Alpha / beta hydrolase as 3-hydroxypalmitate signal molecule degrading enzyme and application of alpha / beta hydrolase

Abstract

The invention discloses an alpha / beta hydrolase as a 3-hydroxypalmitate signal molecule degrading enzyme and application of the alpha / beta hydrolase. Alpha / beta hydrolase is obtained through amplification in pseudomonas nitroreducens, and research finds that the alpha / beta hydrolase is a main quenching enzyme with the capability of degrading 3-hydroxypalmitate signal molecules in the pseudomonas nitroreducens and can effectively degrade quorum sensing signals, namely 3-OH PAME, generated by ralstonia solanacearum, so that the quorum sensing signals generated by ralstonia solanacearum can be effectively degraded, and the quorum sensing signals generated by ralstonia solanacearum can be effectively degraded. Normal expression of ralstonia solanacearum pathogenic factors is interfered, and quorum sensing between ralstonia solanacearum is blocked, so that the pathogenicity of ralstonia solanacearum is inhibited, and plant bacterial wilt is effectively prevented and treated. The invention provides a new target for green prevention and treatment of bacterial wilt, and has a wide application prospect in the field of agricultural biocontrol.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular biological control, and more specifically to α / β hydrolase as a 3-hydroxypalmitic acid methyl ester signal molecule degradation enzyme and application thereof. Background Art

[0002] Ralstonia solanacearum (abbreviated as Ralstonia solanacearum) is a Gram-negative bacterium and a soil-borne plant pathogen that is widely distributed in tropical, subtropical and some temperate regions around the world. Ralstonia solanacearum can infect a variety of plants and crops such as tomatoes, potatoes, peanuts, eggplants, and Casuarinas, causing crops to wilt and die, seriously reducing agricultural production. Currently, there are mainly agricultural control, chemical control, physical control, etc. for the prevention and control of bacterial wilt. These methods can slow down the occurrence of bacterial wilt to a certain extent, but these methods all have major drawbacks, such as (1) agricultural control requires a lot of time, money and manpower costs, and its control efficiency is low and the effective period is long, making it difficult to cope with the characteristics of rapid spread and outbreak of bacterial wilt; (2) long-term use of chemical control will cause environmental pollution, damage to ecological balance, increase pathogen resistance, and endanger human health; (3) physical control alone is difficult to play a role and usually needs to be combined with agricultural control, chemical control, biological control, etc.

[0003] Quorum sensing (QS) refers to the ability of microorganisms to coordinate gene expression, regulate population density, and behavior through self-secretion or induction of external signaling molecules, thereby responding to changing environmental conditions. In 1994, Fuqua et al. first proposed the concept of quorum sensing based on this phenomenon. The PhcBSR QS system is the dominant system in Ralstonia solanacearum. Its primary signaling molecule is 3-OH PAME / 3-OH MAME (3-hydroxypalmitic acid methyl ester), and the methyltransferase PhcA is a global virulence regulator. This dominant system controls approximately 30% of genes and is involved in multiple physiological processes, including cell activity, primary and secondary metabolism, and pathogenicity. When the density increases, the methyltransferase PhcB catalyzes the synthesis of 3-OH PAME / 3-OH MAME, which is sensed by the PhcS-PhcRQ two-component system, phosphorylated by PhcR, and expressed by the PhcA transcriptional regulatory factor, stimulating downstream target genes and regulating the expression of pathogenicity factors EPS (exopolysaccharides) of EP1 (epidermal growth factor receptor (EGFR) family recombinant protein), motility, biofilm, type III secretion system and cell wall degrading enzymes.

[0004] Quorum quenching (QQ) is a new type of biological control method that can inhibit or block the quorum sensing system of microorganisms, making it difficult for pathogenic bacteria to express pathogenicity-related genes. This new type of biological control approach is mainly divided into three types: (1) inhibiting the synthesis of signal molecules by the synthesizing enzymes of the quorum sensing system from the source; (2) degrading signal molecules so that the concentration of signal molecules is below the threshold; (3) interfering with or blocking the binding of signal molecules to the receptor proteins of the quorum sensing system. Quorum sensing quenching provides a new breakthrough for plant disease control and can significantly improve the control effect. Modifying bacteria at the molecular level and constructing biocontrol strains with quorum sensing quenching function has also become an efficient and environmentally friendly plant disease control strategy. However, the current research on the use of quorum quenching to control bacterial wilt in large fields is still in its early stages, and its practical application is relatively limited. Therefore, it is urgent to screen and obtain more biological resources from the natural environment to provide other effective solutions for the control of bacterial wilt. Summary of the Invention

[0005] The purpose of the present invention is to overcome the above-mentioned defects and deficiencies in the prior art and to provide an α / β hydrolase as a 3-hydroxypalmitate signal molecule degrading enzyme or in the preparation of a preparation for degrading 3-hydroxypalmitate signal molecules.

