绿色荧光蛋白基因原核表达载体的构建及其在昆虫肠道短短芽孢杆菌和苏云金芽孢杆菌中的表达

【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(Green Fluorescent Protein)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短短芽孢杆菌CQUBb和苏云金芽孢杆菌CQUBt,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,经电泳分离在凝胶上出现约29kDa的特异蛋白条带;重组菌生长曲线与出发菌没有显著差异,说明外源质粒未对宿主菌的生长带来明显不利影响;抗性条件下传代30次后两菌株外源质粒稳定性仍可达95%、67%;两个菌株比较,CQUBb比CQUBt质粒转化率高、重组菌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。

Construction and expression of prokaryotic expression vector of green fluorescent protein in Brevibacillus brevis and Bacillus thuringiensis isolated from insect larvae intestines

[Objective] To construct a prokaryotic protein expression vector of green fluorescent protein (GFP) with the non-sporulation promoter of cryia gene of Bacillus thuringiensis ( Bt) , and to obtain recombined engineering bacteria with labled gfp gene and non-sporulation promoter of cryia gene the construction of insecticidal engineering strains. [Methods] The promoter of gene cryia and gfp gene was connected by SOE-PCR, The recombined gene pro3a-gfp was inserted into shuttle vector pHT304 at sites BamH I and Sph I Then plasmid pHT3AG was introduced into Brevibacillus brevis CQUBb and Bacillus thuringiensis CQUBt isolated from Apriona gemari larvae intestines. The expression of the recombinants was analyzed with fluorescent microscope,SDS-PAGE, and then the stability of engineering strains was tested under the conditions with or without antibiotics. [ Results ] Two engineering strains labeling with GFP were obtained. They expressed green fluorescent protein from the vegetative growth period and about 29kDa green fluorescent protein band was detected by SDS-PAGE. Heterogenous plasmid replicated and expressed stably in engineering strains and did not lead to significant adverse effects on these two host strains. After 30 generations, 95% and 67% of clones was still bearing recombined plasmids respectively. Compared to CQUBt, CQUBb had higher efficiency of electroporation, and the CQUBb-pHT3AG had longer GFP expression time, expressed more protein than CQUBt-pHT3AG, and the stability of CQUBb-pHT3AG was higher than CQUBt-pHT3AG. [Conclusions] We successfully obtained two transgenic insecticidal engineering strains.