Use of the hir4 gene and / or its encoded protein in modulating plant pathogen resistance

Abstract

This invention belongs to the field of genetic engineering technology, specifically relating to the application of the AtHIR4 gene and / or its encoded protein in regulating plant pathogen resistance. The amino acid sequence of the protein encoded by the AtHIR4 gene described in this invention is shown in SEQ ID No. 1. This invention created transgenic Arabidopsis and rapeseed expressing hypersensitivity response-inducible protein 4 (AtHIR4). In the AtHIR4 transgenic Arabidopsis, the chitin-activated ROS burst and phosphorylation of MAPKs were significantly enhanced. Disease resistance test results showed that the AtHIR4 transgenic plants not only exhibited enhanced resistance to the necrotrophic pathogens *Sclerotinia sclerotiorum* and *Botrytis cinerea*, but also significantly increased resistance to the semi-vitrotrophic pathogen *Pseudomonas syringae*. The AtHIR4 transgenic rapeseed also showed significantly enhanced resistance to *Sclerotinia sclerotiorum* and *Botrytis cinerea*.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to the application of the AtHIR4 gene and / or its encoded protein in regulating plant pathogen resistance. Background Technology

[0002] Sclerotinias clerotiorum is a highly pathogenic fungus that can infect over 700 species of plants across 75 families, including rapeseed, legumes, and sunflowers, causing sclerotinia rot. Rapeseed sclerotinia rot is widespread and occurs frequently in my country, causing serious damage and threatening the safe production of rapeseed. Currently, my country lacks rapeseed varieties with high resistance to sclerotinia rot, and its control mainly relies on chemical control. However, the long-term use (especially overuse) of chemical pesticides easily leads to resistance development, pesticide residues, and environmental pollution. Therefore, identifying disease-resistant proteins and genes is particularly important for breeding rapeseed with resistance. Summary of the Invention

[0003] The purpose of this invention is to provide the application of the AtHIR4 gene and / or its encoded protein in regulating plant pathogen resistance, enhancing the resistance of Arabidopsis thaliana and Brassica napus to Sclerotinia sclerotiorum, Botrytis cinerea, and Pseudomonas syringae, and providing a new approach for the green control of Sclerotinia sclerotiorum and gray mold in rapeseed.

[0004] This invention provides the application of the AtHIR4 gene and / or its encoded protein in regulating plant pathogen resistance, wherein the amino acid sequence of the protein encoded by the AtHIR4 gene is shown in SEQ ID No. 1.

[0005] Preferably, the nucleotide sequence of the AtHIR4 gene is shown in SEQ ID No. 2.

[0006] Preferably, the regulation of plant pathogen resistance includes positively regulating the expression level of the AtHIR4 gene to enhance plant pathogen resistance.

[0007] Preferably, the pathogenic bacteria include one or more of Sclerotinia sclerotiorum, Botrytis cinerea, and Pseudomonas syringae.

[0008] The present invention also provides the application of the AtHIR4 gene and / or its encoded protein in the cultivation of antibacterial plants, wherein the amino acid sequence of the protein encoded by the AtHIR4 gene is shown in SEQ ID No. 1.

[0009] This invention also provides the application of the AtHIR4 gene and / or its encoded protein in the prevention and control of plant diseases, wherein the amino acid sequence of the protein encoded by the AtHIR4 gene is shown in SEQ ID No. 1.

[0010] Preferably, the plant includes Arabidopsis thaliana and / or rapeseed.

[0011] The present invention also provides a method for enhancing plant pathogen resistance, the method comprising the following steps:

[0012] Overexpression of the AtHIR4 gene in plants; the amino acid sequence of the protein encoded by the AtHIR4 gene is shown in SEQ ID No. 1.

[0013] Preferably, the method of overexpressing the AtHIR4 gene in plants includes the following steps:

[0014] The recombinant vector was introduced into the recipient plant using Agrobacterium-mediated transformation; the recombinant vector includes the AtHIR4 gene and the basic vector.

[0015] Preferably, the base vector includes pCNF3; the recipient plant includes Arabidopsis thaliana inflorescence or Brassica napus hypocotyl.

[0016] Beneficial effects

[0017] This invention provides the application of the AtHIR4 gene and / or its encoded protein in regulating plant pathogen resistance, the amino acid sequence of the protein encoded by the AtHIR4 gene being shown in SEQ ID No. 1. This invention created transgenic Arabidopsis and rapeseed expressing hypersensitivity response-inducible protein 4 (AtHIR4). In the AtHIR4 transgenic Arabidopsis, chitin-activated ROS burst and MAPK phosphorylation were significantly enhanced. Disease resistance testing results showed that the AtHIR4 transgenic plants not only exhibited enhanced resistance to the necrotrophic pathogens *Sclerotinia sclerotiorum* and *Botrytis cinerea*, but also significantly increased resistance to the semi-vitrotrophic pathogen *Pseudomonas syringae*. The AtHIR4 transgenic rapeseed also showed significantly enhanced resistance to *Sclerotinia sclerotiorum* and *Botrytis cinerea*. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0019] Figure 1 The results of the detection of the resistance of Arabidopsis thaliana plants overexpressing AtHIR4 to pathogenic fungi;

[0020] Figure 2The results of the detection of the resistance of Arabidopsis thaliana plants overexpressing AtHIR4 to pathogenic bacteria;

[0021] Figure 3 The results of the detection of the resistance of rapeseed plants overexpressing AtHIR4 to pathogenic fungi were obtained. Detailed Implementation

[0022] This invention provides the application of the AtHIR4 gene and / or its encoded protein in regulating plant pathogen resistance. The amino acid sequence of the protein encoded by the AtHIR4 gene is shown in SEQ ID No. 1, specifically: MGNLFCCVQVDQSTVAIKETFGKFEDVLEPGCHFLPWCLGSQVAGYLSLRVQQLDVRCETKTKDNVFVNVVASIQYRALANKANDAYYKLSNTRGQIQAYVFDVIRASVPKLLLDDVFEQKNDIAKAVEEELEKAMSAYGYEIVQTLIVDIEPDEHVKRAMNEINAAARMRLAANEKAEAEKILQIKRAEGEAESKYLSGLGIARQRQAIVDGLRDSVLGFAVNVPGTTAKDVMDMVLVTQYFDTMKEIGASSKSSAVFIPHGPGAVRDVASQIRDGLLQGSSANL.

