Application of AtPEN2 protein and / or coding gene AtPEN2 thereof in prevention of cruciferous plant diseases

Abstract

The invention belongs to the technical field of gene engineering, and particularly relates to application of AtPEN2 protein and / or a coding gene AtPEN2 thereof in prevention of cruciferous plant diseases. The invention provides an application of an AtPEN2 protein and / or a coding gene AtPEN2 thereof in prevention of cruciferous plant diseases. The amino acid sequence of the AtPEN2 protein is as shown in SEQ ID NO. 1. According to the invention, arabidopsis thaliana is used as a model plant to verify the control effect of the overexpressed AtPEN2 protein in a plant on clubroot, and the result shows that the AtPEN2 protein overexpressed plant can improve the resistance to clubroot, the coding gene AtPEN2 of the AtPEN2 protein can be applied to the molecular breeding of clubroot, and a new gene resource is provided for the disease-resistant molecular breeding of cruciferae crops.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and in particular relates to the application of AtPEN2 protein and / or its encoding gene AtPEN2 in preventing diseases of cruciferous plants. Background Art

[0002] The pathogen of the soil-borne disease clubroot, Rhizopus spp., mainly infects cruciferous vegetables, causing the roots of plants to swell and deform, posing a major threat to my country's cruciferous vegetable industry. Therefore, studying the interaction mechanism between Rhizopus spp. and its host, analyzing the pathogenic mechanism of Rhizopus spp., and exploring the gene resources for resistance to clubroot are currently urgent issues to be addressed.

[0003] Discovering disease-resistant genes and clarifying disease-resistant mechanisms are the keys to successful disease-resistant molecular breeding. The myrosinase system exists in cruciferous plants, which can defend against various external biological and abiotic stresses and participate in regulating the growth and development of cruciferous plants. AtPEN2 is a β-O-glucosyl hydrolase, an atypical myrosinase, which can participate in the biosynthesis and secretion of resistance substances. At present, there are few studies on the role of atypical myrosinase AtPEN2 in improving the disease resistance of cruciferous plants. Summary of the invention

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

[0005] The present invention provides the use of AtPEN2 protein and / or its encoding gene AtPEN2 in preventing diseases of cruciferous plants. The amino acid sequence of the AtPEN2 protein is shown in SEQ ID NO.1.

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

[0007] As a preferred solution, the cruciferous plant disease includes clubroot.

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

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

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

[0011] As a preferred solution, the cruciferous plant disease includes clubroot.

[0012] The present invention also provides the use of AtPEN2 protein and / or its encoding gene AtPEN2 in cultivating cruciferous plants resistant to clubroot. The amino acid sequence of the 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, which comprises over-expressing a gene AtPEN2 encoding the AtPEN2 protein in the 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; and the plant disease includes clubroot.

[0015] Beneficial effects:

[0016] The present invention provides an application of AtPEN2 protein and / or its encoding gene AtPEN2 in preventing diseases of cruciferous plants, wherein the amino acid sequence of the AtPEN2 protein is shown in SEQ ID NO. 1. The present invention uses Arabidopsis thaliana as a model plant to verify the control effect of overexpressing AtPEN2 protein in plants on clubroot disease, and the results show that AtPEN2 protein overexpressing plants can improve Arabidopsis thaliana's resistance to clubroot disease, indicating that the encoding gene AtPEN2 of AtPEN2 protein can be applied to molecular breeding of clubroot disease, providing a new gene resource for molecular breeding of disease resistance of cruciferous crops. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

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

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

[0020] Figure 3The phenotype diagram of Arabidopsis seedlings in Example 4; wherein A is the growth phenotype of wild-type Arabidopsis Col-0, AtPEN2-2-3 and AtPEN2-17-3 grown to 31 days in the control group, scale bar = 2 cm; B is the growth phenotype of wild-type Arabidopsis Col-0, AtPEN2-2-3 and AtPEN2-17-3 in the experimental group 17 days after inoculation with root-knot bacteria, scale bar = 2 cm; C is a representative plant diagram of each family of wild-type Arabidopsis Col-0, AtPEN2-2-3 and AtPEN2-17-3 in the experimental group, scale bar = 1 cm;

