A group of quenching lactonase ytnp gene and its application in the prevention and treatment of soft rot

Abstract

This invention discloses a method derived from Bacillus ( Bacillus The population quenching lactonease YtnP gene of sp.) GB-1, the nucleotide sequence of which is shown in SEQ ID NO:1, was introduced into the soft rot pathogen via a two-parent hybridization method. Heterologous overexpression of this gene significantly inhibited biofilm formation, motility, and host pathogenicity of the soft rot pathogen. It was also transferred into Arabidopsis thaliana via Agrobacterium-mediated transformation and overexpressed in Arabidopsis. ytnP Genes can significantly enhance plant resistance to soft rot, and this invention provides new resources and technical approaches for the prevention and control of soft rot.

Description

Technical Field

[0001] This invention belongs to the field of microbial genetic engineering and biological control of plant diseases. Specifically, it discloses a population quenching lactonease YtnP gene and its application in the control of pathogens that depend on AHLs signaling molecules (especially soft rot). Background Technology

[0002] The long-term reliance on and excessive use of chemical pesticides has led to increasing resistance in plant pathogens, exacerbating agricultural ecological and food safety issues. Therefore, developing green alternative strategies based on biological control has become an important direction for plant disease prevention and control.

[0003] Quorum sensing (QS) is a cell communication mechanism in which microorganisms coordinate their collective behavior by secreting and sensing specific signaling molecules. When the concentration of signaling molecules increases to a threshold with increasing bacterial population density, it activates the expression of a series of downstream genes. These genes are usually closely related to key biological functions of pathogens, such as the secretion of virulence factors, biofilm formation, and antibiotic synthesis.

[0004] Among various bacterial signaling molecules, N-acylhomoserine lactones (AHLs) are one of the most representative classes in Gram-negative pathogens. Pathogens continuously synthesize and release AHLs, and when they accumulate to a critical concentration in the microenvironment, they initiate the expression of pathogenic factors. For example, *Carotene soft rot* (*Pectinobacterium carotenoides*) Pectobacterium carotovorum Under this regulation, a large amount of cell wall degrading enzymes are produced, leading to soft rot; while Diggis ( Dickeya zeae The QS system for these pathogens is closely related to their motility and virulence. Bacterial soft rot caused by these pathogens has caused severe economic losses to potatoes, radishes, and other cash crops in many countries and regions around the world, becoming one of the agricultural diseases that urgently needs to be addressed.

[0005] Quorum quenching (QQ), as a novel disease intervention strategy, significantly reduces the pathogenicity of pathogens without directly killing them by interfering with the synthesis, sensing, or signaling molecules of the QS signaling pathway. This strategy does not generate selective pressure, effectively avoids the problem of pathogen resistance, and has outstanding advantages such as being environmentally friendly and highly sustainable, making it an ideal alternative to chemical control.

[0006] Currently known AHL-degrading enzymes mainly fall into three categories: lactoneases, acyltransferases, and oxidoreductases. Among them, AHL lactoneases, which inactivate signaling molecules by hydrolyzing the homoserine lactone ring, have been studied most extensively. However, existing AHL lactoneases still have significant limitations in practical applications: most enzymes have high degradation efficiency for short-chain AHLs but insufficient catalytic activity for long-chain AHLs (such as 3OC12-HSL); some enzymes have poor thermostability and are easily inactivated under the high temperatures of farmland; and some enzymes are sensitive to pH changes and are difficult to maintain stability in the complex plant microenvironment. These shortcomings limit the broad spectrum and field application effectiveness of existing population quenching enzyme preparations. Summary of the Invention

[0007] To address the shortcomings of existing biological control technologies for plant soft rot, this invention provides a method derived from Bacillus (… Bacillus The quorum quenching lactonease YtnP gene of sp.) GB-1, the nucleotide sequence of which is shown in SEQ ID NO:1 and the encoded amino acid sequence is shown in SEQ ID NO:2, can efficiently degrade the quorum sensing signal molecule N-acylhomoserine lactone (AHLs), significantly inhibit the virulence of pathogens that depend on AHLs, and can be used to control plant soft rot.

