大葱细胞质雄性不育基因的克隆及功能分析

Heterosis of Welsh onion is outstanding and male sterility is the basis of heterosis utilization on Welsh Onion. In this study, welsh onion cytoplasmic male sterile 63A and its maintainer line 63B are experimental objects. In order to determine the pollen abortion stage and cytological characterizations of cytoplasmic male sterile（CMS） lines, the CMS lines and its maintainers are used to observe the anther development by means of paraffin section method on welsh onion. By taking advantages of AFLP, RFLP, cDNA-AFLP and isobaric tags for relative and absolute quantification (iTRAQ), the male sterile line and fertile mitochondrial differentially expressed genes will be screened, differential gene expression patterns will be analyzed and the male sterility candidate genes will be determined through Northern, Western technology, whose position will be determined in the mitochondrial genome by Gene walking. In order to determine its subcellular localization, the expression vector of instantaneous male sterility candidate genes will be constructed and transformed into onion epidermal tissue. Sterile candidate genes prokaryotic expression vectors will be constructed and transformed into E. coli to test its toxicity. A mitochondria-targeted expression vector will be firstly constructed and transferred subsequently into Arabidopsis by Agrobacterium-mediated floral dip or spray, so that the anther fertility and developmental morphology of transgenic lines are observed and examined to determine the function of male-sterile genes. By carrying out this project, a new cytoplasmic male sterility gene with independent intellectual property rights will be obtained, which will lay the foundations for directive breeding of CMS on Welsh onion, and definitize the structure and function of it, so as to reveal the molecular mechanism of pollen sterility, which will provide a theoretical basis for the better utilizing of allium vegetables heterosis.

大葱杂种优势明显，雄性不育是大葱优势利用的基础。本研究以大葱胞质不育系63A及其保持系63B为材料，通过花药发育过程的细胞学观察，确定不育系的败育时期；利用AFLP、cDNA-AFLP、同重标签相对与绝对定量（iTRAQ）等技术筛选雄性不育系和可育系线粒体的差异基因；利用Northern、western技术分析差异基因的表达模式，确定雄性不育候选基因；利用基因步移技术确定其在线粒体基因组中的位置。构建不育候选基因原核表达载体，转化大肠杆菌，检测其毒性；构建瞬时表达载体，转化洋葱表皮组织，确定其亚细胞定位；构建线粒体定位表达载体，转化拟南芥，分析转基因植株的花药育性及花药发育特征，明确雄性不育基因的功能。本项目的开展将有望获得具有自主知识产权的雄性不育基因，为通过基因操作技术定向培育大葱CMS奠定基础，同时，明确大葱雄性不育基因的结构特点和功能，揭示大葱雄性不育的分子机制。

大葱杂种优势明显，雄性不育是大葱优势利用的基础。本研究以大葱胞质不育系为材料，通过花药发育过程的细胞学观察，确定不育系的败育时期；克隆雄性不育相关基因；构建不育候选基因原核表达载体，转化大肠杆菌，检测其毒性；构建瞬时表达载体，转化洋葱表皮组织，确定其亚细胞定位；构建线粒体定位表达载体，转化拟南芥，鉴定转基因植株的育性，明确雄性不育基因的功能。本项目研究结果显示：大葱雄性不育系败育时期为小孢子母细胞减数分裂时期；利用RFLP技术、双向电泳、转录组测定获得大葱雄性不育候选基因atpA。不育系与保持系中该基因的CDS均为1524bp；编码508个氨基酸，氨基酸酸序列存在6个氨基酸的差异。Q-PCR结果显示，保持系是该基因的表达丰度为不育系的6.35倍。该基因亚细胞定位于线粒体上。构建不育系atpA的原核表达载体，转化大肠杆菌，未见对大肠杆菌生长发育的抑制。构建AP3启动子、35S启动子驱动的线粒体atpA过表达和RNAi载体转化拟南芥，结果显示转化过表达载体的拟南芥未见异常；转化RNAi载体的拟南芥植物育性显著降低。本项目获得了大葱雄性不系的候选基因，并对其功能进行了的验证，明确了大葱雄性不育与atpA相关，为进一步阐明大葱雄性不育的机制奠定了基础，为深入理解植物雄性不育形成机制提供理论支持。