利用无缝克隆方法构建埃及伊蚊表皮蛋白基因AaCPR100A双向启动子苏云金芽孢杆菌穿梭载体

Construction a shuttle vector containing AaCPR100A epidermal protein gene of Aedes aegypti with two spore-producing-dependent promoters in Bacillus thuringiensis by in-fusion cloning

目的 构建表达dsRNA的双向启动子以及靶基因AaCPR100A的大肠杆菌-苏云金芽孢杆菌穿梭载体pHT315-AaCPR100A。方法 以苏云金芽孢杆菌pSVP27A质粒为模板通过PCR扩增苏云金芽孢杆菌Cry3A正向启动子Pro-1(+);以埃及伊蚊RNA反转为cDNA为模板,扩增靶基因AaCPR100A;将大肠-苏云金穿梭载体pHT315质粒经HindⅢ和SalⅠ双酶切线性化;按照转录方向将正向启动子与靶基因通过无缝克隆插入穿梭载体pHT315中;以生物合成的含有Cry3A反向启动子序列的质粒为模版,经PCR克隆扩增Pro-1(-)反向启动子;将含有正向启动子与靶基因的中间载体通过Eco RⅠ限制性酶切酶线性化,按转录方向在靶基因下游通过无缝克隆插入反向启动子。结果 经琼脂糖凝胶电泳检测,正向启动子、靶基因AaCPR100A和反向启动子PCR产物条带清晰,质量良好,可用于无缝克隆实验;经无缝克隆连接双向启动子以及靶基因片段后,对含有重组载体pHT315-AaCPR100A的重组质粒进行PCR验证,在重组载体中成功扩增正向启动子、靶基因片段以及反向启动子,经核苷酸测序验证双向启动子序列以及靶基因序列测序结果与序列比对结果基本一致,符合构建载体元件的要求,证明重组载体构建成功。结论 本研究利用无缝克隆技术成功构建含有双向启动子以及靶基因的重组穿梭载体,为构建表达伊蚊表皮蛋白基因AaCPR100A dsRNA的苏云金芽孢杆菌工程菌奠定基础。

Objective To construct a shuttle vector pHT315-AaCPR100A with two spore-producing-dependent promoters and the target gene AaCPR100A in Escherichia coli-Bacillus thuringiensis.Methods The forward promoter of Cry3A,named Pro-1(+),was amplified by PCR using pSVP27A plasmid as the template,and the target gene AaCPR100A was amplified using Aedes aegypti RNA reverse conversion cDNA as the template.The plasmid pHT315 was linearized by digestion with HindⅢand SalⅠ.The forward promoter and the target gene were inserted into the linearized vector pHT315 successively by in-fusion cloning according to the transcription direction.The synthesized plasmid containing the Cry3A reverse promoter sequence was used as the template,and the Pro-1(-)reverse promoter was amplified by PCR.The intermediate vector containing the forward promoter and the target gene was linearized by EcoR I restriction enzyme,and the reverse promoter was inserted downstream of the target gene by in-fusion cloning in the direction of transcription.Results By agarose gel electrophoresis,the forward promoter,target gene AaCPR100A and reverse promoter bands were clear and of good quality,which could be used for in-fusion cloning experiments.The two spore-producing-dependent promoters and target gene fragments were connected by In-fusion cloning.The recombinant vector pHT315-AaCPR100A was verified by PCR.The forward promoter,target gene fragment and reverse promoter were successfully amplified in the recombinant vector.Nucleotide sequencing verified that the sequencing results of the bidirectional promoter sequence and the target gene sequence were basically consistent with the sequence alignment results,which met the requirements of the construction of vector elements and proved that the recombinant vector was successfully constructed.Conclusions Based on the above results,this study proves that the recombinant shuttle vector with two spore-producing-dependent promoters can be successfully constructed by infusion cloning technology,laying the foundation for the construction of engineered Bacillus thuringiensis expressing dsRNA of AaCPR100A.