Use of athir4 gene and / or protein coded thereby in regulation of plant pathogen resistance

Abstract

Provided is a use of the hypersensitive induced reaction protein 4 (AtHIR4) gene and / or a protein coded thereby in the regulation of plant pathogen resistance, the amino acid sequence of the protein coded by the AtHIR4 gene being as shown in SEQ ID No. I. Transgenic Arabidopsis thaliana and rapeseed are created by means of over-expression of AtHIR4. Resistance of the AtHIR4 transgenic Arabidopsis thaliana to the necrotrophic pathogenic true fungi Sclerotinia sclerotiorum and Botrytis cinerea is enhanced, and disease resistance to the hemibiotrophic pathogenic bacterium Pseudomonas syringae is also significantly increased. The resistance of the AtHIR4 transgenic rapeseed to Sclerotinia sclerotiorum and Botrytis cinerea is also significantly enhanced.

Description

Application of AtHIR4 gene and / or its encoded protein in regulating plant pathogenic bacteria resistance TECHNICAL FIELD

[0001] The present application belongs to the technical field of genetic engineering, and particularly relates to application of AtHIR4 gene and / or its encoded protein in regulating plant pathogenic bacteria resistance. BACKGROUND

[0002] Sclerotinia sclerotiorum is a serious crop pathogenic fungus, which can infect 75 families and more than 700 species of plants such as Brassica, Leguminosae and sunflower, and cause sclerotinia disease. The sclerotinia disease of Brassica in China has a large area and a high frequency of outbreak, and causes serious damage, which threatens the safe production of Brassica in China. At present, there is a lack of Brassica varieties with high resistance to sclerotinia disease in China, and the prevention and control of sclerotinia disease mainly relies on chemical control, but the long-term use (especially abuse) of chemical pesticides can easily lead to problems such as pesticide resistance, pesticide residues and environmental pollution. Therefore, it is particularly important to excavate disease-resistant proteins and genes for Brassica breeding. SUMMARY

[0003] The present application aims to provide application of AtHIR4 gene and / or its encoded protein in regulating plant pathogenic bacteria resistance, and to enhance the resistance of Arabidopsis and Brassica napus to Sclerotinia sclerotiorum, Botrytis cinerea and Pseudomonas syringae, thereby providing a new idea for green prevention and control of sclerotinia disease and gray mold disease of Brassica.

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

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

[0006] Preferably, the regulation of plant pathogenic bacteria resistance includes positively regulating the expression amount of the AtHIR4 gene to enhance the resistance of the plant pathogenic bacteria.

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

[0008] The application further provides an application of the AtHIR4 gene and / or the encoded protein thereof in cultivating an antibacterial plant, wherein the amino acid sequence of the encoded protein of the AtHIR4 gene is shown as SEQ ID No. 1.

[0009] The application further provides an application of the AtHIR4 gene and / or the encoded protein thereof in preventing and treating plant diseases, wherein the amino acid sequence of the encoded protein of the AtHIR4 gene is shown as SEQ ID No. 1.

[0010] Preferably, the plant comprises Arabidopsis thaliana and / or Brassica napus.

[0011] The application further provides a method for enhancing plant pathogenic resistance, comprising the following steps:

[0012] overexpressing the AtHIR4 gene in the plant, wherein the amino acid sequence of the encoded protein of the AtHIR4 gene is shown as SEQ ID No. 1.

[0013] Preferably, the method for overexpressing the AtHIR4 gene in the plant comprises the following steps:

[0014] introducing the recombinant vector into the recipient plant by using the Agrobacterium transformation method, wherein the recombinant vector comprises the AtHIR4 gene and a basic vector.

[0015] Preferably, the basic vector comprises pCNF3, and the recipient plant comprises Arabidopsis thaliana inflorescence or Brassica napus hypocotyl. Beneficial effects

[0016] The application provides an application of the AtHIR4 gene and / or the encoded protein thereof in regulating plant pathogenic resistance, wherein the amino acid sequence of the encoded protein of the AtHIR4 gene is shown as SEQ ID No. 1. The application creates transgenic Arabidopsis thaliana and Brassica napus overexpressing hypersensitive response-inducing protein 4 (AtHIR4), wherein the ROS burst activated by chitin and the phosphorylation of MAPKs in the AtHIR4 transgenic Arabidopsis thaliana are obviously enhanced, the disease resistance detection result shows that the AtHIR4 transgenic plant not only has enhanced resistance to the necrotrophic pathogenic fungus Sclerotinia sclerotiorum and the gray mold, but also has significantly increased disease resistance to the hemibiotrophic pathogenic bacterium Pseudomonas syringae, and the AtHIR4 transgenic Brassica napus also has significantly enhanced resistance to Sclerotinia sclerotiorum and the gray mold. BRIEF DESCRIPTION OF DRAWINGS

[0017] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the following will briefly introduce the drawings needed in the embodiments.

[0018] Fig. 1 is a disease resistance detection result of the overexpression AtHIR4 Arabidopsis thaliana plant to pathogenic fungi.

[0019] Figure 2 is the result of detecting the disease resistance of overexpressed AtHIR4 Arabidopsis plants to pathogenic bacteria;

[0020] Figure 3 is the result of detecting the disease resistance of overexpressed AtHIR4 Arabidopsis plants to pathogenic bacteria. DETAILED DESCRIPTION

[0021] The present application provides the application of AtHIR4 gene and / or the encoded protein in regulating the resistance of plants to pathogenic bacteria, the amino acid sequence of the protein encoded by the AtHIR4 gene is shown as SEQ ID No. 1, specifically: MGNLFCCVQVDQSTVAIKETFGKFEDVLEPGCHFLPWCLGSQVAGYLSLRVQQLDVRCETKTKDNVFVNVVASIQYRALANKANDAYYKLSNTRGQIQAYVFDVIRASVPKLLLDDVFEQKNDIAKAVEEELEKAMSAYGYEIVQTLIVDIEPDEHVKRAMNEINAAARMRLAANEKAEAEKILQIKRAEGEAESKYLSGLGIARQRQAIVDGLRDSVLGFAVNVPGTTAKDVMDMVLVTQYFDTMKEIGASSKSSAVFIPHGPGAVRDVASQIRDGLLQGSSANL.

