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  • 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
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  • 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
  • C12N15/8285—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 nematode resistance
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/90—Isomerases (5.)
• • 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 invention relates to methods for generating or increasing a pathogen resistance in plants by - preferably pathogen-inducible - expression of a sucrose isomerase.
• Palatinose (isomaltulose) and trehalulose are produced on an industrial scale from sucrose by an enzymatic rearrangement using immobilized bacterial cells.
• the ( ⁇ l-> ⁇ 2-glycosidic bond between the monosaccharides of the disaccharide sucrose isomerized to an ⁇ l-> ⁇ 6 bond with palatinose or an ⁇ l-> ⁇ l bond with trehalulose.
• sucrose isomerase also called sucrose mutase, and corresponding sequences are described, for example, in WO 95/20047 (US Pat. No. 5,786,140; US Pat. No. 5,985,622).
• Sucrose isomerases from Erwinia rhapontici (pall gene, GenBank Acc.-No .: AF279281; Börnke et al.
• WO 01/59136 describes methods for the direct production of non-cariogenic sugars directly in transgenic plants which contain recombinant nucleic acid molecules coding for proteins with the enzymatic activity of a sucrose isomerase. Expression constructs for the said sucrose isomerase for expression in plants and the transgenic plants transformed with the same are described.
• WO 01/59135 describes methods for influencing pollen development using anther, tapetum or pollen-specific expressed sucrose isomerases.
• sucrose isomerase constitutive expression of sucrose isomerase in plants has an adverse effect on the growth of the plant (Börnke F et al.
• the aim of biotechnological work on plants is to produce plants with advantageous new properties, for example to increase agricultural productivity, to improve the quality of food or to produce certain chemicals or pharmaceuticals.
• Plant mechanisms against pathogens are insufficient. Fungal diseases alone result in crop losses of many billions of US dollars a year.
• the introduction of foreign genes from plants, animals or microbial sources can strengthen the immune system. Examples are protection against insect caused by tobacco
• SAR systemic acquired resistance
• SA salicylic acid
• the barley locus has long been described in barley as a negative regulator of pathogen defense.
• the loss or loss of function of the Mio gene results in increased and, above all, race-unspecific resistance to, for example, numerous types of mildew (Büschges R et al.
• Plant pathogenic fungi generally live saprophytically or parasitically. The latter - at least in certain phases of their life cycle - rely on a range of active substances (e.g. a range of vitamins, carbohydrates, etc.), which can only be provided in this form by living plant cells.
• active substances e.g. a range of vitamins, carbohydrates, etc.
• the expert distinguishes parasitic fungi into necrotrophic, hemibiotrophic and biotrophic. In necrotrophic fungal parasites, the infection leads to tissue destruction and thus to the death of the plant.
• These fungi are usually only optional parasitic; they can also multiply saprophytically in dead or dying plant material.
• Biotrophic fungal parasites are characterized in that the parasite and the host live together, at least over longer periods of time. The fungus takes nutrients from the host, but does not kill it. Most biotrophic fungi are obligate parasites. Hemibiotrophic fungi live biotrophically at times and kill the host at a later time, i.e. they change into a necrotrophic phase.
• nematodes Another large group of biotrophic plant pathogens of enormous agro-economic importance are nematodes. Plant pathogens nematodes take their food from the outer plant tissue (ectoparasites) or after penetration into the plant from deeper cell layers (endoparasites). In the case of endoparasitic root nematodes, a distinction is made between two groups according to their lifestyle and diet: cyst-forming nematodes (heterodera and globodera species) and root gall nematodes (meloidogyne species). Both groups are obligatory biotrophic parasites that induce the formation of special nutrient cells in the roots.
• nutrient cells are plant cells whose metabolism has been changed by the nematodes in such a way that they specifically serve to nourish the nematodes that have developed. Endo-parasitic root nematodes are absolutely dependent on these nutrient cells in their development (for an overview see Sijmons et al. (1994) Ann. Rev. Phytopathol. 32: 235-259). Cyst-forming nematodes (heterodera and globodera species) remain at the parasitization site in the root (sessile endoparasites) convert the cells surrounding them into syncytia by protoplast fusion with partial cell wall dissolution.
• the nematodes take their food from these nutrient cells, which are formed in the central cylinder of the root, and swell strongly in the process.
• Root bile nematodes (Meloidogyne species) also remain at the selected parasitization site and cause the formation of nutrient cells, which, however, unlike the cyst-forming nematodes from several, through synchronous core divisions without cell lines. wall-developing multinucleated giant cells exist (Fenoll and Del Campo (1998) Physiol. Mol. Biol. Plants 4: 9-18).
• the formation of the nutrient cell systems is induced by the signaling molecules in the saliva of the nematodes. It is known that a number of plant genes change their expression profile significantly during these differentiation processes.
• Promoters are described in the literature which are specifically induced in the nutrient cell system (syncytia). Examples include the ⁇ 0.3 TobRB7 promoter from tobacco (Opperman et al. (1994) Science 263: 221-223, the Lemmi9 promoter from tomato (Ecobar et al. (1999) Mol Plant Microbe Interact 12: 440-449) , and Geminivirus V-sense promoters (WO 00/01832).
• WO 94/10320 describes DNA constructs for the expression of genes which act as inhibitors of endogenous plant genes (e.g. ATP synthase, cytochrome C, pyruvate kinase) under the control of nematode-induced promoters in the syncytia.
• endogenous plant genes e.g. ATP synthase, cytochrome C, pyruvate kinase
• the present invention has for its object to provide new methods for pathogen defense in plants, which efficiently protect a broad spectrum of pathogens, preferably fungi and nematodes, in as many different plant species, preferably the crop plants used in agriculture. This object is achieved by the method according to the invention.
• a first subject of the invention comprises a method for generating or increasing the resistance to at least one pathogen in plant organisms, the following working steps being included
• transgenic expression of a protein with sucrose isomerase activity in a plant organism or a tissue, organ, part or cell thereof, and b) Selection of the plant organisms in which - in contrast to or compared to the original organism - the resistance to at least one pathogen exists or is increased.
• the method according to the invention can in principle be applied to all plant organisms which produce sucrose. This includes all higher plants. It was surprisingly observed that the growth of the fungus Alternaria was significantly inhibited on potato slices of transgenic potato plants, in the tubers of which tubers are converted to palatinose due to transgenic expression of a sucrose isomerase.
• sucrose isomerase also causes resistance to nematodes. 15
• a syncitia-specific expression of the sucrose isomerase sequence caused by endoparasitic root nematodes results in a significant reduction in the attack of the atodes.
• protein with sucrose isomerase activity means a protein which catalyzes the isomerization of sucrose to other disaccharides as an “essential property”, the ⁇ l-> ⁇ 2-glycosidic bond between glucose and fructose in the sucrose into another
• an HPLC system e.g. from Dionex, which is equipped with a PA-1 (4 x 250 mm) column and a pulsed electrochemical detector can be equipped. Before the injection, the samples can be centrifuged for 2 minutes at 13,000 rpm. The sugars can then be eluted with a 10 minute gradient from 0 to 15 M sodium acetate after 4 minutes at 150 mM NaOH and a flow rate of 1 ml / min.
• the appropriate standards from Sigma can be used to identify and quantify the sugars.
• a protein with sucrose isomerase activity is particularly preferably understood to mean a protein which is capable of isomerizing sucrose to palatinose and / or trehalulose as an essential property.
• the proportion of palatinose and trehalulose in the total disaccharides is that
• At least 2%, preferably at least 20%, particularly preferably at least 50% and most preferably at least 60% are formed by isomerization of sucrose.
• the nucleic acid sequence encoding a protein with sucrose isomerase activity can be isolated from natural sources or synthesized according to conventional methods.
• Containing 25 sequences are in particular microorganisms of the genera Protaminobacter, Erwinia, Serratia, Leuconostoc, Pseudomonas, Agrobacterium, Klebsiella and Enterobacter.
• the following examples of such microorganisms are particularly worth mentioning:
• Protaminobacter rubrum CBS 547, 77
• Erwinia rhapontici NCPPB 1578
• Serratia plymuthica ATCC 15928
• Serratia marcescens NCIB 8285
• Leuconostoc mesenteroides NRRL B-52 If ATCC 1083 0a.
• Pseudomonas mesoacidophila MX-45 (FERM 11808 or FERM BP 3619), Agrobacterium radiobacter MX-232 (FERM 12397 or FERM BP
• the nucleic acid sequence encoding a protein with a sucrose isomerase activity comprises nucleic acid sequences encoding proteins 40 with sucrose isomerase activity, the nucleic acids being selected from the group consisting of
• nucleic acid sequences which code for proteins with sucrose isomerase activity are known in the prior art and are thus available to the person skilled in the art for transfer to plant cells.
• Sequences from Protaminobacter rubrum, Erwinia rhapontici, Enterobacter species SZ 62 and Pseudomonas mesoacidophila MX-45 are described in WO 95/20047. Reference is hereby expressly made to the disclosure of this patent application, both with regard to the sequences disclosed themselves and with regard to the finding and characterization of these and further sucrose isomerase-coding sequences from other sources.
• sucrose isomerases can be known to the person skilled in the art, inter alia. the gene databases using suitable search profiles and computer programs for screening for homologous sequences or for sequence comparisons.
• the person skilled in the art can find further nucleic acid sequences encoding sucrose isomerase from other organisms by means of conventional molecular biological techniques and use them in the context of the present invention.
• the person skilled in the art can derive suitable hybridization probes from the known sucrose isomerase sequences and use them for screening cDNA and / or genomic banks of the desired organism from which a new sucrose isomerase gene is to be isolated.
• the person skilled in the art can rely on common hybridization methods.
• This transformed cell line is able to identify palatinose producers in the gene bank prepared as described above from DNA of the donor organism.
