植物雄性不育相关基因已克隆了不少，但这些不育相关基因的作用机理还有待于进一步验证。前期研究表明，烟草线粒体orf25基因的DNA序列在不育系与保持系间有较大差异，在不育系序列中甚至出现了终止密码子，但其功能至今还不清楚。本项目拟用已克隆的烟草雄性不育相关基因orf25，采用转基因技术，分别利用CaMV35S启动子和花特异启动子PchsA构建植物超表达载体，利用农杆菌介导转化烟草雄性不育系；同时以apt9基因为对照，利用RNAi技术，分别构建干扰orf25和atp9基因的RNAi载体，转化烟草保持系。通过对转基因后代进行分子、形态、生理生化和雄性不育性鉴定，以确定烟草orf25基因的功能及其与atp9基因功能的异同，并探求其与雄性不育性的关系；同时分析不同启动子调控不育相关基因orf25表达的差异。这将为揭示烟草雄性不育的分子遗传机理、深化烟草雄性不育杂种优势利用的理论和应用研究奠定基础。

Though lots of genes related to male sterility have been cloned in plants, the mechanism of action about these genes has yet to be proved. Former research result indicated that the DNA sequences of orf25 gene are different between male sterile line and maintainer line in tobacco. And the DNA sequence of orf25 gene in male sterile line even appeared termination codon. But the function of orf25 gene in tobacco mitochondria was not clear so far. This project will construct plant over expression vectors to transform the tobacco male sterile line suing the induction of Agrobacterium by the transgenic technology. The expression vectors would be constructed with the male sterility correlated gene (orf25) and the CaMV35S promoter and the flower-specific promoter PchsA respectively. While the RNA interference (RNAi) vectors interfered orf25 and atp9 genes will be constructed respectively to transform the tobacco maintainer lines. The function of orf25 gene will be confirmed and be compared with that of atp9 gene by identifying the male sterility, physiology, biochemistry, morphology and molecular characteristic of the transgenic tobacco plants and the wild types. With these results, the relationship between orf25 gene and tobacco male sterility will be discussed. And the expression difference of the orf25 gene regulated by different promoters will be analyzed. All these works will lay a foundation for revealing the molecular genetic mechanism of tobacco male sterility and for deepening the theory and application research of tobacco heterosis utilization with CMS.