[0023] In the present invention, the nucleotide sequence of the AtHIR4 gene is preferably as shown in SEQ ID No.2, specifically: 5'-ATGGGGAATTTGTTTTGTTGTGTGCAAGTGGATCAATCAACGGT AGCGATAAAGGAAACATTCGGGAAATTCGAAGATGTTCTTGAGCCTGGTTGCCATTTTCTTCCATGGTGTCTTGGTAGTCAAGTTGCTGGTTACCTCTCTCTAAGGGTTCAGCAATTGGACGTTCGTTGCGAGACAAAGACTAAGGACAATGTGTTTGTTAATGTTGTTGCATCGATTCAGTACCGTGCTTTAGCTAATAAGGCAAATGATGCGTACTACAAGCTCAGTAACACAAGGGGTCAGATTCAAGCTTATGTGTTTGATGTTATTAGAGCGAGTGTCCCGAAGTTGCTTCTTGATGATGTCTTTGAGCAGAAGAATGATATTGCGAAAGCTGTTGAAGAGGAGCTCGAGAAGGCAATGTCGGCTTACGGTTATGAGATTGTGCAAACTCTCATTGTTGATATCGAGCCTGATGAACATGTCAAACGGGCCATGAACGAAATCAACGCTGCTGCAAGGATGAGATTGGCTGCAAACGAAAAGGCAGAGGCAGAGAAAATCCTACAGATTAAGAGAGCTGAAGGTGAAGCTGAGTCCAAGTACCTCTCTGGTCTTGGTATCGCCCGTCAGAGGCAGGCGATTGTCGATGGATTACGCGACAGTGTTTTGGGTTTCGCTGTGAATGTCCCTGGGACAACTGCTAAAGATGTGATGGACATGGTGCTAGTTACACAGTACTTTGACACAATGAAGGAGATTGGTGCTAGCTCCAAGTCGTCTGCCGTGTTCATACCTCATGGACCAGGAGCGGTTCGTGATGTGGCTTCTCAGATTAGAGATGGCCTTCTTCAAGGCTCGTCCGCAAACCTGTGA-3'.

[0024] In this invention, the regulation of plant pathogen resistance preferably includes positively regulating the expression level of the AtHIR4 gene to enhance plant pathogen resistance; the pathogen preferably includes one or more of Sclerotinia sclerotiorum, Botrytis cinerea, and Pseudomonas syringae.

[0025] The present invention also provides the application of the AtHIR4 gene and / or its encoded protein in the cultivation of antibacterial plants, wherein the amino acid sequence of the protein encoded by the AtHIR4 gene is shown in SEQ ID No. 1.

[0026] This invention also provides the application of the AtHIR4 gene and / or its encoded protein in the prevention and control of plant diseases, wherein the amino acid sequence of the protein encoded by the AtHIR4 gene is shown in SEQ ID No. 1.

[0027] In this invention, the plant preferably includes Arabidopsis thaliana and / or Brassica napus.

[0028] The present invention also provides a method for enhancing plant pathogen resistance, the method comprising the following steps:

[0029] Overexpression of the AtHIR4 gene in plants; the amino acid sequence encoded by the AtHIR4 gene is shown in SQ ID No. 1.

[0030] In this invention, the preferred method for overexpressing the AtHIR4 gene in plants includes the following steps: introducing a recombinant vector into a recipient plant using Agrobacterium-mediated transformation; the recombinant vector contains the AtHIR4 gene and a basic vector. The basic vector preferably includes pCNF3; the recipient material preferably includes an Arabidopsis inflorescence or a Brassica napus hypocotyl; the Agrobacterium-mediated transformation is preferably GV3101. In the embodiments of this invention, Arabidopsis and Brassica napus were used as model plants to verify the effect of the AtHIR4 gene in enhancing plant resistance to Sclerotinia sclerotiorum, Botrytis cinerea, and Pseudomonas syringae. The results showed that overexpression of the AtHIR4 gene in Arabidopsis and Brassica napus effectively enhanced the plant's resistance to Sclerotinia sclerotiorum, Botrytis cinerea, and Pseudomonas syringae.

[0031] To further illustrate the present invention, the application of the AtHIR4 protein provided by the present invention in enhancing plant antibacterial activity is described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0032] Example 1

[0033] 1. Cloning of the AtHIR4 target gene

[0034] By entering the gene number AT5G62740 of AtHIR4 into the TAIR public database website (https: / / www.Arabidopsis.org / index.jsp), the coding sequence (CDS) and protein sequence of the gene were retrieved. Based on the coding sequence, full-length cloning primers for the gene were designed, as follows:

[0035] AtHIR4-F: 5'-agtggatccATGGGGAATTTGTTTTGTTGTGT-3' (SEQ ID No. 3);

[0036] AtHIR4-R: 5'-gacggatccCAGGTTTGCGGACGAGCC-3' (SEQ ID No. 4);

[0037] The lowercase letters represent the BamHI restriction site and its protective bases.

[0038] Using Arabidopsis cDNA as a template, PCR amplification was performed using high-fidelity DNA polymerase Pfu. The total reaction volume is as follows:

[0039] 50μL (cDNA 2μL, 10μM Primer F 2μL, 10μM Primer R 2μL, 5×Pfu Buffer 10μL, 10mM dNTP 1μL, Pfu 1μL, ddH2O 32μL);

[0040] Cycling conditions: (Pre-denaturation: 95℃, 3min; Denaturation: 95℃, 30s; Annealing: 56℃, 30s; Extension: 72℃, 1min / kb; Final extension: 72℃, 10min; Storage: 12℃, 10min) Number of cycles: 35.

[0041] After the PCR reaction, the products were electrophoresed on a 1% agarose gel at 120V for 15 min, stained with EB for 5 min, and then the specificity of the PCR products was detected using a gel imaging system. When the target band in the PCR product was specific, it was purified using a gel extraction kit. When the target band was not specific, the gel needed to be cut and purified using a gel extraction kit. The PCR product was digested with the appropriate restriction endonuclease. The prepared enzyme digestion system was incubated at 37℃ for 6 h. The enzyme digestion system was as follows: 50 μL (10 μL PCR product, 5 μL 10×Buffer, 10.5 μL Enzyme, 0.5 μL Enzyme 2, ddH2O to 50 μL). After incubation, the target gene fragment was purified and recovered using a gel extraction kit and stored at -20℃ for later use.

[0042] 2. Construction of overexpression vectors

[0043] Linearized plant expression vector backbones were obtained using BamHI restriction endonuclease. The target gene was then ligated into the target pCNF3 vector using T4 ligase. The ligation system was 10 μL (4 μL gene fragment, 1 μL vector, 1 μL 10×T4 Buffer, 0.5 μL T4 ligase, 3.5 μL ddH2O). The ligation system was incubated at 16℃ for 6 h. After the reaction, the recombinant plasmid was transformed into *E. coli*. After single colonies grew, single colonies were picked and cultured in the appropriate medium at 28℃ for approximately 8 h. The bacterial cells were then collected, and the recombinant plasmid was extracted according to the method for small-scale plasmid preparation. Single-enzyme digestion verification was then performed using the following enzyme digestion system: 10 μL (1 μL plasmid, 1 μL 10×Buffer, 1 μL BamHI, 3.5 μL ddH2O). 7 μL); the prepared enzyme digestion system was incubated at 37°C for about 30 min, and the double digestion results were detected by agarose gel electrophoresis; the correctly digested plasmids need to be further verified by sequencing, and the correctly sequenced plasmids were stored at -20°C for later use. Sequencing was performed by Tianyi Huiyuan Biotechnology Co., Ltd.

