in particular with a promoter.
  • regulatory DNA elements for example promoter, enhancer, terminator
  • the cells according to the invention can be distinguished from naturally occurring plant cells, inter alia, in that they contain a nucleic acid molecule according to the invention which does not naturally occur in these cells or in that such a molecule is integrated at a location in the genome of the cell where it is not otherwise occurs, ie in a different genomic environment.
  • the transgenic plant cells according to the invention can be distinguished from naturally occurring plant cells in that they contain at least one copy of a nucleic acid molecule according to the invention stably integrated into their genome, optionally in addition to copies of such a molecule or the nucleic acid molecules to be used according to the invention that occur naturally in the cells.
  • the plant cells according to the invention can be distinguished from naturally occurring cells, in particular in that these additional copy (s) are (are) located at locations in the genome where they do not naturally occur. This can be checked, for example, using a Southern blot analysis.
  • Plant cells according to the invention are preferred in which the enzyme activity of individual enzymes involved in starch metabolism is increased and / or decreased by at least 10%, particularly preferably at least 30% and very particularly preferably by at least 50%.
  • the plant cells according to the invention can preferably be distinguished from naturally occurring plant cells by at least one of the following features: If the nucleic acid molecule introduced is heterologous with respect to the plant cell, the transgenic plant cells have transcripts of the nucleic acid molecules introduced. These can e.g. B. by Northem blot analysis.
  • the plant cells according to the invention contain one or more proteins which are encoded by an introduced nucleic acid molecule according to the invention or to be used according to the invention. This can e.g. B. by immunological methods, in particular by Western blot analysis.
  • the cells according to the invention can be distinguished from naturally occurring nucleic acid molecules, for example due to the additional expression of nucleic acid molecules according to the invention or to be used according to the invention.
  • the transgenic plant cells contain, for example, more or fewer transcripts of the nucleic acid molecules according to the invention or to be used according to the invention. This can e.g. B. detected by Northem blot analysis.
  • "More” or “less” means here preferably at least 10% more or less, preferably at least 20% more or less and particularly preferably at least 50% more or less transcripts than corresponding non-transformed cells.
  • the cells preferably also have a corresponding (at least 10%, 20% or 50%) increase or decrease in the content of the protein encoded by the introduced nucleic acid molecules.
  • the transgenic plant cells can be regenerated into whole plants using techniques known to those skilled in the art.
  • the plants obtainable by regeneration of the transgenic plant cells according to the invention and processes for producing transgenic plants by regeneration of whole plants from the plant cells according to the invention are likewise the subject of the present invention.
  • the invention furthermore relates to plants which contain the transgenic plant cells according to the invention.
  • the transgenic plants can in principle be plants of any species, i.e. both monocot and dicot plants. They are preferably useful plants and starch-storing plants, such as e.g.
  • Cereals (rye, barley, oats, maize, wheat, millet etc.), sago, rice, peas, marker peas, cassava, potatoes, tomatoes, rapeseed, soybeans, hemp, flax, sunflower, cow peas, mung beans or arrowroot.
  • the invention also relates to propagation material of the plants according to the invention, for example fruits, seeds, tubers, rhizomes, seedlings, cuttings, calli, protoplasts, cell cultures etc.
  • the change in the enzymatic activities of the enzymes involved in the starch metabolism results in the synthesis of a starch with a different structure in the plants produced by the process according to the invention.
  • a large number of cloning vectors are available to prepare the introduction of foreign genes into higher plants, which a replication signal for E.coli and contain a marker gene for the selection of transformed bacterial cells.
  • examples of such vectors are pBR322, pUC-Ser ⁇ en, M13mp series, pACYC184 etc.
  • the desired sequences can be introduced into the vector at a suitable restriction site.
  • the plasmid obtained is used for the transformation of E. coli cells. Transformed E.co// cells are grown in a suitable medium, then harvested and lysed. The plasmid is recovered.