[0021] Figure 4 It is the disease index of Arabidopsis seedlings in Example 4 17 days after inoculation with root knotweed fungi. DETAILED DESCRIPTION

[0022] The present invention provides an application of AtPEN2 protein and / or its encoding gene AtPEN2 in preventing diseases of cruciferous plants, wherein the amino acid sequence of the AtPEN2 protein is shown in SEQ ID NO.1: *. The AtPEN2 protein of the present invention is an atypical myrosinase in Arabidopsis thaliana, which can improve the resistance of Arabidopsis thaliana to root knot fungi, and provides a new gene resource for molecular breeding of disease resistance of cruciferous crops.

[0023]

[0024] As an embodiment, the cruciferous plant diseases of the present invention include clubroot. As an embodiment, the cruciferous plant of the present invention includes Arabidopsis thaliana.

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

[0026] The present invention also provides the use of AtPEN2 protein and / or its encoding gene AtPEN2 in cultivating cruciferous plants resistant to clubroot, and the amino acid sequence of the AtPEN2 protein is shown in SEQ ID NO.1. As an embodiment, the nucleotide sequence of the encoding gene AtPEN2 is shown in SEQ ID NO.2. In the present invention, the AtPEN2 protein is highly expressed in Arabidopsis plants, which can improve the resistance of Arabidopsis to clubroot, indicating that the encoding gene AtPEN2 of the AtPEN2 protein can be used in molecular breeding of clubroot, providing a new gene resource for molecular breeding of disease resistance of cruciferous crops.

[0027] The present invention also provides a method for improving the disease resistance of cruciferous plants, wherein the gene AtPEN2 encoding the AtPEN2 protein is overexpressed in the cruciferous plants; the amino acid sequence of the AtPEN2 protein is shown in SEQ ID NO.1. As an embodiment, the nucleotide sequence of the gene AtPEN2 encoding the AtPEN2 is shown in SEQ ID NO.2. As an embodiment, the cruciferous plants include Arabidopsis thaliana. As an embodiment, the plant diseases include clubroot.

[0028] As an embodiment, the present invention utilizes a transformant containing the AtPEN2 protein encoding gene AtPEN2 to transform cruciferous plants, thereby achieving overexpression of the AtPEN2 protein encoding gene AtPEN2 in cruciferous plants, thereby improving the disease resistance of cruciferous plants. As an embodiment, the transformant includes a recombinant vector and / or recombinant bacteria containing the AtPEN2 protein encoding gene AtPEN2. The present invention highly expresses the AtPEN2 protein encoding gene AtPEN2 in Arabidopsis plants, thereby improving the resistance of Arabidopsis to clubroot.

[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 preventing cruciferous plant diseases is described in detail below in conjunction with the accompanying drawings and examples, but they 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 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, immediately shake vigorously to mix, and place the homogenized sample on ice for 10 minutes;

[0034] ② Add 0.2 mL of chloroform to every 1 mL of TRIpure, cover the tube tightly, shake vigorously to mix immediately and place on ice for 10 min;

[0035] ③ High-speed refrigerated centrifugation at 12,000 r / min for 20 minutes at 4°C. After centrifugation, the mixture is divided into three layers: a lower red organic phenol-chloroform layer, a middle layer, and an upper colorless water layer. RNA is present in the water layer.

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

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

[0038] ⑥ Place the centrifuge tube in a centrifuge and centrifuge at 12000r / min for 1 minute, then carefully aspirate the remaining liquid with a pipette, let it stand at room temperature for 5-10 minutes, and dry the RNA precipitate;

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

[0040] (2) Cloning of target gene

[0041] ①Use the reverse transcription kit (TransGene, Code#AE311-03) to synthesize the first-strand cDNA from the RNA extracted in step (1). The total reaction system is: Total RNA 4μg, Anchored Oligo (dT) 18 Primer (0.5μg / μL) 1μL, gDNA Remover 1μL, EasyScript RT / RI Enzyme Mix 1μL, 2×ES Reaction Mix 10μL, add RNase-free Water to make up to 20μL. Gently mix the above reaction system and incubate at 42℃ for 30min. Heat at 85℃ for 5sec to inactivate Easyscript RT / RI and gDNA Remover.

