关键词: 大螟, Bt受体蛋白, 氨肽酶N, 基因克隆, 表达谱

Abstract: Aminopeptidase N (APN) is located in the brush border membrane vesicles (BBMV) of insect midgut. It is the major receptor of insecticidal crystal proteins from Bacillus thuringiensis (Bt) and is closely related to the resistance of insects to Bt toxin. To clarify the relationship of APN and the resistance of Sesamia inferens to Bt toxin, the full-length APN-encoding cDNA sequence was cloned from BBMV of S. inferens midgut by degenerate PCR and RACE techniques and named as SiAPN3 (GenBank accession no.: HQ636624). The relative expression levels of SiAPN3 in different larval gut tissues and instars of S. inferens were determined by Quantitative Real-time PCR (qPCR) assays. The results showed that SiAPN3 is 3 411 bp in full-length and consists of an opening reading frame of 3 018 bp that encodes a protein of 1 006 amino acid residues. The predicted molecular weight and isoelectric point are 114 kDa and 4.95, respectively. The putative protein sequence includes one N-linked and twelve O-linked glycosylation sites, a zinc metal binding site motif of HEXXHX18E that is typical of the active sites of zinc-dependent metalloproteases, and a highly conserved GAMEN motif that forms part of the active site. In addition, it contains a cleavable N-terminal signal peptide with 18 amino acids, as well as a glycosylphosphatidylinositol (GPI) anchor signal peptide with 22 amino acids at the C-terminus, which are also the typical characteristics of lepidopteran APN proteins. The expression profiles in different portions of gut of the 4th instar larva revealed that the expression level of SiAPN3 was significantly higher in larval midgut and hindgut compared with that in foregut (P<0.05). The expression profiles in different instar larvae showed that SiAPN3 was expressed in all instars of S. inferens larvae, with the highest level in the 3rd instar larvae and the lowest level in the 1st instar larvae. The expression levels of SiAPN3 at the 3rd and 4th instars were obviously higher than those at other instars, despite lack of statistically significant differences; no significant difference was found in the expression levels among the 1st, 2nd and 5th instars (P>0.05). The results provide experimental evidence for revealing the function of APN gene and the molecular mechanism of resistance to Bt toxin in S. inferens.

Key words: Sesamia inferens, Bt receptor protein, aminopeptidase N, gene cloning, expression profile