[0008] The population-quenching lactonease YtnP of this invention exhibits degradation activity against various AHLs, particularly showing a degradation efficiency of up to 90.6% against long-chain AHL (3OC12-HSL). Enzymatic characterization analysis shows that YtnP maintains activity within a temperature range of 4-70℃, with an optimal reaction temperature of 50℃, and demonstrates good stability below 30℃. It also exhibits stable activity within a wide alkaline range of pH 7.0-11.0, demonstrating excellent environmental adaptability. Functional validation experiments further confirm that heterologous expression... ytnP The gene can significantly inhibit biofilm formation, motility, and host pathogenicity of the soft rot pathogen; overexpression of this gene in Arabidopsis thaliana can significantly enhance plant resistance to soft rot. The YtnP enzyme and its encoding gene provided by this invention offer a novel enzyme resource and technical approach for the green control of bacterial soft rot in plants.

[0009] To achieve the above objectives, the present invention adopts the following technical solution: 1. Through whole-genome sequencing, genome annotation, and homology comparison, from Bacillus ( Bacillus The population quenching lactonease YtnP gene was cloned from sp.) GB-1. Its nucleotide sequence is shown in SEQ ID NO:1. The gene is 855 bp in length, encodes 284 amino acids, and its amino acid sequence is shown in SEQ ID NO:1. The molecular weight is 28.9 kDa, the isoelectric point is 5.58, and it belongs to the metallo-β-lactamase superfamily. The Bacillus ( Bacillus (sp.) GB-1 was disclosed in patent application 202510025692.3; 2. A recombinant expression vector pET-28b-ytnP containing the above genes was constructed. After successful verification, the recombinant expression vector was transformed into Escherichia coli BL21. Positive single clones were screened by colony PCR. Positive clones were verified, and positive clones were selected for expression, isolation, and purification to obtain the population quenching lactone enzyme YtnP. The degradation activity of the recombinant YtnP protein was verified as a substrate for N-hexanoylhomoserine lactone (C6-HSL), N-(3-oxohexanoyl)-L-homoserine lactone (3OC6-HSL), or N-(3-oxododecanoyl)-L-homoserine lactone (3OC12-HSL). 3. Enzymatic characterization of the quorum quenching lactonease YtnP; 4. The ytnP gene was introduced into the soft rot pathogen, *Bacillus carotenoides*, via a two-parent hybridization method. Pectobacterium carotovorum subsp. carotovorum, Pcc) or Diggia ( Dickeya zeae In this study, heterologous expression strains Pcc-ytnP or EC1-ytnP were constructed, and the effect of overexpression of the enzyme YtnP gene on the recombinant strains was examined. 5. Using Agrobacterium-mediated transformation ytnP Genes were transferred into Arabidopsis thaliana and overexpressed. ytnP Transgenic Arabidopsis thaliana, results showed that overexpression in Arabidopsis thaliana ytnP Genes can significantly enhance a plant's resistance to soft rot.

