Chitinase from myxococcus flavus and application of chitinase

Abstract

The invention discloses chitinase derived from myxococcus flavus and application of the chitinase. The amino acid sequence of the chitinase ChitM disclosed by the invention is shown as the 22 -406 amino acid sequence of SEQ ID NO. 2. The chitinase ChitM disclosed by the invention has a remarkable splitting action on plant pathogenic fungi such as banana disc elongispora, banana fusarium oxysporum, tomato fusarium oxysporum, magnaporthe oryzae and botrytis cinerea, and when the encoding gene of the chitinase ChitM is transferred into arabidopsis thaliana, the resistance of the arabidopsis thaliana with the transferred ChitM gene to botrytis cinerea is obviously improved compared with that of wild arabidopsis thaliana; besides, the activity of ChitM is compared with that of commercial chitinase C.Chit, it is found that when the substrates are chitin and shrimp shells, the enzyme activity of the two is not obviously different, when the substrates are chitosan, the activity of ChitM is obviously higher than that of C.Chit, and when the substrates are soluble chitosan and colloidal chitin, the enzyme activity of C.Chit is obviously higher than that of ChitM, and when the substrates are soluble chitosan and colloidal chitin, the enzyme activity of ChitM is obviously higher than that of C.Chit. The results show that the enzyme has great application potential in the aspects of biological prevention and control of plant fungal diseases, disease resistance, breeding and environmental protection.

Description

Technical Field

[0001] The invention relates to the field of genetic engineering and microbial technology, and in particular to a novel chitinase derived from predatory myxobacteria and application thereof. Background Art

[0002] Plant diseases seriously affect the yield and quality of crops. In actual production, about 70% to 80% of diseases are caused by plant pathogenic fungi, causing huge losses to agricultural production. According to statistics, banana wilt infection can reduce banana production by more than 50%, tomato wilt infection can cause tomato production loss of about 30%-60%, and rice blast infection can cause annual rice production loss of about 10%-30%. At present, chemical agents are mainly used in production to prevent and control plant fungal diseases, and have achieved certain results, but it is still very important to improve plant resistance to pathogenic fungi.

[0003] Chitinase has been attracting much attention in recent decades because of its ability to degrade chitin (the main component of the cell wall of plant pathogenic fungi) without any harm to the host plant. At present, many studies have characterized chitinases from plants and microorganisms. For example, chitinase Sschi61 from Streptomyces sampsonii KJ40 treated mycelial cells of Pestalotiopsis trachicarpicola, the black spot pathogen of Eucommia ulmoides, swelled, broke and dissolved; chitinase from Lactobacillus coryniformis 3N11 had a significant inhibitory effect on the growth of Alternaria alternata ET57 on the plate. But overall, there are still few chitinases that have been characterized experimentally, and most of them are from eukaryotic organisms, while there are fewer chitinases from bacteria. In addition, it is difficult to express chitinase efficiently. The production process of commercial chitinase is complicated and expensive, which limits its application range. In-depth exploration of new chitinases with high activity and efficient expression from microbial sources and characterization of their enzymatic properties are key means to solve the current status quo of the industry and have great practical significance.

[0004] Myxobacteria are considered to be "higher prokaryotes" with predatory capabilities. Our research group has isolated a strain of Myxococcus xanthus R31 with strong predatory activity in the early stage. Its genome is rich in various extracellular lytic enzymes, such as chitinase, peptidoglycanase, etc. Based on the evaluation of the predatory activity of Myxococcus xanthus R31 on 5 plant pathogenic fungi, we cloned, expressed, purified, characterized enzymatically, evaluated in vitro antibacterial activity and studied its application potential. We found that the enzyme can lyse the cell walls of a variety of plant pathogenic fungi. After it was transferred into Arabidopsis, the transgenic Arabidopsis had significantly enhanced resistance to gray mold, indicating that the enzyme has great application potential in biological control of plant fungal diseases and disease-resistant breeding. Summary of the invention

[0005] The present invention aims to provide a novel chitinase ChitM derived from predatory myxobacteria and application thereof.

