Function Analysis Of Cation/Ca²⁺ Anti-transporter Protein BrCCX1 In Chinese Cabbage Response To Plasmodiophora Brassicae Infection

Brassica clubroot disease is a kind of disease caused by infection of Plasmodiophora brassicae.Directly hinder nutrients and water to be transported to aboveground parts of the plant,resulting in a significant reduction in yield.In recent years,some of clubroot-disease resistance mechanism has been reported by transcriptome analysis,protein profiling,RNA-seq technology and other methods,and a large number of proteins and genes with different expressions have been screened out,but the function of most of proteins and genes was not clear yet.The study on function and regulaton mechanism of key genes or proteins is expected to provide a new idea for resistant research of Chinese cabbage to clubroot disease.In our previous research,differential transcriptomic analysis was performed using P.brassicae inoculated or uninoculated roots of Chinese cabbage,and a gene with significantly up-regulated expression was found.Its function was predicted as Brassica rapa cation/Ca²⁺exchanger 1 of Chinese cabbage in NCBI,which.So it’s named Br CCX1.In order to further clarify the role of Br CCX1 gene,susceptible’SN742’and resistant’SN205’of Chinese cabbage are used as material;Br CCX1 expression,sequence,bioinformatics and expression sites were analyzed;The function of Br CCX1 gene in resistance to P.brassicae was verified;Upstream transcription factors and interacting functional proteins were screened by yeast single hybridization.The results are as follows:1.Real-time quantitative PCR（RT-q PCR）results showed that the expression of Br CCX1was up-regulated in the roots of all inoculated material,especially in the susceptible material’SN742’;Compared to the expression of Br CCX1 gene among roots,stems and leafs,it was found that Br CCX1 gene was only significantly up-regulated expressed in the infected roots.The results of in situ hybridization showed that dark blue was highly presented on the root sections of the clubroot-diseased’SN742’,which was consistent with RT-q PCR results.The above results indicate that Br CCX1 may have a certain effect on the infection process of P.brassicae root to Chinese cabbage.2.Cloning the full length/promoter sequence of Br CCX1 gene,and using it for sequence and bioinformatics analysis.An amino acid change from C（cysteine）to R（arginine）was found in Br CCX1 protein sequence of Chinese cabbage resistant material’SN205’,and the corresponding protein structure was different between’SN742’and’SN205’;There was no difference in promoter sequences between’SN742’and’SN205’.Bioinformatics results showed that Br CCX1 had 11 transmembrane domains and the homology with PLN03151superfamily protein(cation/ca²⁺exchange protein)is 93.44%.In order to identify the expression site of Br CCX1 in cells,an overexpression vector p BWA（V）BS-Br CCX1-GFP was constructed and used for subcellular localization.The results showed the target gene is mainly located in the plasma membrane.3.Arabidopsis mutant ccx1 was screened by the‘three-primer method’,and inoculated with P.brassicae.Compared with WT,ccx1 delayed the infection process of P.brassicae spores.Furthermore,when Br CCX1 was silenced by VIGS in Chinese cabbage,the pathogenicity of clubroot disease was reduced.4.p Br CCX1-Ab Ai was constructed and used for Y1H library screening.A transcription factors MYB106 and other eight proteins were screened out.One to one validation showed that both MYB106 and TJLP1 proteins could bind to the promoter of Br CCX1.These results suggested that Br CCX1 was regulated by MYB106 and TJLP1 to play role in the progress of the infection of P.brassicae spores to Chinese cabbage.