WO2000009712A1 - Facteurs de transcription regulant l'expression de genes inductibles par ethylene - Google Patents

Facteurs de transcription regulant l'expression de genes inductibles par ethylene Download PDF

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WO2000009712A1
WO2000009712A1 PCT/JP1999/002347 JP9902347W WO0009712A1 WO 2000009712 A1 WO2000009712 A1 WO 2000009712A1 JP 9902347 W JP9902347 W JP 9902347W WO 0009712 A1 WO0009712 A1 WO 0009712A1
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gene
ethylene
transcription factor
teil
expression
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PCT/JP1999/002347
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Japanese (ja)
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WO2000009712A8 (fr
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Yuko Ohashi
Shunichi Kosugi
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Japan As Represented By Director General Of National Institute Of Agrobiological Resources, Ministry Of Agricalture, Forestry And Fisheries
Japan Science And Technology Corporation
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Priority to AU36286/99A priority Critical patent/AU3628699A/en
Publication of WO2000009712A1 publication Critical patent/WO2000009712A1/fr
Publication of WO2000009712A8 publication Critical patent/WO2000009712A8/fr

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    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C12N15/8249—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving ethylene biosynthesis, senescence or fruit development, e.g. modified tomato ripening, cut flower shelf-life
    • 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
    • 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

Definitions

  • the present invention relates to a transcription factor that controls the expression of a group of ethylene-inducible genes.
  • the present invention also relates to a method for imparting resistance to plants against various environmental stresses by regulating the expression level in a plant of a transcription factor that controls the expression of an ethylene-inducible gene group.
  • Ethylene a gaseous plant hormone, affects many processes in plant growth and development. These processes include germination, cell elongation, hair root formation, rhizopium infection, fruit ripening, senescence and abscission (Abelles and Saltvel, 1992).
  • Abeles (1973) proposes “stress ethylene” that mediates resistance to environmental and biological stress. Some stresses, including mechanical damage or pathogen attack, accelerate ethylene production in plants (O 'Domiel et al., 1996). Increased ethylene induces the expression of stress-related genes, which probably confers on the plant resistance to soil fungi and some stress (Morgan and Drew, 1997; Knoster et al., 1998). . These effects are related to the effects of ethylene biosynthesis and ethylene response (ie, the expression of ethylene-responsive genes regulated by putative transcription factors as a result of continuous signaling resulting from ethylene recognition). Controlled by both.
  • ETR1 ethylene resistant 1
  • el-etr-4 ethylene resistant 4
  • ETR1 gene product contains sequences with significant similarity to the response regulators of the bacterial binary control system (Chang et al., 1993).
  • Transgenic yeasts expressing ETR1 have been shown to bind to ethylene, suggesting the function of ETR1 as an ethylene receptor in plants (SchaUer and Bleecler, 1995).
  • CTR1 constitutive triple response 1
  • CTR-1 is a negative regulator of ethylene signaling.
  • the CTR1 gene is superior to the ETR1 gene and encodes a protein with homology to Raf kinase (Kieber et al., 1993). Recently, CTR1 has been found to interact directly with ETR1 (Clark et al., 1998).
  • N3 (ethylene insensitive 3) is genetically higher than ETR1 and CTR1, and is present at the most downstream of the gene pathway at present. Like etrl, the ein3 mutant exhibits a loss-of-fuiiction phenotype for the ethylene response represented by a triple response (Roman et al., 1995; Chao et al., 1997).
  • the amino acid sequence of the protein encoded by EIN3 has been shown to have no significant homology to other amino acid sequences in the database.
  • EIL EIN3-related products
  • EIL EIN-like
  • EIL1 and EIL2 have significant homology to the N-terminal half of EIN3, and the expression of these cDNAs (ie, EIL1 and EIL2) can complement the ein3 mutation like EIN3.
  • EIL and EIN3 appear to be functionally similar to each other (Chao et al., 1997).
  • EIN3 and EIL1 were localized exclusively in the nucleus when expressed as a fusion protein with the GUS reporter in Arabidopsis protoplasts. This suggests that these proteins may be transcription factors (Chao et al., 1997), but their function is not yet clear.
  • ethylene like salicylic acid (SA) and jasmonic acid (JA), is known as a secondary signal substance that is synthesized when plants are subjected to stress such as pathogen infection. I have. When these secondary signal substances are synthesized, the expression of stress-resistant genes that lead to the function of protecting the stress in plants is induced. Examples of stress resistance genes include infection-specific (PR) Genes.
  • SA salicylic acid
  • JA jasmonic acid
  • PR proteins proteins encoded by the PR gene
  • PR proteins have various protective functions.
  • PR proteins are generally classified into two types: acidic PR proteins whose isoelectric point is on the acidic side, and basic PR proteins whose isoelectric point is on the basic side.
  • Acidic PR protein is known to be induced by salicylic acid and is thought to be involved in systemic acquired resistance (SAR) to plant pathogen infection.
  • Basic PR proteins are constitutively expressed in roots and lower leaves. When plants are exposed to stressful conditions such as pathogen infection and wound stress, tobacco basic PR proteins, such as PR-2,3,5, are released by ethylene and PR-1,2,3. , 5, and 6 have been reported to be induced by jasmonic acid.
  • a transcription factor that regulates the expression of ethylene-inducible genes it is possible to regulate the expression level of the target gene, an ethylene-inducible gene, by regulating the expression level of the transcription factor in plants. It is believed that there is.
  • the target gene is a stress resistance gene, it is considered that by regulating the expression of this gene, it is possible to impart resistance to plants to environmental stress. Creating plants that are resistant to environmental stress is an important issue, especially in the agricultural sector. Disclosure of the invention
  • the present invention has been made to solve the above problems, and an object of the present invention is to provide a transcription factor that controls the expression of an ethylene-inducible gene group.
  • a further object of the present invention is to provide a plant with a transcription factor that regulates the expression of ethylene-inducible genes. It is an object of the present invention to provide a method for producing a plant that has been imparted resistance to environmental stress (eg, disease infection and wound stress) by regulating the expression level in the body.
  • Another object of the present invention is to provide a method for screening a transcription factor that controls the expression of a group of ethylene-inducible genes.
