EP0885303A1 - Molecules d'acide nucleique codant pour des enzymes debranchantes issues du ma s - Google Patents

Molecules d'acide nucleique codant pour des enzymes debranchantes issues du ma s

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Publication number
EP0885303A1
EP0885303A1 EP97906182A EP97906182A EP0885303A1 EP 0885303 A1 EP0885303 A1 EP 0885303A1 EP 97906182 A EP97906182 A EP 97906182A EP 97906182 A EP97906182 A EP 97906182A EP 0885303 A1 EP0885303 A1 EP 0885303A1
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EP
European Patent Office
Prior art keywords
nucleic acid
starch
acid molecule
plant
ser
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EP97906182A
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German (de)
English (en)
Inventor
Jens Kossmann
Lothar Willmitzer
Michael Emmermann
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Bayer Bioscience GmbH
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Planttec Biotechnologie GmbH Forschung and Entwicklung
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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
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2451—Glucanases acting on alpha-1,6-glucosidic bonds
    • 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
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2451—Glucanases acting on alpha-1,6-glucosidic bonds
    • C12N9/2457—Pullulanase (3.2.1.41)
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01041—Pullulanase (3.2.1.41)

Definitions

  • the present invention relates to nucleic acid molecules which encode proteins from maize with the enzymatic activity of a debranching enzyme (R enzyme). Furthermore, the invention relates to transgenic plants and plant cells in which an amylopectin with an altered degree of branching occurs as a result of the expression of an additional debranching enzyme activity from maize or the inhibition of an endogenous debranching enzyme activity, and which is obtained from said transgenic plant cells and plants ⁇ strength.
  • R enzyme debranching enzyme
  • Starch plays an important role both as a storage material in a large number of plants and as a renewable, industrially usable raw material and is becoming increasingly important.
  • For the industrial use of the starch it is necessary that it corresponds to the requirements of the processing industry in terms of structure, shape and / or other physico-chemical parameters.
  • the polysaccharide starch is made up of chemically uniform basic building blocks, the glucose molecules, but represents a complex mixture of different molecular forms which have differences in the degree of polymerization and the occurrence of branching.
  • amylose starch an essentially unbranched polymer made from ⁇ -1,4-glycosidically linked glucose molecules
  • amylopectin starch a branched polymer in which the branches are formed by the occurrence of additional c ⁇ -1, 6-glycosidic linkages come about.
  • amylopectin In typical plants used for starch production, e.g. Corn or potato, the two forms of starch occur in a ratio of approx. 25 parts amylose to 75 parts amylopectin.
  • amylopectin there is, for example, a further branched polysaccharide, the so-called phytoglycogen, which differs from amylopectin in that it has a higher degree of branching and a different solubility behavior (see, for example, Lee et al., Aren. Biochem. Biophys. 143 (1971), 365-374; Pan and Nelson, Plant Physiol. 74 (1984), 324-328).
  • amylopectin is used to include the phytoglycogen.
  • starch-producing plants are required which, for example, only contain the component amylopectin or only the component amylose. Plants are required for a number of other uses which synthesize forms of amylopectin with different degrees of branching.
  • Such plants can be produced, for example, by breeding or mutagenesis techniques.
  • mutagenesis can produce varieties which only form amylopectin.
  • a genotype was also generated by chemical mutagenesis in a haploid line that does not form amylose (Hovenkamp-Hermelink, Theor. Appl. Genet. 75 (1987), 217-221).
  • WO 92/11376 discloses DNA sequences which code for a branching enzyme (Q enzyme), the ⁇ -1,6 branches introduces in amylopectin starch. With the aid of these DNA sequences, it should be possible to produce transgenic plants in which the amylose / amylopectin ratio of the starch has changed.
  • Q enzyme branching enzyme
  • the pullulanases which in addition to pullulan also use amylopectin as a substrate, come from microorganisms, e.g. Klebsiella, and before plants. In plants, these enzymes are also called R-enzymes.
  • Isoamylases that do not use pullulan, but do use glycogen and amylopectin as substrates, also occur in microorganisms and plants. Isoamylases have been described, for example, in maize (Manners & Rowe, Carbohydr. Res. 9 (1969), 107) and potato (Ishizaki et al., Agric. Biol. Chem. 47 (1983), 771-779).
  • amylo-1, 6-glucosidases have been described in mammals and yeasts and use border dextrins as a substrate.
  • the object of the present invention is therefore to identify further debranching enzymes which may occur in maize or to isolate corresponding nucleic acid molecules which code for these enzymes.
  • the present invention thus relates to nucleic acid molecules which encode proteins with the biological activity of a debranching enzyme from maize, or to a biologically active fragment thereof, such nucleic acid molecules preferably encoding a debranching enzyme from maize which has the functionality described under Seq ID No. 2 indicated amino acid sequence.
  • Such a nucleic acid molecule particularly preferably comprises the one listed under Seq ID no. 1 indicated nucleotide sequence, in particular the coding region, or a corresponding ribonucleotide sequence.
  • the invention also relates to nucleic acid molecules which encode proteins with the biological activity of a debranching enzyme from maize or biologically active fragments thereof and which hybridize with one of the nucleic acid molecules described above.
  • the present invention relates to nucleic acid molecules whose sequences differ from the sequences of the abovementioned nucleic acid molecules due to the degeneration of the genetic code and which encode a protein which has the biological activity of a debranching enzyme from maize.
  • the invention also relates to nucleic acid molecules, the sequence of which is complementary to all or part of the sequence of the above-mentioned nucleic acid molecules.
