WO2000014242A1 - Nouvelle proteine, gene codant cette proteine et utilisation de la proteine - Google Patents

Nouvelle proteine, gene codant cette proteine et utilisation de la proteine Download PDF

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Publication number
WO2000014242A1
WO2000014242A1 PCT/JP1999/004441 JP9904441W WO0014242A1 WO 2000014242 A1 WO2000014242 A1 WO 2000014242A1 JP 9904441 W JP9904441 W JP 9904441W WO 0014242 A1 WO0014242 A1 WO 0014242A1
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Prior art keywords
sequence
amino acid
seq
protein
antibacterial
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PCT/JP1999/004441
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English (en)
Japanese (ja)
Inventor
Yoshimitsu Takakura
Shigeru Kuwata
Shozo Ohta
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Japan Tobacco Inc.
Corporate Juridical Person, Society For Techno-Innovation Of Agriculture, Forestry And Fisheries
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Priority to AU53009/99A priority Critical patent/AU5300999A/en
Publication of WO2000014242A1 publication Critical patent/WO2000014242A1/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
    • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
    • C07K14/375—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from Basidiomycetes

Definitions

  • the present invention relates to a novel protein having antibacterial activity, a gene encoding the protein, and a method for using the protein and the gene. More specifically, the present invention relates to a mushroom-derived protein having at least an antibacterial activity against rice sheath blight and rice blast, a gene encoding the protein, and a method for using the protein and the gene.
  • landscape technology a novel protein having antibacterial activity, a gene encoding the protein, and a method for using the protein and the gene.
  • lytic enzymes such as chitinase, / 3-1, and 3-glucanase have been conventionally known. In in vitro experiments, these enzymes are effective alone (Schlumbaum et al., (1986) Nature 324: 365-367, Broglie et al., (1991) Science 254: 1194-1197), but in general two or more enzymes It is known that higher effects can be obtained by using in combination (Mauch et al., (1988) Plant Physiol. 88: 936-942, Sela-Buurlage et al., (1993) Plant Physiol. 101: 857-863).
  • the concentration at which these lytic enzymes inhibit the growth of filamentous fungi generally requires several tens to several hundreds / g / ml when used alone, and several g / ml per enzyme even when used in combination. It is known that However, among these lytic enzymes, those that have been demonstrated to act antibacterial against Pyricularia oryzae, an important disease of rice, have not yet been reported to the knowledge of the present inventors.
  • AFPs anti-fungal peptides
  • defensins also have antimicrobial activity.
  • Ca-AMPl Yamahyohei 8-505048
  • CB-1 Oxi
  • Et al. (1996) Biosci. Biotech. Biochem. 60: 481-483) reported that Rs-AFP1 and Rs-AFP2 (Terras et al., (1992) J) have antibacterial activity against Pyricularia oryzae. . Biol. C em.
  • One of the objects of the present invention is to search for and identify novel antibacterial proteins that can inhibit the growth of various plant pathogens, including rice wilt disease and sheath blight, which are two major diseases of rice, at relatively low concentrations. That is.
  • Another object of the present invention is to clone a gene encoding the above novel protein and specify its nucleotide sequence.
  • Still another object of the present invention is to produce a transformant by introducing the gene of the present invention into a host organism (microorganism, animal, plant or the like), and to utilize the gene of the present invention.
  • Still another object of the present invention is to provide an antibacterial agent containing the antibacterial protein of the present invention.
  • the present inventors For the purpose of solving the above-mentioned problems, the present inventors first established an Atsushi system for assaying the antibacterial activity of in vitro against fungus and fungus pathogen. Next, the present inventors extracted proteins from matsutake, one of the edible mushrooms, using ion-exchange column chromatography and gel filtration column chromatography.
  • the antimicrobial dump protein fraction was identified, and the antimicrobial protein was successfully isolated and purified by subjecting each fraction to the above-mentioned Atsushi system. Furthermore, the present inventors determined the partial amino acid sequence of the purified protein, and obtained a partial-length cDNA encoding the protein by the RT-PCR method using an oligonucleotide synthesized based on this amino acid sequence as a primer. . Next, the present inventors screened a cDNA library derived from Matsutake using this partial-length cDNA as a probe to isolate a full-length cDNA encoding the protein, and analyzed the entire nucleotide sequence. Were determined.
  • the present inventors succeeded in isolating a novel antibacterial protein derived from Matsumatsuga and cloning the DNA encoding the same, and also determined the amino acid sequence of the protein and the nucleotide sequence of the DNA. Thus, the present invention has been completed.
  • the first aspect of the present invention can be obtained from a fraction precipitated by an ammonium sulfate precipitation method from an aqueous extract of mushrooms such as matsutake mushrooms, and has at least an antibacterial activity against rice sheath wilt or rice wilt. It has a molecular weight of about 210 kD by gel filtration, and the presence of components of about 50 kD and about 15 kD in the SDS-PAGE method.
  • An antibacterial protein is provided, wherein the antibacterial activity is maintained by heating for minutes, and the antibacterial activity is deactivated by heating at 80 ° C for 10 minutes in a neutral aqueous solution.