  • the present invention relates to a transcription factor that controls the expression of a group of ethylene-inducible genes, and has a specific binding activity to a consensus sequence A (T / c) G (A / T) A (C / T) CT. Having a transcription factor.
  • the transcription factor is the following (a) or (b):
  • (a) has an amino acid sequence containing amino acids from Glu at position 82 to Arg at position 302 in SEQ ID NO: 2, and amino acids from Val at position 482 to Tyr at position 615 of SEQ ID NO: 2 in the sequence listing A transcription factor; or
  • a transcription factor which has an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence (a), and controls the expression of an ethylen-inducible gene group.
  • the transcription factor is the following (c) or (d):
  • a transcription factor that has an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence (c), and controls the expression of an ethylene-inducible gene group.
  • the transcription factor controls the expression of a wound-inducing gene.
  • the group of ethylene-inducible genes includes a group of basic PR genes.
  • the basic PR gene group includes a basic PR-2 gene and a basic PR-5 gene.
  • the present invention also relates to a gene encoding the above-mentioned transcription factor.
  • the present invention relates to a gene encoding a transcription factor that regulates the expression of an ethylene-inducible gene group, comprising a DM of the following (i) or (ii):
  • the present invention provides a method for transforming a plant cell with a polynucleotide containing the above-mentioned gene; and a step of re-differentiating the transformed plant cell to obtain a plant.
  • the present invention relates to a method for imparting resistance to stress.
  • the polynucleotide comprises a DNA having the nucleotide sequence of SEQ ID NO: 1.
  • the environmental stress comprises a pathogen infection.
  • the environmental stress includes wound stress.
  • the present invention provides an ethylene-inducible genetic method comprising the step of identifying a protein having a specific binding activity to consensus sequence A (T / c) G (A / T) A (C / T) CT.
  • the present invention relates to a method for screening a transcription factor that controls the expression of a child group.
  • FIG. 1 is a diagram schematically showing an ethylene response pathway in Arabidopsis thaliana.
  • FIG. 2 shows the nucleotide sequence and the deduced amino acid sequence of TEIL. The deduced amino acid sequence is shown below the nucleotide sequence of the TEIL cDNA. The triangle indicates the position of the 5 'end of the original clone isolated by yeast one-hybrid screening. The predicted helical structure is underlined.
  • Figure 3 shows a comparison of amino acid sequences between TEI and EIN3 and EIL1 polypeptides.
  • FIG. White characters in black boxes indicate that the amino acids are identical in all of TEIL, EIN3, and EIL1.
  • FIG. 4 is a diagram and a photograph showing the analysis of the DNA binding region of TEIL.
  • FIG. 4A schematically shows a deletion mutant of the TEIL protein.
  • a series of N-terminal deletion mutant proteins ( ⁇ 1 to ⁇ 4), or C-terminal deletion mutant proteins (m C1 to m C3) and full-length TE IL (WT) were prepared as fusions with Trx (thioredoxin).
  • FIG. 4 (b) shows the results of electrophoretic mobility shift assay (EMSA) using the ⁇ EIL deletion mutant. Approximately 50 ng of the fusion protein of Trx and TEIL deletion mutant was incubated with the 32 l labeled obsl probe.
  • EMSA electrophoretic mobility shift assay
  • FIG. 5 is a diagram showing optimization of a binding sequence of a TEIL protein.
  • the consensus binding sequence of TEIL was determined by the randobindngsngsecitelection method. Trx-TEIL protein was incubated with a double-stranded oligonucleotide containing a random 18-mer sequence. The DNA / protein complex was separated by acrylamide gel electrophoresis, and the recovered DNA was amplified by PCR for the next round of selection. After four rounds of selection, the amplified DNA was cloned into a plasmid and sequenced.
  • FIG. 5A shows 87 TEIL binding sites sequenced and aligned to give an optimized binding sequence. The TEIL binding affinities of the 39 randomly selected sequences are shown relative to the right of the first 39 sequences. The highest value is shown as 100 and is arranged in order of affinity strength.
  • FIG. 5B shows the results of estimating the consensus sequence of TEIL.
  • FIG. 6 is a diagram and a photograph showing a mutation analysis of the optimized TE IL-binding sequence.
  • FIG. 6A shows the sequence of obsl with the highest binding affinity and the sequence of its mutant sequence, obs2 and obsml-5. Bold letters indicate the consensus sequence of TE IL. Underlines indicate mutated nucleotides. Lower case letters indicate adjacent areas.
  • FIG. 6B shows the results of electrophoretic mobility shift assay (EMSA) using a mutant oligonucleotide probe. An oligonucleotide having the sequence shown in FIG. Labeled with 32 P and incubated with recombinant Trx-TEIL protein. psi is a binding sequence in the promoter of tobacco PRla.
  • ESA electrophoretic mobility shift assay
  • FIG. 7 is an electrophoresis photograph showing increased tebs binding activity in ethylene-treated tobacco leaves.
  • a 25-fold molar excess and a 125-fold molar excess of obsl double-stranded oligonucleotides (lanes 3 and 4), obsm2 double-stranded oligonucleotides (lanes 5 and 6), and obsm3 duplex Oligonucleotides (lanes 7 and 8) were added as antagonistic DNA to the TEI probe DNA binding reaction.
  • FIG. 8 is an electrophoretic photograph showing the results of Northern plot analysis showing tissue-specific accumulation and wound-induced accumulation of the transcript of the TEIL gene.
  • Figure 8A shows the results for RNA isolated from the tissues or cells listed above.
  • FIG. 8B shows the results for RNA isolated from mature leaves cut into small pieces and incubated with water for the indicated period. The total RNA (20 g) was loaded, and the transcript was probed with the cDNA fragment corresponding to the C-terminal region of TEIL (fragment obtained by cutting pGAD-TEIL clone with Dral and BamHI). Detected. It was confirmed by ethidium bromide (EtBr) that equal amounts of RNA in each lane were loaded on the gel.
  • EtBr ethidium bromide
  • FIG. 9 is a graph showing activation of the tebs-reporter gene in tobacco protoplasts and transactivation by overexpression of TEIL.
  • Transient expression assays were performed using tobacco mesophyll protoplasts.
