Eur. J. Entomol. 102 (1): 13-19, 2005 | DOI: 10.14411/eje.2005.002

Gene cloning and sequencing of aminopeptidase N3, a putative receptor for Bacillus thuringiensis insecticidal Cry1Ac toxin in Helicoverpa armigera (Lepidoptera: Noctuidae)

Gui-Rong WANG, Ge-Mei LIANG, Kong-Ming WU*, Yu-Yuan GUO
State Key Laboratory of Plant Disease and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences (CAAS), Yuanmingyuan West Road 2#, Haidian District, 100094 Beijing, P.R. China; e-mail: wgrsun@163.com

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.

Keywords: Helicoverpa armigera, Aminopeptidase N, RACE, gene cloning and expression, ligand binding experiment

Received: June 29, 2004; Revised: September 23, 2004; Accepted: November 3, 2004; Published: March 25, 2005  Show citation

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WANG, G., LIANG, G., WU, K., & GUO, Y. (2005). Gene cloning and sequencing of aminopeptidase N3, a putative receptor for Bacillus thuringiensis insecticidal Cry1Ac toxin in Helicoverpa armigera (Lepidoptera: Noctuidae). EJE, 102(1), 13-19. doi: 10.14411/eje.2005.002
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