Functional Identification And Mapping Of The Clubroot Resistance Gene CRd In Chinese Cabbage(Brassica Rapa Ssp.Pekinensis)

The rapid spread of clubroot disease,which is caused by Plasmodiophora brassicae,threatens Brassicaceae crop production worldwide.Breeding plants that have broadspectrum disease resistance is one of the best ways to prevent clubroot.Previous studies have identified a number of clubroot resistance genes that exhibit specific resistance to P.brassicae.However,there are only a few genes that have been cloned,and the functional verification of genes against P.brassicae and the defense mechanism of virulent vs.avirulent P.brassicae in Chinese cabbage are poorly understood.This study explored and mapped CRd resistance sites,and cloned candidate genes and transgeniced them into Arabidopsis.The main findings are as follows:1.Mapping of CRd gene and screening of candidate genes:Parental materials used in this study were Chinese cabbage inbred line “85-74”(clubroot resistance)and “BJN3-1”(clubroot susceptible).“85-74” inbred line showed resistance to P.brassicae “LAB-19” of physiological species 4.By using the cluster isolation analysis method based on second-generation sequencing combined with genetic mapping,the anti-rhizoma disease gene was located in the F2 isolation population produced by the hybridization of Chinese cabbage inbred lines “85-74” and “BJN3-1”.Genetic analysis showed that its disease resistance was controlled by a single dominant gene,which was located in a region of about 60 Kb(1 c M)between yau389 and yau376 flanking linkage markers on chromosome A03 and named CRd.Within this interval,four genes with tir-sbs-lrr protein domain Bra001160,Bra001161,Bra001162 and Bra001175 were identified as candidate genes through gene functional annotation.2.Cloning and expression analysis of CRd candidate genes:Bra001160,Bra001161,Bra001162 and Bra001175 candidate genes were cloned,and gene sequences with coding region length of 3273 bp,3174 bp,3456 bp and 3273 bp were obtained respectively.According to the sequence results,the structure of Bra001160,Bra001161,Bra001162 and Bra001175 proteins were predicted by NCBI Conserved Domain Search,and the results showed that they all had TIR,NB-ARC and LRR domains,and were typical TIR-NBS-LRR disease-resistant genes.In addition,the expression analysis of candidate genes showed that CRd was not induced by P.brassicae,but because the four candidate genes all had sequence variation in the coding region between disease-resistant and disease-susceptible materials,and caused amino acid variation,so as to induce different disease-resistant and disease-resistant responses to P.brassicae.Therefore,these four genes were further identified as CRd candidate genes.3.Construction Arabidopsis plant expression vector and functional identification of a CRd candidate gene:The over-expression vector of the candidate gene carried herbicide and the red fluorescent screening tag was constructed by enzyme digestion combined and the In-Fusion method,and the Arabidopsis thaliana transgenic was obtained by Agrobacterium-mediated staining.The results of disease resistance identification showed that the disease index and incidence rate of Bra001161 transgenic plants were significantly lower than wild-type Arabidopsis thaliana,so Bra001161 was further confirmed as CRd gene.4.CRd gene phylogenetic analysis:The CRd gene was amplified from the B.rapa which showed resistance to clubroot disease(18 copies)and susceptible to clubroot disease(26 copies),and the sequence difference of CRd gene in 32 amplified materials resulted in '85-74' and ‘ER85B' are on the same branch,indicating that the two materials have the same clubroot resistance gene and are not on the same branch as other CR materials,indicating that the CRd gene is different from other clubroot resistant materials,and the CRd gene is a new CR gene,and the resistance to the physiological race of P.brassicae is also different.5.Subcellular localization of CRd gene and cytological observation of transgenic Arabidopsis root tissue:In order to clarify the role of CRd gene in cells,the present study conducted subcellular localization analysis on tobacco leaf transient expression,and the results indicated that CRd gene was located on the cell membrane,possibly playing a role in identifying pathogens on the cell membrane.In addition,the root cell morphology of arabidopsis thaliana transgenic plants infected by P.brassicae was observed by paraffin section.It was found that no P.brassicae spore was observed in the root tissue of disease-resistant transgenic Arabidopsis thaliana,and the cell morphology was normal.The root cells of the infected and wild-type arabidopsis thaliana have been destroyed and deformed,and the cells have been enlarged,which is full of P.brassicae spores,thus leading to the formation of P.brassicae.This result indicates that the CRd gene transgenic Arabidopsis thaliana has resistance to race 4.6.Sequencing of the transcriptome after “85-74” inoculation of virulent and avirulent P.brassicae:The transcriptome sequencing method was used to explore the defense response pathways of Chinese cabbage containing clubroot resistance gene CRd in response to virulent and avirulent P.brassicae.The results indicate that the effector-triggered immunity in “85-74” is more efficiently activated in response to infection by virulent P.brassicae,and the JA,ET and BR signaling pathways are responsible for the late stage of P.brassicae infection.The reaction is very important.These findings provide a degree of understanding of the pathogenic specific defense mechanisms of P.brassicae in cruciferous crops.In summary,this study mapped and cloned the CRd clubrot resistance gene,and confirmed that CRd was indeed resistant to P.brassicae by disease resistance identification of positive plants after the transformation of CRd gene into Arabidopsis and observation of root tissue cytology.Phylogenetic analysis showed that CRd was a new gene that was different from other known clubroot resistance genes.Through transcriptome sequencing,the main defense response pathways of “85-74” in Chinese cabbage inbred line containing CRd gene in response to virulent and avirulent P.brassicae infection were JA,ET and BR signal transduction pathways.