[0006] The amino acid sequence of the novel chitinase ChitM of the present invention is shown in the amino acid sequence at positions 22 to 406 of SEQ ID NO.1, and the nucleotide sequence of the encoding gene is shown in the base sequence at positions 22 to 406 of SEQ ID NO.2.

[0007] Specifically, the chitinase ChitM is from the predatory myxobacterium Myxococcus xanthus R31, comprising 406 amino acids, the amino acid sequence of which is shown in SEQ ID NO.1 (specifically), the N-terminal 21 amino acids are signal peptides, and the theoretical molecular weight of mature ChitM is 44Kda.

[0008] Furthermore, the glycoside hydrolase ChitM of the present invention also encompasses: the protein used is a mutant of chitinase ChitM, including modification, insertion or deletion of a small amount of amino acids in the protein.

[0009] The present invention also discloses a recombinant vector comprising the coding gene of the chitinase ChitM. Preferably, the recombinant vector is pPiczαA-chitM.

[0010] The present invention also discloses a recombinant engineering bacterium comprising the above recombinant vector. Preferably, the host of the recombinant engineering bacterium is Pichia pastoris. Preferably, the recombinant engineering bacterium is the Pichia pastoris engineering strain KM71H / chitm.

[0011] The chitinase chitM of the invention is obtained by fermentation, culture, induction, secretion, expression, purification and recovery of the recombinant Pichia pastoris engineering strain KM71H / chitm.

[0012] The present invention also provides the use of chitinase ChitM in cracking chitin, especially in preventing and controlling plant pathogenic fungi or degrading shrimp shells or crab shells, preferably in cracking the cell wall of plant pathogenic fungi and inhibiting their growth.

[0013] The plant pathogens are plant pathogenic fungi, including banana wilt pathogen, tomato wilt pathogen, rice blast pathogen and gray mold pathogen.

[0014] The invention also provides the use of chitinase ChitM in degrading solid wastes such as shrimp shells, chitosan, soluble chitosan, etc.

[0015] The chitinase ChitM of the present invention has a significant lysis effect on plant pathogenic fungi such as banana fusarium, banana wilt fungus, tomato wilt fungus, rice blast fungus, tomato gray mold fungus, etc., and the encoding gene thereof is transferred into Arabidopsis thaliana, and it is found that compared with the wild-type Arabidopsis thaliana, the resistance of the Arabidopsis thaliana transferred with the ChitM gene to gray mold is significantly improved; in addition, the activities of ChitM and commercial chitinase (C.Chit) are compared, and it is found that when the substrates are chitin and shrimp shells, there is no significant difference in the enzyme activities of the two, when the substrate is chitosan, the activity of ChitM is significantly higher than that of C.Chit (p<0.05), and when the substrates are soluble chitosan and colloidal chitin, the enzyme activity of C.Chit is significantly higher than that of ChitM (p<0.001, p<0.005), and the above results show that the enzyme has great application potential in biological control of plant fungal diseases, disease-resistant breeding and environmental protection. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Schematic diagram of confrontation culture of Myxococcus xanthus R31 and plant pathogenic fungi

[0017] Figure 2The results of confrontation culture of predatory myxobacterium Myxococcus xhanthus R31 and five plant pathogenic fungi; the first row of a is the colony morphology of the five fungi without confrontation culture after 7 days of culture, and the second row is the colony morphology after confrontation culture with strain R31 for 7 days; b is the statistical results of the colony size of the five fungi and each fungus in the confrontation culture plate, *, *** and **** represent significant differences at p<0.05, p<0.005 and p<0.001 levels, respectively, and the error bars represent the standard errors from three repeated experiments; Gloeosporium musarum Cooke et Mass GDMCC3.403, Fusarium oxysporum f.sp.Cubense GDMCC 3.566, Botrytis cinerea B05.10, Fusarium oxysporum f.sp.lycopersiciFol) and rice blast fungus (Magnaporthe oryzae Guy11).