[0022] In the present application, the nucleotide sequence of the AtHIR4 gene is preferably as shown in SEQ ID No. 2, in particular: 5'-ATGGGGAATTTGTTTTGTTGTGTGCAAGTGGATCAATCAACGGT AGCGATAAAGGAAACATTCGGGAAATTCGAAGATGTTCTTGAGCCTGGTTGCCATTTTCTTCCATGGTGTCTTGGTAGTCAAGTTGCTGGTTACCTCTCTCTAAGGGTTCAGCAATTGGACGTTCGTTGCGAGACAAAGACTAAGGACAATGTGTTTGTTAATGTTGTTGCATCGATTCAGTACCGTGCTTTAGCTAATAAGGCAAATGATGCGTACTACAAGCTCAGTAACACAAGGGGTCAGATTCAAGCTTATGTGTTTGATGTTATTAGAGCGAGTGTCCCGAAGTTGCTTCTTGATGATGTCTTTGAGCAGAAGAATGATATTGCGAAAGCTGTTGAAGAGGAGCTCGAGAAGGCAATGTCGGCTTACGGTTATGAGATTGTGCAAACTCTCATTGTTGATATCGAGCCTGATGAACATGTCAAACGGGCCATGAACGAAATCAACGCTGCTGCAAGGATGAGATTGGCTGCAAACGAAAAGGCAGAGGCAGAGAAAATCCTACAGATTAAGAGAGCTGAAGGTGAAGCTGAGTCCAAGTACCTCTCTGGTCTTGGTATCGCCCGTCAGAGGCAGGCGATTGTCGATGGATTACGCGACAGTGTTTTGGGTTTCGCTGTGAATGTCCCTGGGACAACTGCTAAAGATGTGATGGACATGGTGCTAGTTACACAGTACTTTGACACAATGAAGGAGATTGGTGCTAGCTCCAAGTCGTCTGCCGTGTTCATACCTCATGGACCAGGAGCGGTTCGTGATGTGGCTTCTCAGATTAGAGATGGCCTTCTTCAAGGCTCGTCCGCAAACCTGTGA-3'.

[0023] In the present application, the regulation of plant pathogenic bacteria resistance preferably includes positively regulating the expression amount of the AtHIR4 gene to enhance the plant pathogenic bacteria resistance; the pathogenic bacteria preferably include one or more of Sclerotinia sclerotiorum, Botrytis cinerea and Pseudomonas syringae.

[0024] The present application also provides the application of the AtHIR4 gene and / or the encoded protein thereof in cultivating antibacterial plants, wherein the amino acid sequence of the encoded protein of the AtHIR4 gene is shown in SEQ ID No. 1.

[0025] The present application also provides the application of the AtHIR4 gene and / or the encoded protein thereof in preventing and treating plant diseases, wherein the amino acid sequence of the encoded protein of the AtHIR4 gene is shown in SEQ ID No. 1.

[0026] In the present application, the plants preferably include Arabidopsis thaliana and / or Brassica napus.

[0027] The present application also provides a method for enhancing the plant pathogenic bacteria resistance, which comprises the following steps:

[0028] overexpressing the AtHIR4 gene in plants; the amino acid sequence of the encoded protein of the AtHIR4 gene is shown in SEQ ID No. 1.

[0029] In the present application, the way of overexpressing the AtHIR4 gene in plants preferably comprises the following steps: introducing a recombinant vector into a recipient plant by using an Agrobacterium transformation method; the recombinant vector contains the AtHIR4 gene and a basic vector. The basic vector of the present application preferably includes pCNF3; the recipient material preferably includes Arabidopsis thaliana inflorescences or Brassica napus hypocotyls; the Agrobacterium used in the Agrobacterium transformation method is preferably GV3101. In the embodiments, Arabidopsis thaliana and Brassica napus are used as model plants to verify the effect of the AtHIR4 gene on enhancing the resistance of plants to Sclerotinia sclerotiorum, Botrytis cinerea and Pseudomonas syringae, and the results show that overexpressing the AtHIR4 gene in Arabidopsis thaliana and Brassica napus can effectively enhance the resistance of plants to Sclerotinia sclerotiorum, Botrytis cinerea and Pseudomonas syringae.

[0030] In order to further illustrate the present application, the application of the AtHIR4 protein provided by the present application in enhancing the antibacterial property of plants is described in detail below in combination with the drawings and embodiments, but they should not be understood as limiting the scope of protection of the present application.

[0031] Example 1

[0032] 1. Cloning of the AtHIR4 target gene

[0033] The gene number AT5G62740 of AtHIR4 was input into the public database TAIR website (https: / / www.arabidopsis.org / index.jsp) of Arabidopsis thaliana, and the coding sequence (CDS) and protein sequence of the gene were retrieved, and the full-length cloning primer of the gene was designed according to the coding sequence of the gene, as follows:

[0034] AtHIR4-F: 5'-agtggatcc ATGGGGAATTTGTTTTGTTGTGT-3' (SEQ ID No. 3);

[0035] AtHIR4-R: 5'-gacggatcc CAGGTTTGCGGACGAGCC-3' (SEQ ID No. 4);

[0036] wherein the lowercase letters represent the BamHI restriction site and its protection base.

[0037] The PCR amplification was performed using the Arabidopsis thaliana cDNA as a template and the high-fidelity DNA polymerase Pfu, and the total reaction system was as follows:

[0038] 50 μL (cDNA 2 μL, 10 μM Primer F 2 μL, 10 μM Primer R 2 μL, 5×Pfu Buffer 10 μL, 10 mM dNTP 1 μL, Pfu 1 μL, ddH2O 32 μL);

[0039] Cycle condition: (pre-denaturation: 95°C, 3 min; denaturation: 95°C, 30 s; annealing: 56°C, 30 s; extension: 72°C, 1 min / kb; last extension: 72°C, 10 min; storage: 12°C, 10 min) cycle number: 35.

