Application of brassica juncea BjuWRKY75 in clubroot resistance of brassica napus
By isolating the BjuWRKY75 gene from rapeseed and introducing it into Brassica napus, an overexpression vector was constructed, which solved the problem of insufficient resources for clubroot resistance in Brassica napus and achieved the effects of significantly enhancing resistance and shortening the breeding cycle.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- NORTHWEST A & F UNIV
- Filing Date
- 2025-12-18
- Publication Date
- 2026-05-08
AI Technical Summary
There are few resources for clubroot resistance in Brassica napus, and the existing resistance sources are limited, resulting in insufficient resistance and difficulty in effectively dealing with the rapid spread of clubroot and the variation of diverse pathogens.
The BjuWRKY75 gene was isolated from clubroot-resistant rapeseed, an overexpression vector was constructed, and it was introduced into Brassica napus through Agrobacterium-mediated genetic transformation to enhance its resistance to clubroot.
It significantly improved the resistance of Brassica napus to clubroot disease, reduced the number of root tumors, broadened the genetic basis for clubroot resistance, provided a rapid and precise breeding pathway, and solved the problem of long breeding cycles in traditional breeding.
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Figure CN121991965A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of plant genetic engineering, and in particular to the application of BjuWRKY75, a type of mustard rape, in the resistance of Brassica napus to clubroot disease. Background Technology
[0002] Clubroot is a global soil-borne disease caused by Plasmodiophora brassicae, and is one of the most destructive diseases affecting cruciferous crops such as rapeseed, Chinese cabbage, and kale. In recent years, clubroot has spread rapidly in major rapeseed-producing areas of my country, currently affecting over 667,000 hectares annually, severely impacting rapeseed production and posing a significant new challenge to rapeseed cultivation. Therefore, there is an urgent need to discover clubroot-resistant genes to lay the foundation for breeding new rapeseed varieties with clubroot resistance. However, clubroot-resistant resources are extremely scarce in Chinese rapeseed varieties, while resistant resources exist in its close relatives, Chinese cabbage, kale, and mustard.
[0003] Clubroot fungus primarily damages the roots of plants. After infection, tumors of varying sizes gradually grow on the taproot, lateral roots, and fibrous roots. In later stages of infection, these tumors crack and become rough, making them susceptible to invasion by other fungi and subsequent rot, severely impacting the plant's normal absorption of water and nutrients. Due to its high infectivity, rapid spread, multiple transmission routes, poor efficacy of biological and chemical control, and the lack of plants with broad-spectrum antigens, clubroot has become a devastating soil-borne plant disease worldwide. Currently, some clubroot resistance genes / locuses have been found mainly in vegetables such as European turnip / Chinese cabbage, kale, black mustard, and radish, while fewer resistance sources have been found in rapeseed. At least 28 QTLs associated with clubroot resistance have been mapped to chromosomes A01, A02, A03, A06, and A08 in Chinese cabbage. Of these, 12 resistance QTLs (Crr3, Rcr1, Rcr2, Rcr4, Rcr5, PbBa3.1, CRk, CRd, CRb, CRa, CRaki, PbBp3.3) are located on chromosome A03, and these loci are linked to each other, indicating that the clubroot resistance loci derived from Chinese cabbage are relatively singular. In contrast, clubroot resistance in Brassica napus primarily originates from Chinese cabbage.
[0004] Therefore, this invention artificially synthesizes a clubroot-resistant mustard-type rapeseed by crossbreeding Chinese cabbage and mustard through distant hybridization. Furthermore, by simplifying genome sequencing, the disease resistance gene of this artificially synthesized clubroot-resistant mustard-type rapeseed was located, providing an important theoretical basis and gene resource for breeding new rapeseed varieties with durable resistance. Summary of the Invention
[0005] The purpose of this invention is to provide the application of BjuWRKY75, a type of mustard rape, in the resistance of Brassica napus to clubroot disease, in order to solve the problem mentioned in the background art that the resistance sources of currently created clubroot-resistant Brassica napus germplasm or varieties are relatively singular, resulting in relatively singular disease resistance.
