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EP1095152A2 - Nucleinsäuremoleküle codierend enzyme aus weizen, die an der stärkesynthese beteiligt sind - Google Patents

Nucleinsäuremoleküle codierend enzyme aus weizen, die an der stärkesynthese beteiligt sind

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Publication number
EP1095152A2
EP1095152A2 EP99923557A EP99923557A EP1095152A2 EP 1095152 A2 EP1095152 A2 EP 1095152A2 EP 99923557 A EP99923557 A EP 99923557A EP 99923557 A EP99923557 A EP 99923557A EP 1095152 A2 EP1095152 A2 EP 1095152A2
Authority
EP
European Patent Office
Prior art keywords
nucleic acid
starch
plant
acid molecule
dna
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP99923557A
Other languages
German (de)
English (en)
French (fr)
Inventor
Horst Lörz
Stephanie Lütticke
Martina Block
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Bayer CropScience AG
Original Assignee
Aventis CropScience GmbH
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Aventis CropScience GmbH filed Critical Aventis CropScience GmbH
Publication of EP1095152A2 publication Critical patent/EP1095152A2/de
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1051—Hexosyltransferases (2.4.1)
    • A—HUMAN NECESSITIES
    • A21—BAKING; EDIBLE DOUGHS
    • A21D—TREATMENT OF FLOUR OR DOUGH FOR BAKING, e.g. BY ADDITION OF MATERIALS; BAKING; BAKERY PRODUCTS
    • A21D2/00—Treatment of flour or dough by adding materials thereto before or during baking
    • A21D2/08—Treatment of flour or dough by adding materials thereto before or during baking by adding organic substances
    • A21D2/14—Organic oxygen compounds
    • A21D2/18—Carbohydrates
    • A21D2/186—Starches; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
    • A23L29/20—Foods or foodstuffs containing additives; Preparation or treatment thereof containing gelling or thickening agents
    • A23L29/206—Foods or foodstuffs containing additives; Preparation or treatment thereof containing gelling or thickening agents of vegetable origin
    • A23L29/212—Starch; Modified starch; Starch derivatives, e.g. esters or ethers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C12N15/8245—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving modified carbohydrate or sugar alcohol metabolism, e.g. starch biosynthesis

Definitions

  • the present invention relates to nucleic acid molecules that encode a wheat enzyme that is involved in starch synthesis in plants.
  • This enzyme is a soluble type I starch synthase.
  • the invention further relates to vectors, host cells, as well as plant cells and plants, which contain the nucleic acid molecules according to the invention.
  • polysaccharides are the most important renewable raw materials from plants.
  • Wheat is one of the most important crops, as it produces about 20% of the total starch production in the European Community.
  • the polysaccharide starch is a polymer made up of chemically uniform building blocks, the glucose molecules. However, it is a very complex mixture of different molecular forms, which differ in the degree of polymerization of the occurrence of branches of the glucose chains and their chain lengths, which can also be derivatized, for example phosphorylated. Starch is therefore not a uniform raw material.
  • amylose starch an essentially unbranched polymer composed of 1,4-glycosidically linked glucose molecules, and amylopectin starch, which in turn is a complex mixture of differently branched glucose chains.
  • the branches come about through the occurrence of additional -1, 6-glycosidic linkages.
  • about 1 1 to 37% of the synthesized starch consists of amylose starch.
  • the biochemical synthetic pathways that lead to the building of starch are in the essentially known.
  • the starch synthesis in plant cells takes place in the plastids.
  • starch synthases Important enzymes involved in starch synthase are starch synthases and branching enzymes. Various isoforms are described for starch synthase, all of which catalyze a polymerization reaction by transferring a glucosyl residue from ADP-glucose to ⁇ -1,4-glucans. Branching enzymes catalyze the introduction of ⁇ -2,6 branches into linear ⁇ -1,4-glucans.
  • Starch synthases can be divided into two classes: starch-bound starch synthases ("granule-bound starch synthases”; GBSS) and soluble starch synthases ("soluble starch synthases”; SSS). This distinction cannot be clearly made in every case, since some of the starch synthases are both bound to starch and in soluble form (Denyer et al., Plant J. 4 (1 993), 1 91-1 98; Mu et al., Plant J. 6 (1,994), 1 51-1 59). For different plant species, different isoforms are described within these classes, which differ in terms of their dependence on starter molecules (so-called “primer dependent” (type II) and “primer independent” (type I) starch synthases).
  • At least two isoforms of the starch-bound starch synthase (60 kDA and 100-105 kDA) and another isoform, which may be a soluble starch synthase (Denyer et al., Planta 1 96 (1 995), 256-265; Rahman et al., Aust. J. Plant Physiol. 22 (1 995), 793-803), was identified at the protein level.
  • the presence of several SSS isoforms has previously been demonstrated using chromatographic methods (Rijven, Plant Physiol. 81 (1 986), 448-453).
  • a cDNA encoding GBSS I from wheat has already been described (Ainsworth et al., Plant Mol. Biol. 22 (1 993), 67 to 82).
  • nucleic acid sequences which encode starch synthase isoforms from wheat or partial sequences of such nucleic acids have hitherto been known from WO 97/45545.
