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WO2012063338A1 - Protéine à activité de chitinase et son utilisation - Google Patents

Protéine à activité de chitinase et son utilisation Download PDF

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Publication number
WO2012063338A1
WO2012063338A1 PCT/JP2010/070041 JP2010070041W WO2012063338A1 WO 2012063338 A1 WO2012063338 A1 WO 2012063338A1 JP 2010070041 W JP2010070041 W JP 2010070041W WO 2012063338 A1 WO2012063338 A1 WO 2012063338A1
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Prior art keywords
protein
present
polynucleotide
amino acid
activity
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English (en)
Japanese (ja)
Inventor
佐紀人 北島
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Kyoto Institute of Technology NUC
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Kyoto Institute of Technology NUC
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Priority to PCT/JP2010/070041 priority Critical patent/WO2012063338A1/fr
Publication of WO2012063338A1 publication Critical patent/WO2012063338A1/fr
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
    • A01N37/44—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids containing at least one carboxylic group or a thio analogue, or a derivative thereof, and a nitrogen atom attached to the same carbon skeleton by a single or double bond, this nitrogen atom not being a member of a derivative or of a thio analogue of a carboxylic group, e.g. amino-carboxylic acids
    • A01N37/46—N-acyl derivatives
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
    • A01N65/08—Magnoliopsida [dicotyledons]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C12N15/8286—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for insect 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
    • C12N9/2442—Chitinase (3.2.1.14)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01014—Chitinase (3.2.1.14)
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

  • the present invention relates to a protein having chitinase activity and use thereof.
  • GM plant Genetically modified plant
  • Bt toxin produced by a Gram-positive bacterium Bacillus thuringiensis
  • Such Bt toxins are known to exhibit insect-resistant activity including insecticidal properties at low concentrations (about 1 ppm) (see, for example, Non-Patent Document 1).
  • insect-resistant proteins other than Bt toxins is desired.
  • milky lotion is a milky white liquid that leaks when a plant is damaged, and is accumulated in the milk duct cells of the plant. It is believed that 20,000 species of 40 families have latex.
  • Non-Patent Document 2 describes three types of sugar-like alkaloids (1,4-dideoxy-1,4-imino-D-arabinitol (D-AB1)), 1- deoxynojirimycin (DNJ) and 1,4-dideoxy-1,4-imino-D-ribitol function as inhibitors of glycolytic and sugar-metabolizing enzymes, and as a result, erisan (a predatory lepidopterous insect of the scorpionidae) ) Larvae are shown to exhibit growth inhibitory activity.
  • D-AB1 sugar-like alkaloids
  • DNJ 1- deoxynojirimycin
  • Larvae a predatory lepidopterous insect of the scorpionidae
  • Patent Document 1 discloses that fraction 1 obtained by native PAGE from the emulsion of mulberry (variety: Shinichinoset) exhibits growth inhibitory activity against erysan hatched larvae, At least 56 kDa, 46 KDa, 30 KDa and 18 KDa are disclosed.
  • Non-Patent Document 3 further obtained by further purifying the protein contained in Fraction 1 that was shown to exhibit growth inhibitory activity against Erysan hatching larvae in Patent Document 1, using a DEAE Sepharose column. It is disclosed that one of the obtained proteins is named MLX56.
  • Japanese Patent Publication Japanese Patent Laid-Open No. 2008-245640 (Released Oct. 16, 2008)”
  • Non-Patent Document 2 Fraction 1 disclosed in Patent Document 1 and MLX56 disclosed in Non-Patent Document 3 have insecticidal activity, although they have growth inhibitory activity against insects. Does not have. For this reason, insect-resistant activity is insufficient.
  • sugar-like alkaloids are not proteins, they cannot be used for production of GM plants. Therefore, insect-resistant proteins having sufficient insect-resistant activity including insecticidal properties are desired.
  • Non-Patent Document 3 discloses that one of the obtained proteins is named MLX56. Looking at 2b lane 2, the protein obtained by purification on the DEAE Sepharose column is not a protein with a molecular weight of 56 kDa, but a protein with a molecular weight of 45 kDa, and there is another band with a slightly higher mobility. It is mixed. For this reason, Non-Patent Document 3 cannot prove that MLX56 has a growth inhibitory activity.
  • the present invention has been made in view of the above problems, and an object of the present invention is to provide a protein having sufficient insect-resistant activity including insecticidal properties and use thereof.
  • the present inventor isolated two kinds of novel proteins (LA-a protein and LA-b protein) from the soluble fraction of mulberry (Morisakari varieties) milk and performed functional analysis. As a result, it was found that these proteins have chitinase activity. Furthermore, surprisingly, when these proteins are fed to larvae as a feed, it has been found for the first time that they show remarkable insecticidal properties compared to the case where no protein is given, and the present invention is completed based on such new findings. It came to.
  • mulberry milk contains a protein having chitinase activity and insecticidal properties. Therefore, the present inventor found for the first time that the protein having chitinase activity and insecticidal properties is present in the mulberry emulsion, and is disclosed herein.
  • the protein according to the present invention is characterized by any of the following (a) to (d): (A) a protein comprising the amino acid sequence shown in SEQ ID NO: 1; (B) a protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted and / or added in the amino acid sequence shown in SEQ ID NO: 1 and having chitinase activity; (C) a protein consisting of the amino acid sequence shown in SEQ ID NO: 2; (D) A protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted and / or added in the amino acid sequence shown in SEQ ID NO: 2 and having chitinase activity.
