WO1999010500A1 - Molecules d'acides nucleiques codant pour une cysteine proteinase d'origine vegetale, et leurs regions regulatrices - Google Patents

Molecules d'acides nucleiques codant pour une cysteine proteinase d'origine vegetale, et leurs regions regulatrices Download PDF

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WO1999010500A1
WO1999010500A1 PCT/EP1998/005339 EP9805339W WO9910500A1 WO 1999010500 A1 WO1999010500 A1 WO 1999010500A1 EP 9805339 W EP9805339 W EP 9805339W WO 9910500 A1 WO9910500 A1 WO 9910500A1
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nucleic acid
acid molecule
root
sequence
plants
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PCT/EP1998/005339
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German (de)
English (en)
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Ursula Schlichter
Hans-Henning Steinbiss
John Antoniw
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MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V.
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Priority claimed from DE19737118A external-priority patent/DE19737118C1/de
Application filed by MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. filed Critical MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V.
Priority to AU96225/98A priority Critical patent/AU9622598A/en
Publication of WO1999010500A1 publication Critical patent/WO1999010500A1/fr

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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/63—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from plants
    • 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)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8237—Externally regulated expression systems
    • 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)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8222—Developmentally regulated expression systems, tissue, organ specific, temporal or spatial regulation
    • C12N15/8223—Vegetative tissue-specific promoters
    • C12N15/8227—Root-specific
    • 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)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8237—Externally regulated expression systems
    • C12N15/8239—Externally regulated expression systems pathogen inducible
    • 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
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C12N15/8282—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for fungal resistance

Definitions

  • the present invention relates to nucleic acid molecules encoding a cysteine proteinase from plants, transgenic plants which have been transformed with such nucleic acid molecules, and to the use of such nucleic acid molecules for the production of plants which have an increased resistance to pathogen attack or other stress factors. Furthermore, the present invention also relates to the regulatory regions of the nucleic acid molecules described, which ensure root-specific expression after infestation of the root with pathogens.
  • the present invention is therefore based on the object of providing nucleic acid molecules which are suitable for generating increased resistance to pathogens or other stress factors in plants.
  • the present invention thus relates to nucleic acid molecules comprising a nucleic acid sequence encoding a cysteine proteinase from plants, selected from the group consisting of
  • nucleic acid sequences one strand of which hybridizes with a nucleic acid sequence according to (a), (b), (c) and / or (d) and which encode a cysteine proteinase from plants;
  • the invention also relates to the complementary strands of the molecules described above.
  • the nucleic acid molecules according to the invention encode a cysteine proteinase from plants. These are preferably cysteine proteinases which have structural features of a cysteine proteinase from the papain family. It has been found that these proteins are expressed in connection with the pathogen defense in plants, in particular when barley is infected with the pathogen Gaeumannomyces graminis var. tritici.
  • the nucleic acid molecules now made available can therefore be introduced into plants and brought to expression under the control of suitable promoters, whereby, for example, increased resistance to disease can be achieved.
  • 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).
  • An example of stringent hybridization conditions is hybridization in 50% formamide, 5 x SSC, 5 x Denhardt's solution, 40 mM sodium phosphate pH 6.8; 0.5% (w / v) BSA, 1% (w / v) SDS, 0.1 mg / ml herring sperm DNA at a hybridization temperature of 42 ° C and subsequent washing of the filter in 0.5 x SSC / 0.5% SDS at 60 ° C, preferably in 0.1 x SSC, 0.1% SDS at 65 ° C.
  • nucleic acid molecules which hybridize with the nucleic acid molecules according to the invention can originate from any plant which 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 using standard methods (see, for example, Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2nd edition 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 nucleotide sequence or parts of this sequence or a sequence which exactly or essentially all or part of the in Seq ID No. 3 specified sequence, in particular the coding region, corresponds.
  • 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. If genes which hybridize with the nucleic acid sequences according to the invention have been identified and isolated, the sequence should be determined and the properties of the proteins encoded by this sequence should be analyzed.
  • the molecules which hybridize with the nucleic acid molecules according to the invention include, in particular, fragments, derivatives and allelic variants of the above-described nucleic acid molecules which code for a vegetable protein with the structural features of a cysteine proteinase. Fragments are understood to mean, among other things, parts of the nucleic acid molecules that.
  • fragments includes nucleic acid molecules that contain a protein with amino acids 23 to 365, 146 to 365 or 147 to 365 as in Seq ID No. 1 code shown.
  • the term derivative in this context means that the sequences of these molecules are different 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 over the entire length of at least 70%, in particular an identity of at least 80%, preferably over 90%, particularly preferably over 95% and in particular at least 98%.
  • 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.
  • the proteins which are encoded by the nucleic acid molecules according to the invention preferably have the structural properties of a cysteine proteinase from the papain family. Proteins of this type are understood in the context of the present invention as proteins which have at least one of the following features:
  • a protein encoded by a nucleic acid molecule according to the invention preferably has a sequence which corresponds to amino acid residues 92 to 105 of the sequence shown in Seq. ID No. 2 sequence shown correspond.
  • the encoded proteins also preferably have the enzymatic activity of a cysteine proteinase, i.e. the ability to cleave proteins proteolytically, whereby the amino acid dyad Cys-His catalyzes the reaction.
  • This activity can be determined, for example, as described in Koehler and Ho (Plant Physiol. 87 (1988), 95-103), Phillips and Wallace (Phytochemistry 28 (1989), 3285-3290), Poulle and Jones (Plant Physiol. 88 ( 1988), 1454-1460) or Smith and Gottesman_ (J. Biol. Chem. 264 (1989), 20487-20495).
  • a nucleic acid molecule according to the invention preferably has a sequence that corresponds to that in Seq ID No. 1 corresponds to the sequence contained at positions 77 to 94 with regard to the nucleotide sequence and position.