[0044] 3. Transformation of Agrobacterium

[0045] Remove GV3101 competent cells from the -80℃ freezer and thaw on ice; pipette 1 μL of plasmid into the competent cells, then tap the side of the centrifuge tube with your finger to mix the bacterial culture with the plasmid; transfer the mixed bacterial culture along the side wall to a sterile electroporation cuvette; place the cuvette in a Bio-rad electroporator and perform transformation at 2500V / 6ms; add 1 mL of LB liquid medium, mix well, and then transfer Agrobacterium and LB liquid medium together into a new 1.5 mL centrifuge tube; incubate the centrifuge tube on a shaker at 28℃ for 3 h at 4500 rpm. -1 Centrifuge for 5 min; discard the supernatant, resuspend the remaining bacterial culture, and plate onto LB (50 mg·L⁻¹). -1 Rifampicin + vector resistance) solid plates were placed on LB plates and incubated at 28°C for 2 days. Single colonies were picked up with sterile toothpicks and placed in sterile water, and then bacterial culture PCR was performed for identification. Positive Agrobacterium bacteria were collected for later use.

[0046] The pCNF3 empty vector was transformed into Agrobacterium GV3101 competent cells in the manner described above, at LB (50 mg·L⁻¹). -1 The bacteria were cultured on solid plates containing rifampicin and carrier resistance, and positive colonies were collected to obtain empty vector Agrobacterium for later use.

[0047] Example 2

[0048] Agrobacterium-mediated genetic transformation in Arabidopsis thaliana

[0049] 1. Preparation of Agrobacterium-positive staining solution

[0050] Agrobacterium strain GV3101 containing the AtHIR4 gene was transferred into 2 mL LB liquid medium containing antibiotic (Rif + vector resistance) and incubated at 28 °C and 190 rpm. -1 Agrobacterium was cultured overnight in a shaker to propagate it; then 250 μL of the bacterial culture was transferred to 5 mL of LB liquid medium containing antibiotic (Rif + vector resistance) and incubated at 28 °C and 190 rpm. -1 Incubate Agrobacterium in a shaker for 6–8 hours. When the cells enter the logarithmic growth phase, transfer 5 mL of the bacterial culture to a 15 mL centrifuge tube and incubate at 15°C and 4000 rpm. -1 Centrifuge for 15 min, discard the supernatant, and resuspend the bacterial cells in 3 mL of 5 wt.% sucrose solution. Measure the OD using a spectrophotometer. 600 Dilute the bacterial culture with 5 wt.% sucrose solution to OD. 600 The concentration was 0.8, resulting in a positive Agrobacterium-infected solution.

[0051] 2. Obtaining AtHIR4 transgenic plants

[0052] Wild-type Arabidopsis thaliana Col-0 was infected with the Agrobacterium positivity in step 1: Opened flowers or developed pods of Arabidopsis thaliana Col-0 were cut off, and the plant was thoroughly watered beforehand; before dipping the flowers, 0.002% Silwet L-77 (0.15 μL·mL) was added to the Agrobacterium positivity infusion solution. -1 The inflorescences of the treated Arabidopsis thaliana Col-0 were immersed in the prepared Agrobacterium tumefaciens infection solution for 5 seconds. After all inflorescences were infected, the plants were covered and kept at room temperature for 12 hours without excessive light. The next day, the plants were transferred to a culture room for normal culture and the seeds were harvested.

[0053] In a clean bench, the collected seeds are disinfected with 15% 84 disinfectant for 15 minutes, rinsed 3-5 times with sterile water, and then sown at 1 / 2 MS (50 mg·L⁻¹). -1 Screening was performed using kanamycin sulfate plates, followed by vernalization at 4°C for 3 days, and then transferred to a light-controlled culture room for normal cultivation. Positive seedlings were green, while negative seedlings appeared yellow and wilted. One week later, positive seedlings were selected and transplanted into nutrient soil for normal cultivation. At four weeks of growth, protein immunoblotting was performed on the transgenic Arabidopsis plants. The results of Western blot analysis of transgenic Arabidopsis lines overexpressing AtHIR4 are as follows: Figure 1As shown in Figure A, protein bands of the corresponding size were detected in the leaves of the positive plants after resistance selection using the corresponding antibodies, indicating that transgenic Arabidopsis thaliana lines oxAtHIR4-8 and oxAtHIR4-15 (AtHIR4 transgenic genes) and wild-type Arabidopsis thaliana Col-0 (control group) were successfully obtained by overexpressing AtHIR4. Figure 1 Figure B shows the morphological characteristics of transgenic Arabidopsis lines oxAtHIR4-8 and oxAtHIR4-15 after four weeks of growth. It can be seen that, compared with wild-type Arabidopsis Col-0, transgenic Arabidopsis plants overexpressing AtHIR4 under soil cultivation conditions have slightly smaller aboveground parts and greener leaves after four weeks of growth, indicating that overexpression of AtHIR4 may have a certain impact on the growth of Arabidopsis.

[0054] Test Example 1

[0055] Determination of Arabidopsis thaliana plant resistance to sclerotinia and gray mold

[0056] 1. Seeds from Arabidopsis thaliana Col-0 plants, seeds from oxAtHIR4-8 plants obtained in Example 2, and seeds from oxAtHIR4-15 plants were cultured in a growth chamber for 4 weeks to obtain Arabidopsis thaliana Col-0 plants, oxAtHIR4-8 plants, and oxAtHIR4-15 plants. These plants were then divided into a Sclerotinia sclerotiorum test group and a Botrytis cinerea test group, with three replicates for each group.

[0057] 2. The model strain *Sclerotinia sclerotiorum* strain 1980 was cultured on PDA plates (Potatodextrose broth: 200g peeled potato, 20g glucose, 10g agar, distilled water to 1L). Young mycelia were picked and grown on fresh PDA plates. After 1-2 days of growth, holes were punched at the edge of the colony at the young mycelia using a 2mm or 5mm diameter punch. These holes were then inoculated onto the upper right leaf margins of *Arabidopsis thaliana* Col-0, oxAtHIR4-8, and oxAtHIR4-15 plants, respectively. The plants were then kept in a moist environment for 40 hours before data measurement and photography. The photographic results are shown below. Figure 1 As shown in C, the lesion area caused by the necrotrophic pathogenic fungus *Sclerotinia sclerotiorum* on leaves of *Arabidopsis thaliana* (oxAtHIR4-8 and oxAtHIR4-15) with overexpression of AtHIR4 was significantly smaller than that in wild-type *Arabidopsis thaliana* Col-0.