  • cDNA sequences coding for starch synthases other than GBSS I have so far only been used for pea (Dry et al., Plant J. 2 (1 992), 1 93-202), rice (Baba et al., Plant Physiol 103 (1 993), 565 to 573) and potato (Edwards et al., Plant J. 8 (1 995), 283 to 294).
  • soluble starch synthases have also been identified in a number of other plant species. Soluble starch synthases have been isolated, for example, to homogeneity from pea (Denyer and Smith, Planta 1 86 (1 992), 609 to 61 7) and potato (Edwards et al., Plant J. 8 (1 995), 283 to 294) .
  • any starch-storing plants preferably cereals, in particular wheat, in such a way that they synthesize a modified starch
  • the present invention is therefore based on the object of making available nucleic acid molecules, in particular those from wheat, which encode enzymes involved in starch biosynthesis and with the aid of which it is possible to produce genetically modified plants which are suitable for the production of chemical and / or or physical properties of modified vegetable starches.
  • the present invention therefore relates to nucleic acid molecules which proteins with the activity of a soluble starch synthase from wheat, wherein such molecules preferably encode proteins which essentially have the Seq ID no. Include 2 given amino acid sequence.
  • the invention relates to nucleic acid molecules which are listed under Seq ID No. 1 or a part thereof, preferably molecules which contain the nucleotide sequence specified in Seq ID No. 1 indicated coding region comprise, particularly preferably nucleotide No. 9 to 570 of Seq ID No. 1 as well as corresponding ribonucleotide sequences.
  • the present invention further relates to nucleic acid molecules which hybridized with one of the nucleic acid molecules according to the invention.
  • the invention also relates to nucleic acid molecules which encode a soluble starch synthase from wheat and whose sequence deviates from the nucleotide sequences of the molecules described above due to the degeneration of the genetic code.
  • the invention also relates to nucleic acid molecules which have a sequence which is complementary to all or part of one of the abovementioned sequences.
  • hybridization means hybridization under conventional hybridization conditions, preferably under stringent conditions, as described, for example, in Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd edition (1,989) Cold Spring Harbor Laboratory Press , Cold Spring Harbor, NY). Hybridization is particularly preferably carried out under the following
  • Hybridization buffer 2 x SSC; 1 0 x Denhardt's solution (Fikoll 400 + PEG +
  • Washing temperature T 40 to 75 ° C.
  • nucleic acid molecules which hybridize with the nucleic acid molecules according to the invention can encode starch synthases from any wheat plant which expresses such proteins.
  • Nucleic acid molecules that hybridize with the molecules of the invention can e.g. isolated from genomic or from cDNA libraries of wheat or wheat plant tissue. Alternatively, they can be produced by genetic engineering methods or by chemical synthesis.
  • nucleic acid molecules can be identified and isolated using the 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., 1 989, 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 ones under Seq ID No. 1 indicated nucleotide sequence or parts of this sequence.
  • the fragments used for the hybridization sample can also be synthetic fragments which were produced with the aid of the usual synthetic techniques and whose sequence essentially corresponds to that of a nucleic acid molecule according to the invention.
  • the molecules hybridizing with the nucleic acid molecules according to the invention also include fragments, derivatives and allelic variants of the nucleic acid molecules described above, which encode a starch synthase according to the invention from wheat. Fragments are understood to mean parts of the nucleic acid molecules that are long enough to encode one of the proteins described.
  • 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, substitution, insertion or recombination.
  • nucleic acid molecules in question or the proteins encoded by them are usually variations of these molecules which are 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 act 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. For this, e.g. Enzyme activity, molecular weight, immunological reactivity, conformation etc. belong as well as physical properties such as the running behavior in gel electrophoresis, chromatographic behavior, sedimentation coefficient, solubility, spectroscopic properties, charge properties, stability; pH optimum, temperature optimum etc.
  • a starch synthase Important characteristics of a starch synthase are: i) its localization in the stroma of plastids in plant cells; ii) their ability to synthesize linear - 1, 4-linked polyglucans. This activity can be determined as described in Denyer and Smith (Plante 1 86 (1 992), 606 to 61 7).
  • the protein encoded by the nucleic acid molecules according to the invention is a soluble type I starch synthase from wheat. These proteins have certain areas of homology with previously known soluble starch synthases from other plant species.
  • the nucleic acid molecules according to the invention can be DNA molecules, in particular cDNA or genomic molecules. Furthermore, the nucleic acid molecules according to the invention can be RNA molecules, which can result, for example, from transcription of a nucleic acid molecule according to the invention.
  • the nucleic acid molecules according to the invention can, for. B. obtained from natural sources or produced by recombinant techniques or synthetically.
  • the invention also relates to oligonucleotides which hybridize specifically with a nucleic acid molecule according to the invention. Such oligonucleotides preferably have a length of at least 10, in particular at least 15 and particularly preferably at least 50 nucleotides.
  • the oligonucleotides according to the invention are characterized in that they hybridize specifically with nucleic acid molecules according to the invention, ie not or only to a very small extent with nucleic acid sequences which code for other proteins, in particular other starch synthases.
  • the oligonucleotides according to the invention can be used, for example, as primers for a PCR reaction or as a hybridization sample for the isolation of related genes. They can also be components of antisense constructs or of DNA molecules which code for suitable ribozymes.
  • the invention further relates to vectors, in particular plasmids, cosmids, phagemids, viruses, bacteriophages and other vectors which are common in genetic engineering and which contain the nucleic acid molecules according to the invention described above.