[0006] To achieve the above objectives, the present invention provides the following technical solution:
[0007] A BjuWRKY75 gene for rapeseed of the Brassica oleracea type, the CDS sequence of which is shown in SEQ ID NO.1 and the amino acid sequence encoded by which is shown in SEQ ID NO.2, is used to protect rapeseed of the Brassica oleracea type against clubroot disease, which is caused by Plasmodiophora brassicae.
[0008] Application of the BjuWRKY75 gene from rapeseed in the breeding of clubroot-resistant Brassica napus.
[0009] The method for breeding clubroot-resistant Brassica napus using the BjuWRKY75 gene from mustard-type rapeseed includes the following steps:
[0010] Step (1) Using the genomic DNA of clubroot-resistant mustard R13 as a template, the CDS sequence of the BjuWRKY75 gene was obtained by PCR amplification. The forward primer sequence for the PCR amplification was 5'-tgactccatgtcgacggatccATGGAGGGATATGATGATGG-3', and the reverse primer sequence was 5'-ggtggactcctcttagaattcAAAAGAAGAGTAGATTTGCA-3'.
[0011] Step (2) The CDS sequence obtained in step (1) is constructed into an overexpression vector to obtain a recombinant overexpression vector;
[0012] Step (3) Transform the recombinant overexpression vector into Agrobacterium to obtain a positive Agrobacterium strain containing the target gene;
[0013] Step (4) The BjuWRKY75 gene was introduced into Brassica napus through Agrobacterium-mediated genetic transformation to obtain Brassica napus plants resistant to clubroot disease.
[0014] Preferably, in step (2), the overexpression vector is a 2*35S overexpression vector;
[0015] The method for constructing the overexpression vector includes: linearizing the vector plasmid by double digestion with restriction endonucleases Bgl II and Xbal at 37°C for 2-5 hours, and then ligating the CDS sequence of the BjuWRKY75 gene with homologous arm sequences to the linearized vector using a homologous recombination kit.
[0016] The 10 μl homologous recombination reaction system consisted of: 1 μl Exnase II, 2 μl 5×CEII, 2.5 μl linearized vector, and 4.5 μl BjuWRKY75 gene CDS fragment. The reaction conditions were incubation at 72°C for 30 min.
[0017] Preferably, in step (2), after the recombinant overexpression vector is transformed into Escherichia coli DH5α competent cells, single clones are screened by plating on Amp resistance plates, and the correctness of the recombinant plasmid is verified by colony PCR and Sanger sequencing; the primers for colony PCR are 35S-F / eGFP-R, and the annealing temperature is 58℃.
[0018] Preferably, in step (3), the Agrobacterium is a GV3101 competent cell;
[0019] The Agrobacterium transformation method is as follows: 3 μL of the verified recombinant plasmid is added to GV3101 competent cells with OD600=0.5, ice bath for 30 min, liquid nitrogen flash freeze for 5 min, heat shock at 37℃ for 5 min, ice bath recovery, 600 μL of antibiotic-free LB medium is added, and cultured at 28℃ and 200 rpm for 3 h.
[0020] The positive Agrobacterium strains were obtained through screening with double antibody plates containing 50 μg / mL Kan and 25 μg / mL Rif. The transformation efficiency was verified by SYBR Green quantitative PCR to be (3.2±0.5)×10⁻⁶. 5 cfu / μg DNA.
[0021] Preferably, in step (4), the Agrobacterium-mediated genetic transformation method specifically includes:
[0022] a) After sterilization, Brassica napus seeds were sown on M0 medium and cultured in the dark at 24°C for 7 days to obtain hypocotyls;
[0023] b) Culture the positive Agrobacterium strain to a suitable concentration, then dilute with DM medium to adjust OD600 to 0.2-0.3;
[0024] c) Cut a hypocotyl segment with a length of 0.8-1cm and soak it in the Agrobacterium solution from step b) for 15-30 minutes;
[0025] d) The infected hypocotyls were cultured in the dark on M1 medium for 3 days, selectively cultured on M2 medium for 17 days, germinated on M3 medium (subcultured every 14 days), and rooted on M4 medium. After the roots grew, they were transplanted into nutrient soil.
[0026] e) T0 generation positive Brassica napus plants were obtained by DsRed tag fluorescence screening and PCR identification.
[0027] A recombinant overexpression vector containing the CDS sequence of the BjuWRKY75 gene from Brassica napus, the recombinant overexpression vector being used for genetic transformation of Brassica napus to resist clubroot disease.