[0006] The second object of the present invention is to provide the use of α / β hydrolase or biomaterials related to α / β hydrolase in inhibiting pathogens that rely on 3-hydroxypalmitic acid methyl ester signal molecules to cause disease or in preparing preparations for inhibiting pathogens that rely on 3-hydroxypalmitic acid methyl ester signal molecules to cause disease.

[0007] The third object of the present invention is to provide the use of α / β hydrolase or biological materials related to α / β hydrolase in preventing and controlling plant diseases caused by pathogens that rely on 3-hydroxypalmitate signal molecules to cause pathogenicity or in the preparation of preparations for preventing and controlling plant diseases caused by pathogens that rely on 3-hydroxypalmitate signal molecules to cause pathogenicity.

[0008] The fourth object of the present invention is to provide the use of α / β hydrolase or α / β hydrolase-related biomaterials in improving the ability of nitroreducing Pseudomonas in preventing and controlling plant diseases caused by pathogens that rely on 3-hydroxypalmitate signal molecules to cause disease.

[0009] A fifth object of the present invention is to provide a method for preventing and treating plant diseases caused by pathogenic bacteria that rely on 3-hydroxypalmitic acid methyl ester signal molecules to cause disease.

[0010] The above-mentioned object of the present invention is achieved through the following technical solutions:

[0011] The present invention amplifies α / β hydrolase from Pseudomonas nitroreducens HS-18, the amino acid sequence of which is shown in SEQ ID NO.1, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO.2. The present invention has found that the α / β hydrolase is the main quenching enzyme in Pseudomonas nitroreducens HS-18 that has the ability to degrade 3-hydroxypalmitic acid methyl ester (3-OH PAME) signal molecules. Specifically, the present invention inserts the coding gene of the α / β hydrolase into the pET32a plasmid to construct a recombinant expression vector, transforms the recombinant expression plasmid into the competent BL21 (DE3) pLysS, obtains a prokaryotic expression strain, induces its expression, and obtains in vitro expressed α / β hydrolase. Different concentrations of α / β hydrolase are mixed with 3-OHPAME, and the mixture is extracted after interaction at 37°C. The residual amount of the 3-OH PAME signal molecule is detected by GC-MS technology. The results showed that the residual rate of 3-OH PAME gradually decreased with increasing protease concentration, indicating that the higher the protease concentration, the more significant the degradation effect on 3-OH PAME, indicating that α / β hydrolase is a 3-OH PAME signal molecule degrading enzyme. The present invention further overexpressed α / β hydrolase (24610) in nitroreducing Pseudomonas HS-18 and found that compared with ordinary nitroreducing Pseudomonas HS-18, tomatoes inoculated with HS-18 (24610) did not develop disease until the 10th day, and the tomato survival rate was approximately 55%, indicating that overexpressing α / β hydrolase can effectively enhance the control of bacterial wilt by HS-18. The above results of the present invention indicate that α / β hydrolase interferes with the normal expression of Ralstonia solanacearum pathogenic factors by degrading the 3-OH PAME signaling molecules produced by Ralstonia solanacearum, i.e., by using quorum quenching, thereby blocking quorum sensing among Ralstonia solanacearum and thereby inhibiting the pathogenicity of Ralstonia solanacearum. This provides a new target for the green prevention and control of bacterial wilt disease, and can be used to transform bacteria at the molecular level to construct biocontrol strains with quorum sensing quenching function, which has broad application prospects in the field of agricultural biocontrol.

[0012] Therefore, the present invention provides an α / β hydrolase as a 3-hydroxypalmitate signal molecule degrading enzyme or in the preparation of a preparation for degrading 3-hydroxypalmitate signal molecules. The amino acid sequence of the α / β hydrolase is shown in SEQ ID NO.1.

