The reconstruction and expression of a Bacillus thuringiensis cryIIIA gene in protoplasts and potato plants

Abstract

A Bacillus thuringiensis (B.t.) cryIIIA δ-endotoxin gene was designed for optimal expression in plants. The modified cry gene has the codon usage pattern of an average dicot gene and does not contain AT-rich nucleotide sequences typical of native B.t. cry genes. We assembled the 1.8 kb cryIIIA gene in nine blocks of three oligonucleotide pairs. For two DNA blocks, the polymerase chain reaction was used to enrich for correctly ligated pairs. We compared modified cryIIIA gene with native gene expression by electroporation of dicct (carrot) and monocot (corn) protoplasts. CryIIIA-specific RNA and protein was detected in carrot and corn protoplasts only after electroporation with the rebuilt gene. Transgenic potato lines were generated containing the redesigned cryIIIA gene under the transcriptional control of a chimeric CaMV 35S/mannopine synthetase (Mac) promoter. Out of 63 transgenic potato lines, 58 controlled first-instar Colorado potato beetle (CPB) larvae in bioassays. Egg masses which produced ca. 250 000 CPB larvae were placed on replicate clones of 56 transgenic potatoes. No CPB larvae developed past the second instar on any of these plants. Plants expressing high levels of δ-endotoxin were identified by their toxicity to more resistant third-instar larvae. We show there was good correlation between insect control and the levels of δ-endotoxin RNA and protein.