• the cells of the gene bank are isolated and grown on minimal salt media with galactose and sucrose. After replica stamping of the colonies on plates with the same medium, the cells are killed by evaporation with toluene. Cells from the screening strain are then spread and incubated as a lawn in minimal salt soft agar without addition of C sources over the colonies of the gene bank.
• isomerase clones can also be identified using a PCR fragment. If plasmid DNA of the E. coli clones identified in this way is used as probes for hybridization on filters with immobilized DNA from the donor organism, the gene regions which carry isomerase genes can be detected and made available in a targeted manner.
• Functional equivalents of the proteins with sucrose isomerase activity disclosed in the context of this invention preferably include those from other organisms, for example from microorganisms whose genomic sequence is known in whole or in part, such as, for example, from microorganisms of the genera Protaminobacter, Erwinia, Serratia, Leuconostoc, Pseudomonas, Agrobacterium , Klebsiella and Enterobacter. These can be found, for example, by database searches in sequence databases such as GenBank or by screening gene or cDNA banks - for example using the sequence according to SEQ ID NO: 1 or a part thereof Search sequence or probe - can be found. Mutations include substitutions, additions, deletions, inversions or insertions of one or more amino acid residues.
• the person skilled in the art can additionally introduce various mutations into the DNA sequence encoding the sucrose isomerase by means of routine techniques, which leads to the synthesis of proteins with possibly changed biological properties. So it is e.g. possible to specifically produce enzymes that are localized in certain compartments of the plant cell by adding corresponding signal sequences. Such sequences are described in the literature and are known to the person skilled in the art (see, for example, Braun et al. (1992) EMBO J 11: 3219-3227; Wolter F et al. (1988) Proc Natl Acad Sei USA 85: 846-850; Sonnewald U et al. (1991) Plant J 1: 95-106).
• mutants are produced that are no longer subject to the regulatory mechanisms normally found in the cell via allosteric regulation or covalent modification. Furthermore, mutants can be produced which have an altered substrate or product specificity. Furthermore, mutants can be produced which have a changed activity, temperature and / or pH profile.
• the degeneration of the genetic code offers the skilled worker the possibility of adapting the nucleotide sequence of the DNA sequence to the codon preference ("codon usage") of the target plant, that is to say of the plant or plant cell which is pathogen-resistant due to the expression of the sucrose isomerase nucleic acid sequence, and thereby optimizing the expression.
• the recombinant nucleic acid molecules according to the invention or parts thereof can be introduced into plasmids which permit mutagenesis or a sequence change by recombining DNA sequences.
• base exchanges can be carried out or natural or synthetic sequences can be added.
• adapters or linkers can be added to the fragments where necessary.
• Appropriate restriction sites can also be provided by means of enzymatic and other manipulations or superfluous DNA or restriction sites be removed. Where insertions, deletions or substitutions are possible, in vitro mutagenesis, "primer repair", restriction or ligation can be used. Sequence analysis, restriction analysis and other biochemical-molecular biological methods are generally carried out as analysis methods.
• Said functional equivalents preferably have a homology of at least 40%, particularly preferably at least 50%, particularly preferably at least 70%, most preferably at least 90% to one of the polypeptide sequences with the
• SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 or 36 The homology extends most over at least 30 amino acids, preferably at least 60 amino acids, particularly preferably at least 90 amino acids preferably over the entire length of one of the polypeptides according to SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 or 36.
• GAP Garnier ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇
• Gap Weight 8 Length Weight: 2
• a sequence which has a homology of at least 80% on a protein basis with the sequence SEQ ID NO: 2 is understood to mean a sequence which, when compared with the sequence SEQ ID NO: 2 according to the above program algorithm with the above parameter set, has a homology of has at least 80%.
• Functional equivalents also include those proteins which are encoded by nucleic acid sequences which have a homology of at least 40%, particularly preferably at least 50%, particularly preferably at least 70%, most preferably at least 90% to one of the nucleic acid sequences with the SEQ ID NO: 1 , 3, 5, 7, 9, 11, 13, 15, 17, 19, 21 or 35.
• the homology extends over at least 100 bases, preferably at least 200 bases, particularly preferably at least 300 bases, most preferably over the entire length of one of the sequences according to SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15 , 17, 19, 21 or 35.
• Homology between two nucleic acid sequences means the identity of the two nucleic acid sequences over the respective sequence length, which can be determined by comparison using the program algorithm GAP (Wisconsin Package Version 10.0, University of Wisconsin, Genetics Computer Group (GCG), Madison, USA; Altschul et al . (1997) Nucleic Acids Res. 25: 3389ff) is calculated using the following parameters:
• Gap Weight 50 Length Weight: 3
• a sequence which has a homology of at least 80% on a nucleic acid basis with the sequence SEQ ID NO: 1 is understood to mean a sequence which, when compared with the sequence SEQ ID NO: 1 according to the above program algorithm with the above parameter set, has a homology of has at least 80%.
• Functional equivalents also include those proteins which are encoded by nucleic acid sequences which, under standard conditions, have one of the nucleic acid sequences described by SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21 or 35, hybridize to this complementary nucleic acid sequence or parts of the aforementioned and have the essential properties of a Saacharo isomerase.
• Standard hybridization conditions is to be understood broadly and means stringent as well as less stringent hybridization conditions. Such hybridization conditions are described, inter alia, by Sambrook J, Fritsch EF, Maniatis T et al. , in Molecular Cloning (A Laboratory Manual), 2nd edition, Cold Spring Harbor Laboratory Press, 1989, pages 9.31-9.57) or in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6.).
• the conditions during the washing step can be selected from the range of conditions limited by those with low stringency (with approximately 2X SSC at 50 ° C) and those with high stringency (with approximately 0.2X SSC at 50 ° C, preferably at 65 ° C) (20X SSC: 0.3 M sodium citrate, 3 M NaCl, pH 7.0).
• the temperature during the washing step can be raised from low stringent conditions at room temperature, about 22 ° C, to more stringent conditions at about 65 ° C. Both parameters, salt concentration and temperature, can be varied simultaneously, one of the two parameters can be kept constant and only the other can be varied. Denaturing agents such as formamide or SDS can also be used during hybridization. In the presence of 50% formamide, the hybridization is preferably carried out at 42 ° C.
• Hybridization conditions can be selected from the following conditions, for example:
• Washing steps can be selected, for example, from the following conditions:
• the nucleic acid sequence encoding a protein with a sucrose isomerase activity comprises nucleic acid sequences coding for proteins with sucrose isomerase activity, the nucleic acids being selected from the group consisting of
• nucleic acid sequences encoding an amino acid sequence according to SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 or 36, and
• nucleic acid sequences according to SEQ ID No: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21 or 35, and d) nucleic acid sequences which is degenerate to a nucleic acid sequence of c), and
• nucleic acid sequences which have a homology of at least 40% to a nucleic acid sequence according to SEQ ID No: 1, 3, 5, 7, 9,
• nucleic acid sequences which hybridize with a complementary strand of the nucleic acid sequence according to SEQ ID No: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21 or 35,
• Functionally equivalent fragments means, with respect to a protein with sucrose isomerase activity or a nucleic acid sequence, coding for such a polypeptide or all those coding nucleic acid sequences which, compared to their starting sequence, have a shortening at the 5 'and / or 3' end and / or have one or more deletions, but still have sucrose isomerase activity, or code for a protein with the same.
• deletion mutants in which the synthesis of correspondingly shortened proteins can be achieved by progressive deletion from the 5 'or from the 3' end of the coding DNA sequence.
• sucrose isomerases can also be supplemented by signal sequences which ensure the transport of the gene product, in this case the protein with sucrose isomerase activity, to a specific compartment.
• signal sequences ensure that the sucrose isomerase is transported into the cell wall or the apoplasts of the transformed plant cells, i.e. the transformed plants express a chimeric sucrose isomerase which comprises a signal peptide for transport into the endoplasmic reticulum.
• Suitable signal sequences which ensure inclusion in the endoplasmic reticulum can be found in the relevant literature by the person skilled in the art. For example, the sequence coding for the signal peptide of the proteinase inhibitor II gene from potato (Keil et al. (1996) Nucl Acids Res 14: 5641-5650; Genbank Accession No. X04118).
• Other suitable signal sequences provide e.g. for the inclusion of sucrose isomerase in the
• Vacuole Here is the signal peptide of the patatin gene as an example from potatoes (Sonnewald U et al. (1991) Plant J 1 (1): 95-106).
• Phathogen resistance means the diminution or weakening of 5 disease symptoms of a plant as a result of an infestation by a pathogen.
• the symptoms can be of various types, but preferably include those which directly or indirectly impair the quality of the plant, the quantity of the yield, the suitability for use as feed or food, or else sowing, cultivation, harvesting or processing of the Complicate crops.
• the increased resistance manifests itself preferably in a reduced expression of the disease symptoms, whereby disease symptoms - in addition to the above-mentioned impairments - also, for example, the penetration efficiency of a pathogen in the
• the disease symptoms are preferably at least 10% or at least 20%, particularly preferably by at least 40% or 60%, very particularly preferably by at least 70% or 80%, most preferably by at least
• selection comprises all of the
• 35 drive which are suitable for the detection of existing or increased pathogen resistance.
• These may be symptoms of the pathogen infection (e.g. housorium training in case of fungal infection) but also include the symptoms described above, which indicate the quality of the plant, the quantity of the yield
• pathogen means, for example, non-restrictive fungi, fungus-like pathogens (such as, for example, Chromista; for example Oomycetes) and animal pests such as, for example, nematodes. Nematodes and fungi are particularly preferred. However, it can be assumed that the expression of a Sucrose isomerase protein also causes resistance to other pathogens.
• Fungal pathogens and fungal pathogens (such as, for example, Chromista) 0 comprise biotrophic, hemibiotrophic and necrotrophic fungi and preferably come from the group comprising Plasmodiophora- ycota, Oomycota, Ascomycota, Chytridiomycetes, Zygomycetes, Basidiomycota and Deuteromyceten (Fungi imperfecti).
