钙信号介导的细胞程序化死亡反应中SAD2蛋白的互作因子及互作机理研究

项目名称： 钙信号介导的细胞程序化死亡反应中SAD2蛋白的互作因子及互作机理研究

项目编号： No.31470367

项目类型： 面上项目

立项/批准年度： 2015

项目学科： 生物科学

项目作者： 高英

作者单位： 中国农业科学院作物科学研究所

项目金额： 80万元

中文摘要： 细胞程序化死亡(PCD)是植物防御病原菌的重要机制之一，而胞内钙离子浓度的快速升高对PCD至关重要，但二者之间调控机制尚不明确。我们筛选到一个细胞核质转运受体蛋白基因SAD2的T-DNA插入失活拟南芥突变体sad2-5，呈现出H2O2刺激的钙信号异常升高、伏马毒素B1（FB1）诱导的细胞致死和胞内H2O2异常累积表型，钙离子通道抑制剂LaCl3及H2O2还原剂谷胱甘肽GSH能够回复FB1诱导的PCD表型。Co-IP实验证明SAD2能够和转录因子MYB3、MYB32相互作用。本项目拟研究MYB3、MYB32是否为PCD反应中SAD2的作用底物，通过RNA-seq、CHIP和EMSA技术寻找与MYB3、MYB32互作的与PCD相关的下游基因，并检测这些互作基因突变体的FB1诱导表型、钙信号及二者间的关系，揭示SAD2在PCD反应中的作用机制，为改善和提高作物对不利环境的抵抗能力提供理论依据。

中文关键词： 细胞核质转运受体；SAD2；细胞程序化死亡；钙离子；过氧化氢

英文摘要： Programmed cell death (PCD) is an important mechanism of plants to defense attack of various pathogens. The cytosolic Ca2+ elevation is critical for triggering the PCD, but the regulation mechanism between PCD and Ca2+ release is not unclear. We screened out an Arabidopsis mutant sad2-5 with abnormal enhanced H2O2-specific calcium signaling, which was resulted from a T-DNA insertion in the Nucleo-cytoplasmic trafficking receptor gene SAD2. sad2-5 also showed obvious FB1-induced cell death and abnormal accumulation of H2O2. Lanthanum chloride (LaCl3), a broad spectrum calcium channel blocker, and H2O2 reducing agent GSH can restore the FB1 induced PCD phenotype of sad2-5. Co-IP experiment proved that SAD2 can interact with transcriptional factors MYB3 and MYB32. Based on above results, this project aims to detect whether MYB3 and MYB32 are the direct target of SAD2 during the PCD. Further, we will seek the MYB3 and MYB32-regulated downstream genes involved in the PCD through RNA-seq and Chromatin immunoprecipitation (CHIP) approaches, and test their in vitro interaction with MYB3 and MYB32 by electrophoretic mobility shift assay (EMSA). Also we detect their FB1-induced PCD phenotype, H2O2-specific calcium signaling and their relationship. Thereby, a comprehensive understanding on the mechanism of SAD2 functioning in the PCD is to be achieved. The findings will provide important theoretical basis for improving and enhancing the resistance of crops to adverse environments.

英文关键词： Nucleo-cytoplasmic trafficking receptor;SAD2;PCD;Ca2+;H2O2