A glycoside hydrolase of Rhizoctonia solani GH64 family and its application

Abstract

The invention discloses a glycoside hydrolase of the GH64 family of Rhizoctonia solani and its application, belonging to the field of genetic engineering technology. The enzyme ID is 5093, the nucleotide sequence is shown in SEQ ID NO.1, and the amino acid sequence of the encoded protein is shown in SEQ ID NO.2. The invention uses the above gene to construct a recombinant strain, performs prokaryotic expression, and finds that the glycoside hydrolase 5093 has the ability to induce disease resistance. The enzyme plays an important role in the prevention and control of sheath blight of gramineous crops, laying a foundation for the development of new biological pesticides.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a glycoside hydrolase of Rhizoctonia solani GH64 family and an application thereof. Background Art

[0002] Rhizoctonia solani mainly infects germinating seeds in the soil, and can also infect pods, roots, leaves and stems, often causing sheath blight on gramineous crops such as rice, corn and wheat. The disease starts from the base of the stem and develops upward, forming cloud-like light brown to dark brown spots on the leaf sheaths. In severe cases, it can also infect the stems, causing rot in the stem nodes and symptoms such as lodging and dead ears. In recent years, biological control has become one of the hot topics in domestic and foreign research fields. A full study of the functions and mechanisms of the pathogen's effectors with induced resistance activity is of great significance for studying new strategies for the control of sheath blight. Summary of the invention

[0003] The nucleotide sequence of the GH64 family glycoside hydrolase 5093 of the present invention is shown in SEQ ID NO.1, and the amino acid sequence thereof is shown in SEQ ID NO.2.

[0004] The 5093 nucleotide sequence is shown in SEQ ID NO.1:

[0005] atgggtcgtacattcacggtctataatgcatgcccattcactgtttggcccgctatcttcactgacctgaacgttggctcggctgttcccgcaatcga

[0006] aaacggatgggaagcaccagcgttctcaaagcgctccttcaccgttcctgacaactggaaagccggacgcatctggggtcgcacccaatgtga

[0007] tttctcccagaacccaggaatcaagtcctgtctcactggtggatgtaatggtggattggtgtgcgataacaagactggggttggccttccgccggt

[0008] cacttttgccgagtttacgttgggcgctgatctgcaggactatgttgacgtgagcttggtcgacgggttcaacttgccgatgcgtatctcgaacaat

[0009] ggtggatgcggaactgccgagtgcaccgtgaacctcaacaaggattgccctgagcctcttaggggacctttggacaccaatggggatgtcgct

[0010] gcctgcaagtccgcctgcttggctcagatcgataacccgtccgattctccaagctgctgcaccggcgggttccagaaaccttcaacctgccctgc

[0011] ctccggcgtcttgtattatgccttcttcaagaaccgatgcccgaattcctatgtatatgcttacgacgaatcaagcggtactgcattgtggacttgcccttcgtcgaagaaggtcgattatactgtgactttctgtcct.

[0012] The amino acid sequence of 5093 is shown in SEQ ID NO.2 (specifically MKTAVILAAIAGSALGRTFTVYNACPFT VWPAIFTDLNVGSAVPAIENGWEAPAFSKRSFTVPDNWKAGRIWGRTQCDFSQNPGIKSCLTGGCNGGLVCDNKTGVGLPPVTFAEFTLGADLQDYVDVSLVDGFNLPMRISNNGGCGTAECTVNLNKDCPEPLRGPLDTNGDVAACKSACLAQIDNPSDSPSCCTGGFQKPSTCPASGVLYYAFFKNRCPNSYVYAYDESSGTALWTCPSSKKVDYTVTFCP), the N-terminal 15 amino acids are signal peptide; the molecular weight is 45kD.

[0013] One of the purposes of the present invention is to provide a glycoside hydrolase of the GH64 family of Rhizoctonia solani, the nucleotide sequence of the glycoside hydrolase is shown in SEQ ID NO.1, and the amino acid sequence of the glycoside hydrolase is shown in SEQ ID NO.2.

[0014] The second object of the present invention is to provide an expression vector containing the above-mentioned Rhizoctonia solani GH64 family glycoside hydrolase.

[0015] Preferably, the expression vector is pET28a-sumo.

[0016] The third object of the present invention is to provide a strain containing the above expression vector.

[0017] Preferably, the strain is BL21(DE3).

[0018] The fourth object of the present invention is to provide the use of the above-mentioned Rhizoctonia solani GH64 family glycoside hydrolase in the prevention and treatment of sheath blight of gramineous crops.

[0019] Preferably, the gramineous crop is corn.

[0020] The fifth object of the present invention is to provide a product for preventing and controlling sheath blight of gramineous crops, wherein the product contains the above-mentioned Rhizoctonia solani GH64 family glycoside hydrolase.