[0013] The present invention also provides the use of α / β hydrolase or a biological material related to α / β hydrolase in inhibiting pathogens that are dependent on 3-hydroxypalmitate signal molecules to cause disease or in preparing a preparation for inhibiting pathogens that are dependent on 3-hydroxypalmitate signal molecules to cause disease, wherein the amino acid sequence of the α / β hydrolase is shown in SEQ ID NO.1; the biological material related to the α / β hydrolase comprises (1) an expression cassette containing a nucleotide sequence encoding the α / β hydrolase; (2) a recombinant expression vector containing a nucleotide encoding the α / β hydrolase and / or (1); and (3) a recombinant microorganism containing a nucleotide encoding the α / β hydrolase, (1) and / or (2).

[0014] The present invention also provides the use of α / β hydrolase or a biological material related to α / β hydrolase in preventing and treating plant diseases caused by pathogens that rely on 3-hydroxypalmitate signal molecules to cause disease or in preparing a preparation for preventing and treating plant diseases caused by pathogens that rely on 3-hydroxypalmitate signal molecules to cause disease, wherein the amino acid sequence of the α / β hydrolase is shown in SEQ ID NO.1; the biological material related to the α / β hydrolase comprises (1) an expression cassette containing a nucleotide sequence encoding the α / β hydrolase; (2) a recombinant expression vector containing a nucleotide encoding the α / β hydrolase and / or (1); and (3) a recombinant microorganism containing a nucleotide encoding the α / β hydrolase, (1) and / or (2).

[0015] Overexpression of HS-18 (24610) effectively prevents and controls the occurrence of bacterial wilt by means of quorum quenching, providing an innovative approach for green and sustainable prevention and control of bacterial wilt disease, and has great application value.

[0016] The present invention also provides the use of α / β hydrolase or α / β hydrolase-related biological materials in improving the ability of nitroreducing Pseudomonas in preventing and controlling plant diseases caused by pathogenic bacteria that rely on 3-hydroxypalmitate methyl ester signal molecules for pathogenicity, wherein the amino acid sequence of the α / β hydrolase is shown in SEQ ID NO.1; the α / β hydrolase-related biological materials include (1) an expression cassette containing a nucleotide sequence encoding the α / β hydrolase; (2) a recombinant expression vector containing a nucleotide sequence encoding the α / β hydrolase and / or (1).

[0017] The present invention provides a method for preventing and controlling plant diseases caused by pathogenic bacteria that rely on 3-hydroxypalmitate signal molecules for pathogenicity, wherein the method adopts one or more of the following biological materials for prevention and control: (1) an α / β hydrolase; (2) an expression cassette containing a nucleotide sequence encoding (1); (3) a recombinant expression vector containing a nucleotide encoding (1) and / or (2); (4) a recombinant microorganism containing a nucleotide encoding (1), (2) and / or (3); the amino acid sequence of the α / β hydrolase is shown in SEQ ID NO.1.

[0018] Furthermore, the method comprises inoculating the nitroreducing Pseudomonas overexpressing α / β hydrolase onto plants.

[0019] Furthermore, the above-mentioned pathogenic bacteria is Ralstonia solanacearum.

[0020] Furthermore, the above-mentioned plant disease is bacterial wilt.

[0021] Furthermore, the above nucleotide sequence is shown as SEQ ID NO.2.

[0022] Furthermore, the above-mentioned microorganism is nitroreducing Pseudomonas.

[0023] Furthermore, the nitroreducing Pseudomonas is Pseudomonas nitroreducens HS-18.

[0024] Furthermore, the plant is one or more of tomato, potato, peanut, eggplant, and Casuarina equisetifolia.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] The present invention provides an α / β hydrolase as a 3-hydroxypalmitoyl methyl ester signal molecule degrading enzyme and its application. The present invention amplifies and obtains an α / β hydrolase from nitroreducing Pseudomonas. Studies have found that the α / β hydrolase is the main quenching enzyme in nitroreducing Pseudomonas that has the ability to degrade the 3-hydroxypalmitoyl methyl ester signal molecule. It can effectively degrade the quorum sensing signal produced by Ralstonia solanacearum, namely 3-OH PAME, interfere with the normal expression of Ralstonia solanacearum pathogenicity factors, block quorum sensing between Ralstonia solanacearum, thereby inhibiting the pathogenicity of Ralstonia solanacearum, and effectively preventing and controlling plant bacterial wilt. The present invention provides a new target for the green prevention and control of bacterial wilt and has broad application prospects in the field of agricultural biocontrol. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The results are verified by PCR of prokaryotic expression strains.

[0028] Figure 2 It is used for SDS-PAGE electrophoresis detection of α / β hydrolase protein.

