ZmDim1B基因在玉米CMS-S育性恢复中的功能研究

S type cytoplasmic male sterility (CMS-S) in maize is associated with the 1.6 kb RNA, harboring two adjacent ORFs, orf355 and orf77, encoded in mitochondrial R region. Proteins encoded by orf 355, orf77 and the orf17 derived from orf77 due to RNA editing would disturb the function of mitochondria, causing male sterility of pollen. In the fertile CMS-S maize containing the restorer of fertility, Rf3, 1.6 kb transcript is largely decreased, resulting in fertility restoration of CMS-S maize pollen. RNA degradation of 1.6 kb transcript is related to the deletion of 5'-stem loop structure of 1.6 kb RNA as a result of Rf3 action. Recently, several lines of biochemistry and molecular biology evidence suggested, 5'-stem loop structure of 1.6 kb RNA was modified by adenosine dimethylation rather than cut by RF3 protein. And the dimethylation of 5'-stem loop will be dependent on Rf3 gene. In addition, there would be no protein translated from 1.6 kb RNA. 1.6 kb RNA per se acting as a competitor of 18S rRNA might influence assembly of ribosomes, thereby suppressing the expression of mitochondrial proteins. In this project, maize mitochondrial adenosine dimethylase gene ZmDim1B will be identified, and the relationship between dimethylation of 1.6kb RNA and fertility restoration of CMS-S will be determined. Deciphering the Rf3-dependent restoration of fertility complex will facilitate understanding the mechanism underlying fertility restoration of CMS-S maize.

玉米S型细胞质雄性不育（CMS-S）同线粒体基因组R区转录的1.6 kb RNA相关，其编码orf355和orf77。orf355、orf77和后者因编辑而成的orf17表达的蛋白可能影响线粒体的功能导致雄性不育。恢复基因Rf3存在时，1.6kb转录本量被大大较低导致花粉育性的恢复。1.6 kb转录本的降解同可育花粉内1.6kb RNA 5'-茎环结构的缺失相关。目前，已有生化实验证据显示，恢复系花粉内1.6 kb转录本5'-茎环结构并非被RF3蛋白剪切而是腺苷酸双甲基化修饰，且这一修饰过程是依赖Rf3的。此外，不育基因1.6kb RNA可能不表达蛋白质，而以RNA形式干扰18S rRNA的核糖体组装，进而影响线粒体蛋白表达。本项目将克隆玉米线粒体腺苷酸双甲基化基因ZmDim1B，检测1.6kb RNA双甲基化同育性恢复的关系，解析Rf3介导的育性恢复复合体，阐明CMS-S育性恢复机理。

玉米S型细胞质雄性不育（CMS-S）与线粒体R区编码的1.6 kb RNA有关。含恢复基因Rf3的恢复系花粉内1.6 kb转录本5'-茎环结构的剪切而导致花粉育性恢复。本研究利用5'-Full RACE技术获得1.6 kb RNA的转录起始位点和5'-RNA加工位点，未发现5'-茎环结构loop处的RNA剪切位点，推测CMS-S恢复系内1.6 kb转录本5'-末端茎环结构相邻腺苷酸（AA）受到双甲基化修饰而不是RNA剪切。克隆了玉米ZmDim1B基因并利用大肠杆菌突变体互补实验证明了其腺苷酸双甲基化的分子功能。获得ZmDim1B EMS突变体，但ZmDim1B基因突变并不影响恢复系和不育系花粉的育性。用ZmDim1B筛选恢复系花粉酵母双杂交（Y2H）文库，未鉴定到与ZmDim1B蛋白互作的恢复基因候选基因，同源克隆的Rf3候选基因RF3L也不与ZmDim1B互作，说明1.6 kb转录本5'-末端茎环结构AA的双甲基化修饰与CMS-S恢复基因Rf3介导的育性恢复是相互独立的事件。此外，多聚核糖体实验结果初步证实了1.6 kb RNA通过抑制18S rRNA的核糖体组装导致不育系花粉败育的分子机理。本研究为深入研究玉米CMS-S不育机理和育性恢复机理奠定了基础。