Use of atpen2 protein and / or its encoding gene atpen2 in preventing cruciferous plant diseases

Abstract

The application belongs to the technical field of genetic engineering, and particularly relates to application of AtPEN2 protein and / or its coding gene AtPEN2 in preventing cruciferous plant diseases. The application provides application of AtPEN2 protein and / or its coding gene AtPEN2 in preventing cruciferous plant diseases, and the amino acid sequence of the AtPEN2 protein is shown as SEQ ID NO. 1. The application takes Arabidopsis thaliana as a model plant to verify the prevention and treatment effect of overexpression of AtPEN2 protein on clubroot, and the results show that the AtPEN2 protein overexpression plant can improve the resistance to clubroot, indicating that the coding gene AtPEN2 of the AtPEN2 protein can be applied to molecular breeding of clubroot, and provides a new gene resource for molecular breeding of cruciferous crops.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to the application of AtPEN2 protein and / or its encoding gene AtPEN2 in the prevention of diseases in cruciferous plants. Background Technology

[0002] Clubroot, a soil-borne disease, is caused by the fungus *Platycodon grandiflorus*, which primarily infects cruciferous vegetables, causing swollen and deformed roots. This poses a significant threat to my country's cruciferous vegetable industry. Therefore, researching the interaction mechanism between *Platycodon grandiflorus* and its host, elucidating the pathogenic mechanism of *Platycodon grandiflorus*, and exploring gene resources for resistance to clubroot are urgent problems to be solved.

[0003] Discovering disease-resistance genes and elucidating disease-resistance mechanisms are key to successful molecular breeding for disease resistance. Cruciferous plants possess a black myrosinase system that plays a defensive role against various biotic and abiotic stresses and participates in regulating their growth and development. AtPEN2, a β-O-glucosyl hydrolase and an atypical black myrosinase, is involved in the biosynthesis and secretion of resistance substances. Currently, research on the role of the atypical black myrosinase AtPEN2 in enhancing disease resistance in cruciferous plants is limited. Summary of the Invention

[0004] The purpose of this invention is to provide the application of AtPEN2 protein and / or its encoding gene AtPEN2 in the prevention of diseases in cruciferous plants, and to provide new gene resources for the green control of clubroot disease.

[0005] This invention provides the application of AtPEN2 protein and / or its encoding gene AtPEN2 in the prevention of diseases in cruciferous plants, wherein the amino acid sequence of the AtPEN2 protein is shown in SEQ ID NO.1.

[0006] As a preferred embodiment, the nucleotide sequence of the encoding gene AtPEN2 is shown in SEQ ID NO.2.

[0007] As a preferred embodiment, the cruciferous plant diseases include clubroot.

[0008] As a preferred embodiment, the cruciferous plant includes Arabidopsis thaliana.

[0009] The present invention also provides the application of AtPEN2 protein and / or its encoding gene AtPEN2 in improving the disease resistance of cruciferous plants, wherein the amino acid sequence of AtPEN2 protein is shown in SEQ ID NO.1.

[0010] As a preferred embodiment, the nucleotide sequence of the encoding gene AtPEN2 is shown in SEQ ID NO.2.

[0011] As a preferred embodiment, the cruciferous plant diseases include clubroot.

[0012] The present invention also provides the application of AtPEN2 protein and / or its encoding gene AtPEN2 in the cultivation of cruciferous plants resistant to clubroot disease, wherein the amino acid sequence of AtPEN2 protein is shown in SEQ ID NO.1.

[0013] The present invention also provides a method for improving the disease resistance of cruciferous plants by overexpressing the AtPEN2 protein encoding gene in cruciferous plants; the amino acid sequence of the AtPEN2 protein is shown in SEQ ID NO.1.

[0014] As a preferred embodiment, the cruciferous plant includes Arabidopsis thaliana; the plant disease includes clubroot.

[0015] Beneficial effects:

[0016] This invention provides the application of the AtPEN2 protein and / or its encoding gene AtPEN2 in the prevention of diseases in cruciferous plants. The amino acid sequence of the AtPEN2 protein is shown in SEQ ID NO.1. Using Arabidopsis thaliana as a model plant, this invention verifies the control effect of overexpressing the AtPEN2 protein on clubroot disease. The results show that plants overexpressing the AtPEN2 protein can improve the resistance of Arabidopsis thaliana to clubroot disease, indicating that the AtPEN2 gene encoding the AtPEN2 protein can be applied to molecular breeding for clubroot disease control, providing a new gene resource for molecular breeding of disease resistance in cruciferous crops. Attached Figure Description

[0017] 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.