[0029] Figure 3 3-hydroxypalmitic acid methyl ester (3-OH PAME) residual amount of GC-MS spectrum. Figure 3 a is the control group of protease 24610*; b is the sample group treated with 1 mg / mL protein concentration; c is the sample group treated with 0.125 mg / mL protein concentration.

[0030] Figure 4The following are the statistical results of the degradation rate of 3-hydroxypalmitoyl methyl ester (3-OH PAME). Note: ck means no protease was added, and 24610* means inactivated protease.

[0031] Figure 5 PCR verification results for the construction of the HS-18 (24610) overexpression strain.

[0032] Figure 6 This shows the growth of tomatoes after inoculation.

[0033] Figure 7 is the survival rate of tomatoes after inoculation. DETAILED DESCRIPTION

[0034] The present invention will be further described below with reference to the accompanying drawings and specific examples, but the examples do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0035] Unless otherwise specified, the reagents and materials used in the following examples were commercially available.

[0036] Example 1 Construction of recombinant expression vector

[0037] (1) Amplification of target gene

[0038] Pseudomonas nitroreducens HS-18 was activated and shaken overnight to obtain seed liquid. DNA was extracted using the EasyPure Bacteria Genomic DNA kit. Using the HS-18 genome as a template, specific primers 32a-24610-F (GAGTGCGGCCGCAAGCTTtcaagcatgcagaaagcgca, SEQ ID NO. 3) and 32a-24610-R (ATGGCTGATATCGGATCCatgaccgccacattgtctgtg, SEQ ID NO. 4) were used to amplify the target gene, α / β hydrolase (24610), using the high-fidelity enzyme Phanta Max Master Mix. The amino acid sequence is shown in SEQ ID NO. 1, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO. 2.

[0039] Amplification reaction system: 25 μL of 2× Phanta Max Master Mix, 2 μL of upstream and downstream primers (10 μM each), 1 μL of template DNA, and 20 μL of ddH₂O. PCR protocol: 95°C initial denaturation for 30 seconds, 95°C denaturation for 15 seconds, 64°C annealing for 15 seconds, 72°C extension for 1 minute (34 cycles), and 72°C full extension for 5 minutes.

[0040] The PCR product was detected by 1.0% agarose gel electrophoresis and analyzed under ultraviolet light. After confirming the single band, the target fragment was purified and recovered using the Cycle-Pure Kit (200) from OmegA. The specific steps are as follows:

[0041] ① Add 180 μL of CP Buffer to 45 μL of PCR product, pipette to mix thoroughly, transfer to a centrifuge column, centrifuge at 14,000 rpm for 1 min, and discard the waste liquid;

[0042] ② Add 700 μL Washing Buffer, centrifuge at 14,000 rpm for 1 min, and discard the waste liquid;

[0043] ③ Repeat step ②, then centrifuge at 14000 rpm for 2 min and discard the waste liquid;

[0044] ④ Place the centrifuge column in a new 1.5mL centrifuge tube, open the lid and let it stand for 2 minutes. Add 31μL of ddH2O preheated to 65℃ to the center of the centrifuge column. Let it stand for 2 minutes, then centrifuge at 14000rpm for 60s. Collect the product, detect the concentration, and store it at -20℃ until use.

[0045] (2) Connection conversion

[0046] E. coli containing the pET32a plasmid were activated and streaked onto an LB plate containing Amp resistance. After colonies grew, a single colony was picked with a sterile inoculating loop and incubated in LB liquid medium containing Amp resistance for 12 hours at 37°C and 200 rpm. The plasmid was extracted using a plasmid extraction kit (Xinkailai). The pET32a plasmid was double-digested with the restriction endonucleases Hind III and BamHI. The reaction system consisted of 30 μL of the pET32a plasmid, 5 μL of 10× Cutsmart Buffer, 1 μL of Hind III-HF (20 U / μL), 1 μL of BamHI-HF (20 U / μL), and 13 μL of ddH2O. The digestion was incubated at 37°C for 3 hours and the plate was recovered after digestion. Recombination was performed using a one-step cloning enzyme (Novozymes). The reaction system was as follows: 2×ClonExpress Miss 5 μL, target gene DNA fragment size × 0.04 / DNA concentration, linear pET32a plasmid size × 0.02 / plasmid concentration, and ddH2O was added to 10 μL.