[0018] Figure 3 is the SDS-PAGE analysis of the purified ChitM protein, where M is the molecular weight marker (14-120 kDa); 1, L is the precipitate after crushing, S is the supernatant after crushing; 1-10 purified ChitM;

[0019] Figure 4 The results of ChitM's antibacterial experiment on plates; the first row shows the morphology of banana disk spore fungus (GDMCC 3.403), banana wilt fungus (GDMCC 3.566), gray mold (B05.10), tomato wilt fungus (Fol) and rice blast fungus (Guy11) on PDA plates after PBS treatment; the second row shows the morphology of banana disk spore fungus, banana wilt fungus, gray mold, tomato wilt fungus and rice blast fungus treated with ChitM. The plate diameter is 9 cm.

[0020] Figure 5 The morphology of plant pathogenic fungal spores after ChitM treatment was observed under an optical microscope; the first row shows the spore morphology of GDMCC 3.403, Fusarium wilt (GDMCC 3.566) and Botrytis cinerea (B05.10) treated with PBS for 12 h; the second row shows the spore morphology of GDMCC 3.403, Fusarium wilt and Botrytis cinerea treated with ChitM; the arrows indicate the generated protoplasts; the scale bars in a, e, c and f are 50 μm, the scale bars in b and d are 20 μm, and the scale bars in g and h are 10 μm.

[0021] Figure 6Transmission electron microscopy observation of the morphology of banana disk spores (GDMCC 3.403) after ChitM treatment

[0022] Figure 7 Figure 1 shows the results of resistance test of Arabidopsis thaliana with chitM gene to gray mold; Figure a shows the phenotype of each Arabidopsis plant after spraying and inoculating B05.10 spores; Figure b shows the statistical results of the incidence and disease index of transgenic Arabidopsis thaliana. I, II, III, IV, V and VI represent repeated experiments.

[0023] Figure 8 The statistical analysis results of the phenotype and lesion size of wild-type and PR SP-chitM22-406-transformed Arabidopsis after spot-inoculation with B05.10; Note: Figure a shows the leaves of PR SP-ChitM22-406-transformed Arabidopsis and wild-type Arabidopsis after spot-inoculation with B05.10; Figure b shows the statistical results of the leaf lesion size in Figure a.

[0024] Fig. 9 Comparison of the activities of ChitM and commercial chitinase C.Chit; Figure a is chitin; Figure b is shrimp shell, Figure c is chitosan, Figure d is soluble chitosan; Figure e is colloidal chitin; the values ​​in the figures represent the average of three repeated experiments, the error bars represent the standard error, ns represents no significant difference in t-test analysis, * represents significant difference at the level of p<0.05, *** and **** represent significant differences at the levels of p<0.005 and p<0.001, respectively.

[0025] Fig.10 This is the vector map of plasmid pBA-35S. DETAILED DESCRIPTION

[0026] The following are specific implementation examples of the present invention. It should be noted that these embodiments are merely exemplary and do not constitute any limitation to the scope of the present invention. Modifications and replacements made to the details and forms of the implementation scheme within the scope of the present invention fall within the protection scope of the present invention.

[0027] Unless otherwise defined, the technical terms used in the following examples have the same meanings as those generally understood by those skilled in the art. The experimental reagents used in the following examples are all conventional biochemical reagents unless otherwise specified; the experimental methods are all conventional methods unless otherwise specified.

[0028] Example 1 Determination of the inhibitory activity of predatory myxobacterium R31 against plant pathogenic fungi