• the pathogens mentioned in Tables 15 and 2 and the diseases associated with them should be mentioned as examples, but not by way of limitation.
• Plasmodiophoromycota such as Plasmodiophora brassicae (Kohl's hernia, clubroot of crucifers), Spongospora subterranea (powdery scab of potato tubers), Polymyxa graminis (root disease of cereals and grasses),
• Ascomycota such as Microdochium nivale (snow mold on rye and wheat), Fusarium graminearum, Fusarium culmorum (rotten ears, especially on wheat), Fusarium oxysporum (Fusarium wilt on tomato), Blumeria graminis (powdery mildew on barley (f.sp. ) and wheat (f.sp.
• Basidiomycetes such as Typhula incarnata (Typhula rot on barley, rye, wheat), Ustilago maydis (bump fire on
• fungi imperfecti such as Septoria nodorum (tan) on wheat (Septoria tritici), Pseudocerco-sporella herpotrichoides (broken stalk disease on wheat, barley, rye), Rynchosporium secalis (leaf blotch disease on rye and barley), Alternaria solani (Alternaria solani Potato, tomato), Phoma betae (root fire on beta beet), Cercospora beticola (Cercospora leaf blotch on beta beet), (Alternaria brassicae (black rapeseed on rapeseed, cabbage and cruciferous vegetables, among others), Verticillium dahliae (rapeseed wilt, and columbus rot) Lindemuthianum (focal spot disease on beans), Phoma Hungary - diarrhea (black-legged on cabbage; root neck or stem rot on rapeseed),
• Botrytis cinerea (gray mold on grapevines, strawberries, tomatoes, hops etc.).
• Phytophthora infestans (late blight, late blight in tomatoes etc.), Microdochium nivale (formerly Fusarium nivale; snow mold on rye and wheat), Fusarium graminearum, Fusarium culmorum (ear rot on wheat), Fusarium oxysporum (fusarium wilt on tomato), Blumeria graminis (powdery mildew on barley (f.sp. hordei) and wheat (f.sp.
• Magnaporthe grisea rice blast disease
• Sclerotinia sclerotium white stalk, rape cancer
• Septoria nodorum and Septoria tritici tan on wheat
• Alternaria brassicae black rapeseed on rapeseed, cabbage and cruciferous vegetables, among others
• Phoma Hungary diarrhea, black legs on cabbage; root neck or stem rot on rapeseed.
• Root nematodes (e.g. Pratylenchus, Xiphinema and Longidorus species).
• Wandering nematodes are not tied to a parasitization site, but can change them. They can migrate from one root to another, from one plant to another and sometimes also in the plant tissue. For a long time, their importance as pests was underestimated: today they are among the extremely dangerous nematodes that damage plants. Many growth damage (also known as "soil fatigue") and early yellowing of the crops could be attributed to such root pests. Pratylenchus species in particular are also known to cause violent root damage in ornamental plant cultivation. Diseased roots can be recognized by the fact that they show brown discoloration in places. Rotting organisms subsequently penetrate into the resulting wounds, causing rapid tissue death and deep rotting in these areas. Host plants include: cereals, potatoes, carrots, tomatoes, cucumbers, celery and wine.
• Nematodes producing root galls e.g. Meloidogyne species
• Root gall whales are among the largest pests, especially in greenhouses, but they have been also proven outdoors on carrots, celery and parsley.
• the wheat whale is a specialized parasite of the wheat blossom, which converts it into galls.
• the nematode infestation can already be recognized from the curls or ripples of the leaves in the early stages of the plant.
• Cyst-forming root nematodes (globodera and heterodera species)
• the potato cyst lynx is the number one enemy of potatoes. This species outperforms all other Herderodera species and can destroy up to 80% of the harvest in the event of a massive outbreak. After infestation with cyst-forming nematodes, the plant takes care of it without an externally recognizable cause. Only when you examine the roots do you see pin-sized, brownish, yellow or whitish cysts. The female nematodes burrow into the root and blow up the root through their abdomen, which is filled with eggs and thus swells. The nematode is still in the root with its mouth prick, while the bulging abdomen lies in the ground. The mother dies and its hardening skin becomes a protective cover
• cysts and their contents are very resistant and can last a long time. If the environmental conditions are suitable, the larvae drill into the open and attack new roots.
• the most important cyst-forming nematodes are potato, beet, oat, pea, clover, cabbage, hop, and carrot cyst stalks (for an examination of potato cyst nematodes see also at: http://www.bfl.at/)
• Nematodes are particularly preferred as animal pests.
• the pathogens listed in Table 3 and the diseases associated with them should be mentioned as examples, but not by way of limitation.
• Globodera rostochiensis and G. pallida (cysts on potatoes, tomatoes and nightshade plants), Heterodera schachtii (beet cysts on sugar and fodder beet, oilseed rape, cabbage, etc.), Heterodera avenae (oat cysts on oats and other types of cereals), dityi (chickens) are very particularly preferred.
• Small stem or stick small beet head on rye, oats, corn, clover, tobacco, beet, Anguina tritici (small wheat, disease of wheat on spelled, spelled, rye), Meloidogyne hapla (root gall small on carrot, cucumber, lettuce, tomato, potato, sugar beet , Alfalfa).
• Barley Puccinia graminis f.sp. hordei (barley ste rust), blumeria (Erysiphe) graminis f.sp. Hordei (Barley Powdery Mildew).
• Soybean Phytophthora megasperma fsp.glycinea, Macrophomina phaseolina, Rhizoctonia solani, Sclerotinia sclerotiorum,
• Canola Albugo candida, Alternaria brassicae, Leptosphaeria maculans, Rhizoctonia solani, Sclerotinia sclerotiorum,
• Mycosphaerella brassiccola Pythium ultimum, Peronospora parasitica, Fusarium roseum, Alternaria alternata.
• Alfalfa Clavibater michiganese subsp. insidiosum, Pythium ultimum, Pythium irregulare, Pythium splendens, Pythium debaryanum, Pythium aphanidermatum, Phytophthora megasperma, Peronospora trifoliorum, Phoma medicaginis var. medicaginis, Cercospora medicaginis, Pseudopeziza medicaginumomyidis, fugus aphaginophysiumis, fagus medicaginium, agis trophis, trophic aphidis Stemphylium alfalfae.
• Stenocarpella maydi (Diplodia maydis), Pythium irregulare, Pythium debaryanum, Pythium graminicola, Pythium splendens, Pythium ultimum, Pythium aphanidermatum, Aspergillus flavus, Bipolaris maydis 0, T (Cochliobolus heterostrophus), Helminthobiumus carbonum Exserohilum turcicum I, II & III, Helminthosporium pedicellatum, Physoderma maydis, Phyllosticta maydis, Kabatiella maydis, Cercospora sorghi, Ustilago maydis, Puccinia sorghi, Puccinia polysora, Macrophomina phaseolina, Penicillium oxalicum, Clariusumiaumiaumia, arbariaumiaumia lumbarumumiaumia lumbarumumia,
• Sorghum Exserohilum turcicum, Colletotrichum graminicola (Glomerella graminicola), Cercospora sorghi, Gloeocercospora sorghi, Ascochyta sorghina, Puccinia purpurea, Macrophomina phaseolina, Perconia circinata, Fusarium monilifata, Alternaria alternolahomehapia, Alternaria alternolaunolaisola, Alternaria alternatehumolina, Alternaria alternateunia, Solaria alternate, Ramulispora sorghi, Ramulispora sorghicola, Phyllachara sacchari,
• Sporisorium reilianum (Sphacelotheca reiliana), Sphacelotheca cruenta, Sporisorium sorghi, Claviceps sorghi, Rhizoctonia solani, Acremonium strictum, Sclerophthona macrospora, Peronosclerospora sorghi, Peronosclerospora philippinumararumumarumumomiumaromiumariumumomusium, Faminiciumomaniumumus, Scleromiumiumspariumium, graminiumiumspariumium, gramineromiumium, Famin griumaminomarosum, gramineromium ariumium, graminerium ariumium, graminarium arumium, graminarium arumium, graminarium arumium, graminarium arumium, graminarium arumium, graminarium arumium, graminerium arium, graminerium
• Plant organism or cells derived therefrom generally means any cell, tissue, part or reproductive material (such as seeds or fruits) of an organism which is capable of photosynthesis. Included in the scope of the invention are all genera and species of higher and lower plants in the plant kingdom. Annual, perennial, monocot and dicot plants are preferred. Included are mature plants, seeds, sprouts and seedlings, as well as parts derived from them, propagation material (for example tubers, seeds or fruits) and cultures, for example row or callus cultures. Mature plants mean plants at any stage of development beyond the seedling. Seedling means a young, immature plant at an early stage of development.
• Plant in the context of the invention means all genera and species of higher and lower plants in the plant kingdom. Included under the term are the mature plants, seeds, shoots and seedlings, as well as parts derived therefrom, propagation material, plant organs, tissues, protoplasts, callus and other cultures, for example cell cultures, and all other types of groupings of plant cells to form functional or structural units. Mature plants mean plants at any stage of development beyond the seedling. Seedling means a young, immature plant at an early stage of development.
• Plant includes all annual and perennial, monocotyledonous and dicotyledonous plants and includes, by way of example but not by way of limitation, those of the genera Cucurbita, Rosa, Vitis, Juglans, Fragaria, Lotus, Medicago, Onobrychis, Trifolium, Trigonella, Vigna, Citrus, Linum, Geranium, Manihot, Daucus, Arabidopsis, Brassica, Raphanus, Sinapis, Atropa, Capsicum, Datura, Hyoscyamus, Lycopersicon, Nicotiana, Solarium, Petunia, Digitalis, Majorana, Cichorium, Helianthus, Lactuca, Bromus, Asparagus, Antirrhinum, Heterocallis, Nemesis, Pelargonium, Pelargonium Ranunculus, Senecio, Salpiglossis, Cucumis, Browaalia, Glycine, Pisum, Phaseolus, Loliu, Ory
• Plants from the following plant families are preferred: Amaranth aceae, Asteraceae, Brassicaceae, Carophyllaceae, Chenopodiaceae, Compositae, Cruciferae, Cucurbitaceae, Labiatae, Leguminosae, Papilionoideae, Liliaceae, Linaceae, Malvaceae, Acaceaeaeae, Rosaceaeae, Solaceaeae Tetragoniacea, Theaceae, Umbelliferae.
