Molecular Characterization and Expression Analysis of Toll-like Receptor 9 Gene in Turbot, Scophthalmus Maximus
Journal: Journal of Clinical Medicine Research DOI: 10.32629/jcmr.v1i2.188
Abstract
Toll-like receptors (TLRs) have emerged as key sensors of invading microbes by recognizing pathogen-associated molecular patterns and activating innate immune responses. TLR9 functions as a pattern-recognition receptor that recognizes unmethylated CpG motifs in bacterial and viral DNA. In this study, the full cDNA and genomic sequences of tlr9 in Scophthalmus maximus (smtlr9) were identified and characterized. The full-length cDNA of smtlr9 was of 3754 bp encoding a polypeptide of 1066 amino acid residues. The genome sequence of smtlr9 was composed of 4083 nucleotides, including three exons and two introns. The putative Smtlr9 protein was characterized by a signal peptide sequence, a leucine-rich repeat domain (LRD) containing 15 leucine-rich repeats (LRRs), a transmembrane region, and a Toll/interleukin-1 receptor (TIR) domain. The putative protein shows the highest sequence identity (36.0~73.6%) to human and fish TLR9s. Phylogenetic analysis grouped Smtlr9 with other teleost Tlr9s. The smtlr9 mRNA was constitutively expressed in all tissues examined, with the highest level in the head kidney and kidney. The smtlr9 transcription level was up-regulated by ODN2395 in the muscle, head kidney, spleen, and gills.
Keywords
Scophthalmus maximus, tlr9, molecular characterization, ODN2395, TRBIV, poly I: C, gene expression
Funding
The Fundamental Research Funds for the Central Universities (201822025, 201762003) and Shandong Provincial Natural Science Foundation (ZR2019MC051)
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[2] Koyama S, Ishii KJ, Kumar H, Tanimoto T, Coban C, Uematsu S, et al. Differential role of TLR- and RLR-signaling in the immune responses to influenza A virus infection and vaccination. J Immunol. 2007; 179(7): 4711-4720.
[3] Narayanan KB, Park HH. Toll/interleukin-1 receptor (TIR) domain-mediated cellular signaling pathways. Apoptosis : an international journal on programmed cell death. 2015; 20(2): 196-209.
[4] Xu Y, Tao X, Shen B, Horng T, Medzhitov R, Manley JL, et al. Structural basis for signal transduction by the Toll/interleukin-1 receptor domains. Nature. 2000; 408(6808): 111-115.
[5] Medzhitov R, Preston-Hurlburt, P, Janeway CAJ. A human homologue of the Drosophila Toll protein signals activation of adaptive immunity. Nature. 1997; 388: 394–397.
[6] Kawai T, Akira S. The roles of TLRs, RLRs and NLRs in pathogen recognition. Int Immunol. 2009; 21(4): 317-337.
[7] Rebl A, Goldammer T, Seyfert HM. Toll-like receptor signaling in bony fish. Vet Immunol Immunopathol. 2010; 134(3-4): 139-150.
[8] Boudinot P, Zou J, Ota T, Buonocore F, Scapigliati G, Canapa A, et al. A tetrapod-like repertoire of innate immune receptors and effectors for coelacanths. J Exp Zool B Mol Dev Evol. 2014; 322(6): 415-37.
[9] Baoprasertkul, P., Peatman, E., Abernathy, J., et al. Structural characterisation and expression analysis of Toll-like receptor 2 gene from catfish. FISH AND SHELLFISH IMMUNOLOGY. 2007; 22(4): 418-426.
[10] Akira S, Takeda K. Toll-like receptor signalling. Nat Rev Immunol. 2004; 4(7): 499-511.
[11] Krieg AM. Toll-like receptor 9 (TLR9) agonists in the treatment of cancer. Oncogene. 2008; 27(2): 161-167.
[12] Genova E, Pelin M, Sasaki K, Yue F, Lanzi G, Masneri S, et al. Induced Pluripotent Stem Cells as a Model for Therapy Personalization of Pediatric Patients: Disease Modeling and Drug Adverse Effects Prevention. Curr Med Chem. 2018; 25(24): 2826-2839.
[13] Oshiumi H, Tsujita T, Shida K, Matsumoto M, Ikeo K, Seya T. Prediction of the prototype of the human Toll-like receptor gene family from the pufferfish, Fugu rubripes, genome. Immunogenetics. 2003; 54(11): 791-800.
[14] Jault C, Pichon L, Chluba J. Toll-like receptor gene family and TIR-domain adapters in Danio rerio. Mol Immunol. 2004; 40(11): 759-771.
[15] Franch R, Cardazzo B, Antonello J, Castagnaro M, Patarnello T, Bargelloni L. Full-length sequence and expression analysis of Toll-like receptor 9 in the gilthead seabream (Sparus aurata L.). Gene. 2006; 378: 42-51.
