| Interferon regulatory factor genes encode a family of DNA-binding proteins that are involved in the transcriptional regulation of Interferons and IFN-stimulated genes and play critical roles in viral response, cytokine signalling, cell growth regulation and hematopoietic differentiation. Currently in mammals has identified 10 members of IRF, while 11 members were identified in fish, named IRF-11. But studies of IRFs mainly concentrated in mammals, our study select eel and zebrafish, used the method of RACE we cloned full length cDNA of eel IRF1, IRF4 and IRF 10, and used Real-time PCR research IRF1,2,4,5,7 and 10 immune functions by analyzing their tissue distributions and induced expression. IRF4 plays an important role in the process of development and differentiation of B cells, T cells and dendritic cells in mammals. And it can regulate immune pathway through IRF5, IL21, MyD88, NOD2, pGCla and so on. Then, we study the expression level of IRF4 paralogues and their related genes in zebrafish.The full-length cDNA of eel IRF1 was 1674 bp, consisting of a 5’untranslated region (5’-UTR) of 141 bp,a 3’-UTR of 648 bp and an.open reading frame (ORF) of 885 bp encoding 294 amino acids (aa); Eel IRF2 cDNA sequence is 577 bp, encoding 192 aa; Eel IRF5 cDNA sequence was 888 bp, encoding 295 aa; IRF7 cDNA sequence was 1183 bp, encoding 1183 aa; The full-length cDNA of Eel IRF4 was 1716 bp, consisting of a 5’-UTR of 42 bp, a 3’-UTR of 318 bp and an ORF of 1356 bp encoding 452 aa; The full-length cDNA of Eel IRF 10 was 1724 bp, consisting of a 5’-UTR of 60 bp, a 3’-UTR of and 152 bp and an ORF of 1512 bp, encoding 503 aa. Eel IRF1 has 6 tryptophan residues, IRF4 has 5 tryptophan, IRF 10 has 5 tryptophan, among of the sequence of eel IRF1 and IRF4 and IRF 10 found an ATTTA motif.Real-time PCR detection the expression of ten tissues (head kidney, liver, intestine, blood, skin, brain, heart, center kidney, muscle, spleen) in eel IRF1,2,4,5,7, and 10, the results showed that the eel IRF1 expression was highest in heart and intestine, eel IRF2 expression was highest in skin and spleen, eel IRF5 expression was highest in brain, eel IRF7 expression was highest in skin, eel IRF4 expression was highest in liver, eel IRF 10 expression was highest in intestine and blood.To study IRF 1,2,4,5,7,10 expression patterns,we selected parasites, bacteria and viruses induced eel and head kidney cells of eel to detecting expression of these genes. The results show the expression of eel IRFs was down-regulated in head kidney with the increase of infection series by Neosentis celatus, and their expression was high in intestine at second levels of parasites infection, eel IRF4 expression was highest among these IRFs. To evaluate the expression pattern in IRF1,2,4,5,7 and 10 in response to bacterial-based immune challenges, eels were injected intraperitoneally with Aeromonas hydrophila. The results showed that the level of eel IRF2 was not affected with the stimulation of Aeromonas hydrophila, IRF1 and IRF4 expression were up-regulated in spleen at 6h (46.9-fold), IRF4 expression was up-regulated in spleen at 24 h (87.1-fold), and IRF5 expression up-regulated in kidney at 48 h. Eel head kidney cells were investigated with Aeromonas hydrophila and poly I:C, IRF4 expression was significantly up-regulated by Aeromonas hydrophila at 72 h. In contrast, the expression of IRF1,7 and 10 was significantly down-regulated by Aeromonas hydrophila at 72 h, IRF1 and IRF10 expression were up-regulated at lh and then they were down-regulated. However, IRF5 expression has no significant effect by Aeromonas hydrophila and poly I:C. IRF10 expression was significantly up-regulated at 72 h, IRF4 expression was significantly up-regulated by poly I:C at 6,24 and 48 h,58-fold,37.6-fold and 37.6-fold above control levels respectively.In the present study, the expression pattern of IRF4 paralogues and these related genes, for the first time in teleost, which will provide guidance to further explore the regulatory relationship between them. The results showed that these genes all expressed predominantly in known immune tissues while IRF5 also relatively highly expressed in the non-immune tissue muscle. IRF4b, IRF5, IL21, MyD88, and NOD2 showed maternal expression in the oocyte and the higher expression of IRF4a, Mx and pGC1α before hatching may take part in the embryonic innate defense system. And LPS significantly up-regulated zebrafish IRF4a expression at 48 h, down-regulated IRF4b expression at 4 and 24 h post-stimulation, and it can up-regulate the expression of Mx, IL21, NOD2 and pGCla at ceterain time point. And LPS and poly I:C nearly can not affect IRF5 transcript level. However, poly I:C can up-regulate the expression of Mx, IL21, NOD2 and pGC1α. On the contrary, poly I:C down-regulated the MyD88 transcript at most examined time. As mammals IRF4, teleost IRF4a was refractory to poly I:C treatment while IRF4b can be induced by poly I:C. Inducible expreesion pattern and hylogenetic tree imply that tetrapod IRF4 may came from teleost IRF4a. Furthermore, IRF4a and IRF4b displayed a distinguishing expression pattern in vivo and in vitro, which suggest that IRF4 paralogues might play different roles in immune system. The mechanism in the anti-bacterial and anti-viral response needs further study. |
PDF Full Text Request