Influence Of Bt Cry1Ac Toxin On BBMV Proteins In Helicoverpa Armigera Midgut Through Proteomic Analysis

In order to provide a theoretical basis for the research on the mechanism of action of specific receptor proteins and the insect resistance,brush border membrane vesicles(BBMV) proteins from Fourth instar larvae of Helicoverpa armigera without and 24h after ingestion of Bt Cry1Ac toxin,were prepared by the differential magnesium precipitation method of Wolfersberger,with minor modifications.The BBMV proteins were separated by 2-DE at pH ranges of 3-10 and were analyzed by PDQUEST 2-DE software.After the SDS-PAGE separation of BBMV proteins,ESI-Q-TOF and HCT,tryptic peptide MS/MS were searched for matches in the Swissprot,NCBI and MASCOT databases.Over 149 spots were identified using this approach.These proteins include APNs, H~+-exporting ATPase,actins,ribosomal protein S3a,ADP/ATP translocase,trypsin-like protease,Vacuolar ATP synthase catalytic subunit A etc.After ingestion of Cry1Ac toxin, larvaes of Helicoverpa armigera become anorexy obviously and move slowly,and SDS-PAGE analysis showed a dramatic increase in the expression level of APNs,which was identified as acceptor of Bt toxins.Additionally,we also found Actin and V-ATPase subunit A,both of which were identified as binding proteins of Bt Cry1Ac toxins.Actin was identified as novel proteins that bind Cry1Ac in addition to the previously reported aminopeptidase N.In this study,BBMV proteins from fifth instar larvae of Helicoverpa armigera were prepared,and proteomic approaches based on 2-DE and mass spectrometry were performed to elucidate changes in BBMV proteins without and after ingestion of Cry1Ac toxin.A noticeable increase was found in the level of aminopeptidase N(APN)that is important for detoxification reactions.Additionally,a significant decrease in the expression level of trypsin-like protease is important during early responses and adaptation of the insect to Bt and exposure to its toxins.Furthermore,the increase in V-ATPase subunits indicated elevated cellular energy profile which is necessary to combat toxin stress.The increased level of actin in larvae provided immediate protection by strengthening the midgut epithelium and enhancing cellular defenses in the tissue.This study presented the differences between the BBMV proteins of Helicoverpa armigera without and 24h after ingestion of Cry1Ac toxin,and provided a theoretical basis for research on the action mode of Bt toxin.