[0040] The product after the PCR reaction was electrophoresed on a 1% agarose gel at 120 V for 15 min, and after staining with EB for 5 min, the specificity of the PCR product was detected using a gel imaging system; when the target band in the PCR product was specific, the gel recovery kit was used for purification, and when the target band was not specific, the gel was cut and the gel recovery kit was used for purification; the PCR recovery product was digested using the corresponding restriction endonuclease, and the prepared enzyme digestion system was incubated at 37°C for 6 h, and the enzyme digestion system was as follows: 50 μL (PCR product 10 μL, 10×Buffer 5 μL, Enzyme 10.5 μL, Enzyme 20.5 μL, ddH2O to 50 μL); after incubation, the gel recovery kit was used for purification and recovery, and the target gene fragment was obtained and stored at -20°C for standby.

[0041] 2. Construction of overexpression vector

[0042] The linearized plant expression vector skeleton was obtained using BamHI restriction endonuclease, and then the target gene was connected to the target pCNF3 vector by T4 ligase, the connection system: 10 μL (gene fragment 4 μL, vector 1 μL, 10×T4 Buffer 1 μL, T4 ligase 0.5 μL, ddH2O 3.5 μL); the connection system was placed in 16°C incubation for 6h, and after the reaction was completed, the recombinant plasmid was transformed into E. coli; after single colony growth, a single colony was picked in the corresponding medium and cultured at 28°C for about 8h, then the bacteria were collected and the recombinant plasmid was extracted according to the method of small-scale preparation of plasmid, and then single enzyme digestion verification was carried out according to the following enzyme digestion system, enzyme digestion system: 10 μL (plasmid 1 μL, 10×Buffer 1 μL, BamHI 1 μL, ddH2O 7 μL); the prepared enzyme digestion system was incubated at 37°C for about 30min, and the double enzyme digestion results were detected by agarose gel electrophoresis; the correct plasmid also needs to be further verified by sequencing, and the correct plasmid is stored at-20°C for standby. Sequencing was completed by Tianyihuiyuan Biotechnology Co., Ltd.

[0043] 3. Transformation of Agrobacterium

[0044] The GV3101 competent was taken out from the-80°C refrigerator, and thawed on ice; 1 μL plasmid was taken into the competent, and then the bacterial solution was mixed with the plasmid by flicking the side wall of the centrifuge tube with fingers; the mixed bacterial solution was transferred to a sterile electroporation cup along the side wall; the electroporation cup was placed in a Bio-rad electroporation instrument, and the transformation was carried out under the condition of 2500V / 6ms; 1 mL LB liquid medium was added, mixed, and then the Agrobacterium and LB liquid medium were transferred into a new 1.5 mL centrifuge tube; the centrifuge tube was placed in a 28°C shaker for 3h; 4500r·min -1 centrifugation for 5min; the supernatant was discarded, and the remaining bacterial solution was resuspended and plated on LB (50mg·L -1 Rifampicin+vector resistance) solid plate; the LB plate was placed in a 28°C incubator for 2d; a sterile toothpick was used to pick a single colony into sterile water, and then bacterial liquid PCR identification was carried out, and positive Agrobacterium was collected for standby.

[0045] The pCNF3 empty vector was transformed into Agrobacterium GV3101 competent according to the above method, cultured on LB (50mg·L -1 Rifampicin+vector resistance) solid plate, and positive colonies were collected, and empty vector Agrobacterium was obtained for standby.

[0046] Example 2

[0047] Agrobacterium-mediated genetic transformation of Arabidopsis thaliana

[0048] 1. Preparation of positive Agrobacterium dipping solution

[0049] The Agrobacterium (GV3101) strain containing AtHIR4 gene was transferred into 2 mL LB liquid medium containing antibiotic (Rif+ vector resistance) and cultured overnight at 28℃, 190 r·min -1 of shaker to expand the Agrobacterium; then 250 μL of bacterial solution was transferred into 5 mL LB liquid medium containing antibiotic (Rif+ vector resistance) and cultured for 6-8 h at 28℃, 190 r·min -1 of shaker to culture the Agrobacterium, when the cells grew into logarithmic phase, 5 mL of bacterial solution was transferred into a 15 mL centrifuge tube and centrifuged at 4000 r·min -1 for 15 min at 15℃, the supernatant was discarded, and 3 mL of 5 wt.% sucrose solution was used to resuspend the bacterial cells. The OD 600 was measured by spectrophotometer, and the bacterial solution was diluted with 5 wt.% sucrose solution to OD 600 0.8 to obtain the positive Agrobacterium dipping solution.

[0050] 2. Obtaining of AtHIR4 transgenic plants

[0051] The wild-type Arabidopsis Col-0 was infected with the positive Agrobacterium dipping solution obtained in step 1: the opened flowers or grown fruit pods of Arabidopsis Col-0 were cut off, and sufficient watering was performed in advance; 0.002% Silwet L-77 (0.15 μL·mL -1 ) was added to the positive Agrobacterium dipping solution before dipping the flowers, the inflorescences of treated Arabidopsis Col-0 were immersed in the prepared positive Agrobacterium dipping solution for 5 s, after all the inflorescences were dipped, the plants were covered with a lid, and were kept moist at room temperature for 12 h without excessive light; the next day, the plants were transferred to the culture room for normal culture, and the seeds were collected.

