Molecular Cloning And Expression Analysis Of Toll-like Receptor22(TLR22) In Turbot, Scophthalmus Maximus

Turbot (Scophthalmus maximus) is an important fish cultured all over theworld, and including North China’s coastal areas, but highly vulnerable to theinfection of turbot reddish body irrdovirus (TRBIV). During the last decade，with the development of intensive indoors farming of turbot，various diseases，espeeially viral disease has prevailed leading to a great economic loss in the turbotfarming. As a kind of pathogenic pattern recognition receptors (PRRs), Toll-likerceptors (TLRs) can identify the specific pathogens and activate the downstreamsignaling patnway, which leasing to the expression of antibacterial proteins. Toll-likerceptors not only play an important role in the innate immune response but also activethe acquired immunity. TLRs have a central role in innate immunity and are alsorequired for the development of an adaptive immune response, bridging innate andadaptive immunity. Myeloid differentiation factor88(MyD88) and TIRdomain-containing adaptor inducing interferon-β(TRIF) have been found to be keyadaptors that mediate two individual TLR signaling cascades, respectively: theMyD88-dependent pathway and the TRIF-dependent pathway. Both pathways resultin the activation of nuclear factor kappa B (NF-κB) and mitogen-activated proteinkinase (MAPK), thereby stimulate gene expressions of proinflammatory cytokinesand interferons (IFNs), resulting in the clearance of the microbial infection from hosts.Since TLRs play an important role in host’s anti-pathogen responses, the study ofTLRs in turbot will be beneficial to the understanding of its immune system, anddevelopment of strategies of disease control for this commercially important species.So far, there have been no research reports about turbot TLRs.The objects of this study are turbot, we used the homologous PCR, RapidAmplification of cDNA Ends（RACE）and Genome walking PCR method to isolate the cDNA and genomic sequences of turbot TLR22(SmTLR22). The full-lengthSmTLR22cDNA (GenBank accession no. JF312911) is4183bp long, with an openreading frame (ORF) of2889bp that encodes962amino acid residues, a3’-untranslated region (3’-UTR) of260bp and a5’-UTR of1034bp. The SmTLR22gene is6530bp long, containing a same structure of4-exon and3-intron as some fishTLR22genes. As a major class of PRRs, The SmTLR22belongs to the type Itransmembrane proteins and contain two conserved structures as TLR22from otherfishes, an extracellular leucine-rich repeat (LRR) domain in charge of recognizing andbinding PAMPs, and a cytoplasmic toll/interleukin (IL)-1receptor (TIR) domainresponsible for downstream signaling. The homology search showed that SmTLR22shares high identitiies with other fishes from40.5%to76.7%, with the highestsequence identity to Japenese flounder (76.7%). As shown in the phylogenetic tree，the fish TLR22cluster consists of two distinct clades, marine teleost TLR22andfreshwater teleost TLR22. The turbot TLR22is located in the marine teleost TLR22clade and closely clustered with TLR22from another flat fish Japanese flounder.The tissue distribution of SmTLR22mRNA was examined by qPCR in twelvetissue types including brain, gills, stomach, intestine, heart, head kidney, kidney, liver,spleen, gonad, muscle and skin of healthy turbots. The constitutive expression ofSmTLR22was observed in all tissues examined. Higher levels were detected in theimmune organs including the head kidney, kidney and spleen. Moderate levels wereobserved in the stomach, muscle, intestine, heart, brain, liver and skin. Low levelswere observed in the gills and gonad. Thus, it suggests that SmTLR22plays a rolepredominantly in the immune system.To investigated the expression profile of SmTLR22gene in turbot, the geneexpression of SmTLR22in response to poly I:C, TRBIV and LPS challenges werestudied in a5-day time course in the spleen, head kidney, gills and muscle. Theinduction by poly I:C was earlier and a bit stronger and the spleen exhibited thehighest and earliest increase (Get rid of the influence of low value in muscle tissue).Upon poly I:C challenge, the expression peak of SmTLR22arose at hour6post-injection in the spleen, head kidney and gills and day1in the muscle, with an approximate3.7-,5.5-,2.7-and5.7-fold increase, respectively. Upon challenge withLPS, the maximum induction levels of SmTLR22were3.3-,2.6-,2.8-and2.6-foldand appeared at hour6,6,6and day1post-infection in the spleen, head kidney, gillsand muscle, respectively. In the case of TRBIV challenge, SmTLR22expression wasalso up-regulated, with a maximum increase of6.2-fold arising at day1in the muscleand3.1-,3.1-and2.7-fold arising all at hour6, day1,1in the spleen, head kidneyand gills, respectively. These results provide some evidence for turbot TLR22plays animportant role in immune response.Our findings contribute to an understanding of the TLRs and its signaling pathwayin turbot，thus providing a basis for the breeding of antiviral fish species bytansgenetic technology and for the development of antiviral drug to cure turbotdisease as well.