JP4553126B2 - Matsunomadara beetle chitinase cDNA - Google Patents

Description

  The present invention relates to a DNA encoding a pinewood spotted chitinase for controlling pine worms, and an amino acid sequence of a protein acting as a chitinase deduced from the base sequence.

  It has been clarified that pine wilt caused by pine weevil is mainly caused by pine wood nematodes, which are damaged by Japanese pine species such as Japanese red pine, black pine, and Ryuki pine. The pinewood nematode is mediated by the pinewood beetle, and when the pinewood beetle bites the bark of the pine twig (after-eating), it is said that it will invade the tree from the traces. The pine wood nematode enters and grows in the pine tree and weakens the pine, but the weakened pine is suitable for spawning the pine wood beetle. The hatched pine beetle larvae eat the inner bark and die the pine. In this way, the pine wilt is increased by the symbiosis of the pinewood beetle and the pinewood nematode.

  Conventionally, there are natural enemies and attractants used as a method to restore the pine tree vigor by directly extermination of the pine wood nematode and pine wood beetle larvae that have invaded into the pine tree. Not in. In addition, the soil application of drugs has been elucidated for its preventive effect, but its use is not permitted when safety is carefully considered. Therefore, measures are currently being taken to prevent the invasion of pinewood nematodes by preventing the after-meal of pinewood beetle. In other words, if there is no danger of chemical damage in a large area, spraying of chemicals such as Sumithion and Cevimol is performed from the air, and if there is a risk of danger from a small area or chemical The sprayer is sprayed from the ground with a powerful sprayer that reaches the crown. However, such a control method has an adverse effect on the human body, and environmental pollution of the remaining agricultural chemicals becomes a problem.

  Chitinase is an enzyme that hydrolyzes chitin, and is used for molting of arthropods and crustaceans, biological defense of plants, growth of microorganisms, and the like. The present inventors have already clarified that a chitinase derived from yam is effective as a phytopathogenic fungus control agent. (Patent Document 1)

  The present inventors have clarified the base sequence of the gene encoding the chitinase derived from the yam in Patent Document 2 and found that the chitinase derived from the yam belongs to the family 19.

Several methods for producing chitinase using organisms are already known, which use DNA encoding protozoan-derived chitinase (Patent Document 3), and encode chitinase produced by bacteria by genetic manipulation. There are a method of forming DNA and producing chitinase in large quantities using this (Patent Document 4), a method of producing actinomycetes-derived chitinase (Patent Document 5), and the like.

JP 2000-109405 A Japanese Patent Laid-Open No. 2003-250561 Japanese Patent Laid-Open No. 9-299091 JP 2004-298127 A JP2000-093182A

  As described above, the conventional pine worm control agent is a drug using an organic synthetic compound, and its influence on the human body and the environment has become a problem. The present invention elucidates the base sequence of the pinewood spotted beetle chitinase cDNA, deduces the amino acid sequence of the pine beetle beetle chitinase from the base sequence, and develops a chitinase drug effective as a safe pine worm control agent derived from an organism. Is to make it possible.

  The gist of the present invention is a DNA having the base sequence shown in SEQ ID NO: 1 or 3.

  The above-mentioned base sequence is derived from the Japanese pine beetle, and is the base sequence of the coding region of the chitinase A or chitinase B gene. In this case, the present invention is derived from the pine-nosed beetle shown in SEQ ID NO: 1 or 3. The gist is DNA having the base sequence of the coding region of the chitinase A or chitinase B gene.

  The base sequence shown in SEQ ID NO: 1 or 3 is characterized by encoding a protein consisting of a base sequence in which one or several bases are deleted, substituted or added, and acting as a chitinase, The present invention relates to the nucleotide sequence shown in SEQ ID NO: 1 or 3, more specifically, SEQ ID NO: 1 or 3 derived from the pinewood beetle and having the nucleotide sequence of the coding region of the chitinase A or chitinase B gene. The gist of the present invention is DNA consisting of a base sequence in which one or several bases are deleted, substituted or added, and a base sequence encoding a protein acting as a chitinase.