[0052] The collected seeds were disinfected with 15% 84 disinfectant solution for 15 min in a super-clean bench, and were washed with sterile water for 3-5 times, and the seeds were sowed on 1 / 2MS (50 mg·L -1The positive seedlings are green and the negative seedlings are yellow and withered. After one week, the positive seedlings are selected and transplanted into nutrient soil and cultured normally. After four weeks of growth, the transgenic Arabidopsis plants are detected by Western blotting. The results of Western blotting detection of the transgenic Arabidopsis plant lines overexpressing AtHIR4 are shown in Figure 1A. The protein bands of corresponding size are detected in the leaves of the positive plants after resistance screening using the corresponding antibodies, indicating that the transgenic Arabidopsis oxAtHIR4-8 line and oxAtHIR4-15 line (AtHIR4 transgenic group) overexpressing AtHIR4 and the wild-type Arabidopsis Col-0 (control group) are successfully obtained. Figure 1B shows the morphological characteristics of the transgenic Arabidopsis lines oxAtHIR4-8 and oxAtHIR4-15 after four weeks of growth. It can be seen that compared with the wild-type Arabidopsis Col-0, the transgenic Arabidopsis plants overexpressing AtHIR4 have smaller aboveground morphology and greener leaves under soil culture conditions after four weeks of growth, indicating that overexpression of AtHIR4 may have a certain effect on the growth of Arabidopsis.

[0053] Test Example 1

[0054] Determination of the ability of Arabidopsis plants to resist Sclerotinia sclerotiorum and Botrytis cinerea

[0055] 1. The seeds of Arabidopsis Col-0 plants, the seeds of oxAtHIR4-8 plants and the seeds of oxAtHIR4-15 plants obtained in Example 2 are cultured in a growth chamber for 4 weeks to obtain Arabidopsis Col-0 plants, oxAtHIR4-8 plants and oxAtHIR4-15 plants. The Arabidopsis Col-0 plants, oxAtHIR4-8 plants and oxAtHIR4-15 plants are divided into Sclerotinia sclerotiorum test groups and Botrytis cinerea test groups, respectively, and three sets of repeats are set in each group.

[0056] 2. The model strain S. sclerotiorum strain 1980 was cultured on PDA plates (Potato dextrose broth (PDA): 200 g peeled potato, 20 g glucose, 10 g agar, distilled water to 1 L), and young mycelium was picked and grown on new PDA. After 1-2 days of growth, a puncher with a diameter of 2 mm or 5 mm was used to punch the young mycelium at the edge of the colony, and then the puncher was inoculated on the right upper part of the leaf of the Arabidopsis Col-0 plant, the oxAtHIR4-8 plant and the oxAtHIR4-15 plant, and the culture was kept moist. After 40 h, data measurement and photography were performed, and the results are shown in Fig. 1c. The necrotrophic pathogenic fungus S. sclerotiorum caused significantly smaller lesion areas on the leaves of the Arabidopsis plants overexpressing AtHIR4 (oxAtHIR4-8 and oxAtHIR4-15) than on the leaves of the wild-type Arabidopsis Col-0.

[0057] 3. The model strain Botrytis cinerea strain B05.10 was cultured on PDA plates, and young mycelium was picked and grown on new PDA. After 1-2 days of growth, a puncher with a diameter of 2 mm or 5 mm was used to punch the young mycelium at the edge of the colony, and then the puncher was inoculated on the right upper part of the leaf of the Arabidopsis Col-0 plant, the oxAtHIR4-8 plant and the oxAtHIR4-15 plant, and the culture was kept moist. After 48 h, data measurement and photography were performed, and the results are shown in Fig. 1F. Botrytis cinerea caused significantly smaller lesion areas on the leaves of the Arabidopsis plants overexpressing AtHIR4 (oxAtHIR4-8 and oxAtHIR4-15) than on the leaves of the wild-type Arabidopsis Col-0.

[0058] 4. When measuring data, the lesion diameter was measured using the cross method, and the lesion area was calculated according to the elliptical area formula for statistical analysis. The results are shown in Fig. 1D and Fig. 1G. Fig. 1D shows the lesion area of the wild-type S. sclerotiorum strain inoculated for 40 h, and Fig. 1G shows the lesion area of the wild-type Botrytis cinerea strain inoculated for 48 h. The data represent the mean ± SD, and different letters in the figure indicate statistical significance (p<0.01) in one-way ANOVA. As shown in Fig. 1D and Fig. 1G, the necrotrophic pathogenic fungus S. sclerotiorum and Botrytis cinerea caused significantly smaller lesion areas on the leaves of the Arabidopsis plants overexpressing AtHIR4 (oxAtHIR4-8 and oxAtHIR4-15) than on the leaves of the wild-type Arabidopsis Col-0.

[0059] 5. In each group of Arabidopsis leaves, after measuring the lesion area, a puncher with a diameter of 1.5 cm was used to take an equal area sample from the infection site, and the DNA in the sample was extracted and analyzed by real-time fluorescent quantitative PCR to calculate the relative biomass of the fungal pathogen.

[0060] Primers used to analyze the relative biomass of B. cinerea by RT-qPCR are as follows:

[0061] SsTub-F: 5'-ACCTCCATCCAAGAACTC-3' (SEQ ID No. 5)

[0062] SsTub-R: 5'-GAACTCCATCTCGTCCAT-3' (SEQ ID No. 6);

[0063] AtUBQ5-F: 5'-ACACCAAGCCGAAGAAGA-3' (SEQ ID No. 7);

[0064] AtUBQ5-R: 5'-TCCACAGGTTGCGTTAGG-3' (SEQ ID No. 8);

[0065] Primers used to analyze the relative biomass of B. cinerea by RT-qPCR are as follows:

[0066] BcActin-F: 5'-CTTCGTGTAGCACCAGAGGAG-3' (SEQ ID No. 9)

[0067] BcActin-R: 5'-GAGAGGACGGCTTGAATAGAGA-3' (SEQ ID No. 10);

[0068] AtUBQ5-F: 5'-ACACCAAGCCGAAGAAGA-3' (SEQ ID No. 11)

[0069] AtUBQ5-R: 5'-TCCACAGGTTGCGTTAGG-3' (SEQ ID No. 12);

[0070] Each treatment above includes three replicates (each replicate contains 2 to 4 diseased leaves). The pathogenicity assay is repeated more than 3 times.

