Cloning And Characterization Of Some Genes In TLR Signaling Pathway Of Large Yellow Croaker

Large yellow croaker (Larimichthys crocea), one of the most important maricultured speciesin China, has suffered from serious disease in recent years which results in huge economic losses.Therefore, to understand its immune mechanism is very important for large yellow croakerdiseases control.In the present study, the full-length cDNA of TLR3, TLR5S, TLR5M, IRF3, IRAK4andIL-1β were cloned by RT-PCR and RACE-PCR techniques in large yellow croaker, and thegenomic structure of some of the genes were described. Additionally, the tissue expression andtemporal expression profiles of these genes after stimulation with LPS, poly I:C and one of themain fish pathogens Vibrio parahemolyticus were discribed. The main results are as follows:(1) The full-length cDNA of TLR3was of3384bp, including a5’-terminal untranslatedregion (UTR) of65bp, a3’-terminal UTR of589bp and an open reading frame (ORF) of2730bp encoding a polypeptide of909amino acid residues. The full-length genome sequence ofPcTLR3was composed of5721nucleotides, including five exons and four introns. The putativePcTLR3protein contained three main domains, including LRR motifs, a transmembrane regionand a TIR domain. Quantitative real-time reverse transcription PCR analysis revealed a broadexpression of TLR3in most tissues, with the predominant expression in the liver. PcTLR3transcript level could be induced in the three tissues by injection with poly I:C. In addition, afterLPS and V. parahemolyticus challenge, a moderate up-regulation and down-regulation ofPcTLR3was found in blood cells and liver, respectively.(2) The full-length cDNA of TLR5S was of2286bp, containing an ORF of2730bp thatencoded a polypeptide of642amino acid residues. The full-length genome sequence of TLR5Swas composed of4260nucleotides, including two exons and one intron; The full-length cDNAof TLR5M was of3285bp, containing an ORF of2658bp that encoded a polypeptide of885amino acid residues. The full-length genome sequence of TLR5M was composed of4448nucleotides, including four exons and three introns. The putative TLR5M protein contained threemain domains, including LRR motifs, a transmembrane region and a TIR domain, but theputative TLR5S protein contained LRR motifs only. Both TLR5M and TLR5S expressed in allthe examined tissues, with highest expression in the heart and liver respectively. After injectionwith LPS, TLR5M expression level showed significant increase in spleen, liver and blood, andTLR5S transcript up-regulated in the spleen and liver; the expression of TLR5M in spleen of large yellow croaker injected with V. parahemolyticus were much significantly lower than that ofthe control group at3h; a significant down-regulation of TLR5S transcript was shown in liver at12h and48h post-injection with V. parahemolyticus.(3) The full-length cDNA of IRF3was of2204bp, containing an ORF of1389bp thatencoded a polypeptide of462amino acid residues. The putative IRF3protein contained a IRFdomain. Real-time PCR analysis revealed a broad expression of IRF3in most tissues, with thepredominant expression in the liver. IRF3transcript level could be induced in the three tissues byinjection with LPS or poly I:C.(4) The full-length cDNA of IRAK4was of1817bp, containing an ORF of1398bp thatencoded a polypeptide of465amino acid residues. The putative IRAK4protein contained twomain domains, including a death domain (DD) and a Serine/Threonine protein kinases catalyticdomain (S_TKc). Real-time PCR analysis revealed a broad expression of IRF3in most tissues,with the predominant expression in the liver. After LPS and poly I:C stimulation, IRAK4expression level significantly increased in the spleen and blood, decreased in the liver. The levelof IRAK4expression decreased in the spleen and blood post-injection with V. parahemolyticus.(5) The full-length cDNA of IL-1β was of1295bp, containing an ORF of768bp thatencoded a polypeptide of255amino acid residues. The putative IL-1β protein contained a IL-1domain. The full-length genome sequence of IL-1βwas composed of2702nucleotides, includingfive exons and four introns. Real-time PCR analysis revealed a broad expression of IL-1βin mosttissues, with the predominant expression in the liver. In the spleen and blood, the expression ofIL-1β increased at24h after injection with LPS, poly I:C and V. parahemolyticus. However, theexpression of IL-1βafter injection with poly I:C were higher than that of control group at6h,12h in spleen and at3h,6h,48h in blood.