Expression of a Bacillus thuringiensis cry1C gene in plastid confers high insecticidal efficacy against tobacco cutworm - a Spodoptera insect

Abstract

We have demonstrated that the toxic domain of a wild-type Bacillus thuringiensis (Bt) crylC gene could be expressed efficiently in the chloroplasts of transplastomic tobacco plants, resulting in high resistance to tobacco insects. A DNA fragment with the toxic domain of a wild-type Bt crylC gene was constructed under the control of rbcL promoter and an E. coli thra terminator in a plasmid vector flanked by chloroplast sequences. This plasmid vector was then delivered into the chloroplast through biolistic bombardment technique. Plant transformants that possessed spectinomycin and streptomycin resistance were selected, and the integration of crylC gene in these transformants was confirmed by PCR and Southern analyses. Results from PCR and Southern hybridization indicated that the truncated crylC gene was integrated into the chloroplast genome as we expected through homologous recombination. High expression level of the integrated Bt crylC gene in the chloroplasts was observed through Northern and Western hybridization assays. These transplastomic tobacco plants are highly toxic to the tobacco cutworm Spodoptera litura, causing a mortality rate of 76.9% to 100% after 72 h of feeding. The truncated Bt toxin is expressed in high levels in the chloroplast (about 1% of total proteins) and the plastid transgenes were known not to transmit through pollen. So that, this study will facilitate not only improvement in breeding for insect-resistant plants, but also the prevention of contamination of transgenes among crop plants.