  • the repo overnight plasmid consists of a fusion gene of the CaMV 35S RNA min imal promoter and the GUS gene, and contains 4 copies of obsl (obsl-GUS) or 4 copies of obsm2 (obsm2-GUS). It is.
  • the effector plasmid (35S-TEIL) contained TEILcMA under the control of the CaMV35S promoter.
  • the control effector (35S-NPT II) contained the NPTI I coding sequence instead of the TEIL cDNA.
  • the plasmid (35S-NPTI I) was transfected (white column) or the repo overnight plasmid and the effector plasmid (35S-TEIL) (black column) were electrophoretically transfected. After culturing for 48 hours, GUS activity was measured. The graphs show the average of six measurements and their standard deviation, respectively.
  • FIG. 10 shows three transgenic tobacco plants (35S-TEIL-2,
  • the present inventors have discovered a novel transcription factor that controls the expression of an ethylene-inducible gene group.
  • the present inventors have determined that this transcription factor is (1) inducible when plants are treated with and damaged by ethylene, and (2) as a transcription factor having sequence-specific DNA binding activity. It can function to regulate the expression of ethylene- and wound-inducible genes.
  • the present invention has been completed based on these findings.
  • the “ethylene-inducible gene group” refers to a group consisting of a plurality of genes whose expression is induced by the occurrence of ethylene in a plant.
  • Examples of the gene belonging to the ethylene-inducible gene include a basic PR gene.
  • Base infection-specific (PR) genes refers to a group of genes that are activated when plants are exposed to environmental stresses such as pathogen infection and wound stress.
  • Examples of the basic PR gene include a basic PR-1 gene encoding a protein having an antifungal function, a basic PR-2 gene encoding i3_l, 3-dalkanase (eg, GLA , GLB, and gnl), the basic PR-3 gene encoding class 11 chitinase (eg, CHN48, CHN50, CH5B, and ATHCHTB), the basic PR-4 gene encoding class V chitinase, osmotin And basic PR-6 gene (for example, NT PR0TINH and STP12G) which encodes proteinase inhibitor (PI) and the like (for example, Lc van Loon et al., 1994). See).
  • “Expression” for a gene refers to the transcription of DNA into mRNA.
  • the degree of transcription to mRNA is indicated as the expression level. Therefore, when transcription is suppressed, the expression level decreases, and when transcription is promoted, the expression level increases.
  • Transcription factors are proteinaceous factors that are required in a transcription reaction in addition to RNA polymerase. In eukaryotic cells, transcription factors other than RNA polymerase are required for correct transcription to occur. Transcription factors include those that act by directly binding to DNA and those that function through protein-protein interactions between the factors.
  • the transcription factor contemplated in the present invention is a transcription factor having a sequence-specific DNA binding activity to the consensus sequence A (T / c) G (A / T) A (C / T) CT.
  • parentheses mean that either of the two bases separated by the hatched parenthesis is selected.
  • two bases separated by a diagonal line are both capitalized, it means that they can be used equally.
  • one base is represented in lower case, it may be used in the consensus sequence, but its suitability is lower than that of the other base.
  • (a) has an amino acid sequence containing amino acids from Glu at position 82 to Arg at position 302 in SEQ ID NO: 2 and amino acids from Val at position 482 to Tyr at position 615 in SEQ ID NO: 2 in the sequence listing, A transcription factor (this amino acid sequence is preferably from Ar to position 302 to Arg from position 1 or Ser from position 412 to Glu from position 82, more preferably from Ser to position 412 from position 1 );
  • a transcription factor ((a) that has an amino acid sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sequence (a) and controls the expression of an ethylene-inducible gene group; Mutant) :
  • the transcription factor of the present invention is preferably the transcription factor of (a) or a variant thereof, more preferably the transcription factor of (c) or a variant thereof, and even more preferably (c) Is a transcription factor.
  • deletion, substitution, or addition refers to the number of deletions, substitutions, or additions that can be introduced by site-directed mutation. Such mutations may occur naturally or may be caused by the action of mutagens or artificially by introducing site-directed mutagenesis. Techniques for site-directed mutagenesis are well known in the art. See, for example, Zoller and Smith (1982).
  • Transcription factors of the type that act by directly binding to DNA have a high selectivity for specific DNA sequences on the promoter of the target gene to which they bind. Binding to a DNA sequence is said to be “specific” if it has the same or as high a selectivity as this selectivity. Therefore, the binding between the above transcription factor and the corresponding specific DM sequence is specific. Similarly, variants of the transcription factor that maintain binding activity also bind specifically to the same DNA sequence.
  • a transcription factor To determine whether a DNA sequence has specific binding activity to a DNA sequence, for example, after incubating the DNA sequence labeled with 32 P with a transcription factor for an appropriate time, electrophoretic mobility shift assay (EMSA) ) Can be confirmed. If the formation of a DNA-protein complex is observed, the transcription factor is said to have "specific binding activity" for its DM sequence.
  • ESA electrophoretic mobility shift assay
  • Consensus sequence A (T / c) G (A / T) A (C / T) Protein with specific binding activity to CT is used as a transcription factor that controls the expression of ethylene-inducible genes Can be done. Therefore, a method for screening for a novel transcription factor using a consensus sequence is also included in the scope of the present invention.
  • a method for identifying and isolating a transcription factor having a specific binding activity to a consensus sequence) 6/1 includes, for example, a method of extracting a plant cell nuclear fraction using a DNA binding affinity ram.
  • a library eg, an expression library such as a ⁇ gtll library
  • a directly radiolabeled oligonucleotide containing a consensus sequence as a probe The South- ⁇ Estan Law.
  • the gene encoding the transcription factor of the present invention is the gene intended in the present invention.
  • a method for isolating a naturally-occurring gene encoding the same includes, for example, library screening using the above-mentioned South-Western method. By using this method, cDNA can be directly isolated. Methods for preparing a gene library for isolating a gene of interest, conditions for binding reaction between a protein transcribed from the library onto a membrane and probe DNA, and methods for cloning a gene are well known to those skilled in the art. See, for example, Mani at is et al. (1989).
  • the gene encoding the transcription factor of the present invention not only a naturally occurring gene but also an artificially synthesized gene can be used.
