Cloning and expression of full-length delta-endotoxin cryIA(c) gene in three kinds of prokaryotic systems using...

Two fragments, 6.5kb and 4.3kb encoding 5' end and 3' end of delta-endotoxin cryIA(c) gene, respectively, were selected from the Bacillus thuringiensis kurstaki-HD-73 75kb plasmid gene pool using random primer labelling delta-endotoxin cryIA(b) gene probe. The full-length 3.92kb cryIA(c) gene including 5' end 152 bp promoter sequence and 3' end 198 bp terminater sequence was rebuilt after uncoding sequences were deleted. Three kinds of engineering strains harbouring the same recombinant plasmid pHTY1 were obtained after the cryIA(c) gene had been subcloned in shuttle vector pHT3101 and transformed to E. coli NM522, Bacillus subtilis AS1176 and Bacillus thuringiensis crystal-deficient 4D10(H3ab). SDS-PAGE electrophoresis patterns reveal that the cryIA(c) gene expressed the same 133,300 protoxin proteins in all three host systems with the same molecular weight to the crystal protein from the Bacillus thuringiensis kurstaki HD-73. Immuno-assays indicate that the expression proteins can react with antiserum of HD-73 paraspore crystal protein in the same pattern as the natural crystalprotein. Bioassays using crude expressed products from three host strains reveal that they all showed toxicities to second instar larvae of Plutella xylostella, and their LD50 were calculated to be 0.311 micrograms/cm2, 0.02 micrograms/cm2 and 0.017 micrograms/cm2, respectively.