EP0900277A1 - Molecules d'acide nucleique codant l'enzyme de deramification de pomme de terre - Google Patents

Molecules d'acide nucleique codant l'enzyme de deramification de pomme de terre

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Publication number
EP0900277A1
EP0900277A1 EP97922969A EP97922969A EP0900277A1 EP 0900277 A1 EP0900277 A1 EP 0900277A1 EP 97922969 A EP97922969 A EP 97922969A EP 97922969 A EP97922969 A EP 97922969A EP 0900277 A1 EP0900277 A1 EP 0900277A1
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EP
European Patent Office
Prior art keywords
nucleic acid
starch
plant
acid molecule
gly
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EP97922969A
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German (de)
English (en)
Inventor
Michael Emmermann
Jens Kossmann
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Bayer Bioscience GmbH
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Planttec Biotechnologie GmbH Forschung and Entwicklung
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Publication of EP0900277A1 publication Critical patent/EP0900277A1/fr
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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
    • 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
    • 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
    • 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 that encode potato proteins with the enzymatic activity of a debranching enzyme. Furthermore, the invention relates to transgenic plants and plant cells in which the expression of an additional debranching enzyme activity from potato or the inhibition of an endogenous debranching enzyme activity leads to the synthesis of an amylopectin with an altered degree of branching, and which is obtained from said transgenic plant cells and plants ⁇ strength.
  • 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 is a complex mixture of different molecular forms which have differences in the degree of polymerization and the occurrence of branches.
  • 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 ⁇ -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., Arch. 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).
  • the present invention is therefore based on the object of identifying further debranching enzymes which may occur in potatoes or of isolating 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 potato.
  • Such a nucleic acid molecule preferably encodes a protein with the biological activity of a debranching enzyme from potato, which corresponds to the under Seq ID No. 2 indicated amino acid sequence.
  • Such a nucleic acid molecule particularly preferably comprises those listed under Seq ID No. 1 indicated nucleotide sequence, in particular the coding region.
  • the invention also relates to nucleic acid molecules which encode proteins with the biological activity of a debranching enzyme from potato and which with a hybridize the above-described nucleic acid molecules or their complementary strand.
  • the present invention relates to nucleic acid molecules, the sequences of which 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 potato.
  • from potato means that the debranching enzymes encoded by the nucleic acid molecules according to the invention are typical of the species Soianum tuberosum, i.e. either occur naturally in such plants, for example encoded by genomic or RNA molecules, or molecules derived from them. Derived molecules can be generated, for example, by reverse transcription of RNA molecules, amplification, mutation, deletion, substitution, insertion, etc. That the term also encompasses enzymes encoded by alleles or derivatives of sequences found naturally in potato. These can be generated, for example, by genetic engineering methods in vivo or in vitro.
  • 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 ed. (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 potato 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 or parts of these molecules or the reverse complement of these molecules are carried out, for example by means of hybridization according to standard methods (see, for example, Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY) or by amplification using PCR.
  • 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 include in particular fragments, derivatives and allelic variants of the DNA molecules described above, which encode a protein with the enzymatic activity of a potato debranching enzyme 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.
  • the term derivative 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 70%, in particular an identity of at least 80%, preferably over 90% and particularly preferably over 95%.
  • the deviations from the nucleic acid molecules described above can be determined by deletion, ad- dition, substitution, insertion or recombination have arisen.
  • 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 potato plants or varieties, 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.
  • 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 when protein extracts, for example from potato tubers, are separated in non-denaturing, amylopectin-containing polyacrylamide gels (PAAG) and the gel, after incubation in a suitable buffer, is finally undergoes iodine staining. While unbranched amylose forms a blue complex with iodine, amylopectin gives a reddish-violet color.
  • a staining test as described in WO 95/04826.
  • amylopectin-containing polyacrylamide gels which stain reddish-violet with iodine, there is a shift in color to a blue coloration of the gel at locations where debranching activity is localized, since the branches of the violet-staining amylopectin from this Debranching enzyme are degraded.
  • 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 act as DNA or RNA molecules, for example cDNA, genomic DNA, mRNA etc. They can be naturally occurring molecules or produced by genetic engineering or chemical synthesis methods.
  • the nucleic acid molecules according to the invention encode a previously unknown new protein from potato with the enzymatic activity of a debranching enzyme. So far, only a debranching enzyme has been described for potatoes. So far there has been no evidence in the literature that there are genes in potatoes that encode further debranching enzymes. It has now surprisingly been found that in addition to the previously known debranching enzyme in potatoes there is at least one further enzyme with debranching activity. Thus, the molecules of the invention encode a new type of potato debranching enzyme. With the help of these molecules, it is now possible to specifically intervene in the starch metabolism of potato 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 can be overexpression of the nucleic acid molecules according to the invention in any, preferably starch-storing plants, or by Reduction of the debranching enzyme activity in potato plants by using the nucleic acid sequences according to the invention, for example by means of antisense or ribozyme effects.