  • Restriction analyzes, gel electrophoresis and other biochemical-molecular biological methods are generally used as the analysis method for characterizing the plasmid DNA obtained (Sambrook et al. Loc.cit.). After each manipulation, the plasmid DNA can be cleaved and DNA fragments obtained can be linked to other DNA sequences. Each plasmid DNA sequence can be cloned into the same or different plasmids.
  • a variety of techniques are available for introducing DNA into a plant host cell. These techniques include the transformation of plant cells with T-DNA using Agrobacterium tumefaciens or Agrobacterium rhizogenes as a transformation agent, the fusion of protoplasts using polyethylene glycol (PEG), the injection, the electroporation of DNA, the introduction of DNA using the biolistic method and further possibilities (Gene Transfer to Plants. pp. 24-29, eds .: Potrykus, I. and Spangenberg, G., Springer Verlag Berlin Heidelberg 1995).
  • PEG polyethylene glycol
  • DNA When DNA is injected and electroporated into plant cells, there are no special requirements for the plasmids or DNA used. Simple plasmids such as e.g. pUC derivatives can be used. If whole plants are to be regenerated from such transformed cells, the presence of a selectable marker gene is necessary.
  • Simple plasmids such as e.g. pUC derivatives can be used. If whole plants are to be regenerated from such transformed cells, the presence of a selectable marker gene is necessary.
  • additional DNA sequences may be required. Used for example for the transformation of the plant cell If the Ti or Ri plasmid is used, at least the right boundary, but frequently the right and left boundary of the Ti and Ri plasmid T-DNA as the flank region, must be linked to the genes to be introduced.
  • the DNA to be introduced must be cloned into special plasmids, either in an intermediate vector or in a binary vector.
  • the intermediate vectors can be integrated into the Ti or Ri plasmid of the agrobacteria on the basis of sequences which are homologous to sequences in the T-DNA by homologous recombination. This also contains the ' r region necessary for the transfer of the T-DNA. Intermediate vectors cannot replicate in agrobacteria. Using a helper plasmid, the intermediate vector can be transferred to Agrobacterium tumefaciens (conjugation). Binary vectors can replicate in both E. coli and agrobacteria.
  • T-DNA border region They contain a selection marker gene and a linker or polylinker, which are framed by the right and left T-DNA border region. They can be transformed directly into the agrobacteria (Holsters et al. (1978) Mol. Gen. Genet. 163: 181-187). Serving as host cell should contain a plasmid of Agrobacterium carrying a w 'R region. The v / r region is necessary for the transfer of the T-DNA into the plant cell. Additional T-DNA may be present. The agrobacterium transformed in this way is used to transform plant cells.
  • T-DNA for the transformation of plant cells has been intensively investigated and is described in EP 120516; Hoekema, In: The Binary Plant Vector System Offsetdrukkerij Kanters B.V, Alblasserdam (1985), Chapter V; Fraley et al., Crit. Rev. Plant. Sci., 4: 1-46 and An et al. (1985) EMBO J. 4: 277-287.
  • plant explants can expediently be cultivated with Agrobacterium tumefaciens or Agrobacterium rhizogenes.
  • From the infected plant material e.g. leaf pieces, stem segments, roots, but also protoplasts or suspension cultivated Plant cells
  • a suitable medium which can contain, for example, antibiotics or biocides for the selection of transformed cells.
  • the plants thus obtained can then be examined for the presence of the introduced DNA.
  • Other possibilities of introducing foreign DNA using the biolistic method or by protoplast transformation are known (cf. e.g. Willmitzer, L, 1993 Transgenic plants. In: Biotechnology, A Multi-Volume Comprehensive Treatise (H. Rehm, G. Reed, A. Pühler , P. Stadler, eds.), Vol. 2, 627-659, VCH Weinheim-New York-Basel-Cambridge).
  • EP 292 435 describes a process by means of which fertile plants can be obtained starting from a slimy, soft (friable) granular corn callus. Shillito et al. (Bio / Technology 7 (1989), 581) have observed in this connection that it is also necessary for the regenerability to fertile plants to start from callus suspension cultures, from which a dividing protoplast culture with the ability to regenerate plants, can be produced. Prioli and Sondahl (Bio / Technology 7 (1989), 589) also describe regeneration and Obtaining fertile maize plants from maize protoplasts.