[0047] After incubating the recombination reaction system at 50°C for 30 minutes, quickly place on ice for 5 minutes. Pipette 10 μL of the reaction product into 100 μL of competent E. coli DH5α. Gently flick the tube and quickly place on ice for 30 minutes. Heat shock the tube in a 42°C water bath for 45 seconds, then let it rest on ice for 2 minutes. Add 700 μL of LB medium and shake at 37°C, 200 rpm, to resuscitate the bacteria for 1 hour. Pipette 100 μL of the bacterial solution onto an LB plate containing Gen-resistant strains and incubate overnight at 37°C. Select individual colonies for PCR analysis using detection primers and Taq DNA polymerase. PCR products with bands identical in size to the target gene sequence are analyzed by electrophoresis. Sequencing results are compared with the target gene sequence; correct results are considered positive transformants.

[0048] The pET32a-24610 recombinant plasmid was then transformed into competent BL21(DE3)pLysS by heat shock method and detected by electrophoresis. Figure 1 As shown, the prokaryotic expression strain was successfully obtained.

[0049] Example 2 Expression and purification of α / β hydrolase (24610)

[0050] The BL21 (DE3) pLysS-pET32a-24610 stored at -80 ° C was activated on an LB plate containing Cam (25 μg / mL) and Amp (50 μg / mL) resistance. After inverting and culturing overnight at 37 ° C, single colonies were picked and placed in LB liquid medium containing Cam (25 μg / mL) and Amp (50 μg / mL) resistance. The culture was shaken at 37 ° C and 200 rpm for 12 hours to obtain seed liquid. Then, it was inoculated into 10 mL of fresh LB liquid medium containing Cam (25 μg / mL) and Amp (50 μg / mL) resistance at a ratio of 1:1000, placed in a shaker at 37 ° C and 200 rpm, and cultured until the OD 600 When the pH is 0.4-0.6, add IPTG to a final concentration of 0.1 mmol / L and induce for 4 hours. Then, take 100 μL and add it to a 1.5 mL centrifuge tube. Centrifuge it at 12000 rpm for 1 minute and discard the supernatant. Add 50 μL PBS to resuspend the bacteria, add 10 μL 6× loading buffer and mix well. Boil in water bath for 10 minutes, centrifuge at 12000 rpm for 1 minute, and take 25 μL for SDS-PAGE analysis using Smart PAGE. TM Precast Protein Gel Plus 4-20% Kit (Tiandi Renhe). After staining with Coomassie Brilliant Blue R-250 solution for 10 minutes, the sample was eluted with destaining solution until clear protein bands were visible. The target protein was observed to ensure that the band size was correct and that the protein was expressed normally.

[0051] Pipette 1 mL of seed solution and add it to 1 L of fresh LB liquid medium containing Cam (25 μg / mL) and Amp (50 μg / mL), and culture it in a floor shaker at 37°C and 200 rpm until the OD 600The concentration of IPTG was 0.5, and the final concentration of IPTG was 0.1 mmol / L. The expression was induced at a low temperature of 16°C for 16 hours. The bacterial solution was collected using a floor centrifuge at 4°C, 5000 rpm, and 30 minutes. It was then resuspended in 40 mL of PBS and centrifuged again, and collected in a 50 mL centrifuge tube. It was thoroughly resuspended in 40 mL of PBS (containing 2% Tween 20) and the cells were disrupted using a high-pressure cell disruptor pre-cooled to 4°C until the bacterial solution became transparent. Centrifuge again at 4°C, 5000 rpm, and 15 minutes. The supernatant was collected and filtered through a 0.45 μm bacterial filter into a new centrifuge tube. It was stored in an ice bath and awaited purification. The gravity column was washed with sterile water to check whether it was unobstructed. 2 mL of Ni NTA Beads 6FF (Tian Di Ren He) was added, and after the 20% ethanol had flowed out, it was washed twice with ddH2O and twice with Binding buffer. Combine the filtered supernatant to be purified with beads and incubate on ice for 15 minutes in two separate washes. After all the supernatant has flowed out, wash with washing buffer and measure the concentration using a Nanodrop 2000 until the protein concentration is approximately 0.01. Wash once more with PBS and elute with 10 mL of elution buffer containing 250 mM imidazole.

[0052] Take 25 μL of the eluate containing the target protein, add 5 μL of 6× loading buffer, mix well, boil in a boiling water bath for 10 minutes, centrifuge at 12,000 rpm for 1 minute, and aspirate 25 μL for SDS-PAGE analysis using the Smart PAGE™ Precast Protein GelPlus 4-20% Kit (Tiandi Renhe). After staining with Coomassie Brilliant Blue R-250 for 10 minutes, elute with destaining buffer until clear protein bands are visible.

