WO1999015674A2 - Transgene pflanzen, deren oberirdische teile früher reifen und vollständig absterben - Google Patents
Transgene pflanzen, deren oberirdische teile früher reifen und vollständig absterben Download PDFInfo
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- WO1999015674A2 WO1999015674A2 PCT/EP1998/005973 EP9805973W WO9915674A2 WO 1999015674 A2 WO1999015674 A2 WO 1999015674A2 EP 9805973 W EP9805973 W EP 9805973W WO 9915674 A2 WO9915674 A2 WO 9915674A2
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
- C12N9/1241—Nucleotidyltransferases (2.7.7)
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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
- 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
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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
- 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
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
- Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants
Definitions
- the present invention relates to transgenic plants, the above-ground parts of which ripen earlier and faster than wild-type plants and die completely without this having an effect on the yield of underground plant parts.
- the plants are characterized by a decrease in the activity of GDP-mannose pyrophosphorylase in the cells.
- the invention further relates to the use of nucleic acid olecules which encode a GDP-mannose pyrophosphorylase, or of fragments of such molecules for the production of plants with a reduced activity of this enzyme.
- the present invention also relates to nucleic acid molecules which encode vegetable proteins with the enzymatic activity of a GDP-mannose pyrophosphorylase.
- the present application thus relates to transgenic plants containing plant cells which are genetically modified, the genetic modification leading to a reduction in the activity of an endogenous GDP-mannose pyrophosphorylase (EC 2.7.7.22) occurring in the plant cell in comparison with corresponding non-genetically modified cells of such plants .
- an endogenous GDP-mannose pyrophosphorylase EC 2.7.7.22
- the GDP-mannose pyrophosphorylase is a vegetable protein which is encoded by a nucleic acid molecule selected from the group consisting of:
- nucleic acid molecules encoding a protein comprising the amino acid sequence encoded by the insertion in the plasmid pGMPase (DSM 11746);
- nucleic acid molecules gue ⁇ sen the coding region which is contained in the insertion of plasmid pGMPase (DSM 11746);
- nucleic acid molecules (d) nucleic acid molecules, (d) hy ⁇ brid is with one strand of a nucleic acid molecule of (a), (b), (c) and / or; and (f) Nucleic acid molecules whose sequence deviates from the sequence of a nucleic acid molecule according to (e) due to the degeneration of the genetic code.
- corresponding wild-type plants means that they are plants which served as starting material for the production of the plants according to the invention, i.e. whose genetic information, apart from the introduced genetic modification, corresponds to that of a plant according to the invention.
- Ripening means the ripening and death of the aerial parts of the plants. Ripening and dying preferably means the appearance of dark spots on stems first, the appearance of lesions on leaves in the form of small black dots, and the drying of the leaves. The leaf preferably dries progressively from the tip to the stem. In addition, the leaves of the plant, which begin at the bottom of the stem, preferably dry up first, and the drying of the leaves continues progressively, from bottom to top, on the plant, until finally all above-ground parts of the plant have ripened.
- the fact that the genetically modified plants ripen more quickly means that on the one hand the above-ground parts, in particular the leaves, stems and possibly fruits, start to die earlier than in the corresponding ones Wild-type plants and / or that the entire ripening process proceeds much faster than with corresponding wild-type plants and is completed earlier.
- the fact that the process starts "earlier” preferably means that the ripening and death of the aerial parts begins at least one week, preferably at least 2 to 3 weeks and particularly preferably about 4 weeks earlier than in the case of corresponding wild-type plants.
- the fact that the process of ripening takes place “faster” preferably means that the process of ripening and dying off the aerial parts of the plants is generally faster than in the case of corresponding wild-type plants, preferably by at least one week faster and particularly preferably by at least two weeks more quickly.
- the fact that the process of ripening and dying ends earlier means that the above-ground parts of the plants according to the invention have preferably died off at least 1 to 4 weeks, preferably at least 2 to 4 weeks and particularly preferably at least 3 to 4 weeks earlier than the above-ground parts Parts of corresponding wild-type plants.
- the fact that the ripening process is completed earlier preferably means that the above-ground parts of the plants according to the invention have already completely died off before the ripening process has started on the above-ground parts of corresponding wild-type plants.
- the aerial parts of plants according to the invention die about 4 weeks earlier than those of corresponding wild-type plants if the plants are kept under the following conditions: in 16-liter pots with potting soil (prickly soil) / sand mixture 3: 1; Humidity 60%; Temperature 22 ° C during the day / 15 ° C at night; 16 h light / 8 h dark rhythm; Light intensity approx. 150 ⁇ E.
- plants according to the invention ripen completely means that all above-ground parts of the plant die, in particular that these parts die completely.
- Such plants according to the invention have the advantage, for example, that they are completely based on the use of herbicides. can be pulled in order to achieve a uniform ripening of the aerial plant parts.