  • vectors are suitable for the transformation of pro- or eukaryotic, preferably plant cells.
  • the vectors particularly preferably allow the nucleic acid molecules according to the invention, optionally together with flanking regulatory regions, to be integrated into the genome of the plant cell. Examples of this are binary vectors which can be used in gene transfer mediated by agrobacteria.
  • the integration of a nucleic acid molecule according to the invention in sense or anti-sense orientation preferably ensures the synthesis of a translatable or optionally non-translatable RNA in the transformed pro- or eukaryotic cells.
  • vector generally designates a suitable, the expert Known tool that enables the targeted transfer of a single- or double-stranded nucleic acid molecule into a host cell, for example a DNA or RNA virus, a virus fragment, a plasmid construct, which, in the presence or absence of regulatory elements, may be suitable for nucleic acid transfer in cells can, carrier materials such as glass fiber or metal particles such as can be used for example in the "particle gun” process, but it can also include a nucleic acid molecule that can be brought directly into a cell by chemical or physical methods.
  • the nucleic acid molecules contained in the vectors are linked to regulatory elements which ensure the transcription and synthesis of a translatable RNA in pro- or eukaryotic cells or, if desired, the synthesis of a non-translatable RNA.
  • nucleic acid molecules according to the invention in prokaryotic cells, for example in Escherichia coli, is important for a more precise characterization of the enzymatic activities of the enzymes for which these molecules code.
  • deletions at the 5 'end of the nucleotide sequence make it possible, for example, to identify amino acid sequences which are responsible for the translocation of the enzyme into the plastids (transit peptides). This allows targeted production of enzymes that are no longer localized in the plastids but in the cytosol by removing the corresponding sequences, or are localized in other compartments due to the addition of other signal sequences.
  • mutants can be produced which have a changed K m value or which are no longer subject to the regulatory mechanisms normally present in the cell via allosteric regulation or covalent modification.
  • mutants can be produced which have a modified substrate or product specificity of the protein according to the invention, for example by using ADP-glucose-6-phosphate instead of ADP-glucose. Furthermore, mutants can be produced which have a changed activity-temperature profile of the protein according to the invention.
  • the nucleic acid molecules according to the invention or parts of these molecules can be introduced into plasmids which permit mutagenesis or a sequence change by recombination of DNA sequences.
  • base changes can be made or natural or synthetic sequences added.
  • adapters or linkers can be attached to the fragments.
  • Manipulations which provide suitable restriction sites or which remove superfluous DNA or restriction sites can also be used. Where insertions, deletions or substitutions are possible, in vitro mutagenesis, "primer repair", restriction or ligation can be used. Sequence analysis, restriction analysis or other biochemical-molecular biological methods are generally used as the analysis method.
  • the invention relates to host cells, in particular pro- or eukaryotic cells which are transformed with a nucleic acid molecule according to the invention or a vector according to the invention described above, and cells which are derived from cells transformed in this way and contain a nucleic acid molecule according to the invention or a vector.
  • host cells in particular pro- or eukaryotic cells which are transformed with a nucleic acid molecule according to the invention or a vector according to the invention described above, and cells which are derived from cells transformed in this way and contain a nucleic acid molecule according to the invention or a vector.
  • the invention further relates to recombinantly producible proteins with the activity of a starch synthase, which are encoded by the nucleic acid molecules according to the invention, and methods for their production, in which a host cell according to the invention is cultivated under suitable conditions known to the person skilled in the art, which allow the synthesis of the protein according to the invention, and then it from the
  • Host cells and / or the culture medium is isolated.
  • nucleic acid molecules according to the invention By providing the nucleic acid molecules according to the invention, it is now possible to use genetic engineering methods to specifically intervene in the starch metabolism of plants and to change it in such a way that a modified starch is synthesized, which in its physico-chemical form Properties, for example the amylose / amylopectin ratio, the degree of branching, the average chain length, the phosphate content, the gelatinization behavior, the gel or film formation properties, the starch grain size and / or the starch grain shape is changed compared to known starch.
  • a modified starch which in its physico-chemical form Properties, for example the amylose / amylopectin ratio, the degree of branching, the average chain length, the phosphate content, the gelatinization behavior, the gel or film formation properties, the starch grain size and / or the starch grain shape is changed compared to known starch.
  • nucleic acid molecules according to the invention in plant cells in order to increase the activity of the corresponding starch synthase, or to introduce them into cells which naturally do not express this enzyme. Furthermore, it is possible to modify the nucleic acid molecules according to the invention by methods known to the person skilled in the art in order to obtain starch synthases according to the invention which are no longer subject to the natural cellular regulation mechanisms or which have changed temperature-activity profiles or substrate or product specificities.
  • the synthesized protein can be localized in any compartment of the plant cell.
  • the sequence ensuring localization in plastids must be deleted and the remaining coding region may have to be linked to DNA sequences which ensure localization in the respective compartment.
  • Such sequences are known (see for example Braun et al., EMBO J. 1 1 (1 992), 321 9-3227; Wolter et al., Proc. Natl. Acad. Sci. USA 85 (1 988), 846-850 ; Sonnewald et al., Plant J. 1 (1,991), 95-106).
