THE RECONSTRUCTION AND EXPRESSION OF A BACILLUS-THURINGIENSIS CRYIIIAGENE IN PROTOPLASTS AND POTATO PLANTS

A Bacillus thuringiensis (B.t.) cryIIIA delta-endotoxin gene was desig ned for optimal expression in plants. The modified cry gene has the co don usage pattern of an average dicot gene and does not contain AT-ric h 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 n ative gene expression by electroporation of dicot (carrot) and monocot (corn) protoplasts. CryIIIA-specific RNA and protein was detected in carrot and corn protoplasts only after electroporation with the rebuil t gene. Transgenic potato lines were generated containing the redesign ed cryIIIA gene under the transcriptional control of a chimeric CaMV 3 5S/mannopine synthetase (Mac) promoter. Out of 63 transgenic potato li nes, 58 controlled first-instar Colorado potato beetle (CPB) larvae in bioassays. Egg masses which produced ca. 250000 CPB larvae were place d on replicate clones of 56 transgenic potatoes. No CPB larvae develop ed past the second instar on any of these plants. Plants expressing hi gh levels of delta-endotoxin were identified by their toxicity to more resistant third-instar larvae. We show there was good correlation bet ween insect control and the levels of delta-endotoxin RNA and protein.