  Including the consensus sequence (conserved region 2) of insect chitinase (family 18), in this case, the present invention provides the nucleotide sequence shown in SEQ ID NO: 1 or 3, more specifically, SEQ ID NO: 1 or 3. The base sequence of the coding region of the chitinase A or chitinase B gene, or the base sequence shown in SEQ ID NO: 1 or 3 is deleted, substituted or added in the base sequence shown in SEQ ID NO: 1 or 3 The gist of the present invention is a DNA comprising a base sequence that encodes a protein that acts as a chitinase and that contains a consensus sequence (conserved region 2) of an insect chitinase (family 18).

  In this case, the present invention relates to a base sequence represented by SEQ ID NO: 1 or 3, more specifically, matsunomadara represented by SEQ ID NO: 1 or 3. From the base sequence of the coding region of the chitinase A or chitinase B gene derived from beetle, or the base sequence shown in SEQ ID NO: 1 or 3, wherein one or several bases are deleted, substituted or added A DNA comprising a sequence complementary to a DNA sequence comprising a base sequence encoding a protein acting as a chitinase, preferably a consensus sequence (conserved region 2) of an insect chitinase (family 18) Is the gist.

  The gist of the present invention is a protein encoded by any of the above DNAs and acting as a chitinase.

Furthermore, the gist of the present invention is a protein encoded by any one of the above-described DNAs and acting as a chitinase, which is composed of any one of the following proteins (a) to (d).
(A) A protein comprising the amino acid sequence set forth in SEQ ID NO: 2 in the sequence listing.
(B) A protein having an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence described in SEQ ID NO: 2 in the sequence listing and having chitinase activity.
(C) A protein comprising the amino acid sequence set forth in SEQ ID NO: 4 in the sequence listing.
(D) A protein having an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence described in SEQ ID NO: 4 in the sequence listing and having chitinase activity.

Two types of chitinases (Chitinase A and Chitinase B) were cloned from the larvae of the pine beetle 40 days after birth (after laying eggs). The larva chitinase of the Japanese pine beetle larva did not have a chitin binding domain in its sequence, which both chitinases of other insects have.
According to the present invention, it is possible to provide the nucleotide sequence of the pinewood beetle chitinase cDNA and the amino acid sequence of the pinewood beetle chitinase deduced from the nucleotide sequence. A gene encoding the novel chitinase to which it belongs can be provided. The possibility of producing a pinewood spotted beetle chitinase with an amino acid sequence deduced from its base sequence has emerged, and the first step in the development of a chitinase drug effective as a safe pine worm control agent derived from organisms Was able to contribute.

[Appearance of chitinase activity]
In order to elucidate the expression time of the chitinase gene in the pine beetle, first, the time when the chitinase activity appears was examined. For this reason, larvae of the pine beetle beetle were artificially raised, and daily fluctuations associated with the growth of chitinase activity were followed by activity staining on native PAGE. As a result, it was found that chitinase activity increased rapidly about 40 days after egg laying.

[Isolation of mRNA and cloning of cDNA]
Total mRNA was extracted from the larvae of the pine beetle at the time when the activity of chitinase was observed, and then cDNA was synthesized by RT-PCR using Oligo T binding to Poly A tail peculiar to mRNA as a primer. In order to select a chitinase cDNA, PCR primers were designed from the amino acid sequences of insect chitinases reported so far, particularly beetle chitinases. Using the primers, a full-length cDNA of the pinewood beetle was obtained by the 3′RACE method and the 5′RACE method. Furthermore, the base sequence was decoded according to the information.
As a result, chitinase A and B have an open reading frame of 1116 and 1164 bp, respectively, and their chitinase consists of 371 and 387 amino acid residues, the estimated molecular weights are 41.5 and 42.3 kDa, and the estimated pI is 4.36. It was found to be 4.45 acid chitinase. Conserved regions 1 and 2 unique to Family 18 were also seen. The pinewood beetle was found to express family 18 chitinases as previously reported in insects.

