红莲型水稻育性恢复复合体RFC的鉴定及机理研究

Cytoplasmic male sterility (CMS) and restoration of fertility (Rf) are widely spread in plant kingdom and make important contributions to the utilization of crop heterosis. Massive studies show that the cytoplasmic male sterility gene is located in the mitochondrial genome, and fertility restoration is completed by the nuclear encoded restoration of fertility gene. Therefore, CMS/Rf system is also an excellent material to get insight into the nuclear-cytoplasmic cross-talking. Our lab cloned the cytoplasmic male sterile gene orfH79 and restoration of fertility genes Rf5, Rf6 for Honglian (HL) hybrid rice. Furthermore, we raised the conception of restoration of fertility complex, (RFC), and identified a number of important genes involved in the pathway, but other factors are still not clear. Based on previous studies, this project aims to identify and characterize the candidate interaction factors through subcellular localization, fluorescence quantitative PCR, pulldown, far-western, SPR, EMSA, yeast two hybrid analysis, RNAi, CRISPR-Cas9 and so on. We try to analyze the interaction between the various protein analysis, and provide a theoretical basis for the construction of restoration of fertility complex. Among these candidate genes, an important factor with RNA endonuclease activity were obtained in previous research. Therefore, we will use EMSA, Primer Extension and other methods to elucidate the RNA cleavage activity for HL-CMS rice. It will provide a new insight into the molecular mechanism of RNA cleavage in mitochondria. This research not only benefits to the research and application of Honglian type hybrid rice, but also guides the mechanism research of restoration of fertility in other crops.

植物细胞质雄性不育与育性恢复广泛存在于自然界，在农作物杂种优势利用上做出重要贡献。细胞质雄性不育基因位于线粒体基因组而育性恢复是由细胞核编码的恢复基因完成，因此也是研究核质互作的绝佳材料。课题组克隆了红莲型不育基因orfH79和恢复基因Rf5、Rf6，发现了恢复基因分子复合体，鉴定出通路中多个重要基因，然而其他互作因子仍不清楚。本项目对前期获得的互作因子进行深入研究，通过亚定位、qPCR、pulldown、far-western、SPR、EMSA、酵母双杂、RNAi、CRISPR等方法剖析候选基因生物学功能，解析各因子互作关系，初步构建恢复基因复合体模型。重点关注前期研究获得的剪切因子RFC5，拟利用EMSA、Primer Extension等方法验证其核酸内切酶活性，为阐明线粒体中RNA剪切分子机制奠定基础。本研究既有利于红莲型杂交稻的研究与应用，更有助于指导其他农作物育性恢复机理研究。

植物细胞质雄性不育与育性恢复广泛存在于自然界，在农作物杂种优势利用上做出重要贡献。细胞质雄性不育基因位于线粒体基因组而育性恢复是由细胞核编码的恢复基因完成，因此也是研究核质互作的绝佳材料。课题组克隆了红莲型不育基因orfH79和恢复基因Rf5、Rf6，发现了恢复基因分子复合体，鉴定出通路中多个重要基因，然而其他互作因子仍不清楚。本项目对前期获得的互作因子进行深入研究，通过亚定位、qPCR、pulldown、far-western、SPR、EMSA、酵母双杂、RNAi、CRISPR等方法剖析候选基因生物学功能，解析各因子互作关系，初步构建恢复基因复合体模型。重点关注前期研究获得的剪切因子RFC5，拟利用EMSA、Primer Extension等方法验证其核酸内切酶活性，为阐明线粒体中RNA剪切分子机制奠定基础。本研究既有利于红莲型杂交稻的研究与应用，更有助于指导其他农作物育性恢复机理研究。

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