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枯草芽胞杆菌Bacillus subtilis常被用于表达杀虫和抗菌蛋白。为了探讨苏云金芽胞杆菌B.thuringiensis营养期杀虫蛋白基因(vip3A)在枯草芽胞杆菌中的表达情况,促进杀虫防病工程菌构建,将枯草芽胞杆菌168菌株核糖体小亚基S4蛋白基因的启动子与苏云金芽胞杆菌WB7菌株vip3A基因的编码序列连接,插入大肠杆菌Escherichia coli与枯草芽胞杆菌穿梭载体pAD123,得到重组原核表达质粒pADpvip,将重组质粒转化枯草芽胞杆菌标准菌株168和分离自辣椒体内的生防内生枯草芽胞杆菌BS-2菌株中,获得工程菌株。SDS-PAGE分析表明在枯草芽胞杆菌168菌株的部分工程菌株中有约88kDa大小的VIP条带,而BS-2的工程菌株中未见相应的条带,表明Vip3A蛋白仅在168菌株中表达。生物测定表明有5株168的工程菌株(168vip1-4,6)表现较高的杀虫活性,工程菌株发酵稀释液(约107CFU/mL)处理的小白菜叶片饲喂斜纹夜蛾2龄幼虫72h的杀虫效果可达87.64%～92.13%,但vip3A基因转入内生枯草芽胞杆菌BS-2中不表现杀虫作用。毒力测定表明168vip2菌株对斜纹夜蛾2龄幼虫72h的LC50为0.0194mL/mL。这些结果为进一步研究基因在枯草芽胞杆菌中的表达构建杀虫防病工程菌打下了基础。
Bacillus subtilis Bacillus subtilis is often used to express insecticidal and antimicrobial proteins. In order to investigate the expression of vip3A in Bacillus subtilis of B. thuringiensis and promote the construction of insecticidal and anti-pathogenic engineering bacteria, the Bacillus subtilis 168 isolates of ribosomal small subunit S4 gene The promoter was ligated with the coding sequence of the vip3A gene of Bacillus thuringiensis WB7 strain, and inserted into Escherichia coli and Bacillus subtilis shuttle vector pAD123 to obtain the recombinant prokaryotic expression plasmid pADpvip. The recombinant plasmid was transformed into Bacillus subtilis standard strain 168 and isolated from pepper Of the biocontrol endophytic Bacillus subtilis BS-2 strain obtained engineering strains. SDS-PAGE analysis showed that there were about 88 kDa VIP bands in some of the engineered strains of Bacillus subtilis 168 and no corresponding bands in the engineered strains of BS-2, indicating that the Vip3A protein was only expressed in 168 strains. Bioassay showed that 5 strains of 168 engineering strains (168vip1-4,6) showed higher insecticidal activity. The cabbage leaves of Cabbage treated with fermentation strain (about 107CFU / mL) were fed with the 2nd instar larvae of Spodoptera litura Insecticidal effect of up to 87.64% ~ 92.13%, but vip3A gene into endophytic B. subtilis BS-2 does not show insecticidal effect. Toxicity tests showed that the LC50 of 168vip2 strain to larvae of Spodoptera litura at 72h was 0.0194mL / mL. These results lay the foundation for further study on the expression of genes in Bacillus subtilis and construction of insecticidal and anti-disease engineering bacteria.