甘蓝型油菜-核盘菌酵母双杂交cDNA文库构建及BnJar1互作蛋白筛选

Construction of a Brassica napus-Sclerotinia sclerotiorum yeast two-hybrid cDNA library and screening for BnJar1 interacting proteins

油菜与核盘菌相互作用的过程中,JA(茉莉酸)合成相关基因表达量升高,但是JA信号传递却受到了抑制,推测核盘菌分泌的效应蛋白可能直接或间接作用于JAR1蛋白,使其活性降低,从而抑制依赖JA途径的油菜防御系统。前期通过转录组测序获得了差异表达基因BnJar1全长序列,为进一步揭示BnJar1基因参与油菜-核盘菌相互作用过程的机理,本研究对BnJAR1蛋白进行生物信息学分析和预测,构建了核盘菌-油菜互作表达基因酵母文库,并以BnJar1作为诱饵蛋白对文库进行酵母双杂交筛选,获得了5个来自油菜的互作蛋白:丙二烯氧化物环化酶2(BnAOC2)、COP9信号复合物(BnCOP9)、4a-羟基四氢生物蝶呤脱水酶(BnDcoH)、腈水解酶2(BnNIT2)和茎特异性蛋白(BnTSJT1);2个来自核盘菌的互作蛋白:MFS结构域蛋白(SsMFS)和Rho3 GTP酶(SsRho3)。相关结果为研究油菜-核盘菌的相互作用,挖掘致病或抗病相关基因,进一步解析其机理奠定了基础。

During the interaction between Brassica napus and Sclerotinia sclerotiorum,the expression of JA(jasmonic acid)synthesis-related genes increases,while JA signal transduction is inhibited.It is speculated that the effector proteins secreted by S.sclerotiorum may directly or indirectly interact with JAR1 protein,reducing its activi-ty and thus inhibiting the defense system of B.napus dependent on JA pathway.The full-length sequence of the dif-ferentially expressed gene BnJar1 was obtained by transcriptome sequencing in the early stage of this research.In order to further reveal the mechanism of BnJar1 gene involved in the interaction between B.napus and S.sclerotio-rum,this study analyzed and predicted the bioinformatics of BnJAR1 protein,constructed a yeast expression gene cDNA library of B.napus-S.sclerotiorum interaction,and used BnJar1 as bait protein to screen the library by yeast two-hybrid.Through the study,5 interacting proteins from B.napus were obtained:BnAOC2,BnCOP9,BnDcoH,BnNIT2,and BnTSJT1;and 2 interacting proteins from S.sclerotiorum:SsMFS and SsRho3.The relevant results contributed to our research on the interaction between B.napus and S.sclerotiorum,exploration of pathogenic or re-sistance-related genes,and on further elucidation of their mechanisms.