WO2012063338A1 - Protein having chitinase activity and uses thereof - Google Patents

Abstract

A protein having adequate insect resistance activity and uses of the protein are provided. This protein is any of the following proteins (a) to (d): (a) a protein comprising the amino acid sequence represented by sequence number 1; (b) a protein comprising an amino acid sequence in which one or several amino acids have been deleted, substituted and/or added in the amino acid sequence represented by sequence number 1, and having chitinase activity; (c) a protein comprising the amino acid sequence represented by sequence number 2; (d) a protein comprising an amino acid sequence in which one or several amino acids have been deleted, substituted and/or added in the amino acid sequence represented by sequence number 2, and having chitinase activity.

Description

Protein having chitinase activity and use thereof

The present invention relates to a protein having chitinase activity and use thereof.

Conventionally, as a representative genetically modified plant (GM plant) imparted with insecticidal and insect resistance, a crop into which a gene encoding a Bt toxin produced by a Gram-positive bacterium Bacillus thuringiensis (for example, corn, Have been put to practical use in the United States and the like. 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). However, since there are concerns about the emergence of insects that are resistant to Bt toxins, practical application of insect-resistant proteins other than Bt toxins is desired.

By the way, it is known that plant milk contains substances exhibiting toxicity and growth inhibitory activity against insects. Here, the above “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.

For example, 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.

Furthermore, 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)”

Naoya Wasano et. Al., Canadian Journal of Microbiology 51, 988-995 (2005) Kotaro Konno et. Al., PNAS 103 (5), 1337-1341 (2006) Naoya Wasano et. Al., Phytochemistry 70, 880-888 (2009)

However, the sugar-like alkaloid disclosed in 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. In addition, since 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.

In the first place, since fraction 1 disclosed in Patent Document 1 contains at least four types of proteins having different molecular weights, it is not clear which protein exhibits growth inhibitory activity against insects. . 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.

Until now, it has never been known that 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.

That is, 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.

Further objects, features, and superior points of the present invention will be fully understood from the following description. The benefits of the present invention will become apparent from the following description with reference to the accompanying drawings.

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.

Moreover, since the 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.

In addition, since the vector according to the present invention is configured to contain the polynucleotide according to the present invention, such 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.

Moreover, since 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.

Moreover, since the insecticide according to the present invention has the protein according to the present invention, it is excellent in insect resistance activity.

It is a figure showing the result of having analyzed the soluble fraction of mulberry milk by SDS-PAGE method. It is a figure showing the chromatogram of the result of having used the soluble fraction of mulberry milk for chromatography, (a) is a figure showing the chromatogram of the result of hydrophobic chromatography, (b) is a figure by hydrophobic chromatography. It is a figure showing the chromatogram of the result of having used the obtained LA-b fraction for the cation exchange chromatography. It is a figure showing the result of having analyzed the purified sample of LA-a protein and LA-b protein by SDS-PAGE method. It is a figure showing the result of having performed western blotting using the anti- LA-a antibody. It is a figure showing the result of having analyzed the glucosylation of LA-a protein and LA-b protein, (a) represents the result of having dye | stained the gel using Pro-Q | Emerald | 300 or SYPRO | Ruby | stain, (b) The result of a lectin blot is shown. It is a figure showing the result of having measured the chitinase activity and chitosanase activity of LA-a protein and LA-b protein, (a) showing the measurement result of chitinase activity, (b) showing the measurement result of chitosanase activity . It is a figure which shows the influence which LA-a protein and LA-b protein have on the development of Drosophila, (a) represents the number of Drosophila wings which gave LA-a protein etc., and LA-a Protein etc. are given, and it shows 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.

Hereinafter, embodiments of the present invention will be described in detail. However, the present invention is not limited to this, and can be implemented in a mode in which various modifications are made within the described range. Moreover, all the academic literatures and patent literatures described in this specification are incorporated herein by reference. Unless otherwise specified in this specification, “A to B” representing a numerical range means “A or more and B or less”.

[1. Protein according to the present invention]
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 ".

Here, 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.

The identity of amino acid sequences can be determined using 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.

Typically seen as 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.

For example, 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%.

In addition, 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). However, 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. Here, in this specification, 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”.