[Discussion]
The chitin-binding region found in the lepidopterous chitinases of silkworms and tobacco tin mega was deficient on the C-terminal side of the pinewood spotted chitinase. In addition, the chitinase of the pine beetle beetle lacks the chitin-binding region in the same way as the Spruce beetle chitinase of the same beetle, so in the beetle molting, it is not necessary to have this chitin-binding region in the action of chitinase, etc. Future elucidation is expected. In addition, this finding may be important in the development of bio-pesticides for the pinewood beetle.

  The DNA of the present invention includes DNA having the nucleotide sequence shown in SEQ ID NO: 1 or 3 in the sequence listing, the nucleotide sequence of the coding region of the chitinase A and chitinase B genes, and the nucleotide sequence shown in SEQ ID NO: 1 or 3 A gene having (base) is included. Also included are those that hybridize with the base sequence of the above gene under stringent conditions and have similar activity.

  In addition, the amino acid sequence of the present invention includes the amino acid sequence shown in SEQ ID NO: 2 or 4 in the sequence listing, and the amino acid sequence shown in SEQ ID NO: 2 or 4 in the pine wood beetle-derived kinase activity. Includes amino acid sequences including derivatives obtained by amino acid substitution, deletion, addition, and insertion.

A preferred embodiment of the DNA of the present invention is a novel gene encoding chitinase, and is expected to have high utility as a gene that allows the gene to be expressed in microorganisms.
For example, by incorporating the chitinase gene of the present invention into a microbial plasmid, it can be expected that the gene is expressed by a microorganism. Such a plasmid must have at least an origin of replication that functions (autonomously replicates) in the host microorganism. In addition, it is highly desirable to have a selection marker gene used as a marker when selecting a transformed strain. As selection marker genes, genes that can confer antibiotic resistance and the like, for example, ampicillin resistance genes, tetracycline resistance genes, and the like are well known. Furthermore, the plasmid often has a promoter sequence capable of expressing the structural gene, but the promoter sequence may be inserted together with the structural gene. The above techniques are well known, and those skilled in the art can select and use an appropriate one of these techniques.

  The plasmid is introduced into the microbial cell and functions in the microbial cell. Methods for introducing plasmids into microbial cells are well known, and those skilled in the art can select and use an appropriate one of these known methods. As host microorganisms, Bacillus subtilis, Escherichia coli (hereinafter referred to as “E. coli”), and Saccharomyces cerevisiae are well known and used. In particular, Escherichia coli is often used for gene amplification and selection. Specific examples of hosts such as Bacillus subtilis, Escherichia coli and yeast and specific examples of plasmids to be used are described in many documents, and those skilled in the art can select and use appropriate ones.

  It does not have to be a special method to obtain a chitinase by culturing a microorganism transformed with a plasmid. That is, this microorganism is cultured under a medium and conditions under which the microorganism can grow well, and chitinase in the cell or around the cell membrane is collected in the medium. There are also well-known methods for isolating and purifying polypeptides such as chitinase, and those skilled in the art can isolate and purify them by combining these known methods.

  The chitinase of the present invention obtained as described above has an action of hydrolyzing not only insect epidermis chitin but also nutrient envelope chitin in the midgut, and therefore can be used as an insect control agent. When used as an insect control agent, other materials containing chitinase can be given directly to the pine beetle or applied to pine trunks by treatment methods including high-pressure or low-pressure spraying, pouring, coating, spraying, and dipping. can do.

