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CN-121991965-A - Application of mustard type rape BjuWRKY in cabbage type rape clubroot resistance

CN121991965ACN 121991965 ACN121991965 ACN 121991965ACN-121991965-A

Abstract

The invention relates to the technical field related to plant genetic engineering, in particular to application of mustard type rape BjuWRKY in the clubroot resistance of cabbage type rape, bjuWRKY gene (CDS sequence of which is shown as SEQ ID NO. 1) is successfully cloned from a mustard type rape material R13 with the clubroot resistance, a 2 x 35S over-expression vector is constructed, the gene is introduced into the cabbage type rape by using an agrobacterium-mediated genetic transformation technology, experiments prove that the over-expression of the gene can obviously improve the resistance of the rape to the clubroot No. 4 physiological race, the number of tumors of roots of transgenic plants is obviously reduced, and therefore, a brand-new gene resource and an effective breeding way are provided for overcoming the technical bottlenecks of the existing brassica napus of having less clubroot resistance resources and single resistance source.

Inventors

  • LI KEQI
  • HUANG ZHEN
  • DUN XIAOLING
  • SUN WEINAN
  • YU CHENGYU
  • LI XIAOYAN
  • XU KE
  • NIE JUNWEI

Assignees

  • 西北农林科技大学
  • 中国农业科学院油料作物研究所

Dates

Publication Date
20260508
Application Date
20251218

Claims (8)

  1. 1. A mustard type rape BjuWRKY gene is characterized in that, The CDS sequence of BjuWRKY gene is shown as SEQ ID NO.1, and the coded amino acid sequence is shown as SEQ ID NO. 2; The BjuWRKY gene is used for resisting clubroot of brassica napus, and the clubroot is caused by brassica clubroot (Plasmodiophora brassicae).
  2. 2. Use of the mustard type canola BjuWRKY gene of claim 1 in the cultivation of clubroot resistant brassica napus.
  3. 3. A method for cultivating clubroot-resistant brassica napus by using the mustard type rape BjuWRKY gene as claimed in claim 1, comprising the following steps: The method comprises the following steps of (1) taking genomic DNA of a mustard type rape material R13 resisting clubroot as a template, and obtaining a CDS sequence of BjuWRKY genes through PCR amplification, wherein a forward primer sequence of the PCR amplification is 5'-tgactccatgtcgacggatccATGGAGGGATATGATGATGG-3', and a reverse primer sequence of the PCR amplification is 5'-ggtggactcctcttagaattcAAAAGAAGAGTAGATTTGCA-3'; Step (2) constructing the CDS sequence obtained in the step (1) into an over-expression vector to obtain a recombinant over-expression vector; step (3) transforming the recombinant over-expression vector into agrobacterium to obtain a positive agrobacterium strain containing a target gene; And (4) introducing the BjuWRKY gene into brassica napus by an agrobacterium-mediated genetic transformation method to obtain a brassica napus plant resistant to clubroot.
  4. 4. A method of growing a clubroot brassica napus according to claim 3 wherein in step (2) the over-expression vector is a2 x 35s over-expression vector; The construction method of the over-expression vector comprises the steps of carrying out double enzyme digestion treatment on a vector plasmid by adopting restriction enzymes Bgl II and Xbal for 37 ℃ and 2-5 hours to linearize the vector, and connecting a BjuWRKY gene CDS sequence with a homologous arm sequence with the linearized vector through a homologous recombination kit; The 10. Mu.l reaction system of the homologous recombination is 1. Mu. l Exnase II,2. Mu.l 5X CEII, 2.5. Mu.l linearization vector, 4.5. Mu. l BjuWRKY75 gene CDS fragment, and the reaction conditions are 72 ℃ for 30min.
  5. 5. The method of claim 3, wherein in the step (2), after the recombinant overexpression vector is transformed into E.coli DH 5. Alpha. Competent cells, the recombinant plasmid is verified to be correct by colony PCR and Sanger sequencing by coating an Amp-resistant plate for screening and then the primer of colony PCR is 35S-F/eGFP-R, and the annealing temperature is 58 ℃.
  6. 6. A method of growing a clubroot brassica napus according to claim 3, wherein in step (3) the agrobacterium is GV3101 competent cells; Adding 3 mu verified correct recombinant plasmid into GV3101 competent cells with OD600 = 0.5, carrying out liquid nitrogen quick freezing for 5min after ice bath for 30min, carrying out heat shock for 5min at 37 ℃, adding 600 mu L of antibiotic-free LB medium after ice bath recovery, and culturing for 3h at 28 ℃ and 200 rpm; The positive agrobacterium strain is obtained through double-antibody plate screening containing 50 mug/mL Kan and 25 mug/mL Rif, and the conversion efficiency is verified to reach (3.2+/-0.5) multiplied by 10 5 cfu/mug DNA by fluorescence quantitative PCR (polymerase chain reaction) of SYBR Green method.
  7. 7. A method for breeding clubroot brassica napus according to claim 3, wherein in step (4), the agrobacterium-mediated genetic transformation method is specifically: a) Sowing the sterilized brassica napus seeds into an M0 culture medium, and germinating in the dark at 24 ℃ for 7 days to obtain hypocotyls; b) Culturing positive agrobacterium strain to a proper concentration, and diluting and adjusting OD600 to 0.2-0.3 by using a DM culture medium; c) Cutting a hypocotyl section with the length of 0.8-1 cm, soaking in the agrobacterium tumefaciens bacteria liquid of the step b) and dip-dyeing for 15-30 minutes; d) Culturing the impregnated hypocotyl in M1 culture medium for 3 days in dark, culturing in M2 culture medium for 17 days, culturing in M3 culture medium for bud growth (once every 14 days), culturing in M4 culture medium for rooting, and transplanting to nutrient soil after root system grows out; e) Obtaining T0 generation positive brassica napus plants through DsRed tag fluorescent screening and PCR identification.
  8. 8. A recombinant overexpression vector comprising the CDS sequence of the brassica juncea BjuWRKY gene of claim 1, for use in genetic transformation of brassica juncea against clubroot.

