Full-length cDNA Cloning and Expression Characteristic Analysis of Hatching Enzyme-like Gene in the Chinese Oak Silkworm,Anther-aea pernyi

Hatching enzymes(HEs) are a kind of proteases that play a key role in hatching of metazoan eggs.By means of bioinformatic analysis and RACE technology,we cloned a full length cDNA(998 bp) of the hatching enzyme-like gene from Antheraea pernyi embryo,which was designated as ApHEL(GenBank accession:JN205047).The ApHEL sequence consists of a 5′-UTR of 15 bp,a 3′-UTR of 98 bp,and an ORF of 885 bp which encodes 294 amino acid residues with the predicted molecular weight of 33.51 kD and isoelectric point of 5.50.The deduced ApHEL sequence has a signal peptide of 16 amino acids in its N-terminus.The ApHEL protease's functional domain harbors a zinc-binding sequence(HEWMHILGFLHMQSTHNR),a Met-turn motif(YDYVSCLHY) and other conserved functional domains of hatching enzymes.Sequence identity of ApHEL with hat-ching enzymes of other species ranges between 30.0% and 85.0%.Phylogenetic analysis based on amino acid sequences of hatching enzyme reveals that Antheraea pernyi has close genetic relationship with Bombyx mori,Culex quinquefasciatus and Drosophila melanogaster,all of which form a monophyletic clade.The ApHEL transcripts could not be detected in early embryo,appeared on the 6th day after initiation of egg incubation and maintained at a low level till the 9th day on which the transcription increased dramatically to the maximum level and kept at that level till hatching,indicating that ApHEL plays an important role in hatching process of Antheraea pernyi eggs.Moreover,ApHEL was specifically expressed in midgut of the 5th instar larvae,suggesting that ApHEL has other biological functions.