[0053] Test results such as Figure 2 As shown. Wash the ultrafiltration tube in advance, washing twice with ddH2O and PBS at 3500 rpm for 45 minutes. Then, add 10 mL of the eluate containing the target protein to the ultrafiltration tube and centrifuge at 3500 rpm for 1 hour. Replace the eluate with PBS three times. When the remaining 250 μL is reached, resuspend in PBS containing 10% glycerol. Aliquot 100 μL into different 1.5 mL tubes, snap-freeze in liquid nitrogen, and store in a -80% freezer until ready for use.

[0054] Example 3 Degradation activity of α / β hydrolase (24610) on 3-OH PAME

[0055] Protease 24610 stored at -80°C was thawed on ice and diluted to different concentrations of 1 mg / mL, 0.5 mg / mL, 0.25 mg / mL, 0.125 mg / mL, 0.06 mg / mL, and 0.03 mg / mL using PBS phosphate buffer. 3-Hydroxypalmitic acid methyl ester (3-OH PAME) was dissolved in PBS to a final concentration of 1 mM. The experimental group consisted of 500 μL protease plus 500 μL PBS (containing 1 mM 3-OH PAME). The control group consisted of 500 μL protease inactivated by boiling in water for 10 minutes plus 500 μL PBS (containing 1 mM 3-OH PAME), and 500 μL PBS plus 500 μL PBS (containing 1 mM 3-OH PAME). The reaction was incubated in a metal bath at 37°C for 4 hours. After the reaction is complete, extract with 800 μL of dichloromethane, vortex to mix, and let stand for 10 minutes. Remove the lower layer of dichloromethane and extract three times. Drain the resulting solution with nitrogen and dissolve it in 100 μL of chromatographic methanol. Dispense into a sample vial with an inner tube for later use. The concentration gradient of the standard is 3 mmol / L, 1 mmol / L, 0.5 mmol / L, 0.25 mmol / L, 0.125 mmol / L, 0.0625 mmol / L, and 0.03125 mmol / L.

[0056] The gas chromatography-mass spectrometry (GC-MS) was performed as follows: Agilent 19091S-433:12345 columns, HP-5MS 5% PhenylMethylSilox: 30 m × 250 μm × 0.25 μm. The headspace autosampler syringe was 10 μL per 1 μL injection, with a total flow rate of 14 mL / min. The injection chamber temperature was 300°C, and the column temperature was maintained at 280°C. Data were analyzed using one-way ANOVA with multiple comparisons. The significance level was set as ****, P < 0.0001; ***, P < 0.001; **, P < 0.01; *, P < 0.05; ns, P > 0.05.

[0057] The results are as follows Figure 3 As shown in the figure, compared with the control group, it was found that 24610 protease at different concentrations had a stronger effect on 3-OH PAME, among which 24610* was an inactivated protease with a peak area of ​​about 2.3*10 8 , after losing its activity, it obviously had no degradation effect on 3-OHPAME ( Figure 3 a); 1 mg / mL 24610 protease, the peak area is about 0.4*10 8 ( Figure 3 b); 0.125 mg / mL 24610 protease, peak area is about 0.8*108 ( Figure 3 c).

[0058] The results are as follows Figure 4 As shown in the figure, with the increase of protease concentration, the residual rate of 3-OH PAME gradually decreased, indicating that the higher the concentration of protease added, the more significant the degradation effect of 3-OH PAME, indicating that α / β hydrolase is the 3-OH PAME signal molecule degrading enzyme.

[0059] Example 4 Biological control of Ralstonia solanacearum by overexpressing HS-18 (24610)

[0060] (1) Construction of HS-18(24610) overexpression strain

[0061] To conduct the next pot culture experiment, the HS-18 (24610) strain needed to be constructed. Escherichia coli containing the plasmid pBBR1-MCS5 was activated and streaked onto an LB plate containing Gen (50 μg / mL) resistance. After colonies grew, a sterile inoculation loop was used to pick a single colony in LB liquid medium containing Gen (50 μg / mL) resistance and cultured overnight at 37°C and 200 rpm for 12 hours. The plasmid was extracted using a plasmid extraction kit (Xinkailai), and then the Hind III and BamHⅠ restriction sites were selected to purify and recover the linear vector. Using HS-18 as a template, specific primers: pBBR1-24610-F (GCTCACCATATGTTTTTCCTCCTtcaagcatgcagaaagcgcagc, SEQ ID NO. 5) and pBBR1-24610-R (GCGAGGAGGAGGGTGCAGatgaccgccacattgtct, SEQ ID NO. 6) were used to amplify the target gene. The linearized plasmid and the target gene were homologously recombined, and detection primers were used for detection. The correct bands were sent for detection, and the positive transformants were saved after comparison. The specific method was the same as in Example 1.

