白粉菌诱导簇毛麦叶片酵母双杂交文库构建及CMPG1-V候选互作蛋白筛选  

Construction of yeast two-hybrid cDNA library of Haynaldia villosa leaves induced by Blumeria graminis f.sp.tritici and candidate interaction protein screening for CMPG1-V

作　　者：张恒[1] 陈怡名 张旭[1] 牛影 赵佳 吴承云 郝永利 孙丽[1] 王海燕[1] 肖进[1] 王秀娥[1] ZHANG Heng;CHEN Yiming;ZHANG Xu;NIU Ying;ZHAO Jia;WU Chengyun;HAO Yongli;SUN Li;WANG Haiyan;XIAO Jin;WANG Xiu'e(Cytogenetics Institute/State Key Laboratory of Crop Genetics and Germplasm Enhancement/Jiangsu CollaborativeInnovation Center for Modern Crop Production,Nanjing Agricultural University,Nanjing 210095,China)

机构地区：[1]南京农业大学细胞遗传研究所/作物遗传与种质创新国家重点实验室/江苏省现代作物生产协同创新中心,江苏南京210095

出　　处：《南京农业大学学报》2020年第4期594-604,共11页Journal of Nanjing Agricultural University

基　　金：国家自然科学基金项目(91935304,31971943);国际(地区)合作与交流项目(31661143005);农业部948项目(2015-Z41);江苏省科技厅项目[PZCZ201706,JATS(2019)429];江苏省科技成果转化专项基金(BA2017138);宁夏回族自治区重点研发计划重大项目(2019BBF02022-04)。

摘　　要：[目的]本文旨在使用Mate&PlateTM技术构建簇毛麦受白粉菌诱导的酵母双杂交文库,鉴定正向调节小麦白粉病抗性的簇毛麦E3泛素连接酶蛋白CMPG1-V的互作蛋白,为解析其抗病性机制奠定基础。[方法]提取白粉菌诱导前及诱导后1、2、4、6、12、24和48 h的簇毛麦叶片总RNA,利用SMART技术分离纯化mRNA,合成ds cDNA,将ds cDNA和pGADT7-Rec载体共同转入酵母菌株Y187,构建簇毛麦酵母双杂交文库,明确文库插入片段大小和滴度。以CMPG1-V为诱饵筛选文库,并对部分互作蛋白进行了回补验证,利用BiFC方法验证CMPG1-V与筛选到的1个互作蛋白CMIN6(MYB25-V)的体内互作。[结果]成功构建了受白粉菌诱导的簇毛麦叶片酵母双杂交文库,文库滴度为9.5×107 CFU·mL^-1,插入片段长度为250~2000 bp,重组率为100%。以CMPG1-V为诱饵筛选文库,获得79个互作蛋白,挑选其中10个互作蛋白进行回补验证。通过BiFC试验验证了CMPG1-V与MYB25-V的体内互作。MYB25-V及其在小麦中的同源基因TaMYB25受白粉菌诱导后均上调表达,推测MYB25-V可能参与调控小麦白粉病抗性。[结论]本研究成功构建了白粉菌诱导的簇毛麦酵母双杂交文库,为鉴定CMPG1-V的互作蛋白并解析其抗病性机制奠定了基础。[Objectives]This study aimed to construct the yeast-two-hybrid(Y2H)library of Haynaldia villosa induced by Blumeria graminis f.sp.tritici using Mate&PlateTM technique.The library will provide important platform for identifying the interaction proteins and elucidating the resistance mechanism regulated by E3 ubiquitin ligase protein CMPG1-V that positively regulates wheat powdery mildew resistance in H.villosa.[Methods]Total RNA of H.villosa leaves at 0,1,2,4,6,12,24 and 48 h after inducing with Blumeria graminearum f.sp.tritici(Bgt)was extracted,mRNA was isolated and purified by SMART technique and used for ds cDNA synthesizing.The ds cDNA together with the vector pGADT7-Rec was transformed into the yeast strain Y187.The two-hybrid library was constructed,and the inserted sizes of the cDNA fragments and library titer were evaluated.Y2H library screening was performed using CMPG1-V as a bait,and representative candidate interaction proteins were verified.BiFC further confirmed in vivo interaction between CMPG1-V and a candidate protein CMIN6(MYB25-V).[Results]A Y2H library of H.villosa leaves induced by Bgt was constructed.The library titer was 9.5×107 CFU·mL^-1,the size of insert fragments was 250-2000 bp,and the recombination rate was 100%.Y2H library screening using CMPG1-V as a bait identified 79 candidate interaction proteins.Ten of them were randomly selected for validating their interaction with CMPG1-V by Y2H.In vivo interaction between MYB25-V and CMPG1-V was confirmed by BiFC.Expression analysis showed that MYB25-V and its wheat homologue TaMYB25 were up-regulated in response to Bgt inoculation,indicating the potential involvement of MYB25-V in regulating powdery mildew resistance.[Conclusions]A yeast two-hybrid library of H.villosa leaves induced by Bgt was constructed.This library provides useful platform for identifying CMPG1-V interaction proteins and elucidating the powdery mildew resistance mechanism,especially mediated by CMPG1-V.

关 键 词：小麦白粉病 簇毛麦 酵母双杂交 CMPG1-V基因 互作蛋白 

分 类 号：Q943.2[生物学—植物学] S512.1[农业科学—作物学]