[0071] The results are shown in Figures 1 E and H, where E is the relative biomass of wild-type B. cinerea strain inoculated Col-0, oxAtHIR4-8 and oxAtHIR4-15 Arabidopsis leaves 40h after by RT-qPCR analysis, and H is the relative biomass of wild-type B. cinerea strain inoculated Col-0, oxAtHIR4-8 and oxAtHIR4-15 Arabidopsis leaves 48h after by RT-qPCR analysis; wherein the data represent the mean ± SD, and different letters in the figure indicate statistical significance of p<0.01 in one-way ANOVA.

[0072] As shown in Fig. 1, the biomass of the necrotrophic pathogens S. sclerotiorum and B. cinerea on the leaves of the Arabidopsis plants overexpressing AtHIR4 (oxAtHIR4-8 and oxAtHIR4-15) were significantly less than that of the wild-type Arabidopsis Col-0.

[0073] 6. Chitin triggers MAPKs phosphorylation activation

[0074] Seed sterilization: The seeds of the Arabidopsis Col-0 plants, oxAtHIR4-8 plants and oxAtHIR4-15 plants were sterilized with 70% ethanol for 5 min, washed once with sterile ddH2O, then sterilized with 84 disinfectant solution for 5 min in a clean bench, washed at least 3 times with sterile ddH2O, sowed on 1 / 2MS medium plates (20 mL of medium per plate) in a clean bench, about 35 seeds per plate, dried in the clean bench, but the plates should not be too dry, sealed with medical tape, placed in 4°C for 3 days, then transferred to the lower layer of the culture room (23°C / 20°C) for 10-12 days, then the healthy and uniform seedlings were transferred to 12-well culture plates, taking care not to damage the roots, 4 seedlings per well, 1 mL of sterile ddH2O was added to ensure that the roots were immersed in ddH2O, and the seedlings were recovered overnight, the ddH2O was removed, and 10 μg·mL -1 The chitin solution was incubated for 0 min, 5 min, 15 min and 30 min respectively, ensuring that the roots were immersed in the solution, the seedlings were taken out, the residual solution on the seedlings was quickly removed with absorbent paper and lens paper, and the seedlings were placed in a 1.5 mL centrifuge tube and stored in liquid nitrogen at -80°C.

[0075] Immunoblotting detection using MPK3, MPK4 and MPK6 phosphorylation antibodies: the seedlings stored at -80°C were placed in an ice box containing liquid nitrogen, the seedlings were ground with a grinder, 100 μL of 2×SDS loading buffer (β-mercaptoethanol and DTT were added) was added, and the mixture was denatured at 95°C for 10 min. 12000 r·min -1 centrifuged for 5 min, and the supernatant was subjected to protein electrophoresis; 10% SDS-PAGE electrophoresis was performed, (the molecular weight of MPK3, MPK4 and MPK6 was adjacent to 42 kD-46 kD), the position of the protein marker band was checked, the electrophoresis was stopped to separate the MPK3, MPK4 and MPK6 bands well, and then the membrane was transferred; -1 incubated for 1 h, anti-pERK1 / 2 was added, and the mixture was incubated at 4°C on a shaker at 80 r·min -1 incubated overnight; washed 3 times with 1×TBST for 15 min each time at room temperature on a shaker at 80 r·min-1 Secondary antibody anti-Rabbit-HRP room temperature shaker 80 r·min -1 Incubate for 2h. Continue washing with 1x TBST for 3 times, 15min each time. Choose Clarity Western ECL Substrate or Thermo West Femto chemiluminescent substrate to detect protein according to signal intensity.

[0076] The results of MAPKs phosphorylation in Col-0, oxAtHIR4-8 and oxAtHIR4-15 Arabidopsis seedlings after chitin induction are shown in Figure 1I, the values represent the mean ± SE (n = 12), wherein the upper part is the results of MAPKs phosphorylation detected by immunoblotting using MPK3, MPK4, MPK6 phosphorylation antibodies; the lower part is the staining of PVDF membrane after development using ponceau to show the consistency of loading amount. As can be seen from Figure 1I, the intensity of early immune response of oxAtHIR4-8 and oxAtHIR4-15 Arabidopsis seedlings after chitin treatment is also significantly improved, and the phosphorylation of MAPKs induced by chitin is significantly enhanced in AtHIR4 overexpression plants.

[0077] 7. Chitin triggers ROS burst in Arabidopsis leaves

[0078] After the Arabidopsis Col-0 plants, oxAtHIR4-8 plants and oxAtHIR4-15 plants obtained in Example 2 were grown for 4-5 weeks, 24 leaves were taken from each group of Arabidopsis plants, punched into 0.25 cm 2 diameter leaf discs, each leaf disc was cut into 4 narrow leaves; the leaves were placed in a 96-well plate, 100 μL of ddH2O was added to each well, and the leaves were recovered overnight; the ddH2O was removed, and 100 μL of reaction mixture was added, including 50 μM of Luminol, 10 μg·mL -1 of peroxidase, 10 μg·mL -1 of chitin; the samples were placed in a microplate reader, and the measurement was started immediately, and was measured every 1 minute, repeated for 35 times, and one cycle was 20 min. The number of active oxygen generated by 12 leaf discs after treatment was taken as the relative light unit.

[0079] The results of ROS burst in Col-0, oxAtHIR4-8 and oxAtHIR4-15 Arabidopsis leaves after chitin induction are shown in Figure 1J, wherein the values represent the mean ± SE (n = 12). As can be seen from Figure 1J, oxAtHIR4-8 and oxAtHIR4-15 Arabidopsis leaves show stronger ROS burst after chitin treatment.

[0080] And, from the conclusion in Fig. 1, it can be concluded that the oxAtHIR4-8 strain has a higher protein expression level and a stronger immune response, which also corresponds to its genetic phenotype of antifungal. The above results not only further provide genetic evidence for the positive role of AtHIR4 in Arabidopsis thaliana against necrotrophic pathogenic fungi, but also to some extent clarify that the disease resistance of Arabidopsis thaliana overexpressing AtHIR4 is due to its more rapid and intense early immune response.