  • hybridization means hybridization under conventional hybridization conditions, preferably under stringent conditions, as described, for example, in Sambrock et al., Molecular Cloning, A Laboratory Manual, 2nd edition (1989). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).
  • nucleic acid molecules which hybridize with the nucleic acid molecules according to the invention can originate from any corn plant.
  • Nucleic acid molecules that hybridize with the molecules according to the invention can e.g. can be isolated from genomic or from cDNA libraries.
  • nucleic acid molecules from maize plants can be identified and isolated using the nucleic acid molecules according to the invention or parts of these molecules or the reverse complements of these molecules, e.g. by means of hybridization according to standard methods (see, e.g., Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2nd edition Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).
  • nucleic acid molecules can be used as the hybridization sample that exactly or essentially the Seq ID No. 1 indicated nucleotide sequence or parts of this sequence.
  • the fragments used as a hybridization sample can also be synthetic fragments which were produced with the aid of the common synthetic techniques and whose sequence essentially corresponds to that of a nucleic acid molecule according to the invention. If genes which hybridize with the nucleic acid sequences according to the invention have been identified and isolated, a determination of the sequence and an analysis of the properties of the proteins encoded by this sequence are necessary.
  • the molecules hybridizing with the nucleic acid molecules according to the invention also include fragments, derivatives and allelic variants of the DNA molecules described above which encode a debranching enzyme from maize or a biologically, ie enzymatically, active fragment thereof. Fragments are understood to mean parts of the nucleic acid molecules that are long enough to encode a polypeptide with the enzymatic activity of a debranching enzyme from maize.
  • the term derivative in this context means that the sequences of these molecules differ from the sequences of the nucleic acid molecules described above at one or more positions and have a high degree of homology to these sequences. Homology means a sequence identity of at least 40%, in particular an identity of at least 60%, preferably over 80% and particularly preferably over 90%. The deviations from the nucleic acid molecules described above may have resulted from deletion, addition, substitution, insertion or recombination.
  • nucleic acid molecules which are homologous to the molecules described above and represent derivatives of these molecules are generally variations of these molecules which represent modifications which have the same biological function. These can be both naturally occurring variations, for example sequences from other organisms, or mutations, wherein these mutations can have occurred naturally or have been introduced by targeted mutagenesis. Furthermore, 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 proteins encoded by the different variants of the nucleic acid molecules according to the invention have certain common characteristics.
  • These may include, for example, enzyme activity, molecular weight, immunological reactivity, conformity, etc., and physical properties such as, for example, running behavior in gel electrophoresis, chromatographic behavior, sedimentation coefficients, solubility, spectroscopic properties, stability; pH optimum, temperature optimum etc.
  • the enzymatic activity of the debranching enzyme can be detected, for example, by a staining test, as described in WO 95/04826. This is based on the fact that a protein with a starch-modifying activity can be detected if protein extracts, for example from corn kernels, are separated in non-denaturing, amylopectin-containing polyacrylamide gels (PAAG) and the gel, after incubation in a suitable buffer, is subsequently subjected to iodine staining . While unbranched amylos forms a blue complex with iodine, amylopectin gives a reddish-violet color.
  • a staining test as described in WO 95/04826. This is based on the fact that a protein with a starch-modifying activity can be detected if protein extracts, for example from corn kernels, are separated in non-denaturing, amylopectin-containing polyacrylamide gels (PAAG) and the gel, after incubation
  • amylopectin-containing polyacrylamide gels which stain reddish-violet with iodine, there is a color shift in places where debranching activity is localized, which leads to a blue coloration of the gel, since the branches of the violet-coloring amylopectin are broken down by the debranching enzyme become.
  • the debranching enzyme activity can be detected using the DNSS test (see Ludwig et al., Plant Physiol. 74 (1984), 856-861).
  • the nucleic acid molecules according to the invention can be any nucleic acid molecules, in particular DNA or RNA molecules, for example cDNA, genomic DNA, mRNA etc. They can be naturally occurring molecules, or they can be produced by genetic engineering or chemical synthesis methods.
  • the nucleic acid molecules according to the invention encode a hitherto unknown protein from maize with the enzymatic one Activity of a debranching enzyme. So far, only a locus has been described for maize which encodes a protein with debranching enzyme activity (James et al., See above). So far there has been no evidence in the literature that there are other genes in maize that code for debranching enzymes.
  • nucleic acid molecules according to the invention Homology comparisons of the nucleic acid molecules according to the invention with those in James et al. (see above) show that these sequences show no significant homology and would not hybridize with one another.
  • the molecules according to the invention thus encode a new type of corn debranching enzyme. With the help of these molecules it is now possible to intervene in a targeted manner in the starch metabolism of maize and other starch-storing plants and thus to enable the synthesis of a starch modified in its chemical or physical properties.
  • nucleic acid molecules according to the invention in any, preferably starch-storing plants, or by reducing the debranching enzyme activity in maize plants by using the nucleic acid sequences according to the invention, for example by means of antisense, ribozyme or cosuppression effects.
  • the present invention relates to nucleic acid molecules of at least 15 base pairs in length which hybridize specifically with the nucleic acid molecules according to the invention.
  • Hybridizing specifically here means that these molecules hybridize with nucleic acid molecules which encode the new debranching enzymes from maize, but not with nucleic acid molecules which encode other proteins.
  • Hybridizing preferably means hybridizing under stringent conditions (see above).
  • the invention relates to those nucleic acid molecules which hybridize with transcripts of nucleic acid molecules according to the invention and can thereby prevent their translation. These are preferably RNA molecules complementary to the transcripts.
  • the invention relates to vectors, in particular plasmids, cosmids, viruses, bacteriophages and other vectors common in genetic engineering, which contain the nucleic acid molecules according to the invention described above.
