A cDNA encoding aminopeptidase N3 was cloned by degenerated PCR and RACE techniques. The full-length of APN3harm is 3486bp. Open reading frame is 3042bp in length, encoding 1014 amino acid residues. Its predicted molecular weight and isoelectric point are 117.04 kDa and 5.14, respectively. This deduced amino acid sequence shares some common structural features with aminopeptidase N from Lepidoptera, including the consensus zinc-binding motif HEXXHX18E and the GAMEN motif common to gluzincin aminopeptidases. The full-length of the APN3harm gene from three susceptible and three resistant strains were cloned and sequenced. Comparison analysis revealed fourteen amino acid differences in the APN3harm gene from resistant and susceptible strains and six mutations of amino acids exist in all resistant strains. It is possible that these mutations are related to the resistance of Helicoverpa armigera to Cry1Ac toxin. The results of semi-quantitative RT-PCR showed that the resistance of H. armigera to Cry1Ac is unrelated to the amount of APN3harm mRNA in midgut tissue. In susceptible strains, APN3harm is highly expressed in mid-gut, foregut and hindgut but not in other tissues. To determine if the APN3harm is the receptor of Cry1Ac, recombinant APN3harm protein was successfully expressed in E. coli. A ligand binding experiment showed purified product could bind Cry1Ac toxin. So it is proposed that APN3harm is a putative receptor of Cry1Ac in H. armigera. The sequence of APN3harm was deposited in GenBank with the accession number AY052651.
Using sequence alignment, a conserved domain in the 3' untranslated region (UTR) of the cytoplasmic heat shock protein 90 (HSP90) of Lepidoptera was found. This region is highly variable in other insect groups. Furthermore, universal primers were designed to amplify the complete coding sequence (CDS) of HSP90 from total genomic DNA in Lepidoptera, avoiding the commonly used reverse transcription-polymerase chain reaction (RT-PCR) and 3', 5'-rapid amplification of cDNA ends (RACE) methods based on cDNA. These primers amplified a fragment of about 2.25 kb in the 11 species tested, which represent seven different families of Lepidoptera, including moths and butterflies. The results suggest that the conserved domain of 3'UTR is universal in Lepidoptera and these primers successfully amplify the complete CDS of cytoplasmic HSP90 from genomic DNA. and Peng Jun XU, Tong LI, Jin Hua XIAO, Robert W. MURPHY, Huang DA WEI.