  • the antibacterial protein of the present invention has a component of about 50 kD having an N-terminal amino acid sequence described in SEQ ID NO: 3 in the sequence listing: His Glulie Val His Tyr Thr Asp Val Phe He Ala Gly Ser Gly Pro lie Ser Xaa Thr Tyr Ala Arg His He He Asp Asn Thr Ser Thr Thr: has an N-terminal amino acid sequence of about 15 kD described in SEQ ID NO: 4 in the sequence listing: Arg Glu Trp Glu Ala Gly Val Thr Asp Thr Tyr Gly Met Pro Gin Pro Thr Phe His Val Lys Arg Thr Asn Ala Asp Gly Asp Arg Asp Gin Arg Met Met Asn Asp Met Thr Asn Val Ala Asn Met Leu Gly Gly Tyr Leu Pro Gly Ser Tyr Pro Gin Phe Met Ala Pro Gly Leu Val Leu His lie Thr Has Gly Thr.
  • the antimicrobial protein of the present invention typically comprises an amino acid sequence described in SEQ ID NO: 2 in the sequence listing; or an amino acid sequence having one or more amino acid mutations in this sequence or 50% or more of this sequence. It is an antibacterial protein having an amino acid sequence having homology to and showing antibacterial activity against rice sheath blight or rice blast.
  • the antibacterial protein of the present invention is preferably 60% or more, more preferably 70% or more, still more preferably 80% or more, particularly preferably 90% with the amino acid sequence described in SEQ ID NO: 2 in the sequence listing. As described above, most preferably, it has an amino acid sequence having a homology of 95% or more.
  • An antimicrobial protein comprising the polypeptide having activity; or a combination of any of the polypeptides.
  • a protein having 50% or more homology with each specific amino acid sequence means that the protein has at least 50% or more homology. It means that the homology is preferably 60% or more, more preferably 70% or more, still more preferably 80% or more, particularly preferably 90% or more, and most preferably 95% or more.
  • a protein having an amino acid sequence having is contemplated.
  • a step of recovering a fraction that precipitates from an aqueous extract of mushrooms such as matsutake by a sulfuric acid precipitation method using 75% saturation of ammonium sulfate;
  • a method for producing the antibacterial protein of the present invention is provided.
  • the gene encoding the antimicrobial protein of the present invention is Provided.
  • the gene of the present invention is typically a base sequence described in SEQ ID NO: 1 in the sequence listing, or a base sequence having substitution, deletion, insertion and / or addition of one or more bases in the above base sequence. Or a base sequence that hybridizes with the above base sequence under stringent conditions.
  • the gene of the present invention is generally 50% or more, preferably 60% or more, more preferably 70% or more, still more preferably 80% or more, with the base sequence described in SEQ ID NO: 1 in the sequence listing. Particularly preferably, it has a nucleotide sequence having a homology of 90% or more, most preferably 95% or more.
  • an oligonucleotide for obtaining a mushroom-derived antibacterial protein is provided.
  • each region is 15-30 bases;
  • a single-stranded DNA having the same nucleotide sequence as the above region or a nucleotide sequence complementary to the above region, or reduce the degeneracy of the genetic code so as not to change the amino acid residues encoded by the single-stranded DNA.
  • a mixture of the single-stranded DNAs considered is produced, and if necessary, the single-stranded DNAs modified so as not to lose the binding specificity to the nucleotide sequence of the gene encoding the antibacterial protein are produced.
  • the oligonucleotide produced by the method comprising the steps of:
  • the oligonucleotide of the present invention preferably has the nucleotide sequence of any one of SEQ ID NOS: 5 to 9 in the sequence listing.
  • an amplification reaction is carried out by using the above-mentioned two sets of oligonucleotides as primers for a mushroom fruiting body cDNA library type 1 and encoding the antibacterial protein of the present invention. Amplifying a part of the gene of the present invention, and screening the cDNA library using the resulting amplified product as a probe to isolate a full-length cDNA clone. An isolation method is provided. According to a seventh aspect of the present invention, there is provided a recombinant vector containing the gene of the present invention.
  • the vector is an expression vector.
  • a transformant obtained by introducing the recombinant vector of the present invention into a host organism.
  • an antibacterial agent comprising the antibacterial protein of the present invention as an active ingredient.
  • the antibacterial protein and gene of the present invention were obtained from Matsutake, but are not particularly limited, and can be obtained from desired mushrooms belonging to ascomycetes and basidiomycetes. BRIEF DESCRIPTION OF THE FIGURES
  • FIG. 1 is a photograph showing the growth of the fungus pathogen 24 hours after the start of culture.
  • FIG. 2 is a photograph illustrating the inhibition of the growth of imochi disease fungus by Matsutake protein.
  • the protein concentration was 0.5 g of total extracted protein / m, and the result after 24 hours was shown.
  • FIG. 3 shows the relationship between the matsutake protein separation chart using the MonoQ column and the antibacterial activity.
  • FIG. 4 shows the relationship between the electrophoresis image of the separation of the anti-bacterial protein of Mattake using the MonoQ column and the antibacterial activity.
  • the numbers on the lanes correspond to the fraction numbers in FIG. 3, and M indicates the highest molecular weight.