[0018] Figure 1 The results of WB identification of T0 generation AtPEN2 transgenic Arabidopsis seedlings in Example 3;

[0019] Figure 2 The results of WB identification of T1 generation AtPEN2 transgenic Arabidopsis seedlings in Example 4 are shown; where A is the WB identification result of AtPEN2-2 transgenic Arabidopsis seedlings; and B is the WB identification result of AtPEN2-17 transgenic seedlings.

[0020] Figure 3The images show the phenotypic diagrams of Arabidopsis thaliana seedlings in Example 4. A represents the growth phenotypes of wild-type Arabidopsis thaliana Col-0, AtPEN2-2-3, and AtPEN2-17-3 in the control group, grown to 31 days prior (scale bar = 2cm); B represents the growth phenotypes of wild-type Arabidopsis thaliana Col-0, AtPEN2-2-3, and AtPEN2-17-3 in the experimental group, 17 days after inoculation with *Plasmodium* (scale bar = 2cm); C represents representative plants from each family of wild-type Arabidopsis thaliana Col-0, AtPEN2-2-3, and AtPEN2-17-3 in the experimental group (scale bar = 1cm).

[0021] Figure 4 The disease index is the Arabidopsis seedlings in Example 4 17 days after inoculation with clubroot bacteria. Detailed Implementation

[0022] This invention provides the application of the AtPEN2 protein and / or its encoding gene AtPEN2 in the prevention of diseases in cruciferous plants. The amino acid sequence of the AtPEN2 protein is shown in SEQ ID NO.1:*. The AtPEN2 protein described in this invention is an atypical black myrosinase in Arabidopsis thaliana, which can enhance the resistance of Arabidopsis thaliana to clubroot fungi, providing a new gene resource for molecular breeding of disease-resistant cruciferous crops.

[0023]

[0024] In one embodiment, the cruciferous plant diseases described in this invention include clubroot. In another embodiment, the cruciferous plant described in this invention includes Arabidopsis thaliana.

[0025] This invention also provides the application of the AtPEN2 protein and / or its encoding gene AtPEN2 in improving disease resistance in cruciferous plants, wherein the amino acid sequence of the AtPEN2 protein is shown in SEQ ID NO.1. As one embodiment, the nucleotide sequence of the encoding gene AtPEN2 is shown in SEQ ID NO.2.

[0026] This invention also provides the application of the AtPEN2 protein and / or its encoding gene AtPEN2 in the breeding of clubroot-resistant cruciferous plants, wherein the amino acid sequence of the AtPEN2 protein is shown in SEQ ID NO.1. As one embodiment, the nucleotide sequence of the encoding gene AtPEN2 is shown in SEQ ID NO.2. This invention demonstrates that high expression of the AtPEN2 protein in Arabidopsis thaliana plants can improve the resistance of Arabidopsis thaliana to clubroot, indicating that the encoding gene AtPEN2 can be applied to molecular breeding for clubroot disease, providing a new gene resource for molecular breeding of disease-resistant cruciferous crops.

[0027] This invention also provides a method for improving the disease resistance of cruciferous plants by overexpressing the AtPEN2 protein-encoding gene in cruciferous plants; the amino acid sequence of the AtPEN2 protein is shown in SEQ ID NO.1. As one embodiment, the nucleotide sequence of the AtPEN2 gene is shown in SEQ ID NO.2. As one embodiment, the cruciferous plant includes Arabidopsis thaliana. As one embodiment, the plant disease includes clubroot.

[0028] In one embodiment, this invention utilizes transformants containing the AtPEN2 protein-coding gene to transform cruciferous plants, achieving overexpression of the AtPEN2 protein-coding gene in cruciferous plants and improving their disease resistance. In another embodiment, the transformants include recombinant vectors and / or recombinant bacteria containing the AtPEN2 protein-coding gene. This invention, by highly expressing the AtPEN2 protein-coding gene in Arabidopsis thaliana plants, can improve Arabidopsis thaliana's resistance to clubroot disease.

[0029] To further illustrate the present invention, the application of the AtPEN2 protein and / or its encoding gene AtPEN2 provided by the present invention in the prevention of diseases of cruciferous plants is described in detail below with reference to the accompanying drawings and embodiments, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0030] Example 1

[0031] Gene cloning and vector construction

[0032] (1) Extraction of RNA from Arabidopsis thaliana

[0033] ① Take fresh Arabidopsis thaliana leaves and grind them into powder in a mortar with liquid nitrogen. Quickly transfer 100 mg of frozen tissue powder into a 2 mL centrifuge tube, add 1 mL of RNA extraction solution TRIPure, and immediately shake vigorously to mix. Place the homogenate sample on ice and let it stand for 10 min.

