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

摘要

苏云金芽孢杆菌（Bacillus thuringiensis，Bt）的绝大多数杀虫晶 体蛋白（ insecticidal crystal proteins，ICPs）基因是在芽孢形成期(sporulation phase)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（Bt subsp. tene brions） 中扩增出一个与预期大小（1.1 kb）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pHT3101上，最后构建成一个B t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, wh i le a few of ICP genes such as cry3Aa are non-sporulation-dependent. In thi s stud y, a DNA fragment with the predicated size of 1.1 kb, was obtained by PCR from B t subsp. tenebrions strain with a primer pair (Ep-5s, Ep-3n) designed ac cording to the known sequence of cry3Aa. This DNA fragment contained the completed s eque nce of the cry3Aa promoter, comprising its upstream A+T-rich region, two tr anscr ipts, two Shine-Dalgarno sequences, and two invert-repeats. Through several st ep s of sub-cloning, the promoter was at last inserted into the shuttle vector pHT 3 101 to get a vegetative-expression vector pHPT. And the successful construction of this vector would lay an important foundation for the further research on B t engineering strains.