  • the protein according to the present invention is a protein having chitinase activity and has sufficient insect resistance activity including insecticidal properties. That is, the protein according to the present invention is superior to conventional proteins in killing insects, weakening insects, or inhibiting insect growth. For this reason, the protein which concerns on this invention can be utilized suitably as an insecticide, an insecticide, an insect-proof bait, etc.
  • polynucleotide according to the present invention encodes the protein according to the present invention
  • a vector for expressing the protein according to the present invention can be prepared by using such a polynucleotide.
  • the vector according to the present invention is configured to contain the polynucleotide according to the present invention
  • a vector can be used in a host cell (eg, yeast, Escherichia coli, insect cell, plant cell, mammalian cell, etc.). By introducing, a transformant expressing the protein according to the present invention can be obtained.
  • the transformant according to the present invention expresses the protein according to the present invention as described above, it can be used to prepare the protein. Further, when such a transformant is a transformed plant, the transformed plant has insect resistance activity by expressing the protein according to the present invention. On the other hand, the work of spraying pesticides such as insecticides can be omitted.
  • insecticide according to the present invention has the protein according to the present invention, it is excellent in insect resistance activity.
  • the protein according to the present invention is a protein characterized by any of the following (a) to (d): (A) a protein comprising the amino acid sequence shown in SEQ ID NO: 1; (B) a protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted and / or added in the amino acid sequence shown in SEQ ID NO: 1 and having chitinase activity; (C) a protein consisting of the amino acid sequence shown in SEQ ID NO: 2; (D) A protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted and / or added in the amino acid sequence shown in SEQ ID NO: 2 and having chitinase activity.
  • the protein consisting of the amino acid sequence shown in SEQ ID NO: 1 corresponds to the “LA-a2 protein” shown in the examples described later, and the protein consisting of the amino acid sequence shown in SEQ ID NO: 2 is referred to as “LA in the examples described later”.
  • -B protein ".
  • the above “one or several amino acids are deleted, substituted and / or added” means that the amino acid sequence of the protein before one or several amino acids are deleted, substituted and / or added. 1 or a number such that it has an identity of at least greater than 83%, preferably greater than at least 92%, more preferably greater than at least 95%, most preferably greater than at least 97%. Means that one amino acid has been deleted, substituted and / or added.
  • the site where one or several amino acids are deleted, substituted and / or added is defined in the amino acid sequence if the protein after the deletion, substitution and / or addition of amino acids has chitinase activity. Any site may be used.
  • amino acid sequences can be determined using analysis software Genetyx manufactured by Genetics Co., Ltd.
  • analysis software Genetyx manufactured by Genetics Co., Ltd.
  • Such software is software that uses the program “FASTA” according to the Lipman-Pearson method (Lipman DJ, Pearson WR. Rapid, and sensitive sensitive protein, similarity, searches, Science, 1985, Mar 22, 227 (4693): 1435-41.).
  • Such a mutant protein is not limited to a protein having a mutation artificially introduced by a known mutant polypeptide production method, and may be a protein obtained by isolating and purifying a naturally occurring protein. It is well known in the art that some amino acids in the amino acid sequence of a protein can be easily modified without significantly affecting the structure or function of the protein. Furthermore, it is also well known that there are variants that not only artificially modify, but also do not significantly alter the structure or function of the protein in the native protein.
  • Preferred variants have conservative or non-conservative substitutions, deletions and / or additions.
  • Preferred mutations are silent substitutions, deletions and additions, and particularly preferred are conservative substitutions. These mutations do not change the chitinase activity of the protein according to the invention.
  • conservative substitutions are substitutions of one amino acid for another in the aliphatic amino acids Ala, Val, Leu, and Ile, exchange of hydroxyl residues Ser and Thr, acidic residues Asp and Glu exchange, substitution between amide residues Asn and Gln, exchange of basic residues Lys and Arg, and substitution between aromatic residues Phe, Tyr.
  • the above protein (b) includes “LA-a1 protein” (SEQ ID NO: 3) shown in the Examples described later.
  • the “LA-a1 protein” has 10 amino acid residues substituted, 2 amino acid residues added (inserted), and 9 amino acid residues deleted in the amino acid sequence shown in SEQ ID NO: 1. Protein.
  • the amino acid sequence identity between “LA-a2 protein” and “LA-a1 protein” is 94%.
  • the protein according to the present invention has, for example, an intermolecular and / or intramolecular crosslink (for example, disulfide bond), chemical modification (for example, sugar chain, phosphate, or other functional group).
  • an intermolecular and / or intramolecular crosslink for example, disulfide bond
  • chemical modification for example, sugar chain, phosphate, or other functional group.
  • the present invention is not particularly limited to these, and those to which a label (for example, a histidine tag or the like) or a fusion protein (for example, streptavidin, cytochrome, GFP, or the like) has been added.
  • the protein according to the present invention exhibits chitinase activity.
  • the background chitinase activity measured under the same measurement conditions except that the measurement target protein does not contain the measurement target protein, as measured by a conventionally known chitinase activity measurement method. If it is significantly higher than, the protein to be measured can be said to have “chitinase activity”.
  • the risk rate is less than 5%, preferably dangerous. It is intended that there is a significant difference at a rate of less than 1%, more preferably at a risk rate of less than 0.5%.
  • the protein to be measured when statistical analysis is performed using the tukey test, between the chitinase activity of the protein to be measured and the background chitinase activity measured under the same measurement conditions except that the reaction solution does not contain the protein to be measured.
  • the risk rate is less than 5%, preferably less than 1%, more preferably less than 0.5%, the protein to be measured can be said to have “chitinase activity”.
