哈克尼西棉细胞质陆地棉雄性不育系orf160的克隆及遗传转化  

A Unique orf160 Cloning and Genetic Transformation of Gossypium harknessii Cytoplasmic Male Sterile Line in Upland Cotton

作　　者：李双双[1] 刘国政[1] 陈志文[1] 王玉美[2] 华金平[1] 

机构地区：[1]中国农业大学农学与生物技术学院,杂种优势研究与利用教育部重点实验室/作物遗传改良北京市重点实验室,北京100193 [2]湖北省农业科学院经济作物研究所,湖北武汉430064

出　　处：《作物学报》2013年第9期1538-1547,共10页Acta Agronomica Sinica

基　　金：国家自然科学基金项目(31171591);国家高技术研究发展计划(863计划)项目(2011AA10A102)资助

摘　　要：以哈克尼西棉细胞质陆地棉雄性不育系及其保持系、恢复系为材料,克隆并测序线粒体功能基因的侧翼序列。通过生物信息学分析,在atp4下游发现一个哈克尼西棉细胞质不育系特有的orf160。orf160全长480 bp,N端与atp6序列部分同源,C端序列与核序列部分同源;编码的159个氨基酸序列,与膜蛋白、细胞周期蛋白具有部分同源性。以含有线粒体导肽的GFP瞬时表达载体转化后,在洋葱表皮的细胞膜上观察到绿色荧光,表明线粒体定向载体正常表达。构建酵母和植物表达载体,分别转化酵母、烟草和拟南芥;同对照相比,转基因酵母菌斑畸形且生长缓慢;转基因拟南芥和烟草种子大部分畸形,T_1和T_2代植株的结实率和种子发芽率降低,花粉着色比野生型浅。结果表明,orf160基因的表达影响受体正常发育。The cytoplasmic male sterile （CMS） line with the cytoplasm of Gossypium harknessii has been reported for 50 years, and hybrids have been used in commercial production, but the mechanism remains obscure. The flanking sequences of mitochon- drial functional gene from ＂three lines＂ （CMS line, corresponding maintainer and restorer line） were analyzed. A specific ORF （open reading frame） of 480 bp, orfl60 gene was cloned in CMS line, which located in the downstream of atp4, its N-terminal was homologous with atp6, and the C-terminal was homologous with nuclear sequence, and encoded 159 amino acids that were homologous with membrane proteins and cell cycle protein. The subcellular localization of onion epidermal cells showed the green fluorescence on cell membranes, so the mitochondria-targeting carrier can be normally expressed. The expression vectors were constructed and transformed into yeast, arabidopsis and tobacco, respectively. Compared with control, the modified yeast was abnormal and grew slowly; transformed arabidopsis and tobacco showed that the most seeds were abnormal, small and shriv- eled; lower seed setting rate, germinate activety and pollen grain vitality showed in individuals in T1 and T2 generations. All these showed that orfl60 affects on the development of transformants, and fi.trther research should verify if the function of orfl60 re- lates to the cytoplasmic male sterility of cotton.

关 键 词：棉花 线粒体DNA 细胞质雄性不育 orf160 

分 类 号：S562[农业科学—作物学]