In the present specification, when “significantly” is described, when statistical analysis is performed using a conventionally known tukey test, 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%.

For example, 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. When 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”.

More preferably, when 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. Similarly, assuming that 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. Colloidal chitin not labeled with a dye in place of the dye-labeled chitin CMchitin-RBV (Loewe Biochemica, Munchen, Germany) as a substrate in the method described in the section “Method for measuring chitinase activity” in Examples described later (Colloidal chitin), 4-Nitrophenyl N-acetyl-β-D-glucosaminide (available from Sigma-aldrich), or 4-methylumbellifer N, N'-diacetyl-β-D-chitobioside (obtained from Sigma-aldrich) Possible).

Further, the protein according to the present invention has insect resistance activity. Here, the “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”). .) For example, as shown in the examples described below, 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.

Since the protein according to the present invention has chitinase activity, 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.

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. .

Since 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.

Here, the above “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. Here, in this specification, 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”.

More preferably, when 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. Similarly, when 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.

Examples of the “chitosanase activity measuring method” 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.

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).

In addition, when producing using gene recombination technology, the recombinant protein expression system is not particularly limited as long as the protein according to the present invention can be expressed. For example, 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.

In the case of isolating and purifying a naturally occurring protein, the purification method is not particularly limited. For example, as shown in Examples described later, 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. Examples of 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.

For example, 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. With the above configuration, among the proteins according to the present invention, 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.

In addition, 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 | purified with high purity among the proteins which concern on this invention.

The conditions for cation exchange 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, 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.

[2. Polynucleotide according to the present invention]
The polynucleotide according to the present invention is a polynucleotide encoding the protein according to the present invention.

Here, 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).

Further, the 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.

As 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. As 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.

As one embodiment of the polynucleotide according to the present invention, for example, it 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.

In one embodiment of the present invention, the polynucleotide according to the present invention may be substituted with a chemically synthesized base. Moreover, the site | part by which the polynucleotide based on this invention is substituted is not specifically limited, The protein which the base sequence after substitution has should just have a suitable property. That is, the protein encoded by the base sequence after substitution only needs to have chitinase activity. For example, 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.

In the present specification, the above “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 μg / 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.

[3. Vector and transformant according to the present invention]
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.

As long as 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. For example, 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.

When a plasmid vector is used as the vector according to the present invention, for example, pBR322, pBR325, pUC19, pUC119, pBluescript, pBluescriptSK, pBI vectors, and the like can be used.

To construct the vector according to the present invention, after separating and purifying the polynucleotide according to the present invention, 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.

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. In general, a vector can be amplified in E. coli using E. coli as a host cell. At this time, a preferred E. coli type can be selected according to the type of vector.

Note that 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. For example, 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. For example, when 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. For example, when 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.

Whether or not 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. In addition, when 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.

Since the transformant thus obtained expresses the protein according to the present invention, it can be used to prepare the protein.

Also, plant cells into which a vector has been introduced by the above-described method can be regenerated into a plant body by redifferentiation by a conventional method. Therefore, 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.

Once a transformed plant into which the polynucleotide according to the present invention has been introduced into the genome is obtained, 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.

The plant used for transformation is not particularly limited, and for example, various monocotyledonous plants, dicotyledonous plants and the like can be used. For example, 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.

[4. Insecticide according to the present invention]
The 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.

In the present specification, the term “insect-proofing agent” intends a composition for killing insects, weakening insects, or inhibiting insect growth. In the present specification, 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.

As long as 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, In general, 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. Thus, according to the protein of the present invention, sufficient insect resistance activity is exhibited even in a small amount.

In addition, the 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. For example, 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. For example, 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.

In the above-described embodiment of the present invention, 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.

The present invention is not limited to the above-described embodiments, and various modifications are possible within the scope shown in the claims, and embodiments obtained by appropriately combining technical means disclosed in different embodiments. Is also included in the technical scope of the present invention.

Hereinafter, the present invention will be specifically described with reference to examples, but the present invention is not limited to the examples.

[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.

(1. Purification of LA protein)
Mulberry (Morus alba L.) (Minamisakari varieties) petioles were cut, and the emulsion leaked from the cut surface was collected. The obtained emulsion and buffer A (100 mM potassium phosphate, 10 mM EDTA, pH 6.7) containing 0.1% (v / v) β-mercaptoethanol were rapidly added so that the mixing ratio was 1: 1. Mix, freeze with liquid nitrogen and store at -80 ° C.

