PCR-aided Synthesis and Stable Expression in E.coli of the cryIA (c)Bt Gene.

Insecticidal crystal proteins, known as delta-endotoxins, from a gram-positive bacterium Bacillus thuringiensis have been used as bio-pesticides for over 3 decades. By using a successive PCR method, the 1.8 kb cryIA (c)Bt gene coding for the fragment of protoxin was synthesized. Different from B. thuringiensis subsp. kurstaki Hd 73 cry gene, the synthesized gene has the codon usage pattern of an average Pseudomonas spp gene. 614 nucleotides were changed in the synthesized cryIA (c)Bt gene and the G C content was increased from 37.2% to 64%. The synthesized cryIA (c)Bt gene was cloned into pUT56 vector under the tac promoter and T1-T2 terminator. SDS-PAGE analysis revealed that 66 kD protein of the modified cryIA (c)Bt gene was expressed in E.coli and accounted for about 30% of total protein in the bacterial cells. Bioassays using crude expression products from host strains indicated that they had high toxicity to third instar larvae of the cabbage butterfly (Pieris brassicae). The LD(50) was calculated to be 0.024 &mgr;g/cm(2).