  • the introduced DNA is integrated in the genome of the plant cell, it is generally stable there and is also retained in the progeny of the originally transformed cell. It usually contains a selection marker that gives the transformed plant cells resistance to a biocide or an antibiotic such as kanamycin, G 418, bleomycin, hygromycin or phosphinothricin and others. taught.
  • the individually chosen marker should therefore allow the selection of transformed cells from cells that lack the inserted DNA.
  • the transformed cells grow within the plant in the usual way (see also McCormick et al. (1986) Plant Cell Reports 5: 81-84).
  • the resulting plants can be grown normally and crossed with plants that have the same transformed genetic makeup or other genetic makeup.
  • the resulting hybrid individuals have the corresponding phenotypic characteristics.
  • Two or more generations should be grown to ensure that the phenotypic trait is stably maintained and inherited. Seeds o.a. are harvested to ensure that the appropriate phenotype or other traits have been preserved.
  • the transgenic plant cells and plants according to the invention synthesize a starch whose physicochemical properties have been changed, for example, compared to starch synthesized in wild-type plants.
  • Yet another object of the invention is starch, which can be obtained from a plant cell according to the invention, a plant, its propagation material or by a method according to the invention.
  • Another subject of the invention is a process for the production of starch in a manner known per se, in which host cells, plant cells, plants, plant parts or propagation material according to the invention are processed or integrated into the process.
  • the starch according to the invention is distinguished by the fact that it has one in comparison to a starch which can be obtained from a non-transformed cell or plant (ie the wild type), by at least 30%, preferably at least 50% preferably has at least 70% and very particularly preferably at least 90% reduced phosphate content and their glucan content (see Fraction 3 in Example No. 13) after isoamyiase treatment in the excluded volume of an HPLC column system consisting of 2 TSK-Gel 2000SW- Columns and a TSK-Gel 3000SW column in 10 mM sodium acetate pH 3.0 (at a flow rate of 0.35 ml / min as described in Example No. 13) by at least 50%, preferably at least 150%, particularly preferably at least 300 % and most preferably at least 500% is increased.
  • the starch according to the invention is characterized in that, compared to a starch which can be obtained from a non-transformed cell or plant (ie the wild type), one by at least 10%, preferably at least 30% and particularly preferably has at least 50% increased phosphate content and their glucan content (see Fraction 3 in Example No. 13) after isoamyiase treatment in the exclusion volume of an HPLC column system consisting of 2 TSK-Gel 2000SW columns connected in series and a TSK-Gel 3000SW Column in 10 mM sodium acetate pH 3.0 (at a flow rate of 0.35 ml / min as described in Example No.
  • Another embodiment of the present invention also includes the use of the starch according to the invention in the industrial field, preferably for the production of foods, packaging materials or disposable articles.
  • the starch according to the invention can be chemically and / or physically modified by methods known to those skilled in the art and is suitable in unmodified or modified form for various uses in the food or non-food sector.
  • the possible uses of the starch according to the invention can basically be divided into two large areas.
  • One area includes the hydrolysis products of the Starch, mainly glucose and glucose building blocks obtained by enzymatic or chemical processes. They " serve as a starting material for further chemical modifications and processes, such as fermentation. Of importance here may be the simplicity and cost-effective execution of a hydrolysis process, as is currently carried out essentially enzymatically using amyloglucosidase. Cost savings would be conceivable through a lower use of enzymes
  • a change in the structure of the starch for example an increase in the surface area of the grain, easier digestibility due to a lower degree of branching or a steric structure which limits the accessibility for the enzymes used, could result in this.
  • Starch is a classic additive for many foods, where it essentially takes on the function of binding aqueous additives or increases the viscosity or increases gel formation. Important characteristics are the flow and sorption behavior, the swelling and gelatinization temperature, the viscosity and thickening performance, the solubility of the starch, the transparency and paste structure, the heat, shear and acid stability, the tendency to retrogradation, the ability to form films, the Freeze / thaw stability, digestibility and the ability to form complexes with e.g. inorganic or organic ions.