[0021] Preferably, the product is any one of an aqueous solution, a wettable powder, a dust, and a granule.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The present invention reveals for the first time the biological function of the GH64 family glycoside hydrolase 5093 of Rhizoctonia solani, and uses genomic analysis and bioinformatics analysis to screen for GH64 glycoside hydrolase family domains, thereby determining that it plays a certain role in the process of infecting host plants. The new glycoside hydrolase is a GH64 family, and prokaryotic expression further verifies that 5093 has a good ability to induce disease resistance in corn. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the result of signal peptide function verification in Example 1.

[0025] Figure 2 The SDS-PAGE analysis results of the protein after induced expression in Example 1, wherein M is a marker, the first represents before induction, and the second 1 and second 2 refer to the strains after induced by adding arabinose and IPTG, respectively.

[0026] Figure 3 The results of SDS-PAGE analysis of the protein after ultrasonic disruption in Example 1, wherein 1 represents the 5093 precipitate and 2 represents the supernatant.

[0027] Figure 4 The results of the analysis of the ability to induce disease resistance in Example 2, wherein 46 was inoculated with only the fusion group strain of Rhizoctonia solani AG4HG-III, ck was the control group sprayed with the pET28a-sumo empty vector bacterial solution, and 5093 was the group sprayed with the 5093 protein treatment. DETAILED DESCRIPTION

[0028] Example 1

[0029] (1) Screening of glycoside hydrolase domain proteins

[0030] Our research group isolated the dominant pathogen from millet sheath blight samples collected from Baoding, Cangzhou, Handan and other places in Hebei Province, and identified it as the AG4 HG-III fusion group through morphology and ITS-based molecular biology. The genome was sequenced using PacBio and Illumina sequencing platforms, and 9 proteins with glycoside hydrolase domains were obtained through bioinformatics analysis (see Table 1).

[0031] Table 1 Identified genes with GH64 domains

[0032]

[0033] (2) RNA extraction from Rhizoctonia solani AG4 HG-III fusion strain

[0034] Grind corn leaves at different stages into powder in a pre-cooled mortar and put them in a 1.5 mL EP tube and place them on ice. Add 1 mL of TransZol Up to the EP tube with the sample, mix and shake immediately, place it on ice for 10 min, add 1 mL of chloroform to the EP tube, mix and shake immediately, place it on ice for 10 min, centrifuge it at 4°C and 13,300 rpm for 15 min, transfer the supernatant in the EP tube after centrifugation to a new EP tube, add the same volume of isopropanol, turn it over and mix it, refrigerate it at -20°C for 20 min, at 4°C and 12,000 rpm, centrifuge it for 10 min, discard the supernatant, open the lid and let it stand for 10 min at room temperature to dry, add an appropriate amount of RNA dissolving solution to the precipitate in the previous step, incubate it in a 65°C water bath for 10 min, aspirate 2 μL of sample for electrophoresis detection, perform reverse transcription, and the obtained cDNA is the template for subsequent experiments, and the rest is stored at -80°C for later use.

[0035] (3) Construction of recombinant vector pSUC2-5093

[0036] The cDNA in step (2) was used as a template to amplify the 5093 coding gene sequence, and the signal peptide of the 5093 coding gene sequence was cloned between the EcoR I and Xho I restriction sites of the pSUC2 vector to obtain the recombinant vector pSUC2-5093SP. The recombinant vector pSUC2-5093SP was transformed into the cloning host E. coli DH5α, and the obtained transformants were sequenced to verify whether they were the correct gene clones. The strains with correct sequencing were selected, and the recombinant plasmid pSUC2-5093SP was extracted.

[0037] (4) Validation of yeast secretion system

[0038] On the first day, pick the YTK12 colony on the YPD solid plate to 5mL of YPD liquid medium and culture overnight at 30℃ and 220rpm. On the second day, dilute the culture to a concentration of about 0.2, and continue to culture for 3-4h to reach the logarithmic period of YTK12 yeast growth, that is, the competent cell preparation is complete. Collect the competent bacteria at 3400rpm and wash twice with sterile ddH2O. Prepare the transformation system: 50% PEG3350, 240μL; lithium acetate, 36μL; ssDNA, 10μL; pSUC2 plasmid (pSUC2-5093SP, pSUC2-mg87 or pSUC2-Avr1b), 2μL; ddH2O, 72μL. Pipette and mix the transformation system and let it stand at room temperature for 30min. Incubate the transformation system at 42℃ for 15min and let it stand on ice for 5min. Centrifuge at 3400 rpm for 4 min, discard the supernatant, and suspend the bacteria in about 200 μL of sterile ddH2O. Spread the bacterial solution on a CMD / W solid plate and incubate it upside down in a 30°C incubator for 3 days.

