逆境胁迫下水稻异三聚体G蛋白下游受体效应器的鉴定与功能分析

异三聚体G蛋白通过膜内效应器蛋白质将胁迫信号从膜外传到膜内而促使生物体产生应激反应。但逆境胁迫下水稻异三聚体G蛋白的下游受体－效应器的鉴定与功能分析研究尚不多见。本项目首先应用生物信息学方法从水稻全基因组中筛选出异三聚体G蛋白的互作蛋白质；然后运用双分子荧光分析技术、Far Western-blotting和一对一酵母三杂交技术验证逆境胁迫下水稻异三聚体G蛋白与筛选到蛋白质间的互作，确认水稻异三聚体G蛋白的效应器蛋白质；再从异三聚体G蛋白与效应器蛋白质的共表达、效应器蛋白质活性及其代谢产物的含量，分析效应器蛋白质的功能；最后对经确认的候选效应器蛋白质进行亚细胞定位。目标是鉴定5-6个水稻异三聚体G蛋白的效应器蛋白质，从效应器蛋白质阐明异三聚体G蛋白在水稻抗逆反应中的作用机制.

Heterotrimeric G proteins function as signal mediators in the transduction of numerous external signals in planta. In the canonical heterotrimeric G-protein signaling paradigm, G-protein-coupled receptors (GPCRs) activate Gα and Gβγ dimer of heterotrimeric G proteins, the activated Gα and the Gβγ dimer (freely released) in turns activate downstream effector proteins. However, the effector proteins of heterotrimeric G-proteins in rice are still not well documented. In this study, we will use computational and wet-bench methods to identify several effector proteins of rice heterotrimeric G protein, and analyze the function of effector proteins in response to environmental stresses. First, using diverse computational methods and yeast-two-hybrid, we will perform whole-proteome screening of effector protein candidates, which physically interact with the intracellular subunit of heterotrimeric G protein in rice. Second, the effector proteins will be confirmed by Far western-blotting and one-to-one yeast-two-hybrid. Third, the co-expressing of effector proteins and heterotrimeric G proteins, the activities of effector proteins and their products will also be analyzed. Last, the subcellular localization of effector proteins will also be achieved by using protein localization methods. These results would provide new insight into the regulatory mechanism of heterotrimeric G-protein in rice under environmental stresses.