• Preferred monocotyledonous plants are selected in particular from the monocotyledonous crop plants, such as, for example, the family of the Gramineae such as rice, corn, wheat or other types of cereals such as barley, millet, rye, triticale or oats, and sugar cane and all types of grasses.
• the family of the Gramineae such as rice, corn, wheat or other types of cereals such as barley, millet, rye, triticale or oats, and sugar cane and all types of grasses.
• the invention is particularly preferably applied to dicotyledonous plant organisms.
• Preferred dicotyledonous plants are in particular selected from the dicotyledonous crop plants, such as, for example
• Asteraceae such as sunflower, tagetes or calendula and others
• - Cruciferae especially the genus Brassica, especially the species napus (rape), campestris (turnip), oleracea cv Tastie (cabbage), oleracea cv Snowball Y (cauliflower) and oleracea cv Emperor (broccoli) and other types of cabbage; and the genus Arabidopsis, especially the species thaliana as well as cress or canola and others,
• Cucurbitaceae such as melon, pumpkin or zucchini and others
• - Leguminosae especially the genus Glycine, especially the type max (soybean) as well as alfalfa, peas, beans or peanuts and others Rubiaceae, preferably of the subclass Lamiidae such as, for example, Coffea arabica or Coffea liberica (coffee bush) and others,
• - Solanaceae especially the genus Lycopersicon, especially the species esculentum (tomato), the genus Solanum, especially the species tuberosum (potato) and melongena (eggplant), and the genus Capsicum, especially the species annum (paprika) as well as tobacco and others more,
• Sterculiaceae preferably of the subclass Dilleniidae such as Theobroma cacao (cocoa bush) and others,
• Theaceae preferably of the subclass Dilleniidae, such as, for example, Camellia sinensis or Thea sinensis (tea bush) and others,
• Umbelliferae especially the genus Daucus (especially the species carota (carrot)) and Apium (especially the species graveolens dulce (Seiarie)) and others,
• decorative plants useful or ornamental trees, flowers, cut flowers, shrubs or lawn.
• Examples include, but are not limited to, angiosperms, bryophytes such as hepaticae (liverwort) and musci (mosses); Pteridophytes such as ferns, horsetail and lycopods; Gymnosperms such as conifers, cycads, ginkgo and gnetals, the families of rosaceae such as rose, ericaceae such as rhododendrons and azaleas, euphorbiaceae such as poinsettias and croton, caryophyllaceae such as cloves, solanaceae such as petunias, Gesneriaceae such as the Usamalsaceaeideae such as the Usambaramineae , Iridaceae like gladiolus, iris, freesia and crocus, Compositae like
• agricultural crops which naturally have a high proportion of sucrose or whose roots, tubers or storage roots are used economically, such as potatoes, beets or sugar beets.
• tomato, banana, carrot, sugar cane, strawberry, pineapple, papaya, soy and cereals such as oats, Barley, wheat, rye, triticale, millet and corn.
• potato, beet, sugar beet and sugar cane are preferred.
• expression constructs are used for the expression of proteins with sucrose isomerase activity in plants.
• Expression cassettes of this type are described, for example, in WO 01/59136 and WO 01/59135, to which reference is hereby expressly made.
• a nucleic acid molecule encoding a protein with sucrose isomerase activity (for example described by SEQ ID NO: 2 or a functional equivalent thereof or a functionally equivalent part of the abovementioned) is preferably functionally linked to at least one genetic control element (for example a promoter) , which ensures transgenic expression in a plant organism or a tissue, organ, part or cell thereof.
• a functional link is understood to mean, for example, the sequential arrangement of a promoter with the nucleic acid sequence to be expressed (for example the sequence according to SEQ ID NO: 1) and possibly other regulatory elements such as a terminator such that each of the regulatory elements can perform its function in the transgenic expression of the nucleic acid sequence. This does not necessarily require a direct link in the chemical sense. Genetic control sequences, such as, for example, enhancer sequences, can also perform their function on the target sequence from more distant positions or even from other DNA molecules. Arrangements are preferred in which the nucleic acid sequence to be expressed transgenically is positioned behind the sequence which acts as a promoter, so that both sequences are covalently linked to one another.
• transgenic expression construct consisting of a linkage of promoter and nucleic acid sequence to be expressed, may preferably be integrated in a vector and inserted into a plant genome by, for example, transformation.
• An expression construct is, however, also to be understood as such constructions in which the nucleic acid sequence coding for the protein with sucrose isomerase activity (for example coded by SEQ ID NO: 2 or a functional equivalent thereof or a functionally equivalent part of the aforementioned) - for example by means of a homologous recombination - placed behind an endogenous plant promoter in such a way that this ensures transgenic expression of the said nucleic acid sequence.
• Plant-specific promoters basically means any promoter that can control the expression of genes, in particular foreign genes, in plants or plant parts, cells, tissues or cultures.
• the promoter can be selected so that the expression takes place constitutively or only in a certain tissue or organ, at a certain time in plant development and / or at a time determined by external influences, biotic or abiotic stimuli (induced gene expression).
• the promoter can be homologous or heterologous with respect to the plant to be transformed. Preferred are:
• “Constitutive” promoters mean those promoters which ensure expression in numerous, preferably all, tissues over a relatively long period of plant development, preferably at all times during plant development (Benfey et al. (1989) EMBO J 8: 2195-2202).
• a plant promoter or a plant virus-derived promoter is preferably used.
• the promoter of the 35S transcript of the CaMV cauliflower mosaic virus (Franck et al. (1980) Cell 21: 285-294; Odell et al. (1985) Nature 313: 810-812; Shewmaker et al. (1985) Virology 140 : 281-288; Gardner et al. (1986) Plant Mol Biol 6: 221-228) or the 19S CaMV promoter
• Another suitable constitutive promoter is the LeguminB promoter (GenBank Acc.-No. X03677), the promoter of nopaline synthase from Agrobacterium, the TR double promoter, the OCS (octopine synthase) promoter from Agrobacterium, the Ubi uitin promoter (Holtorf S et al. (1995) Plant Mol Biol 29: 637-649), the ubiquitin 1 promoter (Christensen et al. (1992) Plant Mol Biol 18: 675-689; Bruce et al.
• the constitutive promoter is the promoter of the nitrilase-1 (nitl) gene from A. thaliana (GenBank Acc.-No.: Y07648.2, nucleotides
• Promoters with specificities for the leaves, stems, roots or seeds are also preferred.
• Seed-specific promoters such as the promoter of phaseoline (US 5,504,200; Bustos MM et al. (1989) Plant Cell 1 (9): 839-53; e.g. from Phaseolus vulgari; van der Geest et al. (1996) Plant Mol Biol 32 : 579-588), des 2S albumins (Joseffson LG et al. (1987) J Biol Chem 262: 12196-12201), legumes (Shirsat A et al. (1989) Mol Gen Genet 215 (2): 326-331 ), the USP (unknown seed protein; Bäumlein H et al. (1991) Mol Gen Genet 225 (3): 459-467; Phillips et al.
• seed-specific promoters are those of the genes coding for the "high molecular weight glutenin” (HMWG), gliadin, branching enzyme, ADP glucose pyrophosphatase (AGPase), the napin promoter, the ACP promoter and the FatB3 and FatB4- Promoters, the promoter of starch synthase or other starch-forming / modifying enzymes such as, for example, promoters of genes for branching enzymes code (WO 92/14827, WO 92/11375). Also preferred are promoters which allow seed-specific expression in monocotyledons such as corn, barley, wheat, rye, rice etc.
• the promoter of the lpt2 or lptl gene (WO 95/15389, WO 95/23230) or the promoters described in WO 99/16890 (promoters of the hordein gene, the glutelin gene, the oryzine gene, etc.) can be used advantageously Prolamin gene, gliadin gene, glutelin gene, zein gene, kasirin gene or secalin gene). Further seed-specific promoters are described in WO 89/03887.
• Tuber-, storage root- or root-specific promoters such as the patatin promoter class I (B33), the promoter of the cathepsin D inhibitor from potato.
• the promoter of the class I patatin gene is about 100 to 1000 times more active in tubers than in leaves (Rocha-Sosa et al., Vide supra). Further
• tuber-specific or at least increased expression in tubers are known (e.g. the promoter of the ADP-glucose pyrophosphorylase genes; Müller et al.
• Leaf-specific promoters such as a promoter of the cytosolic FBPase from potato (WO 97/05900), the SSU promoter (small subunit) from Rubisco (ribulose-1, 5-bis-phosphate carboxylase; US 4,962,028) or the ST-LSI promoter from potato ( Stockhaus et al. (1989) EMBO J
• Epidermis-specific promoters are very particularly preferred, such as the promoter of the OXLP gene (“oxalate oxidase like protein”; Wei et al. (1998) Plant Mol Biol 36: 101-112).
• transgenic expression constructs can also contain a chemically inducible promoter (review article: Gatz et al. (1997) Annu Rev Plant Physiol Plant Mol Biol
• promoters such as the PRPl promoter (Ward et al. (1993) Plant Mol Biol 22: 361-366), a promoter induced by salicylic acid (WO 95/19443), a promoter promoted by benzenesulfonamide (EP 0 388 186), a tetracycline-inducible promoter (Gatz et al.
• suitable promoters are, for example, fruit ripening-specific promoters, such as the fruit ripening-specific promoter from tomato (WO 94/21794, EP 409 625).
• Development-dependent promoters partly include the tissue-specific promoters, since the formation of individual tissues is naturally development-dependent.