  • the nucleic acid molecules according to the invention also include, in particular, genomic sequences which comprise a coding region described above, interrupted by intron sequences, and also, if appropriate, flanking regions.
  • the flanking areas include, in particular, (i) 5 'flanking areas which transcribe 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.
  • 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 or only to a very small extent 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 and can thereby prevent their translation.
  • 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 hybridization samples or as primers for amplification by means of PCR. Nucleic acid molecules with a length of 17 to 35 nucleotides are particularly preferred.
  • the nucleic acid molecules described can be DNA or RNA molecules or hybrids of DNA and RNA. Furthermore they can e.g. Contain thioester bonds and / or nucleotide analogs. Such modifications can be advantageous in order to increase the stability of the nucleic acid molecules, in particular with respect to endo- and / or exonucleases occurring in the cells.
  • the invention further relates to vectors, in particular plasmids, cosmids, viruses, bacteriophages and other vectors common in genetic engineering, which contain the various nucleic acid molecules according to the invention described above.
  • the nucleic acid molecules contained in the vectors are linked to regulatory elements which ensure expression in prokaryotic or eukaryotic cells.
  • expression can mean transcription as well as transcription and translation.
  • Preferred vectors are those which are suitable for transforming plant cells, particularly preferred those which ensure stable integration into the plant genome.
  • 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 is suitable (Odell et al., Nature 313 (1985), 810-812), which ensures constitutive expression in all tissues of a plant and the promoter structure described in WO / 9401571.
  • Another example are the promoters of the polyubiquitin genes from maize (Christensen et al., Plant Mol. Biol. 18 (1992), 675-689).
  • promoters can also be used which are only 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. Furthermore, the promoters can be used which lead to an expression of downstream sequences in a specific tissue of the plant (see, for example, Stockhaus et al., EMBO J. 8 (1989), 2245-2251). Examples include promoters that are active in the starch-storing organs of the plants to be transformed. For corn, for example, these are the corn kernels, while the potatoes are the tubers. For example, the bulb-specific B33 promoter (Rochasosa et al., EMBO J.
  • seed-specific promoters have already been described for various plant species.
  • the USP promoter from Vicia faba which ensures seed-specific expression in V. faba and other plants (Fiedler et al., Plant Mol. Biol. 22 (1993), 669-679; Bäumlein et al., Mol Gen. Genet. 225: 459-467 (1991)).
  • promoters of the zein genes ensure 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 ).
  • pathogen-induced promoters can be used, for example the nematode-induced promoter of the TobRB7 gene from tobacco (Opperman et al., Science 263 (1994), 221-223) or the promoter of the PRla gene from tobacco (Beilmann et al ., Europ. J. Biochem. 196 (1991), 415-422).
  • This promoter is also inducible by salicylic acid. It is interesting with regard to a possible systemic induction of the expression of the nucleic acid molecules according to the invention.
  • the regulatory regions of the invention described below can also be used.
  • 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 Agroba et erium turne f aciens 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 (e.g. E. coli) or fungal cells (e.g. yeast, especially 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 present in the cell, possibly 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 cysteine proteinase from plants, in which host cells according to the invention are cultivated under suitable conditions and the protein is obtained from the culture, ie from the cells and / or the culture medium.
  • the invention also relates to proteins with the structural properties of a cysteine proteinase, 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 against proteins according to the invention.
  • proteins according to the invention 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 cysteine proteinase 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 encode a new or an additional plant cysteine proteinase.
  • transgenic plant cells differ from untransformed cells in that the nucleic acid molecule introduced is either heterologous to the transformed cell, i.e. comes from a cell with a different genomic background, or by the fact that the introduced nucleic acid molecule, if it is homologous to the transformed plant species, is located in the genome at a location where it does not naturally occur in non-transformed cells.
  • the nucleic acid molecule introduced can either be under the control of its natural promoter or linked to regulatory elements of foreign genes.
  • the plant cells according to the invention differ from corresponding non-transformed plant cells preferably by having an increased amount of protein according to the invention.
  • This can be determined using detection methods known to the person skilled in the art, such as 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.
  • a possibly occurring DNA sequence that encodes a signal sequence usually has to be removed and the coding region has to be linked to DNA sequences that ensure the 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 protein according to the invention can also be localized in the vacuole. This would be advantageous in connection with the mobilization of amino acid reserves. These are required in order to ensure the strongly induced de novo protein synthesis of PR proteins, among other things, in the case of pathogen attack. In general, this could also result in increased fitness to ward off infections with pathogens or in other stressful situations.
  • 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 monocotyledon or dicotyledon crop, in particular a starch-storing plant, such as, for example, cereal plants, legumes, or cassava, or an ornamental plant.
  • Cereal plants are understood in particular as monocotyledonous plants belonging to the Poales order, preferably those belonging to the Poaceae family. Examples of this are the plants belonging to the genera Avena (oat), Triticum (wheat), Seeale (rye), Oryza (rice), Panicum, Pennisetum, Setaria, sorghum (millet), Zea (corn) etc. Hordeum (barley) is particularly preferred.
  • Starch-storing legumes are e.g. some species of the genus Pisum (e.g. Pisum sativum), Vicia (e.g. Vicia faba), Cicer (e.g. Cicer arietinum), Lens (e.g. Lens culinaris), Phaseolus (e.g. Phaseolus vulgaris and Phaseolus coccineus), etc. Examples of ornamental plants are rose, Rhododendron, pelargonium, orchids.
  • plants according to the invention can also be trees, e.g. Beech, birch, oak, pinus species or fruit trees, e.g. Pear, apple, plum, cherry etc.
  • the invention further 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 the transgenic plant cells described above.
