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WO1998058065A1 - Plantes resistantes aux moisissures et procede de production desdites plantes - Google Patents

Plantes resistantes aux moisissures et procede de production desdites plantes Download PDF

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Publication number
WO1998058065A1
WO1998058065A1 PCT/JP1998/002719 JP9802719W WO9858065A1 WO 1998058065 A1 WO1998058065 A1 WO 1998058065A1 JP 9802719 W JP9802719 W JP 9802719W WO 9858065 A1 WO9858065 A1 WO 9858065A1
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ser
gly
glu
val
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PCT/JP1998/002719
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English (en)
Japanese (ja)
Inventor
Makoto Kakitani
Naoyuki Umemoto
Masayoshi Tsukahara
Isao Ishida
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Kirin Beer Kabushiki Kaisha
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Priority to CA002294652A priority Critical patent/CA2294652A1/fr
Priority to AU80355/98A priority patent/AU8035598A/en
Publication of WO1998058065A1 publication Critical patent/WO1998058065A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants

Definitions

  • the present invention relates to a DNA encoding a protein that causes plant disease resistance, a plant disease-resistant plant, and a method for producing the same. More specifically, a domain containing at least an elicitor binding site of an elicitor receptor, and a plant-derived plant
  • the present invention relates to a DNA encoding a chimeric protein (a chimeric receptor 1) containing a domain containing a signal transmission motif of a resistance gene, a plant disease-resistant plant into which the DNA has been introduced, and a method for producing the same.
  • the elicitor includes degraded products produced by a hydrolase derived from a plant or a power plant, a protein elicitor produced by a power plant, and an AVR gene produced by a power plant.
  • N-acetyl chitooligosaccharide or glucan produced by dalcanase.
  • proteinaceous elicitor examples include mold-derived elicitin belonging to the genus Phytophthora.
  • gene product examples include Avr4 and Avr9 derived from Cladosporium fulvum, and nipl derived from Lincosporium 'Secaris (Rhynchosporium secalis). These elicitors interact specifically with plant resistance gene products, including the elicitor receptor on the plant cell membrane, to produce An intrusion is recognized and a signal is issued.
  • biochemical processes from infection by plant pathogens to resistance are thought to be as follows, taking the synthesis and accumulation of phytoalexin as an example.
  • Elysita is a polysaccharide made from glucose and is a 1,6-linked and
  • this elicitor When this elicitor binds to a receptor present in plant cells, it produces a second messenger that plays a role in signal transduction, and this signaling substance enters the nucleus of the plant cell and transcribes the gene encoding the phytoalexin synthase. Activates phytoalexin synthase. At the same time, the phytolexin degradation system is suppressed, and phytolexin is efficiently accumulated.
  • phytoalexin which plays an important role in soybean resistance, is called glyceroline, and its structure has been determined [M. Yoshikawa et al. (1978) Physiol. Pathol. 12: 73] 0
  • ER a specific receptor for dalcan elicitor from Phytophthora megasperaaf. Sp. Glycinea, a kind of soybean pathogenic fungi, plays an important role in the synthesis and accumulation of the antibacterial glyceolin. It is thought to be a receptor protein that plays This specific receptor for glucan elicitor was separated and purified from the cell membrane fraction of soybean root by Kakitani et al. (JP-A-06-321995). Umemoto et al. [N. Umemoto et al. (1997) Pro Natl. Acad. Sci. Loaned.
  • Plant-derived disease resistance genes that are expected to contain motifs or domains involved in these signal transductions include, for example, the Arabidopsis resistance gene RPS2 [M. Mindrinos et al. (1994) Cell 78: 1089, AF Bent et al. (1994) Science 265: 1856], Arabidopsis resistance gene RPM1 [MR Grant et al.
  • the aforementioned dalcan elicitor receptor lacks characteristic motifs or domains involved in signal transduction, such as those found in plant-derived resistance genes, and may lack signal transduction domains It has been suggested.
  • the present inventors have found that even in plant cells, a domain that recognizes a certain ligand is involved in signal transduction derived from another protein (including a receptor) in a domain that recognizes a certain ligand. Is it possible that signal transduction is carried out by recognizing the ligand even if a quinula receptor linked to a domain is introduced and expressed? I answered if signaling could occur even if they were not of the same plant species.
  • a gene encoding a chimeric receptor in which an elicitor receptor is linked to an intracellular signaling domain of a plant-derived receptor, or an elicitor receptor and a plant-derived disease resistance gene for example, Arabidopsis-derived Cells encoding the resistance gene RPS2, Arabidopsis resistance gene RPM1, tobacco resistance gene N, flax resistance gene L6, tomato resistance gene Pto, rice resistance gene Xa21, etc.
  • a gene encoding a chimeric receptor linked to a domain predicted to be involved in intracellular signal transduction and if it can be expressed in plants regardless of plant type, expresses only the elicitor receptor We thought that the efficiency of signal transmission might be higher than in the case.
  • the result is a distinctly stronger resistance to pathogenic mold It is possible to produce plants with sexual properties, improve the productivity of agricultural crops, and reduce the economic effect and environmental pollution due to the reduction of pesticide use.
  • the present invention relates to a DNA encoding a chimeric protein comprising a domain containing at least an elicitor-binding site of an elicitor receptor and a domain containing a signaling motif of a plant-derived resistance gene, and a plant into which the DNA has been introduced. It is intended to provide a disease resistant plant and a method for producing the same.
  • the present inventors have made intensive efforts to solve the above problems, and as a result, a domain predicted to be involved in signal transduction encoded by a plant-derived resistance gene, and a domain including at least an elicitor binding site of an elicitor receptor.
  • synthesizing a gene encoding the chimeric receptor of the present invention and introducing it into Arabidopsis thaliana and tobacco plants and expressing it the plant succeeded in imparting disease resistance to the plant and completed the present invention. It led to.
  • the present invention is a DNA encoding a chimeric protein containing at least a domain containing an elicitor-binding site of an elicitor-receptor and at least a domain containing a signal transmission motif of a protein conferring plant disease resistance.
  • This chimeric protein functions as a receptor and brings disease resistance to plants.
  • elicitors include glucan, polygalacturonic acid, N-acetyl chitooligosaccharide, elicitin, Cladosporium. Fulbam-derived AVr gene product or lincosporium. Secalis-derived nip 1 gene product. Are their receptors. Further, as the elicitor binding site of the glucan elicitor receptor, a protein containing at least the amino acid sequence represented by SEQ ID NO: 27, or at least one amino acid in the amino acid sequence is deleted, substituted, added or inserted. And a protein having the amino acid sequence of the present invention and having the ability to bind to Dalkan elicitor.