  • the present invention relates to nucleic acid molecules of at least 15, preferably more than 50 and particularly preferably more than 200 base pairs in length, which specifically hybridize with the nucleic acid molecules according to the invention.
  • Hybridizing specifically means that these molecules hybridize with nucleic acid molecules which encode the new potato debranching enzymes, but not with nucleic acid molecules which encode other proteins.
  • Hybridization here preferably means hybridization 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.
  • Such nucleic acid molecules that hybridize specifically with the nucleic acid molecules according to the invention can, for example, be components of mRNA constructs or ribozymes or can be used as primers for amplification by means of PCR.
  • 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 originate 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 potato, 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 protein with the biological activity of a debranching enzyme from potato 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.
  • 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.
  • 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 increased debranching enzyme activity compared to wild-type cells.
  • Such transgenic plant cells differ from non-transformed cells in that the nucleic acid molecule introduced is either heterologous to the transformed cell, ie originates from a cell with a different genomic background, or in that the nucleic acid molecule introduced if it is homologous to the transformed plant species is located in the genome at a location where it is natural in non-transformed cells does not occur.
  • 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 further 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 potato 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 e.g. 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 include the plants belonging to the genera Avena (oat), Triticum (wheat), Seeale (rye), Hordeum (barley), Oryza (rice), Panicum, Pennisetum, Setaria, Sorghum (millet), Zea (corn) etc. belong.
  • 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 (e.g. Lens culinaris), Phaseolus (e.g. Phaseolus vulgaris and Phaseolus coccineus), etc.
  • the present invention also relates to the starch obtainable from the transgenic plant cells or plants.
  • the expression of a new or additional debranching enzyme activity from potato 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 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. In the case of potato plants, the propagation material is preferably the bulbs.
  • the present invention relates to transgenic plant cells of potato in which the activity of the debranching enzyme according to the invention is reduced due to the inhibition of the transcription or translation of endogenous nucleic acid molecules which code for such a new debranching enzyme.
  • This is preferably achieved in that a nucleic acid molecule according to the invention or a part thereof is expressed in the corresponding plant cells in an antisense orientation and the described debranching enzyme activity is reduced due to an antisense effect.
  • a further possibility for reducing the debranching enzyme activity in plant cells consists in the expression of suitable ribozymes which specifically split transcripts of the DNA molecules according to the invention. The production of such ribozymes with the aid of the DNA molecules according to the invention is possible familiar to the expert.
  • the debranching enzyme activity in the plant cells can also be reduced by a co-suppression effect.
  • the method for reducing the activity of enzymes according to the invention in the plant cells by means of a cosuppression effect 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.
  • ribozymes for reducing the activity of certain enzymes in cells is also known to the person skilled in the art and is described, for example, in EP-Bl 0 321 201.
  • the expression of ribozymes in plant cells has been described in e.g. described in Feyter et al. (Mol. Gen. Genet. 250 (1996), 329-338).
  • genomic sequences which code for such enzymes, e.g. by "gene tagging” or transposon mutagenesis or the expression of antibodies which specifically recognize the new debranching enzymes.
  • the mutagenesis of genomic sequences can concern coding regions of the gene (introns or exons) as well as regulatory regions, in particular those necessary for the initiation of transcription.
  • the invention further relates to transgenic potato plants which contain the transgenic plant cells described above with reduced debranching enzyme activity.
  • the invention also relates to the modified starch obtainable from the transgenic cells or plants.
  • the amylopectin starch 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 also relates to propagation material of the transgenic plants described above, in particular seeds and tubers, these containing transgenic plant cells described above.
  • Transgenic plant cells which, owing to the expression of a new or additional debranching enzyme activity, form an amylopectin starch with a different degree of branching compared to amylopectin starch 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 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 starch synthesized in wild-type plants can be produced, for example, by a process which comprises the following steps:
  • a promoter which ensures transcription in plant cells;
  • at least one nucleic acid sequence according to the invention 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;
  • step (iii) optionally a termination signal for the termination of the transcription and the addition of a poly-A tail to the resulting transcript, which is coupled to the 3 'end of the coding region; and (b) transforming plant cells with that in step
  • 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 is the promoters of maize polyubiquitin genes (Christensen et al., Plant Mol. Biol. 18 (1992), 675-689).
  • promoters can also be used which are only activated at a point in time determined by external influences (see, for example, WO / 9307279).
  • Promoters of heat shock proteins that allow simple induction can be of particular interest.
