萝卜NWB细胞质雄性不育关键基因MS1的功能解析

NWB CMS is one of the most important male sterile cytoplasms and is extensively used for the production of hybrid seeds in radish. In our previous research, the inheritance pattern, cytological examination and related gene of NWB-CMS have been studied. Based on the mitochondrial genome sequencing, we found that MS1 was the key candidate gene determining the male sterility in NWB CMS. In this project, we plan to apply the further functional resolution of MS1 at transcriptome level, transgenosis level, regulation level and cellular level. RT-PCR and Western blot strategies will be applied to detect the expression profiles of MS1 and to clarify the space and time distribution of this gene. Then the expression vector, RNAi and overexpression of MS1 will be constructed to verify the biological function. Y2H system and co-immunoprecipitation experiment will be applied to discover the protein interactions between MS1 and the related transcription factors. The subcellular localization will be performed to clarify the spatial distribution in cell level. The clarification of the biological function of the NWB-CMS gene MS1 will make the foundation for further research of the gene network mechanism of male sterility and for efficient precision male sterility breeding.

萝卜NWB细胞质雄性不育（NWB-CMS）是最具应用潜力的不育源。本课题组在国内率先开展了NWB-CMS不育性遗传规律、花器官形态、败育方式和控制基因的研究。线粒体基因组测序发现MS1基因是控制NWB细胞质雄性不育的关键候选基因。本项目拟从基因表达、转基因、调控和细胞水平上对MS1基因进行功能解析。首先利用RT-PCR和Western blot开展基因表达研究，明确MS1基因控制NWB-CMS的时空分布；构建RNAi和过表达载体借助萝卜遗传转化系统验证候选基因的功能；利用酵母双杂交、免疫共沉淀技术筛选与MS1互作的蛋白因子，并通过亚细胞定位技术明确MS1基因在小孢子发育过程的表达模式和位置。萝卜MS1基因的功能解析，将为深入探索NWB-CMS形成的分子机制和杂种优势利用奠定重要的理论基础。

萝卜NWB细胞质雄性不育（NWB-CMS）是韩国育种家发现的最具应用潜力的不育源。与传统的Ogura-CMS相比，NWB-CMS具有败育彻底、配合力高、花器官大、蜜腺发达等优点，已经广泛应用于萝卜杂交制种。但关于NWB-CMS的败育机理尚无报道。本课题组在国内率先开展了NWB-CMS不育性遗传规律、花器官形态和败育方式的研究，并通过线粒体基因组测序发现：MS1（orf463a）是控制NWB细胞质雄性不育的关键候选基因。本项目在前期研究基础上，深入解析MS1引起雄性不育的功能。项目首先利用RT-PCR和Western blot开展基因及蛋白表达研究，研究结果明确了orf463a基因及蛋白主要在花蕾表达，且在单核小孢子期之后，基因表达及蛋白含量均高于单核小孢子期之前。这与组织学观察到的败育是发生在单核小孢子期相一致。拟南芥遗传转化实验证明orf463a确实能够引起雄性不育。免疫沉淀-质谱联用、酵母双杂交筛选表明ATP1是ORF463a的互作蛋白。此外，亚细胞定位实验将ORF463a定位在线粒体上。萝卜orf463a基因的功能解析，将为深入探索NWB-CMS形成的分子机制和杂种优势利用奠定重要的理论基础。