  • examples of the domain containing the signal transduction motif include those containing at least one selected from the group consisting of leucine-rich repeat, leucine zipper, nucleic acid binding site, and serine threonine kinase domain.
  • Derived Pt0 gene, Prf gene, Cf-2 gene or Cf-19 gene Signaling domain of the expression product of the Xa21 gene from rice, the signaling domain of the expression product of the RPS2 gene or the RPM1 gene from the Arabidopsis thaliana (for example, The signaling domain of the expression product of the L6 gene derived from flax or the signaling domain of the expression product of the N gene derived from tobacco.
  • the present invention includes a chimeric protein comprising the amino acid sequence represented by SEQ ID NO: 5, 7 or 9, or an amino acid sequence wherein at least one amino acid is deleted, substituted, added or inserted in the amino acid sequence.
  • DNA encoding a chimeric protein that produces plant disease resistance include those containing the nucleotide sequence represented by SEQ ID NO: 6, 8 or 10.
  • the present invention is a recombinant vector containing the DNA.
  • the present invention is a transformed plant transformed by the recombinant vector or a progeny thereof.
  • the present invention relates to a plant having plant disease resistance into which the DNA is introduced and expressing the plant, or a progeny thereof.
  • the present invention is a method for producing a plant disease-resistant plant, which comprises introducing the recombinant vector into a plant.
  • the present invention is a method for producing a plant having plant disease resistance or progeny thereof by integrating the DNA into a plant chromosome and expressing the DNA.
  • the present invention will be described in detail.
  • the DNA of the present invention is obtained by linking a DNA encoding at least a domain containing at least an elicitor binding site of an elicitor receptor and a DNA encoding a domain containing at least a signal transduction motif of a protein that causes plant disease resistance. It is also referred to as DNA or chimeric DNA encoding a chimeric receptor.
  • the elicitor receptor includes elicitor of a plant degradation product such as polygalacturonic acid, or a receptor that binds to elicitor of a fungal cell wall degradation product such as dalcan or N-acetylchitooligosaccharide, Mold like elicitin A receptor for the proteinaceous elicitor to be produced, or an elicitor receptor that binds to an elicitor such as an AVr gene product derived from Cladosporidium flubum, a nip1 gene product derived from Lincosporium secalis, and the like.
  • Methods for cloning cDNAs (hereinafter referred to as “ERDNA”) encoding these elicitor receptors include the following.
  • the above-mentioned elicitor is labeled with an isotope, and the binding activity to the labeled elicitor is measured from the plant cell membrane fraction to purify the elicitor receptor which specifically binds to elicitor.
  • PCR primers are designed from the partial amino acid sequence information obtained from the purified elicitor receptor, and the cDNA library is used for the purpose from a cDNA library using a probe obtained from a PCR derived from plant cell-derived DNA. C Clean the DNA.
  • the DNA encoding the signal transduction motif in the present invention is known as described above, and can be cloned using PCR or the like.
  • ligation of the ERDN A with the DNA encoding the signaling motif is performed.
  • the above two DNAs can be ligated by digesting these DNAs with an appropriate restriction enzyme and then ligating them using a ligase, or by combining ERDN A with a DNA encoding a signal transduction motif with an appropriate oligonucleotide. It is done by combining.
  • the ligation may be performed by inserting the above DNA into an appropriate plasmid.
  • the position before and after the position where the ERDNA and the DNA encoding the signal transduction motif are linked is not particularly limited, and even if the ERDNA is located upstream and the DNA encoding the signaling motif is located downstream. , And vice versa.
  • the base sequence of the chimeric DNA is determined by a known method (didoxy method, Maxam-Gilbert method, etc.). Usually, the base sequence is determined using a commercially available automatic base sequencer.
  • FIG. 1 shows a schematic diagram of a chimeric DNA of the present invention, taking as an example a chimeric DNA of a DNA encoding a dalcan elicitor receptor (ER) and an RPS2 gene derived from Arabidopsis thaliana.
  • CER is a chimeric DNA consisting of an approximately one-third region (SEQ ID NO: 29) on the 5 ′ side of the RPS2 gene and the entire ERDNA (SEQ ID NO: 2). Cut part and ERDN This is the one in which A is located upstream and the RPS2 gene is located downstream (the arrangement in the CER has been replaced).
  • the elicitor binding site of the dalcan elicitor receptor corresponds to the 239th to 442nd amino acid sequence of the amino acid sequence represented by SEQ ID NO: 1, and has the amino acid sequence represented by SEQ ID NO: 27.
  • a protein comprising at least the amino acid sequence represented by SEQ ID NO: 27, or an amino acid sequence in which at least one amino acid is deleted, substituted, added or inserted in the amino acid sequence, and has a binding ability to glucan elicitor.
  • the length of the elicitor-receptor is not limited as long as it is a protein having the following structure: it may be a protein having an appropriate truncated form (see “2-11” and “2-16” in FIG. 1). , 1-5).
  • the DNA encoding the amino acid sequence of the elicitor-binding site represented by SEQ ID NO: 27 includes a DNA represented by SEQ ID NO: 28.
  • nucleotide sequences of these chimeric DNAs and the amino acid sequences of the chimeric proteins encoded by the DNAs are shown in SEQ ID NOS: 5 and 6 for CER, SEQ ID NOs: 7 and 8 for IER, and The ER is shown in SEQ ID NOs: 9 and 10, and as long as these chimeric proteins have a binding function as an elicitor receptor and bring about plant disease resistance activity, the amino acid sequence (SEQ ID NOs: 5, 7, and 10) In 9), it means that at least one amino acid may cause mutation such as deletion, substitution, addition, or insertion.
  • the DNA of the present invention also includes, in addition to the nucleotide sequence encoding an amino acid contained in the chimeric protein of the present invention, DNA encoding the same polypeptide that differs only in degenerate codons (referred to as degenerate isomers). It is.
  • degenerate isomers DNA encoding the same polypeptide that differs only in degenerate codons. It is.
  • the codon (AAC) corresponding to Asn and the codon (eg, AAT) that has degenerate relation to it are called degenerate isomers.
  • nucleotide sequence is determined, it is then subjected to chemical synthesis, PCR or hybridization using a DNA fragment having the nucleotide sequence as a probe, whereby The DNA of the present invention can be obtained.
  • the DNA sequence encoding the chimeric receptor used in the present invention preferably has at least one stop codon (eg, TAG) adjacent to the 3'-end. Also, if desired, an ATG sequence encoding a methionine for translation initiation along with the translation frame at the 5'-upstream, and other 5'-upstream and 3'-downstream non-translated regions of appropriate length as untranslated regions DNA may bind.