[0010] The present invention has the following beneficial effects: (1) Provides new population quenching enzyme resources: This invention isolates and identifies a new AHL lactone enzyme YtnP from Bacillus GB-1, enriching the gene resource library of population quenching enzymes; (2) Excellent enzymatic properties and broad substrate spectrum: Enzymatic studies show that YtnP exhibits activity in the range of 4-70℃, with an optimal reaction temperature of 50℃. It exhibits good stability in the range of 4-30℃, retaining more than 50% of its activity after incubation at 50℃ for 1 hour. The optimal reaction pH is 7.0, and its activity remains stable at over 90% in a wide alkaline range of pH 7.0-11.0. This enzyme has degradation activity against various AHL signaling molecules (C6-HSL, 3OC6-HSL, 3OC12-HSL), especially showing the highest catalytic efficiency against the long-chain AHL molecule 3OC12-HSL, with a degradation rate of 90.6% and a catalytic efficiency Kcat / Km of 214.8 min. -1 ·mM -1 In addition, Cu² +It has a promoting effect on YtnP activity; (3) Significantly inhibited pathogen virulence: After heterologous expression of the ytnP gene in Pcc and EC1, the biofilm production, swarming ability and pathogenicity to host plants (radish and potato) of the recombinant strains were significantly reduced. (4) Conferring broad-spectrum disease resistance to plants: Overexpression lines obtained by transforming Arabidopsis thaliana with the ytnP gene showed significantly lower leaf disease incidence and disease index after inoculation with Pcc compared to the wild type, with disease indices of 15 and 27.5, respectively. Physiological index measurements showed that the H2O2 content, superoxide anion content and its production rate were significantly reduced in the transgenic lines, soluble sugar content decreased synchronously, SOD activity increased slightly, and CAT activity decreased significantly, indicating that YtnP regulates the ROS metabolic balance in plants through population quenching, thereby alleviating oxidative stress and enhancing the plant's disease resistance. (5) Green and safe: This invention interferes with the pathogenicity of pathogens by degrading signaling molecules, without directly killing pathogens or generating selective pressure, which can reduce the use of chemical pesticides and provides a new way for the green prevention and control of plant soft rot. Attached Figure Description

[0011] Figure 1 SDS-PAGE validation (left) and purification results (right) of recombinant YtnP protein; In the left image, M: Protein Marker; 3: Cell lysis buffer carrying pET-28(b)-ytnP plasmid; 4: Cell lysis precipitate carrying pET-28(b)-ytnP plasmid; In the right image, M: Protein marker; P: Precipitate; S: Supernatant; FT: Flow-through buffer; E1-E5: Elution buffer for protein elution 1-5 times; Figure 2 The graph shows the degradation activity of YtnP protein on C6-HSL, 3OC6-HSL, and 3OC12-HSL. Blue represents the control group, and red represents the experimental group with added YtnP enzyme. Figure 3 The optimal reaction temperature (a) and temperature stability curve (b) of YtnP enzyme are shown. Figure 4 The optimal reaction pH (a) and pH stability curve of YtnP enzyme (b) are shown. Figure 5 Figure showing the effect of different metal ions on YtnP enzyme activity; Figure 6 A statistical graph showing the effect of heterologous expression of the ytnP gene on biofilm production of pathogenic bacteria Pcc or EC1; Figure 7The images show the effects of heterologous expression of the ytnP gene on the motility of pathogens Pcc or EC1. The top left image compares the motility of Pcc-pBBR (control) and Pcc-ytnP, and the top right image shows the corresponding motility region diameter. The bottom left image compares the motility of EC1-pBBR (control) and EC1-ytnP, and the bottom right image shows the corresponding motility region diameter. Figure 8 The graph shows the effect of heterologous expression of the ytnP gene on the pathogenicity of Pcc (symptoms of soft rot in potatoes and radishes). The left graph is a comparison and statistical chart of soft rot symptoms after inoculation of potato slices, and the right graph is a comparison and statistical chart of soft rot symptoms after inoculation of radish slices. Figure 9 The graph shows the effect of heterologous expression of the ytnP gene on the pathogenicity of EC1 pathogen (symptoms of soft rot in potatoes and radishes). The left graph is a comparison and statistical chart of soft rot symptoms after inoculation of potato slices, and the right graph is a comparison and statistical chart of soft rot symptoms after inoculation of radish slices. Figure 10 The disease resistance phenotype of Arabidopsis thaliana overexpressing the ytnP gene after inoculation with Pcc is shown in the following figures: a: disease manifestations of wild-type Arabidopsis thaliana; b: disease manifestations of transgenic Arabidopsis thaliana; c: disease index statistics. Figure 11 To investigate the effects of overexpression of the ytnP gene on ROS metabolism and antioxidant enzyme activity in Arabidopsis leaves, the following parameters were measured: H2O2 content (a); soluble sugar content (b); superoxide anion content (c); superoxide anion production rate (d); POD activity (e); CAT activity (f); and SOD activity (g). WT: wild-type control; ytnP: transgenic line. Detailed Implementation