[0081] Test Example 2

[0082] Detection of resistance of Arabidopsis thaliana plants to pathogenic bacteria

[0083] 1. Using the model bacterium Pseudomonas syringae to streak inoculate on KB plates, sealing the KB plates with plastic wrap and placing them in a 28°C incubator for 2d until the bacteria grow; then selecting single colonies to inoculate 2mL KB+antibiotics (Pst DC3000 is Rif resistant) liquid medium, and shaking culture at 28°C overnight. Centrifuge at room temperature at 4000r·min -1 for 5min to collect the bacterial cells, discard the supernatant, wash twice with ddH2O, and resuspend in 500μL of 10mM MgCl2; measure the OD 600 of the bacteria to calculate the concentration of the bacteria, and dilute the bacteria to an OD 600 value of 5×10 -4 with 10mM MgCl2 to obtain Pseudomonas syringae bacterial solution.

[0084] 2. Selecting 4-week-old leaves of Arabidopsis thaliana Col-0 plants, oxAtHIR4-8 plants and oxAtHIR4-15 plants in step 1 of Test Example 1, using a 1mL needle-free syringe to gently inject 100μL of bacterial solution into the lower epidermis of the leaves, covering them with a transparent cover to keep them moist for 5h-8h, placing the plants in a growth chamber for cultivation, and observing the disease condition every morning by spraying water on the surface of the plants; the plant nutrient soil formula used for cultivation is Finland Kegila peat soil: Jiangsu Peilei substrate: vermiculite = 8:4:1. The growth chamber conditions for cultivating plants for disease resistance experiments are temperature 20℃-23℃, humidity 65%, light intensity 75μE-100μE, and light cycle light / dark: 12h / 12h. The number of bacterial growth can be measured 2d-3d after inoculation. Representative Arabidopsis thaliana leaves photographed 3d after Pseudomonas syringae inoculation are shown in Fig. 2A, which shows that after 3d of Pseudomonas syringae inoculation, the leaves of Col-0 and transgenic Arabidopsis thaliana overexpressing AtHIR4 have obvious yellow spots, but the leaves of oxAtHIR4-8 and oxAtHIR4-15 Arabidopsis thaliana remain healthy green.

[0085] 3. Punch inoculated leaves with puncher, collect 8 leaf discs (6mm in diameter), grade according to disease severity, average and put into 4 1.5mL centrifuge tubes containing 100μL ddH2O, 2 leaf discs per tube; grind the leaf discs, add 900μL ddH2O and mix, serially dilute, use 10 -3 and 10 -4 dilutions to count bacteria, use 10 -2 μL of the diluted bacteria solution to inoculate TSA plates containing corresponding antibiotics (TSA medium formula: tryptone 10g·L -1 , sucrose 10g·L -1 , glutamic acid 10g·L -1 , agar 10g·L -1 ), use a loop to spread evenly, incubate at 28℃ for 2d (inoculate for 3d), then count bacterial colony forming units CFU.

[0086] The results of counting bacteria in Pseudomonas syringae inoculated for 0d and 3d are shown in Fig. 2B, which shows that the number of bacteria in oxAtHIR4-8 and oxAtHIR4-15 Arabidopsis plants is significantly lower than that in wild type Arabidopsis plants, and the number of bacteria in oxAtHIR4-8 Arabidopsis plants is lower due to higher expression of AtHIR4.

[0087] 4. flg22 triggers MAPKs phosphorylation activation

[0088] Seed sterilization: Sterilize the seeds of Arabidopsis Col-0 plants, oxAtHIR4-8 plants and oxAtHIR4-15 plants with 70% ethanol for 5min, wash once with sterile ddH2O, then sterilize with 84 disinfectant solution for 5min in a clean bench, and wash at least 3 times with sterile ddH2O; sow on 1 / 2MS medium plates (20mL of medium per plate) in a clean bench, sow about 35 seeds per plate, dry in the clean bench, but the plates should not be too dry, seal with medical tape; place the plates at 4℃ for germination, then transfer to the lower layer of the culture room (23℃ / 20℃) for 10-12d; then transfer healthy and uniform seedlings to 12-well culture plates, pay attention to prevent root damage, 4 plants per well, add 1mL sterile ddH2O to ensure that the roots are immersed in ddH2O, and recover overnight; remove the ddH2O and add 500μL of 100nM flg22 solution to incubate for 0min, 5min, 15min and 30min, respectively, ensuring that the roots are immersed in the solution; remove the seedlings, quickly remove the residual solution on the seedlings with absorbent paper and lens paper, and store in 1.5mL centrifuge tubes, and store in liquid nitrogen at -80℃.

[0089] 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.

[0090] The phosphorylation results of flg22-induced MAPKs in Arabidopsis plants are shown in Figure 2C. Different letters in the figure represent samples with significant differences (one-way ANOVA, P < 0.01). Data represent mean ± SD, with n = 6 biological replicates. The upper part shows the results of MAPK phosphorylation detected by immunoblotting using phosphorylation antibodies against MAPK3, MPK4, and MPK6. The lower part shows the results of staining the developed PVDF membrane with Ponceau S to show the consistency of sample loading. As shown in Figure 2C, flg22-induced MAPK phosphorylation was significantly enhanced in AtHIR4 overexpression plants.

[0091] 5. flg22 triggers a surge of reactive oxygen species in Arabidopsis leaves.

[0092] 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.

[0093] The results of flg22-induced ROS burst in Arabidopsis plants are shown in Figure 2D. 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.

[0094] 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 relatively broad spectrum of disease resistance.

[0095] Example 3

[0096] Genetic transformation of hypocotyls in Brassica napus

[0097] 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.

[0098] Using sterile forceps, the 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.

[0099] Place the petri dishes in a sterile culture box and incubate at 22-24°C in the dark for 6 days.

[0100] The positive Agrobacterium containing AtHir4 and the empty vector Agrobacterium prepared in Example 1 were streaked to single colonies during this period; single colonies were picked to 2 mL double-antibiotic medium after 5 days of inoculation and shaken overnight for 12 hours; 20 mL glass bottles were used to culture the target bacteria after 6 days of inoculation; 1 mL of the target Agrobacterium was inoculated into 10 mL of resistant liquid LB medium, and the culture was incubated at 28°C for about 4-6 hours on a 180-220 rpm shaker to obtain positive Agrobacterium infection liquid and empty vector Agrobacterium infection liquid.