[0034] ② Add 0.2 mL of chloroform to every 1 mL of TRIPURE, tighten the cap, immediately shake vigorously to mix, and place on ice for 10 min;

[0035] ③ The mixture was centrifuged at 12000 r / min for 20 min at 4℃. After centrifugation, the mixture was divided into three layers: a lower red organic phenol-chloroform layer, a middle layer, and an upper colorless water layer. RNA was present in the water layer.

[0036] ④ Transfer the supernatant of the aqueous sample layer to a clean 1.5 mL centrifuge tube, add an equal volume of isopropanol, mix by inverting, place in a -20℃ refrigerator for 30 min, centrifuge at 12000 r / min at 4℃ for 20 min, and discard the supernatant.

[0037] ⑤ Add 1 mL of 75% (v / v) ethanol prepared with DEPC water to the precipitate, wash the precipitate, centrifuge at 12000 r / min for 5 min at 4℃, discard the supernatant, and be careful not to lose RNA precipitate. Repeat step ⑤ twice.

[0038] ⑥ Place the centrifuge tube in a centrifuge and centrifuge at 12000 r / min for 1 min. Then, carefully aspirate the residual liquid with a pipette and let it stand at room temperature for 5-10 min to dry the RNA precipitate.

[0039] ⑦ Add 50 μL of DEPC water to each tube to fully dissolve the RNA. After determining the concentration of the obtained RNA using Nanodrop 2000, store it at -80℃ to prevent degradation for later use.

[0040] (2) Cloning of the target gene

[0041] ① The RNA extracted in step (1) was used to synthesize first-strand cDNA using a reverse transcription kit (TransGene, Code#AE311-03). The total reaction volume was: Total RNA 4 μg, Anchored Oligo(dT)18 Primer (0.5 μg / μL) 1 μL, gDNARemover 1 μL, EasyScript RT / RI Enzyme Mix 1 μL, 2×ES Reaction Mix 10 μL, and RNase-free Water to make up to 20 μL. The reaction volume was gently mixed and incubated at 42℃ for 30 min. Then, it was heated at 85℃ for 5 sec to inactivate Easyscript RT / RI and gDNARemover.

[0042] ② Enter the gene number AT2G44490 of the Arabidopsis gene AtPEN2 into the TAIR database website (https: / / www.arabidopsis.org / ) to retrieve the coding sequence (CDS) and amino acid sequence of the gene. Combine the retrieved coding sequence and the vector multiple cloning site sequence to design the gene amplification primers PCNF3-AtPEN2-F and PCNF3-AtPEN2-R. The primer sequence design information from 5' to 3' includes the vector terminal homologous sequence (lowercase), restriction enzyme site (lowercase underlined part), and gene-specific sequence (uppercase).

[0043] PCNF3-AtPEN2-F(SEQ ID NO.3):5'-ttacgaacgatagca tctaga ATGGCACATCTTCAAAGAACATTTCCT-3';

[0044] PCNF3-AtPEN2-R(SEQ ID NO.4):5'-gtctttgtagtccat cccggg ATTATTAGCTCCTTTGAAGAACAGAGAAGT-3'.

[0045] ③ Using the Arabidopsis cDNA synthesized in step ① as a template, PCR amplification was performed using the high-fidelity DNA polymerase KOD One. The total PCR amplification system was 40 μL: 10 μM PrimerF 1 μL, 10 μM PrimerR 1 μL, 2×KOD OnePCRMasterMix 20 μL, cDNA 2 μL, and ddH2O 16 μL.

[0046] The PCR cycling conditions were as follows: pre-denaturation, 98°C, 2 min; denaturation, 98°C, 10 sec; annealing, 58°C, 5 sec; extension, 68°C, 5 sec / kb, cycle number 35; final extension, 68°C, 30 sec; storage at 8°C, 5 min.

[0047] After the reaction, 4.4 μL of 10× DNA loading buffer was added to the PCR product and mixed well. The mixture was then run on a 1% (w / v) agarose gel with nucleic acid dye (Biosharp, BS354B; added at a volume ratio of 1:10000) at 130V for 20 min. The correct PCR product was checked using a Tanon 1600 automated digital gel imaging system. The correctly sized bands were excised from the gel and purified using a gel extraction kit to obtain the target gene AtPEN2.