  • the promoters which lead to expression of downstream sequences in a specific tissue of the plant can be used (see, for example, Stockhaus et al., EMBO J. 8 (1989), 2245-2251). Preference is given to using promoters which are active in the starch-storing organs of the plants to be transformed. For corn, for example, these are the corn kernels, while the potatoes are the tubers.
  • the bulb-specific B33 promoter can be used to overexpress the Nucleic acid molecules according to the invention in the potato.
  • the bulb-specific B33 promoter (Rochasosa et al., EMBO J. 8 (1989), 23-29) can be used. Seed-specific promoters have already been described for various plant species.
  • 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 potato is linked in sense orientation to the promoter
  • this nucleic acid sequence can be both native or homologous in origin and foreign or heterologous in origin with respect to the plant species to be transformed, ie Both potato 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 which codes this signal sequence must be removed and the coding region linked to DNA sequences which ensure localization 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. Be. USA 85: 846-850 (1988); Sonnewald et al. , Plant J. 1 (1991), 95-106).
  • nucleic acid sequence from potato mentioned under process step (a) (ii), which encodes a protein with the enzymatic activity of a debranching enzyme is linked in antisense orientation to the promoter this is preferably of a nucleic acid sequence of homologous origin with respect to the plants 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 homology 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 tumefaciens 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 procedure described above for overexpressing a new debranching enzyme from potato can in principle be applied to all plant species. Are of interest both monocot and dicot plants, in particular the starch-storing plants described above.
  • the method described above for reducing the debranching enzyme activity is preferably used on dicotyledonous plants, in particular on potatoes.
  • an RNA is formed in the transformed plant cells. If the nucleic acid sequence encoding a potato debranching enzyme in the expression cassette is linked in sense orientation to the promoter, an mRNA is synthesized, which acts as a template for the synthesis of an additional or new potato debranching enzyme in the plant cells can serve. As a result, these cells have an activity or an increased activity of the potato debranching enzyme, which leads to a change in the degree of branching of the amylopectin formed in the cells. This makes a strength accessible which, in comparison to the naturally occurring strength, is distinguished by a more orderly spatial structure and an increased uniformity. Among other things, this can have favorable effects on the film-forming properties.
  • an antisense RNA is synthesized in transgenic plant cells, which inhibits the expression of endogenous debranching enzyme genes. As a result, these cells show reduced activity of the new potato debranching enzyme, resulting in the formation of a modified starch.
  • the anfcisense technique it is possible to produce plants in which the expression of an endogenous debranching enzyme gene in potatoes is inhibited to varying degrees in a range from 0% to 100%. This enables in particular the production of potato plants which synthesize amylopectin starch with a wide variety of variations in the degree of branching.
  • Potato 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 these 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.
  • the simplicity and cost-effective implementation of a hydrolysis process can be important for reducing the costs. It is currently essentially enzymatic using amyloglucosidase. A cost saving 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 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 paper and cardboard industry should be mentioned in particular.
  • 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 favorable properties of the starch in terms of rigidity, hardness, sound, grip, gloss, smoothness, splitting resistance and surfaces are exploited.
  • 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 sacks, bags and pouches, production of composite materials for paper and aluminum, production of cardboard packaging and rewetting glue for envelopes, stamps, etc.
  • 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 for increasing the abrasion resistance during weaving
  • starch as an agent for textile upgrading, especially after pre-treatments such as bleaching, dyeing etc. which impair quality
  • 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. Combination products made of starch and polymer emulsions are, according to current knowledge, equivalent to the products used hitherto in their erosion and incrustation-reducing effects, but are priced significantly below these. 2.6 Use in crop protection and fertilizers
  • 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 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, strengths gain as a binding of importance, since the addition of 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.
  • 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.
  • the result is polyurethane films which, through the use of starch, obtain the following property profiles: a reduction in the coefficient of thermal expansion, a reduction in shrinkage behavior, an improvement in the pressure / stress behavior, an increase in water vapor permeability without changing the water absorption, a reduction in the flammability and the tear - dense, 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, gelation, 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.
  • 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 starch 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.
  • nucleic acid molecules according to the invention can also be used to produce plants 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 debranching enzymes, GBSS I, SSS I, II or GBSS II Pro or teine 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 of maize (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.
  • 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 usually be preferable because of this If the synthesis of the corresponding proteins should be inhibited to approximately the same extent.
  • molecules which, in addition to sequences coding for debranching enzymes, contain further DNA sequences which code for other proteins involved in starch synthesis or modification. These are each coupled in an antisense orientation to a suitable promoter.
  • the sequences can in turn be connected in series and can be transcribed by a common promoter or else can be transcribed by separate promoters.