- late-ripening potato plants it is now possible to change them in such a way that their herb ripens and dies earlier and completely.
- the herb of the plants according to the invention dies completely. Even with the earlier ripening potato plants used in agriculture, there is the problem that the herb never dies completely and the use of herbicides and herb beaters is therefore always necessary. By cultivating plants where the herb dies completely, the use of these measures could be completely dispensed with.
- transgenic means that cells of the plants according to the invention contain a foreign DNA sequence in their genome which does not occur in corresponding non-genetically modified cells of corresponding wild-type plants.
- Foreign DNA sequence means a DNA sequence that either does not naturally occur in the corresponding plant cells, i.e. which is heterologous to the cells, which in the specific spatial arrangement does not occur naturally in the plant cells or which is located at a location in the genome of the plant cell where it does not occur naturally.
- the foreign DNA sequence is preferably a recombinant molecule which consists of different elements, the combination or specific spatial arrangement of which does not naturally occur in plant cells.
- the presence, the location and the spatial arrangement of the foreign DNA sequence in the genome of the cells of plants according to the invention can be demonstrated, for example, by Southern blot analysis.
- the term "genetically modified" means that the plant cell is changed in its genetic information by the introduction of a foreign nucleic acid molecule and that the presence or expression of the foreign nucleic acid molecule leads to a phenotypic change. Phenotypic change preferably means a measurable change in one or more functions of the cells.
- the genetically modified plant cells of the plants according to the invention show, owing to the presence or expression of the introduced foreign nucleic acid molecule, a reduction in the activity of a GDP-mannose phosphorylase endogenously occurring in the plant.
- GDP-mannose pyrophosphorylase (EC 2.7.7.22) catalyzes the conversion of GTP and mannose-1-phosphate to GDP-mannose and pyrophosphate.
- the term “reduction in activity” means a reduction in the expression of endogenous genes which encode a GDP-mannose pyrophosphorylase, a reduction in the amount of GDP-mannose pyrophosphorylase in the cells and / or a reduction in the enzymatic activity of the GDP-mannose pyrophosphorylase in the cells.
- the reduction in expression can be determined, for example, by measuring the amount of transcripts encoding GDP-mannose pyrophosphorylase, e.g. by Northern blot analysis.
- a "reduction” preferably means a reduction in the amount of transcripts in comparison to corresponding non-genetically modified cells by at least 10%, preferably by at least 30%, in particular by at least 50%, particularly preferably by at least 70% and in particular by at least 90% .
- the reduction in the amount of GDP-mannose pyrophosphorylase can be determined, for example, by Western blot analysis.
- a “reduction” preferably means a reduction in the amount of GDP-mannose pyrophosphorylase protein in comparison to corresponding non-genetically modified cells by at least 10%, preferably by at least 30%, in particular by at least 50%, particularly preferably by at least 70% and in particular by at least 90%.
- the reduction in the enzymatic activity can be determined, for example, by converting GTP and mannose-1-phosphate to GDP-mannose and pyrophosphate.
- the activity of GDP-mannose pyrophosphorylase can be determined, for example, as described in Asua et al. (Biochim. Biophys.
- a "reduction" in the enzymatic activity preferably means a reduction in comparison to corresponding non-genetically modified cells by at least 10%, preferably by at least 30%, in particular by at least 50%, particularly preferably by at least 70% and in particular by at least 90%.
- the genetic modification by means of a foreign nucleic acid molecule which has been introduced into the cells of the plants according to the invention and whose presence or expression leads to a reduction in GDP-mannose pyrophosphorylase activity can be of various types.
- the genetic modification can consist in that an endogenously present gene in the plant cell which encodes a GDP-mannose pyrophosphorylase is changed by the introduction of a foreign nucleic acid molecule in such a way that its expression is reduced or completely suppressed.
- a reduction preferably means a reduction in transcription by at least 10%, preferably by at least 30%, in particular by at least 50%, particularly preferably by at least 70%, in particular at least 90% in comparison to non-genetically modified cells.
- This can be determined, for example, using Northern blot analysis.
- This reduction in transcription preferably brings about a corresponding reduction in the activity of GDP-mannose pyrophosphorylase.
- Such a genetic modification can encode, for example, in an insertion or deletion. the region or promoter region of a GDP-mannose pyrophosphorylase gene.
- the genetic modification can induce the synthesis of a GDP-mannose pyrophosphorylase that no longer exhibits enzymatic activity. This can e.g. are based on mutations in the coding region of the gene which lead to a loss of the enzymatic activity or to incorrect splicing of the mRNA.
- a foreign nucleic acid molecule that encodes an inhibitor of GDP-mannose pyrophosphorylase, e.g. an antibody whose binding to the enzyme leads to inhibition of the enzymatic activity.
- the genetic modification preferably consists in introducing a foreign nucleic acid molecule into the genome of the plant cell, the expression of which leads to the inhibition of the expression of endogenous genes which encode a GDP-mannose pyrophosphorylase, in particular at the transcriptional or translation level.
