Isolation And Characterization Of TLR9,MyD88,IRAK4 And IL-1β In Half-smooth Tongue Sole Cynoglossus Semilaevis

Half-smooth tongue sole (Cynoglossus semilaevis) is a large flatfish species widely distributed alone the coast of China. Because of its good taste and nutrition, C. semilaevis is considered as a commercially important marine fishery resource which is being widely cultured in china. Vibrio anguillarum, a kind of Gram-negative bacterium that causes hemorrhagic septicemia in fishis, is a significant threat to commercial production in marine aquaculture. The cloning of the immune related genes and the research of immune mechanism is helpful to select strains of fish with enhanced resistance to some major diseases.Toll-like receptors (TLRs) are considered as key sensors to trigger the host's innate immune system and adaptive immune responses by recognizing various PAMPs and initiating signal transduction. TLR9, as a member of TLR family, mediates the recognition of unmethylated CpG dinucleotide motifs commonly found in both bacterial and viral genomes. In the current study, the TLR9 gene was isolated from one of flatfish species, C. semilaevis. In the 4588 bp genomic sequence, three exons, two introns, and 5'UTR of 23 bp and 3'UTR of 342 bp were identified. Putative amino acid sequence was 1062 residues long, including a typical conserved cytosolic Toll/interleukin-1 receptor (TIR) domain, 14 leucine-rich repeat (LRR) motifs, with greater than 60% identity to gilthead sea bream Sparus aurata and Japanese flounder Paralichthys olivaceus orthologs. Quantitative RT-PCR analysis indicated a broad expression of csTLR9, especially in spleen and gonads. No statistically significant changes were observed for csTLR9 mRNA levels in spleen and head kidney after inactive V. anguillarum immunisation. In C. semilaevis ontogeny, the expression of csTLR9 appeared to be developmentally regulated. The presence of maternal TLR9 mRNA and the dramatic decrease of TLR9 expression at metamorphic stage indicated TLR9 might be involved in C. semilaevis development. Comparing sequence and expression profile of csTLR9 with mammalian and other piscine TLR9s suggested that the main function of TLR9 might be conserved across vertebrates, although species-specific features were present.Myeloid differentiation factor 88 (MyD88) is a universal and crucial adaptor protein, which plays an essential role in the intracellular signaling elicited by IL-1R/TLR superfamily. In the present study, we report the full-length sequence of MyD88 gene in C. semilaevis. In the 2855 bp genomic sequence, five exons and four introns were identified. The cloned cDNA exhibited 110 bp of 5'UTR, 576 bp of 3'UTR and 858 bp of the entire open reading frame encoding a polypeptide of 285 amino acids. The protein sequence included a typical conserved cytosolic Toll/interleukin-1 receptor (TIR) domain, an intermediate domain (ID) and a death domain (DD), and shared greater than 70% identity with Japanese flounder P. olivaceu ortholog. RT-PCR analysis indicated a broad expression of csMyD88, especially in ovary and spleen. Quantitative RT-PCR analysis indicated that the csMyD88 mRNA levels were significantly increased in the spleen and head kidney after inactive V. anguillarum challenge and the expression of csMyD88 appeared to be developmentally regulated during C. semilaevis ontogeny. Although, species-specific differences were present, the similarity between mammalian and piscine MyD88s suggested that the main function of MyD88 might be conserved across vertebrates.IL-1 receptor-associated kinases (IRAKs) are important mediators in the signal transduction of IL-1R/TLR superfamily members. IRAK4 is able to phosphorylate IRAK1, and overexpression of dominant-negative IRAK4 is blocking the IL-1-induced activation and modification of IRAK1, suggesting a role of IRAK4 as a central element in the early signal transduction of IL-1R/TLR receptors, upstream of IRAK1. The IRAK4 cDNA encodes a protein with 466 amino acids and a calculated molecular mass of 52 kDa. Analysis of the deduced protein sequence revealed an N-terminal death domain and a central kinase domain (S_TKc domain). The overall sequence identity shared between csIRAK4 and other fish orthologs is low, between 40-50%. A broader analysis of a tissue cDNA panel by RT-PCR revealed different level of expressions in a wide variety of tissues. The strongest expression level was observed in spleen,fin,head kidney and gonads samples. The maternal mRNA of IRAK4 in unfertilised eggs and the dramatic decrease of IRAK4 expression level during metamorphosis suggested the important role of IRAK4 in C. semilaevis development. Interleukin-1 (IL-1) is a pleiotropic paracrine and endocrine signaling molecule and is produced by a variety of cell types. The major functions of IL-1βare activation of the proliferation of such lymphocytes as T cells and B cells, activation of cytotoxic activity in macrophage and natural killer (NK) cells, and induction of immunoglobulin (Ig) secretion. The full-length cDNA sequence of IL-1βfrom the C. semilaevis was determined by using PCR with primers designed from known fish IL-1βsequences followed by elongation of the 5'and 3'ends using RACE. The cDNA contains 130 bp 5'UTR, a single ORF of 741 bp that translates into a 247 aa molecule, 417 bp 3'UTR with five cytokine RNA instability motifs (ATTTA), and a polyadenylation signal (AATAAA) at 15 nucleotides upstream of the poly(A) tail. The deduced aa of csIL-1βshowed the highest identity (64%) with Scophthalmus maximus IL-1β. csIL-1βgene was strongly expressed in spleen, fin and head kidney and weakly expressed in muscle. After stimulation with V. anguillarum, we found a significantly increased level of IL-1βexpression in head kidney and spleen compared to that of blank control. In the head kidney and spleen of C. semilaevis stimulated by inactive V. anguillarum showed a peak level of IL-1βexpression at 8 h after intraperitoneal injection, and recovered at 48 h.