miR167-OsARF8调节水稻籽粒灌浆充实的作用机理研究

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.

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。

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。本项目在前期试验基础上，明确了STTM/MIM167转基因植株千粒重、平均灌浆速率和最大灌浆速率较野生型显著增加，大田试验研究结果表明，低表达miR167植株较野生型产量提高幅度为12.22%-25.61%；分析了miR167及4个ARFs靶基因的表达模式和过量表达植株千粒重，结合4个ARFs靶基因进化树分析对miR167调节水稻籽粒灌浆充实主效靶基因进行鉴定，确定了miR167主要通过负向调节OsARF12（与申请书中的OsARF8为同一基因）调节水稻籽粒灌浆充实；进一步通过水稻原生质体亚细胞定位试验证明OsARF12定位于细胞核，符合一般转录因子定位。采用组成型启动子过量表达OsARF12转基因植株(OXARF12)互补STTM/MIM167植株籽粒变大及灌浆速率增加的表型，利用谷蛋白特异启动子低表达OsARF12转基因植株(RNAiARF12)籽粒千粒重及灌浆速率呈现相反趋势，进一步证明了miR167通过负向调节OsARF12调节水稻籽粒灌浆充实。为解析miR167-OsARF12调节水稻籽粒灌浆充实分子机制，对OXARF12花后5天籽粒进行RNA-seq分析，发现生长素和BR生物合成及信号转导通路基因在差异基因中得到显著富集，与STTM/MIM167植株花后5天籽粒中参与生长素和BR代谢通路基因发生显著改变的结果吻合，且OXARF12植株在BR处理后表现超敏感；OsARF12融合GFP标签蛋白植株幼穗ChIP-seq试验结果表明响应激素信号和细胞调节等相关基因得到显著富集。进一步采用酵母单杂、ChIP-qPCR、荧光素酶活性试验，验证了OsARF12通过OsCDKF.2调节胚乳细胞分化，进而调节水稻的灌浆充实。综上，miR167-OsARF12可能通过调节生长素和BR信号转导和胚乳细胞分化，进而控制水稻籽粒灌浆充实。