Determination Of Aminopeptidase N Activity In Helicoverpa Armigera, Expression And Purification Of Bt Toxin Receptor Aminopeptidase N1

Planting of Bt cotton is an important tactics in IPM of Bt cotton.However,as a main target pest,cotton bollworm may have the potential to evolve resistance to Bt cotton with the increasing grown area.Evolution of insect resistance threatens the continued success of transgenic crops.The study of mechanism of Bt resistance is a base of resistance monitoring,risk analysis and resistance delaying,and more important to the effective usage of Bt.Aminopeptidase N(APN) has been identified as a putative Bt Cry toxin receptor on the epithelial cells in the midgut of several lepidopteran species.Therefore, study on the recombinant APN should facilitate understanding insecticidal action of Bt toxins and mechanism of resistance to them.In this study,mutations of APN gene were found in the laboratory-selected Cry1Ac-susceptible and resistant strains of H.armigera.The results of alignment of deduced amino acids sequences of APN between these two strains indicated there were a deletion(Thr45) and five amino acid residues mutation(Thr194→Ser194, Val550→Ile550,Ile861→Thr861,Ala902→Pro902,Thr954→Pro954).In this study,the Bac to Bac Baculovirus Expression System was used to express APN1 gene of susceptible and resistant strains.This expression system is an efficient method for producing recombinant baculovirus for expression testing in insect cells with high expression,easy screening,exact modification after transcription and no endotoxin toxicity.Fistly,APN1 gene was cloned into the pFastBacHTb vector,and the recombinant plasmid was transformed into MAX Efficiency^? DH10Bac^TM competent E. coli to generate a recombinant bacmid.The recombinant bacmid DNA was transfected into the insect cell line(Trichoplusia ni,Tn-5B1-4) to generate a recombinant baculovirus with lipofectin.After amplifying and titer,the baculoviral stock was used to infect Tn cells to express the recombinant protein.The expressed recombinant proteins(APN1 from susceptible and resistant strains) were confirmed to be bound to Cry1Ac toxin by ligand blot analysis.The cells were harvested when the value of MOI is 3.The recombinant protem was purified by HisTrap Ni,immobilized metal affinity chromatography,and then eluted the histidine-tagged protein with 40mM imidazole.The purified protein was determined with SDS-PAGE and ligand blot analysis.The activities of APN enzyme were measured in the anterior and posterior regions of H.armigera midgut.The relations between the activities of APN enzyme and Bt resistance were analyzed.The result indicated that no remarkable differences in the anterior and posterior regions of midgut were found.And these results would be a base of further study of the mechanism of Bt resistance to insects.