Description

Application of mustard type rape BjuWRKY in cabbage type rape clubroot resistance Technical Field The invention relates to the technical field related to plant genetic engineering, in particular to an application of mustard type rape BjuWRKY in cabbage type rape clubroot resistance. Background Clubroot is a worldwide soil-borne disease caused by brassica clubroot (Plasmodiophora brassicae), and is one of the most damaging diseases for cruciferous crops such as rape, cabbage, and the like. In recent years, the clubroot disease rapidly spreads in rape main producing areas in China, the annual hazard area of the clubroot disease in China is more than 66.7 ten thousand hectares, the production of rape in China is seriously influenced, and the method is a new and huge challenge faced by the current rape planting, so that the gene for resisting the clubroot disease is urgently required to be excavated, and a foundation is laid for cultivating new varieties of rape with clubroot disease resistance. However, the resources for resisting clubroot in cabbage type rape in China are very rare, and the resources for resisting clubroot exist in cabbage, cabbage and mustard which are related to the cabbage type rape. The root of the plant is mainly endangered by the plasmodiophora, after the plant is infected, tumors with different sizes grow on the main root, the lateral root and the fibrous root gradually, and the tumors are cracked and rough in the later period of infection, are easy to be invaded by other miscellaneous bacteria to cause decay, and seriously affect the normal absorption of the plant to moisture and nutrients. Because of strong infection, fast transmission speed, multiple transmission ways, poor biological and chemical control effects and lack of plants with broad-spectrum antigens, the clubroot is a worldwide destructive soil-borne plant disease. At present, some clubroot-resistant genes/loci are mainly found in vegetables, such as European turnip/cabbage, black mustard, radish and the like, while fewer sources of resistance are found in brassica napus. Currently, at least 28 QTLs associated with clubroot resistance are mapped to the 5 chromosomes a01, a02, a03, a06 and a08 of chinese cabbageWherein 12 resistance QTL (Crr, rcr1, rcr2, rcr4, rcr5, pbBa3.1, CRk, CRd, CRb, CRa, CRaki, pbBp3.3) are located on A03 chromosome and are interlocked with each other, which indicates that the clubroot resistant locus derived from Chinese cabbage is single. Whereas the clubroot resistance in brassica napus is mainly derived from chinese cabbage. Therefore, the invention artificially synthesizes the mustard type rape with the clubroot resistance by hybridizing the cabbage and the mustard through distant hybridization, and further locates the artificially synthesized disease-resistant gene of the mustard type rape with the clubroot resistance through simplifying genome sequencing, thereby providing important theoretical basis and gene resources for cultivating new varieties of the rape with lasting resistance. Disclosure of Invention The invention aims to provide an application of mustard type rape BjuWRKY in the clubroot resistance of cabbage type rape, so as to solve the problem that the resistance of the cabbage type rape germplasm or variety with the clubroot resistance is single and the disease resistance is single. In order to achieve the above purpose, the present invention provides the following technical solutions: A CDS sequence of the leaf mustard type rape BjuWRKY gene is shown as SEQ ID NO.1, an encoded amino acid sequence of the leaf mustard type rape 3275 gene is shown as SEQ ID NO.2, and the leaf mustard type rape BjuWRKY gene is used for resisting clubroot caused by brassica clubroot (Plasmodiophora brassicae). The application of the mustard type rape BjuWRKY gene in cultivating clubroot-resistant cabbage type rape. The method for cultivating the clubroot-resistant cabbage type rape by using the mustard type rape BjuWRKY gene comprises the following steps: The method comprises the following steps of (1) taking genomic DNA of anti-clubroot brassica juncea R13 as a template, and obtaining a CDS sequence of BjuWRKY genes through PCR amplification, wherein a forward primer sequence of the PCR amplification is 5'-tgactccatgtcgacggatccATGGAGGGATATGATGATGG-3', and a reverse primer sequence of the PCR amplification is 5'-ggtggactcctcttagaattcAAAAGAAGAGTAGATTTGCA-3'; Step (2) constructing the CDS sequence obtained in the step (1) into an over-expression vector to obtain a recombinant over-expression vector; step (3) transforming the recombinant over-expression vector into agrobacterium to obtain a positive agrobacterium strain containing a target gene; And (4) introducing the BjuWRKY gene into brassica napus by an agrobacterium-mediated genetic transformation method to obtain a brassica napus plant resistant to clubroot. Preferably, in the step (2), the overexpression vector is a 2x 35s overexpression vector;