Hydrolase protein P1 from bacillus velezensis HN-2 and application thereof

Abstract

The invention discloses a hydrolase protein P1 from bacillus velezensis HN-2 and application of the hydrolase protein P1. The protein can specifically interact with TMV or CMV and other plant viruses, replication, propagation and other processes of the viruses are inhibited, and the plant virus diseases are effectively prevented and treated. The protein is applied to plants in a manner of spraying or transgenosis expression and the like, so that the virus resistance of the plants can be remarkably improved, the loss of the plants caused by virus infection is reduced, and the protein has a good application prospect.

Description

Technical Field

[0001] The present invention relates to the field of plant biotechnology, and particularly relates to a hydrolase protein P1 derived from Bacillus velezensis HN-2 and its application. Background Art

[0002] Tobacco, as an important cash crop, is widely planted globally. However, tobacco virus diseases seriously affect the yield and quality of tobacco, causing huge economic losses to the tobacco industry. Common tobacco virus diseases include Tobacco Mosaic Virus (TMV), Cucumber Mosaic Virus (CMV), Potato Virus Y (PVY), etc. Tobacco Mosaic Virus (TMV) belongs to the positive single-stranded RNA virus category of Tobamovirus. It has a simple genomic structure and rich genetic information, which makes it regarded as a key model organism in virology research. TMV has a wide host range and can infect nearly 400 plant species in 38 families. When TMV infects these plants, its genetic information will quickly enter tobacco cells and replicate. When a plant is diseased, its chlorophyll will be damaged, which will lead to a decline in the plant's photosynthesis ability, thereby affecting its healthy growth. Eventually, it may cause problems such as deformed, dwarfed or even dead tobacco leaves, making the tobacco leaves lose their original use value. Cucumber Mosaic Virus (CMV) is a single-stranded RNA virus with a tripartite genome. CMV has an extremely wide host range and can infect more than 1000 plant species in 85 families, 365 genera, including many important crops and ornamental plants. On tobacco, the symptoms after CMV infection are complex and diverse. Initially, the leaves show chlorotic yellow spots, which then gradually expand to form a mosaic symptom. The leaves are often distorted and deformed, and the plants are dwarfed. In addition to being transmitted by sap friction, CMV can also be transmitted by insects such as aphids. This makes CMV more likely to spread in tobacco fields. Once it occurs, it often spreads rapidly, causing large-scale tobacco infection.

[0003] In the prevention and treatment of tobacco viruses, although agricultural and physical methods have been tried, these methods are not satisfactory in terms of control effect and economic return. Subsequent chemical control measures, such as using chemical drugs like benzothiadiazole and dufulin, although they can have a certain effect, the use of chemical pesticides may lead to pesticide residues and pose a threat to the soil ecosystem and human health. Therefore, more and more people have begun to pay attention to biological control methods. As a relatively environmentally friendly and sustainable control method, biological control has received extensive attention in recent years. Currently, biological control mainly uses some microorganisms and their metabolites to control plant diseases. Previously, the applicant isolated a biocontrol strain named HN-2 from rhizosphere soil. Through morphological and molecular biological identification, it was identified as Bacillus velezensis, and this bacterium has strong antibacterial ability and a wide antibacterial range. The technical solution of the present invention is designed with B. velezensis HN-2 as the main research material, which is very beneficial for the control of tobacco viruses and is expected to provide a new effective way for the control of tobacco virus diseases. Summary of the Invention

[0004] In view of the deficiencies of the prior art, the purpose of the present invention is to provide a new application of a hydrolase protein P1 derived from Bacillus velezensis HN-2 in the control of tobacco viruses. By inhibiting the replication, transmission and other processes of tobacco viruses with this protein, tobacco virus diseases can be effectively controlled, and the yield and quality of tobacco can be improved.

[0005] The technical solution of the present invention is as follows:

[0006] The application of hydrolase protein P1 or its gene in the control of plant virus diseases or in the preparation of plant virus disease control agents, wherein the amino acid sequence of the hydrolase protein P1 is shown in SEQ ID NO.2, and the nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0007] Further, the plant includes tobacco.

[0008] Further, the virus includes tobacco mosaic virus TMV and / or cucumber mosaic virus CMV.

[0009] The present invention also provides a preparation method of the hydrolase protein P1, which includes the following steps: cloning the hydrolase protein P1 from Bacillus velezensis HN-2, and Bacillus velezensis HN-2 is preserved in the China Center for Type Culture Collection, with the preservation number CCTCC NO: M2018382, the preservation date: June 20, 2018, and the preservation address: Wuhan University, China.