[0028] A clubroot-resistant Brassica napus plant obtained by cultivating clubroot-resistant Brassica napus.
[0029] Compared with the prior art, the beneficial effects of the present invention are:
[0030] 1. The BjuWRKY75 gene was successfully isolated from the disease-resistant mustard-type rapeseed R13 and its key role in regulating clubroot resistance was revealed for the first time. This provides a new resistance gene resource from non-Brassica napus for disease-resistant breeding of Brassica napus, effectively broadening the genetic basis for clubroot resistance breeding and solving the industry bottleneck that the existing resistance source mainly relies on Chinese cabbage, has a single genetic background, and is easily ineffective due to pathogen mutation.
[0031] 2. By constructing an overexpression vector containing the BjuWRKY75 gene and using an optimized Agrobacterium-mediated genetic transformation system, transgenic Brassica napus was successfully bred. It was verified that overexpression of this gene can significantly enhance the plant's resistance to clubroot race 4 and significantly reduce the number of root tumors. This provides an efficient technical pathway for rapidly and accurately creating new disease-resistant germplasm through molecular breeding, which greatly shortens the traditional hybridization breeding cycle. Attached Figure Description
[0032] Schematic diagram.
[0033] Detailed Implementation
[0034] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0035] Please seeFigure 1-2 This invention provides a technical solution: a BjuWRKY75 gene for rapeseed.
[0036] The CDS sequence of the BjuWRKY75 gene is shown in SEQ ID NO.1, and the amino acid sequence it encodes is shown in SEQ ID NO.2;
[0037] The BjuWRKY75 gene is used to protect rapeseed from clubroot disease, which is caused by Plasmodiophora brassicae.
[0038] The method for breeding clubroot-resistant Brassica napus using the BjuWRKY75 gene from mustard-type rapeseed includes the following steps:
[0039] Step (1) Using the genomic DNA of clubroot-resistant mustard-type rapeseed material R13 as a template, the CDS sequence of the BjuWRKY75 gene was obtained by PCR amplification. The forward primer sequence for PCR amplification was 5'-tgactccatgtcgacggatccATGGAGGGATATGATGATGG-3', and the reverse primer sequence was 5'-ggtggactcctcttagaattcAAAAGAAGAGTAGATTTGCA-3'.
[0040] Step (2) The CDS sequence obtained in step (1) is constructed into an overexpression vector to obtain a recombinant overexpression vector;
[0041] Step (3) Transform the recombinant overexpression vector into Agrobacterium to obtain a positive Agrobacterium strain containing the target gene;
[0042] Step (4) The BjuWRKY75 gene was introduced into Brassica napus through Agrobacterium-mediated genetic transformation to obtain Brassica napus plants resistant to clubroot disease.
[0043] Furthermore, in step (2), the overexpression vector is a 2*35S overexpression vector;
[0044] The method for constructing the overexpression vector includes: linearizing the vector plasmid by double digestion with restriction endonucleases Bgl II and Xbal at 37℃ for 2-5 hours, and then ligating the CDS sequence of the BjuWRKY75 gene with the homologous arm sequence to the linearized vector using a homologous recombination kit.
[0045] The 10 μl reaction system for homologous recombination consisted of: 1 μl Exnase II, 2 μl 5×CEII, 2.5 μl linearized vector, and 4.5 μl BjuWRKY75 gene CDS fragment. The reaction conditions were incubation at 72 °C for 30 min.
[0046] Furthermore, in step (2), after the recombinant overexpression vector is transformed into E. coli DH5α competent cells, single clones are screened by plating on Amp resistance plates, and the correctness of the recombinant plasmid is verified by colony PCR and Sanger sequencing; the primers for colony PCR are 35S-F / eGFP-R, and the annealing temperature is 58℃.
[0047] Furthermore, in step (3), Agrobacterium is a GV3101 competent cell;
[0048] The Agrobacterium transformation method was as follows: 3 μL of the verified recombinant plasmid was added to GV3101 competent cells with OD600=0.5, incubated on ice for 30 min, then flash-frozen in liquid nitrogen for 5 min, heat-shocked at 37℃ for 5 min, and after thawing on ice, 600 μL of antibiotic-free LB medium was added, and the cells were cultured at 28℃ and 200 rpm for 3 h.
