Application of chitinase, recombinant plasmid and expression strain in plant control

Abstract

The invention discloses application of chitinase, a recombinant plasmid and an expression strain in plant prevention and control, and relates to the technical field of gene engineering, and the chitinase is chitinase CaChi19B; the chitinase comprises a nucleotide sequence, the nucleotide sequence of the chitinase is coded, and the nucleotide sequence of the chitinase is as follows: cccagccggcg The no-load carrier is a pET22b carrier; the primary bacterial strain is E.coli DH5 alpha. According to the application of the chitinase, the recombinant plasmid and the expression strain in plant control, the chitinase CaChi19B has chitinase activity, can inhibit the growth of fungi and kill nematodes, and has a wide application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, and specifically to the application of chitinase, recombinant plasmid, and expression strain in plant control. Background Art

[0002] Plant pathogenic fungi and plant parasitic nematodes are the main factors causing plant diseases, resulting in huge economic losses. At present, chemical fungicides and drugs are mainly used for prevention and control, but the long-term use of chemical agents will bring serious problems such as environmental pollution and the generation of drug resistance in pathogenic bacteria. Therefore, the development of green and efficient biological control methods is an inevitable trend of social development.

[0003] Chitinase is a general term for a class of enzymes with the function of specifically degrading chitin into chito-oligosaccharides or monosaccharides, and is widely distributed in bacteria, fungi, and plants. Chitinase can hydrolyze chitin in the fungal cell wall, causing the extracellular leakage of cell protoplasm, thereby inhibiting the spore germination and hyphal growth of pathogenic fungi. At the same time, the degradation product chito-oligosaccharide acts as an elicitor to activate the plant defense mechanism, inducing the accumulation of chitinase and other antibacterial substances, achieving the effects of antifungal and antibacterial.

[0004] In recent years, the harm of plant parasitic nematodes has become increasingly serious, and the search for highly efficient and low-toxic nematode biological control factors has attracted more and more attention. Chitinase has been proven to be effective against nematodes and will play an increasingly important role in the biological control of nematodes. For example, extracellular enzymes such as chitinase produced by nematophagous fungi can act on the chitin layer of the nematode body surface cuticle and eggshell, playing a key role in the process of infecting nematodes. Due to the huge application potential of chitinase in biological control, the research and utilization of chitinase will have great economic value and theoretical value. Summary of the Invention

[0005] In view of the deficiencies of the prior art, the present invention provides the application of chitinase, recombinant plasmid, and expression strain in plant control, and solves the problems raised in the above background art.

[0006] To achieve the above objectives, the present invention is achieved through the following technical solutions: A chitinase, wherein the chitinase is chitinase CaChi19B;

[0007] The amino acid sequence of the chitinase CaChi19B is as follows:

[0008] MAADWDAKAVYTAGQTVTYQGKTWKAKWWTQGEVPGSQQWGAWGEVAGTVTPVPTVAPTATP

[0009] TVKPTVAPTATPLPTATPTATPTPKPTVTPTATPLPTATPTVAPTVAPTATPTAVPTQPPVVTGPC

[0010] EAGWSAATAYQGGAKVSYQGFNYSAKWWTQGNDPSQGGVWVSSGACSGGSNGGGGTVTPTPAPGGV

[0011] PTKAEAEAYAATLTNSDIFRKVKASVRTLPNSVVEAVAPGKATNPDNVKRVEALVSQQKFDYFFQV

[0012] RNKAYTYQGFLQAVAKFPGFCDTYTDGRNSDEICRRSLAGMFAHFAQETGGHSIVEFGIPEWRQAL

[0013] VHVREMGWSEGMSGYNAECNDPVFNKTWTCGKLPDGKFKGYFGRGAKQLSYNYNYGPFSQAMFDGD

[0014] QFKLLNEPELVADTWLNLASAVFFFIYPQPPKPSMLHVIDGTWVPNDFDKARQLGNDFPTTIQIIN

[0015] AECQDSPTKAAAQNRLDYYTEFSRELGWDIKKESMKCAGMGRFDGGSSAAFNIYWEKDWKVGGDNK

[0016] CQLVSYQTPYNALIDGQYTKCVEANWGVTLK。

[0017] Optionally, the sequence of the nucleotide is as follows:

[0018] atggccgccgactgggacgccaaagccgtgtataccgctggtcagaccgtgacctaccaggg

[0019] caagacctggaaggccaagtggtggacccagggtgaagttccgggatcgcagcagtggggtgcctg

[0020] gggtgaagtggccggtaccgtgactccggtgcctaccgttgctccgactgcaacaccgaccgtcaa

[0021] gccgacagtggccccgaccgctactccgctgccaaccgcgacaccgacggcaacgccgactcccaa

[0022] gccgaccgtaacaccgacagcaaccccgctgccgactgctactccgaccgttgcaccgacggttgc

[0023] tcctaccgcaacacctactgccgtaccgactcagcccccggtagtgactggtccgtgtgaagccgg

[0024] ctggagcgcggcaaccgcttaccagggcggcgccaaggtttcttaccagggcttcaactacagcgc

[0025] caagtggtggacccagggcaacgatccgtcgcaaggcggcgtatgggtgagcagcggcgcctgctc

[0026] gggcggcagcaatggcggtggcggcacggttactccgaccccggctccgggcggtgtgccgaccaa

[0027] ggccgaagccgaagcctacgcagcaacgctgaccaactccgacatcttccgcaaggtgaaggcgtc

[0028] ggtacgcaccctgcccaatagcgtggtcgaagcggttgcgccgggcaaggcgaccaatccggacaa

[0029] cgtgaagcgtgtcgaagcgctggtttcgcaacagaaattcgactacttcttccaggtgcgcaacaa

[0030] ggcctatacctatcagggcttcctgcaggccgtggccaagttcccgggcttctgcgacacctacac

[0031] cgatggccgcaacagcgacgaaatctgccgccgctcgctcgccggcatgtttgcgcacttcgcgca

[0032] ggaaaccggcggtcacagcatcgtcgagttcggcattccggaatggcgccaggctctggtgcacgt

[0033] gcgtgaaatgggctggtctgagggcatgtccggttacaacgccgagtgcaacgacccggtcttcaa

[0034] caagacctggacctgcggcaagctgccggacggcaagttcaagggctacttcggccgtggcgccaa

[0035] gcagctgtcctacaactacaactatggtccgttctcgcaggccatgtttgacggcgaccagttcaa

[0036] gctgctgaatgagccggagttggtagcggatacctggctgaacctggcatctgccgtgttcttctt

[0037] catctacccgcaaccgccgaagccttcgatgctgcacgtgatcgatggtacctgggtgccgaacga

[0038] cttcgacaaggcacgtcagctgggtaatgatttcccgaccaccatccagatcatcaacgccgagtg

[0039] ccaggacagcccgaccaaggctgctgcacagaaccgccttgactactacaccgagttctcgcgcga

[0040] actgggctgggacatcaagaaggaatcgatgaagtgcgccggcatgggccgcttcgacggtggttc

[0041] gtcggcagcgttcaacatctactgggaaaaggactggaaggtcggcggtgacaacaagtgccagct

[0042] ggtcagctaccagaccccgtacaacgccctgatcgacggtcagtacaccaagtgcgtggaagcaaa

[0043] ctggggcgtaacgctgaagtaa。

[0044] Optionally, the primer set for amplifying the nucleotide sequence is cccagccggcgatggccatggatatggccgccgactgggac, ctcgagtgcggccgcaagcttcttcagcgttacgcccca.

[0045] A recombinant plasmid, comprising a nucleotide sequence and an empty vector;

[0046] The empty vector is pET22b.

[0047] A recombinant cloning strain, comprising a nucleotide sequence or a recombinant plasmid and a primary strain;

[0048] The primary strain is E. coli DH5α.

[0049] A recombinant expression strain, comprising a nucleotide sequence or a recombinant cloning strain and a primary expression strain;

[0050] The primary expression strain is E. coli Rosetta2 DE3.