  • starch is used as an additive for different manufacturing processes or as an additive in technical products.
  • starch is primarily used for retardation (retention of solids), the imaging of filler and fine particles, as a strengthening agent and for drainage.
  • the favorable properties of the starch in terms of rigidity, hardness, sound, grip, gloss, smoothness, splitting resistance and surfaces are exploited.
  • starches A large area of use of starches is in the adhesive industry, where the possible uses are divided into four areas: use as pure starch glue, use with starch glues prepared with special chemicals, use of starch as an additive to synthetic resins and polymer dispersions, and use of starches as an extender for synthetic adhesives.
  • 90% of the starch-based adhesives are used in the fields of corrugated cardboard manufacture, paper sacks, bags and pouches, composite materials for paper and aluminum, cardboard and Remoistening glue used for envelopes, stamps etc.
  • starch as an auxiliary and additive is the area of manufacture of textiles and textile care products.
  • the following four areas of application can be distinguished within the textile industry:
  • starch as a sizing agent, i.e. as an auxiliary for smoothing and strengthening the hook-and-loop behavior to protect against the tensile forces acting during weaving and to increase the abrasion resistance during weaving
  • starch as an agent for textile finishing, especially after quality-reducing pretreatments such as bleaching, dyeing etc.
  • starch as a thickening agent in the manufacture of color pastes to prevent dye diffusion and starch as an additive to chaining agents for sewing threads.
  • the fourth area of application is the use of starches as an additive in building materials.
  • One example is the production of gypsum plasterboard, in which the starch mixed in the gypsum slurry pastes with the water, diffuses to the surface of the gypsum board and binds the cardboard to the board there.
  • Other areas of application are admixing to plaster and mineral fibers.
  • starch products are used to delay setting.
  • starch products lends itself to the manufacture of soil stabilization agents that are used to temporarily protect soil particles from water during artificial earth movements.
  • Combined products made of starch and polymer emulsions are, according to current knowledge, equivalent in their erosion and crust-minimizing effect to the products previously used, but are priced significantly below these.
  • starches are used as binders for tablets or for binder dilution in capsules. Starches also serve as tablet disintegrants since they absorb liquid after swallowing and swell to such an extent after a short time that the active substance is released. Medical lubricant and wound powders are based on starch for qualitative reasons. In the field of cosmetics, starches are used, for example, as carriers for powder additives such as fragrances and salicylic acid. A relatively large area of application for starch is toothpaste.
  • Starch is used as an additive to coal and briquette. Coal can be agglomerated or briquetted with a high-quality addition of starch, which prevents the briquettes from breaking down prematurely.
  • the added starch is between 4 and 6% for barbecued coal and between 0.1 and 0.5% for calorized coal. Furthermore, starches are becoming increasingly important as binders, since their addition to coal and briquette can significantly reduce the emissions of harmful substances.
  • Starch can also be used as a flocculant in ore and coal sludge processing. 2:10. Foundry auxiliary
  • Bentonite which is mixed with modified starches, mostly swelling starches, is predominantly used today as a binder.
  • starch addition is to increase the flowability and improve the binding strength.
  • swelling starches can have other production requirements, such as dispersibility in cold water, rehydration, good miscibility in sand and high water retention capacity.
  • Starch is used in the rubber industry to improve technical and optical quality. The reasons for this are the improvement of the surface gloss, the improvement of the handle and the appearance, for this reason starch is sprinkled on the sticky rubberized surfaces of rubber materials before the cold vulcanization, and the improvement of the printability of the rubber.
  • starch secondary products in the processing process (starch is only a filler, there is no direct link between synthetic polymer and starch) or, alternatively, the integration of starch secondary products in the production of polymers (starch and polymer are one firm bond).
  • starch as a pure filler is not competitive compared to other substances like talc. It is different if the specific starch properties come into play and this significantly changes the property profile of the end products.
  • thermoplastics such as polyethylene.
  • the starch and the synthetic polymer are combined by co-expression in a ratio of 1: 1 to form a 'master batch', from which various products are made using granulated polyethylene using conventional processing techniques.