  • the chitinase activity of the protein (a) is 100%
  • the chitinase activity of the protein (b) under the same measurement conditions as the protein (a) is 10% or more, preferably 15 % Or more, more preferably 25% or more.
  • the protein (c) has a chitinase activity of 100%
  • the protein (d) has a chitinase activity of 10% or more, preferably 15%, under the same measurement conditions as the protein (c). More preferably, it may be 25% or more.
  • Examples of the “chitinase activity measuring method” include the methods described in the “chitinase activity measuring method” section of Examples described later, but the present invention is not limited thereto.
  • the protein according to the present invention has insect resistance activity.
  • insect resistance activity refers to an activity that kills insects (hereinafter referred to as “insecticidal”), an activity that weakens insects, or an activity that inhibits insect growth (hereinafter referred to as “growth inhibitory activity”).
  • growth inhibitory activity an activity that inhibits insect growth
  • a bait containing a protein according to the present invention is given to an insect larvae, and the insect that ingests the bait is significantly killed or weakened compared to the control, or If the growth of the insect that ingested the food is significantly inhibited (delayed) as compared with the control, it can be determined that the protein according to the present invention has insect-resistant activity.
  • the “control” means an insect (or a group of insects) fed with the same conditions except that the protein according to the present invention is not contained in the food.
  • the contact with the protein according to the present invention or ingestion of the protein according to the present invention results in hydrolysis of the chitin on the body of the insect or digestive sputum, resulting in resistance to resistance. It is thought to show worm activity.
  • insect-resistant activity As an insect in which the protein according to the present invention exhibits insect-resistant activity, it is killed, weakened, or inhibited by growth (delay) by contacting the protein according to the present invention or ingesting the protein according to the present invention. If it is a bug to be done, it will not be specifically limited. Examples of such insects include arthropods such as Coleoptera, Lepidoptera, Diptera, Hymenoptera, Hemiptera, Diptera, Lepidoptera, etc., ticks, etc. .
  • the protein according to the present invention has insect-resistant activity including insecticidal properties, it can be suitably used as an insecticide, insecticide, insect-resistant bait, and the like.
  • insect-resistant bait means a bait that exhibits insect-resistant activity when ingested by insects. That is, since the insect that ingested the insect-resistant bait containing the protein according to the present invention is inhibited (delayed) or killed or weakened, the insect can be easily exterminated.
  • the protein according to the present invention may further have chitosanase activity.
  • the background chitosanase activity measured under the same measurement conditions except that the measurement target protein does not contain the measurement target protein, as measured by a conventionally known chitosanase activity measurement method. If it is significantly higher than the above, the protein to be measured can be said to have “chitosanase activity”.
  • the chitosanase activity of the protein of (a) is 100%
  • the chitosanase activity of the protein of (b) under the same measurement conditions as the protein of (a) is 10% or more, preferably 15 % Or more, more preferably 25% or more.
  • the chitosanase activity of the protein (c) is 100%
  • the chitosanase activity of the protein (d) under the same measurement conditions as the protein (c) is 10% or more, preferably 15% More preferably, it may be 25% or more.
  • chitosanase activity measuring method examples include the method described in the section “Method for measuring chitosanase activity” in Examples described later, but the present invention is not limited thereto.
  • the protein according to the present invention may be chemically synthesized using an amino acid synthesizer or the like, may be produced using a gene recombination technique, or is a product obtained by isolating and purifying a naturally occurring protein. May be.
  • the protein when a protein is chemically synthesized using an amino acid synthesizer or the like, the protein can be chemically synthesized by a conventionally known peptide synthesis method (for example, solid phase synthesis method, liquid phase synthesis method).
  • a conventionally known peptide synthesis method for example, solid phase synthesis method, liquid phase synthesis method.
  • the recombinant protein expression system is not particularly limited as long as the protein according to the present invention can be expressed.
  • conventionally known E. coli expression systems, insect cell expression systems, plant cell expression systems, mammalian cell expression systems, cell-free expression systems, and the like can be suitably used.
  • the purification method is not particularly limited.
  • it can be purified from a soluble fraction of plant emulsion.
  • the soluble fraction of plant emulsion can be obtained, for example, by centrifuging the emulsion collected from the plant and collecting the supernatant.
  • means for purifying the protein according to the present invention from the obtained soluble fraction include ion exchange chromatography, hydrophobic chromatography, gel filtration chromatography and the like. These purification means may be performed alone or in combination. When combining a plurality, the combination order is not particularly limited.
  • the protein according to the present invention can be isolated and purified from plant milk by a method including at least a step of subjecting a soluble fraction of plant milk to hydrophobic chromatography.
  • the proteins (a) to (d) can be purified with high purity.
  • the conditions for hydrophobic chromatography can be appropriately set by those skilled in the art according to the type of column used, the type of mobile phase, the flow rate, the amount of sample applied, and the like. For example, as shown in the examples described later, using a HiLoad 16/10 phenyl sepharose HP column (16 mm id ⁇ 100 mm; GE healthcare, Piscataway, NJ, USA), 30% saturated ammonium sulfate to 0% saturated ammonium sulfate The protein may be eluted at a flow rate of 2 ml / min with a linear concentration gradient.
  • the purification method preferably further includes a step of subjecting the sample after the step of subjecting the soluble fraction of the plant emulsion to hydrophobic chromatography, and subjecting the sample to cation exchange chromatography. If it is the said structure, especially the protein of said (e) or (f) can be refine
  • a flow rate is measured by using a linear concentration gradient of 0 to 0.5 M NaCl in a buffer (10 mM calcium phosphate, 1 mM EDTA, pH 6.0) using a UNOS1 cation exchange column (Bio-Rad). It may be eluted at 3.0 ml / min.