  • the present invention thus also relates to a method for producing transgenic plant cells which are transformed with a nucleic acid molecule or vector according to the invention, in which a nucleic acid molecule according to the invention or a vector according to the invention in the genome of a plant cell is integrated, the transgenic plant cells which were transformed by means of a nucleic acid molecule or vector according to the invention and transgenic plant cells which are derived from cells transformed in this way.
  • the cells according to the invention contain one or more nucleic acid molecules or vectors according to the invention, these being preferably linked to regulatory DNA elements which ensure transcription in plant cells, in particular with a suitable promoter.
  • Such cells can be distinguished from naturally occurring plant cells, inter alia, in that they contain a nucleic acid molecule according to the invention which does not naturally occur in these cells or in that such a molecule is integrated at a location in the genome of the cell where it does not otherwise occur , ie in a different genomic environment.
  • transgenic plant cells according to the invention can be distinguished from naturally occurring plant cells in that they contain at least one copy of a nucleic acid molecule according to the invention stably integrated into their genome, optionally in addition to copies of such a molecule that occur naturally in the cells.
  • the plant cells according to the invention can be distinguished from naturally occurring cells in particular in that these additional copy (s) ) is localized in locations in the genome where it does not naturally occur. This can be easily checked, for example, with the aid of a Southern blot analysis according to methods known to the person skilled in the art.
  • the transgenic plant cells have transcripts of the nucleic acid molecules according to the invention, which are e.g. B. simply by Northern blot analysis according to methods known to those skilled in the art. If the introduced nucleic acid molecule according to the invention is homologous with respect to the plant cell, the cells according to the invention can be distinguished from naturally occurring cells, for example on the basis of the additional expression of nucleic acid molecules according to the invention.
  • the transgenic plant cells preferably contain more transcripts of the nucleic acid molecules according to the invention. This can e.g. B. can be detected by Northern blot analysis.
  • “More” preferably means at least 10% more, preferably at least 20% more and particularly preferably at least 50% more transcripts than corresponding, non-transformed cells.
  • the cells preferably also have a corresponding (at least 10%, 20% or 50%) increase in activity or, if appropriate, a reduction in activity of the protein according to the invention.
  • the transgenic plant cells can be regenerated into whole plants using techniques known to those skilled in the art.
  • the present invention also relates to a method for producing transgenic plants, in which one or more nucleic acid molecules or vectors according to the invention are integrated into the genome of a plant cell and a complete plant is regenerated from said plant cell.
  • the plants obtainable by regeneration of the transgenic plant cells according to the invention are also the subject of the present invention.
  • the invention furthermore relates to plants which contain the transgenic plant cells described above.
  • the transgenic plants can in principle be plants of any plant species, ie both monocot and dicot plants.
  • the invention also relates to propagation material of the plants according to the invention, for example fruits, seeds, tubers, rhizomes, seedlings, cuttings, calli, protoplasts, cell cultures etc.
  • the present invention further relates to a method for producing a modified starch comprising the step of extracting the starch from a plant according to the invention described above and / or from starch-storing parts of such a plant.
  • the transgenic plant cells and plants according to the invention synthesize a starch which has its physicochemical properties, for example the amylose / amylopectin ratio, the degree of branching, the average chain length, the phosphate content, the gelatinization behavior, the starch grain size and / or the starch grain shape compared to wild type Plants synthesized starch is changed.
  • a starch can be changed with regard to the viscosity and / or the film or gel formation properties of pastes of this starch compared to known starches.
  • the present invention furthermore relates to a starch which can be obtained from the plant cells, plants and their propagation material according to the invention and starch which can be obtained by the process according to the invention described above.
  • nucleic acid molecules according to the invention it is also possible to use the nucleic acid molecules according to the invention to produce plant cells and plants in which the activity of a protein according to the invention is reduced. This also leads to the synthesis of a starch with changed chemical and / or physical properties compared to starch from wild-type plant cells.
  • Another object of the invention is thus also a transgenic plant cell containing a nucleic acid molecule according to the invention, in which the activity of a starch synthase is reduced compared to a non-transformed cell.
  • the production of plant cells with a reduced activity of a starch synthase can be achieved, for example, by the expression of a corresponding antisense-RNA, a sense-RNA to achieve a cosuppression effect or the expression of a correspondingly constructed ribozyme which specifically cleaves transcripts which code for a starch synthase , using the nucleic acid molecules according to the invention by methods known to the person skilled in the art, cf. Jorgensen (Trends Biotechnol. 8 (1 990), 340-344), Niebel et al., (Curr. Top. Microbiol. Immunol. 1 97 (1 995), 91-103), Flavell et al. (Curr. Top.
  • the number of transcripts encoding it is reduced, e.g. by expression of an antisense RNA.
  • a DNA molecule can be used which comprises the entire sequence coding for a protein according to the invention including any flanking sequences which may be present, and also DNA molecules which only comprise parts of the coding sequence, these parts having to be long enough to to cause an antisense effect in the cells.
  • sequences up to a minimum length of 15 bp, preferably a length of 100-500 bp can be used for efficient antisense inhibition, in particular sequences with a length of more than 500 bp.
  • DNA molecules are used that are shorter than 5000 bp, preferably sequences that are shorter than 2500 bp.
  • DNA sequences which have a high degree of homology to the sequences of the DNA molecules according to the invention, but which are not completely identical.
  • the minimum homology should be greater than approximately 65%.