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

[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, high-fidelity DNA polymerase KOD One was used for PCR amplification. The total PCR amplification system was 40 μL: 10 μM Primer F 1 μL, 10 μM Primer R 1 μL, 2× KOD One PCR Master Mix 20 μL, cDNA 2 μL, and ddH2O 16 μL.

[0046] The PCR cycle 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. The product was electrophoresed 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 PCR product was detected using a Tanon 1600 fully automatic digital gel imager. The band of the correct size was cut from the gel and then purified and recovered using a gel recovery kit to obtain the target gene AtPEN2.

[0048] (3) Enzyme digestion of 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 Sclerotiniasclerotiorum. New Phytologist, 2017) was linearized with two restriction endonucleases, XbaⅠ and SmaⅠ. 70 μL of enzyme digestion system: 50 μL of plasmid, 1 μL of XbaⅠ, 1 μL of SmaⅠ, 7 μL of 10×rCutsmartbuffer (purchased from NEB), 11 μL of ddH2O; mix the above enzyme digestion system and incubate in a 37°C water bath for 2 h; then add 7.7 μL of 10×DNAloadingbuffer and mix well, and run the gel at 130 V for 20 min on 1% (w / v) agarose gel with nucleic acid dye (Biosharp, BS354B; added at a volume ratio of 1:10000); use a Tanon 1600 fully automatic digital gel imager to detect whether the enzyme digestion bands are correct; cut the target bands from the gel, and then purify and recover them using a gel recovery kit.

[0050] (4) Vector construction

[0051] The target gene fragment obtained by gel recovery and purification in step (2) is cloned into the linearized vector obtained in step (3) by seamless cloning technology. Use VAZYME's kit (Cat. No. C112-01) to prepare the following reaction system 10μL on ice: 5×CEⅡbuffer 2μL, ExnaseⅡ 1μL, linearized vector 2μL, target gene fragment 1μL, ddH2O 4μL. Use a pipette to gently mix the above reaction system, centrifuge at 500r / min for 30s, collect the reaction system after centrifugation to the bottom of the PCR tube; place it in a PCR instrument at 37℃ for 30min.

[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, and the specific steps are as follows: take out the competent DH5α from the -80°C refrigerator and thaw it on ice; take 10 μL of the recombinant product and add it to 30 μL of competent cells, gently tap the tube wall to mix, and let it stand on ice for 30 minutes; after heat shock in a 42°C water bath for 45 seconds, immediately place it on ice to cool for 3 minutes; add 1 mL of liquid LB culture medium (without adding antibiotics), shake in a 37°C shaker at 220 r / min for 1 hour; after the shaking is completed, centrifuge at 5000 r / min for 5 minutes, and discard 800 μL of supernatant; resuspend the bacteria with the remaining culture medium, and gently spread it on an LB plate containing Kana resistance with a sterile spreading stick, wherein the final concentration of Kana in the LB plate is 50 μg / mL; place the LB plate in an incubator at 37°C and incubate it upside down.

[0054] (6) Identification of recombinant plasmid

[0055] ① After a single colony grows on the LB plate in step (5), pick several single colonies for PCR identification. Pick a small amount of bacteria and place them in 10 μL of the prepared PCRMix, which contains 10 μM PCNF3-F 0.5 μL, 10 μM PCNF3-R 0.5 μL, 2×Rapid Taq Master Mix 5 μL, and ddH2O 4 μL.

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

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

[0058] The PCR cycle conditions are 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 product obtained in step ① was 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 20min; the PCR product band size was detected using a Tanon 1600 fully automatic digital gel imager.