[0012] The technical solution of the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings, but the present invention is not limited to the following content. Unless otherwise specified, the reagents, methods and equipment used in the present invention are all conventional in the art. Those skilled in the art can refer to commonly used reference books, scientific and technological documents or related instructions and manuals prior to the filing date of this invention for implementation.

[0013] In the following examples, the LB liquid medium consisted of 5.0 g yeast extract, 10.0 g peptone, 10.0 g sodium chloride, and 1000 mL deionized water, pH 7.2, sterilized at 121°C for 20 min; the LB solid medium consisted of 17–20 g agar powder added to each 1 L of liquid medium. MSM liquid medium (1L): (NH4)2SO4 2.0g, CaCl2·2H2O 0.01g, FeSO4·7H2O 0.001g, Na2HPO4·12H2O 1.5g, MgSO4·7H2O 0.2g, KH2PO4 1.5g, pH 6.5.

[0014] Example 1: Cloning of the YtnP gene and construction of the pBBR1-ytnP recombinant expression vector 1. YtnP gene amplification Bacillus ( ) was extracted using a bacterial genomic DNA extraction kit. Bacillus sp .) For GB-1 genomic DNA, follow the kit instructions. Using the extracted genomic DNA as a template, perform PCR amplification using the following primer pairs: Upstream primer: 5'-GCCGCTCTAGAACTAGTGGATCATGGAACAGTTACAAATTGGAG-3' Downstream primer: 5'-CGATAAGCTTGATATCGAATTCCTTATGCTTCTATACTCGTTTTC-3' PCR reaction conditions: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 60℃ annealing for 30 s, 72℃ extension for 30 s, for a total of 35 cycles; final extension at 72℃ for 5 min. The PCR product was detected by 1% agarose gel electrophoresis, showing a specific band at approximately 855 bp, consistent with the expected ytnP gene length. The PCR product was purified and recovered, and then sent for sequencing verification.

[0015] 2. Linearization of pBBR1-MCS5 vector Using pBBR1-MCS5 plasmid as a template, reverse PCR amplification was performed using the following primers to linearize the vector: upstream primer: 5'-GGAATTCGATATCAAGCTTATCG-3', downstream primer: 5'-GATCCACTAGTTCTAGAGCGGC-3'; PCR reaction conditions: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 60℃ annealing for 30 s, 72℃ extension for 3 min (adjusted according to plasmid length), for a total of 35 cycles; final extension at 72℃ for 5 min. PCR products were verified by agarose gel electrophoresis, and the linearized vector fragment was recovered.

[0016] 3. Seamless cloning and construction of pBBR1-ytnP The purified ytnP PCR product was mixed with the linearized pBBR1-MCS5 vector at a molar ratio of 3:1 and reacted at 50°C for 15 min using a homologous recombinase (such as the SoSoo Recombinant Cloning Kit). The ligation product was heat-shock transformed into E. coli S17-1(λ-π) competent cells, plated on LB agar plates containing gentamicin (Gen, 50 μg / mL), and incubated upside down at 37°C overnight.

[0017] 4. Identification of recombinant plasmids Single colonies were selected for colony PCR verification using the ytnP primers from step (1). Plasmids were extracted from positive clones and sequenced; the sequencing results are shown in SEQ ID NO:1. Sequence analysis showed that the protein encoded by this gene consists of 284 amino acids (SEQ ID NO:2), with a theoretical molecular weight of 28.9 kDa, an isoelectric point of 5.58, and an instability coefficient of 46.67. Conserved domain analysis indicated that this protein belongs to the metallo-β-lactamase superfamily and shares 96.8% homology with the previously reported population-quenching lactonease YtnP (CUB52828.1). These results demonstrate the successful construction of the pBBR1-ytnP recombinant expression vector.