  • a gene consisting of a DNA having the base sequence of SEQ ID NO: 1 in the sequence listing, and 00/71
  • a gene consisting of a DNA that hybridizes with a DNA having the nucleotide sequence of SEQ ID NO: 1 in the sequence listing under stringent conditions and encodes a transcription factor that controls the expression of an ethylene-inducible gene group, It is the gene contemplated in the invention.
  • One skilled in the art can easily select a gene encoding a desired transcription factor of the present invention.
  • stringent conditions refers to conditions that hybridize to a specific sequence but do not hybridize to a non-specific sequence.
  • the setting of stringent conditions is well known to those skilled in the art and is described, for example, in Maniatis et al. (1989).
  • Stringent conditions specifically contemplated herein are represented by the following conditions: Hybridization solution containing probe ⁇ 5 XSSC (75 mM trisodium citrate, 750 mM sodium chloride), 1% SDS, 1 Denhar dt solution (0.2% BSA, 0.2% polyvinylpyrrolidone, 0.2% Ficoll 400) ⁇ After incubating overnight at 65 at 0.2XSSC— 0.2. Wash with SDS solution for 65, 30-60 minutes Condition to carry out.
  • Whether the obtained transcription factor controls the expression of the ethylene-inducible gene group is determined by creating a transgenic plant that constantly expresses this transcription factor and determining the expression of the ethylene-inducible gene in the plant ( (For example, by Northern plot).
  • the transcription factor when the expression of an ethylene-inducible gene is significantly enhanced as compared with the expression in a non-transformed plant, the transcription factor is referred to as “regulating the expression” of the ethylene-inducible gene group.
  • TEIL tobacco EIN3-1ike
  • SEQ ID NO: 2 The amino acid sequence of this protein (SEQ ID NO: 2) and the cDNA sequence of the gene encoding TEIL (SEQ ID NO: 1) are shown in FIG.
  • the amino acid sequence of the TEIL protein which is a transcription factor of the present invention, has 92% sequence identity with the amino-terminal half region extending from residue 80 to 300 of the EIN3 and EIL1 amino acid sequences. (Example 1).
  • sequence identity for amino acid sequences is typically determined under the conditions used in GeneWorks software Intel 1 i Genetics, Inc. (based on the method of Smith & Waterman). Say.
  • the above regions also share 60-89 identities between the EIN3 and EIL protein families (EIL1-3) and are highly conserved. This suggests an essential function of the N-terminal region.
  • the conserved region appears to have predominantly DIVA binding activity, as shown in the localization analysis of the DNA binding domain in the present invention.
  • the DNA binding domains of known transcription factors are conserved between their families and species, and recognize identical or similar DNA sequences.
  • the high similarity in the region presumed to have DNA binding activity seen between TEIL and EIN3 and EIL1-3 indicates that the DNA sequence recognized by EIN3 or EIL1-3 is recognized by TEIL. Suggests that the sequences are identical or highly similar. This suggestion is supported by the fact that EIL2, which shows lower similarity to E1N3 than TEIL, can complement the ein3 mutation in the same manner as EIN3 and EIL1 (Chao et al., 1997).
  • the amino acid sequence is also predicted by computer analysis to contain a region rich in ⁇ -helical structure ( Figure 2). This analysis was performed using The Predict Protein server ⁇ http: //dodo.cpmc. Columbia.edu/predictprotein/(based on the method of Rost & Sanger) ⁇ . As inferred by Chao et al., This helical structure is also present in EIN3 and E [L1-3, and is involved in DNA binding and is involved in protein-protein interactions including homodimerization or heterodimer formation. Can be involved.
  • the transcription factors GT-1 and GT-2 have been reported to have a triple-helical DNA-binding domain consisting of three ⁇ -helices (which are unique to higher plants) separated by two short loops (Dehesh et al., 1992; Gilmartin et al., 1992).
  • two conserved clusters of basic amino acids corresponding to the basic domains I and II of EIN3 are found at the N-terminus of TEIL (amino acids of TEIL). Residues 54-67 and 90-96).
  • the ⁇ helix and the basic domain appear to constitute a novel DNA binding domain, since deletion of the region containing these domains leads to complete loss of DNA binding activity.
  • DNA binding domain of TEIL Another salient feature of the DNA binding domain of TEIL is that extensive regions may be required for full activity of DNA binding. Removal of the 203 amino acid residues from the C-terminus results in a constant decrease in binding activity (Example 2). This indicates that the entire region extending from residues 1-412 of the TEIL sequence may be required for full DNA binding activity. Alternatively, there may be a second DNA binding region in the C-terminal region that acts in concert with the action of the ⁇ -terminal binding domain.
  • the optimized DNA binding sequence of TEIL is A (T / C) G (A / T) A (C / T) CT (Example 3).
  • This recognition sequence is unique and not found in sequences recognized by known plant transcription factors.
  • a consensus binding sequence of Oct-1 (ATGCAAT) and a consensus binding sequence of Tit-1 (ATGNATAWWT, which are mammalian transcription factors involved in developmental regulation; where N is any base, and W is A or T (representing any of T) (Herr and Cary, 1995) have similarities to the above recognition sequences.
  • These mammalian transcription factors contain a POU DNA binding domain consisting of two structurally independent segments, a P0U-specific domain and a P0U homeodomain.
  • the P0U-specific domain and the P0U homeodomain consist of four and three alpha helices, respectively. No similarity in amino acid sequence is found between the POU DNA binding domain and TE IL. It should be noted, however, that the N-terminal region of TEIL with DNA binding activity forms some predicted helices as described above.
  • TEIL is functionally similar to EIN3. Even EIL 2, which has only 35% sequence identity with EIN3, can complement the ei ⁇ 3 mutation, as does EIL1. Thus, TEILs with higher similarity to EIN3 would also be able to complement the ein3 mutation.
  • TEIL functions as a transcription factor in tobacco protoplasts. This is consistent with the observations made by the present inventors. Transactivation is thought to occur through direct binding of TEIL to the TEIL binding sequence. When TEIL was expressed in yeast as a fusion with the GAL4 DNA binding domain, the reporter gene could be activated. This transactivation required at least a region of residues 482 to 615 of the TEIL amino acid sequence (Example 7). These indicate that TEIL is a transcription factor with sequence-specific DNA binding and transcriptional activity.
