Cloning And Function Analysis Of Differentially Expressed Proteins Of Bt Cry1Ac Toxin Resistant And Susceptive Trihoplusia Ni Cells

Bacillus thuringiensis (Bt) is a member of the Bacillaceae family and belongs to the Bacillus cereus group. It can produce many kinds of crystalline inclusions, which consist of entomocidal protein protoxins. When activated upon ingestion, these toxinscan weak or kill insects. However, with the widely used of Bt pesticide, several kinds of pests have shown resistance to Bt pesticides. Multi-mechanisms of resistance have been proposed, such as alteration of toxin-binding proteins, changes of proteases in midgut and so on. The other responses of the Cry1Ac-selected insects might also contribute to the evolution of resistance.Here, the Cry1Ac-selected Trichoplusia ni TnH5cells with high resistance were subjected to analysis of proteome and the differentially expressed proteins were identified using mass spectrometry. After checked in Software,49differential spots were identified by MALDI-TOF/TOF and31spots can be characterized in database. Among them,15kinds of protein were up-regulation in R140,16kinds of protein were down regulation in R140. These differential proteins included cytoskeleton proteins, heat-shock proteins, proteins regulating signal pathway and enzymes involved in metabolism, etc. The result suggests that the development of insect resistance to Bt Cry1Ac toxins appears to involve in a complex mechanism involved in the development of insect resistance to Bt Cry1Ac toxins at the cellular levels. The decrease of protein synthesis, changes of signal transduction, more rapid energy production, the enhanced lipid synthesis and the decline of possible Cry1Ac-binding proteins in cytoplasm and other events might respond contribute to the development of resistance in the selected cells. In this study, we cloned3differential expressed proteins (Ras suppressor protein, Fructose1,6-bisphosphate aldolase, Glycerol-3-phosphate dehydrogenase) and studied the relationship between them and Cry1Ac resistance.In addition to this, we found there was a significant difference of the autophagy level (ATG8relative abundance) between TnH5and R140cell. So we deducted that Autophagy may play an important role in Cry1Ac resistance. Consequently, in this study, I focused on the function of Helicoverpa armigera ATG8, cloned H.armigera ATG8mRNA sequence through RACE (Rapid Amplification of cDNA Ends), constructed eukaryotic expressed plasmid with IE2promoter, prepared anti-HaATG8mouse serum, inducted or inhibited of autophagy and detected through western blot or fluorescence microscope, used RNAi technology to study the relationship between Atg8and apoptosis.Our experiment has provided a new system in vitro and novel insight for the research on the mechanism of Bt resistance. Our results would provide some new cues for understanding the mechanism of Bt resistance.