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

[0061] ④ For the bacterial solution with correct sequencing, collect the remaining bacteria and refer to the small amount extraction method of plasmid (public document: Research on the molecular mechanism of CPK18 gene regulating ethylene and disease resistance, Li Hong, Ph.D. dissertation of Huazhong Agricultural University, 2024) to extract the plasmid, and store the extracted plasmid at -20℃.

[0062] Example 2

[0063] Agrobacterium-mediated genetic transformation of Arabidopsis thaliana

[0064] (1) Seed disinfection and germination

[0065] ① In a 1.5mL centrifuge tube, immerse the seeds of Arabidopsis Col-0 in 1mL of 75% (v / v) alcohol for 1min, and discard the alcohol; ② Wash once with sterilized ddH2O, and discard the water; ③ Soak in 1mL of 10% (v / v) 84 disinfectant (100μL 84 disinfectant + 900μL sterilized ddH2O) for 10min, and discard the disinfectant; ④ Wash the seeds 5 times with 1mL sterilized ddH2O; ⑤ Sow the Col-0 Arabidopsis seeds in the antibiotic-free 1 / 2MS culture medium with a pipette, put the culture dish in a constant temperature culture room at 22℃, and select Arabidopsis seedlings with consistent growth after 14 days and move them to the soil. The 1 / 2MS culture medium contains: MS2.22g, MES 0.5g, sucrose 15g, agar 14g, sterile water to 1L, and pH adjusted to 5.8.

[0066] (2) Plasmid transformation of Agrobacterium

[0067] ① Take out the competent Agrobacterium GV3101 from the -80℃ refrigerator and thaw it on ice; ② Take 2μL of the correctly sequenced plasmid in Example 1, add it to 100μL of competent Agrobacterium GV3101 cells, and flick the side of the tube with your finger to mix the plasmid and competent cells thoroughly; ③ Add the mixed liquid to a clean and dried electroporation cup, and put the electroporation cup into the Bio-rad electroporator; select the program for electroporation transformation, then take out the electroporation cup, and add 500μL of liquid LB medium ( No antibiotics were added), and the mixture was transferred to a new 1.5mL centrifuge tube; ④ Place the centrifuge tube in step ③ in a 28℃ shaker, shake at 220r / min for 1h, centrifuge at 5000r / min for 5min, and discard 400μL supernatant; Resuspend the bacteria with the remaining culture medium, and gently spread it on a dual-resistance LB plate containing Kana (final concentration of 50μg / mL) and Rif (final concentration of 50μg / mL) with a sterile coating stick; Incubate inverted in a 28℃ incubator for 48h. ⑤ After single colonies grow, pick several single colonies for PCR identification. The PCR system and cycle program are the same as those in step (6) of recombinant plasmid identification in Example 1. ⑥ Use a white pipette tip to pick up a small amount of colonies identified correctly by PCR, and put them into a 10mL EP tube containing 2mL LB liquid culture medium, and shake culture overnight. The LB liquid culture medium contains Kana and Rif with a final concentration of 50μg / mL respectively; after the shaking culture, take 1mL of bacterial solution, add 1mL 50% glycerol, add it to a 2mL seed tube, and store it in a -20℃ refrigerator.

[0068] (3) Arabidopsis thaliana flower dipping

[0069] A. ① Activate the glycerol bacteria preserved in step (2) by drawing lines on a dual-antibody LB plate containing Kana and Rif (the final concentrations of Kana and Rif are 50 μg / mL, respectively); 2 days later, use a white sterilized pipette tip to pick up a single colony and place it in 2 mL of dual-antibody liquid LB medium (the dual-antibody is Kana+Rif, and the final concentrations of Kana and Rif are 50 μg / mL, respectively) and shake overnight; then inoculate it in a 200 mL conical flask containing 50 mL of dual-antibody liquid LB medium (Kana+Rif) at a ratio of 1:100 (v / v), place it in a 28°C shaker for expansion, and shake at 220 r / min until OD 600= about 1.2; collect Agrobacterium bacterial suspension in a 50mL centrifuge tube, centrifuge at 5000r / min for 10min, and collect Agrobacterium cells; discard the supernatant, resuspend the cells with 50mL of 5% (w / v) sucrose solution, and add 10μL of Silwet L-77 to make the final concentration 0.02% (v / v). ② After the Arabidopsis transplanted in step (1) grows to bolting and flowering, use scissors to cut off all the fruit pods and white flowers on the Arabidopsis plants, immerse all the inflorescences of Arabidopsis in the Agrobacterium cell resuspension obtained in step ①, shake gently for 15s, and drain the excess Agrobacterium bacterial suspension. ③ After step ① is completed for 1h, repeat step ②. ④ Moisturize and darkly culture the Arabidopsis treated with Agrobacterium dipped flowers for 16h, and transfer to normal culture.