[0062] Activate the E. coli containing the plasmid pBBR1-MCS5-24610, streak it on an LB plate containing Gen resistance, and after the colonies grow, use a sterile inoculation loop to pick a single colony in an LB liquid medium containing Gen resistance, and culture it overnight at 37°C and 200rpm for 12 hours. Use a plasmid extraction kit (Xinkailai) to extract the plasmid, and then purify and recover it. Activate the HS-18 strain stored at -80°C on an LB plate, culture it upside down at 30°C overnight, pick a single colony in an LB liquid medium, and culture it on a shaker at 30°C and 200rpm for 12 hours to obtain the seed solution. Mix the seed solution and LB liquid medium at a ratio of 1:100, and shake at 30°C and 200rpm until the OD 600=0.6-0.8. Chill on ice for 10 minutes, transfer 1 mL of bacterial suspension to a 1.5 mL centrifuge tube, centrifuge at 4°C, 5000 rpm, for 5 minutes, and discard the supernatant. Slowly resuspend the cells in 500 μL of 300 mM sucrose solution, centrifuge at 4°C, 5000 rpm, for 5 minutes, and wash again. Slowly resuspend the cells in 500 μL of 10% glycerol, centrifuge at 4°C, 5000 rpm, for 5 minutes, and slowly resuspend in 100 μL of 10% glycerol to obtain HS-18 competent cells. Add 1-2 μL of the extracted pBBR1-MCS5-24610 plasmid to each tube, mix thoroughly, and then add to a pre-chilled 2 mm cuvette. Electroporate using a Bio-Rad electroporator at 2.8 kV. After electroporation, pipette 1 mL of LB liquid medium into the electroporation cuvette. After brief pipetting, transfer the liquid to a 14 mL vent tube. Incubate at 28°C, 200 rpm, and shake for 2 hours. Collect the cells and plate them on LB plates containing Gen (50 μg / mL) for resistance. Once colonies have grown, test them using detection primers. Only the correct bands are tested and, after comparison, save the positive transformants.

[0063] like Figure 5 As shown, the pBBR1-MCS5-24610 vector was successfully introduced into HS-18 cells by PCR verification, and the size was 939 bp.

[0064] (2) Biological control effect on Ralstonia solanacearum EP1

[0065] To verify the biocontrol activity of 24610 quenching enzyme, HS-18 (24610) culture medium and EP1 (Ralstonia solanacearum (pathogen)) culture medium were mixed in a 1:1 ratio. A batch of 15-20 cm tall tomato seedlings with approximately 5-7 leaves and strong growth were prepared. The seedlings were removed from their pots and, during removal, one-third of the root length was trimmed with sterilized scissors. The seedlings were then immersed in the mixed culture medium for 60 minutes before being replanted. Each pot was individually wrapped in plastic to prevent cross-infection and incubated in a tissue culture incubator at 30°C, with 14 hours of light and 10 hours of darkness, and 65% humidity. Watering was performed daily to maintain moisture, and the disease status of the plants was observed and recorded, with photos taken. Three treatment groups were set up. The first group was a control group inoculated with EP1 culture medium alone; the second group was a treatment group inoculated with EP1 and HS-18 culture medium; and the third group was a treatment group inoculated with EP1 and HS-18 (24610) culture medium. Five pots were inoculated in each group, and the experiment was repeated three times. The disease index of tomato seedlings during the disease process was observed and counted, and the calculation method of the disease index grading standard is shown in Table 1.

[0066] Table 1 Disease index grading standard

[0067]

[0068] The results are as follows Figure 6 The figure shows the survival status of tomatoes on the 13th day of culture.

