水稻F-box蛋白OsFBX76调控粒重的分子机理研究

水稻粒重与产量和品质密切相关。F-box参与的蛋白酶体途径在调控水稻粒形和粒重方面发挥重要作用。申请者在前期的研究中克隆了一个编码F-box蛋白的基因，命名为OsFBX76。过表达该基因，粒重变小；RNAi转基因株系及突变体株系表现为粒重增加；酵母双杂交分析表明OsFBX76可以和OSK4、OSK15、OSK20相互作用。以上结果表明OsFBX76可能作为SCF复合体组分参与调控水稻粒重形成。本项目拟在此基础上，运用双分子荧光互补、免疫共沉淀和Pull-down等生化方法进一步验证OsFBX76是否参与形成SCF复合体；进一步对OsFBX76的遗传材料进行生理生化和转录组分析，解析OsFBX76的生物学功能；利用酵母双杂交筛选OsFBX76的靶蛋白，并进行验证。明确OsFBX76通过26S蛋白酶体介导的底物蛋白降解途径调控水稻粒重的分子机制。

Rice grain yield and quality largely depend on grain weight. F-box proteins are components of E3 ubiquitin ligase S-phase kinase-associated protein, cullin, F-box containing complexes (SCF), which play an important role in grain size and weight control. Previously, a gene encoding a rice F-box protein was cloned and named OsFBX76. Transgenic rice plants overexpressing OsFBX76 showed reduced grain weight. The RNAi transgenic plants and CRISPR/Cas9 mutants showed increased grain weight. Yeast two-hybrid analysis confirmed that OsFBX76 could interact with OSK4, OSK15 and OSK20, which suggesting that OsFBX76 acts as a component of SCF complex to regulate grain weight in rice. In this project, the interaction among OsFBX76, OSK and Cullin will be confirmed by Bimolecular Fluorescence Complementation (BiFC), Co-Immunoprecipitation (Co-IP) and Pull-down assays. The biological function of OsFBX76 will be validated through multiple biochemical, physiological and transcriptomics strategies. We will also identity the target substrate of OsFBX76 using yeast two-hybrid. This study will clearly illuminate the biochemical and molecular mechanism for OsFBX76 regulating grain weight by 26S proteosome pathway.