Mutagenesis of Bacillus thuringiensis cry1Ac gene and its insecticidal activity against Plutella xylostella and Ostrinia furnacalis

The cry gene, encoding the Cry protein, has been recently introduced into crops to generate transgenic plants that are resistant to pest insects. In this study, we produced various Cry mutant proteins by converting cry1Ac gene to a novel cry1-44 gene, improving the insecticidal activity and enlarging the host spectrum by multi site-directed mutagenesis. For this mutagenesis, 1857 bp of the modified cry1Ac gene (mod-cry1Ac), which was extensively changed in plant codon usage, was used as a template and amplified by polymerase chain reaction with 20 mutagenic primers, based on cry1-44 sequences. In total, 16 mutant cry genes (mut-cry1Ac) were generated and converted at 32 amino acid residues, located on domain I (1 residue) and domain II (31 residues). Ten mutants were expressed, as a polyhedrin fusion form, using a baculovirus expression system. The expressed proteins were 95 kDa and the trypsin-activated mutant proteins were all stable at 65 kDa. In the bioassay of Plutella xylostella third-instar larvae, LC50 of Mut-2 was 1.5-fold lower than that of Mod-Cry1Ac. In the neonate bioassay of Ostrinia furnacalis, LC50 values of Mut-4, Mut-6, and Mut-8 were 10- to 13-fold lower than that of Mod-Cry1Ac, and even 12- to 15-fold lower than that of Mut-2, with the highest insecticidal activity against P. xylostella. As a result, several mutant cry genes demonstrated higher insecticidal activities, such as Mut-2 to P. xylostella and Mut-4, Mut-6, and Mut-8 to O. furnacalis, which might be expected of desirable cry genes for introduction into transgenic crops.