  • the present invention relates to transgenic plant cells in which the amount of protein according to the invention naturally occurring in the cells 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 be that a nucleic acid molecule according to the invention or a part thereof is expressed in the corresponding plant cells in antisense orientation and there is a reduction in the amount of protein according to the invention due to an antisense effect.
  • a further possibility for reducing the amount of the protein according to the invention in plant cells consists in 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.
  • Methods for reducing the activity of polypeptides according to the invention in the plant cells by a cosuppression effect are known to the person skilled in the art and are described, for example, in Jorgensen (Trends Biotechnol. 8 (1990), 340-344), Niebel et al. , (Curr. Top Microbiol. Immunol. 197 (1995), 91-103), Flavell et al. (Curr. Top. Microbiol. Immunol. 197 (1995), 43-46), Palaqui and Vaucheret (Plant. Mol. Biol.
  • nucleic acid sequence used is preferably a nucleic acid sequence of homologous origin with respect to the plants to be transformed.
  • nucleic acid sequences can also be used which have a high degree of homology to endogenously present nucleic acid molecules according to the invention, in particular homologies higher than 80%, preferably homologies between 90% and 100% and particularly preferably homologies over 95%.
  • Sequences up to a minimum length of 15 bp can be used. An inhibitory effect is not excluded even when using shorter sequences. Longer sequences between 100 and 500 base pairs are preferably used, and sequences with a length of more than 500 base pairs are used in particular for efficient antisense inhibition. As a rule, sequences are used which are shorter than 5000 base pairs, preferably sequences which are shorter than 2500 base pairs.
  • genomic sequences which code for such enzymes, e.g. by "gene tagging” or transposon mutagenesis or the expression of antibodies which specifically recognize the proteins described.
  • the mutagenesis of genomic sequences can affect coding regions of the gene (introns or exons) as well as regulatory regions, in particular those required for the initiation of transcription.
  • the reduction in the amount of protein according to the invention can be determined by methods known to the person skilled in the art, for example by means of Western blot or Northern blot.
  • a reduction in the amount preferably means a reduction in the amount by at least 10%, preferably by at least 20% and preferably by at least 30% compared to non-transformed or genetically modified plants.
  • a reduction in the amount of proteins according to the invention in transgenic plants could, for example, result in certain protein substrates no longer being made available to the pathogen, the pathogen no longer being infectious and the plants surviving an infection better.
  • cysteine proteinase according to the invention is involved in important metabolic pathways of induced cell death, could the non-synthesis of the protein slows down or precludes the process of cell death.
  • 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 Agrobacterium tumefaciens or Agrobacterium rhizogenes as the transformation agent, the fusion of protoplasts, the injection, the electroporation of DNA, the introduction of DNA using the biolistic method and other possibilities.
  • the invention further relates to the use of the nucleic acid molecules according to the invention for the production of plants which have an increased or reduced amount of the protein according to the invention.
  • the present invention further relates to the use of nucleic acid molecules according to the invention for the production of plants with increased disease resistance, in particular in the case of pathogen attack, and increased resistance to other stress factors, such as e.g. Temperature stress (heat / cold), dry stress or salt stress.
  • stress factors such as e.g. Temperature stress (heat / cold), dry stress or salt stress.
  • the present invention also relates to the use of proteins according to the invention for preventing or combating the infestation of plants with pathogens.
  • proteins according to the invention for preventing or combating the infestation of plants with pathogens.
  • Soils can also be treated with the protein according to the invention in order to reduce or eliminate certain pathogen populations.
  • 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 structural features of a cysteine proteinase.
  • the present invention relates to a regulatory region which naturally controls the transcription in plant cells of a nucleic acid molecule according to the invention, which encodes a cysteine proteinase and which is described above, the transcription specifically in the roots after pathogen attack and / or root wounding, especially mechanical wounding is guaranteed.
  • regulatory region is understood to mean a region which influences the specificity and / or the extent of the expression of a gene sequence, for example in such a way that the expression occurs in a cell- or tissue-specific manner and / or that it occurs in Response to certain external stimuli, especially in the case of pathogen attack and / or wounding occurs.
  • regions are usually located upstream of the transcription initiation site, but can also be located downstream of it, for example in transcribed but non-translated leader sequences.
  • Such regulatory regions are usually in an area called a promoter.
  • promoter encompasses nucleotide sequences which are necessary for the initiation of the transcription, ie for the binding of the RNA polymerase, and can also include the TATA box (s), for example.
  • the present invention also relates to regulatory regions of genes which are homologous to a nucleic acid molecule according to the invention, these regulatory regions ensuring root-specific expression after pathogen attack and / or wounding. Regulatory regions of homologous genes therefore include those regions of genes from the same or different plant species which are homologous to a nucleic acid molecule according to the invention and which have essentially the same expression pattern.
  • Homology preferably means a sequence identity in the coding region of at least 40%, preferably at least 60%, particularly preferably at least 80% and particularly preferably of at least 90%. compared to a nucleic acid molecule according to the invention. Homology preferably also means that such a homologous gene encodes a protein which can be classified as cysteine proteinase and in particular as cysteine proteinase from the papain family.
  • the regulatory regions of a nucleic acid molecule according to the invention in winter barley lead to specific expression in the roots after attack by certain pathogens, in particular root-specific pathogens.
  • the regulatory regions according to the invention therefore preferably have the feature that they lead to specific expression in roots in winter barley after infection with a pathogen. It is preferred that such regulatory regions do not lead to expression in other tissues of the plant, for example in the leaves.
  • the pathogen is preferably a nematode, a fungus, a bacterium or a virus, preferably a root-specific pathogen.
  • the pathogen Gaeumannomyces graminis or Polymyxa graminis is particularly preferred.
