水稻孢子体雄性不育系线粒体基因组特异序列鉴定和不育基因克隆研究

Sporophyte male sterility line of rice is a major abortion type which be used in the production of hybrid rice ,so the cloning of those CMS genes has an important role in promoting the development of hybrid rice. In this program, we try to comparatively analyze the mitochondrial genomes of three sporophyte CMS lines DongB51A, XieqingzaoA and Yue4A Which are different from each other in abortion type and restoring and maintaining relationship, to identified mitotype-specific sequences in those abortion types, the researching about transcriptional profile of new ORFs in the regions of mitotype-specific sequences combined the mitochondrial transcriptome sequencing to screening CMS candidate genes. Further to cloning three kind of sporophyte CMS genes which verified by transgenic complementary experiments, we plan to reveal a preliminary abortion mechanism by anaylsis of protein expression between CMS and F1,the toxicity for prokaryotes and the effect on mitochondrial complexes enzyme activity,etc. At the same time, we would also try to explore the influence of composition of mitotype-specific sequences on the abortion type and characteristic of sporophyte male sterility, and provide theoretical basis for creating new sporophyte CMS lines.

水稻孢子体雄性不育系是目前杂交水稻在生产应用上的一个主要败育类型，对其不育基因的克隆鉴定对推动杂交水稻的发展具有重要作用。本项目通过比较3个败育特点和恢保关系有差异的孢子体不育系东B51A、协青早A、岳4A线粒体基因组，分别鉴定3个孢子体不育系特有的线粒体基因组特异序列，结合线粒体转录组测序分析特异序列区新ORFs转录模式，筛选不育候选基因进行转基因验证，进而克隆3种孢子体不育基因并分析不育基因蛋白表达差异、对原核生物的毒性及对线粒体复合体酶活影响等初步揭示其败育机理。同时，探索不同孢子体特有线粒体基因组特异序列组成对孢子体败育特点的影响，为新型质源孢子体不育系选育提供理论依据。

水稻孢子体雄性不育系是目前杂交水稻在生产应用上的一个主要败育类型，对其不育基因的克隆鉴定对推动杂交水稻的发展具有重要作用。D1型不育系为一种来源于东乡野生稻的无花粉型孢子体不育系。在本项目研究中，通过线粒体基因组测序及比较分析，我们在该孢子体不育系鉴定到3个D1型孢子体不育系特异的线粒体特异序列及一个线粒体嵌合基因orf182，Northern blot分析表明在带有恢复基因的同质恢复系DR7中，该基因的表达发生改变，同时orf182只在中国的野生稻品种中鉴定到，western blot分析也表明ORF182能在D1型不育系中表达出一个功能蛋白，通过构建一个融合线粒体导肽和orf182的载体进行转基因验证表明，该基因能导致保持系粤泰B产生无花粉型不育。通过诱导orf182在细菌中表达发现orf182能抑制细菌生长、呼吸率降低、产生大量的ROS和ATP含量降低。因此，本研究成功的克隆了一个水稻新孢子体细胞质雄性不育基因，并阐明了败育机理，同时对于D1型杂交水稻的大面积推广应用具有重要推动作用。本研究同时也分析了孢子体不育系特有的线粒体特异序列在野生稻的分布，为孢子体不育系的选育提供理论支撑。