[0048] (3) Enzyme digestion vector

[0049] The PCNF3-FLAG tag vector (published in the literature: Yang, GG, et al. Acerato-platanin protein SsCP1 targets plant PR1 and contributes to virulence of Sclerotini as clerotiorum. New Phytologist, 2017) was linearized using two restriction endonucleases, XbaI and SmaI. The enzyme digestion system (70 μL) consisted of: 50 μL plasmid, 1 μL XbaⅠ, 1 μL SmaⅠ, 7 μL 10×rCutsmartbuffer (NEB), and 11 μL ddH2O. The digestion system was mixed thoroughly and incubated at 37°C for 2 h. Then, 7.7 μL of 10×DNAloadingbuffer was added and mixed. Electrophoresis was performed on a 1% (w / v) agarose gel with added nucleic acid dye (Biosharp, BS354B; added at a volume ratio of 1:10000) at 130V for 20 min. The correctness of the digested bands was checked using a Tanon 1600 automated digital gel imaging system. The target band was excised from the gel and purified using a gel extraction kit.

[0050] (4) Carrier construction

[0051] The target gene fragment obtained from gel purification in step (2) was directionally cloned into the linearized vector obtained in step (3) using seamless cloning technology. Using a VAZYME kit (catalog number C112-01), the following 10 μL reaction mixture was prepared on ice: 2 μL 5×CEⅡ buffer, 1 μL Exnase Ⅱ, 2 μL linearized vector, 1 μL target gene fragment, and 4 μL ddH2O. The reaction mixture was gently pipetted and centrifuged at 500 rpm for 30 s. The centrifuged mixture was collected at the bottom of a PCR tube and incubated at 37°C for 30 min.

[0052] (5) Transformation of Escherichia coli

[0053] After the reaction in step (4) is completed, the recombinant product is heat-shocked to transform Escherichia coli DH5α. The specific steps are as follows: Take out competent DH5α cells from the -80℃ freezer and thaw them on ice; add 10 μL of recombinant product to 30 μL of competent cells, gently tap the tube wall to mix, and let stand on ice for 30 min; heat shock in a 42℃ water bath for 45 sec, and immediately place on ice to cool for 3 min; add 1 mL of liquid LB medium (without antibiotics), shake at 37℃ for 220 r / min for 1 h; after shaking, centrifuge at 5000 r / min for 5 min and discard 800 μL of supernatant; resuspend the bacterial cells with the remaining medium, and gently spread them evenly on an LB plate containing Kana resistance using a sterile spreader. The final concentration of Kana in the LB plate is 50 μg / mL; place the LB plate in an incubator at 37℃ and invert it for culture.

[0054] (6) Identification of recombinant plasmids

[0055] ① After single colonies grow on the LB plate in step (5), select several single colonies for PCR identification. Select a small amount of bacterial cells and place them in 10 μL of the prepared PCR Mix. The PCR Mix system contains 0.5 μL of 10 μM CNF3-F, 0.5 μL of 10 μM CNF3-R, 5 μL of 2×Rapid Taq Master Mix, and 4 μL of ddH2O.

[0056] PCNF3-F (SEQ ID NO.5): 5'-GTTCATTCATTTGGAGAGGACCT-3';

[0057] PCNF3-R (SEQ ID NO. 6): 5'-GTATTAAATGTATAATTGCGGGACTC-3'.

[0058] The PCR cycling conditions were as follows: pre-denaturation, 95°C, 3 min; denaturation, 95°C, 15 sec; annealing, 58°C, 15 sec; extension, 72°C, 15 sec / kb, cycle number 35; final extension, 75°C, 5 min; storage at 8°C, 5 min.

[0059] ② The PCR products obtained in step ① above were subjected to electrophoresis on a 1% (w / v) agarose gel with added nucleic acid dye (Biosharp, BS354B; added at a volume ratio of 1:10000) at 130V for 20 min; the size of the PCR product bands was checked using a Tanon 1600 fully automated digital gel imaging system.

[0060] ③ Select a single colony with the correct PCR band and inoculate it into 2 mL of liquid culture medium containing Kana antibiotic. Incubate at 37°C with a shaker at 220 r / min for 12 h. After the culture is completed, take a small amount of bacterial solution and send it to Beijing Aoke Dingsheng Biotechnology Co., Ltd. for sequencing to further verify whether the recombinant plasmid is correct.

