棉花细胞质雄性不育恢复基因的克隆与分析

CMS/Rf systems provide both a practical tool for facilitating hybrid seed production and an ideal model for investigating nuclear-cytoplasmic interaction. Although CMS three lines have been developed in cotton, the molecular mechanism of male sterility and fertility restoration, and its application in hybrid seeds production are still lagging behind. In this research, the three-line Upland cotton containing CMS-D2 (Gossypium harknessii) cytoplasm were used for fine mapping, cloning, and functional analysis of the key factor Rf1. Combined second- and third-generation sequencing technologies, the restorer line was used to sequence and assemble, mainly focusing on analysis of the target region of the restorer gene. Through the screening of SNPs, InDel and other polymorphic markers, with the published results of Rf1 mapping and the large segregation population to achieve fine mapping of Rf1. The transcriptome and other results were then used to obtain the candidate gene. After clarifying the expression patterns, subcellular localization and allelic diversity, VIGS and Agrobacterium-mediated transformation were used to verify gene function, then yeast two hybrid and BIFC were used to study the interaction between male sterile related gene and Rf1. This study will lay a foundation for revealing the molecular mechanism of CMS infertility and fertility recovery in cotton, thus expanding the understanding of molecular mechanisms in the field of plant nuclear-cytoplasmic interaction and promoting application of simplified seed production technique in the production of cotton hybrids.

细胞质雄性不育和恢复系统可以实现杂交种简化制种，已成为作物杂种优势利用的主要途径，同时也是研究核质互作的理想材料。棉花虽已实现三系配套，但不育及育性恢复分子机理研究及其生产应用仍显滞后。本研究将以含哈克尼西棉不育胞质的陆地棉三系为材料，针对其应用的核心-恢复基因Rf1开展精细定位、克隆和作用机制等研究。主要利用正在开展的三代加二代恢复系测序和组装，重点对Rf1目标区间分析，通过SNP和InDel等多态性标记的筛选，根据前期定位和分离大群体，实现精细定位；结合已有的转录组等获得候选基因，在明确其表达模式、亚细胞定位和等位位点差异后，通过VIGS和农杆菌介导的遗传转化明确基因功能，并利用酵母双杂交和双分子荧光互补等初步探讨其与不育相关基因的互作。本研究将为揭示棉花CMS不育及育性恢复分子作用机理奠定基础，拓展植物核质互作领域分子机理的了解，并推动三系简化制种技术在棉花杂交种生产中的应用。

细胞质雄性不育和恢复系统可以实现杂交种简化制种，已成为作物杂种优势利用的主要途径，同时也是研究核质互作的理想材料。棉花虽已实现“三系”配套，但细胞质雄性不育及育性恢复分子机理研究及其生产应用仍显滞后。本研究以哈克尼西棉细胞质雄性不育“三系”为材料，针对其应用的核心-恢复基因Rf1开展定位、克隆和作用机制等研究，在前期研究基础上，完成了恢复系基因组组装分析，在候选区间内开发了能同时鉴定恢复基因Rf1和Rf2的紧密连锁分子标记InDel1892，并在遗传群体进行了遗传分析，该标记目前已被用于恢复系分子标记辅助育种。利用三代Pacbio全长转录组分析，结合前期Illumina二代转录组数据，筛选出了2个与棉花育性恢复相关的候选基因GhCCS772和GhCCS61，两个基因在恢复系中特异表达，并位于目标染色体D05目标区间，VIGS技术对其分子功能进行了初步验证，对育性有一定影响。此外，鉴定出哈克尼西棉细胞质雄性不育系（CMS-D2）携带的线粒体不育相关基因orf610a，并初步解析了不育系花粉败育的分子机制，并筛选到能与其互作的蛋白RD22，不育基因-RD22-恢复基因直接是否存在一定关联的研究目前正在进行中。通过本研究，为明确恢复基因功能以及进一步揭示棉花细胞质雄性不育恢复基因与不育基因核质互作分子机理奠定了基础，同时对生产上快速分子选育恢复系和“三系”杂交种具有重要实践意义。