Cloning and sequence analysis of a novel bacillus thuringiensis gene encoding delta endotoxin active against tsetse (Glossina Morsitans) and stemborer (Chilo Partellus)

Abstract

Local isolate of B. thuringiensis, designated L1-2 (Bacillus thuringiensis L1-2) was found to be toxic to adult tsetse, Glossina morsitans morsitans and fourth instar larvae of the stem-borer, Chilo partellus in bioassays. The 5-endotoxin crystals derived from Bacillus thuringiensis L1-2 gave a major protein band of molecular weight of Mr ~ 130,000-140,000 on denaturing polyacrylamide gel electrophoresis. The cloning, expression in E. coli and the determination of the nucleotide sequence of a 3.5-kb DNA segment containing a cry gene from 8. thuringiensis L1-2 has been completed. The cloned gene from 8. thuringiensis L1-2 was found to be similar to that of the 8. thuringiensis subsp. kurstaki HD-73 crylA(c) gene having one amino acid difference at position 148 and 5 additional DNA differences. Bioassays using adult G. m. morsitans and larvae of C. partellus showed that the recombinant 8. thuringiensis L1-2 was as toxic to these insects as the original wild type. 8. thuringiensis L1-2 isolate is unique because, though the genes in this isolate belong to class I (lepidopteran-specific crystal proteins) which are synthesized as Mr 130-to 140,000 protoxins, the obvious advantage 8. thuringiensis L1-2 has over the previously studied 8. thuringiensis, is its wide range of insecticidal activity. Other previously reported 8. thuhngiensis of this class I, have shown only lepidopteran insecticidal activity but this isolate is also active against a adult dipteran. Also other classes which have shown insecticidal activities have so far shown that it is the larvae which suffered heavy mortalities, but with 8. thuringiensis L1-2 the insecticidal activity has been reported against adult Glossina. Amongst these dipteran spp studied so far (mosquitoes and black flies) both have aquatic larvae which offers a wide range of possibilities of infection by 8. thuringiensis. Glossina spp the larvae does not feed and is restricted to a few minutes on the surface of soil before burrowing offering a narrow range of possibilities of infection by 8. thdringiensis. So far no studies have shown crylA gene specific to adult Diptera and show dual specificity and cross-reacts immunologically with dipteran and lepidopteran toxins This work describes the cloning, and expression of Bacillus thuringiensis L1-2 gene as a first step to characterizing the gene.