ZHANG Heng,CHEN Yiming,ZHANG Xu,et al.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[J].Journal of Nanjing Agricultural University,2020,43(4):594-604.[doi:10.7685/jnau.202001036]





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
张恒 陈怡名 张旭 牛影 赵佳 吴承云 郝永利 孙丽 王海燕 肖进 王秀娥
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
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 Collaborative Innovation Center for Modern Crop Production, Nanjing Agricultural University, Nanjing 210095, China
wheat powdery mildewHaynaldia villosayeast-two-hybridCMPG1-V geneinteraction protein
[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250~2 000 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 250-2 000 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.


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更新日期/Last Update: 1900-01-01