- nucleic acid molecules which encode a suitable antisense RNA, a suitable ribozyme or a suitable cosuppression RNA.
- a corresponding DNA sequence is expressed in an antisense orientation, so that an antisense RNA is synthesized in the cells, which is homologous to transcripts of GDP-mannose pyrophosphorylase genes or to parts of such transcripts.
- This preferably has a length of at least 30 nucleotides, preferably of at least 50 nucleotides and particularly preferably of at least 100 nucleotides.
- the expressed antisense RNA should have a high homology to the transcripts which are endogenously expressed in the plant and which encode GDP-mannose pyrophosphorylase.
- the homology is preferably at least 90 %, preferably at least 95% and particularly preferably at least 99%.
- RNA that can specifically cleave transcripts of a GDP-mannose pyrophosphorylase gene.
- the expression of 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 was described, for example, in Feyter et al. (Mol. Gen. Genet. 250 (1996), 329-338).
- the cosuppression effect is based on the expression of a sense RNA which suppresses the expression of endogenous GDP-mannose pyrophosphorylase RNA. The implementation of these techniques is known to the person skilled in the art.
- the reduction in the activity of a GDP-mannose phosphorylase in the cells is achieved in that the foreign nucleic acid molecule comprises the following elements:
- Nucleic acid molecules encoding vegetable GDP-mannose pyrophosphorylase are provided by the present invention.
- the in Seq ID No. 1 nucleic acid sequence encodes a GDP-mannose pyrophosphorylase from potato. With the aid of this sequence, the person skilled in the art is able to isolate corresponding sequences from other plants or plant species using common molecular biological methods (see below).
- nucleic acid molecules according to the invention from potato or parts thereof described below are used for the expression of an antisense RNA.
- all cells or essentially all cells of the plants according to the invention have the genetic modification. In contrast, it is not absolutely necessary for all cells to have a reduction in the activity of GDP-mannose pyrophosphorylase. In a first embodiment it is provided that all or essentially all cells of the plant have a reduced GDP-mannose pyrophosphorylase activity.
- only cells of parts of plants above ground preferably only cells in the leaves or only cells in the leaves and stems, have a reduced GDP-mannose pyrophosphorylase activity due to the expression of the foreign nucleic acid molecule.
- the plants according to the invention can in principle be a plant of any plant species, ie both monocotyledonous and dicotyledonous plants. It is preferably an agricultural crop, particularly preferably cotton or potato, and in particular late-ripening potato plants.
- the present invention also relates to transgenic plant cells which are genetically modified by the introduction of a foreign nucleic acid molecule, the presence or expression of this nucleic acid molecule causing the activity of a GDP-mannose pyrophosphorylase which is endogenously present in the plant cell to be reduced.
- Such plant cells according to the invention can be used for the regeneration of whole plants.
- the present invention also relates to a process for the production of transgenic plants, the above-ground parts of which ripen earlier and faster than in the case of corresponding wild-type plants and die completely, whereby
- a plant cell is genetically modified by the introduction of a foreign nucleic acid molecule and the genetic modification leads to a reduction in the activity of a GDP-mannose pyrophosphorylase which is endogenously present in plant cells;
- step (c) further plants are produced from the plant according to (b).
- the plants can be regenerated in accordance with step (b) by methods known to the person skilled in the art.
- step (c) of the process can e.g. occur through vegetative propagation (for example, through cuttings, tubers or via callus culture and regeneration of whole plants) or through sexual reproduction.
- Sexual reproduction is preferably controlled, i.e. selected plants with certain characteristics are crossed and propagated.
- the method is used to produce transgenic potato plants.
- the introduced foreign nucleic acid molecule enables the synthesis of an antisense RNA which is complementary to transcripts of GDP-mannose pyrophosphorylase genes.
- the present invention also relates to the plants obtainable by the process according to the invention.
- the present invention also relates to propagation material of plants according to the invention and of the transgenic plants produced according to the method according to the invention which contains genetically modified cells according to the invention.
- propagation material includes those components of the plant that are suitable for the production of offspring by vegetative or sexual means. Cuttings, callus cultures, rhizomes or tubers are suitable for vegetative propagation. Other propagation material includes, for example, fruits or seeds.
- the propagation material is preferably seeds and particularly preferably tubers.
- the present invention also relates to the use of nucleic acid molecules which encode a GDP-mannose pyrophosphorylase, of their complements or of parts of such molecules for reducing the GDP-mannose pyrophosphorylase activity in plant cells, for producing plant cells with a reduced activity of the GDP-mannose pyrophosphorylase, for the production of plants containing such plant cells and / or for the production of transgenic plants, the above-ground parts of which mature and die faster than in corresponding wild-type plants and which die off completely.
- the nucleic acid molecules are preferably the nucleic acid molecules according to the invention described below.