  • the symbols (1, 10, ++, +++) below the lane indicate the strength of the antibacterial activity.
  • Antimicrobial proteins 50 kD and 15 kD are indicated by arrows.
  • FIG. 5 shows the relationship between the purification chart of Matsutake antibacterial protein by Superose6 and the antibacterial activity. Arrows indicate the elution positions of protein samples of various molecular weights of Gel Filtration Standard (manufactured by BIO-RAD).
  • FIG. 6 shows the relationship between the electrophoresis image of purification of Matsutake antibacterial protein by Superose 6 and the antibacterial activity.
  • the numbers on the lanes correspond to the fraction numbers in FIG. 5, m39 represents the 39th fraction of monoQ subjected to Superose 6 (see FIG. 4), and M represents the molecular weight marker.
  • the symbols (-1, +, + ten, +++) under the lane indicate the strength of the antibacterial activity.
  • Antimicrobial proteins 50 kD and 15 kD
  • FIG. 7 shows the results of Southern blot analysis of the gene encoding the Matsutake antibacterial protein against the Matsutake genome.
  • X indicates Xhol
  • S indicates Sail
  • P indicates Pstl
  • H indicates Hindin
  • E indicates EcoRI
  • B indicates BamHI.
  • FIG. 8 shows a restriction map of cDNA encoding the Matsutake antibacterial protein and a method of sequencing.
  • the bold line indicates cDNA, and AAAA... Indicates polyA. Restriction enzyme sites on cDNA are shown on the bold line (EcoRI and Xhol are sites for closing to vector). Each arrow indicates the direction and length of the region where the base sequence has been determined.
  • a mushroom-derived protein having an antibacterial effect on plant pathogenic bacteria.
  • the origin and production method of the protein of the present invention are not limited as long as it has the characteristics described herein. That is, the antibacterial protein of the present invention may be any of naturally occurring proteins, proteins expressed from recombinant DNA by genetic engineering techniques, or chemically synthesized proteins.
  • the antibacterial protein and gene of the present invention were obtained from Matsutake, but are not particularly limited, and can be obtained from desired mushrooms belonging to ascomycetes and basidiomycetes.
  • desired mushrooms belonging to ascomycetes and basidiomycetes for example, in addition to mushrooms, shitake mushrooms, enokitake mushrooms, benashimeji mushrooms, agaric mushrooms, moss mushrooms, tengu mushrooms, kunugita mushrooms, hirayake mushrooms, mushrooms, nameko mushrooms, naratake mushrooms, chanterelles, show mouths, etc.
  • it is Matsutake.
  • the protein of the present invention typically has 564 amino acid sequences as shown in SEQ ID NO: 2 in the sequence listing. However, some natural proteins produce one or more amino acids due to differences in varieties of the species producing them, mutations in genes due to differences in ecosystems, or the presence of similar isozymes. It is well known that mutated proteins having mutations exist. As used herein, the term “amino acid mutation” refers to substitution, deletion, insertion, and / or addition of one or more amino acids.
  • the protein of the present invention is a protein having the amino acid sequence of SEQ ID NO: 2 based on an estimate from the nucleotide sequence of the cloned gene. W
  • homologous proteins it is intended to include, but is not limited to, all homologous proteins as long as they have the properties described herein.
  • the homology is at least 5 °% or more, preferably 60% or more, more preferably 70% or more, further preferably 80% or more, particularly preferably 90% or more, and most preferably 95% or more. .
  • substitution of amino acids having similar properties for example, substitution of one hydrophobic amino acid for another hydrophobic amino acid, substitution of one hydrophilic amino acid for another hydrophilic amino acid, When a substitution of an amino acid with another acidic amino acid or a substitution of one basic amino acid with another basic amino acid), the resulting mutated protein often has the same properties as the original protein.
  • Techniques for producing recombinant proteins having such desired mutations using gene recombination techniques are well known to those skilled in the art, and such mutant proteins are also included in the scope of the present invention.
  • the protein of the present invention has a molecular weight of about 50 kD on SDS-PAGE (corresponding to a polypeptide consisting of the amino acid sequence from the 26th to the 435th amino acid in the sequence of SEQ ID NO: 2), and 15 kD.
  • D (equivalent to a polypeptide consisting of the 436th to 564th amino acid sequences), consisting of two polypeptides, whose molecular weight is estimated to be approximately 210 kD on a gel filtration column. Identified.
  • the polypeptide consisting of the 1st to 25th amino acid sequences of SEQ ID NO: 2 is also a subunit of this antibacterial protein.
  • An antimicrobial protein comprising:
  • the above-mentioned polypeptides include homologous polypeptides having a mutation as described above in the present specification.
  • the protein of the present invention can be purified from mushroom fruit bodies using, for example, ammonium sulfate precipitation method and ion-exchange column chromatography according to Examples described later, or the DNA sequence of SEQ ID NO: 1 in the sequence listing according to the present invention can be purified.
  • Purification and isolation of the protein of the present invention can be performed by using ammonium sulfate precipitation, ion exchange chromatography (MonoQ, Q Sepharose or DEAE, etc.), gel filtration column chromatography (Superose6 or Sephacryl S-200HR, etc.). The methods commonly used for purification and isolation can be combined appropriately.
