Molecular Cloning, Expression And Characterization Of Hatching Enzyme GeneⅡ And Its Promoter In The Silkworm, Bombyx Mori

Hatching enzyme (HE) is an enzyme that digests an egg envelop at the time ofembryo hatching. We have reported a kind of B. mori hatching enzyme-like gene, butthe expressed BmHEL presented the weak degradation activity against the eggshells.In medaka, HCE and LCE digested the envelope cooperatively. Whether was thereother hatching enzyme in the silkworm, similar to that of ‘‘HCE-LCE’’ system inmedaka? In the present study, we identified the other kind of hatching enzyme gene(BmHELⅡ)successfully using bioinformatic methods as well as RACE technique,then charactered and expressed BmHELⅡgene and its promoter. These results abovewill be helpful to provide a molecular basis for understanding the mechanismunderlying silkworm hatching as well as spermatogenesis. The researches mainlycontained four parts as following:1. Molecular cloning and characterization of BmHELⅡgeneUsing bioinformatic methods as well as RACE technique, we obtained anotherBmHEL cDNA sequence (BmHELⅡ, GenBank ID: JN627443). The BmHELⅡcDNAwas977bp in length with an open reading frame of885bp which encoded apolypeptide of294amino acids including a putative signal peptide of16amino acidresidues and a mature protein of208amino acids. The deduced BmHELⅡhad apredicted molecular mass of33.62kDa, isoelectric point of5.44and two conservedsignature sequences of astacin family. Bioinformatic analysis results showed that thededuced protease domain amino acid sequence of BmHELⅡhad29.5~87.0%identities to that of HE identified in the other species. The BmHELⅡgene structurewas6-exon-5-intron. The relative level of BmHELⅡtranscript at different stagesduring egg incubation increased with the development of embryos and reached to amaximum just before hatching, hence declined gradually after hatching. Thespatio-temporal expression pattern of BmHELⅡbasically resembled that of hatchingenzyme gene. Moreover, the BmHELⅡtranscript always kept at the high level intestis of silkworm from larvae to moth suggesting that BmHELⅡmay take part in thedevelopment of sperm in the silkworm.2. The verification of the biological function of BmHELⅡThe ORF of BmHELⅡwas subcloned into expression plasmid pET28a, and therecombinant plasmid was transformed into E.coli BL21. The prokaryotic expressive protein existed as inclusion bodies and contained a6×His-Tag sequence, whichfacilitated the target protein purifid through the Ni-NTA affinity chromatography andcould be detected by Western Blot method. After being refolded, the purified proteinBmHELⅡor BmHEL presented the degradation activity against the eggshells. Whilethe mixture of the two proteases at ratio of1:1showed higher enzymatic activity.These results suggested that BmHEL and BmHELⅡplay a role in the silkwormhatching process.3. Cloning and analysis of the promoter of BmHEL and BmHELⅡgeneThe promoter region of BmHEL (BmHELp) and BmHELⅡ(BmHELⅡp) about1.3kb were cloned, respectively. Bioinformatic analysis results suggested that both ofthe promoters harbored some embryo development related transcription factor bindingsites, GATA-1and HSF1. Furthermore, some transcription factor binding sites, forinstance, c-Rel, Tst-1, YY1and Abd-B were only located at BmHELp or BmHELⅡppromoter. A luciferase reporter plasmid driven by BmHELp or BmHELⅡp wasconstructed. Then, the plasmids were transfected to BmN cells, and trancripticalactivities of promoters were investigated at cellular level. These research resultsprovided the preliminary information for screening cis-acting elements related withtissue-specific or regulation of hatching enzyme gene furthermore.4. Comparitive analysis of the promoter of BmHEL in six silkworm varietieswith different hatching rate and wild silkwormSix typical silkworm varieties with highest or lowest hatching rate fromChinese Strain, Japanese Strain and European Strain, and one wild silkworm collectedin Zhenjiang City were selected as experimental materials. The BmHELps andBmandHELp were cloned. The results of multiple sequence alignment showed thatBmHELps had99.5~100%identities among different silkworm variety, suggestingthat the1.3kb promoter might not contain immediate information related withregulation of hatching rate. BmandHELp had87.5~88.0%identities to BmHELpswhich was mainly resulted from the lack of a sequence of81bp in the wild silkworm.