Fusing the vegetative insecticidal protein Vip3Aa7 and the N terminus of Cry9Ca improves toxicity against Plutella xylostella larvae

Abstract

Bacillus thuringiensis insecticidal crystal proteins (ICPs) and vegetative insecticidal proteins (VIPs) have been widely used as a kind of safe bio-insecticides. A problem that has been of concern worldwide is how to improve their insecticidal activities. In this study, to determine the synergism between VIPs and ICPs effect on insecticidal activity, a construct that produces a chimeric protein of the Vip3Aa7 and the N terminus ofCry9Ca, named V3AC9C, was expressed in Escherichia coli BL21 cells. In additional experiments, the V3AC9C chimeric protein, the single Vip3Aa7, and the single N terminus of Cry9Ca were treated with trypsin. SDS-PAGE showed that the V3AC9C could be processed into two single toxins. Bioassays tested on third instar larvae of Plutella xylostella showed that the toxicity of the chimeric protein was markedly better than either of the single toxins. Interestingly, the toxicity of the chimeric protein was 3.2-fold higher than a mixture of the Vip3Aa7 and Cry9Ca toxins (mass ratio of 1:1). The synergism factor (SF) of chimeric protein containing Vip3Aa7 and Cry9Ca was calculated to be 4.79. The SF in mixture of toxins is only 1.46. Hence, the effect was more than the sum of the Vip3Aa7 and Cry9C activities. Analysis of the protein's solubility showed that the Vip3Aa7 helped the N terminus of Cry9Ca to dissolve in an alkaline buffer. It was concluded that the increase in the toxicity of the V3AC9C chimeric protein over the constituent proteins mainly resulted from this increase in solubility. These results lay a foundation for the development of a new generation of bio-insecticides and multi-gene transgenic plants.