[0029] The predatory myxobacterium R31 was inoculated on VY / 2 plates (yeast powder 5 g / L, CaCl2·2H2O 1 g / L, agar 15 g / L, pH 7.2) in advance and cultured at 30°C for 3 days. The five test plant pathogenic fungi (Gloeosporium musarum Cooke et Mass GDMCC 3.403, Fusarium oxysporum f.sp. Cubense GDMCC 3.566, Botrytis cinerea B05.10, Fusarium oxysporum f.sp. lycopersici Fol) and Magnaporthe oryzae Guy11) were inoculated on PDA plates (potato 200 g / L, glucose 20 g / L, agar 15 g / L) and cultured at 30°C for 5-7 days. Schematic diagram of the determination of the predation ability of strain R31 on plant pathogenic fungi is shown in Figure 1 The control group was inoculated with plant pathogenic fungi on VY / 2 plates ( Figure 1 a), the experimental group first used a sterile puncher to inoculate strain R31 at a distance of 1.5 cm from the edge of the VY / 2 plate, and each plate was inoculated with four ( Figure 1 b), and after culturing at 30°C for 2 days, five strains of plant pathogenic fungi were inoculated in the center of the same plate ( Figure 1 The inoculated plates were incubated at 30°C, and the growth diameter of the plant pathogenic fungi was measured and recorded every 24 hours. The experiment was repeated three times.

[0030] The results are as follows Figure 2 As shown in the figure, compared with the control fungus that was not cultured in confrontation, strain R31 inhibited the growth of the five tested plant pathogenic fungi ( Figure 2 a). We further measured the colony diameters of the fungi in the control and confrontation culture plates after 7 days of culture and conducted statistical analysis. The results showed that the colony diameters of the five fungi in the confrontation culture plates were significantly smaller than those of the control without confrontation culture ( Figure 2 b), which indicates that strain R31 has good inhibitory activity against the five tested plant pathogenic fungi.

[0031] Example 2: Construction of recombinant vector pPiczαA-chitm

[0032] First, primers were designed based on the original nucleotide sequence of chitinase ChitM (positions 64-1215 of SEQ ID NO.1) and cloned into the ppiczaαA vector (HonorGene, catalog number: HG-VJI0296) linearized with SacI to obtain the recombinant vector ppiczaαA-chitm, and the recombinant vector ppiczaαA-chitm was transformed into the cloning host E.coil DH5α. The obtained transformants were sequenced to verify whether they were correct gene clones (the same as positions 64-1215 of the nucleotide sequence SEQ ID NO.1), the strains with correct sequencing were selected, and the recombinant plasmid ppiczaαA-chitm was extracted.

[0033] Example 3 Construction of Pichia pastoris engineered strain KM71H / chitm

[0034] Plasmid pPiczαA-chitm was extracted from E.coil DH5α, and electroporated into Pichia pastoris KM71H competent cells. The transformant was spread on a YPDS plate (1% yeast extract, 2% peptone, 2% glucose, 1 mol / L sorbitol, 2% agar powder) containing bleomycin (200 mg / mL), cultured at 30°C for 48 hours, and a single colony was picked for sequencing verification. The correct sequencing verification (same as the 64th to 1518th position of the nucleotide sequence SEQ ID NO.1) was the genetically engineered bacteria KM71H / chitm containing the chitinase ChitM.

[0035] Example 4. Preparation of chitinase ChitM protein

[0036] A single colony of the genetically engineered bacteria KM71H / chitm was inoculated into YPD medium (YPDS without sorbitol) and cultured overnight at 30°C. The next day, it was transferred to 10 mL of BMGY medium (YNB 13.4 g / L, glycerol 10 mL / L, pH 6.0 K2HPO4 / KH2PO4 buffer) and cultured at 30°C, 250 rpm until OD600 = 2-6; 10 mL of the bacterial solution was inoculated into 1 L of BMGY medium and cultured under the same conditions until OD 600 =2-6; centrifuge at 3000g for 5min at room temperature to collect the cells, resuspend the cells with 100mL of BMMY medium (BMGY does not contain glycerol), place in a 1L Erlenmeyer flask and continue to culture, add pure methanol to a final concentration of 0.5 every 24h to induce expression, centrifuge at 3000g for 5min after 7 days of continuous induction, collect the supernatant, and the supernatant is the crude enzyme solution. The crude enzyme solution was filtered with a water filter membrane with a pore size of 0.45μm and filtered with Ni 2+The protein was purified by affinity chromatography column, eluted with imidazole gradient, and the eluent was collected. Then, the solution was replaced with 50mM phosphate buffer using a 10kDa ultrafiltration tube and concentrated. The purity of the protein was tested by SDS-PAGE, and the protein concentration was determined using a Bradford kit. The SDS-PAGE results are shown in Figure 2. Figure 3 The obtained protein is chitinase ChitM, and its amino acid sequence is shown in the amino acid sequence at positions 22-405 of SEQ ID NO.2.