  • TAG stop codon
  • Representative forms of the DNA sequence encoding the chimeric receptor used in the present invention include a plasmid or phage DNA in which this DNA sequence is inserted as a member, and plasmid or phage.
  • the DNA sequence is present in a microorganism (especially a bacterium) or a phage particle or in a plant in which the DNA sequence is inserted into genomic DNA.
  • the bacteria mentioned here include Escherichia coli bacterium.
  • a promoter, a DNA coding for a translation initiation codon (ATG) and a terminator are added to this DNA sequence in an appropriate combination. It is good.
  • the recombinant vector of the present invention can be constructed by incorporating a DNA encoding the chimeric receptor of the present invention (hereinafter also referred to as “chimeric DNA”) into an appropriate vector.
  • chimeric DNA a DNA encoding the chimeric receptor of the present invention
  • the vector for incorporating the chimeric DNA of the present invention is not particularly limited as long as it can replicate in a host, and examples thereof include plasmid DNA and phage DNA.
  • a binary vector, one of the plasmid DNAs can be prepared from Escherichia coli or Agrobacterium by an alkali extraction method (Birnboim, H. & Doly, J. (1979) Nucleic acid Res 7: 1513) or the like. Examples of the binary one vector include ⁇ 121, ⁇ ⁇ ⁇ and the like.
  • PUC118 manufactured by Takara Shuzo
  • PUCI19 manufactured by Takara Shuzo
  • pBluescript SK + manufactured by Stratagene
  • pGEM-T manufactured by Promega
  • Examples of the phage DNA include M13mpl8, M13mpl9, and M13tvl8.
  • a purified chimera of the present invention A method is used in which DNA is cleaved with an appropriate restriction enzyme, inserted into an appropriate restriction enzyme site of vector DNA or a multi-cloning site, and ligated to the vector.
  • the chimeric DNA to be expressed needs to be incorporated into a vector so that the function of the DNA is exhibited. Therefore, in addition to the promoter and the above genes, a signal peptide gene, a terminator, a drug resistance gene and the like can be incorporated into the recombinant vector of the present invention.
  • examples of the signal peptide gene include a tomato (Lycopersicon esculentum) 3-1,3 glucanase-sidinal peptide gene and the like.
  • examples of the enhancer include a tobacco mosaic virus ⁇ sequence and the like. Examples include kanamycin resistance gene and hygromycin resistance gene.
  • promoter examples include a promoter of a gene encoding a small subunit of ribulose-1,5-2 phosphoric acid carboxylase [R. Fluhr et al. Pro Natl, Acad. Sci. USA (1986) 83: 2358], Promoter of nopaline synthase (NOS) gene [WHR Langridge et al. Plant Cell Rep. (1985) 4: 355], Promoter that produces mosquito reflex mosaic virus 19S-RNA [H. Guilley et al. Cell (1982) 30: 763], and a promoter that produces cauliflower mosaic virus 35S-RNA [JT Odell et al. Nature (1985) 313: 810].
  • terminators include the terminator of the nopaline synthase gene [A.
  • a cauliflower mosaic virus 35S promoter (p35S) is ligated upstream of the chimeric DNA, and a terminator (NosT) of the nopaline synthase gene is ligated downstream (referred to as an ER cassette).
  • a cassette in which a kanamycin resistance gene (NPT II) is connected between the promoter and terminator of the nopaline synthase gene (NPT II cassette), and a hygromycin resistance gene is connected between p35S and NosT.
  • cassettes called Hm cassettes.
  • the recombinant vector of the present invention can be obtained by connecting the NPT II cassette upstream of the ER cassette and connecting the Hm cassette downstream of the ER cassette. ( Figure 2).
  • glucan elicitor receptor is important in plants for resistance to a wide variety of forceps, including dalkanes as cell wall components. It is suggested that it plays an important role. Then, the receptor gene was cloned, and the primary sequence was examined, suggesting that the elicitor receptor lacked a domain involved in signal transduction. On the other hand, the reported plant-derived resistance genes are roughly classified into four classes according to their structures as follows [M. S. Dixon et al. (1996) Cell 84: 451].
  • Class 1 Contains leucine-rich repeats and nucleic acid binding sites, some also have leucine zippers and are probably localized in the cytoplasm. Resistance genes that meet these conditions include tobacco-derived resistance gene N, Arabidopsis-derived resistance genes RPS2 and RPM1, flax-derived resistance gene L6, and tomato-derived resistance gene Prf.
  • Class 2 Leucine-ritslipipit, which has a membrane-binding site and is probably located on the cell surface, including tomato-derived resistance genes Cf-2 and Cf-9.
  • Class 3 Has a serine / threonine kinase domain and includes the tomato-derived resistance gene Pto.
  • Class 4 a leucine-rich repeat, a transmembrane site, with a serine / threonine-like kinase domain, and possibly a leucine-rich repeat recognizing elicitor, thereby activating a serine / threonine kinase located in the cell. And a rice-derived resistance gene Xa-21.
  • plant-derived resistance genes contain one or more motifs or domains predicted to be involved in signal transduction, i.e., leucine-rich repeats, leucine zippers, nucleic acid binding sites, and serine / threonine kinase domains. Yes.
  • At least the elicitor binding of the elicitor receptor The domain containing the site and the codes of plant-derived disease resistance genes (RPS2 gene, RPM1 gene, N gene, L6 gene, Pto gene, Xa21 gene, Cf-2 gene, Cf-9 gene, Prf gene, etc.)
  • RPS2 gene, RPM1 gene, N gene, L6 gene, Pto gene, Xa21 gene, Cf-2 gene, Cf-9 gene, Prf gene, etc. A DNA encoding a chimeric protein obtained by linking to a domain involved in signal transduction is produced and expressed.
  • a chimera of a DNA encoding a domain containing at least the elicitor-binding site of the glucan elicitor receptor and a DNA encoding a domain containing a leucine zipper and a nucleic acid binding site of a resistance gene (RPS2) derived from Arabidopsis thaliana The DNA or a fragment thereof is introduced into higher plant cells and expressed according to a known method.
  • the plant can be imparted with stronger and stronger resistance as compared with the case where the glucan elicitor receptor is introduced and expressed.
  • fungi that can infect plants generally have a sublesser, and that plants have acquired the ability to suppress the resistance of plants to fungi.
  • the DNA encoding the chimeric receptor of the present invention or a fragment thereof is introduced and expressed so that the chimeric receptor functions, or these DNAs are modified or the expression level is reduced. By making adjustments, it is possible to create new plants that are more resistant and resistant to power.
  • the DNA of the present invention or a fragment thereof is introduced into a higher plant together with a gene or a trait that enhances resistance to fungi such as dalkanase that exhibits mold resistance, and expressed in higher plants. Strong mold resistance can be imparted to plants.