[0058] 3. The model strain *Botrytis cinerea* strain B05.10 was cultured on PDA plates. Young mycelia were selected and grown on fresh PDA plates. After 1-2 days of growth, holes were punched at the edge of the colony at the young mycelia using a 2mm or 5mm diameter punch. The colonies were then inoculated onto the upper right leaf of *Arabidopsis thaliana* Col-0, oxAtHIR4-8, and oxAtHIR4-15 plants. The plants were then kept in a moist environment for 48 hours. Data were measured and photographs were taken. The photographic results are shown below. Figure 1 As shown in Figure F, the lesion area caused by Botrytis cinerea on leaves of Arabidopsis thaliana (oxAtHIR4-8 and oxAtHIR4-15) with overexpression of AtHIR4 was significantly smaller than that in wild-type Arabidopsis thaliana Col-0.

[0059] 4. During data measurement, the diameter of the lesion was measured using the cross-multiplication method, and the lesion area was calculated using the formula for the area of ​​an ellipse for statistical analysis. The results are as follows: Figure 1 China D and Figure 1 Figure G shows the lesion area determined by the cross-multiplication method for wild-type *Sclerotinia sclerotiorum* strain 40 h after inoculation, where D represents the lesion area determined by the cross-multiplication method for wild-type *Botrytis cinerea* strain 48 h after inoculation. Data represent mean ± SD, and different letters in the figure indicate statistical significance (p < 0.01) in one-way ANOVA. Figure 1 As shown in D and G, the lesion area caused by the necrotrophic pathogens *Sclerotinia sclerotiorum* and *Botrytis cinerea* on leaves of *Arabidopsis thaliana* (oxAtHIR4-8 and oxAtHIR4-15) with overexpression of AtHIR4 was significantly smaller than that in wild-type *Arabidopsis thaliana* Col-0.

[0060] 5. In each group of Arabidopsis leaves, after measuring the lesion area, a sample of equal area was taken from the infected site using a 1.5 cm diameter punch. DNA was extracted from the sample, and the relative content of fungal pathogens and plants was analyzed by real-time quantitative PCR to calculate the relative biomass of fungal pathogens.

[0061] The primers used for RT-qPCR analysis of the relative biomass of Sclerotinia sclerotiorum are as follows:

[0062] SsTub-F: 5'-ACCTCCATCCAAGAACTC-3'(SEQ ID No.5)

[0063] SsTub-R: 5'-GAACTCCATCTCGTCCAT-3' (SEQ ID No. 6);

[0064] AtUBQ5-F: 5'-ACACCAAGCCGAAGAAGA-3' (SEQ ID No. 7);

[0065] AtUBQ5-R: 5'-TCCACAGGTTGCGTTAGG-3' (SEQ ID No. 8);

[0066] The primers used for RT-qPCR analysis of the relative biomass of Botrytis cinerea are as follows:

[0067] BcActin-F: 5'-CTTCGTGTAGCACCAGAGGAG-3' (SEQ ID No. 9)

[0068] BcActin-R: 5'-GAGAGGACCGGCTTGAATAGAGA-3' (SEQ ID No. 10);

[0069] AtUBQ5-F: 5'-ACACCAAGCCGAAGAAGA-3' (SEQ ID No. 11)

[0070] AtUBQ5-R: 5'-TCCACAGGTTGCGTTAGG-3' (SEQ ID No. 12);

[0071] Each of the above treatments consisted of three replicates (each replicate contained 2 to 4 diseased leaves). Pathogenicity tests were repeated at least three times.

[0072] The results are as follows Figure 1 Figures E and H show the relative biomass of wild-type *Sclerotinia sclerotiorum* strains after 40 h of inoculation with *Arabidopsis thaliana* leaves of Col-0, oxAtHIR4-8, and oxAtHIR4-15, as analyzed by RT-qPCR. Figure H shows the relative biomass of wild-type *Botrytis cinerea* strains after 48 h of inoculation with *Arabidopsis thaliana* leaves of Col-0, oxAtHIR4-8, and oxAtHIR4-15, as analyzed by RT-qPCR. The data represent mean ± SD, and different letters in the figure indicate statistical significance of p < 0.01 in one-way ANOVA.

[0073] Depend on Figure 1 As shown in E and H, the biomass of the necrotrophic pathogenic fungi *Sclerotium sclerotiorum* and *Botrytis cinerea* on leaves of *Arabidopsis thaliana* (oxAtHIR4-8 and oxAtHIR4-15) with overexpression of AtHIR4 was significantly lower than that of wild-type *Arabidopsis thaliana* Col-0.

[0074] 6. Chitin triggers phosphorylation activation of MAPKs

[0075] Seed disinfection: Disinfect Arabidopsis thaliana Col-0, oxAtHIR4-8, and oxAtHIR4-15 seeds with 70% ethanol for 5 min, wash once with sterile ddH2O, then disinfect with 84 disinfectant for 5 min in a laminar flow hood, and wash with sterile ddH2O at least 3 times. Sow approximately 35 seeds per 2 / 2 MS medium plate in a laminar flow hood, and let them air dry in the laminar flow hood, but not too dry. Seal with medical tape. Germinate the plates at 4℃ for 3 days, then transfer them to the bottom layer of the culture chamber (23℃ / 20℃) for 10-12 days. Then transfer healthy, uniformly sized seedlings to 12-well plates, taking care to prevent root damage, with 4 seedlings per well. Add 1 mL of sterile ddH2O, ensuring the roots are fully submerged, and let them recover overnight. Aspirate the ddH2O and add 10 μg·mL⁻¹ of sterile ddH2O. -1 Incubate the seedlings with chitin solution for 0 min, 5 min, 15 min and 30 min respectively, ensuring that the roots are submerged in the solution; remove the seedlings, quickly remove any residual solution with absorbent paper and lens paper, place them in 1.5 mL centrifuge tubes and freeze them in liquid nitrogen to -80℃.

[0076] Immunoblot detection was performed using phosphorylated antibodies against MPK3, MPK4, and MPK6, respectively: Seedlings stored at -80℃ were placed in an ice box containing liquid nitrogen, thoroughly ground with a grinder, and then 100 μL of 2×SDS loading buffer (containing β-mercaptoethanol and DTT) was added. The mixture was denatured at 95℃ for 10 min at 12000 rpm. -1 Centrifuge for 5 min, collect the supernatant for protein electrophoresis; perform 10% SDS-PAGE electrophoresis (MPK3, MPK4, and MPK6 have molecular weights close to 42kD-46kD), check the position of protein marker bands, stop electrophoresis to ensure good separation of MPK3, MPK4, and MPK6 bands, then transfer to a membrane; block with 10 mL of 1×TBST + 5% BSA, and incubate at room temperature on a shaker at 80 rpm. -1 Incubate for 1 hour, add primary antibody anti-pERK1 / 2, and incubate at 4°C with a shaker at 80 rpm. -1 Incubate overnight; wash three times with 1×TBST for 15 min each time, at room temperature on a shaker at 80 rpm. -1 Secondary antibody anti-Rabbit-HRP, room temperature shaker at 80 rpm. -1 Incubate for 2 hours. Wash three times with 1×TBST, 15 min each time. Select ClarityWestern ECL Substrate or Thermo West Femto chemiluminescent substrate to detect the protein based on the signal intensity.