  • the incorporation of starch in polyethylene foils can result in increased material permeability in the case of hollow bodies, improved water vapor permeability, improved antistatic behavior, improved antiblocking behavior and improved printability with aqueous inks.
  • Current disadvantages include insufficient transparency, reduced tensile strength, and reduced ductility.
  • starch in polyurethane foams.
  • starch derivatives By adapting the starch derivatives and by optimizing the process, it is possible to control the reaction between synthetic polymers and the hydroxyl groups of the starches.
  • the result is polyurethane foils, which get the following property profiles through the use of starch: a reduction in the coefficient of thermal expansion, a reduction in shrinkage behavior, an improvement in pressure / stress behavior, an increase in water vapor permeability without changing the water absorption, a reduction in flammability and tear density, no dripping of flammable parts, Halogen free and reduced aging.
  • Disadvantages that are currently still present are reduced compressive strength and reduced impact resistance.
  • Solid plastic products such as pots, plates and bowls also have a starch content of over 50%. Furthermore, starch / polymer mixtures can be assessed favorably because they have a much higher biodegradability.
  • starch graft polymer seeds Due to their extreme water-binding capacity, starch graft polymer seeds have become extremely important. These are products with a backbone made of starch and a side grid of a synthetic monomer grafted on according to the principle of the radical chain mechanism.
  • the starch graft polymers available today are characterized by better binding and retention properties of up to 1000 g of water per g of starch with high viscosity.
  • the areas of application for these superabsorbents have expanded significantly in recent years and are in the hygiene area with products of diapers and pads as well as in the agricultural sector, e.g. seed pilling.
  • modified starches by means of genetic engineering methods can, on the one hand, change the properties of the starch obtained, for example, from the plant in such a way that further modifications by means of chemical or physical changes no longer appear necessary.
  • starches modified by genetic engineering processes can also be subjected to further chemical modifications, which leads to further improvements in quality for certain of the areas of use described above.
  • This chemical Modifications are generally known. In particular, these are modifications by heat and pressure treatment, treatment with organic or inorganic acids, enzymatic treatment, oxidations and esterifications, which lead, for example, to the formation of phosphate, nitrate, sulfate, xanthate, acetate and citrate starches.
  • monohydric or polyhydric alcohols can be used in the presence of strong acids to produce starch ethers, so that starch alkyl ethers, O-allyl ethers, hydroxylalkyl ethers, O-carboxylmethyl ethers, N-containing starch ethers, P-containing starch ethers, S-containing starch ethers, crosslinked Starches or starch graft polymers result.
  • the starches according to the invention are used in industrial applications, preferably for food or in the production of packaging materials and disposable articles.
  • Buffer B 50mM Tris-HCl pH 7.6 2.5mM DTT 2mM EDTA
  • Buffer C 0.5 M sodium citrate pH 7.6 50 mM Tris-HCl pH 7.6 2.5 mM DTT 2 mM EDTA
  • Dialysis buffer 50 mM Tris-HCl pH 7.0 50 mM NaCI 2 mM EDTA 14.7 mM beta-mercaptoethanol 0.5 mM PMSF
  • the vector pBluescript II SK (Stratagene) was used for cloning in E. co / V.
  • the E. co // strain DH5a (Bethesda Research Laboratories, Gaithersburgh, USA) was used for the Bluescript vector p Bluescript II KS (Stratagene) and for the pBinAR Hyg and pBinB33 Hyg constructs.
  • the E. co // strain XL1-Blue was used for the in vivo excision.
  • the plasmid pBinAR is a derivative of the binary vector plasmid pBin19 (Bevan, 1984), which was constructed as follows:
  • a 529 bp fragment comprising nucleotides 6909-7437 of the 35S promoter of the cauliflower mosaic virus was isolated as an EcoRI / Kpnl fragment from the plasmid pDH51 (Pietrzak et al., 1986) and between the EcoRI and Kpnl sites of the polylinker of pUC18 ligated and was called plasmid pUC18-35S.
