Effects of different brush border membrane vesicle isolation protocols on proteomic analysis of Cry1Ac binding proteins from the midgut of Helicoverpa armigera

Brush border membrane vesicles （BBMV） isolated from insect midguts have been widely used to study CrylA binding proteins. Sample preparation is important in two- dimensional electrophoresis （2-DE）, so to determine a suitable BBMV preparation method in Helicoverpa armigera for 2-DE, we compared three published BBMV preparation methods mostly used in sodium dodecyl sulfate-polyacrylamide gel electrophoresis （SDS- PAGE）. All methods yielded similar types and numbers of binding proteins, but in different quantities. The Abdul-Rauf and Ellar protocol was the best of the three, but had limitations. Sufficient protein quantity is important for research involving limited numbers of insects, such as studies of insect resistance to Bacillus thuringiensis in the field. Consequently, we integrated the three BBMV isolation methods into a single protocol that yielded high quantities of BBMV proteins from H. armigera larval midguts, which proved suitable for 2- DE analysis.

Gene cloning and expression of aminopeptidase N and cadherin from midgut of the rice stem borer, Chilo suppressalis

The rice stem borer, Chilo suppressalis Walker is one of the most important insect pests on rice in Asia, north Africa and southern Europe. Transgenic Bt rice has been developed in the laboratory with good resistance to this pest and other Lepidopteran insects, which will provide a possible alternative tool for this pest control. The full-length cDNAs encoding an aminopeptidase N （CsAPN） and a cadherin （CsCad） were cloned from C. suppressalis. CsAPN showed common features of, and high identities to, other insect APNs in its deduced amino acid sequence. Although a full-length cDNA encoding cadherin-like protein has been reported in GenBank, the newly isolated cadherin here （CsCad） showed some differences in its amino acid sequence, especially at the 7th cadherin repeat region （CR7）, which indicated the newly isolated CsCad might be another allele. CsAPN and CsCad were successfully expressed in insect Tn cells, and the blot analysis showed these two proteins could bind Bt toxin CrylAb. The results will provide valuable information for the studies of toxin mode of action and the possible toxin resistance mechanisms in this pest.

Capturing the interaction types of two Bt toxins CrylAc and Cry2Ab on suppressing the cotton bollworm by using multi-exponential equations

Transgenic crops are increasingly promoted for their practical effects on suppressing certain insect pests, but all transgenic crops are not equally successful. The insect pests can easily develop resistance against single Bacillus thuringiensis （Bt） toxin trans- genic crops. Therefore, transgenic crops including two or more mixed Bt-toxins can solve this problem by delaying the resistance development and killing the majority of targeted pests before the evolution of resistance. It is important to test the controlling effects of transgenic crops including multiple mixed toxins on a particular insect pest. Previous research has checked the cross-resistance and interactions between Bt toxins CrylAc and Cry2Ab against one susceptible and four resistant strains of cotton bollworm. The results showed that independence was the main interaction type between two toxins for the susceptible strain, whereas synergism was the main interaction type for any one resistant strain. However, the optimal combinations of two toxins were not obtained. In the present study, we developed two multi-exponential equations （namely bi- and tri-exponential equations） to describe the combination effects of two Bt toxins. Importantly, the equations can provide predictions of combination effects of different continuous concentrations of two toxins. We compared these two multi-exponential equations with the generalized linear model （GLM） in describing the combination effects, and found that the bi- and tri-exponential equations are better than GLM. Moreover, the bi-exponential equation can also provide the optimal dose combinations for two toxins.