Molecular Cloning Of Hatching Enzyme Gene In Philosamia Cynthia Ricini And Construction Of Transgenic Recombinant Plasmid

Philosamia cynthia ricini, which feeds on the leaves of castor and breeds indoor, is belonged to the bombycidae family, Lepidoptera. It is an important economic insect. Hatching enzyme cDNAs of several Lepidoptera insects, including Bombyx mori, Antheraea pernyi, Bombyx mandarina Moore, Rondotia menciana Moore and Diamondback moth, have been reported recently.In this paper, based on the conserved sequences of HE of Lepidoptera identified, we designed and synthesized the degenerated primers to clone the part of the PcrHE gene from the bluish-egg of Philosamia Cynthia ricini. According to the method of RACE-PCR and cDNA synthesis technique we obtained the full-length cDNA sequenc of PcrHE. The cDNA sequence is 962 bp in length, containing 3’UTR, 5’UTR and an ORF of 885 bp, encoding 294 amino acids residues which includes a putative signal peptide of 16 amino acid residues in N-Terminal. Homologous analysis results of the deduce protease domain showed that PcrHE has identity of 92.3%,84.2%,83.3%,82.4%,79.5%,76.1%,44.7%,43.6%,33.9%,33.7%,32.3%,32.3%,32.1%,31.7%,31.4% and 30.8% to ApHEL,BmandHE,BmHE,RmHE,PxHE,BmHEⅡ,CuefHCE,DmHEHS,MLCE,XHE,EHE12,ZHCE1,MHCE23,TuHE,Aatacin and QHE, respectively. According to the phylogenetic tree, PcrHE has the nearest relationship with ApHEL compared with other species. We predicted the three-dimensional structure of PcrHE through homology modeling,and the structure is the similarest to the structure of zymogen structure of crayfish astacin metallopeptidase. we also evaluated the three-dimensional structure of PcrHE by PROCHECK, and residues in most favoured regions is 90.2%, indicated that the structure is reliable.By using the RT-PCR method, we investigate the expression pattern of PcrHE in the Philosamia cynthia ricini eggs at different developmental stages during incubation. The result showed that the PcrHE transcripts could be detected and kept at a low level during the early stages, increased dramatically since 8th day of incubation, and reached to the maximum on 9th day, indicating that it plays an important role in hatching processes. Moreover, PcrHE transcripts being detected in the midgut and testis at larval stage suggested that PcrHE might have other biological functions of digestion and spermatogenesis.We cloned the corresponding gene sequence of mature enzyme, removing signal peptide, ORF of PcrHE into prokaryotic expression systems for protein expression. Western Blot results suggested that the proteins of PcrHE were highly expressed. Finally, we cloned the promoters of silkworm hatching enzyme gene(BmHEⅠp, BmHEⅡp), which drive the expression of PcrHE in transgenic recombinant plasmid pSL-3XP3-EGFP/BmHE Ⅰp-PcrHE and pSL-3XP3-EGFP/BmHEⅡp-PcrHE. We are trying to inject the recombinant plasmid into silkworm eggs, to verify the function of PcrHE in vivo. The above results provide basic information for illuminating hatching mechanism of eri-silkworm eggs at molecular level and also provide molecular basis for improving hatchability and the research of other Lepidoptera insects.