Determination of vegetative insecticidal protein (vip) gene contains and insecticidal activities of some entomopathogenic bacteria

In this study, the screening by PCR, the cloning and the characterization of vip genes of local Bacillus thuringiensis kurstaki (BnBt, MnD) and Bacillus thuringiensis tenebrionis (Xd3, Mm2) isolates which isolated and characterized in previous studies were performed. The insecticidal activity of partly purificated Vip3 proteins of BnBt and MnD isolates against the Spodoptera littoralis larvae was determined. As a result of the PCR analysis and the cloning, vip3 gene in MnD and BnBt isolates, vip1 and vip2 genes in Xd3 and Mm2 isolates were determined. Vip3 proteins of BnBt and MnD isolates by obtained from culture supernatants by ion exchange chromatography were subjected to SDS-PAGE analysis and a 90 kDa band of proteins were determined. The obtained proteins were used against the larvae of S. littoralis and according to results, the insecticidal effects of the Vip3 proteins of BnBt and MnD were observed as 40% and 31%, respectively. In the second part of the thesis, the full-length vip3A genes of BnBt and MnD isolates were cloned and the base sequences were determined. vip3A gene was 2,370 bp in length and had 790 amino acids. Then Vip3A proteins were expressed in E. coli BL21(DE3) by cloning into pGEX-2T expression vector and purified. SDS-PAGE analysis showed a band of totally 116 kDa for the Vip3A protein (~90 kDa) with the 26 kDa GST tag attached.