[0077] Phosphorylation results of MAPKs in Arabidopsis seedlings induced by chitin after treatment with Col-0, oxAtHIR4-8, and oxAtHIR4-15 are as follows: Figure 1 As shown in Figure I, the numerical values ​​represent the mean ± SE (n = 12). The upper part shows the phosphorylation results of MAPK detected by immunoblotting using phosphorylation antibodies of MPK3, MPK4, and MPK6; the lower part shows the staining of the developed PVDF membrane with Ponceau S to show the consistency of sample loading. Figure 1 As can be seen from the results, the early immune response of Arabidopsis seedlings treated with chitin was significantly enhanced after treatment with chitin, and the phosphorylation of chitin-induced MAPKs was significantly enhanced in plants with AtHIR4 overexpression.

[0078] 7. Chitin triggers reactive oxygen species bursts in Arabidopsis leaves

[0079] After the Arabidopsis thaliana Col-0, oxAtHIR4-8, and oxAtHIR4-15 plants obtained in Example 2 had grown for 4-5 weeks, 24 leaves were taken from each group of Arabidopsis thaliana plants, and holes of 0.25 cm were punched in them. 2 Leaf discs were collected, and each leaf disc was cut into 4 narrow leaflets. The leaflets were placed in a 96-well plate, and 100 μL of ddH2O was added to each well. The plate was incubated overnight for recovery. The ddH2O was removed, and 100 μL of the reaction mixture, including 50 μM Luminol at 10 μg·mL⁻¹, was added. -1 Peroxidase, 10 μg·mL -1 Chitin; place the sample in an ELISA reader and start the measurement immediately, measuring once every 1 minute, repeating 35 times, for a total cycle of 20 minutes. The reactive oxygen species values ​​produced by the 12 leaf discs after treatment are used as the relative light units.

[0080] The ROS burst results of chitin-induced Arabidopsis thaliana leaves of Col-0, oxAtHIR4-8, and oxAtHIR4-15 are as follows: Figure 1 As shown in J, where the values ​​represent the average ± SE (n = 12). From Figure 1 As can be seen from the results, the leaves of Arabidopsis thaliana oxAtHIR4-8 and oxAtHIR4-15 exhibited a stronger ROS burst after chitin treatment.

[0081] Furthermore, by Figure 1The conclusions suggest that the oxAtHIR4-8 strain exhibits higher protein expression levels and a stronger immune response, which corresponds to its antifungal genetic phenotype. These results not only provide further genetic evidence for the positive role of AtHIR4 in Arabidopsis thaliana's resistance to necrotrophic pathogens, but also clarify to some extent that the disease resistance in Arabidopsis thaliana with overexpression of AtHIR4 is due to a faster and stronger early immune response.

[0082] Test Example 2

[0083] Detection of resistance of Arabidopsis thaliana plants to pathogenic bacteria

[0084] 1. Using the model strain *Pseudomonas syringae*, streak the bacteria onto KB agar plates, seal the plates with plastic wrap, and incubate at 28°C for 2 days until bacterial growth occurs. Then, select single colonies and inoculate them into 2 mL of KB + antibiotic (Pst DC3000 for Rif resistance) liquid medium, and incubate overnight at 28°C with shaking. (Restaurant temperature: 4000 rpm) -1 Collect bacterial cells by centrifugation for 5 min, discard the supernatant, wash twice with ddH2O, and resuspend in 500 μL of 10 mM MgCl2; determine bacterial OD. 600 To calculate bacterial concentration, the bacteria were diluted to OD using 10 mM MgCl2. 600 The value is 5×10 -4 , and obtained the bacterial culture of Pseudomonas syringae.

[0085] 2. Leaves from Arabidopsis thaliana Col-0, oxAtHIR4-8, and oxAtHIR4-15 plants (test example 1, step 1) after 4 weeks of growth were selected. Using a 1mL needleless syringe, 100μL of bacterial suspension was gently injected into the lower epidermis of the leaves. The leaves were covered with a transparent cap to maintain humidity for 5-8 hours. The plants were then placed in a growth chamber for cultivation, and disease development was observed. The plant surface was sprayed with water every morning. The nutrient soil used for cultivation was a mixture of Finnish Kegela peat moss, Jiangsu Peilei substrate, and vermiculite in a ratio of 8:4:1. The growth chamber conditions for cultivating plants for disease resistance experiments were: temperature 20℃-23℃, humidity 65%, light intensity 75μE-100μE, and a photoperiod of light / dark: 12h / 12h. Bacterial growth was measured 2-3 days after inoculation. A representative Arabidopsis thaliana leaf was photographed 3 days after inoculation with *Pseudomonas syringae*. Figure 2 As shown in Figure A, it can be seen that 3 days after inoculation with *Pseudomonas syringae*, the leaves of Col-0 and transgenic Arabidopsis thaliana overexpressing AtHIR4 showed obvious yellow lesions, but the leaves of oxAtHIR4-8 and oxAtHIR4-15 Arabidopsis thaliana remained healthy green.

[0086] 3. Use a punch to punch holes in the leaves for inoculation, collecting 8 leaf discs (6mm in diameter). Grade the leaf discs according to disease severity, average them, and place 2 discs into each of four 1.5mL centrifuge tubes containing 100μL ddH2O. Grind the leaf discs, add 900μL ddH2O, mix well, and perform serial dilutions. Use 10... -3 and 10 -4 Two dilution factors were used for bacterial counting; for 0d samples, a 10⁻⁶ dilution factor was used. -2 Concentration: Using a pipette tip, take 10 μL of the diluted bacterial solution and inoculate it onto a TSA plate containing the corresponding antibiotic (TSA medium formulation: tryptone 10 g·L⁻¹). -1 10g·L of sucrose -1 Glutamic acid 10 g·L -1 10 g / L agar -1 Spread the bacteria evenly with an inoculation loop, incubate at 28°C for 2 days (3 days after inoculation), and calculate the bacterial colony forming units (CFU).

[0087] The bacterial count results of *Pseudomonas syringae* after inoculation for 0 days and 3 days are as follows: Figure 2 As shown in Figure B, the results showed that the number of bacteria in Arabidopsis thaliana oxAtHIR4-8 and oxAtHIR4-15 was significantly lower than that in wild-type Arabidopsis thaliana. Among them, the expression level of AtHIR4 was higher in Arabidopsis thaliana oxAtHIR4-8, and the corresponding number of bacteria was also lower.

[0088] 4. flg22 triggers phosphorylation activation of MAPKs.