[0051] The application of chitinase, recombinant plasmid, and expression strain in controlling plant pathogenic fungi, wherein the fungus is Coniothyrium diplodiella.

[0052] The application of chitinase, recombinant plasmid, and expression strain in controlling nematodes, wherein the nematodes are one or more of Caenorhabditis elegans, a plant parasitic nematode - Meloidogyne.

[0053] A reagent for controlling fungi, comprising one of chitinase, nucleic acid molecule, recombinant plasmid, recombinant cloning strain, and recombinant expression strain.

[0054] A reagent for controlling nematodes, comprising one of chitinase, nucleic acid molecule, recombinant plasmid, recombinant cloning strain, and recombinant expression strain.

[0055] The present invention provides the application of chitinase, recombinant plasmid, and expression strain in plant control, having the following beneficial effects:

[0056] The applications of the chitinase, recombinant plasmid, and expression strain in plant control. The enzymatic hydrolysis products of chitin by chitinase CaChi19B are GlcNAc, (GlcNAc)2, and (GlcNAc)3, and it has endochitinase activity. Chitinase CaChi19B inhibits the growth of the plant pathogenic fungus Coniothyrium diplodiella and has insecticidal effects on Caenorhabditis elegans and Meloidogyne spp., providing a basis for the research of agents that inhibit pathogenic bacteria and pathogenic insects in the field of biological control. Description of the Drawings

[0057] Figure 1 SDS-PAGE detection results of the expressed chitinase; in the figure, M: Color pre-stained protein marker, 1-2: Chitinase CaChi19B;

[0058] Figure 2 Temperature curve of chitinase enzyme activity;

[0059] Figure 3 Temperature stability curve of chitinase;

[0060] Figure 4 pH curve of chitinase enzyme activity;

[0061] Figure 5 pH stability curve of chitinase;

[0062] Figure 6 Inhibitory effect of chitinase on the plant pathogenic fungus C. diplodiella;

[0063] Figure 7 Nematicidal effect of chitinase, (A-D) Effect of chitinase on Caenorhabditis elegans; (E) Effect of chitinase on Meloidogyne spp. Detailed Embodiments

[0064] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments.

[0065] The amino acid sequence of chitinase CaChi19B is shown in SEQ ID No.1;

[0066] SEQ ID No.1:

[0067] MAADWDAKAVYTAGQTVTYQGKTWKAKWWTQGEVPGSQQWGAWGEVAGTVTPVPTVAPTATPTVKPTVAPTATPLPTATPTATPTPKPTVTPTATPLPTATPTVAPTVAPTATPTAVPTQPPVVTGPCEAGWSAATAYQGGAKVSYQGFNYSAKWWTQGNDPSQGGVWVSSGACSGGSNGGGGTVTPTPAPGGVPTKAEAEAYAATLTNSDIFRKVKASVRTLPNSVVEAVAPGKATNPDNVKRVEALVSQQKFDYFFQVRNKAYTYQGFLQAVAKFPGFCDTYTDGRNSDEICRRSLAGMFAHFAQETGGHSIVEFGIPEWRQALVHVREMGWSEGMSGYNAECNDPVFNKTWTCGKLPDGKFKGYFGRGAKQLSYNYNYGPFSQAMFDGDQFKLLNEPELVADTWLNLASAVFFFIYPQPPKPSMLHVIDGTWVPNDFDKARQLGNDFPTTIQIINAECQDSPTKAAAQNRLDYYTEFSRELGWDIKKESMKCAGMGRFDGGSSAAFNIYWEKDWKVGGDNKCQLVSYQTPYNALIDGQYTKCVEANWGVTLK。

[0068] The nucleotide encoding the amino acid sequence of chitinase CaChi19B is shown in SEQ ID No.2;

[0069] SEQ ID No.2:

[0070]

[0071] The primer pair for amplifying the nucleotide sequence encoding the amino acid of chitinase CaChi19B is shown in SEQ ID No. 3-4. SEQ ID No. 3 is: cccagccggcgatggccatggatatggccgccgactgggac, and SEQ ID No. 4 is: ctcgagtgcggccgcaagcttcttcagcgttacgcccca.