[0049] Positive Agrobacterium strains were obtained through screening with double antibody plates containing 50 μg / mL Kan and 25 μg / mL Rif. The transformation efficiency was verified by SYBR Green quantitative PCR to be (3.2±0.5)×10⁻⁶. 5 cfu / μg DNA.
[0050] Furthermore, in step (4), the Agrobacterium-mediated genetic transformation method is specifically as follows:
[0051] a) After sterilization, Brassica napus seeds were sown on M0 medium and cultured in the dark at 24°C for 7 days to obtain hypocotyls;
[0052] b) Culture the positive Agrobacterium strain to a suitable concentration, then dilute with DM medium to adjust OD600 to 0.2-0.3;
[0053] c) Cut a hypocotyl segment with a length of 0.8-1cm and soak it in the Agrobacterium solution from step b) for 15-30 minutes;
[0054] d) The infected hypocotyls were cultured in the dark on M1 medium for 3 days, selectively cultured on M2 medium for 17 days, germinated on M3 medium (subcultured every 14 days), and rooted on M4 medium. After the roots grew, they were transplanted into nutrient soil.
[0055] e) T0 generation positive Brassica napus plants were obtained by DsRed tag fluorescence screening and PCR identification.
[0056] Example 1, such as Figure 1As shown, clubroot-resistant Chinese cabbage and black mustard were hybridized by distant hybridization to synthesize clubroot-resistant mustard-type rapeseed R13. In order to explore the clubroot resistance gene in R13, transcriptome analysis was performed on the resistant material and the susceptible material 135 after inoculation with clubroot fungus. It was found that one gene, BjuWRKY75, belongs to the WRKY transcription factor, and its expression level in the resistant material R13 was significantly higher than that in yellow mustard.
[0057] Example 2, as follows Figure 2 As shown, the CDS sequence of BjuWRKY75 from the resistant parent was constructed into a 2*35S overexpression vector and infected rapeseed. It was found that the rapeseed lines that heterologously overexpressed BjuWRKY75 from the resistant parent showed significantly higher resistance to clubroot disease than the wild type. This indicates that BjuWRKY75 positively regulates resistance to clubroot disease. By transferring this gene into rapeseed, a clubroot-resistant Brassica napus-type rapeseed can be created.
[0058] Example 3: Construction of BjuWRKY75 overexpression vector
[0059] Construction of overexpression vectors
[0060] DNA extracted from R13 using the CTAB method was used as a template. The target sequence was amplified using a high-fidelity enzyme. PCR amplification of the candidate gene was performed. The PCR reaction system consisted of: 25 μl 2×Phanta Maxabuffer, 1 μl dNTP, 2 μl each of forward and reverse primers (as shown in Table 1), 1 μl Phanta Max Super-Fidelity DNA Polymerase, 1 μl template DNA, and 18 μl lddH2O. The vector plasmid was double-digested with restriction endonucleases Bgl II and Xbal at 37°C for 2–5 h to linearize the circular vector. Homologous recombination was performed using a homologous recombination kit to combine the target gene with the digested vector, obtaining an overexpression recombinant vector. The 10 μl homologous recombination reaction system consisted of: 1 μl Exnase II, 2 μl 5×CEII, 2.5 μl linearized vector, and 4.5 μl target gene fragment. All components were gently mixed and incubated at 72°C for 30 min. The ligation product was transformed into E. coli DH5α competent cells, and single clones were screened by plating on Amp resistance plates. After verification by colony PCR (primers 35S-F / eGFP-R, annealing temperature 58℃) and Sanger sequencing, the recombinant plasmid was extracted.