• promoters that are induced by biotic or abiotic stress, such as the pathogen-inducible promoter of the PRPL gene (Ward et al. (1993) Plant Mol Biol 22: 361-366), the heat-inducible hsp70 or hsp80 promoter from tomato (US 5,187,267), the cold-inducing alpha-amylase promoter from the potato (WO 96/12814) or the light-inducible PPDK promoter.
• pathogen-inducible promoter of the PRPL gene Ward et al. (1993) Plant Mol Biol 22: 361-366
• the heat-inducible hsp70 or hsp80 promoter from tomato US 5,187,267
• the cold-inducing alpha-amylase promoter from the potato
• WO 96/12814 the light-inducible PPDK promoter.
• Pathogen-inducible promoters include the promoters of genes that are induced as a result of pathogen attack such as genes from PR proteins, SAR proteins, ⁇ -1, 3-glucanase, chitinase etc. (e.g. Redolfi et al. (1983) Neth J Plant Pathol 89: 245-254; Uknes et al. (1992) Plant Cell 4: 645-656; Van Loon (1985) Plant Mol Viral 4: 111-116; Marineau et al. (1987) Plant Mol Biol 9: 335 -342; Matton et al. (1987) Molecular Plant-Microbe Interactions 2: 325-342; Somssich et al.
• wound-inducible promoters such as that of the pinll gene (Ryan (1990) Ann Rev Phytopath 28: 425-449; Duan et al. (1996) Nat Biotech 14: 494-498; EP-A 375 091), the wunl and wun2 gene (US 5,428,148), the winl and win2 genes (Stanford et al. (1989) Mol Gen Genet 215: 200-208), the systemin gene (McGurl et al. (1992) Science 225: 1570- 1573), the WIPl gene (Rohmeier et al. (1993) Plant Mol Biol 22: 783-792; Eckelkamp et al. (1993) FEBS Letters 323: 73-76), of the MPI gene (Corderok et al. (1994) Plant J 6 2): 141-150) and the like.
• the pinll gene Rost al. (1990) Ann Rev Phytopath 28: 425-449; Duan
• Promoters that are particularly preferred are those that are specifically in nutrient. cell systems (syncytia) after nematode involvement. Examples are to be mentioned
• Gemini virus V-sense promoters (WO 00/01832), in particular the promoters described by SEQ ID NO: 32, 33 or 34.
• nematode-inducible promoters within the scope of this invention are described in WO 98/22599.
• the regulatory areas i.e. the areas upstream of the ATG start codon
• the promoter sequences described in US Pat. No. 6,395,963 are particularly preferred.
• the promoter sequences described in WO 03/033651 are particularly preferred.
• the promoter sequences described in JP 2001508661-A are particularly preferred.
• nemotode-inducible promoters can be derived from genes, the induction of which is described as a result of a nematode attack. Examples include, but are not limited to: The pollenin promoter (Karimi M et al. (2002) J Nematol 34 (2): 75-79) and the promoter of a putative receptor serine / threonine protein kinase (Custers JHHV et al (2002) Mol Plant Pathol 3 (4): 239-249).
• Pathogen- or stress-inducible as well as seed-, tuber-, root-, leaf- and / or stem-specific are particularly preferred, whereby pathogen-inducible (especially the nematode-inducible promoters mentioned above) are most preferred.
• Another - particularly preferred - subject of the invention relates to expression constructs in which a nucleic acid sequence coding for a protein with sucrose isomerase activity in is functional linkage with a stress, pathogen, or wound-inducible promoter.
• Stress, pathogenic or wound-inducible promoters generally mean all those promoters that can be induced by biotic or abiotic stress.
• Abiotic stress means stimuli such as heat, cold, dryness, frost, moisture, salt, UV light, etc.
• Biotic stress means infestation by a pathogen, the term "pathogen" encompassing all of the pathogens mentioned above.
• the stimulus preferably has a strength that leads to a drop in yield of at least 5% compared to average yield values.
• Inducible here means an increase in transcription activity by at least 50%, preferably at least 100%, particularly preferably at least 500%, very particularly preferably at least 1000%, most preferably at least 5000% in comparison to the expression activity of a non-stimulated plant.
• Stress or pathogen inducible promoters include, by way of example, but not by limitation, the pathogen inducible promoter of the PRPl gene (Ward et al.
• wound-inducible promoters such as that of the pinll gene (Ryan (1990) Ann Rev Phytopath 28: 425-449; Duan et al. (1996) Nat Biotech 14: 494-498), the wunl and wun2 genes ( US 5,428,148), the winl and win2 genes (Stanford et al. (1989) Mol Gen Genet 215: 200-208), the systemin gene (McGurl et al. (1992) Science 225: 1570-1573), des WIPl gene (Rohmeier et al.
• Wound-inducible promoters are to be used to advantage when infested with feeding pathogens.
• the average person skilled in the art can also easily find additional examples of genes with stress, pathogen or wound-induced expression patterns in the literature.
• the average person skilled in the art is able to isolate further suitable promoters using routine methods. The person skilled in the art can thus identify appropriate regulatory nucleic acid elements with the aid of common molecular biological methods, for example hybridization experts or DNA-protein binding studies.
• a differential expression library of, for example, pathogen-infected / infected and "normal" tissues is created.
• promoters are isolated which have pathogen-inducible regulatory elements.
• the person skilled in the art also has other methods based on PCR for the isolation of suitable stress-, pathogen- or wound-induced promoters.
• Tissue-specific promoters in particular seed-specific, tuber-specific, fruit-specific and leaf-specific promoters and pathogen-induced promoters are particularly preferred.
• Pathogen-induced promoters in particular nematode-induced promoters, are very particularly preferred.
• promoters can be functionally linked to the nucleic acid sequence to be expressed, which enable transgenic expression in other plant tissues or in other organisms, such as, for example, E. coli bacteria.
• all promoters described above can be used as plant promoters.
• the nucleic acid sequences contained in the expression constructs or expression vectors can be functionally linked to further genetic control sequences in addition to a promoter.
• the term “genetic control sequences” is to be understood broadly and means all those sequences which have an influence on the formation or the function of an expression construct. Genetic control sequences modify, for example, transcription and translation in prokaryotic or eukaryotic organisms.
• the expression constructs preferably comprise a plant-specific promoter 5 'upstream of the respective nucleic acid sequence to be expressed transgenically and a terminator sequence 3' downstream as an additional genetic control sequence, and optionally further customary regulatory elements, each functionally linked to the transgenic nucleic acid sequence to be expressed.
• Genetic control sequences also include further promoters, promoter elements or minimal promoters that can modify the expression-controlling properties. Genetic control sequences can, for example, also result in tissue-specific expression depending on certain stress factors. Corresponding elements are, for example, for water stress, abscisic acid (Lam E and Chua NH (1991) J Biol Chem 266 (26): 17131-17135) and heat stress (Schoffl F et al. (1989) Mol Gen Genetics 217 (2- 3): 246-53).
• Genetic control sequences also include the 5 'untranslated regions, introns or non-coding 3' regions of genes such as the actin-1 intron, or the Adhl-S introns 1, 2 and 6 (general: The Maize Handbook, Chapter 116, Freeling and Walbot, Eds., Springer, New York (1994)). It has been shown that these can play a significant role in regulating gene expression. It has been shown that 5 'untranslated sequences can increase the transient expression of heterologous genes.
• An example of translation enhancers is the 5 'leader sequence from the tobacco mosaic virus (Gallie et al. (1987) Nucl Acids Res 15: 8693-8711) and the like. They can also promote tissue specificity (Rouster J et al. (1998) Plant J 15: 435-440).
• the transgenic expression construct can advantageously contain one or more so-called “enhancer sequences” functionally linked to the promoter, which enable increased transgenic expression of the nucleic acid sequence. Additional advantageous sequences, such as further regulatory elements or terminators, can also be inserted at the 3 'end of the nucleic acid sequences to be expressed transgenically.
• the nucleic acid sequences to be expressed transgenically can be contained in one or more copies in the gene construct.
• Polyadenylation signals suitable as control sequences are plant polyadenylation signals, preferably those which essentially comprise T-DNA polyadenylation signals from Agrobacterium tumefaciens.
• Examples of particularly suitable terminator sequences are the OCS (octopine synthase) terminator and the NOS (nopalin synthase) terminator.
• Control sequences are also to be understood as those which enable homologous recombination or insertion into the genome of a host organism or the removal from the genome allow.
• homologous recombination for example, the coding sequence of a specific endogenous gene can be specifically exchanged for the sequence coding for a sucrose isomerase.
• a transgenic expression construct and / or the transgenic expression vectors derived from it can contain further functional elements.
• the term functional element is to be understood broadly and means all those elements which have an influence on the production, multiplication or function of the transgenic expression constructs according to the invention, the transgenic expression vectors or the transgenic organisms. Examples include, but are not limited to:
• a) Selection markers that show resistance to biocides e.g. Metabolism inhibitors (such as 2-deoxyglucose-6-phosphate (WO 98/45456), antibiotics (such as e.g. kanamycin, G 418, bleomycin, hygromycin) or herbicides (such as gyphosate or phosphinotricin).
• Metabolism inhibitors such as 2-deoxyglucose-6-phosphate (WO 98/45456)
• antibiotics such as e.g. kanamycin, G 418, bleomycin, hygromycin
• herbicides such as gyphosate or phosphinotricin
• Particularly preferred selection markers are those which confer resistance to herbicides. Examples include: DNA sequences which code for phosphinothricin acetyl transferases (PAT) and inactivate glutamine synthase inhibitors (bar and pat gene), 5-enolpyruvylshikimate-3-phosphate synthase genes (EPSP synthase genes) which are resistant to Glyphosat ® (N- (phosphonomethyl) glycine), the gox gene (glyphosate oxidoreductase) coding for the glyphosate ® degrading enzymes, the deh gene (coding for a dehalogenase which inactivates dalapon), sulfonylurea and imidazolinone inactivating acetolactate synthases and bxn genes which degrade nitrodilynase enzymes for bromoxynil enzymes aasa gene conferring resistance to the antibiotic apectinomycin, the strepto
• reporter proteins such as the "green fluorescence protein” (GFP) (Sheen et al. (1995) Plant Journal 8 (5 ): 777-784), chloramphenicol transferase, luciferase (Ow et al. (1986) Science 234: 856-859),
• origins of replication which ensure an increase in the transgenic expression constructs or transgenic expression vectors according to the invention in, for example, E. coli.