  • TEIL transcriptional activation function
  • the reporter gene with tebs (TEIL binding site) linked upstream of the promoter was significantly activated in protoplasts, probably by endogenously activated TEIL or its associated protein (Example 6).
  • stress-inducible genes were released from the cell wall, probably during the preparation of protoplasts (Davis and Hahlbrock, 1987), or due to elimination factors in cell wall digestive enzyme solutions.
  • Activated by direct wounding effects O 'Donnel et al., 1996) that induce Z and ethylene release.
  • nuclear extracts from ethylene-treated leaves showed enhanced tebs binding activity from TEIL or related proteins (Example 4).
  • the enhanced activity appears to depend on the ethylene-mediated post-transcriptional activation of a potentially inactive TEIL protein naturally present in healthy tissue. Chao et al. (1997) have also observed that EIN3 protein levels are not altered by ethylene treatment. These observations suggest that ethylene-induced activation of the TEIL target gene (tebs-containing gene) depends on changes in TEIL DNA binding activity that are regulated by ethylene.
  • the target gene for EIN3 includes a basic PR gene, and that the target gene for TEIL, a homolog of EIN3, also includes a basic gene. Therefore, it was expected that the promoter region of the ethylene-inducible gene including the basic PR gene group would include a binding site for TEIL.
  • TEIL A T / c
  • G A / T
  • C C / T
  • ethylene induction of the CHM8 gene which encodes tobacco basic chitinase, requires a region ranging from -503 to -358 in the promoter region (Shinshi et al., 1995). In this region, there are three putative tebs located adjacent and two GCC boxes (AGCCGCC) required for ethylene-induced expression.
  • the corresponding homologous regions of the CHN50 gene also preserve identical or homologous putative tebs and GCC boxes (Table 1).
  • the ethylene-responsive region of the tobacco motin promoter is defined in a small region located between -248 and -108 (Raghothama et al., 1993). This area also has one tebs and two GCC boxes.
  • this 60 bp region did not contain a GCC box and the sequence (T) TGAC (C). 1 Found in the promoter region of basic PR gene
  • H5B ATG AAA (-372R) ATGAAGTT (-£ B2) -152 BrogliectaU1989
  • AT6TATAC (-480R) ACGAA ( ⁇ 68)
  • the position of the sequence was measured from the starting point of! R.
  • the TGAC element is suggested to be required for elicitor responsiveness of the parsley PR1 gene (Despres et al., 1995), the maize PRms gene (Raventos et al., 1995), and the potato PR-10a gene (Rush ton et al., 1996). ing.
  • the TGAC element is a promoter of the PAL (pheny al ani am amonia ia-lyase) gene known as a stress-responsive gene and the 4CL (4-coumara te: coenzymeA1 igase) gene family. There is no elicitor in one responsive region.
  • the promoter region of many basic PR genes contains the GCC box required for ethylene-induced expression (Ohme-Tagaki and Shinshi, 1990; Eyal et al., 1993; Hart et al., 1993).
  • Ethylene-responsive element binding protein (EREBP) a protein that binds to the GCC box, has been proposed to be a potential regulator of the ethylene-induced basic PR gene (Ohme-Tagaki and Shinsh i, 1995).
  • EEEBP Ethylene-responsive element binding protein
  • Table 1 in most basic PR promoters including the GCC box, the tebs sequence in the present invention is present near the GCC box. Without being bound by a particular theory, it is thought that TEIL can coordinate with EREBP to regulate the expression of the basic PR gene.
  • TEIL is E It may also interact directly with REBP to achieve a cooperative transactivation function.
  • the acidic PR gene activated by salicylic acid may not be the target of TE1L.
  • TEIL was first isolated as a protein that binds to the ps position of the tobacco acidic PR-la promoter; (2) the acidic PR gene is induced by salicylic acid. , Ethylene and salicylic acid, or ethylene and jasmonic acid, are reported to be coordinately induced; and (3) the binding affinity of TEIL to psl is determined by the optimal binding sequence binding. Although substantially lower in affinity, the cis-acting elements defined in many genes are not necessarily strong binding sites for their cognate binding agents.
  • TEIL of the present invention can function as a sequence-specific DNA binding protein that mediates the activation of ethylene-induced transcription
  • its homologues, EIN3 and EIL1-3 also have ethylene-inducible transcription similar to TEIL. It shows that it can function as a sequence-specific DNA binding protein that mediates activation.
  • Each member can target overlapping and different genes through direct or indirect interactions with other proteins, including EREBPs.
  • Ethylene mediates the activation and repression of genes involved in various physiological phenomena.
  • some ethylene-responsive genes are not yet identified transcription factors. (Perhaps including EIN5, EIN6, and EIN7).
  • the gene encoding the transcription factor of the present invention can be introduced into a plant as a polynucleotide containing the gene.
  • the polynucleotide is usually operably integrated with the gene In the form of a suitable plant expression vector. Expression of the introduced gene in a plant can exogenously produce the transcription factor of the present invention.
  • a method for producing a transformed plant by introducing a gene into a plant can be performed according to a conventional method in the art.
  • the “plant” to which the method of the present invention is applied includes both monocotyledonous plants and dicotyledonous plants. Particularly preferred plants include tobacco, peppers, eggplants, melons, tomatoes, sweet potatoes, cabbage, leeks, broccoli, carrots, pomegranates, citrus, Chinese cabbage, lettuce, peaches, rice, potatoes, wheat, apples and apples. . Unless otherwise indicated, a plant means any of a plant, a plant organ, a plant tissue, a plant cell, and a seed. Examples of plant organs include roots, leaves, stems, and flowers. Examples of plant cells include callus and suspension culture cells.
  • the fact that the gene encoding the transcription factor is derived from the same species or a closely related species as the target plant (for example, a species classified into the same genus or the same family) although it may be a preferred embodiment, it is not necessary.
  • polynucleotide used in the method of the present invention has a gene encoding the transcription factor of the present invention, and any additional sequences necessary for achieving a desired transformation.
  • the polynucleotide is typically a plant expression vector.
  • Plant expression vector refers to a recombinant construct of a nucleic acid sequence in which various regulatory elements such as promoters that regulate the expression level of a gene of interest are operably linked in a host plant cell. .
