关键词: 苏云金芽胞杆菌, 枯草芽胞杆菌, 营养期杀虫蛋白, 异源表达, 杀虫活性

Abstract: 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.  

Key words: Bacillus thuringiensis, Bacillus subtilis, vegetative insecticidal protein, heterologous expression, insecticide activity