Characteristics And Immune Responsive Expression Analysis Of MyD88and ISG15in Turbot, Scophthalmus Maximus

Turbot, Scophthalmus maximus, is a commercially important marine fish specie culturedwidely in north of China. Since it was introduced, its farming scale is growing rapidly. Once it isinfected by pathogens, it can cause massive death and seriously harm turbot breeding.The myeloiddifferentiation factor88(MyD88) is an adapter protein that links toll-like receptors (TLRs) signaltransduction that triggers downstream cascades involved in innate immunity. ISG15is one of theISG family which is stimulated by IFNs through the JAK-STAT pathway and it plays a crucial rolein innate antiviral response. ISG15can combine with virus and plays a role in regulation of avariety of physiological processes. Collectively, these results provide a possibility that SmMyD88and SmISG15play a role in immunity response.In this study, we used the homologous PCR and Rapid Amplification of cDNA Ends (RACE)method to isolate the cDNA and genomic sequences of SmMyD88and SmISG15from the turbot,Scophthalmus maximus, respectively. The full length cDNA of SmMyD88is1619bp whichcontains an855bp open reading frame (ORF) that encoding a putative protein of285amino acidresidues. SmMyD88contains a typical death domain at N-terminal and a TIR domain with threehighly conserved boxes (Box1, Box2and Box3) at C-terminal. The full-length cDNA of SmISG15is793bp long, containing an ORF of474bp that encodes158amino acid residues. The deducedSmISG15protein exhibits high homology with other fish ISG15sequences and possesses twoconserved tandem ubiquitin-like (UBL) domain and a C-terminal LRLRGG conjugating motif. Thegene of turbot ISG15is862bp in length and composed of two exons and one intron. The intron islocated in5’ untranslated region.The tissue distribution of SmMyD88and SmISG15mRNA was examined by qRT-PCR intwelve tissue types including brain, gills, stomach, intestine, heart, head kidney, kidney, liver, spleen,gonad, muscle and skin of healthy turbots and flounders, respectively. The constitutive expressionof SmMyD88and SmISG15were observed in all tissues examined, but there is a high level in thelymphomyeloid-rich tissues, such as the spleen, kidney and head kidney. Thus, it suggests thatSmMyD88and SmISG15play a predominant role in the immune system. Gene expression of SmMyD88and SmISG15were analyzed over a7-day time course in thegills, head kidney, spleen and muscle of turbot. The results showed that SmMyD88was upregulatedin all the tested tissues after the injection of LPS, CpG-ODN2395and TRBIV. After stimulation,the expression level reaches the peak earlier and stronger in immune-related organizations. Theseresults provided the evidences of the different MyD88-depended pathways to recognize the specificpathogen. Gene expression of SmISG15was analyzed over a7-day time course in the gills, headkidney, spleen and muscle of turbot challenged with TRBIV and poly I:C. The results showed thatthe SmISG15was upregulated by poly I:C and TRBIV in all the four tissues, but TRBIV had aweaker inductive action than poly I:C. These results provided insights into the roles of sh ISG15inthe antiviral immunity.Our findings contribute to the research of MyD88-dependent pathway of Toll-like receptorsignal way and the roles of ISGs in antiviral pathways in turbot, and help to develop effectiveimmune strategies to protect turbot from pathogens.