miR167-OsARF8调节水稻籽粒灌浆充实的作用机理研究

水稻籽粒灌浆充实的好坏决定了稻米产量的高低和品质的优劣。申请人前期研究已证明，在水稻胚乳中低表达miR167显著增加籽粒的千粒重和灌浆速率，其靶基因OsARF8高表达表现为包括籽粒增大在内的典型BR增强表型。本项目拟在此基础上，借助ChIP-seq、酵母双杂交、酵母单杂交、EMSA、BiFC等生化实验，开展OsARF8下游调节基因的鉴定和互作蛋白的筛选，结合各转基因株系籽粒灌浆过程中生长素和BR合成及信号转导相关基因的表达，探讨miR167-OsARF8调节水稻籽粒灌浆充实的分子机制；进一步检测各转基因株系籽粒的灌浆动态、胚乳细胞的分化和增殖过程、籽粒灌浆过程中生长素和BR的含量，解析miR167调节水稻籽粒灌浆充实的生理机制，阐明miR167-OsARF8介导的生长素和BR信号转导调节水稻籽粒灌浆充实的作用机理，为利用分子改良育种改善水稻籽粒的灌浆充实，提高水稻的产量和品质提供技术支撑。

Grain weight and quality are always determined by the grain filling. Our previous research proved that brown rice weight and grain filling rate of miR167 lower expressed transgenic lines (MIM167 and STTM167) were significantly increased compared with that of wild-type plant. Consistently, BR enhance phenotypes, including larger grain size was observed in one of miR167’s target, OsARF8 overexpressed transgenic rice lines. In this project, ChIP-seq, Yeast two-hybrid, Yeast one-hybrid, EMSA, BiFC and other techniques will be performed to screen and identify the down-stream regulating genes and interaction proteins of OsARF8, combined with the expression of auxin and BR biosynthesis and signaling transduction related genes in the filling rice grains, to explore the molecular mechanism of miR167-OsARF8 module on rice grain filling; followed by detecting the grain filling related parameters, process of endosperm cells proliferation and differentiation, auxin and BR content in the filling rice grains of these transgenic lines, to study the physiological mechanism of miR167-OsARF8 module in regulating rice grain filling, then illuminated the mechanism of miR167-OsARF8 module mediated auxin and BR signaling transduction on rice grain filling. By explaining the mechanism of how miR167-OsARF8 module in regulating rice grain filling, it will be helpful to provide theoretical guide for rice yield and quality improvement by means of molecular breeding.