萝卜细胞质雄性不育育性恢复新机制的分子解析

Through near 10 years of hard work, we identified a unique radish (Raphanus sativus L.) system composed of cytoplasmic male sterility (CMS) and fertility restoration (Rf): loss of function of nuclear gene results in fertility restoration for a new CMS. Using the Next-Generation Genome Sequencing technology, we found that there is no difference between chloroplast genomes of the male sterile line and the male maintain line. However, compared with the male maintain line, the mitochondria genome of the male sterile line contains an extra gene RsorfA. Molecular analysis showed that RsorfA is causative for the identified CMS. Also based on the Next-Generation Genome Sequencing technology, we got draft sequences of the genomes of the male sterile and the fertility restorer lines and identified plenty of polymorphisms. Then, we developed a lot of CAPS (Cleaved Amplified Polymorphic Sequences) and dCAPS (derived CAPS) markers. Using a large backcrossing segregating population composed of 25000 individual plants from the cross between the male sterile line and the fertility restorer line, the new fertility restorer locus RsRf4 was defined into a 3kb DNA region. RT-PCR identified a single gene in the region. Transgenic function complementation test confirmed that the fertility restoration is controlled by the RsRf4 locus. In order to explore the mystery of the new CMS/Rf system, we are going to apply multi-molecular methods to illustrate the molecular mechanism of the fertility restoration to the new CMS. The epigenetic modulation of the RsRf4 locus will be tested by RNA polymerase II (Pol II) and H3K4me3 chromatin immunoprecipitation (ChIP). Proteins interacting with RsRf4-2 from the male sterile line will be identified using HA and FLAG Tag purification system combined with LC-MS/MS analysis. Then the candidate interacting proteins will be checked by the bimolecular fluorescence complementation (BiFC) technique. We will also study the spatial and temporal expression of the RsRf4-2 gene using tissue in situ hybridization method and test whether the RsRf4-2 protein is targeted into the mitochondria. The unique features of the new CMS/Rf system will provide novel insights into the conflict/reconciliation between mitochondria and nuclei in plants. Meanwhile, these cloned genes will be potentially applicable for hybrid production of Brassicaceae plants and could have a significant impact on agriculture.

经过近十年的研究积累，申请者鉴定并构建出一份重要植物种质，即新颖的萝卜细胞质雄性不育(CMS)及育性恢复系统：核基因功能缺失导致CMS育性恢复。通过基因组测序及功能验证，解析获得了植物新CMS基因RsorfA。并精细定位了新育性恢复位点RsRf4，结合转基因功能验证，获得了RsRf4的分子特征。为了深入解析该系统的育性恢复分子机理，本项目拟用RNA聚合酶II和H3K4me3的抗体结合染色质免疫共沉淀(ChIP)技术解析新育性恢复位点RsRf4表观遗传调节式样。采用蛋白质纯化、液相色谱-质谱/质谱及双分子荧光互补技术解析核不育等位基因RsRf4-2编码蛋白的互作因子。同时，用原位杂交技术明确RsRf4-2的时空表达式样及线粒体定位特征。对新CMS的独特育性恢复系统分子机制的深入剖析，将深化植物CMS及育性恢复多样性机理的了解，并为该系统在十字花科作物中的杂种优势育种应用提供强大的理论基础。

解析了新育性恢复位点RsRf4的分子特征。发现育性恢复位点存在插入-缺失现象和启动子区交换现象，从而导致育性的转换。用RNA聚合酶II和表观遗传标记结合ChIP技术分析了新育性恢复位点表观遗传特征。采用蛋白质纯化、液相色谱-串联质谱及双分子荧光互补技术得到了RsRf4-2编码蛋白的互作蛋白。通过切片技术明确了所用材料的花药发育时期与花蕾的长度对应关系，明确了RsRf4-2的时空表达式样，同时发现该蛋白定位于线粒体。这些结果拓宽了对植物育性恢复多样性的认识。部分研究结果发表于国际重要期刊Molecular Breeding(2篇)和International Journal of Molecular Sciences上。