  • finely divided mushrooms are extracted with a buffer solution, filtered, and the supernatant is brought to a suitable concentration of ammonium sulfate (ammonium sulfate), for example, 75% saturation. And the mixture is allowed to stand to obtain a precipitate containing the protein of the present invention.
  • the precipitate may be further dialyzed and then subjected to ion exchange chromatography, eluting with a salt concentration gradient (eg, 50 to 1 M with sodium chloride), and collecting the fraction containing the desired protein.
  • a salt concentration gradient eg, 50 to 1 M with sodium chloride
  • the present invention also provides a gene encoding the antibacterial protein of the present invention.
  • the type of the gene is not particularly limited, and may be any of a naturally occurring DNA, a recombinant DNA, and a chemically synthesized DNA, and may be any of a genomic DNA clone and a cDNA clone.
  • the gene of the present invention typically has the nucleotide sequence of SEQ ID NO: 1 in the Sequence Listing. This is only an example of the present invention, and is a salt of the clone obtained in the following Examples. It is a base sequence. It is important to note that some natural genes have different varieties of the species that produce them, as well as-a small number of mutations due to differences in ecosystems and a small number of mutations due to the presence of similar isozymes. It is well known to traders. Therefore, the gene of the present invention is not limited to only the gene having the nucleotide sequence of SEQ ID NO: 1 in the sequence listing, but includes all genes encoding the antimicrobial protein of the present invention.
  • the basic sequence of genetic engineering such as hybridization and nucleic acid amplification reaction can be performed using this sequence or a part thereof.
  • E. coli a gene encoding a protein having the same physiological activity can be easily isolated from other species. In such a case, such a gene is also included in the scope of the present invention.
  • Hybridization conditions used for screening of homologous genes are not particularly limited, but generally stringent conditions are preferable, for example, 6 XS SC, 5 X Denhardt's, 0.1% SDS 25 ° C or less. It is conceivable to use hybridization conditions such as 68 ° C. In this case, the temperature of the hybridization is more preferably 45 ° C to 68 ° C (without formamide) or 25 ° C to 50 ° C (50% formamide).
  • Nucleic acid amplification reactions include, for example, the replication chain reaction (PCR) (Saiki et al., 1985, Science 230, p. 1350-1354), the Liiges chain reaction (LCR) (Wu et al., 1989, Gen Acomics 4, p. 560—569; Balinger et al., 1990, Gene 89, p. 117-122; Barany et al., 1991, Proc. Natl. Acad. Sci. USA 88, p. 189-193) and transcription-based amplification (Ko et al., 1989, Proc. Nat 1. Acad. Sci. USA 86, p. 1173-11-1.
  • PCR replication chain reaction
  • LCR Liiges chain reaction
  • a strand displacement reaction (SDA) (Walker et al., 1992, Proc. Nat 1. Acad. Sci. USA 89, p. 392-396; Zokars, 1992, Nuc. Ac ids. Res. 20, p. 169 1—1 696), self-retained sequence replication (3 SR) (Guateri et al., 1990, Proc. Natl. Ac ad. S ci. USA 87, p. 1874—1 878) and the isothermal reaction of the Q3 replicaase system (Resaildi et al., 1988, Biotechnolgy 6, p. 1197–1202). including. Further, a nucleic acid sequence-based amplification (Nucleic Acid Sequence Based Amification: NAS ABA) reaction described in European Patent No. 0525882 can also be used. . Preferably, the PCR method is used.
  • the homologous gene to be cloned using the above-described hybridization, nucleic acid amplification reaction, etc. is at least 50% or more, preferably 60% or more with respect to the nucleotide sequence shown in SEQ ID NO: 1 in the sequence listing. , More preferably at least 70%, even more preferably at least 80%, particularly preferably at least 90%, most preferably at least 95%.
  • an oligonucleotide for obtaining a mushroom-derived antibacterial protein is also provided.
  • Each region is 15-30 bases in length
  • a mixture of single-stranded DNA is prepared in consideration of the above, and if necessary, the single-stranded DNA is modified so as not to lose the binding specificity to the nucleotide sequence of the gene encoding the antimicrobial protein.
  • the oligonucleotide of the present invention can be used for, for example, hybridization for detecting or isolating the gene of the present invention, or amplification reaction such as PCR using two suitable primers as a primer pair.
  • the oligonucleotide of the present invention preferably has the nucleotide sequence of any one of SEQ ID NOS: 5 to 9 in the sequence listing. This nucleotide sequence was designed based on the amino acid sequences of SEQ ID NOs: 3 and 4 as a primer for PCR for cloning of a gene fragment encoding each protein, and A primer in which all bases capable of encoding an acid are mixed.
  • the partial fragment of the gene of the present invention is isolated by amplifying the mushroom fruiting body cDNA library by using a suitable combination of the above oligonucleotides and performing a nucleic acid amplification reaction such as PCR using a type III library. can do.
  • a nucleic acid amplification reaction such as PCR using a type III library.