  • the DNA sequence encoding dalcanase may be a protein containing an amino acid sequence represented by SEQ ID NO: 11 or 13, or at least one amino acid in the amino acid sequence is deleted, substituted, or added. Alternatively, there may be mentioned a DNA containing an inserted amino acid sequence and a nucleotide sequence encoding a protein having dalkanase activity.
  • the DNA contains all degenerate isomers. An example of such a degenerate isomer is a DNA containing the nucleotide sequence shown in SEQ ID NO: 12 or 14.
  • Examples of the gene transfer method include a biological method using a virus, an agrobacterium method, an electroporation method, a physical and chemical method, and a polyethylene glycol method. Method, microinjection method, particle gun method, dextran method and the like.
  • the plant into which DNA is to be introduced is a dicotyledon
  • a biological method using an agrobacterium is preferable.
  • a physical-chemical method such as an electroporation method is preferred.
  • the plant material for DNA introduction it is possible to select appropriate materials from leaves, stems, roots, petals, tubers, protoplasts, calli, pollen, seed embryos, shoot primordia, etc., according to the method of introduction, etc. I can do it.
  • DNA when DNA is introduced into cultured plant cells, protoplasts are used as a material, and the DNA is introduced by a physical or chemical method such as an electo-poration method or a polyethylene glycol method.
  • a physical or chemical method such as an electo-poration method or a polyethylene glycol method.
  • DNA when introducing DNA into plant tissue, leaves, stems, roots, petals, tubers, protoplasts, calli, pollen, seed germs, shoot primordia, etc., and preferably leaves and stems are used as materials.
  • DNA can be introduced by a biological method using a viral protein, a physical or chemical method such as a particle gun method, or a microinjection method, or preferably by a biological method using an agrobacterium. Will be
  • such a plant tissue or plant cell can be regenerated using a medium such as an MS medium containing an appropriate plant growth regulator. It is sufficient to culture in.
  • the rooted seedlings can be transformed into plants by transplanting them to soil and cultivating them.
  • a plant By introducing and expressing the DNA sequence encoding the chimeric receptor by the method described above, it is possible to confer resistance to pathogenic fungi or to enhance resistance to pathogenic fungi. I can do it.
  • examples of such a plant include a plant whose cell wall is infected with a pathogenic mold containing glucan.
  • solanaceous plants Examples include leguminous plants, asteraceae plants, dianthus plants, and gramineous plants, and more specifically, tobacco, soybean, potato, rice, chrysanthemum, carnation, and the like.
  • glucan in the cell wall component As the pathogenic fungi, those containing glucan in the cell wall component are preferable.
  • a DNA sequence encoding a chimeric receptor introduced into a plant can be inherited from a plant to a plant and to a progeny via a seed. Therefore, even in seeds formed from pollen or ovary of the plant of the present invention, the introduced DNA sequence is present, and the genetic trait can be passed on to the progeny. Therefore, a plant into which the DNA encoding the chimeric receptor of the present invention has been introduced can grow without losing resistance to pathogenic fungi by growing with seeds.
  • mass growth methods using plant tissue culture methods and conventional methods such as tree cutting, tree cutting, grafting, and stock splitting do not result in loss of resistance to pathogenic fungi, and therefore, growth is not a problem. It is possible.
  • Whether or not the transformed plant has resistance to fungi will be examined based on the presence or absence of a plant resistant response to glucan elicitor and the test for inoculation of vinegar.
  • the resistance reaction test by adding elicitor, add the dalcan elicitor solution to the surface of the leaf, or infill the glucan elicitor solution with the syringe from the back of the leaf, and observe the browning reaction that appears on the leaf surface after a certain period of time. It is performed by examining the accumulation of fluorescent substances (phytoarexins) that are excited by UV light.
  • in the mold inoculation test resistance can be examined by the following inoculation methods. Inoculation methods are broadly classified into two types: inoculation methods for above-ground diseases and inoculation methods for underground diseases.
  • Spray inoculation method Spray a plant with a suspension of pathogenic bacteria or spores and incubate it in an inoculation box at an appropriate temperature and saturation for a certain period of time to check the extent of the disease.
  • Spray inoculation method The spores collected from the lesions can be knocked down onto the plant with a brush or sprayed with a small duster. After inoculating the inoculated plants for 24 hours in an inoculation box adjusted to the appropriate temperature for infection, perform the same procedure as spray inoculation.
  • Wound inoculation method Inoculate a spore suspension or a piece of cultured bacterial flora into the cut end of a branch or the area where the bark has been punched out with a cork boiler.
  • Needle inoculation method Damage the plant with a needle or needle bundle soaked in a suspension of pathogenic bacteria and inoculate it.
  • Cell adhesion inoculation method For pathogenic bacteria that do not form spores on the medium, adhere the cultured mycelial mass or sclerotium to the surface of the plant and inoculate it.
  • Damaged plant inoculation method Instead of inoculating pathogens directly, place the damaged plant on the plant or hang the damaged leaf stem on the plant, and use the mycelium or falling spores that grow from there. Inoculate.
  • Soil irrigation inoculation method Spores or crushed mycelium suspension are irrigated into the soil and inoculated.
  • Soil admixture inoculation method Inoculate the soil with filamentous fungi cultured on bran, grain, rice straw, wheat straw, etc.
  • Contaminated soil inoculation method If there is a frequent site of a specific soil disease, collect the soil, mix well, put it in a pot or vat, sow it there, or transplant a seedling.
  • Root inoculation method Prepare a suspension of bacterial or filamentous fungal spores, immerse the roots of healthy seedlings in this for 3 to 10 minutes, and then transplant them to pots.
  • a resistance test can be performed by the above inoculation method. More specifically, the disease-causing fungus can be detected by directly inoculating mycelium into a plant individual and increasing the number of lesions. In addition, the resistance can be tested by inoculating zoospores of the disease-causing fungus and changing the lesions. In addition, plague bacteria For other types of soil bacteria, the cultured cells are introduced into the soil, seeded or planted in the soil, and the resistance to viability can be tested by observing the phenomenon of withering. However, the present invention is not limited to this. BRIEF DESCRIPTION OF THE FIGURES
  • FIG. 1 is a schematic diagram of the construction of the chimeric DNA of the present invention.
  • FIG. 2 is a schematic diagram of the construction of plasmid pBI (Hm) -ER.
  • FIG. 3 is a schematic diagram of the construction of plasmid pBI (Hm) -LER.
  • FIG. 4 is a schematic diagram of a plasmid containing DNA encoding about 1/3 of the N-terminal side of RPS2.
  • FIG. 5 is a schematic diagram of the construction of plasmid pBI (Hm) -CER.
