水稻红莲型细胞质雄性不育育性恢复蛋白复合体的研究

Cytoplasmic Male Sterility (CMS) and Restoration of Fertility (Rf) is a window to the world of plant mitochondrial-nuclear interactions widely spread in plant kingdom. CMS is determined by mitochondrial genomes and the fertility also can be restored by Rf genes, which encoded by nuclear genomes. It has been successfully used in production of hybrid seeds. On the base of cloning of Rf5, we demonstrated that a Restoration of Fertility Complex (RFC) is responsible for CMS-RNA cleavage. A glycine-rich protein (GRP162) is important for RNA binding, while other factors invovled in restoration are unknown. In this project, the three genes obtained in previous study will be investigated in depth. Several methods such as transient expression, quantity RT-PCR, pull-down, SPR, EMSA, yeast two-hybrid system, RNAi, and so on will be performed to analyze the biological function of these candidate genes. These data are essential for construction of the model of RFC. Meanwhile, we design the Rf5 containing tandem affinity purification tag for isolation of RFC. Combination of the strategies of BN-PAGE and mass spectrometry we will discover the novel genes involved in this pathway, even the key factor for cleavage. This knowledge is useful for understanding of restoration and makes a profound significance for CMS and Rf pathway. Furthermore, this research would contribute to the therotical knowledge of heterosis.

由线粒体基因造成的细胞质雄性不育和由细胞核编码的恢复基因这一系统在杂种优势的利用上得到广泛应用。课题组在克隆红莲型恢复基因Rf5的基础上，发现恢复基因分子复合体(Restoration of Fertility Complex, RFC)，首次鉴定出恢复通路中的重要基因GRP162，然而其他互作因子仍不清晰，尤其是切割因子尚未鉴定。本项目是对前期获得重要互作因子进行深入研究，通过亚细胞定位、荧光定量PCR、pulldown、SPR、EMSA、酵母双杂交、RNAi等方法阐释复合体中各个候选基因的生物学功能，解析各个蛋白之间的互作关系，为构建恢复基因复合体的模型及组装提供理论基础。同时设计含不同标签的恢复基因进行转基因，通过BN-PAGE、免疫共沉淀等方法分离纯化该复合体并进行质谱鉴定，为获得切割因子等新的互作因子奠定基础。同时为阐明细胞质雄性不育与育性恢复及杂种优势的形成机理提供理论基础。

线粒体基因造成的细胞质雄性不育和由细胞核编码的恢复基因这一系统在杂种优势的利用上得到广泛应用。课题组在克隆红莲型恢复基因Rf5的基础上，发现恢复基因分子复合体(Restoration of Fertility Complex, RFC)，首次鉴定出恢复通路中除Rf5外的重要基因GRP162，然而其他互作因子仍不清晰，尤其是切割因子尚未鉴定。本项目是对前期获得重要互作因子（WD40、GI、Vatpase）进行深入研究，通过亚细胞定位、荧光定量PCR、pulldown、SPR、EMSA、酵母双杂交、RNAi等方法阐释复合体中各个候选基因的生物学功能，解析了各个蛋白之间的互作关系，为构建恢复基因复合体的模型及组装提供理论基础。同时设计含不同标签的恢复基因进行转基因，通过BN-PAGE、免疫共沉淀等方法分离纯化该复合体并进行质谱鉴定，获得了切割因子等新的互作因子。为阐明细胞质雄性不育与育性恢复及杂种优势的形成机理提供理论基础。