Molecular cloning and characterization of a novel vip3-type gene from Bacillus thuringiensis and evaluation of its toxicity against Helicoverpa armigera

Abstract

Vegetative insecticidal proteins (Vips) represent the second generation of insecticidal trans-genes that will complement the Bacillus thuringiensis delta endotoxins in future. A new vip3A gene was cloned from the promising native isolate, B. thuringiensis JK37 obtained from the soils of maize field. The entire coding sequence of the gene (2370 bp) was amplified and cloned into pET28a(+) expression vector. The deduced amino acid sequence of the vip3A gene revealed variation of several amino acid residues with that of the known vip3A genes and this gene was designated as vip3Aa61 by the B. thuringiensis nomenclature committee. The recombinant pET28a(+)–vip3Aa61 was transformed and expressed in Escherichia coli strain BL21 (DE3) under the control of T7 promoter. SDS-PAGE and Western blot analysis confirmed the expression of an 89 kDa protein. Insect bioassays with 2nd instar larvae of Helicoverpa armigera, one of the most notorious pest affecting various crops including cotton and chick pea displayed toxicity. The toxicity of Vip3Aa61 was expressed as mean lethal concentration (LC50), which was 169.63 ng cm−2. The novel vip3Aa gene may be used for the construction of transgenic plants expressing insecticidal protein for the control of lepidopteran insect pests.