[16] Takano T, Kondo H, Hirono I, Endo M, Saito-Taki T, Aoki T. Molecular cloning and characterization of Toll-like receptor 9 in Japanese flounder, Paralichthys olivaceus. Mol Immunol. 2007; 44(8): 1845-1853.
[17] Yao CL, Kong P, Wang ZY, Ji PF, Cai MY, Liu XD, et al. Cloning and expression analysis of two alternative splicing toll-like receptor 9 isoforms A and B in large yellow croaker, Pseudosciaena crocea. Fish Shellfish Immunol. 2008; 25(5): 648-656.
[18] Ortega-Villaizan M, Chico V, Falco A, Perez L, Coll JM, Estepa A. The rainbow trout TLR9 gene and its role in the immune responses elicited by a plasmid encoding the glycoprotein G of the viral haemorrhagic septicaemia rhabdovirus (VHSV). Mol Immunol. 2009; 46(8-9): 1710-1717.
[19] Gao Q, Yue Y, Min M, Peng S, Shi Z, Sheng W, et al. Characterization of TLR5 and TLR9 from silver pomfret (Pampus argenteus) and expression profiling in response to bacterial components. Fish Shellfish Immunol. 2018; 80: 241-249.
[20] Han C, Li Q, Liu J, Hao Z, Huang J, Zhang Y. Characterization, evolution, and expression analysis of TLR7 gene subfamily members in Mastacembelus armatus (Synbranchiformes: Mastacembelidae). Dev Comp Immunol. 2019; 95: 77-88.
[21] Qin C, Sun J, He Y, Wang J, Han Y, Li H, et al. Diurnal rhythm and pathogens induced expression of toll-like receptor 9 (TLR9) in Pelteobagrus vachellii. Fish Shellfish Immunol. 2019; 87: 879-885.
[22] Palumbi SR, Martin A, Romano S, Mcmillan WO, Stice L, Grabowski G (1996). The simple fool's guide to PCR. Honolulu: University of Hawaii Honolulu.
[23] Matsushima N, Tanaka T, Enkhbayar P, Mikami T, Taga M, Yamada K, et al. Comparative sequence analysis of leucine-rich repeats (LRRs) within vertebrate toll-like receptors. BMC Genomics. 2007; 8: 124.
[24] Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2001; 25(4): 402-408.
[25] Sachs AB. Messenger RNA degradation in eukaryotes. Cell. 1993; 74(3): 413-421.
[26] Bell JK, Mullen GED, Leifer CA, Mazzoni A, Davies DR, Segal DM. Leucine-rich repeats and pathogen recognition in Toll-like receptors. Trends Immunol. 2003; 24(10): 528-533.
[27] Griebel PJ, Brownlie R, Manuja A, Nichani A, Mookherjee N, Popowych Y, et al. Bovine toll-like receptor 9: a comparative analysis of molecular structure, function and expression. Vet Immunol Immunopathol. 2005; 108(1-2): 11-16.
[28] Skjaeveland I, Iliev DB, Zou J, Jorgensen T, Jorgensen JB. A TLR9 homolog that is up-regulated by IFN-gamma in Atlantic salmon (Salmo salar). Dev Comp Immunol. 2008; 32(6): 603-607.
[29] Zarember KA, Godowski PJ. Tissue expression of human Toll-like receptors and differential regulation of Toll-like receptor mRNAs in leukocytes in response to microbes, their products, and cytokines. J Immunol. 2002; 168(2): 554-561.
[30] Kongchum P, Rexroad CE, 3rd, Hallerman EM, David L, Palti Y. Single nucleotide polymorphism identification, genetic mapping and tissue expression of the rainbow trout TLR9 gene. Anim Genet. 2009; 40(6): 1001.
[31] Klinman DM. Immunotherapeutic uses of CpG oligodeoxynucleotides. Nat Rev Immunol. 2004; 4(4): 249-258.
[32] Rodriguez MF, Wiens GD, Purcell MK, Palti Y. Characterization of Toll-like receptor 3 gene in rainbow trout (Oncorhynchus mykiss). Immunogenetics. 2005; 57(7): 510-519.
[33] Palti Y, Rodriguez MF, Vallejo RL. Mapping of Toll-like receptor genes in rainbow trout. Anim Genet. 2010; 37(6): 597-598.
[34] Meijer AH, Gabby Krens SF, Medina Rodriguez IA, He S, Bitter W, Ewa Snaar-Jagalska B, et al. Expression analysis of the Toll-like receptor and TIR domain adaptor families of zebrafish. Mol Immunol. 2004; 40(11): 773-783.
[35] Byadgi O, Puteri D, Lee YH, Lee JW, Cheng TC. Identification and expression analysis of cobia (Rachycentron canadum) Toll-like receptor 9 gene. Fish Shellfish Immunol. 2014; 36(2): 417-427.
Copyright © 2020 Guanjie Wang, Xuepeng Li, Qiuming Liu, Xianzhi Dong, Guobin Hu
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