Identification and Cloning of vip3A Genes from Isolates of Bacillus thuringiensis and Their Bioactivity Analysis

Abstract:

[Objective] To survey the distribution of vip3A-type genes from isolates of Bacillus thuringiensis from China and clone novel vip3A genes encoded Vip3A proteins with high insecticidal activity against Lepidopteran insect larvae. [Methods] We applied a PCR-RFLP method to identify vip3A-type genes from 171 isolates and cloned novel vip3Aa genes. [Results] The vip3A-type genes appeared in 63 of 117 B. thuringiensis isolates. We cloned two novel vip3Aa genes from isolates of TF9 and Bt16. Then, we subcloned vip3Aa26 and vip3Aa27 into vector pQE30 and transformed into Escherichi coli M15, respectively. The results of SDS-PAGE and Western blot analyses showed that a 88 kDa peptide was expressed in E.coli M15 with 1mmol/L of IPTG induction at 37 centigrade, respectively. The International Nomenclature Committee of Bt nominated these two genes as the novel vip genes of vip3Aa26 and vip3Aa27, respectively. The bioassay results indicated that the Vip3Aa27 proteins was highly toxic to Trichoplusia ni,Spodoptera exigua and Helicoverpa armigera larvae and the LC50 values were 0.125 μg/mL, 0.238 μg/mL and 9.238 μg/mL, respectively. However, the Vip3Aa26 protein only possessed toxicity to T.ni larvae. [Conclusions] The novel Vip3Aa27 protein had higher activity to Lepidopteran insect larvae compared with that for Vip3Aa26 protein. The results demonstrated that some amino acids changes had remarkable effect on the insecticidal activity.