ATP合酶中多基因联合致烟草细胞质雄性不育机理研究

Cytoplasmic male sterility (CMS) had close relation with mitochondrial genes. It is likely that either or both gene products interfere with the function or assembly of the mitochondrial ATPase, thus impairing the highly ATP-dependent process of pollen development. Previous studies focused on analyzing single mitochondrial gene related to CMS, but CMS phenomenon seems so complex that it will not be controlled by single gene. To understand the potential function and significance of multiple genes joint effect on the CMS mechanism, all nine mitochondrial genes coding ATPase from the CMS lines and their maintainer lines will be analyzed as a small system in tobacco. The objectives of this project are: (1) to clone and sequence the target genes and their transcripts by PCR and RT-PCR amplification, and determine their concentration and expression levels; (2) to mine the sterility information in various strctural levels induced by the target genes, their transcripts and their encoded proteins by the bioinformatics analysis; (3) to predict accurately the interaction of ATPase from two lines based on our developed multiple scale component and correlation feature method and our optimized support vector machine; (4) to modify the interaction using yeast two-hybrid system experiment; and (5) to construct the protein interaction networks of ATPase from the CMS lines and their maintainer lines using Cytoscape software (i.e. an open source platform for complex network analysis and visualization). These research results will be helpful for tobacco and related crops in exploring the CMS mechanism caused by ATPase genes. Furthermore, these results will provide the reliable theoretical support for crop molecular breeding.

线粒体基因编码的ATP合酶功能受损，会影响高耗能的花粉发育并导致细胞质雄性不育（CMS）。前人研究偏重ATP合酶中单基因致CMS研究，但CMS现象复杂难以用单基因控制进行解释。为有效解析ATP合酶中多基因联合效应致CMS机制，本项目拟以两对模式作物烟草CMS系及其同型保持系为材料，以编码ATP合酶的全部九个基因为研究体系，测定ATP合酶基因、转录本及其蛋白质序列组成、表达量；利用生物信息学方法挖掘基因、转录、蛋白质水平上ATP合酶多基因在各结构层次致CMS信息；基于支持向量机、实验室自主研发的多尺度组分与关联特征提取法构建高精度蛋白互作网络模型，预测两系中ATP合酶互作关系，利用酵母双杂交系统验证、修正互作信息，进而利用Cytoscape构建两系中ATP合酶互作网络，探索蛋白质互作网络水平上ATP合酶联合致CMS信息。本研究为探索烟草乃至近缘种ATP合酶致CMS机理和分子育种提供可靠理论。

ATP合酶与植物雄性不育关系密切。本研究以3对烟草雄性不育系及其同型保持系为材料，通过分析两系线粒体ATP合酶5个基因（atp9、atp6、orf25、orfB和atp1)DNA序列差异、RNA序列差异、RNA编辑位点差异、蛋白质各级结构差异以及蛋白互作差异，并基于单个基因及线粒体基因组构建进化树，探讨ATP合酶与烟草雄性不育之间的关系。结果显示：1）不育系中atp9、atp6、orf25、orfB和atp1基因序列分别存在3个、12个、2个、1个和1个碱基突变，并分别导致1个、10个、2个、1个和0个氨基酸变化；2）RNA编辑方式均为C→U的转换，其中atp6基因在不育系中发现6个编辑位点造成了6个编码氨基酸变化；3）氨基酸改变引起了蛋白质一级结构、二级结构、超二级结构和三级结构不同程度的差异，其中ATP6差异最显著；4）蛋白相互作用预测推测ATPase中ATP9和ATP6两个最重要蛋白间有相互作用；5）进化分析也支持atp6较好地代表线粒体基因组信息来表征作物的进化关系和时间线索。综上所述，多方面分析均支持atp6为线粒体ATPase中关键基因，推测以atp6为中心的差异最可能与育性相关。