Molecular Cloning And Expression Analysis Of Interferon Regulator Factor In Cynoglossus Semilaevis And Solea Senegalensis

As an improtant part of marine aquaculture, fishery industry has made rapiddevelopment in China. These years, as a new type of Marine aquaculture ofCynoglossus semilaevis and Solea senegalensis have made a rapid development. Atpresent, our country still exist many kinds of problems in Marine aquacultureincluding increasingly serious diseases. Those have become the key factors restrictingthe development of Marine aquaculture development and causing the big economicloss. As transcriptional regulators of type I interferon and IFN stimulated geneswhich form the first line of antiviral defence in vertebrates, interferon regulatoryfactors play critical diverse roles in a wide variety of cellular processes involvingantiviral response, cytokine signaling, cell growth control and lymphocytedevelopment. IRF-1, IRF-2and IRF-7are the key IRF members due to their criticalroles in transcriptional regulation of IFN gene and ISGs. Therefore, the studies ofinterferon regulator factors have an important significance in prevention viral disease.In this study，six full-length cDNAs and two genomic sequences were cloned andidentified in Cynoglossus semilaevis and Solea senegalensis. In Cynoglossussemilaevis, the full-length cDNA sequence of Cynoglossus semilaevis IRF-1(CsIRF-1)is1456bp in length with a105bp5’-UTR and a541bp3’-UTR. CsIRF-2cDNAconsists of1620bp with an89bp5’-UTR and a532bp3’-UTR. CsIRF-7cDNAconsists of1957bp with a48bp5’-UTR and a607bp3’-UTR. The open readingframes (ORFs) of the three cDNAs translate into269,332and433amino acidsrespectively. In Solea senegalensis, the full-length cDNA sequence of Soleasenegalensis IRF-1(SsIRF-1) is1631bp in length with a143bp5’-UTR and a600bp3’-UTR. SsIRF-2cDNA consists of1923bp with an84bp5’-UTR and an852bp3’-UTR. SsIRF-7cDNA consists of2220bp with a64bp5’-UTR and an869bp3’-UTR. The open reading frames (ORFs) of the three cDNAs translate into295,328and428amino acids respectively. The CsIRF-1gene is3008bp long, containing astructure of10-exon and9-intron as other fish IRF-1genes, while SsIRF-1gene is3480bp long, containing the sames tructure with CsIRF-1genes.Amino acid sequence analysis shows that the same with the other fish IRFssequence the IRFs of Cynoglossus semilaevis and Solea senegalensis consived severconservative structure domain. Such as the DNA-binding domain (DBD), IRF associated domain (IAD), Serine-rich domain (SRD), activation domain (AD),repression domain (RD). DBD is a characteristic of all IRF family members withcontaining a typtophan repeats cluster. Through alignment protein sequence of IRFs invertebrate, the IRF-1, IRF-2and IRF-7amino acid sequence have higher homologywith other fish IRFs respectively. Phylogenetic analysis reveals that the CsIRF-1andSsIRF-1is clustered into IRF-1subfamily with other vertebrates while CsIRF-7andSsIRF-7are clustered into IRF-7subfamily.In order to detect IRF-1, IRF-2and IRF-7mRNA in different healthy tissuedistribution, we use RT-qPCR method to test target genes expression level in liver,kidney, spleen, blood, gill, heart and intestine. The results show that IRF-1, IRF-2andIRF-7were constitutively expressed in all tested tissues. The higher expression ofCsIRF-1and SsIRF-1were detected in blood cells, gill and intestine, while theCsIRF-2and SsIRF-2have a lower level in different. In Cynoglossus semilaevis andSolea senegalensis the IRF-7were higher expressed in intestine, heart, kidney, andspleen.In order to explore the protential roles of IRFs of Cynoglossus semilaevis andSolea senegalensis in antiviral response, RT-qPCR were used to understand theinducible expression profiles of IRFs in four tissues after poly(I:C) stimulation. Thetranscription kinetics of IRFs in spleen, kidney, intestine and gill were shown thesame. The expression of IRFs in different tissues rapid significantly up-regulated afterstimulated by poly (I: C). Furthermore, the expression levels of IRFs in all testedtissues starte to increase moderated at6h, and reached the peak value at24h or48hafter injection of poly(I:C). As time increases the IRFs transcription returned to theoriginal level. This phenomenon was similar with other fish IRFs as described. Allresults above indicated that IRFs might be important in Cynoglossus semilaevis andSenegalese sole immune system. The results indicats that IRFs have potentially roleinvolved in response to virus infection.