- the present invention further relates to nucleic acid molecules which encode a vegetable protein, the mature form of which has the enzymatic activity of a GDP-mannose pyrophosphorylase, selected from the group consisting of:
- nucleic acid molecules encoding a protein comprising the amino acid sequence encoded by the insertion in the plasmid pGMPase (DSM 11746);
- nucleic acid molecules comprising the coding region contained in the insertion of the plasmid pGMPase (DSM 11746);
- nucleic acid molecules which hybridize with a strand of a nucleic acid molecule according to (a), (b), (c) and / or (d);
- nucleic acid molecules the sequence due to the Dege ⁇ neration of the genetic code from the sequence of a nucleic acid molecule according to (e) differs.
- the invention also relates to the complementary strands of the molecules described above.
- the in Seq ID No. 1 nucleic acid sequence shown is a complete cDNA encoding a GDP-mannose pyrophosphorylase from potato (Solanum tuberosum).
- a plasmid containing this cDNA was deposited as DSM 11746.
- the nucleic acid molecules according to the invention can code a GDP-mannose pyrophosphorylase from any plant organism, preferably one from a higher plant, in particular a monocotyledonous or a dicotyledonous plant.
- the nucleic acid molecule particularly preferably encodes a protein from an agricultural useful plant, in particular from a cereal plant (for example barley, rye, oats, wheat etc.), or from corn, rice, pea, flax etc.
- the nucleic acid molecule encodes Protein from potato or cotton.
- 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 that can hybridize with the nucleic acid molecules according to the invention in principle come from any plant that expresses the corresponding proteins.
- Nucleic acid molecules that hybridize with the molecules according to the invention can be isolated, for example, from genomic or from cDNA libraries.
- nucleic acid molecules can be identified and isolated using the nucleic acid molecules according to the invention or parts of these molecules or the reverse complements of these molecules, for example by means of hybridization according to standard methods (see for example Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2. 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 ones under Seq ID No. 1 indicated nucleotide sequence or parts of this sequence.
- the fragments used as the hybridization sample can also be synthetic fragments which have been 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.
- the sequence should be determined and the properties of the proteins encoded by this sequence should be analyzed to determine whether it is a GDP-mannose pyrophosphorylase. Homology comparisons at the level of the nucleic acid or amino acid sequence and the determination of the enzymatic activity are particularly suitable for this.
- the molecules hybridizing with the nucleic acid molecules according to the invention include in particular fragments, derivatives and allelic variants of the nucleic acid molecules described above, which encode a vegetable protein with the activity of a GDP-mannose pyrophosphorylase.
- 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 a high degree of homology to them Have sequences.
- Homology means a sequence identity over the entire length of at least 60%, in particular an identity of at least 70%, preferably over 80%, particularly preferably over 90% and in particular at least 95%.
- the deviations from the nucleic acid molecules described above may have arisen, for example, through deletion, addition, 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 plants, in particular other cereal plants or varieties, or mutations, wherein these mutations can have occurred naturally or have been introduced by targeted mutagenesis.
- the variations can be synthetically produced sequences.
- allelic variants can be both naturally occurring variants and also synthetically produced variants or those produced by recombinant DNA techniques.
- the proteins encoded by the different variants of the nucleic acid molecules according to the invention have certain common characteristics. These can include, for example, biological activity, molecular weight, immunological reactivity, conformation etc., and physical properties such as running behavior in gel electrophoresis, chromatographic behavior, sedimentation coefficients, solubility, spectroscopic properties, stability; pH optimum, temperature optimum etc.
- Important characteristics of GDP-mannose pyrophosphorylase are (i) its location in the cytosol of plant cells, (ii) its ability to synthesize GDP-mannose using GTP and mannose-1-phosphate as a substrate. This activity can be determined, for example, according to the in Asua et al. (loc. cit.) or in Szumilo et al. (op. cit.) described methods.
- the molecular weight of the GDP-mannose pyrophosphorylase from potato, derived from the amino acid sequence, is 39,578 daltons.
- the derived molecular weight of a protein according to the invention is therefore preferably in the range from 35 kDa to 45 kDa, preferably in the range from 38 kDa to 42 kDa and particularly preferably approximately 40 kDa.
- the calculated isoelectric point of the protein from potato is 7.21.
- the nucleic acid molecules according to the invention can be any nucleic acid molecules, in particular DNA or RNA molecules, for example cDNA, genomic DNA, mRNA etc. They can be naturally occurring molecules or molecules produced by genetic engineering or chemical synthesis methods. They can be single-stranded molecules that contain either the coding or the non-coding strand, or double-stranded molecules.
- the present invention relates to nucleic acid molecules of at least 15, preferably more than 50 and particularly preferably more than 200 nucleotides in length, which hybridize specifically with at least one nucleic acid molecule according to the invention.
- Hybridizing specifically here means that these molecules hybridize with nucleic acid molecules which encode a protein according to the invention, but not with nucleic acid molecules which encode other proteins.
