Construction of an environmental safe Bacillus thuringiensis engineered strain against Coleoptera.

Abstract

Cloning of new toxic genes from Bacillus thuringiensis (Bt) and construction of Bt engineered strains are two key strategies for bio-control of coleopteran pests in agriculture and forestry. In this study, we cloned a new cry3Aa-type gene, cry3Aa8, from wild Bt strain YC-03 against coleopteran, and constructed a Bt engineered strain, ACE-38, containing insecticidal protein-encoding gene cry3Aa8. The engineered strain, with almost four times of Cry3Aa yield compared with strain YC-03, was an antibiotic marker-free strain. Though no selective pressure was presented in the medium, cry3Aa8 in the engineered strain ACE-38 remained stable. The yield of Cry3Aa by strain ACE-38 reached 2.09mg/ml in the optimized fermentation medium. The activity of strain ACE-38 against Plagiodera versicolora was tested, and the LC50 of ACE-38 cultures in the optimized fermentation medium was 1.13μl/ml. Strain ACE-38 is a non-antibiotic Bt engineered strain with high Chrysomelidae toxicity and exhibits good fermentation property. The modified indigenous site-specific recombination system constructed in this study might be useful for the construction of Bt engineered strains containing genes that cannot be expressed in the indigenous site-specific recombination system using plasmid pBMB1205R.