[0010] Based on the above-mentioned protein and its gene, they can be prepared into various products for preventing and controlling plant virus diseases. For example, they can be prepared into a biological agent, which can be an aqueous solution, suspension or emulsion containing the protein, and is applied to the plant through spraying, smearing or injection, etc., so that the protein can contact and act on tobacco virus. In addition, a transgenic vector can also be constructed to introduce the gene encoding the protein into plant cells, so that the plant itself can express the protein, thereby continuously resisting virus infection.

[0011] On the other hand, the present invention provides a method for preventing and controlling plant virus diseases, which includes the following steps: contacting a plant with the hydrolase protein P1 or an agent containing the hydrolase protein P1, wherein the amino acid sequence of the hydrolase protein P1 is shown in SEQ ID NO.2, and the virus includes tobacco mosaic virus TMV and / or cucumber mosaic virus CMV.

[0012] The contacting methods include conventional agricultural agent usage methods such as root irrigation, smearing, injection and / or spraying, etc.

[0013] The usage concentration of the hydrolase protein P1 is 2 - 20 mg / mL.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0015] The protein of the present invention can specifically interact with plant viruses and precisely inhibit processes such as virus replication and transmission. Compared with traditional prevention and control methods, the prevention and control effect on plant virus diseases is significantly improved, and the yield loss and quality decline caused by virus infection in plants such as tobacco can be effectively reduced.

[0016] This protein belongs to a biologically active substance. Compared with chemical agents, it is environmentally friendly and will not cause environmental pollution, meeting the requirements of the development of green agriculture.

[0017] Compared with some microorganisms used in biological control, the properties of the protein are relatively stable, and it can maintain a relatively stable prevention and control effect, reducing the risk of fluctuations in the prevention and control effect caused by environmental factors. Description of the Drawings

[0018] Figure 1 : The disease situation of tobacco leaves on the 4th day after inoculating TMV, A: Total protein of HN-1, B: Total protein of HN-2; C: SDS-PAGE analysis of total proteins of HN-1 and HN-2, M: Protein marker.

[0019] Figure 2 : SDS-PAGE analysis of P1 protein, M: Protein marker, A: pET-32a, B: P1.

[0020] Figure 3:Disease incidence of tobacco 3 days after inoculation with TMV. A: Mock, B: TMV-GFP, C: TMV-GFP-HN-2P 2 mg / mL, D: TMV-GFP P1 20 mg / mL, E: TMV-GFP P1 10 mg / mL, F: TMV-GFP P1 2 mg / mL.

[0021] Figure 4 :Disease incidence of tobacco leaves 3 days after inoculation with TMV. A: Mock, B: TMV-GFP, C: TMV-GFP-HN-2P 2 mg / mL, D: TMV-GFP P1 20 mg / mL, E: TMV-GFP P1 10 mg / mL, F: TMV-GFP P1 2 mg / mL.

[0022] Figure 5 :Number of green fluorescent spots 3 days after inoculation with TMV. Different letters indicate significant differences between treatments (Duncan's multiple range test P < 0.05).

[0023] Figure 6 :Western blotting analysis of TMV-GFP protein levels in leaves at 3 dpi. A: Mock, B: TMV-GFP, C: TMV-GFP-HN-2P 2 mg / mL, D: TMV-GFP P1 20 mg / mL, E: TMV-GFP P1 10 mg / mL, F: TMV-GFP P1 2 mg / mL.

[0024] Figure 7 :PCR detection results of viruses in leaves after inoculation with CMV.

[0025] Figure 8 :RT-qPCR detection results of viruses in leaves after inoculation with CMV. Detailed implementation manners

[0026] To better understand the technical content of the present invention, the present invention will be further described below in conjunction with specific embodiments and drawings.

[0027] Experimental example 1 Inhibitory effect of hydrolase protein P1 on TMV

[0028] Select a biocontrol bacterium B. velezensis HN-2 with strong antibacterial ability and broad antibacterial spectrum. This bacterium is preserved in the China Center for Type Culture Collection (CCTCC) (preservation number CCTCC M 2018382). After analyzing its total protein by liquid chromatography-mass spectrometry (LC-MS / MS) and other methods, a protein with a size of 29.375 kDa, which is presumed to be related to hydrolase, is obtained and named P1.