  • it may contain a plant promoter, a termine, a marker gene such as a drug resistance gene, and an enhancer. More preferably, it may include an origin of replication.
  • the type of plant expression vector and the preferred type of regulatory element used can vary depending on the host cell. Those skilled in the art will appreciate that, in practicing the present invention, By appropriately selecting a regulatory element such as a hansa, the degree of expression of the introduced gene can be regulated.
  • the plant expression vector used in the present invention may further have a T-DNA region.
  • the T-DNA region increases the efficiency of gene transfer, especially when transforming plants with agrobacterium.
  • Plant promoter refers to a promoter that can function in a plant cell.
  • tobacco PR-1 promoter tobacco infection-specific protein PR-1 promoter
  • heat shock-induced promoters etc.
  • promoters whose expression is induced by certain types of stress cauliflower mosaic Examples include, but are not limited to, the virus (CaMV) 35S promoter, and a constant promoter such as the nopaline synthase promoter (P nos).
  • a “terminator” is a sequence located downstream of a region encoding a protein of a gene and involved in terminating transcription when DNA is transcribed into mRNA and adding a poly A sequence. It is known that evening and evening and evening are related to mRNA stability and affect gene expression levels. Examples of the terminus include the CaMV35S evening and the evening, the nopaline synthase gene evening and the evening (Tnos), and the tobacco PR-1 gene evening and the evening. Not limited.
  • the “drug resistance gene” is preferably a gene that facilitates the selection of a transformed plant.
  • the neomycin phosphotransferase II ( ⁇ ) gene for imparting kanamycin resistance, the hygromycin phosphotransferase gene for imparting hygromycin resistance, and the like can be suitably used.
  • promoters for expressing a drug resistance gene include, but are not limited to, the above-mentioned plant promoters, for example, tobacco PR-1 promoter, CaMV35S promoter, nopaline synthase promoter.
  • Enhancers can be used to enhance the expression efficiency of a target gene. Enhancers include the upstream sequence in the CaMV35S promoter overnight. One region is preferred. A plurality of enhancers can be used for one target gene.
  • a pBI-based vector, a pU-based vector or a pTRA-based vector can be suitably used.
  • the pBI- and pTRA-based vectors can introduce a target gene into a plant via an agrobacterium.
  • a pBI binary vector or an intermediate vector is preferably used.
  • These vectors contain a gene of a region (T-DNA region) that can be introduced into a plant, and an NPTI I gene (which confers kanamycin resistance) expressed as a marker gene under the control of a plant promoter.
  • a pUC-based vector can directly introduce a gene into a plant.
  • pUC18, pUC19, pUC9 and the like can be mentioned.
  • the plant expression vector of the present invention can be produced using a gene recombination technique well known to those skilled in the art.
  • a gene encoding the transcription factor of the present invention is incorporated downstream of the promoter of the above vector.
  • a method well known to those skilled in the art for example, a method via an agrobacterium and a method for direct introduction into a cell can be used.
  • a method via the agrobacterium for example, the method of Nagel et al. (1990) can be used.
  • an agrobacterium is transformed by, for example, electroporation with a plant expression vector, and then the transformed agrobacterium is introduced into plant cells.
  • Methods for directly introducing a plant expression vector into cells include the electoral poration method and the gene gun method, as well as the calcium phosphate method and the polyethylene glycol (PEG) method. These methods are well known in the art, and a method suitable for a plant to be transformed can be appropriately selected by those skilled in the art.
  • Cells transformed by introducing the plant expression vector are first selected using drug resistance, such as kanamycin resistance, or other appropriate phenotype as an indicator. Next It can be redifferentiated into plant tissues, plant organs and / or plants by conventional methods. Further, seeds can be obtained from the regenerated plants. In this way, a transformed plant having the gene encoding the transcription factor of the present invention in a cell is obtained.
  • the transcription factor of the present invention By producing the transcription factor of the present invention in the obtained plant, expression of the ethylene-inducible gene group and Z or the wound-inducible gene group can be promoted, and as a result, resistance to environmental stress can be imparted. .
  • environmental stress refers to any stress that plants can receive in the natural world and hinder their growth.
  • environmental stress include pathogen infection, wound stress, high light, low temperature, freezing, drying, high temperature, high salt, UV irradiation, ozone, insect damage and herbicides.
  • Pathogen infection refers to an infection of a plant with a pathogenic agent, including infection by viruses, viroids, filamentous fungi, and bacteria.
  • Wild stress refers to the external mechanical damage to a plant.
  • “Providing resistance to environmental stress” refers to conferring new resistance on a plant or enhancing the resistance of a plant that already has resistance.
  • Genes that confer plant resistance to environmental stress are called “stress resistance genes”. Examples include, but are not limited to, ethylene inducible genes, salicylic acid inducible genes, including the acid PR gene, and wound inducible genes.
  • wound-inducible gene is a gene whose expression is induced when a plant is exposed to damaging stress.
  • wound-inducing genes include proteinase inhibitors (PI), proteolytic enzymes (eg, polyphenoloxidase and reboxygenase), wound-inducing MAP kinase, and basic PR. Examples include, but are not limited to, various genes encoding proteins (eg, PR-1 to PR_5).
  • the presence or absence of resistance to environmental stress can be confirmed by evaluating the difference that can be observed between the transformed plant and the control plant when the plant is exposed to a certain environmental stress. For example, the disease resistance of a transformed plant to a pathogen infection is assessed as the difference in morphological changes between the transformed and control plants in the pathogen infection. For example, if the degree of a lesion that can be observed in a transformed plant after pathogen infection is significantly suppressed as compared to a control plant, the transformed plant is conferred resistance.
  • wound stress resistance refers to the resistance of a plant to at least one of insect resistance and pathogen resistance after injury.
  • TEIL is a homolog of EIN3
  • EIN3 is encoded by the Arabidopsis thaliana gene and acts downstream of ETR1 (ethylene resistant 1), CTR1 (constitutive triple response 1), and EIN2 (ethylene insensitive 2) in the ethylene signaling pathway (Chao et al., 1997) . Mutations in EIN3 result in an ethylene-insensitive mutant (ein3).
  • the amino acid sequence of TEIL shares 60% overall sequence identity with the amino acid sequence of EIN3. 92% sequence identity is observed in the N-terminal half of positions 80-300 of both amino acid sequences ( Figure 3).