  • the regulatory regions according to the invention are not induced by leaf-specific pathogens, in particular not by BaMMV (barley mild mosaic virus), Erysiphe graminis or Rhynchosporium secalis.
  • the regulatory regions according to the invention also lead to expression in the case of wounding, in particular mechanical wounding, of plant roots, but they preferably do not lead to expression in the mechanical wounding of other parts of plants, for example. B. the leaves.
  • nucleic acid molecules according to the invention which encode a cysteine proteinase
  • the present invention furthermore also relates to regulatory regions which are essentially identical to the regulatory region of a nucleic acid molecule according to the invention or of a homologous gene and which are able to ensure root-specific expression after infestation of the root with pathogens or after wounding. The sequence of such regulatory regions deviates from that of the above-mentioned regulatory regions at one or more positions.
  • Such a region still has the same specificity, ie it causes the cell-specific expression after infestation of the root with the above pathogen or after wounding.
  • Such regulatory regions preferably have such high homology to the above-mentioned regions that they hybridize with at least one of these regions, preferably under stringent conditions.
  • Particularly preferred are regulatory regions that have at least 80%, preferably at least 90% and particularly preferably at least 95% sequence identity to one of the above-mentioned regulatory regions.
  • regulatory regions that e.g. due to deletion (s), insertion (s), substitution (s), addition (s) and / or recombination (s) and / or modification (s) compared to the above-mentioned regulatory regions.
  • regulatory regions are familiar to the person skilled in the art. It is also known to the person skilled in the art that the regulatory regions according to the invention can be linked to further elements which influence the transcription in plant cells, e.g. with enhancer elements.
  • the regulatory region according to the invention comprises a nucleotide sequence selected from the group consisting of:
  • the in Seq ID No. The nucleotide sequence shown in FIG. 5 is the 5 'flanking region of the barley gene which is that of Seq ID no. 1 corresponds to the cDNA shown.
  • the open reading frame begins with the nucleotide at position 1444 in Seq ID No. 5.
  • At the 5 'end of the Seq ID No. 5 shown sequence is an open reading frame (nucleotides 3 to 195), which may be the coding region of another gene. Consensus sequences for TATA boxes can be found at positions 1367 to 1372 and 1380 to 1386.
  • the regulatory region according to the invention preferably has "elicitor responsive elements” (also called W-box; nucleotide sequence TGAC) (see Rushton et al., EMBO J. 15 (1996), 5690-5700). In the Seq ID No. 5 sequence shown, these are for example at positions 1269 to 1272, 1218 to 1221, 957 to 960 (reverse), 734 to 737, 706 to 709 and 689 to 692 (reverse).
  • a regulatory region according to the invention preferably also comprises at least one PR box / GCC box.
  • This element is found in "pathogenesis related protein” genes. Ethylene induces the activation of genes of the pathogen defense cascade via the "ethylene-responsive element-binding protein" (Diekman, Physiologia Plantarum 100 (1997), 561-566; Buttner and Singh, Proc. Natl. Acad. Sei. USA 94 (1997), 5961-5966; Zhou et al., EMBO J. 16 (1997), 3207-3218). In the Seq ID No. 5 sequence shown, this element is in reverse orientation at positions 742 to 747 and in a similar sequence at positions 1331 to 1336.
  • the regulatory region according to the invention preferably contains at least one element with the nucleotide sequence TGACG. This is described as the root-specific sequence of the CaMV 35S promoter (Lam et al., Proc. Natl. Acad Sei. USA 86 (1989), 7890-7894). In the Seq ID No. 5 sequence, such an element is located at positions 1218 to 1222 and coincides with a W-Box. Furthermore, a regulatory region according to the invention can preferably contain a so-called G-Box (Faktor et al., Plant Mol. Biol. 32 (1996), 849-859). In Seq ID No. 5, such a G-box is located at positions 263 to 268.
  • a regulatory region according to the invention can preferably comprise copper-regulated elements (Okamura et al., Plant Mol. Biol. 25 (1994), 705-719). Three of these elements (TCTTTT) are in the Seq ID No. 5 sequence shown in reverse orientation (AAAAGA) located at positions 970 to 985, 1068 to 1073 and 1299 to 1304.
  • the present invention also relates to recombinant DNA molecules which comprise a regulatory region according to the invention.
  • the regulatory region is preferably linked to a heterologous DNA sequence.
  • heterologous means that such a sequence is naturally not linked to the regulatory region.
  • a recombinant DNA molecule according to the invention can contain further regulatory elements which are important for transcription and / or translation in plant cells, e.g. Transcription enhancer, a poly-A signal or translation enhancer.
  • the heterologous DNA is linked in sense orientation to the regulatory region according to the invention and, when expressed, leads to the synthesis of a translatable mRNA, a cosuppression RNA or a ribozyme.
  • Sequences that co- translate a mRNA dieren can in principle come from any organism and encode any desired protein.
  • Gaeumannomyces graminis species have also been demonstrated (Browyer et al., Science 267 (1995), 371-374). Therefore, it might make sense to induce the synthesis of these phytoalexins when the fungus attacks the plant.
  • Other examples of suitable genes are in Lamb et al. (Bio / Technology 10 (1992), 1436-1445) and Dixon et al. (Gene 179 (1996), 61-71).
  • AFP antifungal peptide
  • the regulatory regions can be linked, for example, to DNA sequences which encode non-vegetable lysozymes, e.g. the Lysozy from the T4 phage. This could increase resistance to bacterial pathogens.
  • the regulatory regions can be linked to DNA sequences which induce or cause cell death upon expression.
  • An example of this is the Barnase gene (Strittmacher et al., Bio / Technology 13 (1995), 1085-1089).