  • the protein according to the present invention is preferably a protein derived from a plant latex. If it is derived from a plant emulsion, a protein having a plant-derived sugar chain added thereto can be obtained.
  • the “plant” is not particularly limited as long as it is a plant having an emulsion. Specific examples of such plants include, for example, moraceae plants, asteraceae plants, asteraceae plants, convolvulaceae plants, euphorbiaceae plants, potato family plants, oleander plant plants, cinnamon family plants, poppy family plants, Examples include urushiaceae plants, hypericaceae plants, legumes, cactaceae plants, and liliaceae plants. Among these plants, it is particularly preferable that the plant is a mulberry plant.
  • the polynucleotide according to the present invention is a polynucleotide encoding the protein according to the present invention.
  • the polynucleotide may exist in the form of DNA (for example, cDNA or genomic DNA) or RNA (for example, mRNA).
  • DNA or RNA may be double-stranded or single-stranded.
  • Single-stranded DNA or RNA may be a coding strand (sense strand) or a non-coding strand (antisense strand).
  • polynucleotide according to the present invention may be chemically synthesized, and the codon usage may be changed so that expression of the encoded protein is improved.
  • a method for modifying a polynucleotide according to the present invention a commonly used method for modifying a polynucleotide is used. That is, a polynucleotide having genetic information of a recombinant protein may be prepared by substituting, deleting, and / or adding a specific base of a polynucleotide having protein genetic information.
  • a specific method for converting the base of the polynucleotide for example, use of a polymerase chain reaction method (PCR method) can be mentioned. These methods are known to those skilled in the art.
  • the nucleotide sequence of the polynucleotide according to the present invention is not particularly limited as long as it encodes the protein according to the present invention. Therefore, the present invention includes all polynucleotides having a base sequence corresponding to the amino acid sequence of the protein according to the present invention.
  • polynucleotide may be any of the following polynucleotides (I) to (IV): (I) a polynucleotide having the base sequence represented by SEQ ID NO: 4; (II) a polynucleotide that hybridizes with a polynucleotide having the base sequence shown in SEQ ID NO: 4 under stringent conditions and encodes a protein having chitinase activity; (III) a polynucleotide having the base sequence represented by SEQ ID NO: 5; (IV) A polynucleotide that hybridizes with a polynucleotide having the base sequence shown in SEQ ID NO: 5 under stringent conditions and encodes a protein having chitinase activity.
  • the polynucleotide having the base sequence shown in SEQ ID NO: 4 is a polynucleotide encoding the protein having the amino acid sequence shown in SEQ ID NO: 1, ie, “LA-a2 protein” shown in the Examples described later.
  • the polynucleotide having the base sequence shown in 5 is a polynucleotide encoding the protein having the amino acid sequence shown in SEQ ID NO: 2, that is, the “LA-b protein” shown in the Examples described later.
  • the polynucleotide according to the present invention may be substituted with a chemically synthesized base.
  • part by which the polynucleotide based on this invention is substituted is not specifically limited,
  • such a polynucleotide includes a polynucleotide (SEQ ID NO: 6) encoding “LA-a1 protein” shown in Examples described later.
  • the polynucleotide according to the present invention includes a polynucleotide encoding the protein according to the present invention (for example, a polynucleotide having the base sequence shown in SEQ ID NO: 4 or 5), or a polynucleotide comprising a base sequence complementary thereto.
  • a polynucleotide that hybridizes with a nucleotide under stringent conditions and encodes a protein having chitinase activity is also included.
  • stringent conditions means that only when at least 90% identity, preferably at least 95% identity, most preferably at least 97% identity exists between sequences. It means that hybridization occurs. Specifically, for example, hybridization solution (50% formamide, 5 ⁇ SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5 ⁇ Denhart solution, 10% sulfuric acid The conditions include washing the filter in 0.1 ⁇ SSC at about 65 ° C. after overnight incubation in dextran and 20 ⁇ g / ml denatured sheared salmon sperm DNA).
  • Hybridization can be performed by a known method such as Sambrook et al. (Molecular Cloning, A Laboratory Manual, 3rd Ed, Cold Spring Harbor Laboratory (2001)). Usually, the higher the temperature and the lower the salt concentration, the higher the stringency (harder to hybridize), and a polynucleotide with higher homology can be obtained.
  • the identity of the base sequence can be determined using analysis software Genetyx manufactured by Genetics Co., Ltd.
  • the nucleotide sequence of the polynucleotide according to the present invention can be determined by a conventionally known method.
  • the polynucleotide according to the present invention may be composed only of the polynucleotide encoding the protein according to the present invention, but other base sequences may be added thereto.
  • the base sequence to be added is not limited, but includes a label (for example, histidine tag, Myc tag, and FLAG tag), a fusion protein (for example, GST and MBP), a promoter sequence (for example, yeast-derived promoter sequence, phage) Derived promoter sequences, E. coli-derived promoter sequences, etc.), and base sequences encoding signal sequences (eg, endoplasmic reticulum translocation signal sequences, secretory sequences, etc.).
  • the site to which these base sequences are added is not particularly limited, and may be the N-terminus or the C-terminus of the translated protein.
  • the vector according to the present invention includes the polynucleotide according to the present invention. Since the “polynucleotide according to the present invention” is as described in the above-mentioned section “2. Polynucleotide according to the present invention”, the description thereof is omitted here.
  • the vector according to the present invention includes the polynucleotide according to the present invention
  • other configurations are not particularly limited.
  • Various conventionally known vectors can be used as a base vector constituting the vector according to the present invention.