[0061] ④ For bacterial cultures with correct sequencing, collect the remaining bacterial cells and extract plasmids using the method of small-scale plasmid extraction (publication: Research on the molecular mechanism of CPK18 gene regulating ethylene and disease resistance, Li Hong, doctoral dissertation of Huazhong Agricultural University, 2024). Store the extracted plasmids at -20℃.

[0062] Example 2

[0063] Agrobacterium-mediated genetic transformation in Arabidopsis thaliana

[0064] (1) Seed disinfection and germination

[0065] ① Immerse Arabidopsis thaliana Col-0 seeds in 1 mL of 75% (v / v) ethanol for 1 min in a 1.5 mL centrifuge tube, then discard the ethanol; ② Rinse once with sterile ddH2O, then discard the water; ③ Soak in 1 mL of 10% (v / v) 84 disinfectant (100 μL 84 disinfectant + 900 μL sterile ddH2O) for 10 min, then discard the disinfectant; ④ Wash the seeds 5 times with 1 mL of sterile ddH2O; ⑤ Sow Col-0 Arabidopsis thaliana seeds on antibiotic-free 1 / 2 MS medium using a pipette, place the petri dishes in a constant temperature incubator at 22℃, and after 14 days, select Arabidopsis thaliana seedlings with uniform growth and transplant them into soil. The 1 / 2 MS medium consists of: MS 2.22 g, MES 0.5 g, sucrose 15 g, agar 14 g, sterile water to a final volume of 1 L, and pH adjusted to 5.8.

[0066] (2) Plasmid transformation of Agrobacterium

[0067] ① Remove Agrobacterium competent cells GV3101 from the -80℃ freezer and thaw on ice; ② Take 2 μL of the correctly sequenced plasmid from Example 1 and add it to 100 μL of Agrobacterium GV3101 competent cells. Gently tap one side of the tube wall with your finger to thoroughly mix the plasmid and competent cells; ③ Add the mixed liquid to a clean and dried electroporation cuvette, place the cuvette in a Bio-rad electroporator; select the program for electroporation, then remove the cuvette and add 500 μL of liquid LB medium (…). (Without adding antibiotics), transfer the mixture to a new 1.5 mL centrifuge tube; ④ Place the centrifuge tube from step ③ in a 28℃ shaker, shake at 220 r / min for 1 h, centrifuge at 5000 r / min for 5 min, and discard 400 μL of supernatant; resuspend the bacterial body with the remaining culture medium, and gently spread it evenly on an LB agar plate containing Kana (final concentration 50 μg / mL) and Rif (final concentration 50 μg / mL) using a sterile spreader; incubate upside down in a 28℃ incubator for 48 h. ⑤ After single colonies have grown, pick several single colonies for PCR identification. The PCR system and cycling procedure are the same as the recombinant plasmid identification in step (6) of Example 1. ⑥ Take a small amount of colonies that have been correctly identified by PCR using a white pipette tip and put them into a 10 mL EP tube containing 2 mL of LB liquid medium. Incubate overnight with shaking. The LB liquid medium contains Kana and Rif at final concentrations of 50 μg / mL, respectively. After incubation, take 1 mL of bacterial culture, add 1 mL of 50% glycerol to a 2 mL preservation tube, and store at -20℃.

[0068] (3) Arabidopsis thaliana flower dipping

[0069] A. ①Activate the glycerol bacteria preserved in step (2) by streaking on a double-antibody LB agar plate containing Kana and Rif (the final concentrations of Kana and Rif are 50 μg / mL, respectively); 2 days later, use a white sterile pipette tip to pick up a single colony and place it in 2 mL of double-antibody liquid LB medium (the double antibodies are Kana+Rif, and the final concentrations of Kana and Rif are 50 μg / mL, respectively) and shake overnight; then inoculate it at a ratio of 1:100 (v / v) into a 200 mL Erlenmeyer flask containing 50 mL of double-antibody liquid LB medium (Kana+Rif), place it on a shaker at 28℃ and shake at 220 r / min until OD. 600=Approximately 1.2; Collect Agrobacterium tumefaciens bacterial suspension using a 50 mL centrifuge tube, centrifuge at 5000 r / min for 10 min, and collect Agrobacterium tumefaciens cells; discard the supernatant, resuspend the cells in 50 mL of 5% (w / v) sucrose solution, and add 10 μL of Silwet L-77 to make the final concentration 0.02% (v / v). ② After the Arabidopsis thaliana transplanted in step (1) has grown to the point of bolting and flowering, cut off all the pods and white flowers on the Arabidopsis thaliana plant with scissors, immerse all the inflorescences of Arabidopsis thaliana in the Agrobacterium tumefaciens bacterial suspension obtained in step ①, gently shake for 15 s, and drain the excess Agrobacterium tumefaciens bacterial suspension. ③ Repeat step ② 1 h after step ① is completed. ④ Incubate the Arabidopsis thaliana treated with Agrobacterium tumefaciens in a moist environment in the dark for 16 h, and then transfer it to normal culture.