[0089] Seed disinfection: Disinfect Arabidopsis thaliana Col-0, oxAtHIR4-8, and oxAtHIR4-15 seeds with 70% ethanol for 5 min, wash once with sterile ddH2O, then disinfect with 84 disinfectant for 5 min in a laminar flow hood, and wash with sterile ddH2O at least 3 times. Sow approximately 35 seeds per 2 / 2 MS medium plate in a laminar flow hood, and let them air dry in the laminar flow hood, but not too dry. Seal with medical tape. Germinate the plates at 4℃ for 3 days, then transfer them to the bottom layer of the culture chamber (23℃ / 20℃) for 10-12 days. Then transfer healthy, uniformly sized seedlings to 12-well plates, taking care to prevent root damage, with 4 seedlings per well. Add 1 mL of sterile ddH2O, ensuring the roots are fully submerged, and let them recover overnight. Aspirate the ddH2O and add 500 μL of 100 nM... Incubate the seedlings with flg22 solution for 0 min, 5 min, 15 min and 30 min respectively, ensuring that the roots are submerged in the solution; remove the seedlings, quickly remove any residual solution on the seedlings with absorbent paper and lens paper, place them in 1.5 mL centrifuge tubes and flash freeze them in liquid nitrogen to -80℃.

[0090] Immunoblot detection was performed using phosphorylated antibodies against MPK3, MPK4, and MPK6, respectively: Seedlings stored at -80℃ were placed in an ice box containing liquid nitrogen, thoroughly ground with a grinder, and then 100 μL of 2×SDS loading buffer (containing β-mercaptoethanol and DTT) was added. The mixture was denatured at 95℃ for 10 min at 12000 rpm. -1 Centrifuge for 5 min, collect the supernatant for protein electrophoresis; perform 10% SDS-PAGE electrophoresis (MAPK3, MPK4, and MPK6 have molecular weights close to 42kD-46kD), check the position of protein marker bands, stop electrophoresis to ensure good separation of MAPK3, MPK4, and MPK6 bands, then transfer to a membrane; block with 10 mL of 1×TBST + 5% BSA, and incubate at room temperature on a shaker at 80 rpm. -1 Incubate for 1 hour, add primary antibody anti-pERK1 / 2, and incubate at 4°C with a shaker at 80 rpm. -1 Incubate overnight; wash three times with 1×TBST for 15 min each time, at room temperature on a shaker at 80 rpm. -1 Secondary antibody anti-Rabbit-HRP, room temperature shaker at 80 rpm. -1 Incubate for 2 hours. Wash three times with 1×TBST, 15 min each time. Select ClarityWestern ECL Substrate or Thermo West Femto chemiluminescent substrate to detect the protein based on the signal intensity.

[0091] The results of flg22-induced phosphorylation of MAPKs in Arabidopsis plants are as follows: Figure 2 As shown in Figure C, different letters in the figure represent samples with significant differences (one-way ANOVA, P < 0.01). Data represent mean ± SD, n = 6 biological replicates. The upper part shows the results of immunoblotting detection of MAPK phosphorylation using phosphorylation antibodies against MAPK3, MPK4, and MPK6. The lower part shows the staining of the developed PVDF membrane with Ponceau S to show the consistency of sample loading. Figure 2 As shown in C, flg22-induced phosphorylation of MAPKs was significantly enhanced in AtHIR4 overexpression plants.

[0092] 5. flg22 triggers a surge of reactive oxygen species in Arabidopsis leaves.

[0093] After the Arabidopsis thaliana Col-0, oxAtHIR4-8, and oxAtHIR4-15 plants obtained in Example 2 had grown for 4-5 weeks, 24 leaves were taken from each group of Arabidopsis thaliana plants, and holes of 0.25 cm were punched in them. 2Leaf discs were collected, and each leaf disc was cut into 4 narrow leaflets. The leaflets were placed in a 96-well plate, and 100 μL of ddH2O was added to each well. The plate was incubated overnight for recovery. The ddH2O was removed, and 100 μL of the reaction mixture, including 50 μM Luminol at 10 μg·mL⁻¹, was added. -1 Peroxidase, 100 nM flg22; place the sample in the microplate reader and start the measurement immediately, measuring once every 1 minute, repeating 35 times, for a total cycle of 20 minutes. The reactive oxygen species values ​​produced by 12 leaf discs after treatment are used as relative light units.

[0094] The results of flg22-induced ROS burst in Arabidopsis plants are as follows: Figure 2 As shown in Figure D, the values ​​represent the mean ± SE (n = 12). The results show that treatment of Arabidopsis leaves overexpressing AtHIR4 with 100 nM flg22 significantly enhanced the early immune response, including a stronger ROS burst.

[0095] The above results indicate that AtHIR4 also plays a positive role in Arabidopsis' resistance to bacterial pathogens, and overexpression of AtHIR4 confers Arabidopsis with a broader spectrum of disease resistance.

[0096] Example 3

[0097] Genetic transformation of hypocotyls in Brassica napus

[0098] 1. Seed disinfection and strain preparation: Fast-growing rapeseed Y127 seeds were divided into AtHIR4 transgenic and empty vector groups. 100 seeds were placed in each 10mL centrifuge tube, and 75% v / v ethanol was added. The tubes were inverted and soaked for 1 minute. The ethanol was removed with a pipette, and then an appropriate amount of 50% v / v 84 disinfectant (distilled water: commercial 84 disinfectant volume ratio = 1:1) was added. The tubes were inverted and soaked for 5 minutes. The disinfectant was removed, and the tubes were rinsed 3–5 times with sterile water, capping and inverting each time to maintain a sterile environment inside the centrifuge tube.

[0099] Using sterile forceps, two groups of sterilized seeds were sown into M0 medium (MS 4.4g, sucrose 30g, phytal gel 5.5g, double-distilled water to a final volume of 1L, pH adjusted to 5.84-5.88, sterilized at 121℃ for 20min), with 20-25 seeds sown per dish.

[0100] Place the petri dishes in a sterile culture box and incubate at 22-24°C in the dark for 6 days.

[0101] During this period, single colonies of AtHir4-positive Agrobacterium and Agrobacterium without vector prepared in Example 1 were streaked and cultured. Five days after inoculation, single colonies were picked and cultured in 2 mL of double-antibiotic medium overnight for 12 hours. Six days after inoculation, the target bacteria were cultured in 20 mL glass bottles under the following conditions: 1 mL of target Agrobacterium was inoculated into 10 mL of resistant liquid LB medium and cultured in a shaker at 28°C and 180–220 rpm for about 4–6 hours to obtain Agrobacterium positive and Agrobacterium without vector infection solutions.