  • nucleic acid molecules contained in the vectors are linked to regulatory elements which ensure transcription and translation in prokaryotic or eukaryotic cells.
  • the invention relates to host cells, in particular prokaryotic or eukaryotic cells, which have been transformed with a nucleic acid molecule or a vector described above, and cells which are derived from such host cells and which contain the described nucleic acid molecules or vectors.
  • the host cells can be bacterial or fungal cells, as well as plant or animal cells.
  • the invention also relates to proteins with the biological activity of a debranching enzyme from maize, which are encoded by the nucleic acid molecules according to the invention, or biologically active fragments thereof.
  • the present invention relates to methods for producing a vegetable protein with the biological activity of a debranching enzyme from maize or a biologically active fragment thereof, in which host cells according to the invention are cultivated under suitable conditions and the protein from the culture, i.e. is obtained from the cells and / or the culture medium.
  • nucleic acid molecules By providing the nucleic acid molecules according to the invention, it is now possible to use genetic engineering methods to modify plant cells to have a new or an increased debranching enzyme activity from corn compared to wild-type cells, ie corresponding non-transformed cells.
  • maize cells or plants can be modified such that they have a reduced debranching enzyme activity compared to wild-type cells or plants.
  • the host cells according to the invention are transgenic plant cells which, owing to the presence and expression of an introduced nucleic acid molecule according to the invention, have either a new or an increased debranching enzyme activity compared to non-transformed cells.
  • Such transgenic plant cells differ from non-transformed cells in that the nucleic acid molecule introduced is either heterologous to the transformed cell, i.e. comes from a cell with a different genomic background, or because the nucleic acid molecule introduced, if it is homologous to the transformed plant species, is located in the genome at a location where it does not naturally occur in non-transformed cells.
  • the nucleic acid molecule introduced can either be under the control of its natural promoter or linked to regulatory elements of foreign genes.
  • the invention also relates to transgenic plants which contain the transgenic plant cells described above.
  • the plant which is transformed with the nucleic acid molecules according to the invention and in which a debranching enzyme is synthesized from corn due to the introduction of such a molecule can in principle be any plant. It is preferably a monocotyledon or dicotyledon crop, in particular a starch-storing plant, such as, for example, cereal plants, legumes, potatoes or cassava. Grain plants are understood in particular as monocotyledonous plants belonging to the order Poales, preferably those belonging to the family of the Poaceae. Examples of this are the plants belonging to the genera Avena (oat), Triticum
  • Starch-storing legumes are e.g. some species of the genus Pisum (e.g. Pisum sativum), Vicia (e.g. Vicia faba), Cicer (e.g. Cicer arietinum), Lens
  • Phaseolus e.g. Phaseolus vulgaris and Phaseolus coccineus
  • Phaseolus e.g. Phaseolus vulgaris and Phaseolus coccineus
  • the expression of a new or additional debranching enzyme activity from maize in the transgenic plant cells and plants according to the invention has an influence on the degree of branching of the amylopectin synthesized in the cells and plants.
  • a starch synthesized in these plants therefore has changed physical and / or chemical properties compared to starch from wild-type plants.
  • the invention thus also relates to the starch obtainable from the transgenic plant cells or plants.
  • the invention further relates to propagation material from transgenic plants according to the invention, for example seeds, fruits, cuttings, tubers, rhizomes, etc., this propagation material containing transgenic plant cells described above.
  • the propagation material is preferably the corn kernels.
  • the present invention relates to transgenic plant cells of maize, in which the activity of the debranching enzyme according to the invention is reduced due to the inhibition of transcription or translation of endogenous nucleic acid molecules which code for a debranching enzyme according to the invention.
  • This is preferably achieved in that a nucleic acid molecule according to the invention or a part of it is expressed in the corresponding plant cells in antisense orientation and the described debranching enzyme activity is reduced due to an antisense effect.
  • Another possibility for reducing the debranching enzyme activity in plant cells is the expression of suitable ribozymes which specifically cleave transcripts of the DNA molecules according to the invention.
  • ribozymes with the aid of the DNA molecules according to the invention is familiar to the person skilled in the art. It is also possible to express molecules which have both an antisense and a ribozyme effect in combination. Alternatively, the debranching enzyme activity in the plant cells can also be reduced by a cosupression effect.
  • the method is known to the person skilled in the art and 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.
  • the invention further relates to transgenic maize plants which contain the transgenic plant cells described above with reduced debranching enzyme activity.
  • the amylopectin strength of the transgenic cells and plants has a different degree of branching due to the reduced debranching enzyme activity compared to starch from untransformed plants.
  • the invention therefore also relates to the modified starch obtainable from the transgenic cells or plants.
  • the invention also relates to propagation material of the transgenic plants described above, in particular seeds, wherein this contains transgenic plant cells described above.
  • Transgenic plant cells which, owing to the expression of a new or additional debranching enzyme activity, form an amylopectin strong with a different degree of branching compared to amylopectin strong synthesized in wild-type plants can be produced, for example, by a process which comprises the following steps:
  • nucleic acid sequence which encodes a protein with the enzymatic activity of a debranching enzyme or a biologically active fragment thereof and is coupled in sense orientation to the 3 • end of the promoter;
  • Transgenic maize plant cells which, owing to the reduction in the debranching enzyme activity described, form an amylopectin starch with a different degree of branching compared to amylopectin synthesized in wild-type plants can, for example, be produced by a process which comprises the following steps: (a) Production of an expression cassette which comprises the following DNA sequences:
  • nucleic acid sequence which encodes a protein with the enzymatic activity of a debranching enzyme or a part of such a protein and which is coupled in antisense orientation to the 3 'end of the promoter;
  • any promoter functional in the plants selected for the transformation can be used for the promoter mentioned under (i).
