Molecular and structural characterization of a novel Cry1D toxin from Bacillus thuringiensis with high toxicity to Spodoptera littoralis (Lepidoptera: Noctuidae)

Abstract The investigation of new Bacillus thuringiensis (Bt) insecticidal proteins (Cry) with specific toxicity is one of the alternative measures used for Lepidopteran pest control. In the present study, a new Cry toxin was identified from a promising Bt strain BLB250 which was previously selected for its high toxicity against Spodoptera littoralis . The corresponding gene, designated cry1D-250 , was cloned. It showed an ORF of 3498 bp, encoding a protein of 1165 amino acid residues with a putative molecular mass of 132 kDa which was confirmed by SDS-PAGE and Western blot analyses. The corresponding toxin named Cry1D-250 showed a higher insecticidal activity towards S. littoralis than Cry1D-133 (LC 50 of 224.4 ng cm −2 ) with an LC 50 of only 166 ng cm −2 . Besides to the 65 kDa active toxin, proteolysis activation of Cry1D-133 protein with S. littoralis midgut juice generated an extra form of 56 kDa, which was the result of a second cleavage. Via activation study and 3D structure analysis, novel substitutions found in the Cry1D-250 protein compared to Cry1D-133 toxin were shown to be involved in the protein stability and toxicity. Therefore, the Cry1D-250 toxin can be considered to be an effective alternative for the control of S. littoralis .