[0070] B. After 10 days, repeat step A, dipping the flowers again. The difference is that the step of cutting off all the pods and exposed flowers on the Arabidopsis plant with scissors in step A is omitted. After the Arabidopsis matures, collect the T0 generation seeds in 1.5mL centrifuge tubes, add a few absorbent blue silica gel beads to the centrifuge tubes, and store them in a seed storage cabinet.

[0071] Example 3

[0072] Molecular detection of transgenic Arabidopsis thaliana

[0073] (1) Extraction of total protein from Arabidopsis thaliana

[0074] ① Take some of the transgenic Arabidopsis thaliana T0 generation seeds harvested in Example 2 and place them in a 1.5 mL centrifuge tube. Disinfect the seeds according to the method in step (1) of Example 2. ② Sow the disinfected seeds on a 1 / 2 MS plate containing Kana (final concentration of 50 μg / mL), and then place the petri dish in a constant temperature incubator at 22℃. ③ After 14 days of culture, select positive seedlings and transplant them into the soil. ④ When the positive seedlings in the soil have grown for about one month, take some leaves and place them in a 1.5 mL centrifuge tube with two steel balls attached. Quickly immerse the tube in liquid nitrogen for flash freezing. Then place the centrifuge tube in a grinder and grind the sample at 40 Hz for 1 min. ⑤ After grinding the leaves, add 200 μL of 2×SDS protein extraction buffer (40% (v / v) glycerol, 4% (w / v) SDS (sodium dodecyl sulfate), 0.1% (w / v) bromophenol blue, 0.25M Tris-HCl pH 6.8, 4% (v / v) β-mercaptoethanol) to each tube, vortex to mix, and place in a 98℃ metal bath for 10 min. ⑥ Detect the expression of the target protein using Western blotting (WB). Before loading the WB sample, centrifuge at 12000 r / min for 10 min, and take 15 μL of supernatant from each sample for loading.

[0075] (2) Western blotting of proteins

[0076] ① Protein electrophoresis separation: Prepare a 5% (w / v) SDS-PAGE stacking gel and a 10% (w / v) SDS-PAGE separating gel; assemble the gel plate, first pour in 7 mL of separating gel, then immediately add 1 mL of n-butanol to ensure a smooth interface between the separating gel and the stacking gel, and wait for 30 min; after the separating gel solidifies, remove the added 1 mL of liquid with a syringe, then pour in 3 mL of stacking gel, insert the comb, and wait for 30 min; after the stacking gel solidifies, place the prepared gel plate into a vertical electrophoresis tank, add an appropriate amount of SDS-PAGE electrophoresis buffer, remove the comb, load 15 μL of sample into each well, set the voltage to 100 V, and run the electrophoresis; after 30 min, adjust the voltage to 65 V, and after 2-3 h, according to the marker protein molecular weight indication, when the target protein reaches the middle position of the gel plate, the electrophoresis is complete (the entire electrophoresis process must be completed in an ice water bath), then remove the stacking gel and place the separating gel into the transfer buffer. SDS-PAGE electrophoresis buffer formulation 1L: Tris 3.02g, Glycine 14.4g, SDS 1g.

[0077] ② Transfer: Cut the PVDF membrane to 4cm × 7cm and mark the bottom. Immerse the PVDF membrane in methanol for 30 seconds, wash three times with ddH2O for 5 minutes each time, and then place the PVDF membrane in transfer buffer. Assemble the transfer "sandwich" in the transfer buffer, from bottom to top: fiber pad, filter paper, separating gel, PVDF membrane, filter paper, fiber pad. Place the assembled "sandwich" vertically into the transfer electrophoresis tank, with an ice box on each side of the tank. Fill the tank with transfer buffer and incubate at 60V for 2 hours (the entire transfer process must be completed in an ice-water bath). After transfer, transfer the PVDF membrane to a protein incubation box and wash three times with ddH2O for 5 minutes each time. Transfer buffer formula (1L): Tris 3.02g, Glycine 14.4g, Methanol 200mL.

[0078] ③ Blocking: Add 10 mL of TBST solution containing 5% (w / v) milk powder to the protein incubation box, place it on a shaker at 50 r / min, and incubate overnight at 4°C; wash the PVDF membrane with TBST for 8 min each time.