  • the promoter can be homologous or heterologous with respect to the plant species used.
  • the 35S promoter of the cauliflower mosaic virus (Odell et al., Nature 313 (1985), 810-812) is suitable, which ensures constitutive expression in all tissues of a plant and that described in WO / 9401571 Promoter construct.
  • Another example are the promoters of the polyubiquitin genes from maize (Christensen et al., Plant Mol. Biol. 18 (1992) 675-689.
  • promoters can also be used which are only determined at a time determined by external influences (see for example WO / 9307279) Promoters of heat-shock proteins which allow simple induction can be of particular interest, and promoters which lead to expression of downstream sequences in a particular tissue of the plant can also be used (see, for example, B. Stockhaus et al., 1989, EMBO J. 8: 2245-2251. ren used, which are active in the starch-storing organs of the plants to be transformed. These are the corn kernels in maize, and the tubers in potatoes. For example, the tuber-specific B33 promoter (Rocha-Sosa et al., EMBO J. 8 (1989), 23-29) can be used to overexpress the nucleic acid molecules according to the invention in the potato.
  • Seed-specific promoters have already been described for various plant species. So e.g. the USP promoter from Vicia faba, which ensures seed-specific expression in V. faba and other plants (Fiedler et al., Plant Mol. Biol. 22 (1993), 669-679; Bäumlein et al., Mol Gen. Genet. 225: 459-467 (1991)).
  • promoters of the zein genes ensure a specific expression in the endosperm of the maize kernels (Pedersen et al., Cell 29 (1982), 1015-1026; Quattrocchio et al., Plant Mol. Biol. 15 (1990) , 81-93).
  • nucleic acid sequence mentioned under process step (a) (ii), which encodes a protein with the enzymatic activity of a debranching enzyme from maize is linked to the promoter in se ⁇ se orientation
  • this nucleic acid sequence can be both native or homologous origin as well as foreign or heterologous origin with respect to the plant species to be transformed, ie Both maize plants and any other plants can be transformed with the expression cassette described, preferably the above-mentioned starch-storing plants.
  • the synthesized protein can be localized in any compartment of the plant cell.
  • Vegetable debranching enzymes are usually localized in the plastids and therefore have a signal sequence for translocation into these organelles.
  • the DNA sequence encoding this signal sequence must be removed and the coding region linked to DNA sequences that encode the lo Ensure calibration in the respective compartment.
  • Such sequences are known (see, for example, Braun et al., EMBO J. 11 (1992), 3219-3227; Wolter et al., Proc. Natl. Acad. Sci. USA 85 (1988), 846-850; Sonnewald et al ., Plant J. 1 (1991), 95-106).
  • nucleic acid sequence mentioned under process step (a) (ii), which encodes a protein from maize with the enzymatic activity of a debranching enzyme is linked in an antiseptic orientation to the promoter
  • this is preferably of a nucleic acid sequence of homologous origin with respect to the plant species to be transformed.
  • nucleic acid sequences can also be used which have a high degree of homology to endogenously present debranching enzyme genes, in particular homologies higher than 80%, preferably homologies between 90% and 100% and particularly preferably homologies over 95% .
  • Sequences up to a minimum length of 15 bp can be used. An inhibitory effect is not excluded even when using shorter sequences. Longer sequences between 100 and 500 base pairs are preferably used, and sequences with a length of over 500 base pairs are used in particular for efficient antisense inhibition. As a rule, sequences are used which are shorter than 5000 base pairs, preferably sequences which are shorter than 2500 base pairs.
  • Termination signals for transcription in plant cells are described and can be interchanged with one another as desired.
  • the termination sequence of the octopine synthase gene from Agrobacterium turne faciens can be used.
  • the transfer of the expression cassette constructed according to process step (a) into plant cells is preferably carried out using plasmids, in particular with the aid of plasmids, which ensure stable integration of the expression cassette into the plant genome.
  • the method described above for overexpressing a new corn debranching enzyme can in principle be applied to all plant species. Both monocot and dicot plants are of interest, in particular the starch-storing plants described above.
  • the process described above for reducing the debranching enzyme activity is preferably used on monocotyledonous plants, in particular on maize.
  • an RNA is formed in the transformed plant cells. If the nucleic acid sequence coding for a maize debranching enzyme in the expression cassette is linked to the promoter in the se ⁇ se orientation, an mRNA is synthesized which acts as a template for the synthesis of an additional or new maize debranching enzyme in the plant cells can serve. As a result, these cells have an activity or an increased activity of the corn debranching enzyme, which leads to a change in the degree of branching of the amylopectin formed in the cells. As a result, a strength becomes accessible which is distinguished from the naturally occurring strength by a more orderly spatial structure and an increased uniformity. Among other things, this can have favorable effects on the film-forming properties.
  • Maize is particularly suitable for the production of modified amylopectin using the nucleic acid molecules according to the invention which encode debranching enzymes.
  • the application of the invention is not restricted to this plant species. Any other plant species can be used for the overexpression.
  • the modified starch synthesized in the transgenic plants can be isolated from the plants or from the plant cells using conventional methods and, after purification, can be used for the production of foods and industrial products.
  • starches according to the invention can be modified by processes known to the person skilled in the art and are suitable in unmodified or modified form for various uses in the food or non-food sector.
  • starch can be divided into two large areas.
  • One area comprises the hydrolysis products of starch, mainly glucose and glucon units, which are obtained by enzymatic or chemical processes. They serve as the starting material for further chemical modifications and processes, such as fermentation.