  • a plasmid, phage, cosmid or the like can be used, and a suitable vector can be appropriately selected depending on the host cell into which the vector is introduced and the introduction method.
  • plasmid vector for example, pBR322, pBR325, pUC19, pUC119, pBluescript, pBluescriptSK, pBI vectors, and the like can be used.
  • the fragment of the polynucleotide cleaved using a restriction enzyme treatment or the like and the base vector are cleaved with a restriction enzyme. It can be constructed by binding and closing the linear polynucleotide obtained above. When closing the binding, DNA ligase or the like can be used depending on the nature of the vector and the polynucleotide.
  • the vector according to the present invention After the vector according to the present invention is introduced into a host cell, a transformant containing the polynucleotide according to the present invention can be obtained by screening using the expression of the marker gene of the vector as an index. Therefore, the vector according to the present invention preferably contains a marker gene such as a drug resistance gene. Specific examples of such drug resistance genes include drug resistance genes for hygromycin, bleomycin, kanamycin, gentamicin, chloramphenicol and the like. Thereby, transformed host cells can be easily selected by selecting cells that grow in a medium containing the antibiotic.
  • the vector according to the present invention may further have a promoter for expressing the polynucleotide according to the present invention in a host cell.
  • the promoter is not particularly limited as long as it is a promoter capable of expressing the polynucleotide according to the present invention in a host cell, and a known promoter can be preferably used.
  • the promoter is not particularly limited as long as the promoter is linked so as to express the polynucleotide of the present invention and introduced into the vector.
  • the vector according to the present invention may further contain other DNA segments in addition to the promoter, the marker gene, and the polynucleotide described above.
  • the other DNA segment is not particularly limited, and examples thereof include a terminator, an enhancer, and a base sequence for improving translation efficiency.
  • the vector amplification method (production method) according to the present invention is not particularly limited, and a conventionally known method can be used.
  • a vector can be amplified in E. coli using E. coli as a host cell.
  • a preferred E. coli type can be selected according to the type of vector.
  • the present invention includes a transformant transformed with the vector according to the present invention.
  • the host cell transformed with the vector according to the present invention is not particularly limited.
  • yeast, Escherichia coli, insect cells, plant cells, mammalian cells and the like can be used.
  • the method for introducing the vector according to the present invention into the host cell is not particularly limited.
  • the host cell is a microorganism belonging to the genus Escherichia
  • the recombinant DNA is introduced in the presence of calcium ions.
  • a conventionally known method such as a method of using an electroporation method or the like can be applied.
  • the host cell is a plant cell
  • conventionally known methods such as the Agrobacterium method, the particle gun method, the polyethylene glycol method, and the electroporation method can be applied.
  • the vector according to the present invention has been introduced into the host cell can be confirmed by a conventionally known PCR method, Southern hybridization method, Northern hybridization method or the like. It can also be confirmed by measuring the expression of the protein encoded by the polynucleotide contained in the vector according to the present invention by a conventionally known immunological technique. It can also be confirmed by measuring the enzyme activity (for example, chitinase activity, chitosanase activity) exhibited by the protein encoded by the polynucleotide by a conventionally known biochemical technique.
  • a drug resistance marker gene such as kanamycin resistance or hygromycin resistance is introduced into the vector according to the present invention, a transformed host is selected by selecting cells that grow in a medium containing the antibiotic. Cells can be easily sorted.
  • the transformant thus obtained expresses the protein according to the present invention, it can be used to prepare the protein.
  • the transformant of the present invention includes a transformed plant regenerated from the transformed cell.
  • Regeneration of plant bodies from transformed cells can be performed by methods known to those skilled in the art depending on the type of plant cells. Since the obtained transformed plant body expresses the protein according to the present invention, it can be a plant body to which insecticidal activity is imparted. Therefore, it is possible to omit the work of spraying agricultural chemicals such as insecticides on such transformed plants.
  • offspring can be obtained from the transformed plant by sexual reproduction or asexual reproduction. It is also possible to obtain propagation materials (for example, seeds, protoplasts, etc.) from the transformed plant body, its progeny or clones, and mass-produce the desired transformed plant body based on them.
  • propagation materials for example, seeds, protoplasts, etc.
  • the plant used for transformation is not particularly limited, and for example, various monocotyledonous plants, dicotyledonous plants and the like can be used.
  • examples of monocotyledonous plants include gramineous plants and liliaceous plants.
  • examples of the dicotyledonous plants include mulberry plants, cruciferous plants, legumes, eggplants, cucurbits, convolvulaceae, roses, mallows.
  • insecticide according to the present invention only needs to contain the protein according to the present invention as an active ingredient.
  • the “protein according to the present invention” is as described in the section “1. Protein according to the present invention”, and therefore, the description thereof is omitted here. Since the protein according to the present invention has excellent insecticidal properties, an insecticide containing such a protein as an active ingredient can be an insecticide having excellent insecticidal properties.
  • insect-proofing agent intends a composition for killing insects, weakening insects, or inhibiting insect growth.
  • the above-mentioned “killing insects, weakening insects, or inhibiting insect growth” may be expressed as “extermination of insects”.
  • the “insect” is as described in the section “1. Protein according to the present invention”, and the description thereof is omitted here.
  • the insecticide according to the present invention contains the protein according to the present invention as an active ingredient, the content thereof is not particularly limited, and the content of the protein according to the present invention differs depending on the target insect,
  • the content of the protein according to the present invention may be 0.01% by weight or more, preferably 0.05% by weight or more, with respect to the total weight of the insect repellent according to the present invention.
  • the content is more preferably 0.1% by weight or more, further preferably 0.2% by weight or more, and most preferably 0.5% by weight or more.
