【作者】 李强； 王中康； 袁训娥； 殷幼平；

【Author】 Qiang Li,Zhongkang Wang,Xun’e Yuan,Youping Yin~* (Genetic Engineering Research Center,Bioengineering College of Chongqing University,Chongqing Key Lab of Genetic Function and Regulation,Chongqing 400030,China)

【机构】 重庆大学生物工程学院基因工程研究中心重庆市基因功能与调控重点实验室；

【摘要】 【目的】构建带有苏云金芽孢杆菌cry3a基因非芽孢依赖启动子和绿色荧光蛋白基因gfp的原核表达载体,并转化从桑粒肩天牛幼虫肠道分离的两株常驻细菌,以检测cry3a启动子在昆虫肠道常驻菌中的启动子活性,获得GFP标记菌株,为常驻菌在昆虫幼虫肠道中的定殖情况和杀虫工程菌的构建奠定基础。【方法】采用重叠延伸PCR将cry3a基因启动子和gfp基因进行融合,并与pHT304载体连接构建重组质粒pHT3AG,获得的重组质粒以电脉冲转化肠道常驻菌短短芽孢杆菌CQUBb和苏云金芽孢杆菌CQUBt,于可见光和荧光显微镜下观察荧光并通过SDS-PAGE分析重组菌株的蛋白表达情况,然后对重组菌株进行生长动力学分析和稳定性测试。【结果】重组菌在营养期大量组成型表达GFP,电泳后出现约29KDa的特异蛋白条带;外源质粒未对宿主菌的生长带来明显不利影响;传代30次后外源质粒稳定性较好;CQUBb同CQUBt相比其质粒转化率高、GFP表达时间长、表达量大,并且重组菌株稳定性好。【结论】成功地将cry3a基因核心启动子和gfp基因转入桑粒肩天牛幼虫肠道常驻菌,实现了该启动子在Bt之外的菌株中发挥作用,构建了两个GFP标记菌株;重组基因工程菌株表达量大,稳定性好,可以用作昆虫肠道内微生态研究和芽孢杆菌表达系统以及杀虫菌株的构建。更多还原

【Abstract】 [Objective]To construct a prokaryotic protein expression vector of gfp gene with the non-sporulation promoter of cry3a gene in Bacillus thuringiensis.And then introduce the plasmid to resident bacteria isolated from Apriona gemari larvae intestines by electroporation to detect the activity of pro3a in insect larvae intestines.Lay the foundation for these two strain’s locating and distribution in larvae intestines and the construction of insecticidal engineering strains.[Methods]The promoter of gene cry3a and gfp gene were connected by SOE-PCR,resulting fusion gene pro3a-gfp.Gene pro3a-gfp was inserted into shuttle vector pHT304 at sites BamHⅠand SphⅠ,resulting recombinant plasmid pHT3AG.And then plasmid pHT3AG was introduced into Brevibacillus brevis CQUBb and Bacillus thuringiensis CQUBt respectively.Analyzed the expression with fluorescent microscope、SDS-PAGE,after which were dynamic analysis and stability testing of engineering strains.[Results]There was green fluorescent protein expressed in the nutrition period and about 29KDa protein detected by SDS-PAGE.GFP was constitutive expression in engineering strains.Heterogeneous plasmid can replicate and express stably after 30 generations in engineering strains and did not lead to significant adverse effects on these two host strains. CQUBb had higher efficiency of electroporation than CQUBt.GFP expression in CQUBb-pHT3AG was longer and more than expression in CQUBt-pHT3AG and the stability of CQUBb-pHT3AG was better than CQUBt-pHT3AG.[Conclusions]We successfully made the promoter of cry3a work in non-Bt Strains (CQUBb) and obtained two transgenic strains which can express GFP efficiently and can be used in locating、intestinal microecology and the construction of Bacillus expression system、insecticidal engineering strains.更多还原