[0037] Example 5. Application of chitinase ChitM in lysing plant pathogenic fungi

[0038] Use an Oxford cup to make a PDA plate with holes. Inoculate the well-growing pathogenic fungi in the center of the PDA plate. When the pathogenic fungi grow to 0.5 cm from the hole, add 200 μL of 1 mg / mL ChitM enzyme solution to the hole. Add enzyme solution every 12 hours. Use the plate with phosphate buffer as a control to observe the growth of pathogenic fungi.

[0039] The results showed that purified ChitM had a certain inhibitory effect on the growth of five tested plant pathogenic fungi in vitro ( Figure 4 ), among which the best antibacterial effect was against banana spore fungus and banana wilt fungus.

[0040] Plant pathogenic fungi (Banana spore fungus, banana wilt fungus, gray mold fungus) were activated on PDA plates. When the growth was good, spores were collected and chitinase solution was added to react at 37°C for 12 hours. The control was treated with phosphate buffer. After the reaction, the reaction solution was observed under an optical microscope and a transmission electron microscope for spore morphology changes.

[0041] The spore morphology of pathogenic fungi after ChitM treatment was observed under an optical microscope. The results showed that compared with the control spores ( Figure 5 Compared with a and b), most of the spores of Banana Disk Macrospora GDMCC 3.403 after ChitM treatment turned into protoplasts ( Figure 5 c and d); after ChitM treatment, a small number of spores of banana wilt pathogen GDMCC 3.566 and gray mold pathogen B05.10 also turned into protoplasts ( Figure 5 f and h), indicating that ChitM can lyse the cell wall of pathogenic fungi in vitro.

[0042] The spores of GDMCC 3.403 treated with ChitM were observed by transmission electron microscopy. The results showed that the spores of GDMCC 3.403 treated with PBS had intact morphology and structure, while the spore cell walls of GDMCC 3.403 treated with ChitM became loose or even ruptured, and the cells swelled as a whole ( Figure 6), which is consistent with the results observed under an optical microscope. The results also indicate that ChitM can inhibit the growth of pathogenic fungi in vitro by lysing / destroying the cell wall of pathogenic fungi.

[0043] Example 6. Recombinant vector pBA-35S-chitM 1-406 、pBA-35S-chitM 22-406 and pBA-35S-PR SP-chitM 22-406 Construction

[0044] Plasmid pBA-35S (gift from Professor Zhang Tong of South China Agricultural University, vector map as shown in Figure 1) was cleaved with restriction endonucleases XhoⅠ and SacⅠ. Fig.10 The linearized pBA-35S vector was obtained by double restriction digestion of the chitM gene fragment (shown in SEQ ID NO.1), the full-length chitM gene fragment (shown in SEQ ID NO.1, positions 64-1215) and the gene fragment with the PR gene signal peptide (shown in SEQ ID NO.3) were connected by homologous recombination (i.e., the PR gene signal peptide replaced the signal peptide in SEQ ID NO.1) and transformed into the cloning host E.coil DH5α. The obtained transformants were sequenced to verify whether they were the correct gene clones (the same as positions 1-1218 of the nucleotide sequence SEQ ID NO.1), the strains with the correct sequencing were selected, and the recombinant plasmid pBA-35S-chitM was extracted. 1-406 、pBA-35S-chitM 22-406 and pBA-35S-PR SP-chitM 22-406 .

[0045] Example 7. Construction of chitM-transgenic Arabidopsis

[0046] The three constructed plant expression vectors and the empty vector were transformed into Agrobacterium competent EHA105 by heat shock method, and the transformed bacterial solution was spread on LB medium containing Spec and Rif, cultured at 28°C for 2-3 days, and single clones were picked for sequencing verification. The verified correct strains were transformed into wild-type Arabidopsis thaliana by Agrobacterium-mediated floral dip method. After Arabidopsis thaliana matured, seeds were collected according to different genes, dried at 37°C, and sown on MS medium containing 0.001% Basta. Positive plants grew normally with green leaves.