  • FIG. 6 is a schematic diagram of the construction of plasmid pBI (Hm) -IER.
  • FIG. 7 is a schematic diagram of the construction of plasmid pBI (Hm) -ISER. BEST MODE FOR CARRYING OUT THE INVENTION
  • ER dalcan elicitor receptor
  • dScI, dB Sac I and BamHI sites are crushed, respectively.
  • pNOS N0S promoter
  • p35S Cauliflower mosaic virus 35S promoter
  • Nos-T N0S NPTII: Kanamycin resistance gene, Hm: Hygromycin resistance gene RB, LB: Ti-plasmid right border DNA sequence, Ti-plasmid left border DNA sequence, respectively
  • plasmids were prepared by the following procedure.
  • Reagents used for preparing plasmids such as restriction enzymes and linkers were those manufactured by Takara Shuzo unless otherwise specified.
  • DH5a (manufactured by BRL) was used for amplification and selection of the vector in E. coli.
  • linker DNA was cloned into BamHI and Sail sites of the pBI linker prepared by annealing and ligation to obtain pBI-ER.
  • SEQ ID NO: 2 As a result of determining the base sequence from the obtained vector, the one represented by SEQ ID NO: 2 was obtained.
  • the amino acid sequence recoded by the nucleotide sequence represented by SEQ ID NO: 2 is shown in SEQ ID NO: 1.
  • Hindlll sites at two sites in PER23-1 were cut with Hindlll and self-ligated to delete the Hindlll site and beyond in the ER.
  • the remaining two Notl sites were cut with Notl, filtered in with Klenow fragment, and the BamHI linker was introduced to create a new BamHI site.
  • a plasmid fragment containing the part after Hindlll of the ER gene obtained by cutting pER23-1 with BamHI and Hindlll was ligated to the fragment containing the part before Hindlll of the ER gene cut out with BamHI and Hindi11.
  • DNA for synthetic linker (SEQ ID NOs: 17 and 18) derived from the untranslated 5 'end of soybean 3-glucanase shown below, which was cleaved with Xbal and BamHI and synthesized using an automatic nucleic acid synthesizer, was One ring and ligation were performed to obtain pLER.
  • SEQ ID NO: 4 As a result of determining the base sequence from the obtained vector, the one represented by SEQ ID NO: 4 was obtained.
  • the amino acid sequence encoded by the nucleotide sequence represented by SEQ ID NO: 4 is shown in SEQ ID NO: 3.
  • Arabidopsis thai iana cDNA was obtained by spreading 200,000 plaques of Clontech cDNA library on E. coli C600hfl and spreading them on 10 cm diameter LB plates.
  • the phage was obtained by collecting the phage from the cells. Using the phage collected from the two plates as one pool, five pools of phage library were obtained. Phage DNA was recovered from each pool according to Ishida's method (Gene Expression Experiment Manual, Ishida and Ando, Kodansha Scientific).
  • PCR using Takara Shuzo's EX-Taq kit
  • phage DNA pool # 1 to # 5
  • PCR primer set using Takara Shuzo's EX-Taq kit
  • PCR was performed using about 1 microgram of phage DNA (pool # 1 to # 5) as a template using the following PCR primer set.
  • Antisense primer 5'-GGGAATTCACTCCGCGAGCCGGCGAAT-3 '(SEQ ID NO:
  • Antisense primer 5'-AGCCATGGCTCCTCCTAAAGTGAT-3 '(SEQ ID NO:
  • PCR was performed with the primer set (i) or (ii).
  • the amplified DNA was inserted into the Smal site of pBluescriptSKII (+) after phosphorylation, and plasmid DNAs (pRPS2-6, pRPS2-14 and pRPS2-16) shown in FIG. 4 were obtained. These were confirmed by DNA sequencing to be identical to the sequence reported [M. Mindrinos et al. (1994) Cell 78: 1089] except for a portion of the primer.
  • PRPS2-14 was digested with the restriction enzyme Xbal, and the Xbal fragment was removed by self-ligation.
  • a BaraHI linker was inserted into the EcoRV site of this plasmid, and a Sacl linker was inserted into the Xhol site.
  • This plasmid was cut with BamHI and Sal1, and the entire ER cut out from pER23-l with BamHI and Sail was cloned. From this plasmid, a chimeric DNA containing a part of RPS2 and the entire ER was purified. Ensure that the junction between RPS2 and ER matches the translation frame by DNA sequence. And confirmed.
  • pSpellinker is inserted into the EcoRV site of pRPS2-6.
  • the purified DNA fragment was cloned into the Xbal and Sad cleavage sites of pRPS2-6.
  • the plasmid containing the chimeric gene fused to the 13 'of the 5' end of RPS-2 and the full length of the ER is cut with Spel and Sad, and the chimeric gene is cloned into pIG12 Hm plasmid, and the desired vector pBI ( Hm) -CER was obtained.
  • SEQ ID NO: 6 The amino acid sequence encoded by the nucleotide sequence represented by SEQ ID NO: 6 is shown in SEQ ID NO: 5.
  • ER cDNA encoding 537 amino acids at the N-terminal is purified by BamHI + NspI.
  • RPS2 cDNA is purified using pRPS2-16 EcoRV + Sphl. These two DNA fragments were cloned into the BamHI and Smal sites of pBluescriptSKI I (+) plasmid. This plasmid is called pERRPS-8. It was confirmed that the translation frame was joined by the DNA sequence at the junction between RPS2 and ER.
  • the fragment containing the fusion gene of the part encoding the elicitor-binding region and the part encoding the RPS2 partial length obtained by digesting pERRPS-8 with Spel and Sacl was separated from agarose gel and separated from the plant-expressed binary.
  • the plasmid pIG121-Hm was digested with Xbal and Sacl and ligated with the plasmid fragment to obtain the desired vector pBI (Hm) -IER.
  • the nucleotide sequence was determined from the obtained vector, and as a result, the one represented by SEQ ID NO: 8 was obtained.
  • the amino acid sequence encoded by the nucleotide sequence represented by SEQ ID NO: 8 is shown in SEQ ID NO: 7.
  • the pERRPS-8 plasmid was cut with Nsil + Notl, and the Notl-Nsil adapter shown below was inserted.
  • the DNA sequence confirmed that the translation frames of ATG and ER in the adapter matched. Cut this plasmid with EcoRV + Sall, Purify the RPS2 / ER chimeric DNA.
  • the plasmid prepared here is called pSERRPS-8.
  • a fragment containing the fusion gene for the elicitor-binding region obtained by cutting pSERRPS-8 with Spel and Sacl and the portion encoding the RPS2 partial length was separated from agarose gel and separated from a plant-expressed binary vector plasmid.