Next, 6 ml of the emulsion / buffer A mixture was diluted 4-fold with buffer A and centrifuged at 18000 g for 30 minutes. First, 30% saturated ammonium sulfate is added to the resulting supernatant to remove impurities (a part of it) by precipitation, and ammonium sulfate is further added to the supernatant to make it 80% saturated. Minutes were collected by precipitation. The precipitated protein was dissolved in 4 ml of buffer B (10 mM calcium phosphate, 1 mM EDTA, pH 6.0). 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., Rancho 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 μl of mulberry milk (stock solution)) by the SDS-PAGE method. As shown in FIG. 1, 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”. A protein having a molecular weight of approximately 50 kDa was named LA-a protein, and a protein having a molecular weight of approximately 46 kDa was named LA-b protein.

To purify LA-a protein and LA-b protein, first, cation exchange column equilibrated with buffer B (CM-CellulofineuloC-200; Seikagaku Kogyo KK, Tokyo, Japan; 26 mm id × 100 mm) Load the sample after dialysis and elute at a flow rate of 1.0 ml / min using buffer B containing 0.2 M KCl to contain LA-a protein and La-b protein. The protein fraction was obtained.

Next, 30% saturated ammonium sulfate was added to the eluted protein solution, and the supernatant was subjected to hydrophobic chromatography. For hydrophobic chromatography, a HiLoad 16/10 phenyl sepharose HP column (16 mm id × 100 mm; GE healthcare, Piscataway, NJ, USA) equilibrated with buffer B containing 30% saturated ammonium sulfate was used. A linear concentration gradient from% saturated ammonium sulfate to 0% saturated ammonium sulfate was performed at a flow rate of 2 ml / min. The chromatogram of the result of hydrophobic chromatography is shown in FIG. By this method, a purified preparation of LA-a protein 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. For 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.

5.8 mg 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 (each 1 μg) was analyzed by SDS-PAGE. The protein was detected by silver staining. As a result, as shown in 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.

All the purification steps described above were performed at 4 ° C. All chromatographic steps were performed using a BioLogic Duo-Flow chromatography system (Bio-Rad).

The concentration of the obtained LA protein was determined according to the following formula (1) (see Kalckar DM: J Biol Chem 1947, 167: 461-475).
Protein concentration (mg / ml) = 1.45A ₂₈₀ -0.74A ₂₆₀ (1).

(2. Western blotting)
A rat polyclonal antibody against the purified LA-a protein was prepared by a conventionally known technique, and Western blotting was performed on the LA-a protein and the LA-b protein.

Specifically, 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).

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.

(3. Analysis of N-terminal amino acid sequence)
Protein cysteine residues were alkylated by treatment with iodoacetamide according to the method of Coligan et al. (See Current Protocols in Protein Science John Wiley & Sons 1995). Then, using a C18 reverse phase column (4.6 × 150 mm; TSK gel ODS-100S; Toso, Tokyo, Japan), the flow rate was changed by a linear concentration gradient of 5 to 50% acetonitrile in 0.1% trifluoroacetic acid. Elute at 1.0 ml / min. The eluted protein was concentrated by evaporation, and the N-terminal amino acid sequence was analyzed using an automatic protein sequencer PPSQ-21A (Shimadzu, Japan).

As a result, it was revealed that 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.

(4. Analysis of glycosylation)
LA-a protein and LA-b protein (3 μg per lane) were separated by SDS-PAGE. First, to detect glycosylated protein, 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. As a control, CandyCane glycoprotein molecular weight standards (Invitrogen) were used.

The result is shown in FIG. In FIG. 5 (a), the numbers with asterisks indicate glycoproteins. Staining results with Pro-Q Emerald 300 revealed that the LA-a and LA-b proteins were glycosylated.

For 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. Next, 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). In addition, lectins other than RCA120 were purchased from Seikagaku Corporation.

The result is shown in FIG. LA-a and LA-b proteins reacted strongly with the specific lectin CA120.

From the above results, it was confirmed that the LA-a protein and LA-b protein are glycoproteins.