[0039] The positive transformants pSUC2-5093SP, pSUC2-Mg87 (negative control, a signal peptide lacking secretion function identified in rice blast fungus), and pSUC2-Avr1b (positive control, a signal peptide with strong secretion function identified in soybean phytopathogenes) after secondary screening on CMD / W solid plates were inoculated into 5 mL of liquid CMD / W medium and cultured at 30°C and 250rpm for 36 hours. At the same time, YTK12 was inoculated into 5 mL of YPD liquid medium. The bacterial suspension of different transformants was diluted to the same concentration, and the same amount was spotted on CMD / W, YPRAA and Sucrose medium respectively, and inverted at 30°C to observe the ability of the recombinant yeast strain to utilize the two disaccharides. The results are shown in Figure 1 .

[0040] Collect the cultured cells from the previous step, wash away the culture medium with sterile water, and collect pure cells: resuspend the bacterial solution in 750μL ddH2O, add 250μL 10mM acetic acid-sodium ethyl buffer (pH=4.7) and 500μL 10% sucrose solution, and incubate in a 37℃ water bath for 10min. After centrifugation, take about 100μL of the supernatant and put it into a glass test tube. Add 900μL 0.1% TTC solution to the glass test tube and react at room temperature for 5min. Observe the color change of the solution in the test tube. For the results, see Figure 1 .

[0041] The YTK12 strain carrying pSUC2-5093SP and positive pSUC2-Avr1b grew well on Sucrose and YPRAA medium, indicating that the signal peptide of the candidate effector protein 5093 has the same activity as the Avr1b signal peptide, and can restore the normal secretion pathway of sucrase after fusion expression with the SUC2 gene, thereby decomposing disaccharides in the two media into monosaccharides that can be used by the YTK12 strain. At the same time, the pSUC2-Mg87 negative control lacks the ability to secrete sucrase like the wild-type strain and cannot grow normally on YPRAA and Sourse medium. The growth of the strain on CMD / W medium was used as a control.

[0042] Enzyme activity assay can further verify the secretion activity of the effector protein. 2,3,5-Triphenyltetrazolium chloride (TTC) is a redox indicator that can react with reducing sugars to form brick-red precipitated triphenylformamide (TTF). The YTK12 strain carrying pSUC2-5093SP and positive pSUC2-Avr1b can hydrolyze sucrose into monosaccharides, which react with TTC to turn the colorless solution into brick red. The wild-type and negative control pSUC2-mg87 strains did not produce a color reaction when reacting with TTC. Figure 1 It can be seen that the 5093 signal peptide has secretion function.

[0043] (5) Construction of recombinant vector pET28a-sumo5093

[0044] The 5093 signal peptide encoding gene sequence was cloned between the BamHI and NotI restriction sites of the pET28a-sumo vector to obtain the recombinant vector pET28a-sumo5093, and the recombinant vector pET28a-sumo5093 was transformed into the cloning host E. coli DH5α. The obtained transformants were sequenced to verify whether they were correct gene clones, the strains with correct sequencing were selected, and the recombinant plasmid pET28a-sumo5093 was extracted.

[0045] DH5α competent cell transformation method: The ligation product was transformed into Escherichia coli DH5α by the heat shock method. The specific operation was as follows: 10 μL of the ligation product was added to the thawed competent cells DH5α in the clean bench, and then placed on ice for 25 min. After heat shock at 42°C for 45 s, the cells were immediately cooled on ice for 2 min. 500 μL of LB culture medium was added and the cells were resuscitated in a shaker for 1 h. The cells were centrifuged at 4000 rpm for 1 min, and 100 μL of the bacterial solution was spread on a Kana-resistant LB solid plate for overnight culture.

[0046] (6) Construction of the molecular chaperone BL21(DE3) / pET28a-sumo5093 in Escherichia coli

[0047] Plasmid pET28a-sumo5093 was extracted from E. coli DH5 and transformed into competent E. coli BL21 (DE3) cells. The culture was centrifuged and spread on LB plates containing kanamycin (50 μg / mL), cultured at 37°C overnight, and single colonies were picked for sequencing verification. The correct ones verified by sequencing were recombinant engineered strains containing the glycoside hydrolase 5093 gene.

