Cloning And Expression Analysis Of IRF-5,-10 And CXCR5 In Grass Carp Ctenopharyngoden Idellus

The interferon regulatory factors (IRFs) family are transcription factors that regulate the expression of interferons and interferon-related cytokines. IRF-10 has only been reported in Gallus gallus so far, which can upregulate two primary IFN-y targeted genes, major histocompatibility complex class I and guanylate-binding protein. It has been well documented that in mammals IRF-5 plays an important role in expression of IFN-αand-β, in RIG-Ⅰand Toll-like pathways and also in differentiation of lymphoid cells and apoptosis. However, IRF-5 has not been identified in any other species of fish so far. In the study, the complete cDNA sequence of grass carp Ctenopharyngodon idella IRF-10 (gcIRF10) and IRF-5 (gcIRF5) were cloned using RACE technique.The gcIRF-10 has 1.501 nucleotides with an open reading frame (ORF) of 1191 nt that translates into a 397 amino-acid putative peptide, with a 5'untranslated region (UTR) of 138 bp and 3'UTR of 169 bp. The deduced amino acid sequence shows overall 46.9-79.2% identity with homologous proteins from amphibians, avians and fish, 75.5-94.3% identity with the N-terminal DNA-binding domain (DBD) and 40.4～67.4% identity with the IRF association domain (IAD). The genomic DNA sequence of gcIRF-10 contains 3357 bp and consists of 8 exons and 7 introns. However, the length of IRF-10 genomic DNA in chicken (Gene ID 395243), frog (ENSXETG00000002549), zebrafish (Gene ID 405815), fugu (ENSTRUG00000002917), three-spined stickleback (ENSGACG00000015123) and grass carp (GenBank aceesion no. FJ556997) change from 10.11 kb to 2.662 kb, the difference is mainly due to the variable sizes of their introns, indicating that vertebrate IRF-10 genes are evolutionarily conserved. Analysis the promoter revealed that gcIRF-10 lacks a TATA box. There has sixteen putative binding sites for IRF molecules, GAAA/TTTC motifs and two IRF-8 binding sites, a IRF-2 binding site. gcIRF-10 was observed at a high level of expression in 6 hpf, but its expression declined significantly in larvae of 0-5 dpf, and then increased from 8 dpf onwards. RT-PCR and Western blotting analyses demonstrated that the mRNA and protein of grass carp IRF-10 gene have similar expression patterns, being constitutively expressed in all organs examined from healthy fish, with the highest level in trunk kidney, liver and spleen. By real-time quantitative RT-PCR analysis, gcIRF-10 transcripts were significantly up-regulated in head kidney and spleen infected with grass carp reovirus (GCRV) and the bacterium Flavobacterium columnare, demonstrating the role of IRF-10 in immune response.The gcIRF-5 gene with a full cDNA length of 1821 bp contains an open reading frame (ORF) of 1560 nucleotides, encoding a putative 519 amino acid protein, which showed 34.5～83.9% identity to IRF-5 homologues from mammals, amphibian, avian and fish, and 96.2% and 95.0% identity to zebrafish IRF-5 in the DNA binding domain (DBD) and IRF association domain (IAD), respectively. The genomic DNA sequence of gcIRF-5 contains 6075 bp consisting of 9 exons and 8 introns. The expression of gcIRF-5 was observed in all tissues examined including spleen, liver, head kidney, trunk kidney, heart, gill and muscle. The analysis of real-time quantitative RT-PCR revealed that GCRV and F.columnare can induce the expression of gcIRF-5 in spleen and head kidney.The chemokine receptor CXCR5 and its ligand CXCL13 play an important role in production, migration and homing of B cells. In the present study, CXCR5 was also isolated from the grass carp C.idellus, with the cloning of its full-length genomic sequence. The cDNA of grass carp CXCR5 (gcCXCR5) consists of 1518 bp with a 43 bp 5'-untranslated region (UTR) and a 332 bp 3'UTR. An open reading frame of 1143 bp encodes a 381 amino acid peptide, with seven transmembrane helices. The characteristic residues and/or motifs are located predominantly in the extracellular regions and in the third to seventh transmembrane domains. The deduced amino acid sequence shows 37.6-66.6% identities with CXCR5 of mammals, avian and other fish species. The grass carp gene consists of two exons, with one intervening intron, spaced over 2081 bp of genomic sequence. Phylogenetic analyses clearly demonstrate that the gcCXCR5 resembles the CXCR5s of other vertebrates. Real-time PCR analysis showed that gcCXCR5 was expressed in all tested organs and the expression level of gcCXCR5 was highest in trunk kidney, followed by spleen. Ontogenesis analysis indicated that the expression of gcCXCR5 was observed in 6 hpf to 30 dpf. The expression of gcCXCR5 in tissues was significantly modulated by immunostimulants such as peptidoglycan (PGN), lipopolysaccharides (LPS), polyinosinic-polycytidylic acid sodium salt (Poly I:C) and phytohemagglutinin (PHA). These data provide a base for structure-function analysis of the CXCR5 ligand binding and signal transduction.