  • DNA sequences can also be placed under the control of the regulatory regions according to the invention, which are involved in pathogen defense in plants, in particular in signal perception and signal transduction in pathogen defense. Examples of these are DNA sequences which encode catalase, peroxidases, lipoxygenases or resistance genes. DNA sequences which are generally activated in stressful situations, e.g. Sequences encoding heat shock proteins.
  • the heterologous DNA is linked in antisense orientation to the regulatory region and, when expressed, leads to the synthesis of an antisense RNA.
  • the present invention further relates to vectors which contain a regulatory region according to the invention or a recombinant DNA molecule according to the invention.
  • Such vectors also include, for example, plasmids, cosmids, bacteriophages, viruses, etc., which are conventionally used for molecular genetic methods.
  • Such vectors are preferably suitable for the transformation of plant cells and particularly preferably for the stable integration of sequences contained in them into the plant cell genome.
  • An example of this are binary vectors which can be used in gene transfer mediated by agrobacteria.
  • the present invention further relates to host cells which are transformed with a regulatory region according to the invention, a recombinant DNA molecule or a vector.
  • the host cells are preferably transformed such that the corresponding molecule, in particular the regulatory region, is stably integrated into their genome.
  • the host cells can be any cells, in particular prokaryotic or eukaryotic cells.
  • the host cells are preferably plant cells.
  • Such plant cells can be cells of any plant, in particular monocotyledonous or dicotyledonous plants. It is preferably cells from higher plants, in particular cereal plants, such as e.g. Oats, rye, wheat, rice, corn, millet, sago, particularly preferably from barley, especially winter barley.
  • the present invention also relates to transgenic plants which contain the transformed plant cells described above. Such plants can be regenerated from the transformed plant cells, for example, by methods familiar to the person skilled in the art. Such plants contain a regulatory region according to the invention stably integrated into their genome and thus link a heterologous DNA sequence. This is root-specific when infested with certain Root pathogens and / or expressed in case of mechanical injury to the root.
  • the invention further relates to propagation material of the transgenic plants described above, which contains transformed plant cells according to the invention.
  • propagation material includes any plant material that is suitable for the reproduction of the plants, e.g. Fruits, seeds, tubers, cuttings, rhizomes, calli, cell cultures, protoplasts etc.
  • the present invention further relates to the use of regulatory regions according to the invention for the expression of heterologous DNA sequences in plants, in particular specifically in the roots after the roots have been infected by pathogens or after mechanical damage to the roots. Expression is preferably carried out directly at the site of infection to improve the defense strategies of plants in the case of pathogen attack.
  • the plasmids prcyp and prcyp-prom produced within the scope of the present invention were obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ) in Braunschweig, Federal Republic of Germany, which is recognized as an international depository, in accordance with the requirements of the Budapest Treaty on August 20, 1997 and on August 14 January 1998 under the deposit numbers DSM 11692 and DSM 11922.
  • Figure 1 shows the Northern. Blot analysis of the expression of the rcyp gene in winter barley, which was infected with Gaeumannomyces graminis var. Tritici. 30 ⁇ g of total RNA, which was isolated from barley root (R) and barley leaves (L) at different times after infection (22, 25, 28 and 31 days), were separated out per lane. (+): infected; (-): not infected; RL +: heavily infected regions of the zel; R + remaining tissue of the root that was not so heavily infected. The same blot was hybridized against an a-P labeled actm probe.
  • FIG. 2 shows the Northern blot analysis of the expression of the rcyp gene in winter barley, which was infected with Polymyxa graminis.
  • 10 ⁇ g of total RNA were separated, which was isolated from barley roots infected with Polymyxa graminis.
  • Samples were collected from various experiments: Pol and PoII were collected 30 days after the start of the infection.
  • Total RNA from non-infected barley roots (R-; negative control) and from barley roots infected with Gaeumannomyces gramini (R +; positive control) served as a control.
  • FIG. 3 shows a Northern blot analysis for the expression of the rcyp gene after wounding barley roots. Roots of winter barley were used as a control (R-), treated with carborundum on the surface (CA) or cut into 5 mm pieces (CU). All samples were left in the dark at room temperature in an aqueous solution for 24 h before RNA extraction. In each case 10 ⁇ g of total RNA were separated per lane.
  • FIG. 4 shows a Northern blot analysis for the expression of the rcyp gene in winter barley plants which were infected with BaMMV or Erysiphe graminis, and the detection of BaMMV in plants by means of RT-PCR.
  • A Northern blot analysis, in which 10 ⁇ g total RNA were separated per lane.
  • R + RNA from Gaeumannomyces graminis-infected
  • RNA from roots of healthy plants Barley roots; R-: RNA from roots of healthy plants; L-: RNA from leaves of healthy plants; Po: RNA from plants that were infected with non-virus-containing Polymyxa graminis zoospores; PV: RNA from plants infected with P. graminis zoospores containing viruses; PV *: RNA from plants that contain virus-containing P. graminis zoospores were infected and resistant to BaMMV; M: RNA from plants that had been mechanically infected with BaMMV.
  • B RT-PCR of 0.2 ug total RNA from the same samples as used in A. If BaMMV is present in the sample, a 1256 bp PCR product is expected.
  • Figure 5 shows schematically the vector pUC9-GUS.
  • Figure 6 shows schematically the vector MS23.
  • Figure 7 schematically shows the vector promGUSA.
  • Figure 8 schematically shows the vector promGUSB.
  • Figure 9 schematically shows the vector promCXl.
  • Figure 10 schematically shows the vector promCX4.
  • Figure 11 schematically shows the vector prom34DR.
  • RNA from Gaeumannomyces graminis var. tritici-infected barley roots isolated.
  • the barley pathogen Gaeumannomyces graminis var.tri tici isolated 90 / 3-4 was grown in petri dishes on potato dextrose agar medium (PDA medium, Sigma; Cat.No. P2182) in the dark at 20 ° C until the plates were overgrown with mycelium.