  • a full-length cDNA clone can be isolated.
  • Procedures and conditions for the nucleic acid amplification reaction, plaque hybridization conditions, and the like are well known to those skilled in the art.
  • a recombinant vector containing the gene of the present invention there is also provided a recombinant vector containing the gene of the present invention.
  • a method for incorporating a DNA fragment of the gene of the present invention into a vector such as a plasmid include, for example, "Sambrook, J. et al., Molecular Cloning, A Laboratory Manual, second edition), Cold Spring Harbor Laboratory, 1.53 (1989) J
  • a commercially available ligation kit for example, Takara Shuzo
  • the plasmid is introduced into a host cell (eg, E-coil TBI, LE392 or XL-lBlue, etc.).
  • Methods for introducing a plasmid into a host cell include the calcium chloride method and the chloride method described in “Sambrook, J. et al., Molecular Cloning, A Laboratory Manual, (second edition), Cold Spring Harbor Laboratory, 1.74 (1989)”. Calcium Z rubidium chloride method, electroporation method, electroinjection method, a method using a chemical treatment such as PEG, a method using a gene gun, and the like.
  • the vector can be conveniently prepared by ligating a desired gene to a recombination vector (for example, plasmid DNA or the like) available in the art in a conventional manner.
  • a recombination vector for example, plasmid DNA or the like
  • the vector used include plasmids derived from Escherichia coli such as pBluescript, pUC18, pUC19, and pBR322. It is not limited to these.
  • an expression vector is particularly useful.
  • the type of expression vector is not particularly limited as long as it has a function of expressing a desired gene in various host cells of prokaryotic cells and / or eukaryotic cells and producing a desired protein.
  • expression vectors for Escherichia coli pQE-30, pQE-60, pMAL-C2, pMAL-p2, pSE420 and the like are preferable.
  • yeast expression vectors pYES2 (Saccharomyces), pP I C3.5K, pP I C9K, ⁇ 0815 (above Pichia), pBacPAK8 / 9, pBK283 pVL1392, pBlueBac4.5 and the like as expression vectors for insects are preferred.
  • a transformant can be prepared by introducing a desired expression vector into a host cell.
  • the host cell to be used is not particularly limited as long as it is compatible with the expression vector of the present invention and can be transformed.
  • Natural host cells usually used in the technical field of the present invention or artificially established It is possible to use various cells such as the obtained recombinant cells.
  • bacteria Esscherichia spp., Bacillus spp.
  • Yeast Sacharomyces sp., Pichia spp., Etc.
  • animal cells insect cells, plant cells and the like can be mentioned.
  • the host cell is preferably Escherichia coli, yeast or insect cells, and specifically, Escherichia coli 15, JM109, BL21, etc., yeast (INVScl (Saccharomyces), GS115, M71 (Pichia), etc. ) And insect cells (BmN4, silkworm larvae, etc.).
  • animal cells include cells derived from mouse, African frog, rat, hamster, monkey or human, or cultured cell lines established from these cells.
  • plant cells are not particularly limited as long as cell culture is possible, and examples include cells derived from tobacco, arabidopsis, rice, corn, and wheat.
  • the expression vector When a bacterium, particularly Escherichia coli, is used as a host cell, the expression vector generally comprises at least a promoter region, an initiation codon, a gene encoding a desired antibacterial protein, a stop codon, a terminator, and a replicable unit.
  • the expression vector When yeast, plant cells, animal cells or insect cells are used as host cells, the expression vector generally includes at least a promoter, an initiation codon, and a desired antibacterial protein. Children who combine the gene encoding the protein, the stop codon, and the terminator are preferred. It may also contain a DNA encoding a signal peptide, an enhancer sequence, a 5′- and 3′-side untranslated region of a desired gene, a selectable marker region or a replicable unit, as appropriate.
  • a suitable initiation codon is methionine codon (A).
  • TG is exemplified.
  • stop codon a commonly used stop codon (eg, TA
  • a replicable unit refers to a DNA capable of replicating its entire DNA sequence in a host cell, and can be a natural plasmid, an artificially modified plasmid (prepared from a natural plasmid). Plasmids) and synthetic plasmids. Suitable plasmids include the plasmid pQE30, pET or pCAL or an artificially modified product thereof (a DNA fragment obtained by treating pQE30, pET or pCAL with an appropriate restriction enzyme) in E. coil, and the plasmid in yeast. PYES2 or PPIC9K, and in insect cells, plasmid pBacPAK8 / 9 and the like.
  • sequence of Enhancer and the sequence of Termine those commonly used by those skilled in the art, for example, those derived from SV40 can be used.
  • selection abilities commonly used ones can be used in a conventional manner. For example, tetracycline, ampicillin, or an antibiotic resistance gene such as kanamycin or neomycin, hygromycin, or spectinomycin is exemplified.
  • An expression vector is prepared by linking at least the above-mentioned promoter, initiation codon, gene encoding the desired antibacterial protein, stop codon, and the terminus overnight region to an appropriate replicable unit in a continuous and circular manner. Can be.
  • an appropriate DNA fragment for example, a linker, another restriction enzyme site, etc.