[0047] Example 8. Identification of resistance of transgenic Arabidopsis to gray mold

[0048] The resistance of each transgenic Arabidopsis to Botrytis cinerea was evaluated mainly by spraying and spot inoculation of Botrytis cinerea spores. The specific method was to use a blue gun tip to punch holes along the edge of the colony on a plate of Botrytis cinerea B05.10 that had been cultured for 7 days, inoculate it into a spore-forming medium, and culture it in the dark at 25°C for 5 days, and granular spores could be seen. Prepare a 1% glucose solution, add 0.05% Tween-80 to make a spore wash solution, and oscillate to mix. Take 5 mL of spore wash solution and add it to the spore-forming medium. Shake gently. After all spores are washed, collect the wash solution in a 50 mL EP tube, calculate the spore concentration using a hemocytometer, and finally adjust the spore concentration to 5×10 with the spore wash solution. 5 / mL and 1×10 5 Use a small spray bottle to spray the prepared solution with a concentration of 1×10 5 Spray the spore liquid of 1000 / mL evenly on the wild-type and transgenic Arabidopsis with about 20 leaves, add sufficient water to the square dish to maintain humidity, cover with a plastic lid, and culture in the dark at 22°C for 24 hours, then culture under normal light. After 5 days, observe and count the disease status of each plant.

[0049] The concentration is 5×10 5 6 μL of spore solution was dripped onto Arabidopsis leaves. 22-406 There were 3 pots of genetic Arabidopsis thaliana, with 5 leaves in each pot. Sufficient water was added to the square dish to maintain humidity. The dish was covered with a plastic lid. After culturing in the dark at 22°C for 24 h, normal light cultivation was carried out at 22°C. After 3 days, the disease status of each leaf was observed and counted.

[0050] First, the incidence rate of each plant is counted. The incidence rate is the ratio of the number of diseased leaves to the total number of leaves in the plant. Then the number of diseased leaves at each level is counted to calculate the disease index:

[0051] Disease index = (∑〖(index at each level × number of leaves at each level)〗) / (total number of experimental leaves × 9 (highest disease level)) × 100

[0052] The plant disease grade is divided into levels 0-9, where level 0 means no infection and no visible cell necrosis; level 1 means the size of leaf spots is less than 5% of the total leaf area; level 3 means the size of leaf spots is 5%-15% of the leaf area; level 5 means the size of leaf spots is 15%-25% of the leaf area; level 7 means the size of leaf spots is 25%-50% of the leaf area; level 9 means the size of leaf spots is more than 50% of the leaf area.

[0053] The resistance of transgenic Arabidopsis to Botrytis cinerea was determined by spraying the leaves of wild-type Arabidopsis and each transgenic Arabidopsis plant with spores of Botrytis cinerea B05.10, while the wild-type plant that was not sprayed with the pathogen was used as the control. Figure 7a It can be seen that compared with the control, lesions appeared on the leaves of wild-type Arabidopsis and each transgenic Arabidopsis, but 22-406 The disease was mildest in Arabidopsis plants with the PR SP-chitM gene, with only some water-soaked spots on the leaves with the lowest disease severity, and no obvious necrosis on the leaves with the highest disease severity. We further conducted a statistical analysis of the disease incidence and disease index of each group of Arabidopsis plants. The results showed that the PR SP-chitM gene 22-406 The incidence and disease index of Arabidopsis thaliana expressing chitM gene were significantly lower than those of wild type, empty vector and chitM transgenic plants. 1-406 and chitM 22-406 Arabidopsis thaliana; while the empty vector and chitM 1-406 and chitM 22-406 The incidence and disease index of Arabidopsis thaliana were not significantly different from those of wild type controls ( Figure 7 b).