[0018] Example 2: Induction and purification of YtnP protein (1) Expression vector construction: The purified expression vector was used to construct the expression vector. ytnP Gene fragments were ligated with a linearized pET-28b(+) expression vector using seamless cloning technology to construct the recombinant plasmid pET-28b- ytnP Ligation system: 1 μL of purified PCR fragment, 1 μL of linearized vector, 10 μL of 2×SoSoo Mix, and ddH2O to a final volume of 20 μL. Incubate at 50 °C for 15 min. Transform the ligation product into *E. coli* BL21(DE3) competent cells by heat shock, plate on LB agar plates containing 50 μg / mL kanamycin, and incubate overnight at 37 °C inverted. Single colonies are picked for colony PCR verification, and positive clones are sent for sequencing confirmation.

[0019] (2) Induction of expression: Select positive single colonies and inoculate them into LB liquid medium containing 50 μg / mL kanamycin. Incubate overnight at 37°C with shaking. Transfer to fresh LB liquid medium (containing kanamycin) at a 1% inoculation rate and incubate at 37°C until OD. 600Pre-induce at 15℃ for 30 min with 0.4-0.5 mM IPTG to a final concentration of 0.5 mM, and induce expression at 15℃ for 24 h. After induction, collect bacterial cells by centrifugation at 4000 rpm for 10 min, and wash three times with 50 mM Tris-HCl buffer. Resuspend the bacterial cells at a ratio of bacterial cells:lysis buffer = 1:10 (g:mL), and disrupt the cells using an ultrasonic disruptor under ice bath conditions (power 200W, 5 s operation time, 6 s interval, total time 15 min), repeating twice. Centrifuge the disruption buffer at 12000 rpm for 20 min at low temperature, and collect the supernatant and precipitate separately.

[0020] (3) Protein purification: Recombinant YtnP protein was purified using a Ni-NTA affinity chromatography column. The column was first equilibrated with 5 column volumes of lysis buffer. After loading the supernatant, the sample was allowed to remain in the column for at least 2 minutes. The collected eluent could be loaded again to enhance binding. Non-specific proteins were washed away with 10-15 column volumes of wash buffer. Finally, fractional elution was performed with elution buffer, collecting 1 column volume from each tube. SDS-PAGE analysis showed that the purified protein exhibited a single band at approximately 33 kDa. Figure 1 The concentration was consistent with the theoretical molecular weight. BCA protein concentration determination showed a purified protein concentration of 2.667 mg / mL, which met the requirements for subsequent experiments.

[0021] Example 3: Substrate profile and degradation activity assay of YtnP protein The degradation activity of recombinant YtnP protein was determined using three AHL signaling molecules as substrates. The reaction system consisted of 100 μL purified enzyme solution, 200 μL of AHL substrates (C6-HSL, 3OC6-HSL, 3OC12-HSL), and 10 mM PBS buffer (pH 7.2-7.4), for a total volume of 1 mL. After incubation at 30 °C for 15 min, 200 μL of 10% SDS solution was added to terminate the reaction. The remaining AHL was extracted with 2 volumes of ethyl acetate, and this process was repeated twice. The upper organic phases were combined, evaporated to dryness, and resuspended in 1 mL of chromatographic methanol. The residual substrate was detected by HPLC. HPLC conditions: Elite C18 reversed-phase column (250 mm × 4.60 mm, 5 μm), flow rate 0.5 mL / min, column temperature 30 °C, mobile phase acetonitrile:water = 30:70 (v:v), detection wavelength 210 nm, injection volume 20 μL.