- Hybridization 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. be able to prevent their translation.
- Such nucleic acid molecules which hybridize specifically with the nucleic acid molecules according to the invention can, for example, be components of antisense constructs or ribozymes or can be used as primers for amplification by means of PCR.
- the invention further relates to vectors, in particular plasmids, cosmids, viruses, bacteriophages and other vectors which are common in genetic engineering and which contain the nucleic acid molecules according to the invention described above.
- nucleic acid molecules contained in the vectors are linked in sense orientation to regulatory elements which ensure expression in prokaryotic or eukaryotic cells.
- expression can mean transcription as well as transcription and translation.
- nucleic acid molecules according to the invention in prokaryotic cells, for example in Escherichia coli, enables, for example, a more precise characterization of the enzymatic activities of the encoded proteins.
- mutants can be produced which have a changed ⁇ value or no longer are subject to the regulatory mechanisms normally present in the cell via allosteric regulation or covalent modification.
- mutants can be produced which have an altered substrate or product specificity.
- mutants can be produced which have a changed activity-temperature profile. Genetic engineering manipulation in prokaryotic cells can be carried out according to methods known to the person skilled in the art (cf. Sambrook et al., Loc. Cit.).
- a large number of promoters are available for the expression of a nucleic acid molecule according to the invention in plant cells.
- any promoter that is functional in the plants selected for the transformation can be used.
- 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 constituent 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., Loc. Cit.).
- 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. It is also possible to use the promoters which lead to expression of downstream sequences in a particular tissue of the plant (see, for example, Stockhaus et al., EMBO J. 8 (1989), 2245-2251), for example the ST-LS1- Promoter that is only active in photosynthetically active tissue (Stockhaus et al., Proc. Natl. Acad. Sci. USA 84 (1987), 7943-7947).
- promoters which are active in the starch-storing organs of plants to be transformed.
- these are the corn kernels, while the potatoes are the tubers.
- the tuber-specific B33 promoter (Rocha-Sosa et al., EMBO J. 8 (1989), 23-29) can, for example, be used in the potato to precess the nucleic acid molecules according to the invention.
- Seed-specific promoters have already been described for various plant species. For example, the USP promoter from Vicia faba, which ensures seed-specific expression in V. faba and other plants (Fiedler et al., Plant Mol. Biol.
- promoters of the zein genes ensure specific expression in the endosperm of the maize kernel (Pedersen et al., Cell 29 (1982), 1015-1026; Quattrocchio et al., Plant Mol. Biol. 15 (1990), 81-93 ).
- prokaryotic organisms e.g. E. coli are sufficiently described in the literature, as are those for expression in yeast, in particular in Saccharomyces cerevisiae.
- Termination signals for transcription in plant cells are described and can be interchanged as desired.
- the termination sequence of the octopine synthase gene from Agro acterium tumefaciens can be used.
- the invention relates to host cells, in particular prokaryotic or eukaryotic cells, which have been transformed with a nucleic acid molecule or a vector described above, and cells which are derived from such host cells and contain the nucleic acid molecules or vectors described.
- the host cells can be bacterial (eg E. coli) or fungal cells (eg yeast, in particular S. cerevisiae), as well as plant or animal cells.
- the term "transformed” means that the cells of the invention are genetically modified with a nucleic acid molecule of the invention insofar as they contain at least one nucleic acid molecule of the invention in addition to their natural genome. This can be freely in the cell, if present as a self-replicating molecule, or it can be stably integrated into the genome of the host cell.
- the present invention further relates to methods for producing a GDP-mannose pyrophosphorylase, in which host cells according to the invention are cultivated under conditions which allow expression of the protein, and the protein from the culture, i.e. is obtained from the cells and / or the culture medium.
- the invention also relates to proteins which are encoded by the nucleic acid molecules according to the invention or which are obtainable by a method according to the invention.
- the present invention further relates to antibodies which specifically recognize a protein according to the invention.
- These can e.g. be monoclonal or polyclonal antibodies. It can also be fragments of antibodies that recognize proteins according to the invention. Methods for producing such antibodies or fragments are known to the person skilled in the art.
- the host cells according to the invention are transgenic plant cells which, owing to the presence of an introduced nucleic acid molecule according to the invention, express a GDP-mannose pyrophosphorylase according to the invention.
- 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 modify plant cells to the extent that they express a new or an additional plant GDP-mannose pyrophosphorylase.
- transgenic plant cells differ from untransformed cells in that the nucleic acid molecule introduced is either heterologous to the transformed cell, that is to say from a cell with a different genomic background, or in that the introduced one Nucleic acid molecule, if it is homologous to the transformed plant species, is located in the genome at a location where it is not naturally found in non-transformed cells.
- the nucleic acid molecule introduced can either be under the control of its natural promoter or linked to regulatory elements of foreign genes.
- the plant cells according to the invention preferably differ from corresponding non-transformed plant cells in that they have an increased amount of protein according to the invention.