  • the plasmid pIGI2Hra was ligated with a plasmid fragment cut with Xbal and Sacl to obtain the desired vector pBI (Hm) -ISER.
  • SEQ ID NO: 10 As a result of determining the base sequence from one of the obtained vectors, one represented by SEQ ID NO: 10 was obtained.
  • the amino acid sequence encoded by the nucleotide sequence represented by SEQ ID NO: 10 is shown in SEQ ID NO: 9.
  • Transformation vector containing the re-chimeric receptor gene [pBI (Hm) -CER, pBI (Hm) -ISER] and the control vector containing the ER gene as a control [pBI (Hm) -ER] -introduced Agrobacterium tumefaciens EHAIOI was inoculated into 3 ml of YEB medium, cultured at 28 ° C for 16 hours, collected by centrifugation, suspended in 10 ml of infection medium, and used for transformation. Agrobacterium infection solution.
  • the medium compositions of the YEB medium and the infection medium are described below.
  • YEB medium 5g / l beef extract, lg / 1 yeast extract, 5g peptone, 5g / l skull —S, 2mM magnesium sulfate
  • Infection medium 1/2 concentration of MS [Murashige & Skoog (1962) Physiol. Plant. 15: 473] Inorganic salts and vitamins of the medium, 15 g / l sucrose, lOg / 1 glucose, lOmM MES (pH5.4)
  • the root pieces were transplanted to an arabid regeneration medium and cultured for 2 days, and then transplanted to an arabid selection regeneration medium and cultured for 1 week. Thereafter, the cells were transplanted to a fresh regeneration medium for selection of arabido every week, and the culture was continued until a regenerated seedling was obtained.
  • Seedlings whose shoots and leaves reached about 1 cm were transplanted to arabid rooting medium and continued to be cultured.
  • the presence of the transgene in the plant whose roots extended to 1-2 cm was confirmed by PCR, and the transformant was used.
  • the culture was performed under the conditions of 22t: 16 hours lighting and 8 hours no lighting unless otherwise specified. The components of the medium used for the culture are described below.
  • MSHF medium MS [Murashige & Skoog (1962) Physiol. Plant. 15: 473] Inorganic salts and vitamins of the medium, 20 g / l sucrose, 2 g / l gellan gum, 5 mM MES (pH 6.2)
  • Callus induction medium Inorganic salts and vitamins of MS medium, 20 g / l sucrose, 0.5 mg / 12,4-dichlorofluorophenoxyacetic acid, 0.1 mg power ricetin, 2 g / l gellan gum, 5 mM MES (pH 6.2)
  • Arabide co-culture medium Inorganic salts and vitamins of MS medium, 20 g / l sucrose, 0.5 mg / 12,4-dichlorophenoxyacetic acid, 0.1 mg / l strength ricetin, 0.2 mM acetosyringone, 2 g / l gellan gum, 5mM MES (pH 6.2)
  • Arabido regeneration medium Inorganic salts and vitamins of MS medium, 20 g / l sucrose, lmg / 1 2-isopentenylamine, 0.15 mg / 1 indole-250 mg / 1 Cefotaxime, 2g / l gellan gum, 5mM MES (pH 6.2)
  • Regeneration medium for selection of arabide Inorganic salts and vitamins of MS medium, 20 g sucrose, lmg / 1 2-isopentenylamine, 0.15 mg / l indoleacetic acid, 2 g / l gellan gum, 50 mg / 1 kanamycin, 250 mg / l Cefotaxime , 5mM MES (pH 6.2)
  • Arabidopsis root medium Inorganic salts and vitamins of MS medium, 20 g / l sucrose, 2 mg / l naphthalene acetic acid, 2 g / l gellan gum, 250 mg / l Cefotaxime, 5 mM MES (pH 6.2)
  • a vector for transformation containing the chimeric receptor gene [pBI (Hni) -IER] and a vector for transformation containing the ER gene [pBI (Hm) -LER] were used as controls by electroporation.
  • the introduced Agrobacterium tumefaciens EHA101 was inoculated into 3 ml of YEB medium, cultured at 28 ° C for 16 hours, collected by centrifugation, suspended in 10 ml of infection medium, and transformed into an agrobacterium for transformation. This was a terium infection solution.
  • the medium compositions of the YEB medium and the infection medium are described below.
  • YEB medium 5g / l beef extract, lg / 1 yeast extract, 5g / l peptone, 5g / l sucrose, 2mM magnesium sulfate
  • Infection medium 1/2 concentration of MS [Murashige & Skoog (1962) Physiol. Plant. 15: 473] Inorganic salts and vitamins of the medium, 15 g / l sucrose, lOg / 1 glucose, 10 mM MES (pH 5.4)
  • Leaves of the samsun line of tobacco (Nicotiana tabacum L.) grown in a greenhouse were sterilized and cut into approximately 1 cm squares.
  • the leaf cuttings were floated in an Agrobacterium infection solution for 10 minutes, and excess infection solution attached to the leaf pieces was wiped off on a filter paper, then transplanted to a tobacco co-culture medium, and cultured for 2 days in the dark.
  • the leaf pieces were transplanted to a tobacco regeneration medium and cultured for 1 week, and then transplanted to a regeneration medium for tobacco selection and cultured for 2 weeks. After that, transfer to fresh regeneration medium for tobacco selection every two weeks and continue culturing until regenerated seedlings are obtained. I did.
  • Seedlings whose stems and leaves became about lcm were transplanted to tobacco rooting medium and cultured.
  • the presence of the transgene in the plant whose roots extended to l-2 cm was confirmed by PCR and used as a transformant.
  • the culture was performed under the conditions of 25 ° C, 16 hours of illumination and 8 hours of no illumination unless otherwise specified. The components of the medium used for the culture are described below.
  • Tobacco co-culture medium MS [Murashige & Skoog (1962) Physiol. Plant. 15: 473] Inorganic salts and vitamins of the medium, 30 g sucrose, O.lmg / 1 naphthalene acetic acid, lmg benzyladenine, 0.2 mM acetocillingone, 8 g / l Agar, 5mM MES (pH5.8)
  • Tobacco regeneration medium MS medium inorganic salts and vitamins, 30 g / l sucrose, 0.1 mg / 1 naphthalene acetic acid, lmg / 1 benzyl adenine, 250 mg cefotaxime, 8 g / l agar, 5 mM MES (pH 5.8)
  • Regeneration medium for tobacco selection MS medium inorganic salts and vitamins, 30 g / l sucrose, 0.1 mg / 1 naphthalene acetic acid, lmg / 1 benzyladenine, 100 mg / 1 kanamycin, 250 mg cefotaxime, 8 g agar, 5raM MES (pH5 .8)
  • Tobacco rooting medium Inorganic salts and vitamins in MS medium, 30g / l sucrose, 250rag / l Cefotaxime, 8g agar, 5mM MES (pH5.8)
  • an Arabidopsis transformant in which high expression of the gene containing the nucleotide sequence represented by SEQ ID NO: 6 was confirmed, and an Arabidopsis trait in which high expression of ER protein 'was confirmed as a control
  • the induction of a hypersensitive response by soybean elicitor was examined for the leaves of the transformant (ER; SEQ ID NO: 2) and untransformed Arabidopsis thaliana (control).