  • sufficient insect resistance activity is exhibited even in a small amount.
  • insecticide according to the present invention may appropriately contain components other than the protein according to the present invention that do not inhibit the effect of the protein according to the present invention, if necessary.
  • stabilizers, thickeners, extenders, binders and the like that are commonly used in this technical field may be further added.
  • the form of the insecticide according to the present invention is not particularly limited, and may be used in the form of liquid, powder, granule, tablet or the like.
  • the insecticide according to the present invention can be applied to a variety of monocotyledonous plants, dicotyledonous plants and other general plants in order to combat insects.
  • monocotyledonous plants include gramineous plants and liliaceous plants.
  • dicotyledonous plants include mulberry plants, cruciferous plants, legumes, eggplants, cucurbits, convolvulaceae, roses, mallows.
  • the insecticide can be read as a method for controlling insects using the protein according to the present invention, or the use of the protein according to the present invention for controlling insects.
  • the protein according to the present invention is preferably a protein derived from plant milk.
  • the plant is preferably a mulberry plant.
  • the polynucleotide according to the present invention is a polynucleotide encoding the protein according to the present invention.
  • the polynucleotide according to the present invention is characterized by any of the following (I) to (IV): (I) a polynucleotide having the base sequence represented by SEQ ID NO: 4; (II) a polynucleotide that hybridizes with a polynucleotide having the base sequence shown in SEQ ID NO: 4 under stringent conditions and encodes a protein having chitinase activity; (III) a polynucleotide having the base sequence represented by SEQ ID NO: 5; (IV) A polynucleotide that hybridizes with a polynucleotide having the base sequence shown in SEQ ID NO: 5 under stringent conditions and encodes a protein having chitinase activity.
  • the vector according to the present invention is characterized by including the polynucleotide according to the present invention.
  • the transformant according to the present invention is characterized by being transformed with the vector according to the present invention.
  • the insecticide according to the present invention is characterized by containing the protein according to the present invention.
  • Example 1 Isolation and purification of novel protein and functional analysis
  • Two novel proteins were isolated and purified from the soluble fraction of mulberry milk, and the functions of these novel proteins were analyzed.
  • the solution was dialyzed overnight against buffer B using a dialysis membrane with a cutoff molecular weight of 25 kDa (Spectra / Por 7; Spectrum Laboratories Inc., Collinso Dominguez, CA, USA).
  • the sample after dialysis was diluted to 50 ml.
  • FIG. 1 shows the result of analyzing the soluble fraction of mulberry milk (corresponding to 0.025 ⁇ l of mulberry milk (stock solution)) by the SDS-PAGE method.
  • the soluble fraction of mulberry milk was rich in two proteins with molecular weights of approximately 50 kDa and 46 kDa. These two kinds of proteins were named “LA (Latex-abundant) proteins”.
  • LA-a protein A protein having a molecular weight of approximately 50 kDa
  • LA-b protein a protein having a molecular weight of approximately 46 kDa
  • CM-CellulofineuloC-200 Seikagaku Kogyo KK, Tokyo, Japan; 26 mm id ⁇ 100 mm
  • the protein fraction was obtained.
  • the fraction containing LA-b protein after hydrophobic chromatography was further subjected to gel filtration chromatography using a PD10 column (GE healthcare) and 10DG column (Bio-Rad, Hercules, CA, USA). Repeatedly desalted and replaced with buffer B.
  • the sample after gel filtration chromatography was further subjected to cation chromatography to obtain a purified preparation of LA-b protein.
  • cation chromatography a UNOS1 cation exchange column (Bio-Rad) was used, and elution was performed at a flow rate of 3.0 ml / min with a linear concentration gradient of 0 to 0.5 M NaCl in buffer B.
  • a chromatogram of the result of cation exchange chromatography is shown in FIG.
  • LA-a protein and 1.1 mg LA-b protein could be purified from 3 ml mulberry milk, respectively.
  • the purity of the obtained LA-a protein and LA-b protein was analyzed by SDS-PAGE.
  • the protein was detected by silver staining.
  • FIG. 3 since only a single band was detected by the SDS-PAGE method, it was confirmed that the LA-a protein and the LA-b protein could be purified with high purity. did.
  • the purified preparations of LA-a protein and LA-b protein were concentrated to a concentration of 1.0 mg / ml using Microcon YM-10 (Millipore Corp., Bedford, MA, USA), respectively. It was frozen using liquid nitrogen and stored at ⁇ 80 ° C.
  • protein samples were separated by SDS-PAGE and electrically transferred to PVDF membrane using a Kyhse-Anderson buffer system (Kyhse-Andersen J: J Biochem Biophys Methods 1984, 10: 203- 209).
  • the protein-transferred membrane was reacted with an anti-LA-a polyclonal antibody, and then reacted with an anti-rat antibody conjugated with alkaline phosphatase (Coligan JE, Dunn BM, Ploegh HL, Speicher DW, Wingfield PT: Current Protocols in Protein Science John Wiley & Sons 1995).
  • the film was then developed with a mixture of 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and nitroblue tetrazolium (NBT).
  • BCIP 5-bromo-4-chloro-3-indolyl phosphate
  • NBT nitroblue tetrazolium
  • Figure 4 shows the results of Western blotting. As shown in FIG. 4, the anti-LA-a protein antibody recognized not only LA-a protein but also LA-b protein. This result indicates that LA-a protein and LA-b protein are similar proteins, although the molecular weights are different from each other.
  • the LA-a protein and the LA-b protein have similar amino acid sequences on the N-terminal side and both have a hevein domain.
  • LA-a protein and LA-b protein (3 ⁇ g per lane) were separated by SDS-PAGE.
