芥菜oxa胞质雄性不育基因的发掘与功能解析

Cytoplasmic male sterility (CMS) is one of the major approach of heterosis utilization in Brassica juncea. Previously, cytological and molecular methods have been performed to characterization the oxa CMS phenotype. It was found that the oxa CMS is significantly distinguished from any other CMS type that has been reported in cruciferous crops. However, the CMS gene that is responsible for the male sterility in oxa CMS line is urged to be identified. And the mechanism of oxa CMS are largely unknown in Brassica juncea. In this project, differential mitochondrial genes or ORFs between the sterile line and its maintainer through sequencing are planning to obtain by comparing the two mitochondrial genomes. The male sterility associated genes will be selected for further study. The candidate gene will introduced into Arabidopsis and B. juncea by the transgenic modification methods for functional analysis; To reveal the mechanism underlying infertility of oxa CMS, yeast two hybrid technology will be used to identify the proteins that interact with the CMS protein. Subsequently, Pull-down and BiFC methods will be further used to confirm the protein interaction. By implementation of this project, we should not only identify the sterile gene of oxa CMS in B. juncea, but also clarify its structure and function. Morever, the mechanism of oxa CMS in B. juncea should be preliminarily illustrated. This research may help to promote the use of this novel oxa CMS in heterosis and increase its application in the breeding project about cruciferous vegetables. For this reason, this project may have great theoretical value, economic worth and broad application future.

细胞质雄性不育（CMS）是芥菜杂种优势利用的主要途径之一。本课题组前期研究发现，芥菜oxa CMS无论是在细胞学水平还是在分子水平上均与已报道的十字花科作物细胞质雄性不育类型不同，为一种新的细胞质雄性不育源，但其不育基因的功能及其作用机制尚不明晰。本研究拟以芥菜oxa胞质不育系、保持系和恢复系为材料，通过线粒体基因组测序与构图，并结合转录本分析，筛选并验证不育系和保持系之间的差异基因或者ORFs，克隆获得不育候选基因。转化拟南芥和芥菜验证该不育基因的功能，并通过酵母双杂交、Pull down、BiFC等技术手段分离和验证其互作蛋白。本项目的实施，将获得新的芥菜细胞质雄性不育基因，明确芥菜oxa CMS不育相关基因的结构和功能，初步揭示该不育基因的作用机制，促进新型细胞质雄性不育源在十字花科蔬菜杂种优势的利用，具有重要的理论意义和应用前景。

细胞质雄性不育/育性恢复 (CMS/Rf) 系统是利用杂种优势的主要途径，目前广泛应用于大田作物和蔬菜作物的育种中。本研究以实验室创制的芥菜oxa胞质不育系、保持系和恢复系为材料，利用高通量测序技术、投射电镜、扫描电镜、qRT-PCR及生理生化检测等方法开展研究，并取得以下重要进展:（1）芥菜oxa CMS不育系201A线粒体基因组大小为 219,775bp,GC含量45.24%，含有99个基因，基因总长占基因组的29.19%；芥菜oxa CMS 不育系201A叶绿体基因组大小为153, 483bp,GC含量36.36%，含有135个基因，其中编码蛋白基因有89个、核糖体RNA基因有8个、转运RNA基因有38个。（2）对花蕾中28个线粒体基因进行表达分析，结果表明复育系201A(Rf)相对不育系201A 表达上调2倍以上基因有cox2-1、cox3、nad3、atp8、rps3，其中rps3上调表达4.8倍。这表明rps3基因的差异表达可能影响花粉育性。（3）通过芥菜oxa CMS花蕾活性氧鉴定和花药细胞中H2O2亚细胞定位，证实不育系花粉败育时期，在小孢子和绒毡层细胞内的线粒体周围和内部H2O2大量积累，导致线粒体的大小和形态异常，进而对小孢子的发育产生一定影响。（4）细胞学实验表明不育系在花药发育的第10期小孢子出现异常，大部分小孢子呈畸形皱缩，处在三核期的小孢子几乎不存在，表现为细胞核的消失。（5）构建了F2遗传分离群体，利用BSA-seq 技术和常规图位克隆相结合的方法，在芥菜A05染色体上23-kb的区域内定位了一个新的 Rf基因BjuA017917。该基因编码一个鸟苷核苷酸解体抑制物 (GDI)的非PPR蛋白，被确定为控制芥菜oxa CMS育性恢复的候选基因。