[0070] B. After 10 days, repeat step A and dip the flowers again, except that the operation of cutting off all the fruit pods and white flowers on the Arabidopsis plants with scissors in step A is omitted. When the Arabidopsis matures, collect the T0 generation seeds in a 1.5 mL centrifuge tube, add some water-absorbing blue silica gel beads to the centrifuge tube, and store it in a seed storage cabinet.

[0071] Example 3

[0072] Molecular Detection of Transgenic Arabidopsis

[0073] (1) Arabidopsis total protein extraction

[0074] ① Take some of the transgenic Arabidopsis T0 generation seeds harvested in Example 2 into a 1.5mL centrifuge tube, and sterilize the seeds according to the method of step (1) in Example 2. ② Sow the sterilized seeds on a 1 / 2MS plate containing Kana (final concentration of 50μg / mL), and then place the culture dish in a constant temperature culture room at 22°C for cultivation. ③ After 14 days of cultivation, select the positive seedlings and move them to the soil. ④ When the positive seedlings in the soil grow to about 1 month, take some leaves and put them into a 1.5mL centrifuge tube with two steel balls, and quickly put them into liquid nitrogen for quick freezing; then put the centrifuge tube into a grinder at 40Hz for 1min to grind the sample. ⑤ After the leaves were ground, 200 μL of 2×SDS protein extract (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) was added to each tube, vortexed and mixed, placed in a 98°C metal bath and boiled for 10 minutes. ⑥ Western Blot (WB) was used to detect whether the target protein was expressed. Before WB loading, centrifuged at 12000 r / min for 10 minutes, and 15 μL of supernatant was taken for each spot.

[0075] (2) Protein immunoblotting

[0076] ① Protein electrophoresis separation: Prepare 5% (w / v) SDS-PAGE stacking gel and 10% (w / v) SDS-PAGE separation gel; assemble the gel plate, first inject 7mL of separation gel, immediately add 1mL of n-butanol to ensure that the interface between the separation gel and the stacking gel is flat, and wait for 30 minutes; after the separation gel solidifies, remove the added 1mL of liquid with a syringe, then inject 3mL of stacking gel, insert a comb, and wait for 30 minutes; after the stacking gel solidifies, put the prepared gel plate into the vertical electrophoresis tank, add an appropriate amount of SDS-PAGE electrophoresis fluid, remove the comb, load 15μL of sample into each well, and run the gel at a voltage of 100V; after 30 minutes, adjust the voltage to 65V, and after 2-3 hours, according to the indicator maker protein molecular weight, when the target protein is electrophoresed to the middle of the gel plate, the electrophoresis is completed (the entire electrophoresis process must be completed in an ice water bath), then remove the stacking gel, and put the separation gel into the transfer solution. SDS-PAGE electrophoresis buffer formula 1L: Tris 3.02g, glycine 14.4g, SDS 1g.

[0077] ② Transfer: Cut the PVDF membrane into 4cm×7cm and mark the bottom of the membrane; soak the PVDF membrane in methanol for 30s, wash it with ddH2O 3 times, 5min each time, and then put the PVDF membrane into the transfer solution; assemble the transfer "sandwich" in the transfer solution, from bottom to top: fiber pad, filter paper, separation gel, PVDF membrane, filter paper, fiber pad; put the assembled "sandwich" vertically into the transfer electrophoresis tank, put an ice box on each side of the tank, fill it with transfer solution, 60V, 2h (the entire transfer process must be completed in an ice water bath). After the transfer is completed, transfer the PVDF membrane into the protein incubation box and wash it with ddH2O 3 times, 5min each time. Transfer solution formula 1L: Tris 3.02g, Glycine 14.4g, methanol (Methanol) 200mL.