[0061] Table 1 Primer Table
[0062] Genes Forward primers (5'-3') Reverse primers (5'-3') BjuWRKY75 tgactccatgtcgacggatccATGGAGGGATATGATGATGG ggtggactcctcttagaattcAAAAGAAGAGTAGATTTGCA
[0063] Agrobacterium transformation
[0064] 3 μL of the validation plasmid was added to GV3101 competent cells (OD600=0.5), incubated on ice for 30 min, then flash-frozen in liquid nitrogen for 5 min, heat-shocked at 37℃ for 5 min, and after ice-incubation, 600 μL of antibiotic-free LB medium was added (28℃, 200 rpm, 3 h). The cells were plated on plates containing Kan (50 μg / mL) and Rif (25 μg / mL) antibiotics (28℃, 48 h). Single colonies were picked and amplified, and colony PCR was performed for retesting. Positive strains were mixed with sterile glycerol at a 1:1 (v / v) ratio and stored at -80℃. The transformation efficiency was verified by quantitative real-time PCR (SYBR Green method) to be (3.2±0.5)×10⁻¹⁰. 5 The cfu / μg DNA level meets the requirements for subsequent genetic transformation experiments.
[0065] Example 4: Genetic transformation of Brassica napus
[0066] Agrobacterium-mediated genetic transformation of Brassica napus
[0067] First, rapeseed seeds were sterilized, and mature, plump seeds were selected and sown on M0 medium. After germination in the dark at 24℃ for 7 days, hypocotyls were obtained. Before transformation, Agrobacterium containing the target gene was cultured to a suitable concentration and diluted with DM medium to adjust OD600 to 0.2-0.3. Hypocotyl segments of 0.8-1 cm in length were cut and soaked in Agrobacterium solution for 15-30 minutes. Then, they were transferred to M1 medium for dark culture for 3 days, followed by sequential culture in M2 medium for selection for 17 days, M3 medium for germination culture (subcultured every 14 days), and M4 medium for rooting culture. After the roots grew, they were transplanted into nutrient soil. The obtained T0 generation seedlings were identified by fluorescent screening (using DsRed tags) and PCR to identify positive plants, and the transformation efficiency was calculated.
[0068] Plasmodium inoculation test
[0069] Since physiological race 4 is widely prevalent in my country, the test strain was collected from Mian County, Shaanxi Province (identified as physiological race 4 by the ECD system). It was washed and stored at -20℃. Before inoculation, the swollen roots were thawed at room temperature, and a suspension of dormant spores was prepared, following the method described in (Xiao Chonggang and Guo Xianghua, 2002). Inoculation was performed using the injection method, injecting 2-4 ml of the bacterial suspension close to the root of each plant. The spore concentration was 1×10⁻⁶. 8 CFU / ml; inoculation identification is performed approximately 14 and 21 days after inoculation.
[0070] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.
[0071] >BjuWRKY75's CDS sequence
[0072] SEQ ID NO.1
[0073] ATGGAGGGATATGATGATGGATCGTTGTATGCTCCGTTTCTGTCGCTGAAACCACATCAAAGCCTTTCTAAGTCGGAGCTAGAAGAAGGCAAAGAAGAAGCCTCGAAGGTTAGAGAAGGATCTTCGAGAAGCAGGGAGTTGAAAAAGAAGGGGAAGAAACAAAAGTTTGCGTTTCAGACAAGGAGCCAAGTTGATATTCTTGATGATGGCTATCGTTGGA GAAAATATGGCCAAAAAGCCGTCAAAAACAACAAGTTCCCTAGGAGTTACTATAGGTGCACATATGGAGGATGCAATGTAAAGAAGCAAGTGCAAAGATTAACAGCAGACCAAGAAGTTGTAGTCACGACCTACGAAGGAGTACACTCGCATCCCATCGAAAAATCTACAGAAAATTTCGAGCATATTCTCACCCAAATGCAAATCTACTCTTCTTTTTAA
[0074] >Amino acid sequence of BjuWRKY75
[0075] SEQ ID NO.2MEGYDDGSLYAPFLSLKPHQSLSKSELEEGKEEASKVREGSSRSRELKKKGKKQKFAFQTRSQVDILDDGYRWRKYGQKAVKNNKFPRSYRCTYGGCNVKKQVQRLTADQEVVVTTYEGVHSHPIEKSTENFEHILTQMQIYSSF*
Claims
1. A BjuWRKY75 gene for rapeseed, characterized in that, The CDS sequence of the BjuWRKY75 gene is shown in SEQ ID NO.1, and the amino acid sequence it encodes is shown in SEQ ID NO.2; The BjuWRKY75 gene is used to protect rapeseed from clubroot disease, which is caused by Plasmodiophora brassicae.