[0022] The results are as follows Figure 2 As shown, the degradation rates of recombinant YtnP protein on C6-HSL, 3OC6-HSL, and 3OC12-HSL were 10.8%, 44.8%, and 90.6%, respectively, indicating that YtnP has the highest degradation activity on long-chain AHL (3OC12-HSL).

[0023] Example 4: Enzymatic Characterization of YtnP Protein 1. Optimal reaction temperature and temperature stability (1) Determination of optimal reaction temperature: The purified YtnP enzyme solution (2.667 mg / mL) from Example 3 was preheated to 4, 20, 30, 40, 50, 60, and 70 °C for 5 min each in 50 mM phosphate buffer at pH 7.0. Then, 100 μL of the enzyme solution was mixed with 100 μL of 100 μM 3OC12-HSL substrate, and the reaction was continued at the corresponding temperature for 15 min. After the reaction was completed, 50 μL of 10% SDS was immediately added to terminate the reaction. The substrate degradation was detected by HPLC, and the relative enzyme activity at each temperature was calculated. The results are as follows: Figure 3 The optimal reaction temperature for YtnP is 50℃.

[0024] (2) Temperature stability determination: Equal volumes of enzyme solution were incubated at 4, 20, 30, 40, 50, 60, 70, and 80 °C for 1 h, respectively, and immediately cooled in an ice bath after removal. The enzyme activity was then measured under optimal reaction conditions (pH 7.0, 50 °C, substrate 100 μM 3OC12-HSL, reaction time 15 min). The results are as follows: Figure 3 b. YtnP exhibits good stability at 4-30℃, and retains more than 50% of its activity after incubation at 50℃ for 1 hour.

[0025] 2. Optimal reaction pH and pH stability (1) Determination of optimal reaction pH: Buffer solutions with different pH values ​​were prepared: Na2HPO4 / citric acid buffer for pH 2.0-7.0, Tris-HCl buffer for pH 8.0-9.0, and Gly / NaOH buffer for pH 10.0-11.0. 100 μL of enzyme solution was mixed with 100 μL of 100 μM 3OC12-HSL substrate, and the reaction was carried out at 50℃ for 15 min. After terminating the reaction, enzyme activity was measured. The results are as follows: Figure 4 a. The optimal reaction pH for YtnP is 7.0.

[0026] (2) pH stability determination: The enzyme solution was placed in buffer solutions with different pH values ​​(3.0-11.0) and incubated at 50℃ for 1 h. After incubation, the enzyme activity was measured under optimal reaction conditions (pH 7.0, 50℃, substrate 100 μM 3OC12-HSL, reaction time 15 min). The results are as follows: Figure 4 b. YtnP enzyme activity remains stable at over 90% within the pH range of 7.0-11.0.

[0027] 3. The effect of metal ions on enzyme activity Under optimal reaction conditions (pH 7.0, 50℃, substrate 100μM 3OC12-HSL, reaction time 15 min), 1 mM Na was added to the reaction system. + Li + Zn 2+ Cu 2+ K + Mg 2+ Cd 2+ Common metal ions such as Ca2+ were added, and residual enzyme activity was measured after incubation for 5 minutes. The system without added metal ions served as a control (enzyme activity 100%), and the relative enzyme activity of each group was calculated. Results are as follows: Figure 5 Cu 2+ It has a slight promoting effect on YtnP activity (relative enzyme activity 107.77%); Zn 2+ K + Mg 2+ Na + Cd 2+ Li + and Ca 2+ It inhibits YtnP activity to varying degrees.

