Functional Verification Of CRb Candidate Gene Conferring Resistance To Clubroot Disease In Brassica Rapa

Clubroot disease caused by Plasmodiophora brassicae is one of the most serious soil-borne diseases in cruciferous crop production.Clubroot has a wide range of occurrence,and is almost distributed in the main cultivation areas of cruciferous crops in China,leading to a significant reduction in crop yield or even extinction.In recent years,clubroot disease has been controlled by chemical agents,but this method is easy to pollute the environment and the control effect is not stable.It is one of the most economical and effective ways to control clubroot disease by breeding new varieties with clubroot resistance genes.It is of great significance to verify the function of target genes by transgenic technology.In the early stage,our laboratory identified two genes Bra012540 and Bra012541 that encode TIR-NBS-LRR protein as CRb candidate genes,and analyzed the differential expression of the two genes in different tissues at different time points of infection.Furthermore,overexpression in Arabidopsis thaliana can enhance the resistance of transgenic plants to clubroot.On the basis of previous studies,this study verified the functions of two candidate genes Bra012540 and Bra012541 through transgenic technology.Firstly,the recombinant plasmids driven by the 35S promoter to express Bra012540 and Bra012541 genes were transformed into Brassica napus‘Westar’for disease resistance identification.Secondly,Bra012540 and Bra012541 were connected in series to construct a transgenic plant expression vector driven by the gene’s own promoter and transformed into Chinese cabbage inbred line‘C-24’.And the disease resistance of the T₁ transgenic Chinese cabbage was identified.The main results are as follows:1.The overexpression recombinant plasmids p CAMBIA1305.1-35S-Bra012540-Bra012541,p CAMBIA1300S-35S-Bra012541 and p CAMBIA1300S-35S-Bra012540 were successfully introduced into wild type‘Westar’by agrobacterium-mediated method and overexpression transgenic plants were obtained.Using PCR technology for screening the positive plants as clubroot disease resistance identification,found that overexpression transgenic plants root morphology was much better than that of wild type controls,the incidence and disease index and P.brassicae DNA content were significantly lower than that of wild type controls.At the same time analyzing the overexpression transgenic plants under P.brassicae stress related physiological and biochemical indicators change,we found that H₂0₂ and MDA contents in transgenic Brassica napus roots were significantly lower than those in wild-type controls,and CAT,SOD and POD enzyme activities in transgenic Brassica napus roots were significantly higher than those in wild-type controls.These results indicated that overexpression Bra012540 and Bra012541 genes in Brassica napus increased the resistance of transgenic plants to clubroot disease.2.To further verify the functions of Bra012540 and Bra012541 genes,a plant expression vector p CAMBIA1301-Bra012541-Bra012540 driven by its own promoter was successfully constructed by in-fusion seamless cloning technique.Meanwhile,agrobacterium tumefaciens（GV3101）was used as vector and hygromycin was used as a screening marker to introduce the recombinant plasmid into Chinese cabbage inbred line‘C-24’.After PCR identification and screening,a total of 8 T₀ generation positive transgenic plants were obtained and T₁ generation seeds were harvested.It was found that the root morphology,incidence and disease index of transgenic Chinese cabbage at 0 d,15 d,30 d and 40 d after inoculation with P.brassicae were nearly consistent with those of the susceptible wild-type control‘C-24’.In addition,the relative expression of Bra012541 and Bra012540 CRb candidate genes of transgenic Chinese cabbage after P.brassicae infection was very low.The results showed that the phenotype of the offspring of transgenic plants was not significantly changed after the wild-type‘C-24’of susceptible Chinese cabbage was transferred into the CRb resistance gene driven by the gene’s own promoter.