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

Experimental method (see Fig. 1)
1. Identification of the time of appearance of chitinase in the Japanese pine beetle The Japanese pine beetle larvae were bred on artificial diet for 60 days after laying. In order to isolate chitinase mRNA, the appearance time of chitinase was examined. Therefore, pine spotted beetle larvae were homogenized with 10 mM phosphate buffer (pH 7.5) at 4 ° C., 3 times the body weight, and the supernatant was collected by centrifugation (10,000 rpm, 10 minutes). The supernatant (10 microL) was separated by neutral PAGE for medium acidity, and then electroblotted onto a gel containing a substrate (glycol chitin) to perform activity staining of chitinase. The active band of chitinase is
Detection was performed by staining with Fluostain (a fluorescent dye that binds to chitin and emits blue fluorescence when irradiated with ultraviolet light). The detection method of chitinase activity was based on the following paper (D. Koga et al .: Induction pattern of chitinases in yam callus by inoculation with autoclaved Fusarium oxysporum, ethylene, and chitin and chitosan oligosaccharides. Biosci. Biotechnol. Biochem., 56, 280-285 (1992)).

  The detection results are shown in FIG. High chitinase activity (more than 2 bands) was observed 39 to 40 days after egg laying.

2. Extraction of mRNA for chitinase in Japanese pine beetle 20 mg of larvae frozen with liquid nitrogen on the 40th day after egg laying where chitinase was expressed was ground in a mortar and total RNA was extracted using RNAeasy Mini Kit (QIAGEN).

3. Sequencing of cDNA by 3'RACE method (3'Rapid amplification of cDNA ends) First, in order to clarify the 3'-side sequence of the pinewood spotted beetle chitinase mRNA, Using an oligo dT complementary to a common poly A tail with an adapter added, and using the total RNA prepared in 2 above as a template, Omniscript RT kit (QIAGEN) and RT-PCR The strand cDNA was synthesized (see FIG. 3).

Furthermore, using the cDNA as a template, a degenerate primer was created from several insect chitinase (family 18) consensus sequence (conserved region 2) as a 5′-side primer (see FIGS. 4 and 5), As the 3 'primer, PCR (2.5U Taq
DNA polymerase, 10 mM Tris-HCl. PH 8.3, 1.5 mM MgCl2, 50 mM KCl, 0.2 mM dNTPs, 100 p mole each primer) (reaction in the lower part of FIG. 3). Then, the PCR product (the result of the agarose electrophoresis is FIG. 6) was subcloned using E. coli, and the base sequence was determined. As a result, a base sequence that was considered to be the mRNA of the pinewood spotted beetle chitinase could be obtained (FIG. 7).

4. Sequencing of cDNA by 5'RACE method (5'Rapid amplification of cDNA ends)
In order to clarify the 5 ′ base sequence of mRNA, 5′RACE method was used (FIGS. 8 and 9). 8 and 9, the total RNA extracted in the previous item 2 was used as a template, and Omni-script RT kit (QIAGEN) was used to perform RT-PCR and double-stranded cDNA synthesis until 3 '. Same as the RACE method, but the differences are. After RT-PCR, oligo dC was added to the 5 ′ side using terminal deoxynucleotidyl transferase. This was used as a template, 'the side primer, obtained by adding an adapter to its complementary nucleotide sequence (2 ^nd from PCR adapter only) as primer, whereas the 3' 5 Elucidation primer side in 3'RACE method PCR was carried out using a product prepared on the basis of the prepared base sequence.

  The PCR product (the result of the agarose electrophoresis shown in FIG. 10) was subcloned into Escherichia coli, and the nucleotide sequence was analyzed (FIGS. 11 and 12).

5). cDNA full-length sequencing To analyze the total length of the pinewood swordfish chitinase mRNA, the 5 'primer synthesized on the basis of the 5' RACE method and the adapter used in the 3 'RACE method are used on the 3' side. PCR was carried out using the highly reliable DNA polymerase Elongase Enzyme Mixture (GIBCO BRL) using the 3'RACE cDNA (first strand cDNA) as a template (Fig. 13). .

