Cloning, Expression And Insecticidal Activity Of Novel Vip3 Genes From Bacillus Thuringiensis

The pests are very important in agricultural production. A large number of chemical pesticides were utilized to control pests. However, the disadvantages of chemical pesticides are seriously harm to human health and the environment, and inducing chemical-resistant pests results in using more chemical pesticide increasing the cost. Biocontrol is an effective alternative. Bacillus thuringiensis (Bt) is the most widely used as insecticidal micriobe for its specific toxicity and safety for animal and enviroment. Its insecticidal activity is derived from the toxic protein, such as insecticidal crystal proteins (ICPs) and vegetative insecticidal proteins (VIPs).The Vip protein is secreted by Bt strains during vegetative growth. It shows no homologous to Cry protein and its insecticidal mechanism is different from Cry proteins. The Vip protein can expand the Bt insecticide spectrum and provent the resistance production.The vip3-type genes of 72 Bt standard strains identified by PCR were found in 18 strains among them. High-resolution melting analysis (HRMA) system was utilized to identify vip3 gene-type in these strains by using of PCR products. In our method, vip3 genes can be identified on the basis of the differences in melting-peak curve shapes, which result from differences in DNA sequences of PCR products. In the HRMA of the 18 isolates, melting-peak curves were divided into 8 types. HRMA systems was utilized to high-throughput and rapidly identify vip3-type genes in these strains. Both known and novel vip3-type genes in Bacillus thuringiensis isolates can be detected by using of this method. The primers were designed based on the published vip3 genes in GeneBank. Five vip3 genes were cloned and designated as vip3Aa38, vip3Aa39, vip3Af3, vip3Ag3 and vip3Ba2 by the Nomenclature Commitee of Bt toxins, respectively.The vip3 gene was inserted into the vector pEB and a peptide of 88kDa was expressed in Escherichia coli Rosetta(DE3) by IPTG induction at low temperature. Insecticidal activities of soluble expressed products of vip3Aa39 gene were tested against Agrotis ipsilon, Plutella xylostella, Helicoverpa armigera and Spodoptera exigua. Vip3Aa39 had insecticidal activity against four insects. 39 amino acid residues were found to be different between the Vip3Aa39 and Vip3Aa11 proteins expressed in our previous study. The bioassay results indicated that Vip3Aa39 had insecticidal activity against A. ipsilon with 50% lethal concentration (LC50) of 5.43μg/ml compared to 73.62μg/ml for Vip3Aa11; Vip3Aa39 showed insecticidal activity against P.xylostella with LC50 of 140.64μg/ml while Vip3Aa11 protein had no activity against P.xylostella; Vip3Aa11 demonstrated insecticidal activity against H.armigera with LC50 of 35.18μg/ml compared to 286.99μg/ml for Vip3Aa39; Vip3Aa39 had insecticidal activity against S. exigua with LC50 of 2.02μg/ml similar to 2.04μg/ml for Vip3Aa11. The results indicate that insecticidal activity of Vip3Aa39 and Vip3Aa11 is different from each other against A.ipsilon, P.xylostella and H.armigera.