Functional Analysis Of Non-heading Chinese Cabbage BcmiR400b-3p And BcCPR1 Genes In Botrytis Cinerea And Hyaloperonospora Brassicae

Non-heading Chinese cabbage[Brassica campestris（syn.Brassica rapa）ssp.chinensis]is a vegetable crop and belongs to the genus Brassica of Brassicaceae.Because of its high nutritional value and versatility,it is widely cultivated.However,it is susceptible to a variety of viruses,bacteria and fungi,which causes serious economic losses.In order to reduce the yield loss caused by various diseases,disease resistance has become one of the important breeding goals.In this paper,we used non-heading Chinese cabbage’Suzhouqing’and Arabidopsis thaliana as the main experimental materials and used VIGS,q RT-PCR,transgenic technology and other methods to explore the effects of BcmiR400b-3p and BcCPR1 in downy mildew and gray mold infestation.The results are as follows:1.Functional analysis of BcmiR400b-3p and its target gene BcBAK7 under pathogen infection in non-heading Chinese cabbageIn this study,RNAfold web server was used to predict the secondary structure of pre-BcmiR400b-3p,which confirmed that it had a typical stem-loop structure and the secondary structure is stable.Plant CARE was used to analyze the cis-elements of the BcmiR400b-3p promoter,and the promoter sequence was found that contain stress,hormone and light response elements.The expression of BcmiR400b-3p was up-regulated and the expression of the predicted target gene BcBAK7 was negatively correlated with BcmiR400b-3p after downy mildew and gray mold infection in non-heading Chinese cabbage.In the tobacco co-expression experiment,BcBAK7 was verified as a target gene of BcmiR400b-3p by GUS staining.The pre-BcmiR400b-3p was cloned using’Suzhouqing’as the material and was inserted into the pRI101overexpression vector.The 35S::BcmiR400b-3p overexpressing transgenic A.thaliana was obtained by Agrobacterium-mediated method.Compared with the wild type,the 35S::BcmiR400b-3p transgenic lines were resistant to gray mold,the disease index decreased by 4.6～18.It was found that the expression of At BAK7 was down-regulated in the transgenic lines compared with the wild type.The study of BcBAK7,the target gene of BcmiR400b-3p,found that BcBAK7 overexpressing transgenic A.thaliana was susceptible to gray mold,the disease index increased by 4.6～9.3.BcBAK7 gene silencing plants obtained by VIGS were resistant to gray mold and downy mildew,and the silencing of BcBAK7 activated the expression of SA and JA pathway-related genes.2.Cloning and function analysis of BcCPR1 gene involved in disease resistance in non-heading Chinese cabbageThe full-length CDS sequence of BcCPR1 gene was cloned by homologous cloning method using’Suzhouqing’as the material.Homology analysis used MEGA7software.BcCPR1 gene contained a 1221 bp open reading frame（ORF）,encoding406 amino acids.The homology between B.oleracea var.oleracea and B.rapa was up to 97.54%and 92.66%,based on the close relationship.By the Agrobacterium-mediated method,the sub-cellular localization was researched in tobacco leaves and transgenic A.thaliana was acquired.Subcellular localization results showed that the localization was on the cell membrane.BcCPR1-overexpressing A.thaliana was more resistant than wild-type plants to gray mold,the disease index decreased by 30.6～32.6,while the accumulation of H₂O₂ decreased and the JA pathway marker gene PDF1.2 increased.