  • hydrolysis products of starch mainly glucose and glucon units
  • They serve as the starting material for further chemical modifications and processes, such as fermentation.
  • amyloglucosidase amyloglucosidase.
  • 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 cause this.
  • Starch is a classic additive for many foodstuffs, in which it essentially takes on the function of binding aqueous additives or causes an increase in viscosity or increased 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 for film formation, freeze / thaw stability, digestibility and the ability to form complexes with eg inorganic or organic ions.
  • the starch can be used as an auxiliary for different manufacturing processes or as an additive in technical products.
  • the starch is used primarily for retardation (retention of solids), the setting of filler and fine particles, as a strengthening agent and for drainage.
  • the cheap Properties of the starch in terms of rigidity, hardness, sound, grip, gloss, smoothness, splitting resistance and surfaces.
  • the requirements for the starch in relation to the surface treatment are essentially a high degree of whiteness, an adapted viscosity, high viscosity stability, good film formation and low dust formation.
  • the solids content, an adapted viscosity, a high binding capacity and high pigment affinity play an important role.
  • a rapid, uniform, loss-free distribution, high mechanical stability and complete restraint in the paper flow are important.
  • an adapted solids content, high viscosity and high binding capacity are also important.
  • starches A large area of use of the 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 the use of starches as extenders for synthetic adhesives.
  • 90% of the starch-based adhesives are used in the fields of corrugated board production, production of paper bags, bags and pouches, production of composite materials for paper and aluminum, production of cardboard and rewetting glue for envelopes, stamps etc. used.
  • starch as a sizing agent, i.e. as an auxiliary for smoothing and strengthening the Velcro behavior to protect against the tensile forces acting during weaving as well as to increase the abrasion resistance during weaving, starch as an agent for textile upgrading, especially after quality-reducing pretreatments such as bleaching, dyeing etc., starch as a thickening agent during production of color pastes to prevent dye diffusion and starch as an additive to chain 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 plasterboard, in which the starch mixed in the gypsum paste pastes with the water, diffuses to the surface of the plasterboard and binds the cardboard to the plate there.
  • Other areas of application are admixing to plaster and mineral fibers.
  • starch products are used to delay setting.
  • starch Another market for starch is in the manufacture of soil stabilizers that are used to temporarily protect soil particles from water during artificial earthmoving. Combinations of starch and polymer emulsions are, according to today's knowledge, in their erosion and The crust-reducing effect of equating the previously used products is, however, significantly lower in price.
  • starch in crop protection agents to change the specific properties of the preparations.
  • the starch can be used to improve the wetting of crop protection agents and fertilizers, for the metered release of the active substances, for converting liquid, volatile and / or malodorous substances into microcrystalline, stable, moldable substances, for mixing incompatible compounds and for Extension of the duration of action by reducing the decomposition can be used.
  • starch can be used as a binder for tablets or for binder dilution in capsules.
  • the starch can furthermore serve as a tablet disintegrant, since after swallowing it absorbs liquid and swells to such an extent after a short time that the active substance is released.
  • Medical sliding and wound powders are based on starch for qualitative reasons.
  • starches are used, for example, as carriers for powder additives such as fragrances and salicylic acid.
  • a relatively large area of application for the starch is toothpaste.
  • Starch is used as an additive to coal and briquette. Coal can be agglomerated or briquetted with a high-quality additive, which allows the briquettes to disintegrate at an early stage is prevented.
  • 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 emission of harmful substances.
  • the starch can also be used as a flocculant in ore and coal sludge processing.
  • Another area of application is as an additive to foundry additives.
  • Various casting processes require cores that are made from binder-mixed sands.
  • Bentonite which is mixed with modified starches, mostly swelling starches, is predominantly used today as a binder.
  • the purpose of the starch addition is to increase the flow resistance and to improve the binding strength.
  • the swelling starches can have other production requirements, such as dispersibility in cold water, rehydration, good miscibility in sand and high water-binding capacity.
  • the starch can be used in the rubber industry to improve the technical and optical quality.
  • the reasons for this are the improvement of the surface gloss, the improvement of the grip 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.
  • a further sales opportunity for the modified starches is in the production of leather substitutes.
  • starch secondary products in the processing process (starch is only filler, there is no direct bond between synthetic polymer and starch) or alternatively the integration of starch secondary products in the production of polymers (starch and polymer form a firm bond).
  • starch as a pure filler is not competitive compared to other substances such as talc. The situation is different when the specific starch properties come into play and the property profile of the end products is thereby significantly changed.
  • An example of this is the use of starch products in the processing of 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 produced using granulated polyethylene using conventional process techniques.
  • 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 hydroxy groups of the starches in a targeted manner.
  • the result is polyurethane films which, through the use of starch, obtain the following property profiles: a reduction in the Coefficients of thermal expansion, reduction in shrinkage behavior, improvement in pressure / stress behavior, increase in water vapor permeability without changing water absorption, reduction in flammability and tear density, no dripping of flammable parts, freedom from halogen 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 can also be manufactured with a starch content of over 50%.
  • starch / polymer mixtures can be assessed favorably, since they have a much higher biodegradability.
  • starch graft polymers Because of their extreme water-binding capacity, starch graft polymers have also become extremely important. These are products with a backbone made of starch and a side lattice grafted on according to the principle of the radical chain mechanism of a synthetic monomer.
  • 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 superabsorbers have expanded considerably in recent years and are in the hygiene area with products of diapers and underlays as well as in the agricultural sector, e.g. seed pilling.