  • an appropriate DNA fragment can be used by a conventional method such as digestion with a restriction enzyme or a ligase using T4 DNA ligase.
  • the protein of the present invention has an extremely strong antibacterial activity.For example, germination of spores is extremely low at a very low concentration of 20 ng / m1 against rice blast fungus.
  • the protein of the present invention can be used as a drug such as an antibacterial agent or a pesticide, and as a preparation containing it in an active form.
  • the DNA sequence encoding the protein of the present invention is used, as described above, the DNA can be mass-produced by incorporating the DNA into an expression vector that functions in E. coli or yeast.
  • the antimicrobial protein of the present invention can be used for prevention and treatment of plant diseases involving fungi or bacteria. That is, according to the present invention, there is provided an antibacterial agent containing the antibacterial protein of the present invention as an active ingredient.
  • the antibacterial agent of the present invention can be usually applied systemically or locally on plants.
  • the amount of application depends on the type of plant, growth stage, symptom, application method, treatment time, application Types of proteins (full-length protein, substituting, deleting, inserting
  • a solution, a suspending agent, an emulsifier and the like can be mixed and sprayed as necessary.
  • one or more active substances are mixed as at least one inert diluent.
  • the aqueous diluent include distilled water and saline.
  • Non-aqueous diluents include, for example, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and alcohols such as ethanol.
  • Such antimicrobial compositions may also contain adjuvants such as preserving, wetting, emulsifying, dispersing, or stabilizing agents (eg, arginine, aspartic acid, and the like).
  • adjuvants such as preserving, wetting, emulsifying, dispersing, or stabilizing agents (eg, arginine, aspartic acid, and the like).
  • These may be sterilized by filtration through a bacteria-retaining filter, blending of a bactericide, or irradiation as necessary. They can also be produced in the form of a sterile solid composition by, for example, a freeze-drying method and dissolved in sterile distilled water or another solvent before use.
  • the dosage form of the antibacterial agent thus obtained may be determined according to the intended use, and is mixed with the above-mentioned additives, and is in the form of tablets, pills, powders, granules, liquids, emulsions, etc. Can be sprayed.
  • Rice sheath blight fungus (strain JT872) is cultured for 2 days in a 1/2 potato decoction medium (PD medium: Difco), and 3 hyphae masses of about 5 mm are mixed with a 1/2 PD medium using a Teflon homogenizer. The fragmented mycelium obtained by lightly grinding was used as an inoculum.
  • PD medium Difco
  • Teflon homogenizer Teflon homogenizer
  • the above inoculation source is about 1,000 blast conidia of Blast fungus and about 300 fragmented hyphae of Bacterial wilt fungus per 96-well micro-well plate (made by Koingu). Were added together with the IOO I 1 / 2PD medium, and cultured in a 28 ° C incubator. Bacterial growth was measured using a microplate reader (Bio-Rad Benchmark) for the absorbance of 595 bandages. The effects of salts and buffers on bacterial growth were determined by adding a certain amount of NaCl, phosphate buffer, Tris buffer, Hepes buffer, bovine serum albumin, dithiothreitol, etc. to the medium.
  • dialysis was performed using a benzoylated dialysis tube (manufactured by SIGMA) against 10 mM Hepes buffer, pH 7.5, and insoluble materials were removed by centrifugation to obtain a matsutake protein sample.
  • the protein concentration of the Matsutake protein sample was measured by the Bradford method using bovine serum albumin (BSA) as a standard protein.
  • BSA bovine serum albumin
  • the total growth inhibitory concentration against the fungus disease was estimated to be 0.7 g or less of total extracted protein / ml. It was found that the substance contained an extremely high antibacterial activity. When the concentration was high, germination was completely suppressed, and when the concentration was low, hyphal growth was inhibited. The degree of inhibition of hyphal elongation was clearly concentration-dependent. In addition, the cells of the fungus bacillus had a cytoplasm separated from the cell wall, and had a plasma-like appearance. Fig.
  • FIG. 1 shows the growth rate of the hyphae of the cultivars of the cultivars in the plot without matsutake protein, 24 hours after the start of the culture.
  • FIG. 2 shows the plots of the phytopathogens in the plot with 0.5 g / m1 of total matsutake protein added. It indicates the degree of hyphal growth 24 hours after the start of culture.
  • the antibacterial protein was purified.
  • 3.6 mg / 10 ml of the protein sample was applied to an ion exchange column MonoQ HR5 / 5 (Pharmacia) to partially purify the antibacterial protein.
  • the flow rate is 1 ml / min
  • the basic buffer is 50 mM Mes pH 6.0
  • 50 mM NaCK elution buffer is 50 mM Mes pH 6.0, 1 M NaCl
  • gradient 50 mM to 1 M with NaCl It took 40 minutes from 20 minutes after hitting the sample.
  • An aliquot of each fraction (1 ml) was subjected to SDS-PAGE electrophoresis and an antibacterial assay against imochi disease.
  • Figure 4 shows the relationship between the electrophoretic image and the strength of the antibacterial activity.
