Research On BT Toxin Oligomer Formation Induced By Lipid Raft And The Immunodetection Explore Of Cadherin

Bacillus thuringiensis (Bt) is a kind of insect pathogenic Gram-positive bacteria, it can produce a specific Insecticidal Crystal Proteins( ICPs) during its sporulation phase. CrylAc toxin is a kind of Bacillus thuringiensis insecticidal crystal protein, it can induce a pre-pore in epithelial cells of insect midgut, eventually leading to cell dehydration and death. Lipid rafts is a specific micro-structure locate in brush border membrane lipid bilayer vesicles that abundant with cholesterol, sphingolipids and glycosyl phosphatidylinositol (GPI) anchoring protein, it also provide a platform for activities of cell biology and it has played an important role in many biological activities. Incubated with BBMV at 37℃for 1h Cry1 Ac toxin monomer can form a 250kDa oligomers structure, this structure is the key to membrane perforation and result to insects'death. The formation of toxic oligomer is due to Cry1 Ac toxin monomer interaction with the receptor of cadherin.then the oligomer binding on the GPI-anchored protein which help for the formation of pre-pore which leading to insect died eventually.Lipid rafts is a special area of BBMV, whether isolated BBMV and isolated Lipid Raft can form oligomeric structure by incubat with CrylAc toxin monomers, this is an important point of our study. Whether the mode of action of Bt toxin to sensitive and resistant strains of Helicoverpa armigera is alike,Whether the resistant strain of Helicoverpa armigera can form oligomers and is that different Lepidoptera insects'have different mechanism of oligomer formation,All these problems we are intend to research. We have studied five kinds of insects,including resistant and susceptible strains of Helicoverpa armigera, Imported cabbage worm, Chilo suppressalis and Spodoptera litura. Form our research we have proved that the formation of CrylAc toxin oligomer has no direct relation with vitro Lipd Raft, in vitro CrylAc toxin can not form oligomer structure unless incubate with BBMV, but the integrity of lipid rafts is play a crucial role. We use the cholesterol inhibitor, methyl-β-cyclodextrin and saponin treatment BBMV. centrifuged to remove cholesterol which rich in lipid rafts, and then the the treated BBMV was incubatted with Cry1 Ac toxin.the result is that it can not form oligomers, this results indicated that the integrity of the lipid rafts play an important role.It can not formation the oligomeration after lipid raft incubatted with Cry1 Ac toxin in vitro.the results indicated that the action of their Biological function must rely on the environment of BBMV; At the same time, it also was confirmed that GPI-anchored protein which locate on Lipid Rafts micro-area is a key factor to the form of oligomers. BBMV was treatmented by PIPLC which can remove GPI-anchored protein. then oligomers structure can not be formed; Toxin-binding result show that the Cry1 Ac toxin monomer mainly combined in Cadherin receptors and Aminopeptidase receptor with little or almost no binding.We have used Dot-blotting. ELISA to detect the Cadherin protein of resistant and susceptible strains of Helicoverpa armigera, Chilo suppressalis and Spodoptera litura. Actually, using the Methods of Dot-blotting and ELISA are qualitative and quantitative detect the circumstance of cadherin protein's presence in insects midgut through the different direction, respectively.The purpose is to find a easy way to detect the resistant of field strain of Helicoverpa armigera.ln Dot-blotting detection, the result of field strain of Helicoverpa armigera is not obvious with room strains of Helicoverpa armigera.:In the ELISA test, the OD values of sensitive and resistant strains of Helicoverpa armigera were small and the sensitive strain was little higher, but the the OD value of Chilo suppressalis and Spodoptera litura were much higher than the two strains of Helicoverpa armigera and can significantly distinguish, the two kinds of detection methods results are consistent.