蚕豆叶绿体atpE基因的克隆、测序和表达  

Cloning, Sequencing and Expression of the atp E Gene of Broad Bean Chloroplast

作　　者：顾莹[1] 杨颖贞[1] 史瑾[1] 魏家绵[1] 

机构地区：[1]中国科学院上海植物生理研究所

出　　处：《生物化学与生物物理学报》1998年第5期477-482,共6页

基　　金：国家自然科学基金;植物分子遗传国家重点实验室基金

摘　　要：蚕豆叶绿体的ａｔｐＥ和ａｔｐＢ基因在叶绿体ＤＮＡＫｐｎＩ酶切的第九条（约４．０ｋｂ）片段上，此片段被克隆到载体ｐＵＣ１８中，构成重组质粒ｐＵＫ１。用玉米叶绿体ａｔｐＥ基因作探针对ｐＵＫ１的ＣｌａＩ、ＥｃｏＲＩ等的酶切产物进行杂交，确定了蚕豆叶绿体的ａｔｐＥ在ＣｌａＩ酶切的约０．９ｋｂ的片段上。根据ｐＢｌｕｅｓｃｒｉｐｔＫＳ（＋）ＤＮＡ多聚接头Ｆ引物和Ｒ引物测序，得到ε亚基基因的完整核苷酸序列。此后，ε亚基基因被克隆到载体ｐＪＬＡ５０５的多聚接头处，构建了表达质粒ｐＪＬＡ５０５－ａｔｐＥ，转化Ｅ．ｃｏｌｉ，进行温敏诱导表达。产物经电泳扫描分析，表达量占全菌蛋白的３８％以上。蛋白质印迹分析结果表明此表达产物可特异性地与玉米叶绿体ＣＦ１的ε亚基抗体反应。大肠杆菌中表达的蚕豆叶绿体ａｔｐＥ基因产物ε亚基蛋白以包含体形式存在于细菌中，经纯化后测定其具有天然ε亚基蛋白的生物功能。The ninth Kpn I fragment (about 4.0 kb) of broad bean chloroplast DNA contains the entire chloroplast atp E gene and atp B gene. This fragment was inserted into the pUC18 polylinker region, yielding plasmid pUK1. Southern hybridization using atp E gene from maize chloroplast as probe revealed that atp E gene encoding ε subunit of broad bean chloroplast was localized in 0.9 kb Cla I fragment of pUK1. The whole sequence of atp E gene of broad bean chloroplast was determined by using primer F and primer R of the polylinker region of pBluescript KS (+) DNA. The entire atp E gene of broad bean chloroplast was inserted into the polylinker region of vector pJLA505 to form recombinant plasmids pJLA505 atp E. The expression plasmid was transformed into E.coli DH5α cells which were then induced at 42℃. By analysis with SDS PAGE, the product of interest accounted for more than 38% of total E.coli cell proteins. Identification of the expressed product by Western blot analysis demonstrated that it can react specifically with anti ε serum of maize chloroplast CF 1. The expressed product aggregated to form insoluble inclusion bodies and was purified. The purified product had the same function as that of the native ε subunit.

关 键 词：叶绿体CF1 Ε亚基 克隆 测序 表达 蚕豆 

分 类 号：Q78[生物学—分子生物学] S643.603.2[农业科学—蔬菜学]