- This can be determined using detection methods known to the person skilled in the art, e.g. Western blot analysis.
- An increased amount preferably means an increase of at least 5%, preferably at least 10% and particularly preferably at least 20%.
- the synthesized protein can be localized in any compartment of the plant cell.
- the 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. 11 (1992), 3219-3227; Wolter et al., Proc. Natl. Acad. Sci. USA 85 (1988), 846-850; Sonnewald et al ., Plant J. 1 (1991), 95-106).
- the invention further relates to transgenic plants which contain the transgenic plant cells described above.
- the plant which contains plant cells according to the invention can be any plant. It is preferably a monocot or dicot plant, in particular an agricultural crop, for example a starch-storing plant, such as, for example, cereal plants (rye, barley, oats, wheat, etc.), rice, corn, legumes (for example pea), cassava or potato, or an ornamental plant .
- Cereal plants are understood in particular as monocotyledonous plants belonging to the Poales order, preferably those belonging to the Poaeeae family. Examples of this are the plants belonging to the genera Avena (oat), Triticum
- Vicia e.g. Vicia faba
- Cicer e.g. Cicer arietinum
- Lens e.g. Lens culinaris
- Phaseolus vulgaris and Phaseolus coccineus e.g. Phaseolus vulgaris and Phaseolus coccineus
- the invention furthermore relates to propagation material from transgenic plants according to the invention, for example seeds, fruits, cuttings, tubers, rhizomes, calli, cell cultures etc., this propagation material containing transgenic plant cells described above.
- the present invention further relates to transgenic plant cells in which the activity of the protein 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 protein.
- This can be achieved, for example, by expressing a nucleic acid molecule according to the invention or a part thereof in the corresponding plant cells in an antisense orientation and by reducing the amount of protein according to the invention due to an antisense effect.
- Another possibility for reducing the amount of the protein according to the invention in plant cells is the expression of suitable ribozymes which specifically cleave transcripts of the DNA molecules according to the invention. It is also possible to express molecules which have both an antisense and a ribozyme effect in combination.
- the amount of protein according to the invention in the plant cells can also be reduced by a cosupression effect. Such methods are known to the person skilled in the art (see above).
- the reduction in the activity of the protein according to the invention can be determined as described above.
- the invention further relates to transgenic plants which contain the transgenic plant cells described above.
- the invention also relates to propagation material of the transgenic plants described above, for example seeds, fruits, cuttings, tubers, rhizomes, calli, cell cultures, etc., which contains the transgenic plant cells described above.
- 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 Agrobacteriu tumefaciens or Agrobacterium rhizogenes as a transformation agent, the fusion of protoplasts, the injection, the electroporation of DNA, the introduction of DNA using the biological method and other possibilities.
- 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 GDP-mannose pyrophosphorylase.
- FIG. 1 shows schematically the plasmid pGMPase.
- the fine line corresponds to the sequence of pBluescript II SK (-).
- the strong line represents the cDNA encoding the GDP mannose pyrophosphorylase from Solanum tuberosum. Restriction sites of the insertion are given.
- the cDNA insert is ligated between the EcoRI and Xhol sites of the polylinker of the plasmid.
- the DNA sequence of the cDNA insertion is under Seq ID No. 1 specified.
- Figure 2 shows schematically the plasmid p35S-antiGMPase.
- A Fragment A: CaMV35S promoter of the cauliflower mosaic virus, 540 bp.
- C fragment C: ocs terminator of the octopine synthase gene from Agrobacterium turne f ciens.
- FIG. 3 shows a comparison of the amino acid sequences of GDP-mannose pyrophosphorylases from Saccharomyces cerevisiae (bottom line in each case and Solanum tuberosum (top line in each case).
- FIG. 4 shows a Northern blot analysis of the expression of the GMPase in tubers of GDP-mannose pyrophosphorylase "antisense" plant (lines 6, 28 and 37) in comparison to corresponding non-transformed wild-type plants (WT).
- FIG. 5 shows a comparison of 3-month-old transgenic GMPase “antisense” plants (right and middle) and a wild type plant of the same age (left).
- FIG. 6 shows the appearance of dark spots on the stems and leaves of 3-month-old transgenic GMPase “antisense” plants and the progressive drying of the leaves.
- 6.5 x 10 phage plaques from a cDNA library were prepared from leaves of potato plants (Koßmann et al., Planta 188 (1992), 7- 12) , screened for fragments contained therein that hybridize specifically with the probe used.
- the probe was derived from an EST (expressed sequence tag) from Arabidopsis thaliana, the sequence of which has homology to a yeast mannose-1-phosphate guanyl transferase (EC 2.7.7.13) (Gen Bank accession number T 21688).
- phage plaques were transferred to Hybond N nylon membranes (Amersham, UK) and the DNA was denatured by NaOH treatment, the filters were neutralized and the DNA was fixed on the filters by UV treatment.