  • the gel was stained with Pro-Q Emerald 300 (Invitrogen, Carlsbad, CA, USA), and then SYPRO Ruby stain ( Invitrogen, Carlsbad, CA, USA) was used to stain the gel.
  • SYPRO Ruby stain Invitrogen, Carlsbad, CA, USA
  • lectin blotting protein samples were separated by SDS-PAGE and electrically transferred to a PVDF membrane.
  • the protein-transferred membrane was reacted with biotinylated RCA120 (Vector (Laboratories, Burlingame, CA, USA), and then reacted with streptavidin to which alkaline phosphatase was bound.
  • the film was developed using a mixture of BCIP and NBT. Similar experiments were performed with other lectins (ConA, DBA, LCA, PHA-E4, PNA, UEQ-I, WGA, ABA, DSA, Lotus, MAM, PHA-L4, SBA, and SSA).
  • lectins other than RCA120 were purchased from Seikagaku Corporation.
  • LA-a protein and LA-b protein are glycoproteins.
  • CM-chitin-RBV is a product of the chitin modified carboxymethyl (CM) chitin (which is colloidalized by chemical modification since it is insoluble) and labeled with RBV (a dye having absorption at 550 nm).
  • RBV a dye having absorption at 550 nm.
  • the amount of RBV solubilized is regarded as chitinolytic activity.
  • Soluble chitosan (Chitosan, low molecular weight; Sigma-Aldrich, MO, USA) as a substrate according to the method of Osswald et al. (See Anal Biochem 1992, 204: 40-46) except that the enzymatic reaction was extended from 30 minutes to 2 hours. ) was used to perform a chitosanase assay by fluorescence analysis.
  • As a buffer 0.1 M citrate-Na 2 HPO 4 buffer (pH 3 to 7) was used.
  • the “soluble chitosan” is a compound solubilized by deacetylating insoluble chitin.
  • FIG. 6 is a diagram showing the results of measuring the chitinase activity and chitosanase activity of LA-a protein and LA-b protein, (a) showing the measurement result of chitinase activity, and (b) showing the measurement result of chitosanase activity. Represents.
  • the LA-a protein and LA-b protein had chitinase activity.
  • the chitinase activity of LA-a protein and LA-b protein showed the maximum value at pH 5.
  • LA-a protein and LA-b protein had chitosanase activity.
  • the chitosanase activity of LA-a protein and LA-b protein showed the maximum value at pH 5.
  • Example 2 Measurement of insecticidal properties
  • Drosophila melanogaster (Canton-S strain) was bred at 25 ° C. on a standard yeast agar medium.
  • To test the insecticidal properties of LA-a and LA-b proteins 15 second-instar larvae were placed in a vial containing 200 mg of Formula 4-24 instant Drosophila medium (Carolina Biological Supply, Burlington NC, USA). The rats were fed with 0.8 ml of PBS containing 0.1% (w / w) LA protein and 10 mg of Bacto Yeast Extract (Difco, Detroit, MI, USA) as a nitrogen source for 6 days.
  • Formula 4-24 instant Drosophila medium Carolina Biological Supply, Burlington NC, USA
  • control group is given the same diet as that described above except that it contains the same amount of bovine serum albumin (BSA) instead of the LA protein, or the control group described above except that it does not contain the protein instead of the LA protein.
  • BSA bovine serum albumin
  • the same bait was given.
  • the vial was incubated at 25 ° C. For each test group, three vials were prepared for the experiment.
  • LA-a protein administration group 0.1% (w / w) containing LA-a protein
  • LA-b protein administration group containing 0.1% (w / w) LA-b protein
  • Control group 1 Contains 0.1% (w / w) BSA instead of LA protein.
  • Control group 2 Does not contain protein instead of LA protein.
  • FIG. 7 is a diagram showing the influence of LA-a protein and LA-b protein on the development of Drosophila.
  • A shows the number of Drosophila pupae given LA-a protein and the like, and 15 larvae LA-a protein and the like were given to the animals, and how many of them became pupae after 6 days.
  • B shows life and death and developmental stage of Drosophila 6 days after feeding LA-a protein, etc., feeding 15 larvae, how many became pupae 6 days later, Or whether they died.
  • the values shown in the graphs of (a) and (b) of FIG. 7 are shown as the average value ⁇ standard deviation of three vials.
  • “No protein” represents the control group 2
  • BSA represents the control group 1
  • LA-a represents the LA-a protein administration group
  • LA-b It represents the LA-b protein administration group.
  • control group 1 or control group 2
  • LA-a protein in any of pupa Pupa
  • living larvae Larva (live)
  • dead larvae Larva (dead)
  • LA-a protein and LA-b protein have strong growth inhibitory activity and insecticidal activity against insects. Since both LA-a protein and LA-b protein have chitinase activity, LA-a protein and LA-b protein hydrolyze chitin on the body of Drosophila larvae or the surface of digestive sputum. It was thought that the growth of Drosophila larvae was inhibited and the larvae died.
  • MLX56 a protein derived from mulberry milk
  • has no chitinase activity and when MLX56 is ingested by several larvae of lepidopterous insects, it has been reported to show growth inhibition but not insecticidal properties.
  • Patent Document 1 and Non-Patent Document 3 From this, it was confirmed that the LA-a protein and the LA-b protein are novel proteins having completely different functions from MLX56, which is a protein derived from mulberry milk.
  • Example 3 Determination of amino acid sequence and nucleotide sequence
  • the amino acid sequence and nucleotide sequence of LA-a protein and LA-b protein derived from mulberry milk were determined.