  • sequences with homologies between 95 and 100% is preferred.
  • the invention also relates to a method for producing a modified starch, comprising the step of extracting the starch from a cell or plant according to the invention and / or from starch-storing parts of such a plant.
  • the invention furthermore relates to starch which can be obtained from the cells, plants and propagation material or parts thereof according to the invention and starch which can be obtained by a process according to the invention.
  • starches according to the invention can be modified by processes known to those skilled in the art and are suitable in unmodified or modified form for various uses in the food or non-food sector.
  • the possible uses of the starches according to the invention can be divided into two large areas.
  • One area comprises the hydrolysis products of starch, mainly glucose and glucan building blocks, which are obtained via enzymatic or chemical processes. They serve as the starting material for further chemical modifications and processes, such as fermentation.
  • the simplicity and cost-effective execution of a hydrolysis process can be important for reducing the costs.
  • it is essentially enzymatic using amyloglucosidase. It would be conceivable to save costs by using fewer enzymes.
  • a structural change in strength, e.g. Surface enlargement of the grain, easier digestibility through e.g. A lower degree of branching or a steric structure that limits the accessibility for the enzymes used could cause this.
  • Starch is a classic additive for many foods, where it essentially takes on the function of binding aqueous additives or increases the viscosity or increases gel formation.
  • the important properties are the flow and Sorption behavior, the swelling and gelatinization temperature, the viscosity and thickening performance, the solubility of the starch, the transparency and paste structure, the heat, shear and acid stability, the tendency to retrogradation, the ability to form films, the freeze / thaw stability, the viscosity stability in Salt solutions, digestibility and the ability to form complexes with, for example, inorganic or organic ions.
  • starch can be used as an additive for different manufacturing processes or as an additive in technical products.
  • starch When using starch as an auxiliary, the paper and cardboard industry should be mentioned in particular.
  • the starch primarily serves 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.
  • rapid, even, loss-free distribution, high mechanical stability and complete restraint in the paper flow are important.
  • At the Use of the starch in the spray area is also of importance to an adapted solids content, high viscosity and high binding capacity.
  • 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 use of starches as an extender for synthetic adhesives.
  • 90% of the starch-based adhesives are used in the fields of corrugated cardboard, paper bags, bags and pouches, composite materials for paper and aluminum, cardboard packaging and rewetting glue for envelopes, stamps, etc.
  • starch as a sizing agent, i.e. as an auxiliary for smoothing and strengthening the hook-and-loop behavior to protect against the tensile forces that occur during weaving and to increase the abrasion resistance during weaving, starch as a means of textile upgrading after poor-quality pre-treatments such as bleaching, dyeing, etc., starch as a thickening agent in the manufacture of color pastes to prevent dye diffusion and starch as an additive to chaining agents for sewing threads.
  • the fourth area of application is the use of starches as an additive in building materials.
  • One example is the production of gypsum plasterboard, in which the starch mixed in the gypsum slurry pastes with the water, diffuses to the surface of the gypsum board and binds the cardboard to the board there.
  • Other areas of application are admixing to plaster and mineral fibers.
  • starch products are used to delay setting.
  • starch Another market for starch is in the manufacture of soil stabilizers that are used to temporarily protect soil particles from water during artificial earthmoving. Combined products made of starch and polymer emulsions are, according to current knowledge, to be equated with the previously used products in terms of their erosion and incrustation-reducing effects, but are significantly less expensive than these.
  • 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, to release the active ingredients in a dosed manner, to convert liquid, volatile and / or malodorous active ingredients into microcrystalline, stable, moldable substances, to mix incompatible compounds and to extend the duration of action by reducing the Decomposition can be used.
  • starch can be considered Binder for tablets or for binder dilution in capsules can be used.
  • 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 lubricant 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 starch is toothpaste.
  • Starch is used as an additive to coal and briquette. Coal can be agglomerated or briquetted with a high-quality addition of starch, which prevents the briquettes from breaking down prematurely.
  • the added starch is between 4 and 6% for barbecued coal and between 0, 1 and 0.5% for calorized coal. Furthermore, starches are becoming increasingly important as binders, since their addition to coal and briquette can significantly reduce the emissions of harmful substances.
  • 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.
  • starch addition is to increase the flow resistance and to improve the binding strength.
  • source strengths have other production-related requirements, such as dispersibility in cold water, rehydration, good miscibility in sand and high water retention.
  • the starch can be used to improve the technical and optical quality.
  • the reasons for this are the improvement of the surface gloss, the improvement of the handle and the appearance, for this reason starch is sprinkled on the sticky rubberized surfaces of rubber materials before the cold vulcanization, and the improvement of the printability of the rubber.
  • Another way of selling the modified starches is in the production of leather substitutes.
  • starch secondary products in the processing process (starch is only a filler, there is no direct link between synthetic polymer and starch) or, alternatively, the integration of starch secondary products in the production of polymers (starch and polymer are one firm bond).
  • starch as a pure filler is not competitive compared to other substances such as talc. It is different if the specific starch properties come into play and this significantly changes the property profile of the end products.
  • 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 co-expressed in a 1: 1 ratio to form a 'master batch' combined, from which various products are made with granulated polyethylene using conventional processing 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 hydroxyl groups of the starches.