[0069] The results of tomato survival rate statistics for each time period are as follows Figure 7 As shown, by the seventh day of cultivation, both tomatoes inoculated with EP1 alone and those inoculated with EP1 and HS-18 began to develop disease. Tomatoes inoculated with EP1 / HS-18 (24610) developed disease by the tenth day. The survival rate of HS-18 tomatoes introduced with the 24610 gene was approximately 55%. This indicates that overexpressing HS-18 (24610) can effectively enhance the control of bacterial wilt by HS-18.

[0070] The above results show that α / β hydrolase can effectively degrade the quorum sensing signal produced by Ralstonia solanacearum, namely 3-OHPAME, interfere with the normal expression of Ralstonia solanacearum pathogenicity factors, block the quorum sensing between Ralstonia solanacearum, thereby inhibiting the pathogenicity of Ralstonia solanacearum, and effectively preventing and controlling plant wilt disease. Bacteria can be modified at the molecular level to construct biocontrol strains with quorum sensing quenching function, which has broad application prospects in the field of agricultural biocontrol.

Claims

1. Use of α / β hydrolase as a 3-hydroxypalmitoyl methyl ester signal molecule degrading enzyme or in the preparation of a preparation for degrading 3-hydroxypalmitoyl methyl ester signal molecule, characterized in that: The amino acid sequence of the α / β hydrolase is shown in SEQ ID NO.

1.

2. Use of α / β hydrolase or a biomaterial related to α / β hydrolase in inhibiting pathogens that are dependent on 3-hydroxypalmitate signaling molecules or in preparing a preparation for inhibiting pathogens that are dependent on 3-hydroxypalmitate signaling molecules, characterized in that: The amino acid sequence of the α / β hydrolase is shown in SEQ ID NO.1; the biological material related to the α / β hydrolase includes (1) an expression cassette containing a nucleotide sequence encoding the α / β hydrolase; (2) a recombinant expression vector containing a nucleotide encoding the α / β hydrolase and / or (1); and (3) a recombinant microorganism containing a nucleotide encoding the α / β hydrolase, (1) and / or (2).

3. Use of α / β hydrolase or a biomaterial related to α / β hydrolase in preventing and treating plant diseases caused by pathogens that rely on 3-hydroxypalmitate signal molecules to cause disease, or in the preparation of a preparation for preventing and treating plant diseases caused by pathogens that rely on 3-hydroxypalmitate signal molecules to cause disease, characterized in that: The amino acid sequence of the α / β hydrolase is shown in SEQ ID NO.1; the biological material related to the α / β hydrolase includes (1) an expression cassette containing a nucleotide sequence encoding the α / β hydrolase; (2) a recombinant expression vector containing a nucleotide encoding the α / β hydrolase and / or (1); and (3) a recombinant microorganism containing a nucleotide encoding the α / β hydrolase, (1) and / or (2).

4. Use of α / β hydrolase or α / β hydrolase-related biomaterials to enhance the ability of nitroreducing Pseudomonas to control plant diseases caused by pathogens that rely on 3-hydroxypalmitate signaling molecules, characterized in that: The amino acid sequence of the α / β hydrolase is shown in SEQ ID NO.1; the α / β hydrolase-related biological material includes (1) an expression cassette containing a nucleotide sequence encoding the α / β hydrolase; and (2) a recombinant expression vector containing a nucleotide sequence encoding the α / β hydrolase and / or (1).

5. A method for preventing and treating plant diseases caused by pathogenic bacteria that rely on 3-hydroxypalmitic acid methyl ester signal molecules for pathogenicity, characterized in that: The control is carried out using one or more of the following biological materials: (1) α / β hydrolase; (2) an expression cassette containing a nucleotide sequence encoding (1); (3) a recombinant expression vector containing a nucleotide encoding (1) and / or (2); (4) a recombinant microorganism containing a nucleotide encoding (1), (2) and / or (3); the amino acid sequence of the α / β hydrolase is shown in SEQ ID NO.

1.

6. The use according to any one of claims 2 to 4 or the method according to claim 5, characterized in that: The pathogen is Ralstonia solanacearum.

7. The use according to any one of claims 3 to 4 or the method according to claim 5, characterized in that: The plant disease is bacterial wilt.

8. The use according to any one of claims 2 to 4 or the method according to claim 5, characterized in that: The nucleotide sequence is shown in SEQ ID NO.

2.

9. The use according to any one of claims 2 to 3 or the method according to claim 5, characterized in that: The microorganism is nitroreducing Pseudomonas.

10. The use according to any one of claims 3 to 4 or the method according to claim 5, characterized in that: The plants are one or more of tomato, potato, peanut, eggplant and casuarina.

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