[0054] At the same time, we also used the dot-grafting method to clone wild-type and transgenic PR SP-chitM 22-406 The results showed that after the non-transgenic wild-type Arabidopsis was inoculated with B05.10 spores, the leaves at the inoculation site showed obvious necrosis, while the PR SP-chitM transgenic 22-406 Although lesions appeared at the inoculated site of transgenic Arabidopsis, the degree of necrosis and the area of ​​lesions were smaller than those of the wild-type control without transgenic genes ( Figure 8 a), Statistical analysis of lesion diameter showed that the PR SP-chitM 22-406 The lesion diameter of Arabidopsis thaliana with the gene was significantly smaller than that of the wild-type control ( Figure 8 b) These results indicate that the secreted chitM gene can indeed enhance the resistance of Arabidopsis to Botrytis cinerea.

[0055] Example 9 Comparison of the activity of chitinase ChitM and commercial chitinase (C.Chit)

[0056] The purified ChitM and commercial chitinase (C.Chit) were adjusted to the same concentration with 50mM phosphate buffer. 50mg of chitin, shrimp shell, chitosan, soluble chitosan and colloidal chitin were taken respectively, and 1mL of 50mM phosphate buffer was added to prepare a mother solution with a concentration of 50mg / mL. 100μL of the mother solution was added with 100μL of ChitM and C.Chit of the same concentration respectively, and the control was a heat-inactivated enzyme solution. After reacting at 50℃ for 2h, the activities of the two on different substrates were measured to compare the activity differences between the two.

[0057] The results showed that there was no significant difference in activity between chitin and shrimp shells. Fig. 9a and b); when the substrate is chitosan, the activity of ChitM is higher than that of C.Chit( Fig. 9 c); When the substrate is soluble chitosan and colloidal chitin, the activity of C.Chit is higher ( Fig. 9 d and e). The above results indicate that ChitM has certain potential for commercial application.

[0058] The above are only preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be regarded as limiting the present invention, and the protection scope of the present invention should be based on the scope defined by the claims. For ordinary technicians in this technical field, several improvements and modifications can be made without departing from the spirit and scope of the present invention, and these improvements and modifications should be regarded as the protection scope of the present invention.

[0059] 1. chitM gene sequence (1218 bp, the underlined part indicates the signal peptide sequence, SEQ ID NO.1)

