水稻细胞质雄性不育及其恢复性是水稻杂种优势利用的遗传基础。本实验室克隆了水稻野败型细胞质雄性不育基因WA352及其恢复基因Rf4，阐明了不育的分子机理，但是Rf4的分子机理还不清楚。在前期的工作中，我们通过酵母双杂交筛选到RF4的两个互作因子F-A和F-B，并利用CRISPR/Cas9对水稻野败型细胞质雄性不育Rf4单基因复育系珍汕R5(含野败细胞质和Rf4)中的F-A或F-B进行敲除，敲除突变体能导致花粉败育。本项目拟用F-A和F-B的突变体分析其功能以及与恢复途径的关系；另外，利用酵母双杂交、双分子荧光互补以及Co-IP验证F-A、F-B和RF4是否为同一个恢复复合体的成员；并通过原核表达F-A、F-B、RF4的蛋白以及体外转录WA352，利用EMSA研究F-A、F-B及RF4与WA352的结合试验。本项目将阐明恢复基因Rf4的分子机理，为进一步利用水稻杂种优势进行杂交稻育种奠定基础。

Cytoplasmic male sterility (CMS) and its fertility restoration are important genetic bases for utilization of heterosis. Recently, our team has cloned a novel mitochondrial gene, WA352, causing CMS-WA in rice and its restorer gene Rf4. WA352 causes CMS by its hijacking interaction with the nucleus-encoded mitochondrial protein COX11, However, the molecular mechanism of Rf4 is unclear. In previous works, F-A and F-B were found to interaction with RF4. we used CRISPR/Cas9 to knockout the F-A or F-B gene in ZSR5 caused pollen abortion, indicating that F-A, F-B might work alone with Rf4 to restore fertility in rice. In this study, we want to analyze the biological functions of the F-A and F-B by genetic transformation. Furthermore, yeast two-hybrid assays, BiFC and Co-IP would be carried out to confirm whether the F-A, F-B and Rf4 work as a complex and EMSA would be performed to test whether F-A, F-B and RF4 interact with WA352 transcripts directly. Through this project, we aim to elucidate the molecular mechanism of Rf4, which may offers opportunities to further exploit rice heterosis in hybrid rice production.