Cloning and expression analysis of APN1 gene from Lymantria xylina

  Objective  Aminopeptidase N(APN) is a class of important kind of BT receptor protein in insect midgut. The mechanism of Cry toxin produced by Bt bacteria to kill insects has been controversial in the academic research, but it is generally believed that the binding of toxin and Bt receptor protein is the necessary link to its virulence. The APN1 gene of Lymantria xylina was studied by gene cloning, biological information analysis and expression patterns, with the purpose to provid a reference for the subsequent study of APN gene family, other Bt receptor proteins and the mechanism of Cry toxin.

  Method  APN1 was cloned by cDNA from midgut of the moth as template in a PCR system. Biological analysis and real time quantitative PCR(qRT-PCR) were performed to analyze the expression pattern of LxAPN1 gene in different ages and tissues of Lymantria xylina.

  Result  The full-length DNA of APN1 gene was cloned from midgut of Lymantria xylina and named LxAPN1. The full-length sequence of LxAPN1 was 3 159 bp, ORF was 3 054 bp, encoding 1 017 amino acids. Sequence alignment and phylogenetic tree analysis showed that LxAPN1 and LdAPN1 were highly homologous, with signal peptide at N-terminal, zinc binding site HEXXH (X₁₈) E and conserved region GAMENWG, and GPI binding site at the end. LxAPN1 was not expressed in egg stage, and expressed in 1−7 instar larvae, the expression of LxAPN1 decreased after larval stage; the expression of LxAPN1 in intestine was significantly higher than that in head and cuticle.

  Conclusion  LxAPN1 was successfully cloned in the midgut of Lymantria xylina. LxAPN1 and LdAPN1 are highly homologous, and the distribution of phylogenetic tree is also very similar, which not only indicates the similarity between Lymantria xylina and Lymantria dispar, but also the similarity of APN1 function between them; The expression of LxAPN1 was the highest in the second instar larvae of Lymantria xylina. And the expression was the highest in the gut from 6 instar larvae, as an Bt receptor in the intestinal of Lymantria xylina.