[0072] The technical solutions provided by the present invention will be described in detail below in conjunction with the embodiments, but they cannot be construed as limiting the protection scope of the present invention;

[0073] In the embodiments of the present invention, Coniothyrium diplodiella IVF134 (deposit number ACCC36140) comes from the China Center for Agricultural Culture Collection; Caenorhabditis elegans and Meloidogyne are preserved in the laboratory.

[0074] Example 1: Construction of recombinant plasmid 22b-CaChi19B;

[0075] (1) Primer design. The obtained primer pair is shown in Table 1;

[0076] Table 1 Primer pair

[0077]

[0078] (2) Amplify the chitinase gene. Using the nucleotide sequence of the CaChi19B gene as a template (as shown in SEQ ID No. 2), PCR amplify the chitinase gene;

[0079] The reaction system for PCR amplification is: upstream primer 0.3 μl; downstream primer 0.3 μl; 2×Taq Master Mix 10 μl; ddH2O 9.4 μl; the total volume of the reaction system is 20 μl;

[0080] The reaction conditions for PCR amplification are: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 65°C for 30 s; extension at 72°C for 2 min, a total of 30 cycles; post-extension at 72°C for 10 min;

[0081] (3) After the PCR amplification is completed, recover the amplification product; the obtained product is shown in SEQ ID No. 2;

[0082] (4) Use the restriction endonucleases NcoⅠ and HindⅢ to double-digest the vector pET22b, and recover the product after enzymatic digestion and purification;

[0083] (5) The amplified product obtained in step (3) and the product after double digestion and purification in step 4 are subjected to homologous recombination ligation using 2×ClonExpress homologous recombination enzyme.

[0084] The reaction system for PCR homologous recombination is as follows: 2 μl of purified DNA product; 2 μl of digested vector; 5 μl of 2×ClonExpress Mix; 1 μl of ddH2O; the total volume of the reaction system is 10 μl.

[0085] After mixing the homologous recombination reaction system, it is placed in a 50°C water bath for 5 min, and then immediately cooled on ice; the recombinant plasmid 22b-CaChi19B is obtained.

[0086] Example 2: Expression of chitinase gene;

[0087] (1) Construction of recombinant cloning strain 22b-CaChi19B-E.coli DH5α:

[0088] The recombinant plasmid obtained in Example 1 is chemically transformed into E.coli DH5α respectively, screened by colony PCR and sequenced to obtain the cloning strain 22b-CaChi19B-E.coli DH5α.

[0089] (2) Construction of recombinant expression strain 22b-CaChi19B-E.coli Rosetta2 DE3;

[0090] The recombinant plasmid 22b-CaChi19B is extracted by StarPrep rapid plasmid miniprep kit (Beijing Kangrun Chengye Biotechnology Co., Ltd.), chemically transformed into the expression strain E.coli Rosetta2 DE3, and cultured overnight in a 37°C incubator to obtain the recombinant expression strain 22b-CaChi19B-E.coli Rosetta2 DE3.

[0091] (3) Expression of the recombinant expression strain;

[0092] The recombinant expression strain 22b-CaChi19B-E.coli Rosetta2 DE3 is inoculated into 10 ml of LB medium containing ampicillin (100 mg / ml), cultured overnight at 37°C, and then inoculated into 500 ml of LB medium containing ampicillin (100 mg / ml) at an inoculation amount of 1% v / v, and cultured at 37°C until OD 600 = 0.7, isopropyl-β-D-thiogalactoside (IPTG) is added to make its final concentration in the LB medium 0.1 mM, and induced at 16°C and 200 rpm for 20 h.

