苏云金芽孢杆菌cry3Aa启动子的克隆和营养期表达载体的构建  

Cloning of the cry 3Aa Promoter from Bacillus Thuringiensis and Construction of the Bt Non-Sporulation Dependent Expression Vector

作　　者：余健秀[1] 蒙国基[1] 曾少灵[1] 谢瑞瑜[1] 谭乐[1] 庞义[1] 

机构地区：[1]中山大学生物防治国家重点实验室昆虫学研究所,广州510275

出　　处：《高技术通讯》2001年第3期5-8,14,共5页Chinese High Technology Letters

基　　金：86 3计划!(86 3 10 1 0 3 0 1 0 1);国家自然科学基金!(3990 0 0 98)资助项目

摘　　要：苏云金芽孢杆菌 (Bacillusthuringiensis ,Bt)的绝大多数杀虫晶体蛋白 (insectici dalcrystalproteins,ICPs)基因是在芽孢形成期 (sporulationphase)表达的 ,而只有少数基因如cry3Aa是在营养期表达的。在本研究中 ,根据已知的cry3Aa基因启动子序列设计了一对引物 (Ep 5s和Ep 3n) ,利用这对引物从拟步虫甲亚种 (Btsubsp .tenebrions)中扩增出一个与预期大小 ( 1.1kb)一致的DNA片段。序列测定及分析结果表明 ,这个DNA片段含cry3Aa启动子全序列 ,包括上游AT富含区、两个启动子区、两个SD序列及两组反向重复序列。经过一系列的克隆之后将这个片段克隆到穿梭载体pHT310 1上 ,最后构建成一个Bt的营养期表达载体 pHPT。The insecticidal activity of Bacillus thuringeinsis (Bt) resides in the parasporal crystalline inclusion body, consisting of a group of insecticidal crystal proteins (ICPs). Most ICP genes are normally sporulation dependent, while a few of ICP genes such as cry 3Aa are non sporulation dependent. In this study, a DNA fragment with the predicated size of 1.1 kb, was obtained by PCR from Bt subsp. tenebrions strain with a primer pair (Ep 5s, Ep 3n) designed according to the known sequence of cry 3Aa. This DNA fragment contained the completed sequence of the cry 3Aa promoter, comprising its upstream A+T rich region, two transcripts, two Shine Dalgarno sequences, and two invert repeats. Through several steps of sub cloning, the promoter was at last inserted into the shuttle vector pHT3101 to get a vegetative expression vector pHPT. And the successful construction of this vector would lay an important foundation for the further research on Bt engineering strains.

关 键 词：苏云金芽孢杆菌 cry3Aa基因 启动子 营养期 表达载体 生物杀虫剂 

分 类 号：Q786[生物学—分子生物学]