  • Winter barley Hordeum vulgäre of the 'Maris Otter 1 variety was used for infection experiments with the fungus Gaeumannomyces graminis var. Tri tici.
  • PDA plates overgrown with mycelium were chopped into pieces about 5 mm in size and mixed with about 100 ml of sterilized sand. This mixture serves as an inoculum. Pots were filled as follows: An approximately 2 cm high layer of sterilized sand was covered with the inoculum. About 1 cm of sterilized sand was placed on the inoculum and each pot was equipped with 4 seedlings. These were then overlaid with sterilized vermiculite. Control plants were grown in the same way, but using fertilization of fungus-free PDA plates. The plants were cultivated as described above.
  • a method based on PCR was used in the second round of screening:
  • the cDNA insertion fragments were amplified directly from the phage lysate by means of PCR: 5 ⁇ l of lysate were used as a template, the insertion fragment using the 'reverse' and the 'M13- 20 1 primers amplified under the specified reaction conditions (Taq Po- lymerase, Life Technologies).
  • the PCR was carried out as follows: 5 min. Denaturation at 95 ° C; 30-35 cycles of 1 min. at 95 ° C, 1.5 min. at 55 ° C, and 2 min. at 72 ° C.
  • Equal amounts of PCR products of a phage lysate were separated electrophoretically on two parallel gels and the DNA was transferred to Hybond N filter (Amersham) using the Southern blot method (Sambrock et al., Op. Cit.).
  • the filters were hybridized with radioactively labeled cDNA probes either from healthy or infected roots (see above) and the signal strengths of an amplified PCR fragment were compared between the two filters hybridized with different probes. Differences in signal strength indicate the identification of a differently induced cDNA clone.
  • the corresponding PCR fragment was radioactively labeled (using the High Prime Labeling Kit, Boehringer) and used as a probe to screen the cDNA library.
  • the phagemids of the corresponding positive lambda phage clones were cut out in vivo as described in the protocol of the cDNA synthesis kit (Stratagene).
  • the corresponding PCR fragment of a putative positive cDNA clone was eluted (QIAquick gel extraction kit, Qiagen) and used as a template for the probe (High Prime Labeling Kit, Boehringer Mannheim) in the Northern blot analysis (carried out with 40 ⁇ g total RNA; according to Sambrock et al., loc. cit .; washing the filters after hybridization under stringent conditions: 2x SSC, 0.1% SDS, lx SSC, 0.1% SDS, 0, lx SSC, 0.1% SDS each for 20 min at 65 ° C).
  • the phagemids of the corresponding positive lambda phage clones were cut out in vivo as described in the protocol of the cDNA synthesis kit (Stratagene).
  • Plasmid DNA was isolated using the Mini or Medi 'DNA preparation kit' from Qiagen according to their information. The DNA sequence of PCR products and plasmid DNA was used using the cycle sequencing protocol the 'Taq dye sequencing' kit and the automated laser fluorescence DNA sequencer from Applied Biosystems. The sequence of a clone obtained is under Seq ID No. 1 shown. The clone, prcyp, is registered under number DSM 11692. The corresponding gene is called rcyp in the following.
  • the isolated cDNA is 1286 nucleotides long and includes an open reading frame encoding a 365 amino acid polypeptide (nucleotides 4 to 1098).
  • the encoded polypeptide has a molecular weight of 39,630 daltons, calculated based on the amino acid sequence.
  • the 3 'untranslated region comprises two putative polyadenylation signals. Homology comparisons with known sequences showed a similarity to members of the papain superfamily of cysteine proteinases.
  • the first 30 amino acids of the polypeptide form a hydrophobic part of the protein. This characteristic is typical of signal sequences of secreted proteins synthesized on the ER.
  • the polypeptide also contains two putative N-glycosylation sites at positions Asn-74 and Asn-134 in the amino acid sequence derived from the cDNA.
  • the encoded polypeptide is synthesized as a preprotein. So far, however, the cleavage sites in the pre-protein can only be postulated on the basis of theoretical data, since the N-terminus of the mature protein has not yet been determined. On the basis of a computer analysis (program Sig Cleave of the GCG Software Analysis Package; see above) it can be postulated that a first cleavage site between amino acid residues 22 and 23 of the sequence shown in Seq ID No. 2 specified amino acid sequence. A second interface (between preprotein and the mature one Protein) can be postulated between amino acid residues at positions 145 and 146 or between those at positions 146 and 147.
  • the protein encoded by the isolated cDNA sequence has the catalytic triad Cys-His-Asn typical of cysteine proteinases. This triad is formed by the amino acid residues Cys 171 -His 307 -Asn 3 . The flanking regions of these amino acid residues, which form the active center, are also conserved, in particular the residues Gin and Asn - Ser 330 -Trp 331 .
  • the encoded polypeptide also has cysteine residues which can form the following intramolecular cysteine bridges: Cys 168 -Cys 209 , Cys 202 -Cys 242 and Cys 301 -Cys 354 .
  • the data obtained indicate that the encoded protein is a vegetable cysteine proteinase.
  • cysteine proteinases An important difference to the previously known cysteine proteinases is, however, that the protein encoded by the cDNA described contains the sequence GGALGPSGSQVHTF (amino acid residues 92 to 105 in Seq ID No. 2), which does not appear in any of the previously known cysteine proteinases.
  • GGALGPSGSQVHTF amino acid residues 92 to 105 in Seq ID No. 2
  • a corresponding genomic sequence was then isolated using the sequence information obtained from the cDNA.
  • two oligonucleotides were used as primers for a PCR, which are based on the in Seq ID no. 1 cDNA shown at positions 126-156 and 1045-1072 (reverse) hybridized.
