基于cry3aa启动子的苏云金芽孢杆菌表达载体的构建与-biopublisher.docVIP

新载体pBUC3苏云金芽孢杆菌 刘辰1,2 张文飞1,2 1 海南热带农业资源研究所三亚572025，中国 2 广西大学生命科学与技术学院, 南宁, 530005 通讯作者 摘要 苏云金芽孢杆菌(Bacillus thuringiensis简称Bt)能够伴晶体(parasporal crystal proteins)。为了实现Bt Cry基因的表达依赖于芽孢形成苏云金芽孢杆菌cry3Aa启动子基因为模板，从苏云金芽孢杆菌拟步虫甲亚种(Bt subsp. tenebrions)中扩增出与目的大小一致的DNA片段测序将该DNA片段平末端连接到穿梭载体pBU4上，构建了一个新载体pBUC3。cry1Ac31基因随后通过电转化，将含有cry1Ac31基因的载体转Bt无晶体突变株IPS。利用PCR、镜检和SDS进行验证结果表明cry1Ac31基因得到了表达。苏云金芽孢杆菌关键词 苏云金芽孢杆菌, cry3Aa启动子, 营养期表达载体 cry1Ac31表达 Vector pBUC3 can Express Bt Cry1Ac31 during the Vegetative Phase in Bacillus thuringiensis Chen Liu 1,2 Wenfei Zhang 1,2 Yan Zhou1 Youzhi Li2* 1. Hainan Institute of Tropical Agricultural Resources, Sanya, 572025, P.R. China 2. College of Life and Technology Science, Guangxi University, Nanning, 530004, P.R. China * Corresponding author, Abstract The feature of Bacillus thuringiensis (B. t.) is the ability to generate parasporal crystal protein during the spore-forming process. In order to realize the expression of the Bt Cry genes independent on spore formation, we attempted to design new vector that could express Bacillus thuringiensis insecticidal crystal protein in the vegetable growth phase in this research. First, we designed a pair of specific primers based on cry3Aa promoter gene sequence to amplify cry3Aa promoter from B.t. subsp. Tenebrions; then the DNA fragment with blunt-end was ligated to the cross-species vector pBU4 to build new pBUC3 vector which would be non-dependent sporulation vector expressing in the vegetative growth phase of Bacillus thuringiensis. In order to validate the vector whether express B.t cry gene in the vegetative phase, The B.t cry1Ac31 gene was ligated to the pBUC3 vector to be transformed into B.t non crystal mutant IPS strain by electronic transformation approach. Through the examinations by the PCR, microscopic observation and SDS, the results showed that B.t cry1Ac31 was expressed in cell growth vegetable phase. Our study suggested that the feature of cry3Aa promoter being non-dependent on spore