[0029] To express the P1 protein, the P1 gene was amplified from the chromosomal DNA of B. velezensis HN-2 (the nucleotide sequence of the P1 gene is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2), cloned into the EcoR I and Hind III sites of the expression plasmid pET-32a, and transformed into the host Escherichia coli BL21(DE3). The BL21(DE3) cells containing the expression plasmid were cultured with shaking at 180 rpm in LB medium containing 100 μg / ml ampicillin at 37 °C until the OD 600 reached 0.5 - 0.7. The bacterial liquid in the culture flask was supplemented with isopropyl β-D-thiogalactoside (final concentration 1 mM). After culturing with shaking at 37 °C and 180 rpm for 12 h, the cells were collected by centrifugation at 10,000 g for 20 min. The cells were resuspended in PBS (pH = 7.3), lysed with a cell disruptor, and the cell debris was removed by centrifugation at 10,000 g for 20 min. The hydrolytic enzyme protein P1 expressed in the supernatant was collected and diluted with PBS (pH = 7.3). Five to six leaves of Nicotiana benthamiana with consistent growth conditions were selected and the following treatments were set: Nicotiana benthamiana treated with water, Nicotiana benthamiana with its leaves sprayed with P1 protein at a concentration of 20 mg / mL, Nicotiana benthamiana with its leaves sprayed with P1 protein at a concentration of 10 mg / mL, Nicotiana benthamiana with its leaves sprayed with P1 protein at a concentration of 2 mg / mL, and Nicotiana benthamiana with its leaves sprayed with the total protein of HN-2 at a concentration of 2 mg / mL. After 48 h of treatment with the agents, tobacco mosaic virus (TMV+GFP) carrying green fluorescent protein was inoculated. The negative control was Nicotiana benthamiana treated with water without virus inoculation, and the positive control was Nicotiana benthamiana pretreated with water and then inoculated with virus. According to the number of lesions on the leaves of Nicotiana benthamiana and the content of green fluorescent protein in the leaves, the control effect of P1 on TMV was judged.

[0030] The inhibitory effects of the total protein of HN-2 and the total protein of HN-1 on TMV were compared (HN-1 is another strain of Bacillus velezensis preserved in our laboratory). Figure 1 -A is the inhibitory effect of tobacco on TMV after treatment with HN-1 protein, Figure 1 -B is the inhibitory effect of tobacco on TMV after treatment with HN-2 protein. It can be seen that the inhibitory effect of the total protein of HN-2 on TMV is significantly better than that of the total protein of HN-1. SDS-PAGE protein gel electrophoresis analysis of the extracted total proteins of HN-2 and HN-1 showed that a specific band appeared in the total protein of HN-2, with a molecular weight of approximately 25 - 35 kDa. The results are shown in Figure 1-C. Proteomic analysis was performed on the top 100 differential proteins in the total proteins of HN-2 and HN-1 using LC-MS / MS. GO classification analysis (P-adjust < 0.05) was carried out on HN-1 protein and HN-2 protein to obtain the functional information of DEGs. The DEGs of the top 100 differential genes of both were enriched in 25 secondary classification terms under 3 primary classification names of biological process, cellular component, and molecular function. Among the biological processes, there were mainly 10 parts including D-alanine metabolic process, D-alanine family amino acid metabolic process, D-alanine biosynthesis process, D-alanine family amino acid biosynthesis process, alanyl-tRNA aminoacylation, D-amino acid biosynthesis process, the process of L-threonine catabolism to glycine, D-amino acid metabolic process, mRNA catabolic process, and alanine biosynthesis process; among the cellular components, there were mainly 6 parts including cytoplasm, intracellular organelles, intracellular, organelles, cell, and cell membrane, with the first 5 having a relatively large proportion; among the molecular functions, there were mainly catalytic activity, ligase activity, uridine kinase activity, alanine racemase activity, glycine C-acetyltransferase activity, polyribonucleotide nucleotidyltransferase activity, alanine-tRNA ligase activity, 3'-5'-exoribonuclease activity, UMP kinase activity, acyl-CoA dehydrogenase activity, among which catalytic activity and ligase activity had a relatively large proportion, especially catalytic activity. According to the KEGG annotation results and official classification, the differential genes were classified into biological pathways. The results showed that there were mainly 10 pathways including vancomycin resistance, RNA degradation, pyruvate metabolism, pyrimidine metabolism, purine metabolism, pentose phosphate pathway, microbial metabolism in different environments, glyoxylate and dicarboxylate metabolism, glycine, serine and threonine metabolism, and D-alanine metabolism. Among them, the Pvalue of the microbial metabolism pathway in different environments was the smallest, and the number of enriched genes was also relatively large. It can be seen that the differentially expressed genes of HN-1 protein and HN-2 protein ranked in the top 100 were involved in many life processes such as metabolism, cell composition, transcriptional regulation of stimulus response, and biological regulation. By comprehensively analyzing the results of GO and KEGG analysis, the differential protein data table, and the SDS-PAGE gel results, there was a protein at 29.375 kDa that was presumed to be a hydrolase involved in biofilm formation, named P1. P1 might be the reason for the difference in tobacco's resistance to TMV infection between the total proteins of HN-1 and HN-2.

