Cloning and characterization of a novel cry8Ab1 gene from Bacillus thuringiensis strain B-JJX with specific toxicity to scarabaeid (Coleoptera: Scarabaeidae) larvae

Abstract

Isolation of Bacillus thuringiensis (Bt) strain or its cry gene encoding insecticidal crystal protein (ICP) with specific toxicity is of great importance to biological control of insect pests. In this study, by screening 66 strains of Bt isolated from soil samples collected in Shandong Province, China, a new cry8-type gene from Bt strain B-JJX was identified via PCR-RFLP method. This novel gene, cry8Ab1, was cloned from the Bt strain B-JJX and expressed in an acrystalliferous mutant strain HD-73(-). The open reading frame of the cry8Ab1 gene consists of 3543bp with a G+C content of 37.99% and encodes a protein of 1180 amino acids with a putative MW of 133.3kDa which was confirmed by SDS-PAGE analysis. The Cry8Ab1 protein was expressed and released as spherical parasporal crystals from Bt acrystalliferous mutant strain HD-73(-) along with the presence of spores. In bioassays, this protein was toxic to 3-day-old larvae of the scarabaeid pests, Holotrichia oblita and H. parallela, with an LC50 of 5.72 and 2.00μgtoxing(-1)soil, respectively. The results are in accordance with the insecticidal activities of the original Bt strain B-JJX, which had an LC50 of 1.72 and 0.96μgtoxing(-1)soil against H. oblita and H. parallela, respectively.

Keywords: Biological control; Insecticidal activity; PCR-RFLP; Scarabaeid larvae; cry8 gene.