Identification And Characterization Of A Novel Phage-type Like Lysozyme Gene From Manila Clam Ruditapes Philippinarum

Manila clam，Ruditapes philippinarum，are cultured in Europe and Asia becauseof their important commercial value. Nevertheless, with the expanded production andenvironmental deterioration in recent years, various diseases caused by a wide rangeof microorganisms, from viruses to metazoan parasites resulted in enormous losses tothe clam aquaculture. Like other invertebrates, the R.philippinarum lack acquiredimmunity, and exclusively rely on its innate immunity. Consequently,a betterunderstanding of the clam immune response mechanism is instrumental for givingnew insights into health management and disease control in clam aquaculture.In our study, a phage-type like lysozyme gene was fisrt identified andcharacterized in Manila clam，Ruditapes philippinarum. To our knowledge, there wasno experimental evidence for the existence of phage-type like lysozyme in theeukaryotes until now. A phage-type lysozyme gene (named as RpLysBp) which onlyreported in bacteriophages was fisrt identified in the eukaryocytes.The present study intended to determine and characterize the phage-type likelysozyme cDNA in Manila clam, the expression of RpLysBp in various tissues wasidentified. The expression pattern of RpLysBp challenged with immune stimulantswas also determined in clam, and the lytic activity of the express and purifyrecombinant clam phage-type like lysozyme protein was also analysised.The mainresults were as follow:Firstly，The full-length cDNA of Manila clam phage-type lysozyme consisted of828bp (GenBank accession no. KJ427777), and contained a462bp open readingframe (ORF) coded for a154-amino acids.The5’ untranslated region (UTR) and3’UTR was27bp and339bp respectively, and there were a stop codon (TAA) and apolyadenylation signal (AAATAAA) and a poly (A) tail in the3’UTR. The calculatedmolecular mass and isoelectric point (PI) of deduced protein were17.46kDa and7.10,respectively. SignalP program analysis showed that no signal peptide was identified atthe N-terminal of deduced protein.Secondly，The RT-PTR was employed to determine the express pattern ofRpLysPh mRNA, and the relative RpLysPh mRNA expression in gill, hemocyte,digestive tract，mantle, foot，muscle and haemocyte was calculated using tubulin asreference gene. The hightes level of RpLysPh mRNA expression was detected inhemocytes (P<0.01).To examine the effect of xenobiotics on living organisms, thetranscriptional levels of RpLysPh gene in Manila clam exposed to seawater artificially contaminated with immune stimulants was examined. The resluts showed that theamount of RpLysPh transcripts in all treated group increased gradually afterpost-injection, and the highest level expression(P<0.05) of RpLysPh was observed inLPS treated group at12h, in PGN challenged group at3h, in Poly(I:C) and WGPinjected group at6h, compared to the control group(0h). In the control group, nosignificant difference was observed in the expression of RpLysPh mRNA during theexperiment.Thirdly，coding sequence of RpLysPh was amplifed with specific primersdesigned with the corresponding restriction enzyme sites of EcoRI and NotI at theN-terminus and C-terminus, respectively. The PCR pruduct was cloned into pMD19-Tsimple vector (Takara, China), then digested completely along with the pET30a vector(Novagen, Germany) by restriction enzymes EcoRI and Not I (Takara, China), andligated to construct the recombinant plasmid, which was transformed into DH5αcompetent cells. The expression vector was transformed into the cells of BL21andcultured for4h under the optimal conditons(1mmol/L IPTG, transformationtemperature35℃). The recombinant protein was confirmed by SDS-PAGE, and themolecular weight of the recombinant protein was17.46kDa, which was accord withthe predicting outcomes.Finally, we studied in single nucleotide polymorphisms of five color clam shelltype phage lysozyme gene by three-primer sequencing, resulting genotype data usePopGen32statistical analysis showed, SNP235, SNP285, SNP352, SNP46four SNPloci contain two alleles, and four polymorphic loci are intentionally mutation, whichprovides the basis for future research lysozyme gene mutations and functionalrelationships.