Isolation and sequencing of the cryIC-like delta endotoxin gene from Egyptian strains of Bacillus thuringiensis toxic against Lepidoptera

Abstract

The aim of the present work was to explore the diversity of Bacillus thuringiensis (Bt) among local Egyptian isolates that produce parasporal crystalline inclusions. On the basis of bioactivities, nine isolates were collected from different ecological environments within Egypt. The Bt2K isolate was effective against Lepidoptera as compared to B. thurigiensis ssp. kurstawi that was used as a reference standard. The gene isolated from the strain was shown to be homologous to the Lepidoptera active cryIC gene from B. thurigiensis ssp. ansawai as shown by PCR and alignment of the complete nucleotide sequences (98% identical). Therefore, a cryIC insecticidal toxin coding gene was cloned from an Egyptian isolate of B. thuringiensis. Sequence analyses of the cryIC gene showed the absence of potential polyadenylation signal sequences within the coding region and a less biased codon usage than most cryIC like genes. Therefore, the gene may be a suitable candidate for expression in plants without extensive modification. This possibility was examined by subcloning the cryIC gene into a plant expression vector and then transferring it to tobacco and potato through Agrobacteriummediated transformation. Tobacco and potato plants with a stably integrated native cryIC gene were completely protected from predation by Lepidoptera. It was concluded that novel insecticidal genes exist in nature that may not require extensive modifications for efficient expression in plants.

Key words: Bacillus thuringiensis, cryIC gene, plant transformation, Bt toxins, DNA sequencing.