[0101] 2. Preparation of explants and infection:

[0102] The co-culture medium M1 (M1 medium: MS 4.4 g, sucrose 30 g, mannitol 18 g, 2,4-D (1 mg·mL -1 ) 1 mL, KT (1 mg·mL -1 ) 0.3 mL, double-distilled water to 1 L, adjust pH to 5.84-5.88, agar powder 5.5 g, sterilize at 121°C for 20 min, add acetosyringone when the medium is quickly cooled, final concentration 100 μM) and DM liquid (DM medium: MS 4.4 g, sucrose 30 g, double-distilled water to 1 L, adjust pH to 5.84-5.88, sterilize at 121°C for 20 min) were prepared, and acetosyringone was added to the DM liquid to a final concentration of 100 μM for standby use.

[0103] The OD 600 values of the positive Agrobacterium infection liquid and the empty vector Agrobacterium infection liquid were adjusted to about 0.5 with DM liquid. 2 mL of the cultured positive Agrobacterium infection liquid and empty vector Agrobacterium infection liquid were respectively taken into sterile centrifuge tubes, centrifuged at 3000 rpm for 3 min, and the supernatant was discarded; 2 mL of DM liquid was added for suspension, centrifuged at 3000 rpm for 3 min, and the supernatant was discarded; 2 mL of DM liquid was added again for suspension to obtain positive Agrobacterium DM bacterial liquid and empty vector Agrobacterium DM bacterial liquid, which were stored in a 4°C refrigerator for standby use.

[0104] After 6 days of inoculation, the AtHIR4 group, empty vector group, and control group were prepared for cutting explant dishes; 18 mL of DM medium was added to each cutting explant dish in the AtHIR4 group and empty vector group in advance, and 20 mL of DM medium was added to each cutting explant dish in the control group in advance; the hypocotyls of the rape Y127 seeds were cut, and the cut explants were respectively transferred to the cutting explant dishes; 2 mL of positive Agrobacterium DM bacterial liquid was poured into each cutting explant dish in the AtHIR4 group, 2 mL of empty vector Agrobacterium DM bacterial liquid was poured into each cutting explant dish in the empty vector group, and no bacterial liquid was added to the cutting explant dishes in the control group; the liquid volume in each dish was 20 mL at this time, and the explants were immersed for 15 min, with shaking at intervals for 4-5 times.

[0105] When the 10thmin of infection, start with pipette suction DM bacteria liquid, with sterile forceps respectively to the explant on sterile filter paper placed for a while, and on the sterile filter paper to spread the explant, blow in the clean bench for about 3min, the purpose is to suck and evaporate the excess bacteria liquid on the explant. Then each group of explants are transferred to M1 medium (MS 4.4g, sucrose 30g, mannitol 18g, 2, 4-D (1mg·mL -1 ) 1mL, KT (1mg·mL -1 ) 0.3mL, agar powder 5.5g, double distilled water to 1L, adjust pH to 5.84-5.88, 121℃ sterilization 20min, when the medium is cooled quickly, add acetosyringone, so that its final concentration is 100μM) in the dark at 22-24℃ (placed in the light culture room in the dark place) for 22-26h. After the explant is transferred to M2 medium (MS 4.4g, sucrose 30g, mannitol 18g, 2, 4-D (1mg·mL -1 ) 1mL, kinetin (1mg·mL -1 ) 0.3mL, agar powder 5.5g, double distilled water to 1L, adjust pH to 5.84-5.88, 121℃ sterilization 20min. When the medium is cooled quickly, add 150μL silver thiosulfate, timentin (200mg·mL -1 ) 1.5mL, kanamycin sulfate (50mg·mL -1 ) 300μL) for 14-18d, induce callus formation, normal culture under light (24℃ light 16h / dark 8h). The callus-forming explants are transferred to M3 medium (MS 4.4g, glucose 10g, xylose 0.25g, MES 0.6g, double distilled water to 1L, adjust pH=5.84-5.88, Phytal gel 5.5g. When the medium is cooled quickly, add zeatin (2mg·mL -1 ) 1mL, auxin (1mg·mL -1 ) 0.1mL, timentin (200mg·mL -1 ) 1.5mL, kanamycin sulfate (50mg·mL -1 ) 300μL, 0.1M silver nitrate 150μL.) and then subculture on M3 every 2-3 weeks until green shoots appear in callus. The green shoots with complete growth points are transferred to M4 medium (MS 4.4g, sucrose 10g, double distilled water to 1L, adjust pH=5.84-5.88, agar powder 8g, 121℃ sterilization 20min. When the medium is cooled quickly, add timentin (200mg·mL -1)1.5mL) and induced rooting. The plants after rooting were transplanted into soil for normal culture, and identified by PCR to obtain transgenic Brassica napus plants oxAtHIR4-1 and oxAtHIR4-2, empty vector transgenic plants (EV) and Y127 plants of the control group.

[0106] The cDNA of the transgenic Brassica napus plants oxAtHIR4-1 and oxAtHIR4-2, empty vector transgenic plants (EV) and Y127 plants of the control group were used as templates for PCR experiments, and the results are shown in Figure 3A. The primers used are as follows:

[0107] AtHIR4-F: 5'-ATGGGGAATTTGTTTTGTTGTGT-3' (SEQ ID No. 13);

[0108] AtHIR4-R: 5'-TCGAGGTCCTCCTCGGAGATG-3' (SEQ ID No. 14);

[0109] BnUBQ5-F: 5'-ACACCAAGCCGAAGAAGA-3' (SEQ ID No. 15);

[0110] BnUBQ5-R: 5'-ACACCAAGCCGAAGAAGA-3' (SEQ ID No. 16).

[0111] The morphological characteristics of the oxAtHIR4 transgenic Brassica napus plants are shown in Figure 3B. The growth phenotype of the AtHIR4 transgenic Brassica napus plants had no obvious difference compared with the starting plants Y127 and the empty vector transgenic plants.

