Abstract
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 fea- tures 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 sus- ceptible 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, recom- binant 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.
Highlights
Bacillus thuringiensis, a Gram-positive bacterium, produces inclusion bodies of insecticidal proteinaceous crystals during sporulation
Because the growers have used large quantity of chemical insecticides to control cotton bollworm in the past it became resistant to insecticides and since the 1990s has became a major threat to cotton production (Wu et al, 1997)
Three Bt-resistant strains (Rdu, Rsh, Rm) were selected from the SS1 strain by incorporating Cry1Ac toxin, Bt insecticides and Cry1Ac transgenic cotton into the diet separately for 46 generations
Summary
A Gram-positive bacterium, produces inclusion bodies of insecticidal proteinaceous crystals during sporulation. CDNAs for APN genes from many Lepidoptera have been cloned and sequenced (Gill et al, 1995; Knight et al, 1995; Hua et al, 1998; Chang et al, 1999; Garner et al, 1999; Yaoi et al, 1999; Zhu et al, 2000; Emmerling et al, 2001; Rajagopal et al, 2003; Wang et al, 2003a) but only from Bt-susceptible strains. * Author for correspondence; e-mail: wkm@caascose.net.cn midgut of H. armigera and found mutations of the APN1 gene in a Cry1Ac-resistant strain. We clone and express a new APN gene named as APN3harm.
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