2. The application of the BjuWRKY75 gene of rapeseed as described in claim 1 in the cultivation of clubroot-resistant Brassica napus.
3. A method for cultivating clubroot-resistant Brassica napus using the BjuWRKY75 gene of Brassica oleracea as described in claim 1, characterized in that, Includes the following steps: Step (1) Using the genomic DNA of clubroot-resistant mustard-type rapeseed material R13 as a template, the CDS sequence of the BjuWRKY75 gene was obtained by PCR amplification. The forward primer sequence for the PCR amplification was 5'-tgactccatgtcgacggatccATGGAGGGATATGATGATGG-3', and the reverse primer sequence was 5'-ggtggactcctcttagaattcAAAAGAAGAGTAGATTTGCA-3'. Step (2) The CDS sequence obtained in step (1) is constructed into an overexpression vector to obtain a recombinant overexpression vector; Step (3) Transform the recombinant overexpression vector into Agrobacterium to obtain a positive Agrobacterium strain containing the target gene; Step (4) The BjuWRKY75 gene was introduced into Brassica napus through Agrobacterium-mediated genetic transformation to obtain Brassica napus plants resistant to clubroot disease.
4. The method for cultivating clubroot-resistant Brassica napus according to claim 3, characterized in that, In step (2), the overexpression vector is a 2*35S overexpression vector; The method for constructing the overexpression vector includes: linearizing the vector plasmid by double digestion with restriction endonucleases Bgl II and Xbal at 37°C for 2-5 hours, and then ligating the CDS sequence of the BjuWRKY75 gene with homologous arm sequences to the linearized vector using a homologous recombination kit. The 10 μl homologous recombination reaction system consisted of: 1 μl Exnase II, 2 μl 5×CEII, 2.5 μl linearized vector, and 4.5 μl BjuWRKY75 gene CDS fragment. The reaction conditions were incubation at 72°C for 30 min.
5. The method for cultivating clubroot-resistant Brassica napus according to claim 3, characterized in that, In step (2), after the recombinant overexpression vector is transformed into Escherichia coli DH5α competent cells, single clones are screened by plating on Amp resistance plates, and the correctness of the recombinant plasmid is verified by colony PCR and Sanger sequencing; the primers for colony PCR are 35S-F / eGFP-R, and the annealing temperature is 58℃.
6. The method for cultivating clubroot-resistant Brassica napus according to claim 3, characterized in that, In step (3), the Agrobacterium is a GV3101 competent cell; The Agrobacterium transformation method is as follows: 3 μL of the verified recombinant plasmid is added to GV3101 competent cells with OD600=0.5, ice bath for 30 min, liquid nitrogen flash freeze for 5 min, heat shock at 37℃ for 5 min, ice bath recovery, 600 μL of antibiotic-free LB medium is added, and cultured at 28℃ and 200 rpm for 3 h. The positive Agrobacterium strains were obtained through screening with double antibody plates containing 50 μg / mL Kan and 25 μg / mL Rif. The transformation efficiency was verified by SYBR Green quantitative PCR to be (3.2±0.5)×10⁻⁶. 5 cfu / μg DNA.
7. The method for cultivating clubroot-resistant Brassica napus according to claim 3, characterized in that, In step (4), the Agrobacterium-mediated genetic transformation method is specifically as follows: a) After sterilization, Brassica napus seeds were sown on M0 medium and cultured in the dark at 24°C for 7 days to obtain hypocotyls; b) Culture the positive Agrobacterium strain to a suitable concentration, then dilute with DM medium to adjust OD600 to 0.2-0.3; c) Cut a hypocotyl segment with a length of 0.8-1cm and soak it in the Agrobacterium solution from step b) for 15-30 minutes; d) The infected hypocotyls were cultured in the dark on M1 medium for 3 days, selectively cultured on M2 medium for 17 days, germinated on M3 medium (subcultured every 14 days), and rooted on M4 medium. After the roots grew, they were transplanted into nutrient soil. e) T0 generation positive Brassica napus plants were obtained by DsRed tag fluorescence screening and PCR identification.
8. A recombinant overexpression vector, characterized in that, The recombinant overexpression vector contains the CDS sequence of the BjuWRKY75 gene of Brassica napus as described in claim 1, and is used for genetic transformation of Brassica napus to resist clubroot disease.