Molecular Cloning And Functional Analysis Of Fas/FasL In Gadus Macrocephalus

Pacific cod nervous necrosis virus(PCNNV)is the main virus that infected the pacific cod(Gadus macrocephalus)and led to high mortality of the cod fry.In this study,Fas and FasL genes were cloned from G.macrocephalus and sequence characterizations were analyzed using bioinformatics.The prokaryotic expression,subcellular localization and effect on EPC of Fas/FasL were carried out.We tried to explore the immunomodulatory mechanism of the pacific cod larval and juveniles during PCNNV outbreak from the perspective of apoptosis,and provide theoretical basis for artificial breeding of the pacific cod.The full-length of FasL cDNA has 1388 bp containing a 315 bp 5′-UTR,a 500 bp 3′-UTR and an open reading frame(ORF)of 573 bp.The ORF encodes a protein of 190 amino acids and contains a TNF family homology domain(THD).The prokaryotic expression vector pET-32 aFasL was constructed,and the recombinant protein FasL with the molecular weight of 39 KDa was purified.Result of mass spectral analysis of recombinant protein FasL was consistent with the expected.MTT test results showed that EPC can be induced to death by high concentration of recombinant protein FasL.Fas is the receptor of FasL,the full-length of Fas cDNA has 1975 bp,including a 5′-UTR of 106 bp,a 3′-UTR of 867 bp and an ORF of 1002 bp.The ORF encodes a protein of 333 amino acids.The predicted protein contained a signal peptide,a transmembrane region,a conserved Fas family signature death domain(DD),and three Cysteine-rich domains.Phylogenetic tree analysis showed that the evolutionary directions of Fas and FasL are consistent with that of species,and the functional domain is highly conserved.The mRNA of Fas and FasL was detected by qPCR.The expression of Fas and FasL could be examined in all tested tissues,and there were higher expressions in spleen and gill.The expression of Fas and FasL was not synchronized during the development of Pacific cod larval.In addition,the expression of Fas gene was up-regulated after the larval was infected by PCNNV,but the expression of FasL was inhibited by high concentration of virus.In this study,the eukaryotic expression vectors,GFP-Fas and pDsRed-FasL were constructed respectively.Results of subcellular localization observation showed that FasL was mainly expressed in the cytoplasm,but FasL was mainly located on cell membrane as star-shaped.The synergistic effect of recombinant protein FasL and Fas overexpression was detected by MTT assay.Results showed that Fas overexpression could increase the mortality of EPC cells induced by FasL recombinant protein.QPCR analysis showed that the overexpression of Fas could significantly upregulated the expression of genes related to Fas/FasL signaling pathway,including bcl-2,bax,RIP3,MLKL,PGAM,while the induction of caspase3,caspase9 gene was not significant.The overexpression of FasL significantly up-regulated expression of bax,caspase3,caspase9,RIP3,MLKL from the Fas/FasL signaling pathway,while the induction of PGAM gene was downregulated.Results of flow cytometry showed that Fas overexpression in EPC cells could induce apoptosis and necrosis,and FasL overexpression resulted in apoptosis of EPC cells,which was consistent with the results of qPCR assay.