[0112] Test Example 2

[0113] Determination of the ability of Brassica napus plants to resist Sclerotinia sclerotiorum and Botrytis cinerea

[0114] The Brassica napus plants oxAtHIR4-1, oxAtHIR4-2, EV and Y127 obtained in Example 3 were respectively cultured in a growth chamber for 4 weeks. The transgenic Brassica napus plants and wild-type Brassica napus plants were respectively divided into Sclerotinia sclerotiorum test groups and Botrytis cinerea test groups, and three sets of repeats were set in each group.

[0115] Activated Sclerotinia sclerotiorum strain 1980 and Botrytis cinerea strain B05.10 were prepared in the manner described in Test Example 1.

[0116] The activated S. sclerotinum strain 1980 was cultured on PDA plates (Potato dextrose broth (PDA): 200 g peeled potato, 20 g glucose, 10 g agar, and distilled water to 1 L), and young mycelium was picked and grown on new PDA. After 1-2 days of growth, a puncher with a diameter of 2 mm or 5 mm was used to punch the young mycelium at the edge of the colony, and then the puncher was inoculated on the right upper part of the leaf of the Brassica napus oxAtHIR4-1 plant, oxAtHIR4-2 plant, EV plant, and Y127 plant, and the culture was kept moist. Data measurement and photography were performed 48 h later. The photography results are shown in Fig. 3C. When measuring the data, the cross-intersection method was used to measure the lesion diameter, and the lesion area was calculated according to the elliptical area formula for statistical analysis. The results are shown in Fig. 3D. As shown in Figs. 3C and 3D, the lesion area caused by S. sclerotinum on the AtHIR4 transgenic Brassica napus leaf was smaller.

[0117] The activated Botrytis cinerea strain B05.10 was cultured on PDA plates, and young mycelium was picked and grown on new PDA. After 1-2 days of growth, a puncher with a diameter of 2 mm or 5 mm was used to punch the young mycelium at the edge of the colony, and then the puncher was inoculated on the right upper part of the leaf of the Brassica napus oxAtHIR4-1 plant, oxAtHIR4-2 plant, EV plant, and Y127 plant, and the culture was kept moist. Data measurement and photography were performed 48 h later. The photography results are shown in Fig. 3F. When measuring the data, the cross-intersection method was used to measure the lesion diameter, and the lesion area was calculated according to the elliptical area formula for statistical analysis. The results are shown in Fig. 3G. As shown in Figs. 3F and 3G, the lesion area caused by Botrytis cinerea on the AtHIR4 transgenic Brassica napus leaf was smaller.

[0118] In each group of leaves, after measuring the lesion area, a square puncher with a side length of 2.5 cm was used to take an equal-area sample from the infection site, and the DNA in the sample was extracted and analyzed for the relative content of fungal pathogens and plants by real-time fluorescent quantitative PCR. Each treatment included three replicates (each replicate contained 2 to 4 diseased leaves). The pathogenicity determination test was repeated more than 3 times.

[0119] The primers used for analyzing the relative biomass of S. sclerotiorum and B. cinerea by RT-qPCR were the same as those in Test Example 1. The results are shown in Figure 3E and Figure 3H, wherein E is the relative biomass of S. sclerotiorum at 40 h after inoculation of the wild-type S. sclerotiorum strain analyzed by RT-qPCR; H is the relative biomass of B. cinerea at 48 h after inoculation of the wild-type S. sclerotiorum strain analyzed by RT-qPCR; the data in the figure represent the mean ± SD, and different letters indicate statistical significance (p < 0.01) in one-way ANOVA. As can be seen from Figure 3E and H, the biomass of the necrotrophic pathogenic fungi S. sclerotiorum and B. cinerea on the leaves of the AtHIR4 transgenic oilseed rape plants (oxAtHIR4-1 and oxAtHIR4-2) was significantly less than that on the empty vector transgenic oilseed rape plants (EV) and the wild-type oilseed rape plants (Y127).

[0120] As can be seen from the above results, the AtHIR4 transgenic oilseed rape plants have significantly enhanced resistance to the necrotrophic pathogenic fungi, and the lesion area caused by S. sclerotiorum and B. cinerea on the leaves of the AtHIR4 transgenic oilseed rape plants is smaller (Figure 3C, D, F, G), and the relative biomass analysis of the pathogenic fungi also shows that the leaves of the AtHIR4 transgenic oilseed rape plants exhibit significant resistance to the infection of S. sclerotiorum and B. cinerea (Figure 3E, H).

[0121] As can be seen from the above examples, the AtHIR4 constitutively expressing transgenic Arabidopsis and oilseed rape are successfully constructed, and the disease resistance detection shows that overexpression of AtHIR4 in the Arabidopsis and oilseed rape plants not only enhances their resistance to the necrotrophic pathogenic fungi S. sclerotiorum and B. cinerea, but also significantly increases the disease resistance of the transgenic Arabidopsis to the semi-biotrophic pathogenic bacteria. It is shown that AtHIR4 mediates broad-spectrum disease resistance to multiple pathogenic bacteria in plants, and the AtHIR4 gene can provide a new gene resource for green control of Sclerotinia sclerotiorum and B. cinerea diseases of oilseed rape.

[0122] Although the above examples have made a detailed description of the present application, it is only a part of the embodiments of the present application, but not all the embodiments, and other embodiments can be obtained by people according to the present embodiments without creativity, which all belong to the protection scope of the present application.

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.

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 according to claim 1 or 2, characterized in that, The pathogens include one or more of Sclerotinia sclerotiorum, Botrytis cinerea, and Pseudomonas syringae.

5. 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.

6. 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.

7. The application according to claim 5 or 6, characterized in that, The plants include Arabidopsis thaliana and / or rapeseed.

8. 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.

9. The method according to claim 8, 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.

10. The method according to claim 9, characterized in that, The basic vector includes pCNF3; the recipient plant includes Arabidopsis thaliana inflorescence or Brassica napus hypocotyl.

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