The Functional Study Of BrUFO Gene In Chinese Cabbage Responsed To Plasmodiophora Brassicae Infection

Chinese cabbage is an important vegetable crop in China.But its yield is seriously affected by clubroot disease.It is expected to fundamentally resist the infection of Plasmodiophora brassicae by excavating the gene of resistance to cultivate clubroot-disease resistant varieties.At present,a large number of disease resistance genes have been excavated,but the resistance function and mechanism of most genes have not been verified.In our previous research,a gene Bra039878 was found to be significantly up-regulated in the roots of Chinese cabbage after inoculation with P.brassicae by difference transcriptome.In this study,it was annotated as BrUFO.The spatio-temporal expression pattern of BrUFO gene was analyzed by RT-qPCR,in situ hybridization and subcellular localization.The function of BrUFO gene on resistance to clubroot disease was analyzed by virus-induced gene silencing(VIGS).The interacting protein of BrUFO was screened by yeast double-hybrid experiment,and the resistance mechanism of BrUFO gene to clubroot disease was predicted.The results are as follows:1.The cDNA sequence of the BrUFO gene was amplified.It was found that the candidate gene has a high homology with Arabidopsis UNUSUAL FLORAL ORGAN(UFO),so it is named BrUFO.Prediction of its functional domain found that it is a nucleoprotein contained a conserved protein domain family F-box.2.The expression pattern of BrUFO gene was analyzed by RT-qPCR.The results showed that BrUFO gene expression was higher in the inoculated materials than in the uninoculated control materials at either infection stage or onset stage.In the control group,the expression level of BrUFO gene in ’SN742’ was higher than that in ’SN205’,suggesting that BrUFO gene played a role in Chinese cabbage’s response to P.brassicae infection.3.The analysis results of in situ hybridization found that blue hybridization signal of BrUFO gene was highly showed in the clubroot-diseased roots of ’SN742’ and ’SN205.In uninoculated control group,hybridization signal of BrUFO gene was stronger in the susceptible material ’SN742’ than in resistant material ’SN205’.As described above,the results were consistent between in situ hybridization and RT-qPCR.4.Vector p BWA(V)BS-BrUFO-GFP was constructed and used to express instantaneously and detect the fluorescence localization of BrUFO translation products in tobacco cell.The results showed that fluorescence signals were showed in the nucleus,cell membrane and cytoplasm of tobacco leaf cells in the plants injected with p BWA(V)BS-GFP blank vector,while there were only fluorescence signals in the nucleus of tobacco leaf cells in the plants injected with p BWA(V)BS-BrUFO-GFP recombinant vector.Therefore,it is inferred that BrUFO gene is located in the nucleus.5.pTRV2-BrUFO gene silenced plant expression vector was constructed,and used to predict the function of BrUFO gene response in P.brassicae by virus-induced gene silencing(VIGS).pTRV2-00 and Wild type plants were used as control.RT-qPCR analysis showed that BrUFO gene was silenced significantly(60%)in pTRV2-BrUFO treated lines on day 40 after infection.The disease index of Wild Type control group,pTRV2-00 control group and pTRV2-BrUFO treatment group were 10,41.25 and 6.25,respectively on day 40 after infection.The occurrence of clubroot disease was reduced in BrUFO silenced plants,thus confirming that the reduction of BrUFO gene expression could enhance the resistance of plants to clubroot disease.6.pGBKT7-BrUFO bait vector was constructed and used for screening the interaction proteins of BrUFO from P.brassicae induced yeast library.Nine proteins were screened BrU-1,BrU-2,BrU-3,BrU-5 and BrU-9 was verified to interact with BrUFO respectively by one to one verification.Bru-1,BrU-2 and BrU-3 belonged to S-phase kinase associated protein 1(SKP1),and BrU-5 belonged to GDSL esterase/acyltransferase protein.Bru-9 belongs to B cell receptor-associated 31-like protein.SKP1 protein is a regulatory factor for the validation of many genes’ resistance to disease.GDSL lipase genes are also involved in plant disease resistance and stress responses.In conclusion,BrUFO plays a key role in the response mechanism of P.brassicae infection,possibly through interaction with SKP1 protein and GDSL lipase.