[0031] Protein P1 has 264 amino acids and a size of 29.375 kDa. It was predicted that protein P1 has no signal peptide, no transmembrane domain, is a single subcellular localization, and the subcellular localization is extracellular. The hydrolase protein P1 was expressed in vitro by prokaryotic expression and SDS-PAGE was carried out, and the results are as Figure 2As shown in the figure, the prokaryotically expressed P1 protein was diluted with PBS (pH = 7.3), and the infection of TMV among different treatment groups was compared by inoculating TMV after treatment with TMV-GFP alone and 2 mg / mL total HN-2 protein. The results are as Figure 3 , Figure 4 shown. Three days after treatment, the inhibitory effect of tobacco treated with the hydrolase protein P1 on TMV was significantly better than that of the total HN-2 protein. The lesion conditions are as Figure 3 and Figure 4 , and the number of lesions is as Figure 5 shown. Among them, the treatment effect of 10 mg / mL P1 was the best, and the number of lesions was reduced by 1.57 times compared with the treatment with the total HN-2 protein, and the inhibition rate was 79.07%. The content of GFP in tobacco protein after treatment was determined by Western Blot method, and the results are as Figure 6 shown. It was found that the band color of the P1 treatment was significantly lighter than that of other treatment groups. The hydrolase protein P1 can delay the infection of TMV and is superior to the total HN-2 protein.

[0032] In summary, through exogenous expression and activity detection, it was found that P1 plays an important role in inducing resistance and delaying the infection of TMV. This scheme provides a basis for the future application of Bacillus and its proteins in the prevention and control of TMV.

[0033] Experimental Example 2 Inhibitory effect of hydrolase protein P1 on CMV

[0034] The P1 protein obtained in Experimental Example 1 was taken, and Nicotiana benthamiana with 5 - 6 leaves in the same growth condition was selected. The leaves were sprayed with 10 mg / mL P1 protein. After 12 h of treatment with the agent, cucumber mosaic virus (CMV) was inoculated, and the CP protein of CMV virus was detected after 192 h. The positive control was Nicotiana benthamiana inoculated with the virus after being treated with water. Viral RNA was extracted from the leaves of each group, and the viral load was detected by PCR and RT-qPCR after reverse transcription. The results are as Figure 7 and Figure 8 shown. The results showed that the P1 protein could significantly inhibit the viral load of CMV, reducing the viral load by 99.93%.

[0035] The above are only some embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall fall within the protection scope of the present invention.

Claims

1. Use of hydrolase protein P1 in preventing and treating plant virus diseases or in preparing medicaments for preventing and treating plant virus diseases, characterized in that, The amino acid sequence of the hydrolase protein P1 is shown in SEQ ID NO.

2.

2. The application according to claim 1, wherein The plant includes tobacco.

3. The application according to claim 1, wherein The virus includes tobacco mosaic virus TMV and / or cucumber mosaic virus CMV.

4. Use of the gene encoding the hydrolase protein P1 in preventing and treating plant virus diseases or in preparing agents for preventing and treating plant virus diseases, characterized in that, The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

5. The application according to claim 4, wherein The virus includes tobacco mosaic virus TMV and / or cucumber mosaic virus CMV.

6. The application according to claim 4, wherein The plant includes tobacco.

7. The application according to any one of claims 1 to 6, characterized in that, The preparation method of the hydrolase protein P1 includes the following steps: cloning the hydrolase protein P1 from Bacillus velezensis HN-2, and Bacillus velezensis HN-2 is deposited in the China Center for Type Culture Collection with the deposit number CCTCC NO:M 2018382.

8. A method for preventing and controlling plant virus diseases, characterized in that, Including the following steps: contacting the plant with the hydrolase protein P1 or an agent containing the hydrolase protein P1, the amino acid sequence of the hydrolase protein P1 is shown in SEQ ID NO.2, and the virus includes tobacco mosaic virus TMV and / or cucumber mosaic virus CMV.

9. The method according to claim 8, wherein The contacting methods include root irrigation, smearing, injection and / or spraying.

10. The method according to claim 8, wherein The use concentration of the hydrolase protein P1 is 2-20 mg / mL.

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