[0093] (4) Collect the disrupted cells to obtain the crude chitinase solution;

[0094] After the expression is completed, centrifuge the induced culture solution at 4°C and 7000 rpm for 10 min to collect the cells. Resuspend the cells with citrate-sodium dihydrogen phosphate buffer and disrupt them by ultrasonic treatment on ice. Each ultrasonic treatment lasts for 1 s, with a 2-s interval, and the ultrasonic treatment lasts for 20 min. Then centrifuge at 4°C and 12000 rpm for 30 min to collect the supernatant, which is the crude chitinase solution;

[0095] (5) Purification of the crude chitinase solution;

[0096] Use a His-tag column to purify the crude chitinase solution obtained in step (4). After SDS-PAGE of the purified recombinant protein solution (i.e., chitinase), a protein band of about 60 kDa is obtained (as Figure 1 shown);

[0097] The amino acid sequence of the chitinase CaChi19B obtained after sequencing is shown in SEQ ID No.1.

[0098] Example 3: Determination of chitinase activity;

[0099] Using 20 g / l colloidal chitin as the substrate, determine the enzyme activity of the chitinase CaChi19B prepared in Example 2. Take 400 μl of the substrate (pH 6.5), add 100 μl of the purified chitinase solution, react in a 40°C water bath for 30 min, then transfer it to a boiling water bath and boil for 10 min to inactivate the enzyme. Then add 1 ml of potassium ferricyanide reagent and boil in boiling water for 5 min. Centrifuge at 12000 rpm for 10 min in a centrifuge, using an equal amount of citrate-sodium hydrogen phosphate (100 mM, pH 6.5) as the blank control, and take the supernatant to measure its absorbance at 420 nm. The enzyme activity of the purified CaChi19B is 156 mU / mg;

[0100] The definition of the enzyme activity unit (U) is: under the above conditions, the amount of enzyme required to release 1 μmol of reducing sugar per minute;

[0101] After determination, the products of the chitinase CaChi19B hydrolyzing colloidal chitin are GlcNAc, (GlcNAc)2, and (GlcNAc)3, showing endochitinase activity.

[0102] Example 4: Enzymatic properties of chitinase;

[0103] Determine the enzymatic properties of the chitinase in Example 2, including the optimal temperature, optimal pH, temperature stability, pH stability, the effects of metal ions and chemical reagents on enzyme activity, and measure the enzyme activity using 20 g / l colloidal chitin as the substrate;

[0104] 1. Optimal temperature: The reaction system includes 400 μl of colloidal chitin (20 g / l) and 100 μl of pure chitinase enzyme solution; the pH value is 7.0 (citric acid - sodium dihydrogen phosphate, 100 mM); reactions are carried out for 0.5 h within the temperature gradient ranges of 25°C, 30°C, 40°C, 45°C, 50°C, 60°C, 70°C, and 80°C respectively. Using the inactivated crude enzyme solution as the blank, after the reaction ends, transfer it to a boiling water bath and boil for 10 min to inactivate the enzyme. Then add 1 ml of potassium ferricyanide reagent to the reaction system, boil in boiling water for 5 min, and then measure the absorbance at OD 420 to determine the enzyme activity of chitinase at different temperatures; draw a curve based on the relative enzyme activities of the enzyme at different temperatures to determine the optimal reaction temperature of chitinase; as Figure 2 , the optimal reaction temperature of chitinase CaChi19B is 40°C;

[0105] 2. Temperature stability study: Place the pure chitinase enzyme solution at different temperatures (25°C, 30°C, 40°C, 45°C, 50°C, 60°C, 70°C, 80°C) and incubate for 60 min and 90 min respectively, then detect the residual enzyme activity, compare it with the enzyme activity of the untreated sample, and calculate the relative enzyme activity; the results are as Figure 3 shown; the results indicate that chitinase CaChi19B has a certain tolerance to temperature and has good thermal stability at 25 - 50°C;

[0106] 3. Optimal pH: The substrate is colloidal chitin at 20 g / l, and the enzyme activity is measured in 100 mM citric acid - sodium dihydrogen phosphate buffer solutions with pH values of 3.0, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, and 8.0; the reaction system is the same as that in the optimal reaction temperature experiment, and the reaction temperature is 40°C; draw a curve based on the relative activities of the enzyme at different pH values, as Figure 4 , the optimal reaction pH value of chitinase CaChi19B is determined to be 6.5;

