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作物学报 ›› 2013, Vol. 39 ›› Issue (09): 1538-1547.doi: 10.3724/SP.J.1006.2013.01538

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

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

李双双1,刘国政1,陈志文1,王玉美2,华金平1,*   

  1. 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
  • 收稿日期:2012-12-04 修回日期:2013-04-22 出版日期:2013-09-12 网络出版日期:2013-07-09
  • 通讯作者: 华金平, E-mail: jinping_hua@cau.edu.cn, Tel: 01 0-62734748
  • 作者简介:华金平, E-mail: jinping_hua@cau.edu.cn, Tel: 010-62734748
  • 基金资助:

    本研究由国家自然科学基金项目(31171591)和国家高技术研究发展计划(863计划)项目(2011AA10A102)资助。

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

LI Shuang-Shuang1,LIU Guo-Zheng1,CHEN Zhi-Wen1,WANG Yu-Mei2,HUA Jin-Ping1,*   

  1. 1 College of Agronomy and Biotechnology / Key Laboratory of Crop Heterosis and Utilization of Ministry of Education / Beijing Key Laboratory of Crop Genetic Improvement, China Agricultural University, Beijing 100193, China; 2 Institute of Cash Crops, Hubei Academy of Agricultural Sciences, Wuhan 430064, China
  • Received:2012-12-04 Revised:2013-04-22 Published:2013-09-12 Published online:2013-07-09
  • Contact: 华金平, E-mail: jinping_hua@cau.edu.cn, Tel: 01 0-62734748
  • Supported by:

    华金平, E-mail: jinping_hua@cau.edu.cn, Tel: 010-62734748

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orf160orf160全长480 bpN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,T1T2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。

关键词: 棉花, 线粒体DNA, 细胞质雄性不育, orf160

Abstract:

) of480 bp, orf160gene 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 shriveled; lower seed setting rate, germinate activety and pollen grain vitality showed in individuals in T1 and T2 generations. All these showed that orf160 affects on the development of transformants, and further research should verify if the function of orf160 relates to the cytoplasmic male sterility of cotton.

Key words: Gossypium, Mitochondrial DNA, Cytoplasmic male sterility, orf160

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