• a selectable marker which gives the successfully recombined cells resistance to a biocide (for example a herbicide), a metabolism inhibitor such as 2-deoxyglucose-6-phosphate (WO 98 / 45456) or an antibiotic.
• a biocide for example a herbicide
• a metabolism inhibitor such as 2-deoxyglucose-6-phosphate (WO 98 / 45456) or an antibiotic.
• the selection marker allows the selection of the transformed cells from untransformed (McCormick et al. (1986) Plant Cell Reports 5: 81-84).
• an expression construct according to the invention into an organism or cells, tissues, organs, parts or seeds thereof (preferably in plants or plant cells, tissues, organs, parts or seeds) can advantageously be implemented using vectors in which the transgenic Expression constructs are included.
• Vectors can be, for example, plasmids, cos ide, phages, viruses or even agrobacteria.
• the transgenic expression construct can be inserted into the vector (preferably a
• Plasmid vector can be introduced via a suitable restriction site or a recombinase att sequence.
• the resulting transgenic expression vector is first introduced into E. coli. Correctly transformed E. coli are selected, grown and the recombinant vector with those familiar to the person skilled in the art
• transformation or transduction or transfection
• the DNA or RNA can be introduced directly by microinjection or by bombardment with DNA-coated microparticles.
• the cell can also be chemically permeabilized, for example with polyethylene glycol, so that the DNA can pass through
• the DNA can also be obtained by protoplast fusion with other DNA-containing units such as minicells, cells, lysosomes or liposomes. Electroporation is another suitable method for introducing DNA, in which the cells are reversible by an electrical
• 20 impulse can be permeabilized.
• Appropriate methods are described (for example in Bilang et al. (1991) Gene 100: 247-250; Scheid et al. (1991) Mol Gen Genet 228: 104-112; Guerche et al. (1987) Plant Science 52: 111- 116; Neuhause et al. (1987) Theor Appl Genet 75: 30-36; Klein et al. (1987) Nature
• a transformation can also be carried out by bacterial infection using Agrobacterium tumefaciens or Agrobacterium rhizogenes. 45
• the Agrobacterium -mediated transformation is best suited for dicotyledonous plant cells. The procedures are described for example by Horsch RB et al. (1985). Science 225: 1229f).
• the transgenic expression construct has to be integrated into special plasmids, either into a shuttle or intermediate vector or a binary vector. If a Ti or Ri plasmid is used for the transformation, at least the right boundary, but mostly the right and the left boundary of the Ti or Ri plasmid T-DNA as flanking region, is connected to the transgenic expression construct to be introduced.
• Binary vectors are preferably used. Binary vectors can replicate in both E.coli and Agrobacterium. They usually contain a selection marker gene for the
• transformed plants e.g. the nptll gene, which confers resistance to kanamycin
• linker or polylinker flanked by the right and left T-DNA delimitation sequence In addition to the T-DNA restriction sequence, they also contain a selection marker that enables selection of transformed E. coli and / or agrobacteria (e.g. the nptlll gene, which confers resistance to kanamycin).
• Corresponding vectors can be transformed directly into Agrobacterium (Holsters et al. (1978) Mol Gen Genet 163: 181-187).
• the Agrobacterium which acts as the host organism in this case, should already contain a plasmid with the vir region. This is necessary for the transfer of T-DNA to the plant cell.
• An Agrobacterium transformed in this way can be used to transform plant cells.
• the use of T-DNA for the transformation of plant cells has been intensively investigated and described (EP 120 516; Hoekema, In: The Binary Plant Vector System, Offsetdrukkerij Kanters BV, Alblasserda, Chapter V; An et al. (1985) EMBO J 4: 277-287).
• Various binary vectors are known and some are commercially available, for example pBI101.2 or pBIN19 (Clontech Laboratories, Inc. USA).
• Direct transformation techniques are suitable for every organization and cell type.
• no special requirements are placed on the plasmid used.
• Simple plasmids such as the pUC series can be used.
• Stably transformed cells ie those which contain the inserted DNA integrated into the DNA of the host cell, can be selected from untransformed cells if a selectable marker is part of the inserted DNA.
• Any gene that can confer resistance to antibiotics or herbicides can act as a marker (see above).
• Transformed cells that express such a marker gene are able to survive in the presence of concentrations of a corresponding antibiotic or herbicide that kill an untransformed wild type. Examples are mentioned above and preferably comprise the bar gene which confers resistance to the herbicide phosphinotricin (Rathore KS et al.
• Generations should be cultivated to ensure that genomic integration is stable and inheritable.
• the above-mentioned methods are described, for example, in Jenes B et al. (1993) Techniques for Gene Transfer, in: Transgenic Plants, Vol. 1, Engineering and Utilization, edited by SD Kung and R Wu, Academic Press, p.128-143 and in Potrykus (1991) Annu Rev Plant Physiol Plant Molec Biol 42: 205-225.
• the expression construct is preferably cloned into a vector which is suitable for transforming Agrobacterium tumefaciens, for example pBin19 (Bevan et al. (1984) Nucl Acids Res 12: 8711f).
• a whole plant can be obtained using methods known to those skilled in the art. This is based on callus cultures, for example. The formation of shoots and roots can be induced in a known manner from these still undifferentiated cell masses. The sprouts obtained can be planted out and grown.
• nucleic acid sequence coding for a protein with sucrose isomerase activity a) the nucleic acid sequence coding for a protein with sucrose isomerase activity
• Natural genetic environment means the natural chromosomal locus in the organism of origin or the presence in a genomic library.
• SEQ ID NO: 1 nucleic acid sequence coding for sucrose isomerase from Protaminobacter rubrum 5
• SEQ ID NO: 2 amino acid sequence coding for sucrose isomerase from Protaminobacter rubrum
• SEQ ID NO: 3 nucleic acid sequence coding for sucrose isomerase from sucrose isomerase from Erwinia rhaponthici (N-terminal fragment)
• SEQ ID NO: 4 amino acid sequence coding for sucrose isomerase from sucrose isomerase from Erwinia 15 rhaponthici (N-terminal fragment)
• SEQ ID NO: 5 nucleic acid sequence coding for sucrose isomerase from Erwinia rhaponthici
• SEQ ID NO: 6 amino acid sequence coding for sucrose isomerase from Erwinia rhaponthici
• SEQ ID NO: 7 nucleic acid sequence coding for sucrose isomerase from Protaminobacter rubrum (variant) 25
• SEQ ID NO: 8 amino acid sequence coding for sucrose isomerase from Protaminobacter rubrum (variant)
• SEQ ID NO: 9 nucleic acid sequence coding for sucrose isomerase from Enterobacter species SZ62
• SEQ ID NO: 10 amino acid sequence coding for sucrose isomerase from Enterobacter species SZ62
• SEQ ID NO: 11 Nucleic acid sequence coding for sucrose isomerase from Serratia plymuthica
• SEQ ID NO: 12 amino acid sequence coding for sucrose isomerase from Serratia plymuthica 40
• SEQ ID NO: 13 nucleic acid sequence coding for fusion protein from sucrose isomerase from Erwinia rhapontici (pall) and signal peptide sequence of the proteinase inhibitor II gene 14.
• SEQ ID NO: 15 nucleic acid sequence (complete cDNA with untranslated region) coding for sucrose isomerase (iso involvedose synthase) from Klebsiella sp. LX3 10
• SEQ ID NO: 17 nucleic acid sequence (open reading frame) coding for sucrose isomerase (isomaltulose synthase) from Klebsiella sp. LX3
• SEQ ID NO: 19 nucleic acid sequence coding for sucrose isomerase from Enterobacter species SZ62 25 (fragment)
• SEQ ID NO: 20 amino acid sequence coding for sucrose isomerase from Enterobacter species SZ62 (fragment) 30
• SEQ ID NO: 21 nucleic acid sequence coding for sucrose isomerase from Pseudomonas mesoacidophila MX45 (fragment)
• SEQ ID NO: 23 nucleic acid sequence coding for
• SEQ ID NO: 24 nucleic acid sequence coding for ⁇ 0.3TobRB7
• SEQ ID NO: 26 oligonucleotide primer FB84
• SEQ ID NO: 27 oligonucleotide primer FB 97
• SEQ ID NO: 28 oligonucleotide primer Leml
• SEQ ID NO: 29 oligonucleotide primer Lem2
• SEQ ID NO: 30 oligonucleotide primer Tobl
• SEQ ID NO: 31 oligonucleotide primer Tob2
• SEQ ID NO: 32 nucleic acid sequence coding for V-sense
• Wheat Dwarf Virus promoter (GenBank Acc.-No .: AX006849; Sequence 1 from WO 00/01832) 25
• SEQ ID NO: 33 nucleic acid sequence coding for V-sense
• Maize streak virus promoter (GenBank Acc.- No .: AX006850; Sequence 2 from WO 00/01832)
• SEQ ID NO: 34 nucleic acid sequence coding for V-sense
• Pepper huasteco virus promoter (GenBank Acc.-No .: AX006851; Sequence 3 from WO 00/01832)
• SEQ ID NO: 35 nucleic acid sequence coding for sucrose 35 isomerase from Serratia plymuthica
• SEQ ID NO: 36 amino acid sequence coding for sucrose isomerase from Serratia plymuthica
• Fig. 1 Schematic representation of the expression cassette in the plasmid p35S-cwIso.