[0078] ③ Blocking: Add 10 mL of TBST solution containing 5% (w / v) milk powder to the protein incubation box, place in a shaker at 50 r / min, and place in a 4°C refrigerator overnight; 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 in a shaker at 50 r / min, and place in a 4°C refrigerator overnight; 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 on 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 300μL Peroxide solution and 300μL Luminol / enhancer solution and incubate at room temperature for 3 minutes; finally, detect the protein bands using a Tanon 5100 chemiluminescence imager.

[0082] The present invention tested 20 T0 generation Arabidopsis seedlings, and the WB test results are shown in Figure 1 , where the antibody used in WB is Anti-Flag, and CBB means Coomassie Brilliant Blue staining to show the loading amount of each sample. Figure 1 It can be seen that a total of 12 Arabidopsis seedlings were identified to express the target protein, numbered 1, 2, 3, 4, 5, 7, 11, 15, 16, 17, 18 and 19, and the other numbered plants were negative seedlings ( Figure 1 A and B). According to the thickness of the protein bands detected by WB, the expression level of the target protein in a single Arabidopsis plant can be distinguished. The AtPEN2 expression level in the Arabidopsis plants numbered 1, 2, 5, and 7 is high, the AtPEN2 expression level in the Arabidopsis plant numbered 17 is at a medium level, and the AtPEN2 expression level in the Arabidopsis plants numbered 3, 4, 11, 15, 16, 18, and 19 is the lowest.

[0083] Example 4

[0084] Transgenic Arabidopsis thaliana inoculated with Plasmodium

[0085] (1) Screening of T1 positive seedlings

[0086] ① According to the WB test results in Example 3, numbers 2 and 17 were selected from the T0 generation family, recorded as AtPEN2-2 and AtPEN2-17, and continued to breed the T1 generation.

[0087] ② The 5 Arabidopsis seedlings in the T1 generation of the AtPEN2-2 family and the 13 Arabidopsis seedlings in the AtPEN2-17 family in step ① were subjected to WB detection. The detection method was the same as in Example 3. The detection results are shown in Figure 2 , where the antibody used in WB is Anti-Flag, and CBB means Coomassie Brilliant Blue staining to show the loading amount of each sample.

[0088] according to Figure 2 It can be seen that all five Arabidopsis seedlings of the AtPEN2-2 family were positive seedlings ( Figure 2 Middle A), all 13 Arabidopsis seedlings of AtPEN2-17 family were positive seedlings, and the protein expression of individual Arabidopsis seedlings was slightly different ( Figure 2 (B) Two families of T1 generation, AtPEN2-2-3 and AtPEN2-17-3, were selected for subsequent experiments.

[0089] (2) Preparation of crude extract of dormant spores of root knotweed

[0090] Take out the swollen rape root sample stored in the -30℃ refrigerator (public literature: Ying Zhao, Kai Bi et, al. Transcriptome Analysis of Arabidopsis thaliana in Response to Plasmodiophora brassicae during Early Infection. Frontiers in Microbiology, 2017), wash it with running water, cut the root sample into small pieces with a kitchen knife for easy crushing, put it into a juicer, add appropriate amount of water to grind; after being fully ground, filter it with 8 layers of gauze to obtain the crude extract of dormant spores of root knot fungus, then take 100 μL of the crude extract and dilute it 1000 times with ddH2O, count it with a hemocytometer under a microscope, calculate the concentration of the spore suspension, and dilute the concentration of the crude spore extract to 1×10 7 spores / mL.

