水稻细胞质雄性不育相关基因orf290的作用机理研究

Cytoplasmic male sterility (CMS), a universal phenomenon characterized with pollen sterility which is caused by dysfunction of mitochondria in plant kingdom. Discovery and exploration of plant CMS greatly push the utilization of heterosis of crop plants. However, the complexity of mitochondrial genome caused by frequent rearrangement of repeat sequence, bring us great challenge to our understanding which and how to control the occurrence of CMS. . Rice, one of the most important cereal crop plants, is presented as the model of plant heterosis exploration based on a great varieties of CMS. Generally, rice CMS can be categorized into three types, i.e. Wild-abortive type, Honglian type and Botai type. Interestingly, CMS-related alleles of orf79 and orf(H)79 were detected in Honglian and Baotai CMS lines respectively, and were suggested as their respective CMS genes. However, this discovery make us confused, and it is hard for us to make clear the difference of male sterile mechanism between these two CMS systems at molecular level.. In order to further understand the mechanism of Honglian CMS, we sequenced the mitochondrial genome of Honglian CMS line, and found a new chimeric gene named orf290, which is homological to the WA-CMS related gene orf352, and express mainly in the anthers. Importantly, the transcripts of orf290 in the hybrid rice lines are cleaved by the restorer genes Rf5 and Rf6, ectopic expression of orf290 can lead to male sterility. In this study, we employed approaches of genetics, cytology and molecular biology to elucidate how orf290 control the pollen sterility in Honglian CMS lines.

细胞质雄性不育是由线粒体基因突变或重组所导致的一种高等植物雄蕊发育异常的表现，它是作物杂种优势利用的基础。植物细胞质雄性不育的发现，极大地促进了农作物杂种优势的开发利用。但植物线粒体基因组由于大量重组，导致结构变异巨大，为细胞质雄性不育研究带来了巨大挑战。水稻细胞质雄性不育分为野败型、红莲型和包台型三种遗传不同类型，前期研究表明，红莲型和包台型CMS都携带有orf(H)79，二者都与atp6共转录，然而这不足以阐释红莲型和包台型CMS的遗传差异。为了进一步阐明红莲型CMS的机理，我们测序了红莲型CMS线粒体基因组，发现一个新的嵌合基因orf290，它与野败型CMS基因orf352同源，受恢复基因Rf5/Rf6的调控，且异位表达能导致小孢子败育。因此，本研究拟借助细胞、分子、遗传等手段，解析该基因的功能及其与CMS的关系。

本研究综合运用免疫组化、扫描电镜、透射电镜、冷冻电镜、同位素示踪结合质谱等细胞和分子生物学技术手段，比较分析红莲型水稻不育系和保持系花药、小孢子发育过程的细胞学特征，线粒体电子传递链复合物的结构、线粒体蛋白质组的转换规律和小孢子胞壁代谢的分子生物学研究。对红莲型水稻不育系和保持系线粒体蛋白质组的转换特征分析发现，不育系ETCIII中核心亚基C9和C10与ORFH79的转换速率类似且明显高于保持系，而其他蛋白质在不育系和保持系之间的转换速率基本一致。与此相对应的是，在红莲型水稻不育系线粒体电子传递链复合物ETCIII的结构中发现ORFH79亚基的存在，且在不育系ETCIII上形成一个尾状结构，导致ETCIII的结构改变，活性显著下降。同时，发现红莲型雄性不育水稻成熟期小孢子萌发孔内陷，接近成熟时，花粉内壁发育不完整，这可能是导致红莲型水稻小孢子雄性不育的关键。