  • nucleotide sequences of polynucleotides encoding LA-a protein and LA-b protein were determined. Specifically, mRNA was extracted and purified from mulberry milk according to a conventional method, and mRNA-seq analysis was performed on this using a GS-FLX DNA sequencer (Roche). From the obtained EST database, the nucleotide sequences shown in SEQ ID NOs: 4, 5 and 6 were found. The reason why these are LA-a and b is as described later. Cloning to express the LA-a protein and LA-b protein in E. coli was performed by reverse transcription from mRNA by Superscript III First strand Synthesis Supermix (Invitrogen) and the following specific primers prepared based on the coding region.
  • Superscript III First strand Synthesis Supermix Invitrogen
  • LA-a protein isolated from mulberry milk contains two isoforms. Therefore, the protein encoded by the polynucleotide having the base sequence shown in SEQ ID NO: 4 is named “LA-a2 protein”, and the protein encoded by the polynucleotide having the base sequence shown in SEQ ID NO: 6 is “LA-a1 protein”. Named. Further, it was revealed that the LA-b protein is a protein encoded by a polynucleotide having the base sequence shown in SEQ ID NO: 5.
  • the protein encoded by the polynucleotide having the base sequence shown in SEQ ID NO: 4 and the protein encoded by the polynucleotide having the base sequence shown in SEQ ID NO: 6 have almost the same estimated molecular weight, and are distinguished by SDS-PAGE.
  • the protein encoded by the polynucleotide having the base sequence shown in SEQ ID NO: 5 is smaller than these, the protein encoded by the polynucleotide having the base sequence shown in SEQ ID NO: 4, The protein encoded by the polynucleotide having the base sequence shown in SEQ ID NO: 6 is referred to as “LA-a protein”, and the protein encoded by the polynucleotide having the base sequence shown in SEQ ID NO: 5 is referred to as “LA-b protein”. I concluded. The reason why the molecular weight estimated from the amino acid sequences of these three proteins was smaller than the molecular weight estimated from the SDS-PAGE mobility was thought to be due to the post-translational sugar chain modification.
  • amino acid sequence of the LA-a2 protein (SEQ ID NO: 1)
  • amino acid sequence of the LA-b protein (SEQ ID NO: 2)
  • amino acid sequence of the LA-a1 protein (SEQ ID NO: 3) are respectively SEQ ID NOs: 4-6. It determined from the base sequence of polypeptide shown in.
  • the protein according to the present invention has chitinase activity and exhibits sufficient insect resistance activity including insecticidal properties. Therefore, the protein according to the present invention is suitably used as an insecticide.

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Abstract

La présente invention concerne une protéine possédant une activité de résistance aux insectes et l'utilisation de la protéine. Cette protéine est l'une quelconque des protéines (a) à (d) suivantes : (a) une protéine comprenant la séquence d'acides aminés représentée par la séquence numéro 1 ; (b) une protéine comprenant la séquence numéro 1 dans laquelle un ou plusieurs acides aminés ont été supprimés, substitués et/ou ajoutés et possédant une activité de chitinase ; (c) une protéine comprenant la séquence d'acides aminés représentée par la séquence numéro 2 ; (d) une protéine comprenant la séquence numéro 2 dans laquelle un ou plusieurs acides aminés ont été supprimés, substitués et/ou ajoutés et possédant une activité de chitinase.
PCT/JP2010/070041 2010-11-10 2010-11-10 Protéine à activité de chitinase et son utilisation Ceased WO2012063338A1 (fr)

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Cited By (1)

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CN109258693A (zh) * 2018-09-30 2019-01-25 大连理工大学 重组几丁质酶在杀虫或抑菌方面的应用

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WO2008108345A1 (fr) * 2007-03-02 2008-09-12 National Institute Of Aglobiological Sciences Protéine résistant aux insectes et gène de résistance aux insectes codant pour la protéine résistant aux insectes.
WO2010075143A1 (fr) * 2008-12-22 2010-07-01 Monsanto Technology Llc Gènes et leurs utilisations en amélioration des plantes

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WO2008108345A1 (fr) * 2007-03-02 2008-09-12 National Institute Of Aglobiological Sciences Protéine résistant aux insectes et gène de résistance aux insectes codant pour la protéine résistant aux insectes.
WO2010075143A1 (fr) * 2008-12-22 2010-07-01 Monsanto Technology Llc Gènes et leurs utilisations en amélioration des plantes

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NAOYA WASANO ET AL.: "Arata na Kuwa Taichusei Tanpakushitsu no Screening", DAI 53 KAI JAPANESE SOCIETY OF APPLIED ENTOMOLOGY AND ZOOLOGY TAIKAI KOEN YOSHI, 12 March 2009 (2009-03-12), pages 17 *
NAOYA WASANO ET AL.: "Kuwa Taichusei Tanpakushitsu no Sho Seijo", SANSHI-KONCHU KINO RIYO GAKUJUTSU KOENKAI, THE JAPANESE SOCIETY OF SERICULTURAL SCIENCE 79 KAI TAIKAI KOEN YOSHISHU, 21 March 2009 (2009-03-21), pages 90 *
WASANO, N. ET AL.: "A unique latex protein, MLX56, defends mulberry trees from insects.", PHYTOCHEMISTRY, vol. 70, no. 7, May 2009 (2009-05-01), pages 880 - 888 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109258693A (zh) * 2018-09-30 2019-01-25 大连理工大学 重组几丁质酶在杀虫或抑菌方面的应用
CN109258693B (zh) * 2018-09-30 2021-03-16 大连理工大学 重组几丁质酶在杀虫或抑菌方面的应用

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