• 35S 35S Cauliflower Mosaic Virus (CaMV) promoter SP: signal peptide of the proteinase inhibitor II gene pall: sucrose isomerase gene from Erwinia rhapontici
• OCS polyadenylation signal of the octopine synthase gene EcoRI, Asp718, BamHI, Sall, Hindlll: restriction sites
• Fig. 2 Schematic representation of the expression cassette in the plasmid pB33-cwIso. Abbreviations:
• B33 promoter of the class I patatin gene
• B33 SP signal peptide of the proteinase inhibitor II gene pall: sucrose isomerase gene from Erwinia rhapontici
• OCS polyadenylation signal of the octopine synthase gene EcoRI, Asp718, BamHI, Sall, Hindlll: restriction sites Detailed description of the individual Elements see below.
• Fig. 3 Western blot analysis of pall expressing potato tubers of various transgenic lines. 20 ⁇ g of soluble protein per lane were applied to an SDS gel, separated and transferred to nitocellulose. The filter was then hybridized with a polyclonal Pall antibody. The expression in tubers of wild-type potato plants (wt) was compared with that in potato lines 5, 12, 26 and 33.
• Fig. 4 HPLC analysis of soluble carbohydrates in plants expressing sucrose isomerase.
• A Sugar standards.
• B Extract of a transgenic tuber.
• C Extract of a wild-type tuber.
• Fig. 5 Content of palatinose, sucrose, glucose and starch in wild-type potato tubers (wt) and potato tubers of various transgenic lines (3 to 37) that express the chimeric pall gene in the cell wall.
• the values of the wilt type (wt; striped columns) and the transgenic potato tubers (3 to 37; black columns) correspond to the mean values of four measurements + standard deviation.
• a transgenic but pall non-expressing line was analyzed.
• Fig. 6 Infection of potato tubers with Alternaria solani. Potato slices of wild-type tubers and tubers of pall expressing transgenic lines 5 and 33 at the time
• Fig. 7 Schematic representation of the expression cassette in the plasmid pLemmi9-cwIso. Abbreviations:
• Lemmi9 Lemmi9 promoter from tomato (Lycopersicon esculentum) SP: signal peptide of the proteinase inhibitor II gene pall: sucrose isomerase gene from Erwinia rhapontici OCS: polyadenylation signal of the octopine synthase gene EcoRI, Asp718, BamHI, Sall, Hindlll: Description of the restriction sites individual elements see below.
• Fig. 8 Schematic representation of the expression cassette in the plasmid p ⁇ 0.3TobRB7-cwIso.
• OCS polyadenylation signal of the octopine synthase gene EcoRI, Asp718, BamHI, Sall, Hindlll: restriction sites. Detailed description of the individual elements see below.
• oligonucleotides can be carried out, for example, in a known manner using the phosphoamidite method (Voet, Voet, 2nd edition, Wiley Press New York, pages 896-897).
• the cloning steps carried out in the context of the present invention such as, for example, restriction cleavages, agarose gel electrophoresis, purification of DNA fragments, transfer of nucleic acids to nitrocellulose and nylon membranes, linking of DNA fragments, transformation of E. coli cells, cultivation of bacteria, Multiplication of phages and sequence analysis of recombinant DNA - as with Sambrook et al. (1989) Cold Spring Harbor Laboratory Press; ISBN 0-87969-309-6.
• Agrobacterium tumefaciens was carried out according to the method of Hofgen and Willmitzer ((1988) Nucl. Acids Res. 16: 9877).
• the Agrobacteria were grown in YEB Medium (Vervliet et al. (1975) Gen Virol 26: 33-33).
• the sequencing of recombinant DNA molecules is carried out using a laser fluorescence DNA sequencer from MWG-Licor using the method of Sanger (S nger et al. (1977) Proc Natl Acad Sei USA 74: 5463-5467).
• Example 1 PCR amplification of a subfrag of sucrose isomerase from Erwinia rhaponitici
• sucrose isomerase A subfragment of sucrose isomerase was cloned by means of polymerase chain reaction (PCR). Genomic DNA from E. rhapontici (DSM 4484) was used as template material and was isolated according to the standard protocol. The amplification was carried out using the following specific primers, which were derived from a sucrose isomerase sequence of the prior art:
• Primer FB83 comprises bases 109 to 127 and primer FB84 bases 1289 to 1306 of the coding region of the sucrose isomerase gene from E. rhapontici.
• the PCR reaction mixture (100 ⁇ l) contained:
• dNTPs dATP, dCTP, dGTP, dTTP
• the mixture was heated to 95 ° C. for 5 min.
• the polymerization steps (30 cycles) were carried out in an automatic T3 thermal cycler (Biometra) according to the following program: denaturation 95 ° C (1 minute), attachment of the primers at 55 ° C (40 seconds), polymerase reaction at 72 ° C ( 2 minutes) .
• the fragment obtained was cloned into the vector pCR blunt (Invitrogen). The identity of the amplified DNA was verified by sequence analysis.
• the amplified subfragment can also be used as a hybridization probe for the isolation of further sucrose isomerase DNA sequences from other organisms or as a probe in the analysis of transgenic cells and plants.
• Example 2 PCR amplification of a sucrose isomerase from Erwinia rhaponitici
• a Erwinia rhapontici genomic library was screened according to standard methods. Subsequent sequence analyzes allowed the determination of the open reading frame of sucrose isomerase.
• the oligonucleotide primers FB83 and FB97 were derived from this sequence.
• sucrose isomerase The complete open reading frame of sucrose isomerase was cloned by means of polymerase chain reaction (PCR). Genomic DNA from E. rhapontici (DSM 4484) was used as template material and was isolated according to the standard protocol. The amplification was carried out using the following specific primers
• Primer FB83 comprises bases 109 to 127 and primer FB97 bases 1786 to 1803 of the coding region of the sucrose isomerase gene.
• the primers additionally carry the following for cloning the amplified DNA into expression vectors
• the PCR reaction mixture (100 ⁇ l) contained:
• dNTPs dATP, dCTP, dGTP, dTTP
• Pfu DNA polymerase dATP, dCTP, dGTP, dTTP
• dATP, dCTP, dGTP, dTTP dATP, dCTP, dGTP, dTTP
• Pfu DNA polymerase - 2.5 units
• the mixture was heated to 95 ° C. for 5 min.
• the polymerization steps (30 cycles) were carried out in an automatic T3 thermal cycler (Biometra) according to the following program: denaturation 95 ° C (1 minute), attachment of the primers at 55 ° C (40 seconds), polymerase reaction at 72 ° C ( 2 minutes) .
• the amplified sucrose isomerase fragment was cloned into the vector pCR blunt (Invitrogen), whereby the plasmid pCR-SucIso2 (without translation start) was obtained. The identity of the amplified DNA was verified by sequence analysis.
• the PCR fragment thus contains the sequence of a E. rhapontici sucrose isomerase extending from nucleotide 109-1803 of the sucrose isomerase gene.
• a DNA sequence coding for a sucrose isomerase was isolated from the plasmid pCR-SucIso2 and provided with the 35S promoter of the Cauliflower Mosaic Virus, which mediates a constitutive expression in transgenic plant cells and a plant termination signal.
• the plant termination signal contains the 3 'end of the polyadenylation site of the octopine synthase gene.
• sucrose isomerase gene Before the coding sequence of the sucrose isomerase gene, a signal peptide of a vegetable gene (proteinase inhibitor II gene from potato (Keil et al. (1986) Nucl Acids Res 14: 5641-5650; Genbank) necessary for inclusion in the endoplasmic reticulum was also added Acc. -No.: X04118) by cutting out the sucrose isomerase fragment from the construct pCR-SucIso2 via the restriction sites BamHI and Sall and ligating it into a BamHI / Sall-opened pMA vector.
• the vector pMA represents a modified form of the Vector pBinAR (Höfgen and Willmitzer (1990) Plant Sei. 66: 221-230).
• the plant termination signal contains the 3 '- End of the polyadenylation site of the octopine synthase gene.
• the expression cassette in the plasmid p35S-cwIso thus consists of fragments A, B and C (FIG. 1):
• Fragment A contains the 35S promoter of the Cauliflower Mosaic Virus (CaMV). It contains a fragment which comprises the nucleotides 6909 to 7437 of the CaMV (Franck (1980) Cell 21: 285).
• Fragment B contains nucleotides 923 to 1059 "of a proteinase inhibitor II gene from the potato (Keil el al., Supra), which is linked via a linker with the sequence ACC GAA
• TTG GG are fused to the sucrose isomerase gene from Erwinia rhapontici, which comprises nucleotides 109 to 1803.
• a signal peptide of a vegetable protein necessary for the uptake / on proteins into the endoplasmic reticulum (ER) is fused N-terminally to the sucrose isomerase sequence.
• Fragment C contains the polyadenylation signal of the octopine synthase gene (Dhaese et al. (1983) EMBO J. 2: 419-426. GenBank Acc.-No .: Z37515, nucleotides 1344 to 1533).
• sucrose isomerase the coding region of sucrose isomerase from E. rhapontici is under constitutive control, the gene product is taken up in the ER and then secreted.
• the plasmid pB33-cwIso was prepared using the binary plasmid p35S-cwIso.
• the 35S promoter was exchanged for the promoter of the class I patatin gene (Rocha-Sosa et al (1989) EMBO J 8: 23-29).
• the expression cassette of this plasmid pB33-cwIso thus consists of the three fragments A, B and C (see FIG. 2):
• Fragment A contains the region -1512 to +14 relative to the transcription initiation site of the class I patatin gene.
• the promoter region was ligated as a Dral fragment into the vector pUCI8 cut with SstI, the ends of which had been filled in using the T4 DNA polymerase and thus smoothed.
• the fragment with the restriction enzymes EcoRI and Asp718 was then cut out again from the vector pUC18 and cloned into the plasmid p35S-cwIso, from which the 35S CaMV promoter had previously been deleted after partial restriction with the enzymes EcoRI and Asp718.
