Characterization Analysis Of Hatching Enzyme Gene From Diaphania Pyloalis And Evolution Investigation Based On Gene Structure Of HE In Insect

Diaphania pyloalis is one of main pests in the mulberry fields, belonged to the family pyralidae, Lepidoptera. In this paper, we took Diaphania pyloalis as materials, and cloned hatching enzyme gene of Diaphania pyloalis(Diaphania pyloalis hatching enzyme, DpyHE) full-length cDNA sequence by RACE. The length of DpyHE cDNA sequence was 983 bp, containing 5'UTR 21 bp, 3'UTR 44 bp and a 918 bp open reading frame, which encoded 305 amino acids. The deduced DpyHE consisted of the signal peptide sequences of 16 amino acids in N-terminal and harbored 222 C-terminal amino acids as mature enzyme. Homologous analysis results of the deduced mature enzyme showed that DpyHE has 76%–70.9% identities to that of HE identified in the other lepidoptera insects. The highest was Antheraea pernyi. There was a zinc-dependent metalloproteinase domain in amino acid sequence of DpyHE between Pro90 and Ser240. Using Astacin as a template, we predicted the three dimensional structure of DpyHE by homology modeling. The result showed that the three-dimensional structure of the protease functional domain and the active center of DpyHE were similar to those of Astacin, respectively. Furthermore, we cloned the corresponding gene sequence of mature enzyme, removing signal peptide, and ORF of DpyHE into prokaryotic expression systems for protein expression. Western Blot results verified that the proteins of DpyHE was expressed, which provided the basic data to further analysis of activity of DpyHE.Based on the intron loss of hatching enzyme gene in fish, meaningful results of evolutionary analysis have been obtained recently. The hatching enzyme gene of the Plutella xylostella has only one exon, which is different from the six-exon gene structure of the silkworm hatching enzyme gene. In this paper, we investigated homologous sequence and information of intron in HE structure of representative insects, then analyzed the state of intron loss of hatching enzyme gene and relationship in insect by Notung 2.8 software. We found that hatching enzyme genes in 5 species of Lepidoptera were 6-exon-5-intron structure, while 2 species of hatching enzyme genes in Diptera branches were 3-exon-2-intron structure and only Plutella xylostella in Lepidoptera was single-exon structure. The similar results were reported in the medaka HE structure, which LCE was 9-exon-8-intron and HCE was single-exon structure. To expand the candidates for evolution analysis, we selected 18 representative insects, which had genome sequence in NCBI corresponding to conservative domain of hatching enzyme. Based on the information of intron in HE structure corresponding to conservative domain, eighteen insects could be divided into 0 and 1 categories. The evolution path of investigated insect species maybe existed intron-loss. Nodes of intron-loss and species classification according to molecular phylogeny analysis were coincident. The above preliminary results indicated that the intron-loss in insects hatching enzyme gene might be an ideal candidate to elucidate the evolutionary relationship among insects.