[0102] 2. Preparation and infection of explants:

[0103] Prepare co-culture medium M1 (M1 medium: MS 4.4g, sucrose 30g, mannitol 18g, 2,4-D (1mg·mL)). -1 1 mL, KT (1 mg·mL) -1 Add 0.3 mL of MS medium to a final volume of 1 L with double-distilled water, adjust the pH to 5.84–5.88, add 5.5 g of agar powder, sterilize at 121°C for 20 min, and add acetylsuccinone when the medium is almost cooled to a final concentration of 100 μM. Add acetylsuccinone to DM solution (DM medium: MS 4.4 g, sucrose 30 g, double-distilled water to a final volume of 1 L, adjust the pH to 5.84–5.88, sterilize at 121°C for 20 min) to a final concentration of 100 μM for later use.

[0104] The OD values ​​of the positive Agrobacterium-infected solution and the empty vector Agrobacterium-infected solution were adjusted using DM solution, respectively. 600 Adjust the concentration to approximately 0.5. Transfer 2 mL of the cultured positive Agrobacterium-positive and empty vector Agrobacterium-positive inoculum to sterile centrifuge tubes, centrifuge at 3000 rpm for 3 min, and discard the supernatant. Add 2 mL of DM solution to resuspend the culture, centrifuge at 3000 rpm for 3 min, and discard the supernatant. Resuspend again with 2 mL of DM solution to obtain positive Agrobacterium-positive DM culture and empty vector Agrobacterium-positive DM culture, and store at 4℃ for later use.

[0105] Six days after sowing, three groups of explant cutting dishes were prepared: AtHIR4 group, empty vector group, and control group. 18 mL of DM medium was added to each explant cutting dish in the AtHIR4 group and the empty vector group beforehand, and 20 mL of DM medium was added to each explant cutting dish in the control group beforehand. Hypocotyls of rapeseed Y127 seeds were cut, and the cut explants were transferred into each explant cutting dish. Then, 2 mL of positive Agrobacterium DM bacterial solution was poured into each explant cutting dish in the AtHIR4 group, and 2 mL of empty vector Agrobacterium DM bacterial solution was introduced into each explant cutting dish in the empty vector group. No bacterial solution was added to the explant cutting dishes in the control group. At this time, the liquid volume in each dish was 20 mL. The dishes were soaked for 15 min, and the mixture was shaken once at intervals. This process was repeated 4 to 5 times.

[0106] At 10 minutes into infection, pipette the DM bacterial solution and use sterile forceps to pick up the explants and place them on sterile filter paper. Let them rest briefly, then spread them out on the filter paper and blow them in a laminar flow hood for about 3 minutes to remove and evaporate excess bacterial solution. Then, transfer each group of explants to M1 medium (MS 4.4g, sucrose 30g, mannitol 18g, 2,4-D (1mg·mL⁻¹)). -1 1 mL, KT (1 mg·mL) -1 Add 0.3 mL of agar powder, 5.5 g of agar powder, and double-distilled water to a final volume of 1 L. Adjust the pH to 5.84–5.88, sterilize at 121°C for 20 min, and add acetylsuccinone to the medium while it is cooling rapidly to a final concentration of 100 μM. Incubate at 22–24°C in the dark (place in a light-protected incubator). After co-culturing for 22–26 h, transfer the explants to M2 medium (MS 4.4 g, sucrose 30 g, mannitol 18 g, 2,4-D (1 mg·mL⁻¹)). -1 1 mL, kinetin (1 mg / mL) -1 Add 0.3 mL of agar powder and 5.5 g of agar powder to a final volume of 1 L with double-distilled water. Adjust the pH to 5.84–5.88 and sterilize at 121°C for 20 min. While the culture medium is cooling rapidly, add 150 μL of silver thiosulfate and termethin (200 mg / mL). -1 1.5 mL, kanamycin sulfate (50 mg / mL) -1 Explants were cultured in 300 μL of medium for 14–18 days to induce callus formation. The culture was then carried out under normal light conditions in a culture room (24℃, 16 h light / 8 h dark). The callus-forming explants were then transferred to M3 medium (MS 4.4 g, glucose 10 g, xylose 0.25 g, MES 0.6 g, double-distilled water to 1 L, pH adjusted to 5.84–5.88, Phytal gel 5.5 g). When the medium was almost cooled, zeatin (2 mg / mL) was added. -1 1 mL, auxin (1 mg·mL) -1 0.1 mL, Termetidine (200 mg / mL) -1 1.5 mL, kanamycin sulfate (50 mg / mL) -1 300 μL of 0.1 M silver nitrate and 150 μL of 0.1 M silver nitrate were added to M3 medium. Subculture was repeated every 2–3 weeks until green shoots appeared on the callus. Green shoots with intact growth points were transferred to M4 medium (MS). The medium was then sterilized at 121°C for 20 min with 4.4 g of MS, 10 g of sucrose, double-distilled water to a final volume of 1 L, pH adjusted to 5.84–5.88, and 8 g of agar powder. The medium was then cooled. Temmetine (200 mg / mL) was added. -1Rooting was induced in 1.5 mL of the solution. Rooted plants were then transplanted into soil for normal culture. PCR identification yielded transgenic rapeseed plants expressing AtHIR4, including oxAtHIR4-1 and oxAtHIR4-2 plants, empty vector plants (EV), and the control group Y127 plant.

[0107] Using cDNA from transgenic rapeseed plants oxAtHIR4-1 and oxAtHIR4-2, empty vector-transformed plants (EV), and the control group Y127 plants as templates, PCR experiments were performed, and the results are as follows: Figure 3 As shown in Figure A, the primers used are as follows:

[0108] AtHIR4-F: 5'-ATGGGGAATTTGTTTTGTTGTGT-3' (SEQ ID No. 13);

[0109] AtHIR4-R: 5'-TCGAGGTCCTCCTCGGAGATG-3' (SEQ ID No. 14);

[0110] BnUBQ5-F: 5'-ACACCAAGCCGAAGAAGA-3' (SEQ ID No. 15);

[0111] BnUBQ5-R: 5'-ACACCAAGCCGAAGAAGA-3' (SEQ ID No. 16).

[0112] Morphological characteristics of oxAtHIR4 transgenic rapeseed plants, such as Figure 3 As shown in Figure B, the growth phenotype of AtHIR4 transgenic rapeseed showed no significant difference compared with the starting plant Y127 and the empty vector transgenic plant.

[0113] Test Example 2

[0114] Determination of resistance to sclerotinia stem rot and gray mold in Brassica napus plants

[0115] The *Brassica napus* plants oxAtHIR4-1, oxAtHIR4-2, EV, and Y127 obtained in Example 3 were cultured in a growth chamber for 4 weeks. The transgenic *Brassica napus* plants and wild-type *Brassica napus* plants were divided into *Sclerotinia sclerotiorum* experimental group and *Botrytis cinerea* experimental group, with three replicates in each group.

[0116] Activated Sclerotinia sclerotiorum strain 1980 and Botrytis cinerea strain B05.10 were prepared using the method described in Test Example 1.