[0060] ATGTCCGGCAAGTTCGTGTCACGCAGTCTGGCCCTGCTGGGAATCTGTGGGGGCCTGAG

[0061] CGCC CTGGGAGGCTGTGGCGACGCCGAGCCAGTGCCCGCCGCCCTCGAACCGGTGGGC

[0062] CAGGTGGAAGGCGCCGCCATTGTCGACACCATCACCGAAGGCACCTACGTCATCCGCTC

[0063] GGTGATGACGAACAAGTGCATCGACGTCGCCTCGTCGAGCACGGCGGACGGCGCCAAG

[0064] GTGCAGCAATGGGATTGCAACGGCACGAATGCGCAGCGCTTCCGCGTCACGCCCACGT

[0065] CCGGGGGCTACTTCAGCATCATCAACGTGAACAGCAACAAGGCGCTCGACATCAAGGA

[0066] GGCGAGCACCGCGGCGAACGCGCTGGTCCACCAGTGGGGTTATGGCGGAGGAGCGAAC

[0067] CAGCAGTTCCCGCTTCGTGAAGGAAGTGGGCAGCGAGTTCAGCATCCGCGCGCGTCACA

[0068] CGGACATGGCCATTGACGTGTATTGGGGCAACACGGCCAACGGCACGGAGCTGGTGCA

[0069] GTACCCCTACGAGCAGCGCACGAACCAGCGCTGGACGTTCGACCGCATCGACGGGGGC

[0070] GGTGGAAATCCCGGCACGGGGCTGGCCGCCATCCTGAGCGAGTCCACGTTCAACGCCA

[0071] TGTTCCCGAACCGGAACCCCTTCTACACGTACAGCAGCCTCATCGCCGCCGCGAGCACC

[0072] TTCCCGGCCTTCGCCAACACGGGCTCGCTGGAGACGCGCAAGCGCGAGGTGGCGGCCT

[0073] TCTTCGCCAACACGGCCCATGAGACGGGCAACTACGTGTACGTGGAGGAGATCAATCGC

[0074] GGTGACTACTGCGGCTCATGGGGTCCGCCGGGCTGCTACTGCGTCGCAGGCAAGCAGTA

[0075] CTACGGACGTGGGCCCATCCAGTTGTCCTGGAACGGCAACTACTGCGCCGCCGGCGCCG

[0076] CGCTGGGGCTGCCGCTTCACACCAACCCGGACCTGCTGGCCCAGGACGCCAACGCCGC

[0077] GTGGCGCTCCGCGTTCTGGTTCTGGACCACGCAGGCCGGCGCCGGAACCATGTCCGCG

[0078] CACCGCGCCATGGTCGACGGCTACGGCTTCGGTGAGACCATCCGCACCATCAATGGCTC

[0079] GCTGGAGTGCAATGGTGGCAACCCGGGCCAGGTGCAGAGCCGCATCAACAACTATGAG

[0080] CGCATCACCAACATGCTCGGCGTGAGCCCTGGCGGCAACCTGGGCTGCTGA

[0081] 2. ChitM protein sequence (the underlined part indicates the signal peptide sequence, SEQ ID NO.2)

[0082] MSGKFVSRSLALLGICGGLSA LGCGDAEPVPAALEPVGQVEGAAIVDTITEGTYVIRSVM

[0083] TNKCIDVASSSTADGAKVQQWDCNGTNAQRFRVTPTSGGYFSIINVNSNKALDIKEASTAA

[0084] NALVHQWGYGGGANQQFRFVKEVGSEFSIRARHTDMAIDVYWGNTANGTELVQYPYEQR

[0085] TNQRWTFDRIDGGGGNPGTGLAAILSESTFNAMFPNRNPFYTYSSLIAAASTFPAFANTGSL

[0086] ETRKREVAAFFANTAHETGNYVYVEEINRGDYCGSWGPPGCYCVAGKQYYGRGPIQLSWN

[0087] GNYCAAGAALGLPLHTNPDLLAQDANAAWRSAFWFWTTQAGAGTMSAHRAMVDGYGF

[0088] GETIRTINGSLECNGGNPGQVQSRINNYERITNMLGVSPGGNLGC

[0089] 3. PR SP (Pathogenesis-related protein 1A signal peptide) sequence, SEQ ID NO.3

[0090] ATGGGATTTGTTCTCTTTTCACAATTGCCTTCATTTCTTCTTGTCCTACACTTCTCTTATTCCTAGTAATATCCCACTCTTGCCGTGCC.

Claims

1. A chitinase ChitM, characterized in that Its amino acid sequence is shown in the amino acid sequence at positions 22 to 406 of SEQ ID NO.

2.

2. A gene encoding the chitinase ChitM according to claim 1.

3. The gene according to claim 2, characterized in that The nucleotide sequence of the gene is shown in the 64th to 1215th base sequence of SEQ ID NO.

1.

4. A recombinant plasmid containing the gene according to claim 2 or 3.

5. An engineered bacterium expressing the recombinant plasmid according to claim 4.

6. The engineered bacteria according to claim 5, characterized in that The host bacteria of the engineering bacteria is Pichia pastoris KM71H.

7. Use of the chitinase ChitM according to claim 1 in cleaving chitin.

8. The use according to claim 7, characterized in that The invention is used in controlling plant pathogenic fungi or degrading shrimp shells or crab shells, preferably in breaking down the cell walls of plant pathogenic fungi and inhibiting their growth.

9. The use according to claim 8, characterized in that: The plant pathogenic fungi are Gloeosporium musarum Cooke et Mass GDMCC 3.403, Fusarium oxysporum f.sp.Cubense GDMCC 3.566, Botrytis cinereaB05.10, Fusarium oxysporum f.sp.lycopersici Fol and Magnaporthe oryzaeGuy11.

10. Use of the chitinase ChitM according to claim 1 in disease resistance breeding, preferably introducing it into plants as a disease resistance gene.

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