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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。
Pid3 is a blast disease resistance gene encoding CC-NBS-LRR type protein cloned from rice. In order to further study the molecular regulation mechanism of Pid3-encoding protein PID3-mediated resistance to blast, we used GST pull-down combined with mass spectrometry to identify and analyze the interaction proteins of the rice blast-resistant protein PID3. First, a GST-tagged fusion protein expression vector, pGEX6P1-PID3CC-NBS, capable of expressing the CC-NBS domain of PID3 was successfully constructed, and GST-PID3CC-NBS fusion protein was obtained after being induced and purified by GST beads. We Pid3 transgenic rice homozygous Pid3-TP309 as the research materials, respectively, to their pathogenic and pathogenicity of the Magnaporthe grisea physiological races ZB13 and ZHONG-10-8-14 inoculation, and then take the leaves were extracted Two sets of total proteins were obtained. After co-cultured with two sets of total proteins respectively using the purified protein GST-PID3CC-NBS, two sets of candidate proteins capable of interacting with PID3CC-NBS in resistance and susceptibility reactions were obtained by GST pull-down technique. The amino acid sequences of these proteins were identified by mass spectrometry. The differences in their binding to PID3 in the pathogenic and susceptible pathways were compared and analyzed. We screened out 31 of them that could specifically bind only in PID3-mediated blast resistance Of PID3 interacting proteins. These interacting proteins include receptor kinases, LRRs, ATPase, phosphorylase and ion channel proteins. They are widely involved in the regulation of programmed cell death, defense response, metabolism of matter and energy, Signal transduction and many other biological processes. This study explored the key factors of PID3-mediated resistance signal transduction, and the results laid a good foundation for revealing the molecular mechanism of PID3-mediated resistance to rice blast disease.