应用GST pull-down技术筛选水稻抗稻瘟病蛋白PID3互作蛋白的研究  

Screening of rice proteins interacting w ith the rice blast resistance protein PID3 byST pull-dow n

作　　者：赵文[1,2] 王静[2] 李伟滔[1] 王静[1] 李晓兵[2] 朱立煌[2] 陈学伟 

机构地区：[1]四川农业大学水稻研究所,成都611130 [2]中国科学院遗传与发育生物学研究所,北京100101

出　　处：《植物病理学报》2015年第5期476-485,共10页Acta Phytopathologica Sinica

基　　金：国家转基因重大专项(2013ZX08009-001;2014ZX0800903B);国家重点实验室开放项目(2011A0525-02)

摘　　要：Pid3是从水稻中克隆获得的编码CC-NBS-LRR类蛋白的稻瘟病抗病基因。为了更深入地研究Pid3编码蛋白PID3介导的稻瘟病抗性的分子调节机制,我们应用GST pull-down结合质谱技术,对水稻抗稻瘟病蛋白PID3的互作蛋白进行了鉴定和分析。首先,成功构建了带有GST标签且能够表达PID3的CC-NBS结构域的融合蛋白表达载体p GEX6P1-PID3CC-NBS,经诱导表达和GST beads纯化后获得GST-PID3CC-NBS融合蛋白。我们以Pid3的转基因水稻纯合株系Pid3-TP309为研究材料,分别用对其致病和不致病的稻瘟病菌生理小种ZB13和ZHONG-10-8-14接种,然后取叶片分别提取获得两组总蛋白。用纯化后的蛋白GST-PID3CC-NBS分别与两组总蛋白共培养后,利用GST pull-down技术获得了分别在抗病、感病反应中能与PID3CC-NBS互作的两组候选蛋白。通过质谱技术鉴定这些蛋白的氨基酸序列,比对分析其在抗病、感病途径中与PID3结合的差异性,我们共筛选出31个只在PID3介导的抗稻瘟病反应中能与其特异结合的PID3互作蛋白,这些互作蛋白包括受体激酶、LRR类蛋白、ATP酶、磷酸羧化酶和离子通道蛋白等,广泛参与了调控细胞程序化死亡、胁迫防御反应、物质与能量代谢和信号传导等多个生物学过程。本研究探寻了PID3介导的抗病信号转导过程的关键因子,研究结果为揭示PID3介导的稻瘟病抗病分子机理奠定了良好基础。Rice Pid3, encoding a CC-NBS-LRR protein （PID3）, confers resistance to blast disease caused by Magnaporthe oryzae. However, the molecular events in PID3-mediated signaling are poorly known. Here, we report the identification of PID3 interacting proteins （P3IPs） by using GST pull-down approach followed by mass spectrometry analysis. We successfully constructed the expression vector of pGEX6P1-PID3^CC-NBS and purified the expressing protein PID3^CC-NBS with GST antibody. We then co-incubated the GST-PID3^CC-NBS protein with total proteins prepared from the Pid3 transgenic plants inoculated respectively with the incompatible blast isolate ZHONG-10-8-14 and the compatible blast isolate ZB13. The protein complexes of GST-PID3^CC-NBS were pulled down by GST antibody and then identified by mass spectrometry analysis. We compared the pulled-down proteins between the incompatible and compatible interactions of Pid3-M. oryzae and obtained 31 specific P3IPs from the incompatible interaction treatment. These P3IPs, mainly including receptor kinases, kinases, LRR proteins, ATPases, Ribulose bisphosphate carboxylases, and potassium channel AKT2/3 proteins, are involved in many molecular processes, such as signaling transduction, programmed cell death, response to stresses, and metabolism of mass and energy. Taken together, our study uncovers some key factors that regulate PID3-mediated rice disease resistance and will provide insight into NBS-LRR protein-mediated immune signaling.

关 键 词：抗稻瘟病 PID3 GST pull—down 互作蛋白 稻瘟病菌 

分 类 号：S435.111[农业科学—农业昆虫与害虫防治]