  • the result is polyurethane foils that get the following property profiles through the use of starch: a reduction in the coefficient of thermal expansion, a reduction in shrinkage behavior, an improvement in pressure / stress behavior, an increase in water vapor permeability without changing the water absorption, a reduction in flammability and tear density, no dripping of flammable parts, Halogen free and reduced aging.
  • Disadvantages that are currently still present are reduced compressive strength and reduced impact resistance.
  • Solid plastic products such as pots, plates and bowls can also be manufactured with a starch content of over 50%.
  • starch / polymer mixtures can be assessed favorably because 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 grid of a synthetic monomer grafted on according to the principle of the radical chain mechanism.
  • the starch graft polymers available today are characterized by better binding and retention properties of up to 1000 g of water per g of starch with high viscosity.
  • the areas of application for these superabsorbents have expanded considerably in recent years and are in the hygiene sector with products such as diapers and pads as well as in the agricultural sector, for example in seed pilling.
  • Decisive for the use of the new, genetically modified starches are on the one hand the structure, water content, protein content, lipid content, fiber content, ash / phosphate content, amylose / amylopectin ratio, molar mass distribution, degree of branching, grain size and shape as well as crystallinity, and on the other hand also the properties flow into the following characteristics: flow and sorption behavior, gelatinization temperature, viscosity, viscosity stability in salt solutions, thickening performance, solubility, paste structure and transparency, heat, shear and acid stability, tendency to retrogradation, gel formation, freeze / thaw stability, complex formation, iodine binding, film formation, adhesive strength , Enzyme stability, digestibility and reactivity.
  • modified starches by means of genetic engineering methods 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 methods no longer appear 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.
  • These chemical modifications are generally known. In particular, these are modifications by heat treatment, treatment with organic or inorganic acids, oxidation and esterifications, which, for example, lead to the formation of phosphate, nitrate, sulfate, xanthate, acetate and Lead citrate starches.
  • monohydric or polyhydric alcohols can be used in the presence of strong acids to produce starch ethers, so that starch alkyl ether, O-allyl ether, hydroxyl alkyl ether, O-carboxylmethyl ether, N-containing starch ether, P-containing starch ether), S-containing starch ether, cross-linked starches or starch graft polymers result.
  • a preferred use of the starches according to the invention is in the production of packaging material and disposable articles on the one hand and as food or intermediate food product on the other hand.
  • nucleic acid molecules according to the invention are linked to regulatory DNA elements which ensure transcription in plant cells. These include in particular promoters, enhancers and terminators. In general, any promoter active in plant cells can be used for the expression.
  • the promoter can be selected so that the expression is constitutive or only in a certain tissue, at a certain time in plant development or at a time determined by external influences.
  • the promoter can be homologous or heterologous to the plant. Suitable promoters are, for example, the 35S RNA promoter of the Cauliflower Mosaic Virus and the ubiquitin promoter from maize for constitutive expression, the patatin promoter B33 (Rocha-Sosa et al., EMBO J. 8 (1 989), 23-29 ) for a tuber-specific expression or a promoter which ensures expression only in photosynthetically active tissues, for example the ST-LS1 promoter (Stockhaus et al., Proc. Natl. Acad.
  • the present invention provides nucleic acid molecules that encode a protein with the function of a soluble starch synthase from wheat.
  • the nucleic acid molecules according to the invention allow the production of this enzyme, its functional identification within the starch biosynthesis, the production of genetically modified plants in which the activity of this enzyme is changed and thus enables the synthesis of a starch with a modified structure and changed physicochemical properties in such modified plants .
  • nucleic acid molecules according to the invention can also be used to produce plants in which the activity of the starch synthase according to the invention is increased or decreased and at the same time the activities of other enzymes involved in starch synthesis are changed.
  • the change in the activities of a starch synthase in plants leads to the synthesis of a starch with a different structure.
  • nucleic acid molecules which encode a starch synthase or corresponding antisense constructs can be introduced into plant cells in which the synthesis of endogenous GBSS I, SSS or GBSS II proteins is already inhibited due to an antisense effect or a mutation or the synthesis of the Branching enzyme is inhibited (such as in WO 92/14827 or Shannon and Garwood, 1,984, in Whistler, BeMiller and Paschall, Starch: Chemistry and Technology, Academic Press, London, 2nd Edition: 25-86).
  • DNA molecules can be used for the transformation which simultaneously contain several regions coding for the corresponding enzymes in antisense orientation under the control of a suitable promoter.
  • each sequence can be under the control of its own promoter, or the sequences can be transcribed as a fusion from a common promoter or under the control of 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.
  • the resulting transcript should preferably not exceed a length of 10 kb and in particular a length of 5 kb.
  • Coding regions which are located in such DNA molecules in combination with other coding regions in antisense orientation 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 (SSS I and II), branching enzymes (isoamylases, pullulanases, R enzymes, "branching” enzymes, “debranching” enzymes), starch phosphorylases and disproportionation enzymes.
  • GBSS I and II starch-bound
  • SSS I and II soluble Starch synthases
  • branching enzymes isoamylases, pullulanases, R enzymes, "branching” enzymes, "debranching” enzymes
  • starch phosphorylases starch phosphorylases and disproportionation enzymes.