[0079] ④ Primary antibody incubation: Add 10 mL of TBST solution containing 3% (w / v) milk powder to the protein incubation box, add 3.3 μL of Anti-Flag antibody at a ratio of 1:3000 (v / v), place on a shaker at 50 r / min, and incubate overnight at 4°C; wash the PVDF membrane with TBST for 8 min each time.

[0080] ⑤ Secondary antibody incubation: Add 10 mL of TBST solution to the protein incubation box, add 1 μL of Anti-Mouse antibody at a ratio of 1:10000 (v / v), place in a shaker at 50 r / min, and incubate at room temperature for 1 h; wash the PVDF membrane with TBST for 8 min each time.

[0081] ⑥ Development: Transfer the PVDF membrane into a mixture of 300 μL peroxide solution and 300 μL uminol / enhancer solution, and incubate at room temperature for 3 min; finally, detect the protein bands using a Tanon 5100 chemiluminescence imaging system.

[0082] This invention tested 20 T0 generation Arabidopsis thaliana seedlings; the Western blot results are shown below. Figure 1 In this context, the antibody used for WB is Anti-Flag, and CBB indicates that Coomassie Brilliant Blue staining shows the amount of sample loaded for each sample. According to... Figure 1 It was found that 12 Arabidopsis thaliana seedlings expressed the target protein, numbered 1, 2, 3, 4, 5, 7, 11, 15, 16, 17, 18, and 19. The other numbered plants were negative. Figure 1 (A and B in the text). Based on the thickness of the protein bands detected by Western blotting, the expression levels of the target protein in individual Arabidopsis plants can be distinguished. Among them, the expression level of AtPEN2 was relatively high in Arabidopsis plants numbered 1, 2, 5, and 7, the expression level of AtPEN2 was at a moderate level in Arabidopsis plant numbered 17, and the expression level of AtPEN2 was the lowest in Arabidopsis plants numbered 3, 4, 11, 15, 16, 18, and 19.

[0083] Example 4

[0084] Transgenic Arabidopsis thaliana inoculated with clubroot bacteria

[0085] (1) Screening for positive T1 generation seedlings

[0086] ① Based on the WB test results in Example 3, select numbers 2 and 17 from the T0 generation family, and record them as AtPEN2-2 and AtPEN2-17, and continue to propagate the T1 generation.

[0087] ② Five Arabidopsis thaliana seedlings from the T1 generation of the AtPEN2-2 family and 13 Arabidopsis thaliana seedlings from the AtPEN2-17 family were subjected to Western blotting (WB) analysis, using the same method as in Example 3. The results are shown in [link to example]. Figure 2 In this context, the antibody used for WB is Anti-Flag, and CBB indicates that Coomassie Brilliant Blue staining shows the amount of sample loaded for each sample.

[0088] according to Figure 2 It can be seen that all five Arabidopsis thaliana seedlings from the AtPEN2-2 family were positive. Figure 2 In the case of AtPEN2-17, all 13 Arabidopsis thaliana seedlings were positive, with slight differences in protein expression among individual seedlings. Figure 2 (B) Two families, AtPEN2-2-3 and AtPEN2-17-3, from generation T1, were selected for further experiments.

[0089] (2) Preparation of crude extract of dormant spores of *Plasmodiophora stearothermiae*

[0090] The swollen rapeseed root samples preserved at -30℃ were taken out of the freezer (publication: Ying Zhao, Kai Bi et al. Transcriptome Analysis of Arabidopsis thaliana in Response to Plasmodiophora brassicae during Early Infection. Frontiers in Microbiology, 2017), washed thoroughly with running water, and cut into small pieces with a knife for easy crushing. They were then placed in a juicer with an appropriate amount of water and ground. After thorough grinding, the samples were filtered through 8 layers of gauze to obtain a crude extract of dormant spores of *Plasmodiophora brassicae*. 100 μL of the crude extract was then serially diluted 1000 times with ddH2O. The spores were counted under a microscope using a hemocytometer to calculate the concentration of the spore suspension. The concentration of the crude spore extract was then diluted to 1 × 10⁻⁶. 7 spores / mL.

