Cloning And Characterization Of The Lysozyme Gene In The Sea Cucumber Stichopus Japonicus

Lysozyme, also called muramidase, is an antibacterial factor for innate immunity. Itexists in many tissues for plant, animal as well as microorganism.A method based on degenerate PCR and rapid amplification of cDNA ends (RACE) forcloning a full-length cDNA was described. This method was more simple and inexpensivethan screening cDNA library, and can be easily adapted to clone other genes. The degenerateprimes were designed on the basis of echinoderm (including starfish and sea urchin) lysozymegenes, lysozyme-like genes and other invertebrate lysozyme genes conserved sequences. A222bp fragment of lysozyme cDNA from Stichopus japonicus was obtained using RT-PCRtechnique. Based on the known fragment of lysozyme, primers were designed for the5’ and3’extensions by RACE techniques. The complete sequence of lysozyme gene cDNA was713bp.The accession number of this gene in GeneBank was EF036468. The complete sequence oflysozyme gene cDNA revealed an open reading frame encoding145amino acids. Ahexanucleotide AATAA consensus signal for polyadenylation was found in the3’ untranslatedregion. Lysozyme gene contained a246bp5’ untranslated region and29bp3’ untranslatedregion; the encoded protein was13.8kDa with isolectric point7.63. There were26specieslysozyme sequences which had high similarity with the Stichopus japonicas lysozyme, andthe Stichopus japonicas lysozyme had the highest similarity with Asterias rubens lysozyme upto72%. Genomic DNA extracted from Stichopus japonicas was used for PCR-amplificationto get the complete sequence of lysozyme gene DNA. Lysozyme gene DNA sequence had a1139bp intron compared to the full lysozyme cDNA sequence.Genomic DNA extracted from Stichopus japonicas was used for PCR-amplification. Thefragment was cloned, sequenced and used as a probe in Southern blots. Stichopus japonicasgenomic DNA was digested by BamH I, EcoR I and Xho I, respectively. The digested DNAwas transferred onto nylon membrance with positive charge through upwards capillarymethod, following0.8%agarose gel electrophoresis. The probe was labled with digoxigenin.The process of hybridization was in a hybridization oven. Only one band was detected in the lane digested by BamH I and Xho I, respectively, suggesting that the Stichopus japonicaslysozyme was coded by a single gene.Total RNA extracted from Stichopus japonicas was used for RT-PCR amplification. Thefragment was cloned, sequenced and used as a probe in Northern blots. The probe was labledby the DIG-PCR method. The total RNA was transferred onto nylon membrance throughupwards capillary method, following1.0%agarose denaturing gel electrophoresis. Theprocess of hybridization was in a hybridization oven. No hybridization signal showed in anylane, suggesting that the Stichopus japonicas lysozyme has a low expression.Comparing with other i-type lysozymes, Stichopus japonicus lysozyme deduced aminoacid sequence has well-conserved active residues (E34and S50) and well-conserved motifsMDVGSLSCG(P\Y)(Y\F)QIK, so it is predicted that Stichopus japonicus lysozyme belongsto the i-type. Analysis of the deduced amino acid residues of the lysozyme gene in ConservedDomain Database shows that the Stichopus japonicas lysozyme has a conserved domain ofHirudo medicinalis destabilase. The putative three-dimensional model of Stichopus japonicaslysozyme also has a remarkably similarity with the Hirudo medicinalis destabilase. And theStichopus japonicas lysozyme also has the active sites of isopeptidase (S81and H117),compared to destabilase and bifunctional enzymes with lysozyme and isopeptidase activities.Therefore, it is predicted that the Stichopus japonicas lysozyme is a bifunctional enzyme withlysozyme and destabilase characteristics, which has glycosidase and isopeptidase activities.