- the filters were prehybridized in 0.25 M NaHP0 4 , pH 7.2, 0.25 M NaCl, 7% SDS, 1 mM EDTA, 25% formamide, 10% PEG for 2 hours at 42 ° C.
- the filters were then in 0.25 M NaHP0 4 , pH 7.2, 0.25 M NaCl, 7% SDS, 1 mM EDTA, 25% formamide, 10% PEG after addition of the corresponding radioactively labeled probe for 10 h at 42 ° C hybridizes.
- phage clones were further purified using standard procedures. With the help of the in vivo excision measurement methods were obtained from positive phage clones E. coli clones which contain a double-stranded pBluescript plasmid with the respective cDNA insert. After checking the size and restriction pattern of the inserts, a suitable clone, cGMPase, was further analyzed.
- the plasmid pGMPase (FIG. 1) was isolated from the clone and its 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 1524 kb long and represents a complete cDNA.
- a part of the nucleotide sequence (1478 bp) is under Seq ID No. 1 specified.
- the deduced amino acid sequence is in Seq ID No. 2 specified. Sequence comparisons showed that this DNA sequence and the deduced amino acid sequence in different areas showed strong homology to the cDNA from Saccharomyces cerevisiae, which encodes a GDP-mannose pyrophosphorylyase, or to the corresponding amino acid sequence (see FIG. 3).
- Fragment A (540 bp) contains the 35S promoter from the cauliflower mosaic virus.
- fragment B contains the protein-coding region of a GDP-mannose pyrophosphorylase from Solanum tuberosum. This was isolated from pGMPase as described above and fused in antisense orientation to the 35S promoter in pBinAR.
- Fragment C (215 bp) contains the polyadenylation signal of the octopine synthase gene from Ag-robacte ium tumefaciens.
- the size of the plasmid p35S-antiGMPase is approximately 13.5 kb.
- the plasmid was transferred into potato plants using Agrobacteria.
- the leaves for callus induction were then placed on MS medium with 1.6% glucose, 5 mg / 1 naphthylacetic acid, 0.2 mg / 1 benzylaminopurine, 250 mg / 1 claforan, 50 mg / 1 kanamyeine and 0.8% Bacto Agar placed. After incubation for one week at 25 ° C. and 3000 lux, the leaves were induction on MS medium with 1.6% glucose, 1.4 mg / 1 zeatin ribose, 20 ⁇ g / 1 naphthylacetic acid, 20 ⁇ g / 1 giberellic acid, 250 mg / 1 Claforan, 50 mg / 1 kanamyein and 0.8% Bacto Agar. Whole plants were regenerated from the transformed cells.
- the transgenic potato plants obtained were kept in the greenhouse under the following conditions: light period: 16 h at 25,000 lux and 22 ° C. dark period: 8 h at 15 ° C. atmospheric humidity: 60%.
- transgenic potato plants showed a decrease in the activity of a GDP-mannose pyrophosphorylase and a decrease in the GDP-mannose pyrophosphorylase RNA (see FIG. 4).
- RNA was isolated from leaf tissue of plants according to standard protocols. 50 ⁇ g of RNA were separated on an agarose gel (1.5% agarose, 1 ⁇ MEN buffer, 16.6% formaldehyde). After electrophoresis, the RNA was capillary blotted with 20 x SSC onto a nylon membrane Hybond N
- RNA was fixed on the membrane by UV radiation.
- the membrane was in phosphate hybridization buffer
- Transgenic plants showed no differences in their appearance compared to untransformed plants both in sterile culture and up to two months after transfer to greenhouses. After two months, dark spots formed on the stem. Lesions in the form of small black dots were visible on the leaves (FIG. 6).
- the leaves of the plants started to dry up. First the tip of the terminal pinnate of a leaf was stretched until the whole leaf was brown and dried up to the stem. This ripening of the leaves continued, progressing from bottom to top, until finally all parts of the plant above ground had ripened (FIG. 5).