  • the numbers shown above each lane are the same as the numbers of the fractions in FIG. 3, and the strength of the antibacterial activity is also shown in accordance with FIG. A closer look at the protein bands that might be linked to antimicrobial activity identified two bands at approximately 50 kD and approximately 15 kD as potential candidates (arrows in Figure 4). Since the concentrations of these two bands and the degree of antimicrobial activity were positively correlated, it was strongly suggested that these bands might be the main antimicrobial proteins.
  • the amount of antibacterial protein was estimated from the molecular weight of the protein (67 kD albumin) by densitometry overnight, and the total growth inhibitory concentration against the fungus was calculated to be approximately 20 ng / ml.
  • MT50F1 (5′-gtigaigtrttrtcdatdatrtg-3 ′ (72)) (SEQ ID NO: 6)
  • MT15R1 (5'-acigayacitayggiatgcc-3 '(4)) (SEQ ID NO: 7),
  • MT15F-2 (5'-aaicciggigccatraaytg-3 '(4)) (SEQ ID NO: 9) was synthesized (where i is inosine, y is c or t, r is a or g, w is a or t, h is c or a or t, and d indicates g or a or t, respectively).
  • Matsutake mRNA was prepared therefrom using an mRNA purification kit (Pharmacia). About 5 to 10 g of mRNA was obtained from fruiting bodies. Of these, 5 g were subjected to a ZAP cDNA synthesis kit (Stratagene) to synthesize cDNA. The cDNA of 1 to 5 kb was fractionated by a gel filtration column, ligated to Uni-ZAP XR vector-1 (Stratagene), and packaged with Gigapack III (Stratagene). All procedures followed the instructions attached to the kit. The evening time of the constructed Matsutake cDNA library was calculated to be about 1 million pfu.
  • the reaction conditions were as follows. In the reaction mixture of 50, 60 ng of cDNA, 51 taq buffer solution 5, 1 dNTPs ⁇ 1, 10 pmoles I of each primer, Ex taq (Takara) + Taq START antibody (Clontech) 1 / il Using the program temperature control system PC-700 (A STEK), once at 94 ° C for 3 minutes, and then at 94 ° C for 1 minute, 50 ° C for 1 minute and 72 ° C for 2 minutes.
  • PC-700 program temperature control system
  • Total DNA was extracted from matsutake mushroom, 5 g of DNA was digested with 6 kinds of restriction enzymes, fractionated by agarose gel electrophoresis, and then alkaline blotted on Zeta-probe blotting membrane (BI-RAD). did.
  • the cloned RT-PCR product of 4) was RI-labeled with the Redi-prime labeling kit (Amarsham) and used as a probe for genomic Southern analysis. Hybridization and washing conditions were in accordance with the manufacturer's instructions. As a result, one band was detected in EcoRI and Xhol digestion, two bands were detected in BamHI and Pstl digestion, and three bands were detected in Hindlll digestion.
  • the clone obtained in 4) was excised from the vector, and this was used as a probe to screen the Matsutake cDNA library constructed in 3).
  • a phage of about 15,000 pfu was plated on a 14 ⁇ 10 cm square petri dish along with the host bacterial XL blue MRF 'according to the instructions of the ZAP cDNA synthesis kit (Stratagene).
  • Plastic The membrane was contacted with a Hybond-N + nylon membrane filter (manufactured by Amersham), alkali-treated according to the manufacturer's instructions to denature the DNA, and immobilized on the membrane. Hybridization and washing conditions were performed under conditions of high stringency according to the manufacturer's instructions for the Zeta-probe membrane.
  • the base sequence at the 5 'end was determined to be approximately 500 bp.
  • the obtained nucleotide sequence was analyzed using Genetyx ver. 9.0 analysis software (manufactured by Software Development). As a result, it was suggested that # 1 (2.0 kb) and 14 (1.9 kb) were derived from the same gene, and that # 5 (2.3 kb) was different from this group.
  • the clones # 1 and 14 contain a sequence very similar to the 31 amino acids determined in 1) (29 out of 31 amino acids are identical), and 3
  • the translation initiation codon ATG exists 25 amino acids upstream from the N-terminus of one amino acid.
  • the presence of the translation stop codon TAA 27 bp (same reading frame) upstream thereof strongly suggested that this ATG was the translation initiation site.
  • # 4 (1.8 kb) and 1 l (1.7 kb) have shorter cDNA insert sequences, and both clones retain part of the 50 kD 31 amino acids. The grouping was difficult because there was no area with the problem.
  • the cDNA encoding the mushroom-derived antibacterial protein of the present invention consisted of 1946 bases in total and encoded 564 amino acids (SEQ ID NOS: 1 and 2 in the sequence listing).
  • the molecular weight estimated from the amino acid sequence was calculated to be about 61.9 kD, and the isoelectric point was calculated to be 5.68.
  • two putative sugar chain addition sites were present (amino acids 53 to 56 in SEQ ID NO: 2 in the sequence listing, and Asn in 287 to 290 in SEQ ID NO: 2).
  • the amino acids at the N-terminus determined from the purified protein are as follows: the 50-kD N-terminal 32 amino acids are the amino acid sequence at positions 26 to 57 in the amino acid sequence of SEQ ID NO: 2 in the sequence listing; It corresponded to the 502nd amino acid sequence, respectively.
