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

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.

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.

首先比较分析7种特征提取策略和4种分类器对异三聚体G蛋白下游效应器的预测效果，结果表明CKSAAP_PROSITE_200效果最佳，其准确性（ACC）和马修斯相关系数（MCC）分别达74.62% 和 0.49。据此，我们开发出异三聚体G蛋白互作蛋白质的预测工具G-Effectors，并获得15个高得分的潜在G蛋白下游效应器，其中一个恰好在前人的试验研究中得以验证。其二，项目组采用蛋白质操作技术，包括BiFC和Y2H技术，对逆境胁迫下水稻异三聚体G蛋白与高得分潜在效应器的互作进行试验验证，结果发现Gα与HSP70间的互作，进一步确认HSP70是逆境胁迫下水稻异三聚体G蛋白的下游效应器。且发现在稻瘟病菌胁迫下水稻HSP70上调表达。然后，继续挖掘异三聚体G蛋白与HSP70互作后激发表达的抗性基因。将来自15个作图群体共89个与水稻稻瘟病抗性相关的QTLs信息，应用Biomercator 4.0工具整合到水稻参考基因组图谱上，发现水稻稻瘟病抗性QTLs位点分布在第1、3、5、6、7、8和第11号染色体上，最小图距为0.8 cM，平均图距为6.7 cM，极大缩小了水稻抗稻瘟病基因的筛选范围。最后，从两种栽培稻（粳稻和籼稻）的基因组中分别鉴定出554和597个NBS-LRRs型抗性基因，MCscan工具分析发现NBS-LRR基因存在有234个的P/A (Present/Absent) 多态性，这可能是导致不同水稻品种对逆境具有不同抗性能力的原因。下一步将从中确认稻瘟病菌胁迫下由水稻异三聚体G蛋白与HSP70互作激发表达的抗性基因。