- Transgenic GDP-mannose antisense plants do not differ in their tuber yield from untransformed potato plants. Tubers from 15-week-old untransformed potato plants weighed an average of 169.7 g per plant. The yields for three independently transformed lines averaged 168.6 g (line 37), 162.5 g
- 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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Abstract
Description
Claims
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AU10241/99A AU1024199A (en) | 1997-09-19 | 1998-09-18 | Transgenic plants having early-ripening and completely withering aerial parts |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
DE19741375.7 | 1997-09-19 | ||
DE19741375A DE19741375C2 (de) | 1997-09-19 | 1997-09-19 | Transgene Pflanzen, deren oberirdische Teile früher reifen und vollständig absterben |
Publications (2)
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WO1999015674A2 true WO1999015674A2 (de) | 1999-04-01 |
WO1999015674A3 WO1999015674A3 (de) | 1999-05-20 |
Family
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Family Applications (1)
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PCT/EP1998/005973 WO1999015674A2 (de) | 1997-09-19 | 1998-09-18 | Transgene pflanzen, deren oberirdische teile früher reifen und vollständig absterben |
Country Status (3)
Country | Link |
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AU (1) | AU1024199A (de) |
DE (1) | DE19741375C2 (de) |
WO (1) | WO1999015674A2 (de) |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
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WO1996027670A2 (de) * | 1995-03-03 | 1996-09-12 | Forschungszentrum Jülich GmbH | ENZYMATISCHES VERFAHREN ZUR HERSTELLUNG VON GDP-α-D-MANNOSE, DAFÜR GEEIGNETE GDP-MANNOSE-PYROPHOSPHORYLASE UND PHOSPHOMANNOMUTASE UND DEREN GEWINNUNG SOWIE ENZYMTEST |
Family Cites Families (7)
Publication number | Priority date | Publication date | Assignee | Title |
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NL8300698A (nl) * | 1983-02-24 | 1984-09-17 | Univ Leiden | Werkwijze voor het inbouwen van vreemd dna in het genoom van tweezaadlobbige planten; agrobacterium tumefaciens bacterien en werkwijze voor het produceren daarvan; planten en plantecellen met gewijzigde genetische eigenschappen; werkwijze voor het bereiden van chemische en/of farmaceutische produkten. |
EP0846771A1 (de) * | 1987-05-20 | 1998-06-10 | Novartis AG | Zea mays-Pflanzen und transgene Zea mays-Pflanzen, die aus Protoplasten oder aus von Protoplasten erhaltenen Zellen regeneriert wurden |
ES2065919T5 (es) * | 1987-12-15 | 2005-06-16 | Gene Shears Pty Limited | Ribozimas. |
US7705215B1 (en) * | 1990-04-17 | 2010-04-27 | Dekalb Genetics Corporation | Methods and compositions for the production of stably transformed, fertile monocot plants and cells thereof |
DK0873682T3 (da) * | 1990-06-23 | 2002-12-30 | Bayer Cropscience Gmbh | Forbedrede genotyper af Zea mays (L.) med evne til langvarig, højeffektiv planteregenerering |
JPH05199877A (ja) * | 1991-10-03 | 1993-08-10 | Sumitomo Chem Co Ltd | バレイショおよびイネの誘導型植物防御遺伝子の制御領域、その用途およびアッセイ法 |
DE4222407C1 (de) * | 1992-07-08 | 1993-10-07 | Max Planck Gesellschaft | Modulartiges Promotor-Konstrukt |
-
1997
- 1997-09-19 DE DE19741375A patent/DE19741375C2/de not_active Expired - Fee Related
-
1998
- 1998-09-18 AU AU10241/99A patent/AU1024199A/en not_active Abandoned
- 1998-09-18 WO PCT/EP1998/005973 patent/WO1999015674A2/de active Application Filing
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1996027670A2 (de) * | 1995-03-03 | 1996-09-12 | Forschungszentrum Jülich GmbH | ENZYMATISCHES VERFAHREN ZUR HERSTELLUNG VON GDP-α-D-MANNOSE, DAFÜR GEEIGNETE GDP-MANNOSE-PYROPHOSPHORYLASE UND PHOSPHOMANNOMUTASE UND DEREN GEWINNUNG SOWIE ENZYMTEST |
Non-Patent Citations (5)
Title |
---|
DUWENIG E ET AL: "Antisense inhibition of cytosolic phosphorylase in potato plant ( Solanum tuberosum L.) affects tuber sprouting and flower formation with only little impact on carbohydrate metabolism." PLANT JOURNAL, (1997 AUG) 12 (2) 323-33. JOURNAL CODE: BRU. ISSN: 0960-7412., XP002093023 ENGLAND: United Kingdom * |
LAGRIMINI M. ET AL.: "Peroxidase-induced wilting in transgenic tobacco" THE PLANT CELL, Bd. 2, Januar 1990, Seiten 7-18, XP002093025 * |
OOMS G ET AL: "Phenotypic changes in T-cyt-transformed potato plants are consistent with enhanced sensitivity of specific cell types to normal regulation by root-derived cytokinin." PLANT MOLECULAR BIOLOGY, (1991 OCT) 17 (4) 727-43. JOURNAL CODE: A6O. ISSN: 0167-4412., XP002093024 Netherlands * |
ROUNSLEY S. ET AL.: "AC O22287" EMBL DATABASE,1. Januar 1998, XP002093110 Heidelberg * |
SASAKI T. ET AL.: "Rice cDNA from shoot, AC D48687" EMBL DATABASE,9. M{rz 1995, XP002093022 hEIDELBERG * |
Also Published As
Publication number | Publication date |
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DE19741375C2 (de) | 1999-10-21 |
WO1999015674A3 (de) | 1999-05-20 |
AU1024199A (en) | 1999-04-12 |
DE19741375A1 (de) | 1999-04-01 |
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