Identification Of Midgut HcAPN3 Gene From Hyphantria Cunea And Binding To Bt Cry Toxin Of HcAPN3

The special primers of aminopeptidase N were designed by BLAST and the result of ITRAQ of Hyphantria cunea midgut, a fragment of APN was obtained by PCR and the full lenth gene of APN was obtained by RACE-PCR method. This obtained sequence was blasted on NCBI and the result showed that it belongs to the family of APN3, named hcapn3, its Gen Bank accession number is KJ013598. Hc APN3 contains Peptidase Gluzincin family region and ERAP1_ like C-terminal domain, the typical structural features of aminopeptidase. The Hc APN3 was expressed in insect cells showed as a 108 k Da protein by SDS-PAGE analysis as well as the Peptidase Gluzincin family domain and ERAP1_C-like C-terminal domain showed as a 58 k Da and 49 k Da protein by SDS-PAGE respectively which was expressed in E. coli. Anti- Hc APN3 was prepared using the recombination proteins of ERAP1_C domain. The expression of APN3 in H.cunea larvae was determined by immunolocalization. The result showed that Hc APN3 was mainly located in the porterior of midgut but little in the anterior of midgut, it did not distributed in other tissues.The protein of Cry1 Ac and Cry9Ea6 were extracted and digested by trypsin. The result showed that the effectiveness was the best when the mass ratio of Cry1 Ac and trypsin was 40:1, Cry9Ea6 and trypsin was 20:1. BBMV proteins of H. cunea was extracted and analysis by SDS-PAGE. Ligand blot analysis showed that Cry9Ea6 could bind to BBMV, Hc APN3 and the two domains, but the signal from Cry9Ea6 toxin binding to Gluzincin domain was more intense than that of ERAP1_C domain. So the synergistic effect of Gluzincin domain and ERAP1_C domain to Cry9Ea6 was measured. The result showed that Gluzincin domain could enhance the activity of cry9Ea6 toxin to insects, but ERAP1_C domain had no effect. Also, ligand blot analysis showed that Cry1 Ac could bind to both Hc APN3 and peptidase Gluzincin family domain, but not bind to ERAP1_C-like C-terminal domain. This is the first report on the aminopeptidase N of H. cunea, studied the ability to bind to Cry9Ea6 and Cry1 Ac by ligand blot assay, which established the foundation for further research.