  • the molecular weights of the 26th to 435th amino acid sequences and the 436th to 564th amino acid sequences of the amino acid sequence of SEQ ID NO: 2 were calculated, each was approximately 4%.
  • the mushroom-derived antibacterial protein of the present invention is first translated as a precursor form, then the N-terminal leader sequence (25 amino acids) is cut off in an appropriate intracellular organ, and the amino acid number between 435 and 436 is reduced. It is presumed that the two polypeptides, approximately 50 kD and 15 kD, which are cleaved at the same time, associate with each other and exert antibacterial activity.
  • the cloned cDNA was derived from the gene encoding the mushroom-derived antibacterial protein of the present invention.
  • the amino acid sequence of the mushroom-derived antibacterial protein of the present invention and its gene DNA sequence were subjected to a homology search (BLAST) against a database (DD BJ). Pyranosoxidase has only 35% and 46% homology, respectively, when compared with the entire amino acid sequence and the entire DNA sequence, and there are other homologous sequences. Therefore, this protein is considered to be a novel protein.
  • SEQ ID NO: 2 in the sequence listing of the present invention or the entire sequence excluding the first to 25th sequences thereof, or the polypeptide and 436th amino acid sequence consisting of the 26th to 435th amino acid sequences
  • a pharmaceutical preparation containing as an active ingredient a protein component characterized by containing a polypeptide consisting of the amino acid sequence from the 1st to the 564th amino acid sequence, it can be expected to be used as a powerful antibacterial agent.
  • the DNA sequence is the 101st to 92nd sequence, or the 176th to 1792th sequence.
  • the DNA sequence is introduced into E. coli, yeast, insects, or certain animal cells using an expression vector that can be amplified in each host, and expressed. It is expected that it can be obtained.

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Abstract

L'invention concerne une nouvelle protéine antibactérienne capable d'inhiber la croissance de champignons pathogènes des plantes, tels que Pyricularia orizae ou Rhizoctonia solani (principaux champignons pathogènes du riz), et pouvant être détectée et identifiée à une concentration relativement faible. Le gène de cette protéine est également cloné. La protéine se caractérise en ce qu'elle obtenue à partir d'une fraction d'un extrait aqueux d'un champignon précipité selon une technique employant un sulfate d'ammonium; qu'elle a une activité antibactérienne au moins sur Pyricularia orizae ou Rhizoctonia solani; qu'elle présente un poids moléculaire d'environ 210 kD lorsqu'elle est déterminée selon la technique de la filtration en milieu gélifié; qu'elle révèle la présence de constituants d'environ 50 kD et d'environ 15 kD selon la technique de l'électrophorèse sur gel-SDS; qu'elle maintient ladite activité antibactérienne lorsqu'elle chauffée dans une solution aqueuse neutre à 60 °C pendant 10 minutes; et qu'elle perd cette activité antibactérienne lorsqu'elle chauffée dans une solution aqueuse neutre à 80 °C pendant 10 minutes.
PCT/JP1999/004441 1998-09-08 1999-08-19 Nouvelle proteine, gene codant cette proteine et utilisation de la proteine WO2000014242A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1369476A4 (fr) * 2001-03-12 2004-07-28 Japan Tobacco Inc Nouvelle proteine, gene codant ladite proteine et procede d'utilisation correspondant
US7525014B2 (en) 2000-09-07 2009-04-28 Japan Tobacco Inc. Disease-resistant plants and method of constructing the same

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH10313876A (ja) * 1997-02-13 1998-12-02 Natl Food Res Inst 抗腫瘍タンパク質およびその遺伝子

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH10313876A (ja) * 1997-02-13 1998-12-02 Natl Food Res Inst 抗腫瘍タンパク質およびその遺伝子

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7525014B2 (en) 2000-09-07 2009-04-28 Japan Tobacco Inc. Disease-resistant plants and method of constructing the same
US7851671B2 (en) 2000-09-07 2010-12-14 Japan Tobacco Inc. Disease-resistant plants and method of constructing the same
EP1369476A4 (fr) * 2001-03-12 2004-07-28 Japan Tobacco Inc Nouvelle proteine, gene codant ladite proteine et procede d'utilisation correspondant
AU2002236300B2 (en) * 2001-03-12 2007-10-11 Japan Tobacco Inc. Novel protein, gene encoding the same and method of using the same
EP1865059A1 (fr) * 2001-03-12 2007-12-12 Japan Tobacco, Inc. Nouvelle protéine, son codage génétique et son procédé d'utilisation
US7713531B2 (en) 2001-03-12 2010-05-11 Japan Tobacco, Inc. Protein, a gene encoding therefor and a method of using the same
US7776333B2 (en) 2001-03-12 2010-08-17 Japan Tobacco Inc. Protein, a genes encoding therefor and a method of using the same
US7855282B2 (en) 2001-03-12 2010-12-21 Japan Tobacco Inc. Protein, a gene encoding therefor and a method of using the same
US7989610B2 (en) 2001-03-12 2011-08-02 Japan Tobacco Inc Protein, a gene encoding therefor and a method of using the same

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