苏云金芽胞杆菌营养期杀虫蛋白基因vip3A在枯草芽胞杆菌中的表达

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88 kDa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72 h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72 h的LC₅₀为0.0194 mL/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.

Expression of a vegetative insecticidal protein gene vip3A from Bacillus thuringiensis in B. subtilis

Bacillus subtilis is commonly engineered to express insecticidal and antagonistical proteins. In order to develop a B. subtilis strain that expresses Vip3A protein and construct insecticidal and antagonistical strains, we fused a vip3A gene isolated from a B. thuriginensis strain WB7 to a promoter derived from the ribosomal subunit protein S4 promoter of B. subtilis strain 168, and then inserted this expression cassette into pAD123, an Escherichia coi-B. subtilis shuttle vector. An obtained plasmid （pADpvip） was then introduced into B. subtilis strain 168 and an antagonistic endophytic strain BS-2 which was isolated from chili pepper （Capsicum annum L.）. Detection of vip3A expression by SDS-PAGE showed that an 88 kDa band appeared in the protein samples prepared from the pADpvip transferred cells of the strain 168, but was not shown in the samples of strain BS 2 that contained the same plasmids, indicating that the vip3A was only expressed in strain 168. Fermentation broths of engineered strains derived from strain 168 and BS-2 were subsequently tested for their insecticidal activities. Second-instar larvae of oriental leafworm （Spodoptera litura） were fed with Chinese cabbage leaves treated with the bacterial suspensions at a concentration of 10⁷CFU/mL. The suspension of five engineered strains （168vip1-4, 6） derived from strain 168 resulted in 87.64%-92.13% larval mortality at 72 h after treatment, while the fermentation broths of the transferred strain BS-2 showed no toxicity to the larvae. The results of toxicity test showed that the 72-h LC₅₀ value of strain 168vip2 for the 2nd- instar larvae was 0.0194 mL/mL. These results lay the foundation for further work to improve expression of the Vip3A proteins in B. subtilis strains and to construct a B. subtilis strain that expresses both Vip3A and antagonistical proteins.