  • 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 in an antisense orientation behind a suitable promoter can originate from DNA sequences which code for the following proteins: Starch grain-bound (GBSS I and II) and soluble starch synthases (eg SSS I and II), branching enzymes, other debranching enzymes, disproportionation enzymes and starch phosphorylases. This is only an exemplary list. 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.
  • GBSS I and II Starch grain-bound
  • SSS I and II soluble starch synthases
  • the constructs can furthermore be introduced into classic mutants which are defective for one or more genes of starch biosynthesis. These defects can relate, for example, to the following proteins: starch-grain-bound (GBSS I and II) and soluble starch synthases (eg SSS I and II), ver branching enzymes (BE I and II), debranching enzymes, disproportionation 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 the 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 pUC derivatives can be used. If whole plants are to be regenerated from cells transformed in this way , a selectable marker gene should advantageously be present.
  • the Ti or Ri plasmid is used for the transformation of the plant cell, at least the right boundary, but advantageously the right and left boundary of the Ti and Ri plasmid T-DNA as the flank region, should be linked to the genes to be introduced ⁇ the.
  • the DNA to be introduced should 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 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 should contain a plasmid carrying a vir region. The vir 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 for the transformation of 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 BV, 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 (e.g. 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 gives the transformed plant cells resistance to a biocide or an antibiotic such as kanamycin, G 418, bleomycin, hygromycin or phosphinotricin and others. averages.
  • 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 vector pBluescript II SK (Stratagene) was used for cloning in E. coli.
  • 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 radioactive labeling of DNA fragments was carried out using a DNA random primer labeling kit from the company Boehringer (Germany) according to the manufacturer's instructions.
  • a cDNA library based on polyA + RNA from tuber material was created in the vector Lambda ZAPII (Stratagene) and packed in phage heads. E. coli cells of the XLl-Blue strain were then infected with the phages containing the cDNA fragments (1 ⁇ 10 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 turf, which were removed after one minute.
  • the filters were placed in 0.5 M NaOH for 2 min; 1.5 M NaCl, then incubated in 0.5 M Tris / HCl pH 7.0 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 48 ° C. for 3 hours before radioactively labeled sample was added.
  • a maize cDNA sequence encoding a debranching enzyme was used as a sample (see James et al., Plant Cell 7 (1995), 417-429, nucleotides 1150-2128)]. The hybridization was carried out at 48 ° C.
  • 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 2295 bp long and the nucleotide sequence of 2133 bp of this insertion as well as the derived amino acid sequence is in Seq ID No. 1 specified. Homology comparisons showed that the encoded protein is a new debranching enzyme from potato.
  • the Seq ID No. 1 indicated nucleotide sequence represents a partial cDNA which encodes a previously unknown de-branching enzyme from potato. With the aid of this sequence, it is possible to isolate a complete cDNA sequence or a genomic sequence from suitable cDNA or genomic libraries using conventional methods.
  • MOLECULE TYPE cDNA to mRNA
  • HYPOTHETICAL NO
  • ANTISENSE NO
  • GCC AAA CAA TAT TCT CAT TTT CTG GAC GCG AAC CAG TAT CCG ATG CTC 1774 Ala Lys Gin Tyr Ser His Phe Leu Asp Ala Asn Gin Tyr Pro Met Leu 580 585 590
  • MOLECULE TYPE Protein

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Abstract

L'invention concerne des molécules d'acide nucléique codant l'enzyme de déramification de pomme de terre, ainsi que des cellules végétales transgéniques et des plantes dans lesquelles l'expression d'une enzyme de déramification de pomme de terre ou l'inhibition d'une telle activité enzymatique de déramification endogène entraîne la synthèse d'une amylopectine aux propriétés modifiées.
EP97922969A 1996-05-06 1997-05-06 Molecules d'acide nucleique codant l'enzyme de deramification de pomme de terre Withdrawn EP0900277A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DE19618125A DE19618125A1 (de) 1996-05-06 1996-05-06 Nucleinsäuremoleküle, die neue Debranching-Enzyme aus Kartoffel codieren
DE19618125 1996-05-06
PCT/EP1997/002292 WO1997042328A1 (fr) 1996-05-06 1997-05-06 Molecules d'acide nucleique codant l'enzyme de deramification de pomme de terre

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AU724164B2 (en) 2000-09-14
CA2253234A1 (fr) 1997-11-13
WO1997042328A1 (fr) 1997-11-13
JP2000509286A (ja) 2000-07-25
US6670525B2 (en) 2003-12-30
US20030167527A1 (en) 2003-09-04
US6255563B1 (en) 2001-07-03
HUP9902317A2 (hu) 1999-10-28
AU2891797A (en) 1997-11-26
DE19618125A1 (de) 1997-11-13
HUP9902317A3 (en) 2002-01-28

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