  • a 192 bp fragment was isolated from the plasmid pAGV40 (Herrera-Estrella et al., 1983) with the aid of the restriction endonucleases Hindlll and Pvull, which comprises the DNA of the Ti plasmid pTiACH5 (Gielen et al, 1984) (nucleotides 11749-11939 ). After adding Sphl linkers to the Pvull site, the fragment was ligated between the SpHI and HindIII sites of pUC18-35S and was designated plasmid pA7.
  • the promoter of the Patatin gene B33 from Solanum tuberosum was inserted as a Dral fragment (nucleotides -1512 - +14) in the vector pUC19 cut with Sst I, the ends of which were obtained with the aid of T4-DNA polymerase had been smoothed, ligated. This resulted in the plasmid pUC19-B33.
  • the B33 promoter was cut out from this plasmid with EcoRI and Smal and ligated into the correspondingly cut vector pBinAR. This gave rise to the plant expression vector pBinB33.
  • the DNA was transferred by direct transformation using the Höfgen & Willmitzer method (1988, Nucleic Acids Res. 16: 9877).
  • the plasmid DNA of transformed agrobacteria was isolated by the method of Birnboim & Doly (1979, Nucleic Acids Res. 7: 1513-1523) and analyzed by gel electrophoresis after a suitable restriction cleavage. 4 Transformation of potatoes
  • the leaves were used to induce shoots on MS medium with 1.6% glucose, 1.4 mg / l zeatin ribose, 20 mg / l naphthylacetic acid, 20 mg / l gibberellic acid, 250 mg / l claforan , 50 mg / l kanamycin, and 0.80.% Bacto agar.
  • the radiocative labeling of DNA fragments was carried out using a DNA random primer labeling kit from Boehringer Mannheim (Germany) according to the manufacturer's instructions.
  • the starch synthase activity was determined by determining the incorporation of 14 C-glucose from ADP [ 14 C-glucose] into a product insoluble in methanol / KCI as described in Denyer & Smith, 1992, Planta 186: 609-617.
  • tissue samples from potato tubers were disrupted in 50 mM Tris-HCl pH 7.6, 2 mM DTT, 2.5 mM EDTA, 10% glycerol and 0.4 mM PMSF.
  • the electrophoresis was carried out in a MiniProtean II chamber (BioRAD).
  • the monomer concentration of the 1.5 mm thick gels was 7.5% (w / v), 25 mM Tris-Glycine pH 8.4 served as gel and running buffer. Equal amounts of protein extract were applied and separated for 2 h at 10 mA per gel.
  • the activity gels were then incubated in 50 mM Tricine-NaOH pH 8.5, 25 mM potassium acetate, 2 mM EDTA, 2 mM DTT, 1 mM ADP-glucose, 0.1% (w / v) amylopectin and 0. 5 M sodium citrate. Glucans formed were stained with Lugol's solution. 9. Starch analysis
  • Starch was isolated from potato plants using standard methods and the amylose to amylopectin ratio according to the method described by Hovenkamp-Hermelink et al. described method (Potato Research 31 (1988) 241-246).
  • glucose units may be phosphorylated at the carbon atoms of positions C2, C3 and C6.
  • 100 mg of starch in 1 ml of 0.7 M HCl were hydrolyzed for 4 hours at 95 C (Nielsen et. Al. (1994) Plant Physiol. 105: 111-117). After neutralization with 0.7 M KOH, 50 ml of the hydrolyzate were subjected to an optical-enzymatic test to determine glucose-6-phosphate.
  • the change in the absorption of the test mixture (100 mM imidazole / HCl; 10 mM MgCl 2 ; 0.4 mM NAD; 2 units of glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides; 30 C) was followed at 334 nm.
  • the total phosphate was determined as described in Arnes, 1996, Methods in Enzymology VIII, 115-118.
  • the grain size was determined using a "Lumosed" photo sedimentometer from Retsch GmbH, Germany. For this, 0.2 g of starch was suspended in approx. 150 ml of water and measured immediately. The program supplied by the manufacturer calculated the average diameter of the starch granules on the assumption of an average density of the starch of 1.5 g / l.