[0028] Example 5: Inhibitory effect of YtnP enzyme gene on pathogen virulence 1. Construction of heterologous expression strains The recombinant plasmid pBBR1-ytnP was constructed according to the method in Example 1. The verified recombinant plasmid pBBR1-ytnP was transformed into the donor bacterium *E. coli* S17-1(λ-π). The donor and recipient bacteria were inoculated separately into LB liquid medium. The donor bacteria were cultured at 37°C with shaking for 12 h, and the recipient bacteria (soft rot pathogen Pcc or EC1) were cultured at 28°C for 24 h. 500 μL of each bacterial suspension was mixed, centrifuged at 8000 rpm for 5 min, and the supernatant was discarded. The bacterial pellet was resuspended in 1 mL of sterile water, centrifuged again, and then resuspended in 100 μL of sterile water. All bacterial suspensions were added dropwise to the surface of LB agar plates and co-cultured at 28°C for 8 h. 1 mL of sterile water was added to each plate to scrape off the bacterial growth, which was then serially diluted and spread onto LB selection plates containing 50 μg / mL gentamicin and 50 μg / mL clindamycin to obtain the recombinant strains Pcc-ytnP or EC1-ytnP. Pcc-pBBR1 and EC1-pBBR1 carrying the empty vector pBBR-MCS5 were used as controls.

[0029] 2. Biofilm yield determination Add 100 μL of LB liquid medium to each well of a 96-well plate, and inoculate each well with 1 μL of OD. 600Adjust the bacterial suspension to pH 1.0 and incubate at 28°C for 10 h. For assay, discard the bacterial suspension from each well, rinse each well four times with sterile water, and air dry. Add 150 μL of 0.1% crystal violet and stain for 15 min, then wash four times and air dry. Add 300 μL of 95% ethanol to dissolve the crystal violet for 15 min, and measure the absorbance at 595 nm using a microplate reader.

[0030] The results are as follows Figure 6 As shown, compared with the empty vector control, the biofilm production of both Pcc-ytnP and EC1-ytnP was significantly reduced.

[0031] 3. Performance testing Prepare a 0.5% agarose semi-solid culture medium. Pour 20 mL into a 90 mm Petri dish and allow it to solidify. Take the OD... 600 3 μL of bacterial suspension adjusted to 1.0 was inoculated onto the surface and incubated at 30℃ for 24 h. The diameter of the motile area was then measured.

[0032] The results are as follows Figure 7 As shown, ytnP After heterologous gene expression, the swarming ability of both Pcc and EC1 was weakened to varying degrees.

[0033] 4. Pathogenicity test Cut white radish and potato into 0.4cm thick slices, sterilize with 90mg / L hypochlorous acid, air dry, and place in petri dishes lined with moistened absorbent paper. OD values ​​of Pcc-pBBR1, Pcc-ytnP, EC1-pBBR1, and EC1-ytnP bacterial cultures were then calculated. 600 Adjust to 1.0, take 5 μL of bacterial suspension and inoculate it in the center of the slice, incubate at 28℃ for 24 h, observe the symptoms of soft rot and take pictures, repeat each treatment 3 times.

[0034] The results are as follows Figure 8 , 9 As shown, compared with the empty vector control, the area of ​​soft rot in plant tissues inoculated with Pcc-ytnP and EC1-ytnP was significantly reduced, indicating that... ytnP Gene expression significantly reduced the pathogenicity of pathogens Pcc and EC1.

[0035] Example 6: ytnP Experiment on the effect of gene overexpression on disease resistance in Arabidopsis thaliana 1. Construction of transgenic Arabidopsis thaliana: ytnP The gene was transferred into Arabidopsis thaliana Col-0 via Agrobacterium-mediated transformation, and genetic transformation was carried out using the inflorescence infection method. The process was as follows: Arabidopsis thaliana sowing and transplanting → Agrobacterium activation and expansion → collection of bacterial cells → resuspension of the infusion solution → inflorescence infection → dark culture for 2 days → screening and detection of positive seedlings to obtain transgenic Arabidopsis thaliana T1 generation seeds overexpressing ytnP.