  • Ash / phosphate content, amylose / amylopectin ratio, molar mass distribution, degree of branching, grain size and shape as well as crystallinity on the other hand also the properties that result in the following characteristics: flow and sorption behavior, gelatinization temperature, viscosity, thickening performance, solubility, paste structure and transparency , Heat, shear and acid stability, tendency to retrogradation, gel fertilizer, freeze / thaw stability, complex formation, iodine binding, film formation, adhesive strength, enzyme stability, digestibility and reactivity.
  • modified starches by means of genetic engineering interventions in a transgenic plant can on the one hand change the properties of the starch obtained from the plant in such a way that further modifications by means of chemical or physical processes no longer appear to be necessary.
  • the starches modified by genetic engineering processes can be subjected to further chemical modifications, which leads to further improvements in quality for certain of the fields of application described above.
  • nucleic acid molecules according to the invention can in principle also be used to produce plants. len in which the activity of the debranching enzyme according to the invention is increased or decreased and at the same time the activities of other enzymes involved in starch biosynthesis are changed. All combinations and permutations are conceivable.
  • nucleic acid molecules which encode a protein according to the invention or corresponding antisense constructs can be introduced into plant cells in which the synthesis of endogenous GBSS I, SSS I, II or GBSS II proteins or the su gene is already carried out is inhibited due to an antisense effect or a mutation, or the synthesis of the branching enzyme is inhibited (as described, for example, in WO92 / 14827 or the ae mutant (Shannon and Garwood, in Whistler, BeMiller and Paschall, Starch: Chemistry and Technology, Academic Press, London, 2nd Edition (1984), 25-86)).
  • DNA molecules can be used for the transformation which simultaneously contain several regions coding for the corresponding debranching enzymes in antisense orientation under the control of a suitable promoter, or which encode a corresponding cosuppression RNA or a corresponding ribozyme.
  • each sequence can be under the control of its own promoter, or the sequences can be transcribed as a fusion from a common promoter. The latter alternative will generally be preferable since in this case the synthesis of the corresponding proteins should be inhibited to approximately the same extent.
  • DNA molecules which, in addition to DNA sequences which code for debranching enzymes, contain further DNA sequences which code for other proteins involved in starch synthesis or modification. These can encode an antisense RNA, a corresponding ribozyme or a cosuppression RNA.
  • the sequences can in turn either be connected in series and transcribed by a common promoter or but each with its own promoter. There is no upper limit to the number of antisense fragments transcribed from a promoter in such a DNA molecule. However, the resulting transcript should generally have a length of no more than 20 kb, preferably no more than 5 kb.
  • Coding regions which are located in such DNA molecules in combination with other coding regions behind a suitable promoter can originate from DNA sequences which code for the following proteins: starch-bound (GBSS I and II) and soluble starch synthases (eg SSS I and II), branching enzymes, disproportionation enzymes and starch phosphorylases. This is only an example. The use of other DNA sequences in the context of such a combination is also conceivable. With the help of such constructs it is possible to inhibit the synthesis of several enzymes simultaneously in plant cells which have been transformed with them.
  • the constructs can be introduced into classic mutants which are defective for one or more genes in starch biosynthesis (Shannon and Garwood, see above). These defects can e.g. relate to the following proteins: starch grain-bound (GBSS I and II) and soluble starch synthases (eg SSS I and II), branching enzymes (BE I and II), "debranching” enzymes (see locus), disproportionation fermentation enzymes and starch phosphorylases. Again, this is only an exemplary list.
  • cloning vectors which contain a replication signal for E. coli and a marker gene for the selection of transformed bacterial cells.
  • examples of such vectors are pBR322, pUC series, M13mp series, pACYC184 etc.
  • the desired sequence 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. coli 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.
  • 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, the injection, the electroporation of DNA, the introduction of DNA using the biolistic method and other possibilities.
  • plasmids When injecting and electroporation of DNA into plant cells, no special requirements are made of the plasmids used. Simple plasmids such as e.g. pUC derivatives can be used. However, if whole plants are to be regenerated from such transformed cells, the presence of a selectable marker gene is necessary.
  • the Ti or Ri plasmid is used for the transformation of the plant cell, at least the right boundary, but often 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 owing to sequences homologous to sequences in the T-DNA are integrated into the Ti or Ri plasmid of the agrobacteria by homologous recombination. This also contains the vir region necessary for the transfer of the T-DNA.
  • Intermediate vectors cannot replicate in agrobacteria.
  • the intermediate vector can be transferred to Agrobacterium tumefaciens by means of a helper plasmid (conjugation).
  • Binary vectors can replicate in both E. coli and agrobacteria.
  • the agrobacterium serving as the host cell is said to contain a plasmid which carries a vir region. The vir region is necessary for the transfer of the T-DNA into the plant cell. Additional T-DNA can 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 sufficient in EP 120 516; Hoekema, In: The Binary Plant Vector System Offset- drukkerij Kanters B.V., Alblasserdam (1985), Chapter V; Fraley et al. , Crit. Rev. Plant. Sci., 4, 1-46 and An et al. EMBO J. 4 (1985), 277-287.
  • plant explants can expediently be cultivated with Agrobacterium tumefaciens or Agrobacterium rhizogenes.
  • Whole plants can then be regenerated from the infected plant material (for example leaf pieces, stem segments, roots, but also protoplasts or suspension-cultivated plant cells) in a suitable medium, which can contain antibiotics or biocides for the selection of transformed cells.
  • the plants obtained in this way 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. for example Willmitzer, L., 1993 Transgenic plants. In: Biotechnology, A Multi-Volume Comprehensive Treatise (HJ 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.
  • 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 normally contains a selection marker which shows the transformed plant cells resistance to a biocide or an antibiotic such as kanamycin, G 418, bleomycin, hygromycin or phosphinotricin and others. mediated.
