Molecular Cloning And Expression Analysis Of Toll-like Receptor3in Turbot, Scophthalmus Maximus

Turbot, Scophthalmus maximus, is a kind of important marine fish species widelycultured in northern China’s coastal areas. In recent years, the increasing farming ofturbot industry of China has been suffered a huge damage from viral epidemics.Especially, turbot reddish body iridovirus (TRBIV) has caused enormous economicloss. Toll-like receptors (TLRs), belonging to the type Ⅰ proteins, are at the front linein the fight against invading microorganisms, they are key mediators of innateimmunity in both vertebrates and invertebrates. The responses of TLRs to pathogenassociated molecular patterns (PAMPs) serve as a universal trigger for the immunesystem, the most crucial result of which is the induction of inflammation responsefactors and interferons (IFNs) via myeloid differentiation factor88(MyD88)-dependent or MyD88-independent signaling pathways. The TLR proteins arecomposed of two main domains, an extracellular leucine-rich (LRR) domain and acytoplasmic Toll/interleukin-1receptor (TIR) domain, connected by a linker knownas transmembrane (TM) domain. The cytoplasmic TIR domain has been shown to beinvolved in the signaling as well as in the localization of TLRs, while the LRR isinvolved in pathogen recognition. TLR3, the first identified antiviral TLR, is anindispensable recognition receptor for host defense against viral infection. TLR3inmammals is involved in recognizing double-stranded RNA (dsRNA) produced duringthe replication of viruses. Each of the TLRs except TLR3triggers activation of theMyD88-dependent signaling pathway. In contrast, TLR3triggers a MyD88-independent signaling pathway via TIR domain-containing adapter inducing IFN-β(TRIF). However, the information of TLR3in turbot is scarce.The full-length SmTLR3cDNA (GenBank accession number KJ194173) is3482bp long and contains an open reading frame (ORF) of2751bp which encodes apolypeptide of916amino acid residues, a5’-terminal untranslated region (UTR) of244bp and a3’-UTR of487bp. Similar to other TLR3in teleost and mammals,SmTLR3contains three domains: a N-terminal LRR domain, TM domain and a C- terminal TIR domain. The SmTLR3protein shares a high sequence identity of52.8%-78.5%to other teleost TLR3s and a lower identity of43.7%to human TLR3. Thelength of the genomic sequence of SmTLR3gene is4483bp (GenBank Accession no.KJ194174), including five exons and four introns. A number of transcription factorbinding sites were identified in the1538-bp flanking region of SmTLR3gene,including ISRE and those for IRFs and STATs. The tissue distribution of SmTLR3was examined by qPCR in various tissues including brain, gills, stomach, intestine,heart, head kidney, kidney, liver, spleen, gonad, muscle and skin of healthy turbots.The results showed that SmTLR3transcript was constitutively expressed in all tissuesexamined. The highest level was observed in the head kidney, followed by stomach,intestine and heart. Low levels were observed in the brain, spleen, gonad, muscle andskin. In the other tissues, a moderate expression was seen.In order to explore the functions of SmTLR3in antiviral immune response, its geneexpression in response to poly I:C and TRBIV challenges were investigated in fourtissues, gills, head kidney, spleen and muscle. Upon challenge with poly I:C, the up-regulation of SmTLR3started at hour3post-injection in the spleen and hour6in thegills, head kidney and muscle. It continued until the end of the experiment, embracingtwo waves of induced expression, in the four organs. The first expression peak ofSmTLR3arose at day1in the gills and muscle with an approximate3.7-and8.7-foldincrease, respectively, and at hour12in the head kidney and spleen with anapproximate8.4-and3.2-fold increase, respectively, while the second peak arose atday5in all four organs with an approximate4.4-,3.6-,10-and3.3-fold increase,respectively. Challenge of animals with TRBIV caused a later and a weaker inductionof SmTLR3, with a single expression peak observed during the5-day time course thatarose very suddenly in most organs. The expression peak was detected at day3,2,1and1in the gills, head kidney, spleen and muscle with an approximate3.3-,4.3-,2.1-and4.6-fold increase, respectively. After peak time, the SmTLR3expressiongradually decreased, but at a level still over control on the end date in all four organs.Our findings may provide a new perspective for better understanding the functions ofthe SmTLR3in antiviral responses in teleost fish, and help to improve strategies forprotection turbot from viral diseases.