[0117] Activated *Sclerotinia sclerotiorum* strain 1980 was cultured on PDA plates (Potatodextrose broth: 200g peeled potato, 20g glucose, 10g agar, distilled water to 1L). Young mycelia were picked and grown on fresh PDA plates. After 1-2 days of growth, holes were punched at the edge of the colony at the young mycelia using a 2mm or 5mm diameter punch. These holes were then inoculated onto the upper right leaf of *Brassica napus* plants oxAtHIR4-1, oxAtHIR4-2, EV, and Y127, respectively. The plants were then kept in a moist environment for 48 hours before data measurement and photography. The photographic results are shown below. Figure 3 As shown in Figure C. During data measurement, the diameter of the lesion was measured using the cross-multiplication method, and the lesion area was calculated using the ellipse area formula for statistical analysis. The results are shown below. Figure 3 As shown in D; by Figure 3 As shown in C and D, the lesion area caused by Sclerotinia sclerotiorum on AtHIR4 transgenic rapeseed leaves is smaller.

[0118] Activated Botrytis cinerea strain B05.10 was cultured on PDA plates. Young mycelia were picked and grown on fresh PDA plates. After 1-2 days of growth, holes were punched at the edge of the colony at the young mycelia using a 2mm or 5mm diameter punch. The inoculated colonies were then placed on the upper right side of the leaves of Brassica napus plants oxAtHIR4-1, oxAtHIR4-2, EV, and Y127. The plants were then cultured under moist conditions. Data were measured and photographs were taken after 48 hours. The photographic results are shown below. Figure 3 As shown in Figure F. During data measurement, the diameter of the lesion was measured using the cross-intersection method, and the lesion area was calculated using the ellipse area formula for statistical analysis. The results are shown in Figure F. Figure 3 As shown in G, by Figure 3 As shown in F and G, Botrytis cinerea caused smaller lesion areas on AtHIR4 transgenic rapeseed leaves.

[0119] In each group of leaves, after measuring the lesion area, an equal-area sample was taken from the infected site using a square punch with a side length of 2.5 cm. DNA was extracted from the sample, and the relative content of fungal pathogen and plant was analyzed by real-time quantitative PCR. Each treatment included three replicates (each replicate contained 2 to 4 diseased leaves). Pathogenicity assays were repeated at least three times.

[0120] The primers used for RT-qPCR analysis of the relative biomass of *Sclerotinia sclerotiorum* and *Botrytis cinerea* were the same as in test example 1. The results are as follows: Figure 3 China E and Figure 3Figure H shows the relative biomass of *Sclerotinia sclerotiorum* 40 h after inoculation with wild-type *Sclerotinia sclerotiorum* strain, analyzed by RT-qPCR; and the relative biomass of *Botrytis cinerea* 48 h after inoculation with wild-type *Sclerotinia sclerotiorum* strain, analyzed by RT-qPCR. Data in the figure represent mean ± SD, and different letters indicate statistical significance (p < 0.01) in one-way ANOVA. Figure 3 As shown in E and H, the biomass of the necrotrophic pathogenic fungi Sclerotinia sclerotiorum and Botrytis cinerea on the leaves of AtHIR4 transgenic rapeseed plants (oxAtHIR4-1 and oxAtHIR4-2) was significantly less than that of rapeseed plants with empty vector (EV) and wild-type rapeseed plants (Y127).

[0121] The results above show that the AtHIR4 transgenic rapeseed plants exhibit significantly enhanced resistance to necrotrophic pathogenic fungi, and the lesion area caused by *Sclerotinia sclerotiorum* and *Botrytis cinerea* on the leaves of AtHIR4 transgenic rapeseed is smaller. Figure 3 The relative biomass analysis of pathogenic fungi (C, D, F, G) also showed that AtHIR4 transgenic rapeseed leaves exhibited significant resistance to infection by Sclerotinia sclerotiorum and Botrytis cinerea. Figure 3 (E, H).

[0122] As demonstrated by the above embodiments, this invention successfully constructed transgenic Arabidopsis and rapeseed plants constitutively expressing AtHIR4. Disease resistance tests showed that overexpression of AtHIR4 in Arabidopsis and rapeseed plants not only enhanced their resistance to the necrotrophic pathogens *Sclerotinia sclerotiorum* and *Botrytis cinerea*, but also significantly increased the resistance of transgenic Arabidopsis to semi-vitrotrophic pathogens. This indicates that AtHIR4 mediates broad-spectrum resistance to multiple pathogens in plants, and the AtHIR4 gene can provide a new gene resource for the green control of *Sclerotinia sclerotiorum* and *Botrytis cinerea* in rapeseed.

[0123] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. The application of the AtHIR4 gene and / or its encoded protein in regulating plant pathogen resistance, wherein the amino acid sequence of the protein encoded by the AtHIR4 gene is shown in SEQ ID No. 1; The pathogenic bacteria are one or more of the following: Sclerotinia sclerotiorum, Botrytis cinerea, and Pseudomonas syringae. The plants mentioned are Arabidopsis thaliana and rapeseed.

2. The application according to claim 1, characterized in that, The nucleotide sequence of the AtHIR4 gene is shown in SEQ ID No.

2.

3. The application according to claim 1 or 2, characterized in that, The regulation of plant pathogen resistance includes positively regulating the expression level of the AtHIR4 gene to enhance plant pathogen resistance.

4. The application of the AtHIR4 gene and / or its encoded protein in the cultivation of antibacterial plants, wherein the amino acid sequence of the protein encoded by the AtHIR4 gene is shown in SEQ ID No. 1; The pathogens targeted by the antibacterial agents are one or more of the following: Sclerotinia sclerotiorum, Botrytiscinerea, and Pseudomonas syringae. The plants mentioned are Arabidopsis thaliana and rapeseed.

5. Application of the AtHIR4 gene and / or its encoded protein in the prevention and control of plant diseases, wherein the amino acid sequence of the protein encoded by the AtHIR4 gene is shown in SEQ ID No. 1; The pathogens of the disease are one or more of the following: Sclerotinia sclerotiorum, Botrytiscinerea, and Pseudomonas syringae. The plants mentioned are Arabidopsis thaliana and rapeseed.

6. A method for enhancing resistance to plant pathogens, characterized in that, The method includes the following steps: Overexpression of the AtHIR4 gene in plants; the amino acid sequence of the protein encoded by the AtHIR4 gene is shown in SEQ ID No. 1; The pathogenic bacteria are one or more of the following: Sclerotinia sclerotiorum, Botrytis cinerea, and Pseudomonas syringae. The plants mentioned are Arabidopsis thaliana and rapeseed.

7. The method according to claim 6, characterized in that, The method of overexpressing the AtHIR4 gene in plants includes the following steps: The recombinant vector was introduced into the recipient plant using Agrobacterium-mediated transformation; the recombinant vector includes the AtHIR4 gene and the basic vector.

8. The method according to claim 7, characterized in that, The basic vector includes pCNF3; the recipient plant includes Arabidopsis thaliana inflorescence or Brassica napus hypocotyl.

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