[0091] (3) Arabidopsis thaliana inoculated with Plasmodium fungi

[0092] The AtPEN2-2-3 and AtPEN2-17-3 positive seedlings from step (1) were designated as OE-2-3 and OE-17-3, respectively. Arabidopsis seedlings from wild-type Col-0, OE-2-3, and OE-17-3 families were randomly divided into a control group and an experimental group, with 20 Arabidopsis seedlings from each family in each group. After 14 days of growth, each wild-type Col-0, OE-2-3, and OE-17-3 plant in the experimental group was irrigated with 1 mL of the crude spore extract prepared in step (2); each wild-type Col-0, OE-2-3, and OE-17-3 plant in the control group was irrigated with an equal amount of water. Photos were taken 17 days after inoculation, and the disease index results are shown in the attached table. Figure 3 , Figure 4 And Table 1, Figure 4 The numbers 0 to 5 indicate the degree of swelling in the roots of Arabidopsis thaliana.

[0093] The disease index (DI) for Arabidopsis thaliana is calculated based on the degree of root swelling, categorized into five levels: Level 0: No root symptoms; Level 1: Small tumors on lateral roots, none on the taproot; Level 2: Small tumors on both taproot and lateral roots; Level 3: Medium to large tumors distributed on the taproot; Level 4: Root deformity, severe tumors on both taproot and lateral roots, with the taproot completely swollen; Level 5: Both taproot and lateral roots are swollen and rotten, resulting in impaired plant growth. The disease index (DI) is calculated using the following formula: DI = (1×n1 + 2×n2 + 3×n3 + 4×n4 + 5×n5) × 100 / 5 × n t Where n1 to n5 represent the number of Arabidopsis seedlings at each level, n t This represents the total number of Arabidopsis thaliana seedlings in the experiment.

[0094] Table 1. Statistical results of disease incidence in Arabidopsis seedlings.

[0095] Family lineage Col-0 OE-2-3 OE-17-3 Grade 1 (plant) 0 0 0 Grade 2 (plant) 0 3 5 Grade 3 (plant) 5 9 11 Grade 4 (plant) 9 7 4 Level 5 (plant) 6 1 0 Disease index 81 66 59

[0096] according to Figure 3 As shown in section A, compared with the two transgenic families OE-2-3 and OE-17-3 and Col-0, which were not inoculated with *Cladophora* after 31 days of growth, overexpression of AtPEN2 had virtually no impact on the normal growth and development of *Arabidopsis thaliana*. According to the statistical results of *Arabidopsis thaliana* growth phenotype and disease index after inoculation with *Cladophora*, both transgenic families OE-2-3 and OE-17-3 were more resistant to disease than the wild-type Col-0. The disease index of wild-type Col-0 was 81, while the disease indices of transgenic families OE-2-3 and OE-17-3 were 66 and 59, respectively, both lower than those of wild-type Col-0. Figure 3 B, Figure 3 C and Figure 4 It is evident that overexpression of AtPEN2 in Arabidopsis thaliana can enhance its resistance to clubroot bacteria.

[0097] In summary, the AtPEN2 protein positively regulates the resistance of Arabidopsis thaliana to pathogens. Overexpression of the AtPEN2 protein in plants can enhance their resistance to clubroot disease and can be used to control clubroot disease, providing a new approach to the control of clubroot fungal diseases.

[0098] 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 AtPEN2 protein and / or its encoding gene AtPEN2 in the prevention of diseases in cruciferous plants, wherein the amino acid sequence of the AtPEN2 protein is shown in SEQ ID NO.1; The disease affecting the cruciferous plant is clubroot; the cruciferous plant is Arabidopsis thaliana.

2. The application according to claim 1, characterized in that, The nucleotide sequence of the encoding gene AtPEN2 is shown in SEQ ID NO.

2.

3. The application of AtPEN2 protein and / or its encoding gene AtPEN2 in improving disease resistance in cruciferous plants, wherein the amino acid sequence of the AtPEN2 protein is shown in SEQ ID NO.1; The disease affecting the cruciferous plant is clubroot; the cruciferous plant is Arabidopsis thaliana.

4. The application according to claim 3, characterized in that, The nucleotide sequence of the encoding gene AtPEN2 is shown in SEQ ID NO.

2.

5. Application of AtPEN2 protein and / or its encoding gene AtPEN2 in the breeding of cruciferous plants resistant to clubroot disease, wherein the amino acid sequence of the AtPEN2 protein is shown in SEQ ID NO.1; and the cruciferous plant is Arabidopsis thaliana.

6. A method for improving the disease resistance of cruciferous plants, characterized in that, The AtPEN2 gene encoding the AtPEN2 protein was overexpressed in cruciferous plants; the amino acid sequence of the AtPEN2 protein is shown in SEQ ID NO.

1. The plant disease is clubroot; the cruciferous plant is Arabidopsis thaliana.

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