枯草杆菌启动子－信号肽序列的克隆及序列分析  

Cloning and Sequencing of Promoter and Signal Sequence Coding Regions from Bacillus subtilis

作　　者：罗进贤[1] 李文清[1] 张添元[1] 柴戍杰 王红革[1] 

机构地区：[1]中山大学生物系及生物工程研究中心

出　　处：《Acta Genetica Sinica》1994年第1期74-80,共7页

基　　金：国家"七五"重点科技攻关项目

摘　　要：利用含红霉素抗性基因和缺启动子－信号肽序列的氨苄青霉素抗性基因的双功能质粒ｐＧＰＢ１４为探针载体，克隆了枯草杆菌的启动子－信号肽序列并对克隆的片段进行序列分析。枯草杆菌染色体ＤＮＡ经Ｓａｕ３Ａ酶解后与ＢｏｍＨＩ酶切的质粒ｐＧＰＢ１４连接，转化大肠杆菌Ｃ６００，筛选抗氨苄青霉素及抗红霉素的转化子，从双抗性转化子中提取重组质粒并经酶切分析，显示克隆的ＤＮＡ片段在０．２７－１．５ｋｂ之间。用Ｓａｎｇｅｒ的双脱氧链终止法测定了１０个克隆片段的ＤＮＡ顺序，结果表明，克隆的片段都含有启动子、核糖体结合优点及信号肽序列。克隆片段可以在大肠杆菌和枯草杆菌中恢复氨苄青霉素抗性的表型。β－内酰胺酶活力测定结果证明：大肠杆菌的酶活力主要积累在周质空间内而枯草杆菌的酶活力主要分泌到胞外。Promoter and signal sequence coding regions from B. subtilis were cloned in E. coli using a bifunctional and signal sequence selection plasmid pGPB14 as a vector, The Sau3A digested chromosome DNA was ligated with BamH1 digested pGPB14. The ligated mixture was used to transform E, coli C600. Ampicillin and erythromycin resistant clones were selected. Recombinant plasmids were isolated from double resistant transformants. Restriction analysis showed that inserts of various length had been cloned and these inserts rendered the E. coli cells resistant to different concentrations of ampicillin.The recombinant plalsmids were transformed and showed the same secretion function in B. subtilis.The amount and localization of β-lactamase were determined in transformed E. coli and B. subtilis.The results indicate that the β-lactamase activities of E. coli mainly in periplasm while the enzyme produced by B. subtilisis secreted extracellularly.10 of the cloned fragments were sequenced by Sange's dideoxy chain termination method. The sequencing data show that all fragments contain promoter, ribosom binding site and signal sequence coding region.

关 键 词：枯草杆菌 启动子 信号肽 序列 分析 

分 类 号：Q939.103[生物学—微生物学]