  The PCR product was subcloned into E. coli and the nucleotide sequence was analyzed. As a result, it was possible to clarify the nucleotide sequences of the cDNAs of the two pine swordfish chitinase shown in FIGS. The amino acid sequence could also be estimated (SEQ ID NO: 2, 4). From the amino acid sequence of this chitinase, it was reconfirmed that both were family 18 chitinases. However, as a feature of this spotted chitinase, it did not have two chitin-binding regions that are additionally present on the C-terminal side, which are found in other insect chitinases (FIG. 16).

  We were able to clone the pine wood beetle chitinase cDNA related to “pine weevil”. This gene is necessary for bio-pesticides that we are developing as a measure against “pine pine worms”. The pine wood beetle chitinase produced by this gene is originally an enzyme required for molting itself, but it is expected to digest (decompose) its own digestive tract when it is ingested, leading to death. ("Control poison with poison"). For this reason, the cDNA of the present invention is expected to be valuable as the world's first biopesticide in “control of pinworms”.

It is drawing explaining the flow of chitinase cDNA cloning. It is a photograph replaced with drawing which shows the result of the chitinase activity dyeing | staining using the beetle larvae 37-42 days after birth (post-laying). It is drawing explaining the principle of reverse transcription (Reverse transcription) and 3'RACE method. BRIEF DESCRIPTION OF THE DRAWINGS It is drawing explaining the structure of typical sugar hydrolase Family18 chitinase. It is a drawing explaining the sequence comparison of Region 2 part and the design of Degenerate primer. It is the photograph replaced with drawing which shows the result of 3'RACE using the beetle larvae 37 to 40 days after birth. It is drawing which shows the result 1 of the sequence of 3'RACE product in the beetle larvae 40 days after birth. It is drawing explaining the principle of 5'RACE method. FIG. 9 is a continuation of FIG. 8 for explaining the principle of the 5 ′ RACE method. It is a photograph replacing a drawing showing the results of 5'RACE of each of Chitinase A and Chitinase B in a 40-day-old beetle larva. It is drawing which shows the sequencing result of 5'RACE product of Chitinase A. It is drawing which shows the sequence result of 5'RACE product of Chitinase B. It is the figure explaining RT-PCR for obtaining full-length Chitinase A, and the photograph replaced with a drawing. It is a drawing showing the amino acid sequence deduced as the entire base sequence of Chitinase A. It is a drawing showing the deduced amino acid sequence of the entire nucleotide sequence of Chitinase B BRIEF DESCRIPTION OF THE DRAWINGS It is drawing explaining the structure comparison of typical Family18 chitinase and pine wood beetle chitinase (Chitinase A, Chitinase B).

Claims (7)

  DNA having the base sequence shown in SEQ ID NO: 1 or 3.   The DNA according to claim 1, wherein the base sequence is derived from a pine beetle, and is a base sequence of a coding region of a chitinase A or chitinase B gene.   The base sequence shown in SEQ ID NO: 1 or 3, comprising a base sequence in which one or several bases are deleted, substituted or added, and encodes a protein that acts as a chitinase 2. DNA.   4. DNA according to claim 1, 2 or 3, characterized in that it comprises a conserved region 2 of insect chitinase (family 18).   DNA comprising a sequence complementary to the DNA sequence of any one of claims 1 to 4.   A protein encoded by the DNA of any one of claims 1 to 5 and acting as a chitinase. The protein according to claim 6, comprising any one of the following proteins (a) to (d):
(A) A protein comprising the amino acid sequence set forth in SEQ ID NO: 2 in the sequence listing.
(B) A protein having an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence described in SEQ ID NO: 2 in the sequence listing and having chitinase activity.
(C) A protein comprising the amino acid sequence set forth in SEQ ID NO: 4 in the sequence listing.
(D) A protein having an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence described in SEQ ID NO: 4 in the sequence listing and having chitinase activity.