  • 500 ng to 1 ⁇ g genomic DNA from young barley leaves were used as a template in the PCR.
  • the reaction was carried out according to the manufacturer's instructions (Taq Polymerase, Life Technologies) in the following program:
  • Amplification 30 cycles of 30 seconds at 94 ° C
  • the PCR products were purified using High Pure PCR Purification Kits (Boehringer Mannheim, Germany). The PCR resulted in a fragment of approximately 1300 bp. Sequencing of the fragment obtained was carried out as described above for the cDNA. The sequence obtained corresponds to the range of nucleotides 1566 to 2862 that in Seq ID No. 3 sequence shown. The deduced amino acid sequence is in Seq ID No. 4 shown. Sequence analysis revealed that the gene has an intron that is located between nucleotides 508 and 509 of that shown in Seq ID no. 1 cDNA sequence shown is localized. Typical splice points are located at the 5 'and 3' ends of the intron.
  • Example 1 Leaves and roots from control and infected plants were harvested at different times (day 22, 25, 28, 31) after planting the seedlings. In the case of infected roots, a distinction was made between heavily infected, already browned tissue (RL +) and less heavily infected, not yet discolored tissue (R +). At no time could a transcript of the cDNA identified in Example 1 be detected in leaves of infected or healthy plants. In healthy roots of the control plant weak basic expression was shown in some samples. A strong signal was observed in the infected tissue, with the expression being generally stronger in heavily infected tissue than in the hardly infected tissue. In both cases a very strong signal can be seen on day 22 and 25, but its intensity decreases towards day 31. The results of this experiment are shown in Figure 1.
  • Barley plants were, as in Hahn et al. (Molecular Plant - Microbe Interaction 6 (6) (1953), 745-754) with the fungus Rhynchosporium secal is inoculated and the total RNA isolated as indicated there. Both the resistant barley variety Atlas 46 and the susceptible variety Atlas were examined. Leaf samples were also taken from control plants of the two varieties that were not infected. In a Northern blot analysis with 20 ⁇ g of the total RNA from the respective samples, no expression of the rcyp gene could be found in the resistant or susceptible barley.
  • the cDNA (Seq ID No. 1) was first amplified by means of PCR using a partial fragment which comprised nucleotides 442 to 1098.
  • the primers used for the PCR were designed in such a way that Ncol interfaces were introduced at the 5 'and 3' ends of the amplified fragment.
  • the amplified fragment was inserted into the expression vector pRSET (Invitrogen) cut with Ncol.
  • the Resulting construct in which the cDNA fragment is arranged in sense orientation to the promoter, was transformed in E. coli cells.
  • the expressed protein is purified and can be used, for example, for antibody production.
  • the coding region is cloned into the vector pRT104 (Nucl. Acid Res. 15 (1987), 5890). This also contains the 35S promoter.
  • the vector is particularly suitable for the transformation of barley and maize and is usually co-transfected with the vector pAHC20, which contains a bar gene under the control of the ubiquitin promoter.
  • the cDNA is cloned in sense orientation behind the 35S promoter.
  • the cDNA or fragments thereof are cloned in the antisense orientation behind the 35S promoter.
  • the genomic DNA was produced as follows: About 200-400 mg of leaf material were ground in liquid nitrogen and then taken up in 1 ml of CTAB extraction buffer (0.02 M EDTA, 0.1 M Tris HCl pH 8.0, 1.4 M NaCl , 2% CTAB (Hexadecyltrimethylammoniumbromid)), with 0.4% mercaptoethanol was added fresh before the extraction. After adding 100 ⁇ l of chloroform: octanol (24: 1), the phases were mixed. An incubation for 30 min at 65 ° C was followed by an incubation for 15 min at room temperature.
  • RNA-wrapped glass hook was immersed in 50-200 ⁇ l H2O and the DNA was dissolved in water.
  • 7U of RNAse A were added and incubated at 37 ° C. for one hour.
  • the DNA was precipitated by adding 1/10 vol. 4 M LiCl and 2 vol. Abs. Ethanol and subsequent incubation at -20 ° C overnight.
  • the DNA was sedimented by centrifugation at 15000 rpm (Sigma laboratory centrifuge 1K15). The precipitate was washed in 70% ethanol and, after drying in air, taken up in 100 ⁇ l of H 2 O.
  • gene-specific primers were produced, which have the following sequence sequence:
  • Primer GSP1 5 '-GGA AGG TGT GGA CCT GCG ACC CAG ATG-3' (Seq. ID No. 6) (binds to the cDNA clone rcyp at position 319-291)
  • Primer GSP2 5'-CCG GCC CCG GGT AAG TTG CCG CAG TCA-3 '(Seq. ID No. 7) (binds to the cDNA clone rcyp at position 100-74)
  • PCR products which potentially contained the promoter were purified.
  • the High Pure PCR Purification Kit from Boehringer Mannheim was used.
  • the purified PCR products were cloned into pGEM ® -T-vector system from Promega according to the manufacturer. The successful cloning of the promoter fragments was confirmed by sequencing the corresponding clones and subsequent sequence analysis (methods as previously indicated).
  • the sequence of one of the isolated clones is in Seq ID No. 5 shown.
  • the search for open reading frames showed that there is an open reading frame at the 5 'end of the sequence shown in the region of nucleotides 3 to 195 (start of the stop codon). This could be part of the coding region of another gene.
  • the sequence of the cDNA sequence described in Example 1 begins at position 1441.
  • the start codon for translation comprises nucleotides 1444 to 1446. Sequence analysis showed that the sequence of the regulatory region shown includes the following elements, among others:
  • TGACG At position 1218 to 1222 is the element TGACG, which is described as the root-specific sequence of the CaMV 35S promoter (Lam et al., Proc Natl. Acad. Sci. USA 86 (1989), 7890-7894).