[0107] 4. pH stability: Place the pure chitinase solution in buffers with different pH values (3.0, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0) at 100 mM and let it stand at 4°C for 3 h, then detect the residual enzyme activity at the optimal reaction temperature (40°C), using the untreated enzyme activity as the blank control, and calculate the relative enzyme activity, as Figure 5 shown; when the pH value range of chitinase CaChi19B is 6.0 - 6.5, the enzyme activity remains above 80% after incubation for 3 h, showing high pH tolerance;

[0108] 5. Effects of metal ions on chitinase CaChi19B. Add Fe with a final concentration of 10 mM to the reaction system (the same reaction system as in the optimal reaction temperature experiment), 3+ Ca 2+ Co 2+ Zn 2+ Cu 2+ K + Ni 2+ Na + Mn 2+ Li + Mg 2+ Ba 2 + NH4 + Tris, EDTA, SDS, urea, and then measure the enzyme activity under standard conditions (40 °C, pH 6.5). Taking the enzyme activity of the untreated chitinase enzyme solution as 100%, calculate the effects of metal ions on chitinase, and the results are shown in Table 2;

[0109] Table 2 Effects of metal ions and chemical reagents on chitinase CaChi19B

[0110]

[0111]

[0112] As can be seen from Table 2, most metal ions have an inhibitory effect on the enzyme activity of chitinase CaChi19B.

[0113] Example 5: Study on the antifungal activity of chitinase CaChi19B;

[0114] Measure the antifungal activity of purified CaChi19B through the mycelial growth inhibition test; Add the chitinase solution in Example 2 to the PDA medium, mix well and pour plates. Place the mycelial discs of the same diameter in the center of the PDA plate containing 1 U of chitinase, and use 100 mM citric acid - sodium dihydrogen phosphate buffer (pH 6.5) as a control (CK); After culturing for 3 days, the estimation formula for the mycelial growth inhibition rate (GI) is as follows: GI = (R - r) / R × 100%, where R and r refer to the average diameters of fungal mycelia on the control plate and the experimental plate, respectively; As Figure 6 shown, the enzyme CaChi19B inhibits the growth of the plant pathogenic fungus C. diplodiella, and the inhibition rate is 22.3 ± 1.4%;

[0115] Example 6: Study on the nematicidal effect of chitinase CaChi19B;

[0116] The chitinase solution of Example 2 was used to study the effect of CaChi19B on the mortality of Caenorhabditis elegans; a mixture of OP50 bacterial suspension, L1 nematode solution (16 synchronized L1 worms), and different amounts of chitinase solution was dropped at the center of the plate; after 3 days of observation, it was found that when the enzyme concentration was 0.1 mg / ml, Caenorhabditis elegans grew in groups but away from the enzyme droplet area ( Figure 7 B); when the enzyme concentration was 1 mg / ml, the number of Caenorhabditis elegans was significantly less than that of the control group ( Figure 7 C); the semi-lethal concentration of chitinase CaChi19B on Caenorhabditis elegans was determined using a 12-well culture plate, and its semi-lethal concentration was 1.66 mg / ml ( Figure 7 D);

[0117] To further determine the effect of CaChi19B on nematodes causing plant diseases, the effect of CaChi19B on Meloidogyne incognita was studied; the semi-lethal concentration of chitinase CaChi19B on Meloidogyne incognita was determined using a 12-well culture plate, and its semi-lethal concentration was 0.36 mg / ml ( Figure 7 E);

[0118] As can be seen from the above examples, the present invention provides a chitinase, a recombinant plasmid, an expression strain, and their applications in degrading chitin, controlling plant pathogenic fungi, and plant parasitic nematodes; the products of the enzymatic hydrolysis of colloidal chitin by the chitinase CaChi19B provided by the present invention are mainly GlcNAc, (GlcNAc)2, and (GlcNAc)3, and have endochitinase activity; the chitinase CaChi19B inhibits the growth of the plant pathogenic fungus Coniothyrium diplodiella and has an insecticidal effect on Caenorhabditis elegans and Meloidogyne incognita.

[0119] The above is only a preferred specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention, according to the technical solution and inventive concept of the present invention, making equivalent substitutions or changes, should be covered by the protection scope of the present invention.

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