不结球白菜BcFBN6a参与叶绿素积累的功能分析OA

[Objectives]The paper aimed to clone BcFBN6a gene and explore its function in chlorophyll accumulation in non-heading Chinese cabbage.[Methods]The CDS sequence of BcFBN6a was homologously cloned using the cDNA of non-heading Chinese cabbage'Suzhouqing'as a template,and the analysis for conserved structural domains,phylogeny and conserved motifs with homologous protein sequences from other species were performed.The BcFBN6a-pRI101 vector was transformed into Agrobacterium tumefaciens and infiltrated into tobacco leaves for subcellular localization analysis of BcFBN6a.RT-qPCR technology was used to analyze the expression pattern of BcFBN6a in different tissues.Overexpression transgenic lines of BcFBN6a in Arabidopsis thaliana and BcFBN6a-silenced lines in non-heading Chinese cabbage,obtained by Agrobacterium-mediated floral dip transformation and virus-induced gene silencing(VIGS)respectively,were utilized to study the function of BcFBN6a on chlorophyll accumulation.Interacting proteins of BcFBN6a were predicted using the GeneMANIA database and further validated by yeast two-hybrid(Y2H),bimolecular fluorescence complementation(BiFC)and luciferase complementation imaging(LCI)assays.[Results]The ORF of BcFBN6a,containing a typical PAP domain,was 714 bp.The BcFBN6a had the closest evolutionary relationship with Brassica rapa and was localized in chloroplast.BcFBN6a gene was highly expressed in leaves.Heterologous overexpression lines of BcFBN6a in A.thaliana significantly increased chlorophyll accumulation in leaves,and the expression levels of chlorophyll biosynthesis-related genes,such as AtHEMA1,AtCHLH,AtGUN4 and AtPORB,were significantly upregulated.The chlorophyll content in BcFBN6a-silenced plants induced by VIGS was significantly lower than that in pTY empty control,and the expression levels of chlorophyll synthesis related genes were downregulated.The Y2H,BiFC and LCI assays revealed that BcFBN6a could interact with BcGUN4.[Conclusions]BcFBN6a could positively regulate chlorophyll accumulation in non-heading Chinese cabbage,and a protein BcGUN4 was screened,which interacted with BcFBN6a.