  • the element here coincides with a W-Box.
  • G-box At position 263 to 268 there is a so-called G-box (Faktor et al., Plant Mol. Biol. 32 (1996), 849-859), which together with so-called H-boxes is important for root-specific expression.
  • Seq ID No. 3 the genomic sequence of the rcyp gene, which is flanked by the promoter region located in the 5 'region and the polyadenylation signals located in the 3' region.
  • This example describes the investigation of different partial areas of the promoter isolated according to example 5 with regard to their inducibility by pathogens.
  • To the corresponding DNA fragments were amplified using sequence-specific primers and cloned into the promoterless vector pUC9-GUS or into the vector MS23, which contains the minimal promoter of the 35S promoter and the reporter gene GUS (see FIGS. 5 and 6).
  • the constructs obtained were then transformed into plant cells and the transient expression of the GUS gene was measured via blue staining or fluorometric determination.
  • parsley protoplast system The test system for elicitor inducibility that was used in Rushton et al. (EMBO J. 20 (1996), 5690-5700) described parsley protoplast system. Parsley protoplasts are isolated from a suspension culture. The protoplasts are then incubated with Elicitor of the fungus Phythophthora sojae, harvested after 8 hours and the GUS activity measured when the promoters are induced.
  • the inducibility of the promoter is investigated by biolistic bombardment of barley roots with the promoter-GUS constructs. In comparison to the parsley protoplast system, this ensures that gene activity can be induced in the homologous system of the barley root.
  • the roots infected with the fungus Gaeumannomyces graminis var. Tritici are removed from the seedlings before bombardment. After bombardment, the root pieces are cultivated on PDA plates for 2-3 days and then the inducibility of the promoter fragments is determined by determining the GUS activity.
  • the promoter was amplified over its entire length to the end of the 5 'untranslated region by means of PCR (nucleotides 1-1444 in Seq ID No. 3).
  • the primers had the following sequence: Forward primer 'pro-sph':
  • the entire length of the promoter was amplified by the rcyp gene by means of PCR (nucleotides 1 to 1517 in Seq ID No. 3).
  • the primers had the following sequence: Forward primer 'pro-sph':
  • a GUS fusion protein is formed after translation: the first 21 amino acids of the RCYP protein and a further 12 amino acids - coded by the polylinker of the vector - are fused N-terminally to the GUS protein.
  • Fragments of the promoter were cloned into the vector MS23. This contains the 35S minimal promoter, which is upstream of a GUS gene. The PCR fragments can be placed upstream of the minimal promoter, which contains the TATA box, via the cleavage sites Spei and Xbal. If transcription factors are bound by the examined fragment, the gene expression of the GUS gene is induced and can be detected accordingly. To enable cloning, a Spel interface was integrated in the forward primer and an Xbal interface in the reverse primer.
  • This area includes the W boxes at positions 1296 to 1272 and 1218 to 1221.
  • the fragments are obtained with the following primers by means of PCR: Forward primer 'CSpe':
  • construct CXI contains the promoter fragment in a single version, while the construct CX4 contains the fragment in a double version directly connected in series (see FIGS. 9 and 10).
  • Construct 34DR see Figure 11

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Abstract

L'invention concerne des molécules d'acides nucléiques qui codent pour une protéinase d'origine végétale, des cellules végétales et des végétaux transformés au moyen desdites molécules, ainsi que l'utilisation desdites molécules pour produire des plantes présentant une plus grande résistance aux maladies. L'invention concerne également les régions régulatrices qui régulent l'expression des molécules d'acides nucléiques de l'invention et qui sont responsables d'une expression caractéristique de racine après attaque par des pathogènes ou blessure mécanique des racines.
PCT/EP1998/005339 1997-08-26 1998-08-21 Molecules d'acides nucleiques codant pour une cysteine proteinase d'origine vegetale, et leurs regions regulatrices WO1999010500A1 (fr)

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WO2000070066A1 (fr) * 1999-05-14 2000-11-23 Dekalb Genetics Corporation Promoteur rs324 du mais et procedes d'utilisation de ce promoteur
WO2006066054A2 (fr) * 2004-12-17 2006-06-22 Pioneer Hi-Bred International, Inc. Promoteur de l'oxygenase 2-oxoglutarate-dependante induit par la blessure ou par des insectes, destine preferentiellement aux racines et issu du mais et son utilisation

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Cited By (6)

* Cited by examiner, † Cited by third party
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WO2000070066A1 (fr) * 1999-05-14 2000-11-23 Dekalb Genetics Corporation Promoteur rs324 du mais et procedes d'utilisation de ce promoteur
US6194636B1 (en) 1999-05-14 2001-02-27 Dekalb Genetics Corp. Maize RS324 promoter and methods for use thereof
US6426446B1 (en) 1999-05-14 2002-07-30 Dekalb Genetics Corporation Maize RS324 promoter and methods for use thereof
WO2006066054A2 (fr) * 2004-12-17 2006-06-22 Pioneer Hi-Bred International, Inc. Promoteur de l'oxygenase 2-oxoglutarate-dependante induit par la blessure ou par des insectes, destine preferentiellement aux racines et issu du mais et son utilisation
WO2006066054A3 (fr) * 2004-12-17 2006-08-24 Pioneer Hi Bred Int Promoteur de l'oxygenase 2-oxoglutarate-dependante induit par la blessure ou par des insectes, destine preferentiellement aux racines et issu du mais et son utilisation
US7193135B2 (en) 2004-12-17 2007-03-20 Pioneer Hi-Bred International, Inc. Root-preferred, wound- and insect-inducible 2-oxoglutarate-dependent oxygenase promoter from maize and its use

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