  • the present invention recognizes the contact and invasion of plant pathogens such as molds having a glucan structure on the cell wall, etc., and thereby induces a resistance reaction that is deeply involved in disease resistance more strongly than when only the ER protein is expressed. And a plant that can be used. By producing a plant that strongly induces the resistance response caused by such dalcan elicitor, it is possible to breed plants that exhibit strong resistance to phytopathogenic fungi such as mold.
  • transgenic Arabidopsis thaliana in which high expression of the gene containing the nucleotide sequence represented by SEQ ID NO: 10 was confirmed, and a non-transformed Arabidopsis thaliana (control) The induction of a hypersensitive response by soybean elicitor was examined on the leaves.
  • LOMM MgC l 2 fungal cells was dissolved in a solution wall from Dar cans elicitor (lOO ⁇ g / ral), and infill the 10 mM MgC l 2 solution in a syringe without a back or we needle leaves of Arabidopsis plants grown in a greenhouse
  • the cells were cultured at 22 ° C for 7 days on a 16-hour light, 8-hour dark cycle, and the changes appearing on the leaf surface were observed.
  • the mold cell wall-derived dalcan elicitor used here was obtained according to the method of Umeki et al. [N. Umemoto et al. (1997) Pro Natl. Acad. ScI. USA 94: 1029].
  • Expression of a chimeric receptor composed of the N-terminal domain of Arabidopsis thaliana RPS2 protein, which is expected to contain, in Arabidopsis thaliana proved that glucan elicitor, which cannot be recognized by the host plant, was recognized.
  • the present invention provides a plant which can recognize contact and invasion of plant pathogens such as mold having a glucan structure on a cell wall or the like. By producing a plant that strongly induces the resistance response caused by such a glucan elicitor, it becomes possible to produce a plant that exhibits strong resistance to phytopathogenic bacteria such as power plants.
  • a tobacco transformant in which high expression of the gene containing the nucleotide sequence represented by SEQ ID NO: 8 was confirmed, and as a control, tobacco transformation in which high expression of ER protein was confirmed.
  • the induction of the hypersensitive response by soybean elicitor was examined for the leaves of the tobacco (LER; SEQ ID NO: 4) and the untransformed tobacco (control).
  • the leaves of tobacco plants grown in the greenhouse are cut off with a petiole, and plastic that transmits light is used. Place it in the box. Place a cut silicon tube with a diameter of 5 mm and a height of 5 mm on the front side of the leaf so that it can hold the solution.
  • mold cell wall-derived dalcan elicitor UOO g / ml dissolved in 10 mM MgCl 2 solution, chemically synthesized dalcan elicitor-(100 mg / ml), and 10 mM MgCl 2 solution as a control
  • the cells were cultured in a humid condition at 25 ° C. for 7 days in a light-dark and 8-hour dark cycle to prevent drying.
  • Mold cell wall-derived chemical synthesis 10 mM MgCl glucan elicita glucan elicita control 1
  • the present invention provides a DNA encoding a chimeric receptor capable of recognizing contact and invasion of plant pathogens such as mold having a glucan structure in a cell wall or the like, and a plant body, whereby a firefly lexin which is deeply involved in disease resistance is provided. Resistance reactions such as induction are more intense than when the ER protein alone is expressed. By producing a plant that strongly induces the resistance response caused by such a glucan elicitor, it is possible to breed plants that exhibit strong resistance to phytopathogenic fungi such as mold. Industrial applicability
  • a DNA encoding a chimeric protein (a chimeric receptor-1) comprising a domain containing at least an elicitor-binding site of an elicitor-receptor and a domain containing a signaling motif of a plant-derived resistance gene. And a method for producing the plant disease-resistant plant.
  • the chimeric receptor of the present invention can be used for elucidating the mechanism of disease resistance to mold.
  • the DNA containing the base sequence encoding the quinula receptor of the present invention and fragments thereof are useful as materials for establishing a technique for breeding mold-resistant plants. That is, when the DNA of the present invention or a fragment thereof is introduced into various plants and expressed, the resistance of the plants to mold can be increased.
  • Glu Asp Leu Lys Tyr Lys Ser lie Asp Gly Asp Leu Val Gly Val Val
  • Leu Leu Trp Trp lie His Ser Arg Ser Asp Glu
  • Sequence type nucleic acid
  • TTC CAA AAC TTT GTC CTA AAA AAT GGT GAC CAA CAA GAA TAC ATT CAT 196 Phe Gin Asn Phe Val Leu Lys Asn Gly Asp Gin Gin Glu Tyr lie His His
  • GAT TTT GGA TTT GGA ATT TAC AAT GAT CAC CAC TAT CAT TTG GGG TAC 1300 Asp Phe Gly Phe Gly lie Tyr Asn Asp His His Tyr His Leu Gly Tyr
  • Glu Asp Leu Lys Tyr Lys Ser lie Asp Gly Asp Leu Val Gly Val Val
  • Leu Leu Trp Trp lie His Ser Arg Ser Asp Glu
  • Sequence type nucleic acid
  • CTTCTTTCCT CAACCTTCTT TCTTCTTATA TATTCGAACG ATCCGGGCCG CTCGAATTAA 60 ACCACCTTCA GCAACAATG GTT AAC ATC CAA ACC AAT ACA TCT TAC ATC TTC 112
  • ATC AAA TCC TCC AAC TCT TCC CTC TCT CTC TCA TAC CCT TCT CGC CAA 304 lie Lys Ser Ser Asn Ser Ser Leu Ser Leu Ser Tyr Pro Ser Arg Gin
  • GGC ATT ATT ACC CAA AAG GGG TCC ACT GAT GCT GGT GGT GAT TTT GGA 1312 Gly lie lie Thr Gin Lys Gly Ser Thr Asp Ala Gly Gly Asp Phe Gly