  • 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., Plant Cell Reports 5 (1986), 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 properties. Seeds can be obtained from the plant cells. Two or more generations should be grown to ensure that the phenotypic trait is stably maintained and inherited. Seeds should also be harvested to ensure that the appropriate phenotype or other characteristics have been preserved.
  • the invention further relates to the use of the nucleic acid molecules according to the invention for the production of plants which synthesize an amylopectin strong with a different degree of branching compared to wild-type plants.
  • Another object of the present invention is the use of the nucleic acid molecules according to the invention or parts of these molecules or the reverse complements of these molecules for the identification and isolation of homologous molecules which encode proteins with the enzymatic activity of a de-branching enzyme or fragments of such products teine, from plants or other organisms.
  • homologous molecules which encode proteins with the enzymatic activity of a de-branching enzyme or fragments of such products teine, from plants or other organisms.
  • Protoplast isolation medium (100 ml)
  • Protoplast washing solution 1 like protoplast insulating solution, but without cellulase, pectolyase and BSA Transformation buffer
  • PEG 6000 is added to the above buffer under b) shortly before use of the solution (40% by weight PEG).
  • the solution is filtered through a 0.45 ⁇ m sterile filter.
  • Protoplast culture medium (in mg / 1)
  • the vector pBluescript II SK (Stratagene) was used for cloning in E. coli. 2nd Bacterial strains
  • the E.coli strain DH5 ⁇ (Bethesda Research Laborato ⁇ ries, Gaithersburgh, USA) was used for the Bluescript vector and for the pUSP constructs.
  • the E.coli strain XLl-Blue was used for in vivo excision.
  • the liquid medium is aspirated and the remaining cells are rinsed with 50 ml protoplast washing solution 1 and sucked dry again.
  • 10 ml of protoplast isolation medium are added to 2 g of the harvested cell mass.
  • the resuspended cells and cell aggregates are incubated at 27 ⁇ 2 ° C with gentle shaking (30 to 40 rpm) for 4 to 6 h in the dark.
  • the suspension is sieved through a stainless steel and nylon sieve of 200 or 45 ⁇ m mesh sizes.
  • the combination of a 100 ⁇ m and a 60 ⁇ m sieve enables the cell aggregates to be separated just as well.
  • the protoplast-containing filtrate is assessed microscopically. It usually contains 98-99% protoplasts. The rest are undigested single cells. Protoplast preparations with this degree of purity are used for transformation experiments without additional gradient centrifugation. By centric The protoplasts are sedimented at 100 rpm in the swinging rotor (100 ⁇ g, 3 min). The supernatant is discarded and the protoplasts are resuspended in washing solution 1. The centrifugation is repeated and the protoplasts are then resuspended in the transformation buffer.
  • the protoplasts resuspended in transformation buffer are filled into 50 ml polyahomer tubes with a titer of 0.5-1 x 10 6 protoplasts / ml in 10 ml portions.
  • the DNA used for the transformation is dissolved in Tris-EDTA (TE) buffer. 20 ⁇ g of plasmid DNA are added per ml of protoplast suspension. A plasmid imparting resistance to phosphinetricin is used as the vector (cf., for example, EP 0 513 849).
  • TE Tris-EDTA
  • a plasmid imparting resistance to phosphinetricin is used as the vector (cf., for example, EP 0 513 849).
  • the protoplast suspension is gently shaken to distribute the DNA homogeneously in the solution. Immediately afterwards, 5 ml of PEG solution is added dropwise.
  • the PEG solution is distributed homogeneously. Then another 5 ml of PEG solution are added and the homogeneous mixing is repeated. The protoplasts remain at ⁇ 2 ° C. for 20 min of the PEG solution. The protoplasts are then sedimented by centrifugation for 3 minutes (100 g; 1000 rpm). The supernatant is discarded. The protoplasts are washed by shaking gently in 20 ml of W5 solution and then centrifuged again. Then they are resuspended in 20 ml protoplast culture medium, centrifuged again and resuspended in culture medium.
  • the titer is set to 6 - 8 x 10 5 protoplasts / ml and the protoplasts in 3 ml portions in petri dishes ( ⁇ 60 mm, height 15 mm). activated. The petri dishes sealed with Parafilm are placed in the dark at 25 ⁇ 2 ° C.
  • the protoplasts are cultivated without the addition of fresh medium. As soon as the cells regenerated from the protoplasts have developed into cell aggregates with more than 20-50 cells, 1 ml of fresh protoplast culture medium is added which contains sucrose as an osmoticum (90 g / 1).
  • the cell aggregates formed from protoplasts can be plated on agar media with 100 mg / 1 L-phosphinothricin.
  • N6 medium with the vitamins of the protoplast culture medium, 90 g / 1 sucrose and 1.0 mg / 1 2.4D is just as suitable as an analog medium, for example with the macro and micronutrient salts of the MS medium (Murashige and Skoog (1962), see above).
  • the calli resulting from stably transformed protoplasts can continue to grow unhindered on the selective medium.
  • the transgenic calli can be transferred to fresh selection medium which also contains 100 mg / 1 L-phosphinothricin but which no longer contains auxin.
  • the transgenic corn calli that have integrated the L-phosphinothricin acetyltransferase gene into their genome differentiate first plants on this medium in the presence of L-phosphinothricin.
  • the embryogenic transformed maize tissue is on hormone-free N6-medium (Chu CC. Et al., Sci. Sin. 16 (1975), 659) in the presence of 5xl0 ⁇ 4 M L-Phosphi ⁇ nothricin.