  • TEIL also shows high homology (58% -35% sequence identity) to the amino acid sequence of EIU-3 from Arabidopsis thaliana, which was isolated as a protein related to EIN3.
  • the relevance for TEIL was closest for EIL1, and lower for EIL2 and EIL3.
  • the degree of overall similarity Considering the extent and the presence of the basic domain IV characteristic of these proteins other than EIL2 (the domain corresponding to amino acids 267-276 in TEIL), TEIL is one of the EIN3 and EIL1 members of the Arabidopsis EIN3 family. Seems to be closest to
  • TEIL DNA binding activity
  • Recombinant fusions of full-length TEIL or N-terminal or C-terminal deletion mutants with Trx (thioredoxin) were made. These fusions were then purified using affinity columns (FIG. 4A). The purified product was subjected to electrophoretic mobility shift assay (EMSA) using obsl (FIG. 4B). obsl is a probe DNA with high affinity for TEIL binding, as shown below (see Figure 6A for obsl sequence).
  • the probes used in the EMSA are all double-stranded oligonucleotides bonded to complementary chains.
  • Some recombinant proteins particularly recombinant proteins containing the N-terminal region, were used in a partially purified state because it was difficult to express them in large amounts in E. coli.
  • the protein of interest in the sample was quantified by Western blot analysis using the S protein (data not shown).
  • Trx- ⁇ is a variant in which the N-terminal region covering residues 1 to 81 of the TEIL amino acid sequence has been deleted. Tnc- ⁇ retained the ability to bind to probe DNA, but the affinity was reduced by approximately 30% compared to full-length TEIL (WT) ( Figure 4B; lane 2 compared to lane 1) . Further deletions of the N-terminal region up to residues 93, 132, and 160 (corresponding to ⁇ 2, ⁇ 3, and ⁇ ⁇ ⁇ 4, respectively) resulted in a complete loss in DNA binding capacity (FIG. 4 ;; lanes 3 to 4). Five) .
  • ACl and AC2 which lack the C-terminal region covering residues 413-615 and 303-615, respectively, have a DNA binding activity of 10-15% compared to full-length TEIL (WT).
  • Figure 4B Lanes 6 and 7 compared to Lane 1.
  • An additional deletion of the amino acid sequence spanning residues 226-615 (corresponding to AC3) resulted in a complete loss in avidity ( Figure 4B: lane 8) ).
  • C-terminal deletion mutants were also tested by in vivo experiments using the yeast one hybrid assay using the (psl) 4- CYCl-HIS3 repo.
  • the AC3 construct cloned into pGBT9 (Clontech) did not activate the repo overnight gene.
  • the similarly cloned AC2 construct activated the reporter gene, although its activation ability was weaker than that of full-length TEIL (WT) (data not shown).
  • WT full-length TEIL
  • TEIL binds to DS1 in the tobacco PRla promoter, but it appears that there is a more favorable sequence than psl for binding to TEIL.
  • Trx thioredoxin
  • Trx- ⁇ EIL fusion protein The fusion protein was incubated with a double-stranded oligonucleotide consisting of a random 18-mer with an annealing site bound at both ends for the PCR primers. After separating the DNA protein complex by polyacrylamide electricity and icing, the band of the oligonucleotide bound to Trx-TEIL was amplified by PCR.
  • oligonucleotides were cloned into a plasmid (PGEM-3Zf, Promega Corporation). A total of 87 oligonucleotides were sequenced from each clone (FIG. 5A). Comparison of these sequences indicated that the consensus sequence for TEIL was A (T / c) G (A / T) A (C / T) CT (FIG. 5B). This consensus sequence matches 6 bp with the sequence of psl (ATGAATAA).
  • Variants of the third or fifth nucleotide resulted in a significant decrease in TEIL binding (FIG. 6B; corresponding to lanes 4 or 5). This indicates that the third and fifth nucleotides may be absolutely required for binding.
  • the mutant in which the eighth nucleotide was changed from T to G did not affect the binding affinity.
  • the G to T mutation of the ninth oligonucleotide also had no effect on binding selection (FIG. 6B; lane 7).
  • Example 4 Nuclear extracts from ethylene-treated leaves were replaced with TEIL binding sites (tebs). With enhanced binding activity to
  • the recombinant TEIL protein showed strong binding to obsl, a high affinity sequence of the TEIL binding site (tebs).
  • tebs a high affinity sequence of the TEIL binding site
  • the present inventors used a labeled DNA probe containing 4 copies of obsl to perform electrophoretic mobility shift assay (EMSA).
  • ESA electrophoretic mobility shift assay
  • TEIL gene transcripts show tissue-specific and wound-induced accumulation
  • the obsl report construct (obs GUS) is composed of the CaMV 35S (-54) promoter (CaMV minimal promoter) and the bacterial j3-dalcuronidase (G (US) It is a fusion with the messenger.
  • the control reporter construct (obsm2-Gus)
  • the four repeats of obs-1 in obsl-GUS were replaced with the four repeats of obsm2.
  • Each of these constructs was transfected into tobacco mesophyll protoplasts. 7-10 times higher GUS activity was observed with obsl-GUS than with obsm2-GUS (data not shown). This observation indicates that an active form of TEIL or a related protein was endogenously present in tobacco mesophyll protoplasts and activated the repo allele through sequence-specific binding to the obsl sequence. Suggests.
  • 35S-TEIL the effector plasmid
  • 35S-II the control effector plasmid
  • 35S-TEIL and 35S-NPTII were the TEIL cDNA and neomycin phosphotransfer operably linked to the CaMV 35S RNA promoter, respectively. It is a construct containing the sferase gene.
  • the GUS activity was further enhanced by about 2 to 3 times as much as that of the combination with 35S-NPTII due to the expression of the repo overnight gene (Fig. 9).
  • TEIL In tobacco protoplasts, overexpression of TEIL activated reporter gene transcription, so the TEIL protein may itself contain a transcription activation domain.
  • yeast one-eight hybrid system To identify this transcriptional activation domain, we utilized the yeast one-eight hybrid system. Yeast systems are advantageous because they allow lower basal expression of the reporter gene and are more reproducible than plant systems.