  • constructs can be introduced into plant mutants which are defective for one or more genes of starch biosynthesis (Shannon and Garwood, 1,984, in Whistler, BeMiller and Paschall, Starch: Chemistry and Technology, Academic Press, London, 2nd Edition: 25 -86). These defects can relate to the following proteins: starch-bound (GBSS I and II) and soluble starch synthases (SSS I and II), branching enzymes (BE I and II), "debranching" enzymes (R-enzymes), disproportionation enzymes and starch phosphorylases . This is only an example.
  • a large number of cloning vectors are available to prepare the introduction of foreign genes into higher plants, 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, M 1 3mp series, pACYC1 84 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 transformation plant cells with T-DNA using Agrobacterium tumefaciens or Agrobacterium rhizogenes as transformation agents, 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, there are no special requirements for 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 generally necessary.
  • the Ti or Ri plasmid is used for the transformation of the plant cell, at least the right boundary, but frequently the right and left boundary of the Ti and Ri plasmid T-DNA as the flank region, must be linked to the genes to be introduced.
  • the DNA to be introduced must be cloned into special plasmids, either in an intermediate vector or in a binary vector.
  • the intermediate vectors can be integrated into the Ti or Ri plasmid of the agrobacteria on the basis of sequences which are homologous to sequences in the T-DNA by homologous recombination. This also contains the vir region necessary for the transfer of the T-DNA. Intermediate vectors cannot replicate in agrobacteria. Using a helper plasmid, the intermediate vector can be transferred to Agrobacterium tumefaciens (conjugation). Binary vectors can replicate in both E. coli and agrobacteria.
  • 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 may be present.
  • the agrobacterium transformed in this way can be used to transform plant cells.
  • T-DNA for the transformation of plant cells has been intensively investigated and is sufficient in EP 1 20 51 6; Hoekema, In: The Binary Plant Vector System Offsetdrukkerij Kanters B.V., Alblasserdam (1 985), Chapter V; Fraley et al., Crit. Rev. Plant. Sci., 4, 1-46 and An et al. EMBO J. 4 (1 985), 277-287.
  • plant explants can expediently be cultivated with Agrobacterium tumefaciens or Agrobacterium rhizogenes.
  • the infected plant material e.g. leaf pieces, stem segments, roots, but also protoplasts or suspension-cultivated plant cells
  • a suitable medium which, among other things. certain sugars, amino acids, antibiotics or biocides for the selection of transformed cells can contain, whole plants can be regenerated again.
  • the plants thus obtained can then be examined for the presence of the introduced DNA.
  • Other possibilities of introducing foreign DNA using the biolistic method or by protoplast transformation are known (cf. for example Willmitzer, L., 1 993 Transgenic plants.
  • the first transgenic, fertile wheat plant which after bombardment with microprojectile bound DNA could be regenerated by Vasil et al. (Bio / Technology 10 (1,992), 667-674).
  • the target tissue for the bombardment was an embryogenic callus culture (type C callus).
  • the bar gene which encodes a phosphinothricin acetyltransferase and thus imparts resistance to the herbicide phosphinothricin was used as the selection marker.
  • Weeks et al. Plant Physiol. 102 (1,993), 1077-1084
  • Becker et al. Plant J. 5 (2) (1 994), 299-307.
  • the target tissue for DNA transformation here is the scutellum of immature embryos, which was stimulated in an introductory in vitro phase to induce somatic embryos.
  • the efficiency of the transformation lies with that of Becker et al. (loc cit.) developed system with 1 transgenic plant per 83 embryos of the "Florida" variety significantly higher than that of Weeks et al. established system with 1 to 2 transgenic plants per 1000 "Bohwhite” embryos.
  • 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 one of the selection markers mentioned above, which e.g. Resistance to a biocide such as phosphinothricin or an antibiotic such as Kanamycin, G 41 8, bleomycin or hygromycin mediated or the selection of the presence or absence of certain sugars or amino acids allowed.
  • the individually selected marker should therefore allow the selection of transformed cells over 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 (1 986), 81-84).
  • the resulting plants can be grown normally and with plants that have the same transformed genetic makeup or other genetic makeup, are crossed.
  • 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 Laboratories, Gaithersburg, USA) was used for the Bluescript vector and for the antisense constructs.
  • the E. coli strain XL1-Blue was used for the in vivo excision.
  • the specified media were adjusted to pH 5.6 with KOH and solidified with 0.3% Gelrite.
  • ears with caryopses of developmental level 1 are harvested 2 to 14 days after anthesis and surface sterilized.
  • the isolated scutella are plated with the embryo axis facing the medium on induction medium # 30.
  • the target DNA is added to the precipitation mixture in a ratio of 1: 1, consisting of the target gene and a resistance marker gene (bar gene).
  • DNA fragments that were used as screening probes were labeled using a specific PCR with the incorporation of DIG-labeled dUTP (Boehringer Mannheim, Germany).
  • the plasmid pTaSSI 8/1 was deposited at the DSMZ in Braunschweig, Federal Republic of Germany in accordance with the Budapest Treaty under number DSM 1 2794.
  • Example 1 Identification, isolation and characterization of a cDNA encoding a soluble starch synthase (SS I) from wheat (Triticum aestivum L., cv. Florida)
  • SS I soluble Starch synthase
  • the wheat cDNA bank was synthesized from poly (A) + RNA from approx. 20 day old caryopses (endosperm) in a Lambda Zap II vector analogous to the manufacturer's instructions (Lambda ZAP 11-cDNA Synthesis Kit, Stratagene GmbH, Heidelberg, Germany ). After determining the titer of the cDNA bank, a primary titer of 1.26 ⁇ 10 6 pfu / ml could be determined.