[0048] (7) Preparation of glycoside hydrolase pET28a-SUMO5093 protein

[0049] The genetically engineered bacteria BL21(DE3) / pET28a-sumo5093 were inoculated into LB liquid medium containing kanamycin (50 μg / mL) and cultured overnight at 37°C and 220 rpm. The next day, they were inoculated into fresh medium and cultured at 25°C with shaking until the OD 600 The final concentration of arabinose was 0.4-0.6, and arabinose was added at a final concentration of 2mM. The mixture was incubated at 25°C for 6h. IPTG was added again for induction for 8h (28°C, 200rpm). The cells were collected by centrifugation at 12000rpm for 10min. The results of SDS-PAGE are shown in the figure. Figure 2 As shown in the figure, the protein obtained was produced under the optimal conditions for glycoside hydrolase induction. The cells were washed twice with 1× Lysis buffer, resuspended and ultrasonically disrupted, and centrifuged at 4°C and 12000rpm to remove the precipitate. The supernatant was the crude enzyme solution. The SDS-PAGE results were shown in the figure. Figure 3 As shown, the obtained protein is a crude enzyme solution of glycoside hydrolase.

[0050] (8) GH64 family glycoside hydrolase activity detection

[0051] Fill each well of the 48-well plate with washing solution, let it stand for 1 minute, shake off the liquid in the well, and pat it dry on absorbent paper (repeat 5 times). Take out the required strips from the aluminum foil bag after equilibration at room temperature for 20 minutes, set up standard wells and sample wells, add 50μL of different concentrations of standard wells, add 10μL of the sample to be tested to the sample well, and then add 40μL of sample diluent, add 100μL of horseradish peroxidase-labeled detection antibody to the standard wells and sample wells, seal the reaction wells with a sealing film, and bathe at 37℃ for 60min; discard the liquid, pat dry on absorbent paper, fill each well with washing solution, let it stand for 1min, shake off the washing solution, and pat dry on absorbent paper (repeat 5 times); add 50μL of substrate A and B to each well, incubate at 37℃ in the dark for 15min; add stop solution, and measure the OD value at 450nm within 15min.

[0052] The results showed that the empty enzyme activity of pET28a-sumo was 0.11334U / L, and the enzyme activity of pET28a-sumo5093 was 0.2424U / L.

[0053] Example 2

[0054] Application of glycoside hydrolase pET28a-SUMO5093 protein:

[0055] Seeds of corn inbred line B73 were sterilized with sodium hypochlorite for 5 minutes, rinsed with sterile distilled water 3 times, soaked overnight, and planted in a flower pot with a ratio of nutrient soil: vermiculite = 3:1. The crude enzyme solution obtained under the optimal conditions in Example 1 was sprayed on corn leaves. After 12 hours, the fusion group strain of Rhizoctonia solani AG4 HG-III grown on PDA medium for 5 days was hit with a sterile blue gun tip and inoculated on the above corn leaves. The leaves were wrapped with plastic wrap to keep them moist for 24 hours. The disease was observed after one week. The experiment was repeated 3 times.

[0056] The results are as follows Figure 4 As shown, compared with the control group, the disease resistance of corn leaves treated with GH64 family 5093 was enhanced, indicating that glycoside hydrolase 5093 has a certain ability to induce disease resistance against the pathogenicity of Rhizoctonia solani.

[0057] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A glycoside hydrolase of Rhizoctonia solani GH64 family, characterized in that The amino acid sequence of the glycoside hydrolase is MGRTFTVYNACPFTVWPAIFTDLNVGSAVPAIENGWEAPAFSKRSFTVPDN WKAGRIWGRTQCDFSQNPGIKSCLTGGCNGGLVCDNKTGVGLPPVTFAEFTLGADLQDYVDVSLVDGFNLPMRISNNGGCGTAECTVNLNKDCPEPLRGPLDTNGDVAACKSACLAQIDNPSDSPSCCTGGFQKPSTCPASGVLYYAFFKNRCPNSYVYAYDESSGTALWTCPSSKKVDYTVTFCP.

2. A gene encoding the GH64 family glycoside hydrolase of Rhizoctonia solani according to claim 1, characterized in that: The gene sequence encoding the glycoside hydrolase is shown in SEQ ID NO.

1.

3. An expression vector containing the gene of the Rhizoctonia solani GH64 family glycoside hydrolase as claimed in claim 2.

4. The expression vector according to claim 3, characterized in that The starting vector of the expression vector is pET28a-sumo.

5. A strain containing the expression vector according to claim 4.

6. The strain according to claim 5, characterized in that The starting strain of the strain is BL21 (DE3).

7. Use of the Rhizoctonia solani GH64 family glycoside hydrolase according to claim 1 in the prevention and treatment of sheath blight in gramineous crops.

8. The use according to claim 7, characterized in that: The gramineous crop is corn.

9. A product for preventing and controlling sheath blight of gramineous crops, characterized in that: The product contains the glycoside hydrolase of the Rhizoctonia solani GH64 family as described in claim 1.

10. The product according to claim 9, characterized in that The product is any one of an aqueous solution, a wettable powder, a dust, and a granule.

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