  • GAL4bd GAL4 DNA binding domain
  • GAL4bd GAL4 DNA binding domain for fusion with TEIL and GAL1-LacZ or GAL1-HIS3 as a reporter gene are used (Clontech).
  • pGBT-TEIL a plasmid producing a fusion of GAL4db and TEIL protein activated the repo overnight gene (lacZ or HIS3) in yeast.
  • the level of activation was determined by using pCLl
  • PGBT9 indicates a control construct having only the GAL4 DNA binding domain.
  • His + or His-I was used for transforming yeast in a histidine-free medium.
  • the value represents the average value of the activity of lactosidase from three independent colonies, and the unit is defined as the amount that hydrolyzes 1 / mol ofo-nitrophenyl] 3-D-force lactobyranoside / min. You.
  • a series of TE-terminal deletion mutants of TEIL were created as a fusion partner with GAL4bd.
  • pGBT-C556 a plasmid that produces a TEIL mutant lacking the region spanning residues 557-615 of the amino acid sequence, retained about half the activity of wild-type TEIL.
  • pGBT-C481 and pGBT-C412 which produce TEIL variants lacking the region spanning residues 482-523 and 413-523, respectively, completely lost activity.
  • the transcription activation region of TEIL is located in a region extending from residues 482 to 615 of the amino acid sequence (particularly, a region extending from residues 482 to 556). However, in the region extending from residues 482 to 615, no known transcription activation domain (glutamine-rich sequence, proline-rich sequence, or acid amino acid-rich sequence) was found.
  • Example 8 Transgenic plants with high expression vector of TEIL constantly express TEIL gene
  • PBI121 (Cl on tech) was used as a starting material to construct a vector that highly expresses TEIL.
  • PBI 121 is under the control of the N0S promoter overnight (Pnos) and the evening minerals (Tnos) ⁇ ⁇ gene, a multiple cloning site, and a 35S CaMV promoter and has Tnos It is a plasmid containing the i3-GUS gene derived from Escherichia coli.
  • PBI121 was first cut with Xbal and BamHI and the large fragment was recovered.
  • the plasmid (pBSK-TEIL) was digested with restriction enzymes (Spel and Bglll), ligated with a fragment of TEIL cDNA cut out, ligated to this fragment, and then introduced into E. coli JM109.
  • the kanamycin resistant strain was recovered to obtain the desired high expression vector (PBI-35S-TEIL). Restriction enzyme analysis confirmed that the TEIL gene had been introduced in the correct direction.
  • the resulting expression construct was introduced into Agrobacterimn tumefaciens LBA4404 (Ooms et al., 1981)) by electroporation (Wen-Jun and Forde, 1989). Transformation of Nicotiana bacta cv. Samsun NN was performed by a leaf coexisting culture method (Horsch et al., 1985). The leaf pieces are immersed in a bacterial solution, which is then Transfer medium (3% sucrose, 85% bismuth! ⁇ 1 511 ⁇ 6-51 «) ( ⁇ (MS) basic medium). 2 days, 25 ° C, the continuous illumination of a white fluorescent lamp, at an intensity of 120 iE / m z / s, were co-cultured.
  • the leaf pieces were transferred to a medium containing 80 ⁇ g / ml kanamycin to remove the bacteria. Every three weeks, the shoots formed in a medium containing kanamycin were transferred to a hormone-reduced selection medium, subcultured on a selection medium. After root formation, the plants were transferred to pots containing soil. Fourteen independent transformed tobacco individuals (35S-TEIL-1 to 14) were obtained.
  • the expression of the TEIL gene in the obtained transformant was confirmed as follows. From the leaves of three transformants obtained (35S-TEIL-2, 35S-TEIL-10 and 35S-TEIL-14 strains), mRNA was extracted by a conventional method, and TEIL cDNA was used as a probe. A Northern plot analysis was performed. As controls, intact leaves (SNN) of untransformed plants (Nicotian a tabacum cv. Samsun NN) and leaves (SN N-wound) one day after wounding were used. The results are shown in FIG.
  • the present invention provides a transcription factor that controls the expression of an ethylene-inducible gene group.
  • a transcription factor that controls the expression of an ethylene-inducible gene group resistance to environmental stress (eg, disease infection and wound stress) is imparted.
  • a method for producing a plant is provided.
  • the present invention provides a method for screening a transcription factor that controls the expression of an ethylene-inducible gene group.

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Abstract

L'invention concerne des facteurs de transcription régulant l'expression de gènes inductibles par éthylène, des gènes codant ces facteurs, un procédé destiné à doter des plantes d'une résistance aux contraintes environnementales par transfert dans celles-ci des gènes ci-dessus, ainsi qu'un procédé de criblage de ces facteurs. Ces facteurs de transcription, qui régulent l'expression de gènes inductibles par éthylène et possèdent une activité de liaison de l'ADN spécifique d'une séquence consensus A(T/C)G(A/T)A(C/T)CT, peuvent être soit (a), soit (b): (a) un facteur de transcription possédant une séquence d'acides aminés contenant les acides aminés à partir de Glu au niveau de la position 82, jusqu'à Arg au niveau de la position 302 dans la séquence représentée par SEQ ID NO: 2, et contenant les acides aminés à partir de Val au niveau de la position 482, jusqu'à Tyr au niveau de la position 615 dans la séquence représentée par SEQ ID NO: 2; (b) un facteur de transcription dérivé du facteur (a) par délétion, substitution ou addition d'un ou de plusieurs acides aminés dans la séquence d'acides aminés telle que décrite en (a), et régulant l'expression de gènes inductibles par éthylène.
PCT/JP1999/002347 1998-08-11 1999-05-06 Facteurs de transcription regulant l'expression de genes inductibles par ethylene WO2000009712A1 (fr)

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WO2000046383A3 (fr) * 1999-02-05 2001-09-07 Univ Leiden Methode de modulation de la biosynthese de metabolite dans des cellules recombinees
US7393946B1 (en) 1999-02-05 2008-07-01 Rijksuniversiteit Leiden Method of modulating metabolite biosynthesis in recombinant cells
EP3186380A1 (fr) * 2014-08-26 2017-07-05 Københavns Universitet Plantes à sensibilité réduite à l'éthylène

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