Mechanism Of Resistance To Bt Toxin Cry1Ac In Helicoverpa Armigera Hübner And Research Of Lipid Raft From Larval Midgut

Helicoverpa armigera,the cotton bollworm,is one of the most important pests of cotton in China.Transgenic cotton that express Bacillus thuringiensis(Bt) insecticidal crystal protein genes has been deployed for controlling cotton bollworm since 1997. However,despite the widespread use of Bt cotton,the mechanism of action of Bt toxins is not yet fully understood.Detailed understanding of Bt resistance mechanisms can help delay or reduce the evolution of resistance in insect populations.There were several steps for the mode of action of Bt toxins in insect.Any change of these steps may cause resistance(altered proteolytic processing,binding site modification, damaged midgut cells repaired).Following the ingestion of Bt crystals by susceptible insects,protoxins are solubilized and hydrolyzed by gut proteinases to an active toxin form.Activated Cry proteins pass through the peritrophic membrane and bind to proteins in brush border membrane of midgut epithelial cells.Toxin binding is followed by events that lead to cell lysis and disintegration of the brush border membrane and eventually insect death.The enzymes from midgut juice and brush border membrane vesicle(BBMV) from larvae were compared between Bacillus thuringiensis-resistant(YCR) and -susceptible(YCS) Helicoverpa armigera.Serine proteinase from midgut juice and BBMV of the susceptible strain were more active than those of the reisistant strain. Zymogram analysis showed that midgut juice of resistant strain lost a activity band when comparaed with susceptilbe strain.Aminopeptidase activity from BBMV of the susceptible strain were more active than that of the resistant strain,and there was no difference in activity of midgut juice.Susceptible strain BBMV had more alkaline phosphatase activity than that of the resistant strain,but lower activity in midgut juice. Esterase activity of midgut juice and BBMV were higher in resistant strain than that of susceptible strain.The number and qualitative of soluble or membrane esterase were different among two strains.There was no difference in CrylAc protoxin activation of midgut juice from resistant and susceptible strain.But the degradation of CrylAc toxin revealed that resistant strain was more faster than susceptible strain.The midgut juice of Helicoverpa armigera larvae showed more intensely degrade the vitro-activated toxin. Fast degradation of CrylAc toxin may be a potential resistance factor in this strain. Transgenic cotton producing full-length protoxin may increase the toxicity to H. armigera.The plasma membrane is made of a lipid bilayer that contains sphingolipids, cholesterol and glycosylphosphatidylinositol-anchored proteins organized in microdomains termed lipid rafts.Lipid rafts are characterized by their insolubility in detergents at 4℃.We use non-ionic detergent(Triton X-100) to treat with BBMV from midgut of Helicoverpa armigera larval,the non-raft membrane would be solubilized.Then we utilize OptiPrep gradient centirfugation,preparations of lipid rafts could be obtained.Glycosphingolipid(GM1) was used as markers of lipid raft. GM1 dot revealed by probing with cholera toxin subunit B coupled to horse radish peroxidase(CTB-HRP) and followed by chemiluminiscent HRP substrates.Our results suggest that lipid rafts in brush border membrane vesicle from Helicoverpa armigera larvae midgut were isolated and identificated successfully.