`Cloning of vip1/vip2 genes and expression of Vip1Ca/Vip2Ac proteins in Bacillus thuringiensis

Forty-one Bacillus thuringiensis (Bt) standard reference strains and 118 Bt local isolates were screened for vip1/vip2 genes by PCR amplification, with only three strains (HD201, HD109 and HD12) producing the desired bands. Southern blot showed that vip1/vip2 genes were located on a 10 Kb EcoRV fragment of their total DNAs. Furthermore, the vip1Ca/vip2Ac genes were cloned from a partial genomic library of HD201. Sequence homologous analysis revealed that vip2Ac gene was highly conserved and encoded a protein possibly having ADP-ribosyltransferase activity, and that vip1Ca gene was of low homology, especially at its 3-terminus. Western blot showed that Vip1Ca and Vip2Ac proteins could be detected from middle logarithmic phase to the stationary phase in Bt HD201. However, bioassays of HD201 supernatants exhibited no activity against Culex quinquefasciatus, Spodoptera exigua, S. litura, Helicoverpa amigera and Tenebrio molitor larvae. Whether Vip1Ca and Vip2Ac proteins have any toxicity to other susceptible targets still needs to be investigated.