• Fragment B contains nucleotides 923 to 1059 one
• Proteinase inhibitor II gene from the potato which is linked to the sucrose isomerase gene from E. rhapontici via a linker with the sequence ACC GAA TTG GG
• Nucleotides 109 to 1803 comprises, are fused. This makes it necessary for the inclusion of proteins in the ER Signal peptide of a vegetable protein fused N-terminal to the sucrose isomerase sequence.
• Fragment C contains the polyadenylation signal of the octopine synthase gene (Dhaese et al. (1983) EMBO J 2: 419-426; GenBank Acc.-No .: Z37515, nucleotides 1344 to 1533).
• Example 5 Potato transformation and selection of transgenic plants
• 25 lines could be detected pall expression in the Western blot.
• a Western blot of representative lines is shown in FIG. 3.
• Example 6 HPLC analysis of the transgenic pB33-cwIso potatoes
• tuber extracts of the transgenic lines were examined by means of HPLC with regard to their content of soluble carbohydrates.
• the HPLC analysis was carried out according to the in Börnke et al. (2002) Planta 214: 356-364.
• the production of the tuber extracts is described in Sonnewald et al. (1992) Plant J 2: 571-581.
• the results of the HPLC analysis are shown in FIG. 4.
• the functionality of the "Feeding cell” specific Lemmi9 promoter has already been demonstrated (Escobar C et al. (1999) 45 Mol Plant Microbe Interact 12: 440-449).
• the plasmid pLemmi9-cwlso contains three fragments A, B and C (see FIG. 7):
• A) Fragment A contains the Lemmi9 promoter from tomato (Lycopersicon esculentum).
• the fragment contains the sequence of 1417 bp before the translation start (ATG) of the Lemmi9 gene and was characterized as a functional promoter fragment (Escobar et al. (1999) Mol Plant Microbe Interact 12: 440-449, Accession Z69032). It was amplified by PCR from tomato genomic DNA (Lycopersicon esculentum). The amplification was carried out using the following specific primers:
• Lem2 5 'atcGGTACCTGCTTCTGGAACGAAAGGG 3' (SEQ ID NO: 29)
• the primers additionally carry the following restriction sites: primer Leml, EcoRI; Primer Lem2, Asp718.
• the PCR reaction mixture (100 ⁇ l) contained:
• dNTPs dATP, dCTP, dGTP, dTTP
• Pfu DNA polymerase dATP, dCTP, dGTP, dTTP
• dATP, dCTP, dGTP, dTTP dATP, dCTP, dGTP, dTTP
• Pfu DNA polymerase - 2.5 units
• the mixture was heated to 95 ° C. for 5 min.
• the polymerization steps (30 cycles) were carried out in an automatic T3 thermocycler (Biometra) according to the following program: denaturation 95 ° C. (1 minute), attachment of the primers at 56 ° C. (40 seconds), polymerase reaction at 72 ° C (3 minutes).
• the amplicon was digested with the restriction enzymes EcoRI and Asp718 and cloned into the corresponding restriction sections of the polylinker from pBluescript (Stratagene). The identity of the amplified DNA was verified by sequence analysis.
• the fragment was then digested with the restriction enzymes EcoRI and Asp718 and cloned into the plasmid pB33-cwIso, from which the B33 promoter had previously been deleted after partial restriction with the enzymes EcoRI and Asp718.
• Fragment B contains nucleotides 923 to 1059 of the
• Proteinase inhibitor II gene from the potato (Keil et al. (1986) Nucl Acids Res 14: 5641-5650; Genbank Acc.No .: X04118), which is linked to the sucrose isomerase via a linker with the sequence ACC GAA TTG GG. Gene from E. rhapontici, which the Nucleotides 109 to 1803 comprises, are fused. As a result, a signal peptide of a vegetable protein necessary for the incorporation of proteins into the ER is fused N-terminally to the sucrose isomerase sequence.
• Fragment C contains the polyadenylation signal of the octopine synthase gene (Dhaese et al. (1983) EMBO J 2: 419-426. Accession Z37515, nucleotides 1344 to 1533).
• Potato cells were transformed as described above using Agrobacterium -mediated gene transfer with the construct pLemmi9-cwIso or pLemmi9-GUS and whole potato plants were regenerated.
• the promoter of the class I patatin gene B33 in the plasmid pB33-cwIso was replaced by the ⁇ 0.3TobRB7 promoter (Opperman et al. (1994) Science 263: 221-223) and the fusion protein from proteinase inhibitor signal peptide and the sucrose isomerase thus placed under feeding cell-specific control.
• the functionality of the "Feeding cell” specific ⁇ 0.3TobRB7 promoter has already been demonstrated (Opperman et al. (1994) Science 263: 221-223).
• the plant termination signal includes the 3 'end of the polyadenylation site of the octopine synthase gene.
• the plasmid p ⁇ O .3TobRB7-cwIso contains three fragments A, B and C (Fig. 8):
• Fragment A contains the ⁇ 0.3TobRB7 promoter from Nicotiana tabacum. The fragment contains the region from -298 bp to +76 of the TobBR7 gene and has been characterized as a functional promoter fragment (Opperman et al. (1994) Science. 263: 221-223, Acc.-No .: S45406). It was generated from genomic DNA from Nicotiana tabacum Var. Samsun NN amplified. The amplification was carried out using the following specific primers:
• Tob2 5 '-GGGTACCAGTTCTCACTAGAAAAATGCCCC-3' (SEQ ID NO: 31)
• the primers additionally carry the following restriction sites: primer Tobl, EcoRI; Primer Tob2, Asp718.
• the PCR reaction mixture (100 ⁇ l) contained:
• dNTPs dATP, dCTP, dGTP, dTTP
• the mixture was heated to 95 ° C. for 5 min.
• the polymerization steps (30 cycles) were carried out in an automatic T3 thermocycler (Biometra) according to the following program: denaturation 95 ° C. (1 minute), attachment of the primers at 56 ° C. (40 seconds), polymerase reaction at 72 ° C (3 minutes).
• the amplicon was digested with the restriction enzymes EcoRI and Asp718 and cloned into the corresponding restriction sites of the polylinker from pBluescript (Stratagene). The identity of the amplified DNA was verified by sequence analysis.
• the fragment was then digested with the restriction enzymes EcoRI and Asp718 and cloned into the plasmid pB33-cwIso from which the B33 promoter had previously been deleted after restriction with the enzymes EcoRI and Asp718.
• Fragment B contains nucleotides 923 to 1059 one
• Proteinase inhibitor II gene from the potato (Keil et al. (1986) Nucl. Acids Res. 14: 5641-5650; Genbank Acc. O.: X04118), which binds to the sucrose isomerase gene from E. rhapontici, which comprises nucleotides 109 to 1803 are fused.
• a signal peptide of a vegetable protein necessary for the uptake of proteins in the ER is fused N-terminally to the sucrose isomerase sequence.
• C) Fragment C contains the polyadenylation signal - of the octopine synthase gene (Dhaese et al. (1983) EMBO J 2: 419-426. Accession Z37515, nucleotides 1344 to 1533).
• Potato cells were analyzed as described above
• Agrobacterium-mediated gene transfer was transformed with the construct p ⁇ 0.3TobRB7-cwIso or p ⁇ O .3TobRB7-GUS and potato plants were regenerated
• Transformed plants are confirmed with the help of npt-specific primers via PCR.
• the cuttings from transgenic lines which express the sucrose isomerase under the control of a "feeding cell" -specific promoter are first grown on medium with kanamycin and later transferred to pots with sterile earth. The plants are grown at 22 ° C. (16 h day / 8 h night) The infection of the plants is carried out as follows: 3 ml of a suspension (approx.
• Root bile nematodes 500 J2 larvae of root bile nematodes (Meloidogyne species) are inoculated into the soil directly next to the stems of the plants Plants are removed from the pots after 2 to 3 weeks and the roots are washed, then the entire root of each plant is examined using a stereo microscope and the number of galls on the root system of transgenic plants and wild-type plants is compared.
• Transgenic plants which express the sucrose isomerase under the control of a "feeding cell" -specific promoter show a marked resistance to endoparasitic root nematodes. The number of galls on the root system of these plants after nematode attack is significantly reduced compared to non-transformed plants.
• Example 11 In vitro nematode resistance test
• Plants Potato (Solanum tuberosum L. cv. Solara)
• Nematodes Meloidogyne incognita
• Murashige & Skoog medium (MS; solidified with agar) consisting of micro and 1/2 macro elements including vitamins, sucrose and diachin agar (0.7%) pH 5.8.
• Plants Sterile transgenic potato plants (Solanum tuberosum L. cv. Solara transformed with p ⁇ 0.3TobRB7-cwIso or pLemmi9-cwIso) and corresponding transgenic control plants (Solanum tuberosum L. cv. Solara transformed with p ⁇ O .3TobRB7-GUS or pLemmi9 ) were provided in jars with several plants each. Starting from each plant, three lines were generated using stem sections and subsequent cultivation on modified Murashige & Skoog medium (MSm; solidified with agar). Each line was planted on a separate 9 cm petri dish. The plants were grown for 2 to 3 weeks under a light / dark regime of 16h light / 8h dark at 25 ° C.
• MSm Murashige & Skoog medium
• Nematodes were obtained from sterile cultures. M. incognita was grown monoxenically in the dark at 25 ° C. on root explants of Cucumis sativus, as described by Wyss et al. (Wyss U et al. (1992) Nematologica 38: 98-111). Egg bags were collected from the sterile cultures and placed on a sieve in a glass funnel with sterile water. The funnels were connected with a plastic tube, which was closed with a clamp. Hatched hatchlings were obtained by opening the clamp and draining the suspension into small vessels. The viscosity of the suspension was increased by adding a suspension of sterile "Gel Rite". The density of the nematodes in the suspension was determined and normalized by adding sterile water.
• the potato lines transformed with p ⁇ O .3TobRB7-cwIso or pLemmi9-cwIso show a significant reduction in the formation of bile. This means a significant reduction in the damage caused by nematodes.

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