棉花细胞质雄性不育基因的鉴定与功能分析

The cytoplasmic male sterility (CMS) is widespread in plant, and some genes and orfs of plant mitochondrial genome are involved in the sterility and sterile recovering in many plants. Cotton is one of the crops that have heterosis and can be used CMS system to develop hybrids but the molecular mechanism of sterility remains unclear. To uncover the molecular mechanism of cytoplasmic male sterility in cotton, our research will base on mitochondrial genome sequences newly obtained. Comparing the completely mitochondrial genome sequences of CMS lines, maintainer and restorer lines of upland cotton, we aim to identify cytoplasmic male sterility-related candidate gene and proteins in cotton, combining with analyzing the mitochondrial transcriptome, mitochondrial proteome. The CMS associated genes/orfs are further targeted, and the candidate CMS-associated genes/orfs with complete reading frame are constructed into mitochondria targeted vectors for transformation to validate the functions. Using the information of transcriptome as well as mitochondrial genomes, we analyze the function of regulation molecules, such as lncRNA and miRNA, and how these molecules regulate the CMS-associated genes/orfs, then to select the target candidate genes and using to construct vectors, and to transform cotton plants; Based on the evaluation results on the positive transformed plants and the offspring, we try to explore the function of sterile genes, and to outline the metabolism regulatory network in cotton CMS line, and to clarify the molecular mechanism of cotton cytoplasm male sterility.

植物细胞质雄性不育现象普遍存在。研究证实，线粒体编码基因的表达参与细胞质雄性不育形成的调控。棉花杂种优势显著，能够利用细胞质不育系培育三系杂交种，但其雄性不育的分子机制仍不清楚。本研究利用棉花细胞质雄性不育三系材料，基于三系线粒体基因组分析，确定候选基因/orf；结合线粒体转录组差异表达分析，进一步缩小候选基因范围，对候选基因构建线粒体靶向载体进行遗传转化及转基因验证。同时，利用转录组测序、lncRNA和miRNA测序数据，结合线粒体蛋白质组分析，筛选棉花细胞质雄性不育形成相关候选基因和蛋白，确定不育基因转基因验证其功能；根据转基因后代表现，分析不育相关基因表达及调控机制，构建棉花细胞质雄性不育分子代谢调控网络，阐明棉花细胞质雄性不育形成的分子机制。

研究棉花细胞质雄性不育机理，具有重要理论与实践意义。前期研究证实，线粒体基因组变异是造成细胞质雄性不育的重要原因，而miRNA也参与不育发生调控。本项目比较研究了哈克尼西棉胞质不育系2074A、陆地棉胞质不育系2074S、陆地棉胞质保持系2074B及哈克尼西棉胞质恢复系E5903线粒体基因组序列，发现大片段重复序列是导致四个线粒体基因组大小差异、基因含量变异及重排的主要因素。对比2074A与2074B线粒体基因组差异ORF，筛选得到育性相关orf46及orf183；功能验证发现过表达orf183烟草植株花器官异常，花药干瘪且没有花粉粒散出；过表达orf46导致烟草花期推迟、花粉粒减少。对比不育系2074A与保持系2074B花蕾miRNA表达谱，发现miR827a/GhPPR、miRn25/GhARF10、miRn75/GhAP2、miR393/GhAFB2参与调控棉花花器官发育；过表达miRn25的烟草植株表现出节间缩短、生长受到抑制、生殖生长期延迟等表型性状。对比分析2074A与2074B花蕾线粒体蛋白质组，发现大量蛋白质参与碳水化合物代谢、抗氧化胁迫反应、物质转运等生物学过程，表明能量和物质供应不足以及抗氧化胁迫能力下降是导致败育发生的主要因素。此外，分析24个植物种的内源基因转移，发现线粒体基因组在进化过程中，很多序列转移到细胞核和叶绿体基因组中，也接收了大量来自核基因组的转座子和来自叶绿体基因组的tRNA基因，这些是发生在某些共同祖先中的单一事件。此外，雷蒙德氏棉(D5)、亚洲棉(A2)、哈克尼西棉（D2-2）、三裂棉（D8）和陆地棉（AD1）的PPR基因家族基因进化分析，启动了棉花细胞质雄性不育育性恢复机制探索。