[0036] 2. Select plump and uniform transgenic Arabidopsis thaliana T1 generation seeds, disinfect with 75% ethanol for 10 min, disinfect with 10% sodium hypochlorite for 1 min, and wash 10 times with sterile water. Sow in 1 / 2 MS medium containing Hygromycin B, vernalize at 4℃ for 3 days, then transfer the culture dishes vertically to an artificial climate chamber at 22℃, with 16h light / 8h dark and a light intensity of 120 µmol·m⁻¹. -2 ·s -1 After 7 days of cultivation, seedlings with roots and true leaves were transplanted into nutrient soil. The Pcc strain was activated on LB agar plates, and single colonies were cultured in LB liquid until OD... 600 =0.6-1.0, collect bacterial cells by centrifugation, and resuspend in 10mM MgCl2 to OD. 600 =0.2. Use a needle-free syringe to inoculate the bacterial suspension onto the underside of Arabidopsis leaves, keep them moist for 2-3 days, observe the disease incidence and take photos.

[0037] 3. Disease Index Statistics: Disease Grading Standards: Grade 0 (No symptoms), Grade 1 (Slight yellowing), Grade 2 (Extensive yellowing), Grade 3 (Extensive yellowing + slight rot), Grade 4 (Complete rot). Disease Index (DI) = ∑(Disease Grade × Number of leaves affected at each grade) / (Total number of leaves × Highest grade) × 100; The results are as follows Figure 10 Three days after Pcc inoculation, overexpression ytnP The leaf morbidity and disease index (DI=15) of the transgenic Arabidopsis thaliana were significantly lower than those of the wild-type control (DI=27.5), indicating that overexpression of the transgenic Arabidopsis thaliana was significantly higher than that of the wild-type control. ytnP The gene significantly enhanced Arabidopsis' resistance to soft rot.

[0038] 4. Determination of antioxidant system-related substances and enzyme activities: Arabidopsis leaves inoculated with soft rot pathogen Pcc for 3 days were used as experimental materials. The leaf surface was gently rinsed with deionized water and the moisture was blotted dry with filter paper. Commercial reagent kits (Beijing Solarbio Science & Technology Co., Ltd., Suzhou Greens Biotechnology Co., Ltd.) were used for determination. Specific procedures were performed according to the instructions of each kit.

[0039] The results are as follows Figure 11As shown, the hydrogen peroxide (H2O2) content was 0.38 μmol / g fresh weight in the transgenic lines and 0.51 μmol / g fresh weight in the wild-type control. Soluble sugar content was 8.8 mg / g fresh weight in the transgenic lines and 14.3 mg / g fresh weight in the wild-type control. Superoxide anion content was 0.17 μmol / g fresh weight in the transgenic lines and 0.21 μmol / g fresh weight in the wild-type control. Superoxide anion production rate was approximately 0.008 μmol / min / g fresh weight in the transgenic lines and approximately 0.011 μmol / min / g fresh weight in the wild-type control. Peroxidase (POD) activity was 5414 U / g fresh weight in the transgenic lines and 5225 U / g fresh weight in the wild-type control, with no significant difference between the two. Catalase (CAT) activity was 481 U / g fresh weight in the transgenic lines and 1500 U / g fresh weight in the wild-type control. Superoxide dismutase (SOD) activity: 854 U / g fresh weight in transgenic lines and 677 U / g fresh weight in wild-type controls. These results indicate that the ytnP gene regulates ROS metabolic balance in plants through population quenching, alleviating oxidative stress and thus enhancing plant disease resistance.

Claims

1. A population quenching lactonease YtnP gene, the nucleotide sequence of which is shown in SEQ ID NO:

1.

2. The application of the population-quenched lactonease YtnP gene as described in claim 1 in improving plant resistance to soft rot.

3. The application according to claim 2, characterized in that: The pathogen causing soft rot is *Carotene carotenoides* subsp. *carotene* (…). Pectobacterium carotovorum subsp. carotovorum, Pcc), Digib ( Dickeya zeae ).

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