  • Corn embryos that express the Phsphinothricin acetyl transferase gene (PAT gene) sufficiently strongly develop into plants on this medium. Untransformed embryos or those with only very weak PAT activity die. As soon as the leaves of the in vitro plants have reached a length of 4-6 mm, they can be transferred into soil.
  • the plants After washing off agar residues at the roots, the plants are planted in a mixture of clay, sand, vermiculite and uniform earth in a ratio of 3: 1: 1: 1 and more relatively during the first 3 days after transplanting Humidity adapted to earth culture.
  • the cultivation takes place in a climatic chamber with a light period of 14 h approx. 25000 lux at plant height at a day / night temperature of 23 ⁇ 1/17 ⁇ 1 ° C.
  • the adapted plants are cultivated at a humidity of 65 ⁇ 5%.
  • a cDNA library based on polyA + RNA from maize leaves was created in the vector Lambda ZAPII (Stratagene) and packaged in phage heads. E. coli cells of the XLl-Blue strain were then infected with the phages containing the cDNA fragments (1 ⁇ 10 6 pfu) and plated out on medium in petri dishes at a density of approximately 30,000 per 75 cm 2 . After about 8 hours of incubation, nitrocellulose membranes were placed on the lysed bacterial lawn, which were removed after one minute.
  • the filters were placed in 0.2 M NaOH for 2 min; 1.5 M NaCl, then incubated in 0.4 M Tris / HCl pH 7.5 for 2 min and then in 2 x SSC for 2 min. After the DNA had been dried and fixed by UV crosslinking, the filters were incubated in hybridization buffer at 42 ° C. for 3 hours before radioactively labeled sample was added.
  • a potato cDNA encoding a potato debranching enzyme was used as a sample (see Seq ID No. 3). This had previously been isolated with the aid of degenerate oligonucleotides which had been derived from the partial amino acid sequence of a debranching enzyme from potato.
  • Hybridizing phage clones were isolated and further purified using standard procedures. With the aid of the in vivo excision method, E. coli clones were obtained from positive phage clones which contain a double-stranded pBluescript plasmid with the respective cDNA insert. After checking the size and the restriction pattern of the insert, suitable clones were used for plasmid DNA isolated. Such a plasmid isolated, pREM-53, had an insertion of 1195 bp.
  • the nucleotide sequence of the cDNA insertion was determined by standard methods using the dideoxy method (Sanger et al., Proc. Natl. Acad. Sci. USA 74 (1977), 5463-5467). The insertion is 1995 bp long and the nucleotide sequence and the derived amino acid sequence are shown in Seq ID No. 1 specified.
  • the Seq ID No. 1 indicated nucleotide sequence represents a partial cDNA encoding a previously unknown debranching enzyme from maize. With the aid of this sequence it is possible, using conventional methods, to isolate a complete cDNA sequence or a genomic sequence from suitable cDNA or genomic libraries.
  • ORGANISM Zea mays
  • F TISSUE TYPE: leaf tissue
  • GGC ACG AGG TCA AAA CTC CCT CCA GGG TCA GAT TTG CAA CAA GCT GCA 48 Gly Thr Arg Ser Lys Leu Pro Pro Gly Ser Asp Leu Gin Gin Ala Ala 1 5 10 15
  • AAATGATGTT ATAGAGGTAC AAAAGCATTG GAACATTTCT TTATAGAGGT GAACCACCCT 1915
  • MOLECULE TYPE cDNA TO mRNA
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • GAG AAA CTC AAC TCT TTT CCA CCA GAT TCT GAG GAG CAG CAG GCT CTT 384 Glu Lys Leu Asn Ser Phe Pro Pro Asp Ser Glu Glu Gin Gin Ala Leu 675 680 685
  • ATC ACA GCC ATC CAA GAT GAA GAT GGC TAT AAT TGG GGG TAT AAT CCT 432 Ile Thr Ala Ile Gin Asp Glu Asp Gly Tyr Asn Trp Gly Tyr Asn Pro 690 695 700 GTT CTC TGG GGA GTT CCA AAG GGA AGC TAT GCT GGT AAT GCA AAT GGT 480 Val Leu Trp Gly Val Pro Lys Gly Ser Tyr Ala Gly Asn Ala Asn Gly 705 710 715

Abstract

L'invention concerne des molécules d'acide nucléique codant pour des enzymes débranchantes issues du maïs. Elle concerne également des cellules végétales et des végétaux transgéniques dans lesquels une amylopectine avec des caractéristiques modifiées est synthétisée suite à l'expression d'une enzyme débranchante issue du maïs ou à l'inhibition d'une activité d'enzyme débranchante endogène de ce type.
EP97906182A 1996-03-07 1997-03-06 Molecules d'acide nucleique codant pour des enzymes debranchantes issues du ma s Withdrawn EP0885303A1 (fr)

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DE19608918A DE19608918A1 (de) 1996-03-07 1996-03-07 Nucleinsäuremoleküle, die neue Debranching-Enzyme aus Mais codieren
DE19608918 1996-03-07
PCT/EP1997/001141 WO1997032985A1 (fr) 1996-03-07 1997-03-06 Molecules d'acide nucleique codant pour des enzymes debranchantes issues du maïs

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CA2248535A1 (fr) 1997-09-12
AU718730B2 (en) 2000-04-20
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AU2096097A (en) 1997-09-22
JP2001501450A (ja) 2001-02-06
KR19990087684A (ko) 1999-12-27
US6762346B2 (en) 2004-07-13
DE19608918A1 (de) 1997-09-11
US6255561B1 (en) 2001-07-03
US20020162138A1 (en) 2002-10-31

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