  • the gelatinization or viscosity properties of the starch were recorded with a Viscograph E from Brabender OHG, Germany, or with a Rapid Visco Analyzer, Newport Scientific Pty Ltd, Investment Support Group, Warriewood NSW 2102, Australia.
  • a suspension of 20 g starch in 450 ml water was subjected to the following heating program: heating from 50 C to 96 C at 3 / min., Keeping constant for 30 minutes, cooling to 30 C at 3 / min. and keep it constant for another 30 minutes.
  • the temperature profile provided characteristic gelatinization properties.
  • RVA Rapid Visco Analyzer
  • a suspension of 2 g starch in 25 ml water was subjected to the following heating program: suspend for 60 s at 50 C, heat from 50 C to 95 C at 12 / min., 2.5 minutes keep constant, cool to 50 C at 12 C / min. and keep it constant for another 2 minutes.
  • the RVA temperature profile provided the viscometric parameters of the starches examined for the maximum (max) and final viscosity (fin), the gelatinization temperature (T), the minimum viscosity (min) that occurs after the maximum viscosity and the difference between the minimum and final viscosity (setback, set) (see Table 1 and Fig. 1) .
  • the residue was weighed after centrifugation (10 min at 10000 x g) of the starch swollen at 70 ° C. after the soluble portion had been separated off.
  • the water absorption capacity of the starch was based on the weight of the starch corrected for the soluble mass.
  • a 2384 bp EcoRI fragment containing a cDNA coding for SS I from potato (Abel 1995, loc.cit.) was smoothed and introduced in the vector pBinAR precut with Smal in a "sense" orientation with respect to the 35S promoter.
  • a 4212 bp Asp718 / Xbal fragment containing a cDNA coding for the SS III from potato was in "antisense" orientation with respect to the 35S promoter between the Asp718 and the Xbal interface of the vector pBinAR-Hyg introduced.
  • a 1650 bp HindIII fragment which contains a partial cDNA coding for the BE enzyme from potato was smoothed and introduced in "antisense" orientation with respect to the B33 promoter into the vector pBinB33 precut with Smal.
  • the plasmid obtained was cut open with BamHI.
  • a 1362 bp BamHI fragment containing a partial cDNA coding for the SS HI enzyme from potato was also inserted into the cleavage, also in an “antisense” orientation with respect to the B33 Promotors introduced.
  • a 2384 bp EcoRI fragment containing a cDNA coding for SS I from potato (Abel, 1995, loc.cit.) was smoothed and cloned into the Smal site of the pBinB33 vector in an "antisense" orientation with respect to the B33 promoter .
  • On 1362 bp BamHI fragment containing a partial cDNA coding for the SS in from potato (Abel et al., 1996, loc.cit.) was also inserted into the BamHI site of the resulting vector in an “antisense” orientation with respect to the B33 Promotors introduced.
  • Example 10 B Preparation of the plasmid pB33 ⁇ R1 -Hyg
  • a 1.9 kB fragment of R1 from S. tuberosum (WO 97/11188) was obtained from the vector pBluescript by digestion with Asp718. The fragment was cloned into the Asp718 site behind the B33 promoter in the antisense orientation of the vector pB33-Binar-Hyg. This vector contains hygromycin resistance.
  • Example 11 Introduction of the plasmids into the genome of potato cells
  • the plasmids listed in Examples 1 to 10 were transferred individually and / or successively into agrobacteria, with the aid of which the transformation of potato cells was carried out as described above. Whole plants were then regenerated from the transformed plant cells.
  • Transgenic plant cells of the genotype asSSI-asSSII-asSSIII were generated by transformation with the plasmid p35S ⁇ SSI-Hyg described in Example No. 1 and subsequent retransformation with the plasmid described in Example No. 8 p35S ⁇ SSII- ⁇ SSIII-Kan.
  • Transgenic plant cells of the genotype asSSII-asSSI-asSSIII were generated by transformation with the plasmid p35S ⁇ SSII-Hyg described in Example No. 10 and subsequent retransformation with the plasmid pB33 ⁇ SSI ⁇ SSIII-Kan described in Example No. 9.