  • poly (A) + RNA of approximately 20 days old caryopses was transcribed into single-stranded cDNA and used in a tailing reaction.
  • the resulting cDNA provided in the 5 'region with the oligo (dA) anchor # 9 (kit) was amplified in a first reaction with the primers oligo (dT) # 8 (kit) and B2F5 according to a modified protocol as follows: 5 ⁇ l of thawed cDNA, 5 ⁇ l of 10 ⁇ reaction buffer (Life Technologies), 0.25 ⁇ M B2F5 primer, 0.75 ⁇ M oligo (dT) # 8, 0.2 mM dNTP's and 5U Taq polymerase (recombinant , Life Technologies).
  • the PCR profile was: 94 ° C 3794 ° C 45 "/ 56 ° C 1 772 ° C 1 '30", 29 cycles / 72 ° C 5'. This was followed by a further PCR with the primers Oligo (dT) # 8 (kit),
  • B2F5 primer 0.25 ⁇ M B2F6 primer, 0.75 ⁇ M oligo (dT) # 8, 0.2mM dNTP's and
  • 5U Taq polymerase (recombinant, Life Technologies) used.
  • the PCR profile was:
  • B2F5 5'CCTCCCAATTCAAGGATTAGTG 3 '(Seq ID No. 3)
  • B2F6 5'CCTCGCATGCAGCATAGCAA 3 '(Seq. ID No. 4)
  • PCR products obtained by the above methods were separated in an agarose gel and the DNA fragments with a size greater than 800 bp were isolated.
  • the PCR fragments were cloned using the pCR-Script SK (+) cloning kit from Stratagene (Heidelberg). Sequence analysis of the cloned subfragments identified approximately 1 50 bp previously unknown sequence of the SS I clone.
  • the oligonucleotides B2R00 and B2F6.2 were selected for the amplification of a DNA fragment (SS I probe), which was then labeled with digoxygenin-1 1 -UTP as described and as a probe for screening the wheat cDNA bank was used.
  • the SS I probe was labeled by means of a PCR reaction with the primers B2R00 and B2F6.2 analogously to the information in "The DIG System Users Guide for Filter Hybridization" (Boehringer Mannheim).
  • the clone TaSSI 8/1 was further analyzed.
  • the plasmid DNA was isolated from the clone TaSSI 8/1 and the sequence of the cDNA insertions was determined using the dideoxynucleotide method (Sanger et al., Proc. Natl. Acad. Sci. USA 74 (1 977), 5463-5467).
  • the insertion of the clone TaSSI 8/1 is 2805 bp long and represents a complete cDNA.
  • the nucleotide sequence is shown under Seq ID No. 1 specified.
  • the corresponding amino acid sequence can be found under Seq ID No. 2 specified.
  • a comparison with previously published sequences showed that the under Seq ID No. 1 sequence is new and includes a complete coding region.
  • Example 3 Preparation of the plant transformation vector pTa-gamma-SSI-8/1
  • the expression of the cDNA isolated under Example 1 was based on constructed from pUC1 9 as the base plasmid the plant transformation vector pTa-gamma-SSI-8/1.
  • the cDNA insertion of the plasmid TaSSI 8/1 is connected completely in sense orientation to the 3 'end of the ubiquitin promoter.
  • This promoter consists of the first untranslated exon and the first intron of the ubiquitinl gene from maize (Christensen AH et al., Plant Molecular Biology 1 8 (1 992), 675-689).
  • Parts of the polylinker and the NOS terminator originate from the plasmid pACT1 .cas (CAMBIA, TG 0063; Cambia, GPO Box 3200, Canberra ACT 2601, Australia). Vector constructs with this terminator and constructions based on pActl .cas are described in MCElroy et al. (Molecular Breeding 1 (1 995), 27-37). The resulting vector was called pUbi.cas.
  • the expression vector was cloned by restricting a fragment from the clone TaSSI 8/1 with the restriction enzymes Xba I and Ssp. I. The fragment was filled in at the ends using a Klenow reaction and then ligated into the Sma I cloning site of the expression vector pUbi.cas. The resulting expression vector was called pTA-gamma-SSI 8/1.
  • the 5'-untranslated leader of the clone TaSSI-8/1 was first removed by treatment with exonnuclease. The cloning into the expression vector pUbi.cas was then carried out. This construct was called Ta-gamma-SSI-8/1 -2.
  • microorganism referred to under I has been received by this international depository on (date of first deposit) and an application for conversion of this first deposit into a deposit under the Budapest Treaty has been received on (date of receipt of the application for conversion)

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HUP0101833A2 (hu) 2001-10-28
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NO20005614L (no) 2001-01-04
SK15772000A3 (sk) 2001-06-11
JP2002514427A (ja) 2002-05-21
US6890732B1 (en) 2005-05-10
CA2328508A1 (en) 1999-11-18
AU4039199A (en) 1999-11-29
NO20005614D0 (no) 2000-11-07
WO1999058688A3 (de) 2000-01-13
KR20010043457A (ko) 2001-05-25
CN1299415A (zh) 2001-06-13
DE19820607A1 (de) 1999-11-11

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