  • GCT CCT ATT TCT GAA GCC ATT TTC TCC AAT GTT GAC TTT GTA AAG GAG 1888 Ala Pro lie Ser Glu Ala lie Phe Ser Asn Val Asp Phe Val Lys Glu
  • 165 170 175 lie lie lie Gly Val Tyr Gly Pro Gly Gly Val Gly Lys Thr Thr Leu Met
  • Gin Ser lie Asn Asn Glu Leu lie Thr Lys Gly His Gin Tyr Asp Val
  • Thr Thr Arg Ser lie Ala Leu Cys Asn Asn Met Gly Ala Glu Tyr Lys
  • Leu Ala Glu lie lie Val Ser Lys Cys Gly Gly Leu Pro Leu Ala Leu
  • 340 345 350 lie Thr Leu Gly Gly Ala Met Ala Gly Leu Gin Glu Phe Asp Pro Gly
  • Lys lie Leu Glu Asp leu Lys Tyr Lys Ser lie Asp Gly Asp Leu Val
  • Gin Ala Tyr Ser lie Val Gin Asp Phe Leu Asn Leu Asp Thr Lys Leu
  • Sequence length 3405 Sequence type: nucleic acid
  • ATC GTT GGC TGT GCT CAG GTG TTG TGT GAA TCT ATG AAT ATG GCG GAG 101 lie Val Gly Cys Ala Gin Val Leu Cys Glu Ser Met Asn Met Ala Glu
  • ATC CAA CAA GAC GGT CTA GAG GGA CGA AGC TGC TCA AAT CGT GCC AGA 245 lie Gin Gin Asp Gly Leu Glu Gly Arg Ser Cys Ser Asn Arg Ala Arg
  • ATC AAG TCC GTT GTC GGA AAT ACC ACG ATG ATG GAA CAG GTT TTG GAA 533 lie Lys Ser Val Val Gly Asn Thr Thr Met Met Glu Gin Val Leu Glu
  • ATA TAC AGA GCT TTG AGA CAG AAA CGT TTC TTG TTG TTG CTA GAT GAT 821 lie Tyr Arg Ala Leu Arg Gin Lys Arg Phe Leu Leu Leu Leu Asp Asp 250 255 260
  • Glu Asp Leu Lys Tyr Lys Ser lie Asp Gly Asp Leu Val Gly Val Val
  • Val Ser Ala lie Leu Lys Ser lie Gly Glu Leu Arg Glu Arg Ser Glu
  • Lys lie Tyr Arg Ala Leu Arg Gin Lys Arg Phe Leu Leu Leu Leu Asp 785 790 795 800
  • Sequence type nucleic acid
  • TTC CAA AAC TTT GTC CTA AAA AAT GGT GAC CAA CAA GAA TAC ATT CAT 196 Phe Gin Asn Phe Val Leu Lys Asn Gly Asp Gin Gin Glu Tyr lie His His

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Abstract

On décrit des ADN codant des protéines chimères qui présentent chacune un domaine contenant au moins le site de liaison avec l'éliciteur d'un récepteur de l'éliciteur, et un autre domaine contenant au moins le motif de transduction de signal d'une protéine capable de conférer à des plantes la résistance à la maladie. On décrit en outre des vecteurs recombinants contenant ces ADN, l'utilisation de ces vecteurs ou de leurs descendants pour préparer des plantes transformées, ainsi qu'une méthode de production de plantes résistantes à la maladie qui se caractérise par un transfert desdits vecteurs dans des plantes hôtes.
PCT/JP1998/002719 1997-06-18 1998-06-18 Plantes resistantes aux moisissures et procede de production desdites plantes WO1998058065A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001070988A3 (fr) * 2000-03-23 2002-04-04 Eden Bioscience Corp Recepteurs d'eliciteurs a reponse hypersensible et leur utilisation
US8857043B2 (en) 2002-12-30 2014-10-14 Varian Medical Systems, Inc. Method of manufacturing an implantable marker with a leadless signal transmitter
WO2023227912A1 (fr) * 2022-05-26 2023-11-30 Cambridge Enterprise Limited Protéine de liaison au glucane pour améliorer la fixation de l'azote dans des plantes

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102754591B (zh) * 2012-07-03 2013-10-23 福建农林大学 一种多抗水稻种质的选育方法

Citations (6)

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Publication number Priority date Publication date Assignee Title
WO1995028423A1 (fr) * 1994-04-13 1995-10-26 The General Hospital Corporation Famille, amorces et sondes de genes rps et methodes de detection associees
WO1995035371A1 (fr) * 1994-06-17 1995-12-28 Kirin Beer Kabushiki Kaisha Recepteur de l'eliciteur du glucan et adn le codant
WO1996032007A1 (fr) * 1995-04-13 1996-10-17 Yeda Research And Development Co. Ltd. Plants de tomates transgeniques contenant un gene de resistance au fusarium
WO1997022242A1 (fr) * 1995-12-15 1997-06-26 Kirin Beer Kabushiki Kaisha Vegetaux resistant a la moisissure et leur procede d'obtention
EP0823481A1 (fr) * 1996-08-09 1998-02-11 Keygene N.V. Résistance contre les nématodes
WO1998006750A2 (fr) * 1996-08-09 1998-02-19 Keygene N.V. Resistance aux nematodes et/ou aux aphides

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995028423A1 (fr) * 1994-04-13 1995-10-26 The General Hospital Corporation Famille, amorces et sondes de genes rps et methodes de detection associees
WO1995028478A1 (fr) * 1994-04-13 1995-10-26 The General Hospital Corporation Gene rps2 et utilisations de ce dernier
WO1995035371A1 (fr) * 1994-06-17 1995-12-28 Kirin Beer Kabushiki Kaisha Recepteur de l'eliciteur du glucan et adn le codant
WO1996032007A1 (fr) * 1995-04-13 1996-10-17 Yeda Research And Development Co. Ltd. Plants de tomates transgeniques contenant un gene de resistance au fusarium
WO1997022242A1 (fr) * 1995-12-15 1997-06-26 Kirin Beer Kabushiki Kaisha Vegetaux resistant a la moisissure et leur procede d'obtention
EP0823481A1 (fr) * 1996-08-09 1998-02-11 Keygene N.V. Résistance contre les nématodes
WO1998006750A2 (fr) * 1996-08-09 1998-02-19 Keygene N.V. Resistance aux nematodes et/ou aux aphides

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001070988A3 (fr) * 2000-03-23 2002-04-04 Eden Bioscience Corp Recepteurs d'eliciteurs a reponse hypersensible et leur utilisation
US8857043B2 (en) 2002-12-30 2014-10-14 Varian Medical Systems, Inc. Method of manufacturing an implantable marker with a leadless signal transmitter
WO2023227912A1 (fr) * 2022-05-26 2023-11-30 Cambridge Enterprise Limited Protéine de liaison au glucane pour améliorer la fixation de l'azote dans des plantes

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