(5. Chitinase assay and chitosanase assay)
(Measurement method of chitinase activity)
Except for extending the enzyme reaction from 10 minutes to 2 hours, using the dye-labeled chitin CMchitin-RBV (Loewe Biochemica, Munchen, Germany) as a substrate according to the method of Mayer et al. (See Planta 1996, 200: 289-295) A chitinase assay by colorimetry was performed. As a buffer, 0.1 M citrate-Na ₂ HPO ₄ buffer (pH 5 to 8) was used. Hydrolyzed chitin was recovered and measured by absorbance at 550 nm. The chitinase activity was expressed as “Δ550 nm g protein ⁻¹ min ⁻¹ ”.

Note that “Δ550 nm” in the unit “Δ550 nm g protein ⁻¹ min ⁻¹ ” represents how much the absorption at 550 nm has changed before and after the reaction. 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). The amount of RBV solubilized (that is, how much chitin has been solubilized together) is regarded as chitinolytic activity.

(Measurement method of chitosanase 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.

As described in the method of Osswald et al., The control consisting of the complete reaction mixture was stopped immediately after the addition of the enzyme. The reaction product glucosamine (GlcN) was recovered, labeled with fluoresamine (Fluka, Buchs, Switzerland) and observed fluorometrically (excitation: 395 nm; emission: 493 nm). Chitosanase activity was calculated based on the amount of GlcN calculated using a GlcN standard curve generated under the same conditions as the assay. The chitosanase activity was expressed in units of “μmol GlcN g protein ⁻¹ min ⁻¹ ”. The unit “μmol GlcN g protein ⁻¹ min ⁻¹ ” represents the amount (μmol) of glucosamine produced from chitosan by 1 g of protein per minute.

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.

As shown in FIG. 6 (a), 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.

As shown in FIG. 6 (b), 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. In addition, the 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. 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. In the graph, “No protein” represents the control group 2, “BSA” represents the control group 1, “LA-a” represents the LA-a protein administration group, and “LA-b” It represents the LA-b protein administration group.

As shown in FIG. 7 (a), most larvae hatched in the control group 1 and control group 2, whereas in the LA-a protein administration group or the LA-b protein administration group, Delay was observed. As a result of tukey test, there is a significant difference (p <5%) between the control group 1 (or control group 2) and the LA-a protein-administered group on any of the third, fourth, fifth and sixth days. There was also a significant difference between the control group 1 (or control group 2) and the LA-b protein administration group (p <5%).

Furthermore, as shown in FIG. 7 (b), in the LA-a protein administration group, 80% of larvae died after 6 days of breeding, and in the LA-b protein administration group, 40% of larvae after 6 days of breeding. Died. As a result of tukey test, control group 1 (or control group 2), LA-a protein in any of pupa (Pupa), living larvae (Larva (live)) and dead larvae (Larva (dead)) There was a significant difference (p <5%) between the three groups: the administration group and the LA-b protein administration group.

From these results, it was revealed that 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.

Note that 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. (See 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.

First, the 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. By PCR using:
CTACAAATAAGCGGCTGTACTGCC (SEQ ID NO: 7)
RRTGAGCCACARTTGTGGAAGGGATGCAG (SEQ ID NO: 8) (wherein, R is A or G in SEQ ID NO: 8).

As a result, it was revealed that 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.

In addition,
-Having the N-terminal hevein domain obtained by Edman analysis, as described in the section "3. Analysis of amino acid sequence at N-terminal" above, and-these cDNAs in which the anti-LA-a antibody is expressed in Escherichia coli It was concluded that the obtained cDNA encodes LA-a protein or LA-b protein, for example, by reacting with the protein product (data not shown).

Also,
-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. Since 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.

Furthermore, the amino acid sequence of the LA-a2 protein (SEQ ID NO: 1), the amino acid sequence of the LA-b protein (SEQ ID NO: 2), and the 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.

Claims (8)

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 according to claim 1, wherein the protein is derived from a plant emulsion. The protein according to claim 2, wherein the plant is a mulberry plant. A polynucleotide encoding the protein according to claim 1. The polynucleotide according to claim 4, characterized by any one 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. A vector comprising the polynucleotide according to claim 4. A transformant transformed with the vector according to claim 6. An insecticide comprising the protein according to claim 1.

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