Studies on the cryV-type insecticidal protein gene in bacillus thuringiensis

Abstract

Bacillus thuringiensis is a gram-positive soil bacterium that produces the insecticidal proteins during the sporulation. The insecticidal protein genes have been classified as cryⅠ, cryⅡ, cryⅢ, cryⅣ, and cryⅤ genes according to their toxic spectrums. Since the cryⅤ genes are apparently silent in B. thuringiensis strains, it is possible that some B. thuringiensis strains that do not show detectable insecticidal activity may carry cryⅤ-type genes that encode insecticidal proteins with highly specific activities. In this study, we tried to screen the cryⅤ-type genes from a number of different B. thuringiensis strains to find a novel cryⅤ-type gene that may have a new insecticidal activity spectrum. DNA dot blot hybridizations with a cryⅤ-specific probe indicated that the cryⅤ-type genes are widely distributed in B. thuringiensis strains. Two cryⅤ-type genes, $cryⅤ_1$ and $cryⅤ_465$, were cloned from B. thuringiensis subsp. kurstaki HD-1 and B. thuringiensis subsp. entomocidus BP465 strains respectively, and nucleotide sequences were determined. Both $cryⅤ_1$ and $cryⅤ_465$ genes were highly homologous to known cryⅤ gene. Purified inclusions of the $cryⅤ_1$ protein was toxic to Plutella xylostella and Bombyx mori, whereas the $cryⅤ_465$ protein was only toxic to P. xylostella. Toxicity of both $cryⅤ_1$ and $cryⅤ_465$ protein may prove to be useful additions to the responsible management strategy for control of P. xylostella that is resistant to commercial preparations of B. thuringiensis. The cryⅤ-type genes have been characterized as linkage with a cryⅠ gene. We detected a cryID-like gene located approximately 0.5-kb 5`` of the $cryⅤ_465$ gene, and this type of cryⅠ-cryⅤ linkage seems to be common in other cryⅤ-type genes. We also cloned a cryⅠA(a)-$cryⅠ_1$ gene linkage from. thuringiensis subsp. kurstaki HD-1 and a cryIE-$cryⅤ_1$ gene linkage from B. thuringiensis subsp. tolworthi HD-537. These cryⅠ-cryⅤ linkages showed common characteristics, such as a distanc...