  • the transgenic potato plants synthesized modified starch varieties.
  • the starch formed from the transgenic plants produced according to Example 11 differs, for example, from starch synthesized in wild type plants (potato) in its phosphate or amylose content and in the viscosities and gelatinization properties determined by means of RVA.
  • the results of the physico-chemical characterization of the modified starches are shown in Table 1.
  • the enzyme activities of the suppressed soluble starch synthases were reduced by up to 85% compared to the non-transformed control plants.
  • SSI starch synthase isoform I
  • SSII starch synthase isoform II
  • SSIII starch synthase isoform III
  • BE branching enzyme
  • as anti-sense
  • oe overexpressed (sense)
  • cos cosuppressed (sense)
  • the separation of the glucan chains was carried out after separation of the amylose by means of thymol precipitation (Tomlinson et al. Loc. Cit.) By a high-performance anion exchange chromatography system with an amperometric detector (HPEAC-PAD, Dionex).
  • HPEAC-PAD amperometric detector
  • the samples (10 mg / ml amylopectin) were dissolved in 40% DMSO and 1/10 volume part of 100 mM sodium acetate pH 3.5 and 0.4 U isoamylase (Megazyme) were added. After incubation, 10 ⁇ l of the sample were applied to the column system and according to the information by Tomlinson et al. (loc. cit.) eluted.
  • the results of the HPEAC-PAD analysis on the length and distribution of the side chains of starch samples Nos. 1, 7, 8, 13 and 14 are shown in FIG. 2.
  • the glucans were detected using a refractive index detector (Gynkotek), the chain lengths of the eluted linear glucans were determined both by mass spectrometry and by iodometry (Hizukuri (1986) loc.cit.).
  • Fraction 1 represents the proportion of the A and B1 chains (according to Hizukuri (1986) loc.cit.)
  • Fraction 2 represents the proportion of the B2, B3 and B4 chains (according to Hizukuri (1986) loc.cit.)
  • Fraction 3 gives the proportion of high molecular weight glucan molecules eluting in the exclusion volume.

Abstract

L'invention concerne des molécules d'acide nucléique recombinées contenant au moins deux séquences nucléotidiques codant pour des enzymes participant au métabolisme de l'amidon, des procédés pour la préparation de cellules végétales et de végétaux transgéniques synthétisant un amidon qui est modifié quant à sa teneur en phosphates et à sa structure de chaîne latérale. L'invention concerne en outre des vecteurs et des cellules hôtes contenant les molécules d'acide nucléique décrites, les cellules végétales et les végétaux résultant des procédées décrits, l'amidon synthétisé par les cellules végétales et les végétaux décrits, ainsi que des procédés pour la préparation de cet amidon.
EP99936576A 1998-07-31 1999-07-21 Vegetaux synthetisant un amidon modifie, leurs procedes de preparation, leur utilisation, ainsi que l'amidon modifie Ceased EP1100937A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DE19836098 1998-07-31
DE19836098A DE19836098A1 (de) 1998-07-31 1998-07-31 Pflanzen, die eine modifizierte Stärke synthetisieren, Verfahren zur Herstellung der Pflanzen, ihre Verwendung sowie die modifizierte Stärke
PCT/EP1999/005182 WO2000008184A1 (fr) 1998-07-31 1999-07-21 Vegetaux synthetisant un amidon modifie, leurs procedes de preparation, leur utilisation, ainsi que l'amidon modifie

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US6596928B1 (en) 2003-07-22
HUP0102998A3 (en) 2003-07-28
AU772364B2 (en) 2004-04-22
AU5161999A (en) 2000-02-28
US7385104B2 (en) 2008-06-10
US20070074310A1 (en) 2007-03-29
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HUP0102998A2 (hu) 2001-11-28
US20030167529A1 (en) 2003-09-04
CA2338002C (fr) 2013-02-19
US7247769B2 (en) 2007-07-24
BR9912665A (pt) 2001-05-02
PL345829A1 (en) 2002-01-14
JP2002525036A (ja) 2002-08-13

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