[0091] (3) Arabidopsis thaliana inoculated with root-knot bacteria

[0092] The positive seedlings of AtPEN2-2-3 and AtPEN2-17-3 in step (1) were recorded as OE-2-3 and OE-17-3, and the Arabidopsis seedlings of the 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 of each family in each group. When the Arabidopsis seedlings grew to 14 days, 1 mL of the crude spore extract prepared in step (2) was inoculated into the roots of each wild-type Col-0, OE-2-3 and OE-17-3 plant in the experimental group; the same amount of clean water was inoculated into the roots of each wild-type Col-0, OE-2-3 and OE-17-3 plant in the control group. Photos were taken 17 days after inoculation, and the disease index results were statistically analyzed. Figure 3 , Figure 4 and Table 1, Figure 4 The numbers 0 to 5 indicate the degree of root swelling of Arabidopsis thaliana.

[0093] Statistical standard of Arabidopsis root root disease index: According to the degree of root swelling of Arabidopsis, it is divided into five levels: Level 0, no disease symptoms on the root; Level 1, there are very small tumors on the lateral roots, but not on the main root; Level 2, small tumors on both the main root and the lateral roots; Level 3, medium to large tumors distributed on the main root; Level 4, root deformity, serious tumors on both the main root and the lateral roots, and the main root is completely swollen; Level 5, both the main root and the lateral roots are swollen and rotten, and the plant growth is impaired. Disease index (DI) is calculated according to the following formula: DI = (1×n1+2×n2+3×n3+4×n4+5×n5)×100 / 5×n t , where n1~n5 represents the number of Arabidopsis seedlings corresponding to each level, n t Represents the total number of Arabidopsis seedlings in the experiment.

[0094] Table 1 Statistical results of disease of Arabidopsis seedlings

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

[0096] according to Figure 3 As shown in Figure A, compared with the two transgenic lines OE-2-3, OE-17-3 and Col-0 that were not inoculated with root-knot fungi for 31 days, overexpression of AtPEN2 did not affect the normal growth and development of Arabidopsis thaliana. According to the statistical results of the growth phenotype and disease index of Arabidopsis thaliana after inoculation with root-knot fungi, the two transgenic lines OE-2-3 and OE-17-3 were more disease-resistant than the wild type Col-0. The disease index of the wild type Col-0 was 81, and the disease index of the transgenic lines OE-2-3 and OE-17-3 were 66 and 59, respectively, which were lower than the wild type Col-0 ( Figure 3 Middle B, Figure 3 Middle C and Figure 4 ). It can be seen that overexpression of AtPEN2 in Arabidopsis can enhance the resistance of Arabidopsis to root-knot fungi.

[0097] In summary, AtPEN2 protein positively regulates Arabidopsis resistance to pathogens. Overexpression of AtPEN2 protein in plants can improve the ability of plants to resist clubroot disease and can be used to prevent and treat clubroot disease, providing a new idea for the prevention and control of clubroot fungal diseases.

[0098] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Use of AtPEN2 protein and / or its encoding gene AtPEN2 in preventing diseases of cruciferous plants, wherein the amino acid sequence of the AtPEN2 protein is shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that: The nucleotide sequence of the coding gene AtPEN2 is shown as SEQ ID NO.

2.

3. The use according to claim 1 or 2, characterized in that: The cruciferous plant diseases include clubroot.

4. The use according to claim 1 or 2, characterized in that: The cruciferous plants include Arabidopsis thaliana.

5. Use of AtPEN2 protein and / or its encoding gene AtPEN2 in improving disease resistance of cruciferous plants, wherein the amino acid sequence of the AtPEN2 protein is shown in SEQ ID NO.

1.

6. The use according to claim 5, characterized in that: The nucleotide sequence of the coding gene AtPEN2 is shown as SEQ ID NO.

2.

7. The use according to claim 5 or 6, characterized in that: The cruciferous plant diseases include clubroot.

8. Use of AtPEN2 protein and / or its encoding gene AtPEN2 in cultivating cruciferous plants resistant to clubroot, wherein the amino acid sequence of the AtPEN2 protein is shown in SEQ ID NO.

1.

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

1.

10. The method according to claim 9, characterized in that The cruciferous plants include Arabidopsis thaliana; and the plant diseases include clubroot.

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