| Yellow catfish(Pelteobagrus fulvidraco)is a highly valuable fish species of increasing commercial interest in China.However,the intensive culture led to a parallel increase of infectious diseases caused by several pathogens,resulting in severe economic losses.The interferon(IFN)response is the first line of immunre defense in vertebrates.The interferon-regulatory factor(IRF)family,identified as transcriptional regulators of the type I interferon system,plays an important role in the innate and adaptive immune response.In this study,13 members of the IRF family were identified from yellow catfish.The deduced amino acid sequences of PfIRFs were analyzed.The basal expression profiles of PfIRFs in various healthy tissues and their modulation expression following ligands exposure were determined by quantitative PCR(qPCR).Furthermore,the subcellular localization of PfIRF3,PfIRF7 and PfIRF9 were investigated.The expression level of IFN was measured in PfIRF-overexpressing cells.1.Characterization of the full-length cDNA of PfIRFsThe full-length cDNA sequences of 13 PfIRFs were cloned,namely PfIRFl,PfIRF2A,PfIRF2B,PfIRF3,PfIRF4A,PfIRF4B,PfIRF4C,PfIRF5,PfIRF6,PfIRF7,PfIRF8,PfIRF9 and PfIRF10.The deduced PfIRF proteins exhibit a characteristic IRF domain architecture,including a DNA-binding domain(DBD)in the N tenninus and a IRF-binding domain in the C terminus.PfIRF3 contains a serine-rich C-terminal domain.All the three IRF4 genes(PfIRF4A,PfIRF4B and PfIRF4C)possess two nuclear localization signals(NLSs)and only PfIRF4A has a serine-rich domain.PfIRF5 comprises two NLSs but no viral activation domain(VID).PfIRF7 contain a serine-rich domain.PfIRF8 and PfIRF9 harbor two NLSs.PfIRF9 have a proline-rich domain.Phylogenetic analysis revealed that these genes fell into four IRF groups identified in all vertebrate taxam,namely IRF1 subfamily(P/IRF1,PfIRF2A and PfIRF2B),IRF3 subfamily(PfIRF3 and PfIRF7),IRF4 subfamily(PfIRF4A,PfIRF4B,PfIRF4C,PfIRF8,PfIRF9 and PfIRF10),and IRF5 subfamily(PfIRF5 and PfIRF6).2.Tissue distribution of PfIRFsThe basal expression profile of each IRF in eight tissues from healthy yellow catfish was determined by qRT-PCR assay.The 13 IRF transcripts were detected in all tested tissues,but the expression patterns varied among the tissues.PfIRF1 was highly expressed in spleen and trunk kidney,whilst expressed at a very low level in skin.PfIRF2A and PfIRF2B were highly expressed in liver.PfIRF3 was expressed predominantly in trunk kidney,with very weak expression in intestine.PfIRF4A,PfIRF4B and P/IRF4C were abundant in head kidney,trunk kidney,gill and spleen.PfIRF5 was expressed predominantly in head kidney,skin and heart.The highest level of PfIRF6 was found in gill,but the lowest level was observed in skin.PfIRF7 was highly expressed in liver,and PfIRF8-10 were highly expressed in gill.3.Modulation of PfIRFs expression by polyI:C,LPS and Edwardsiella ictaluriIn-vivo modulations of the 13 IRF transcripts were measured in spleen and head kidney following stimulation with polyI:C,LPS and Edwardsiella ictaluri.In the head kidney stimulated with polyI:C,the expression levels of PfIRFI,PfIRF3,PfIRF4B,PfIRF4C,PfIRF6 and PfIRF7 reached a peak at 3h.PfIRF8 expression reached maximum at 6h.The highest induction of PfIRF4A,PfIRF5 and PfIRF10 were observed at 12h,whereas the significant up-regulation of PfIRF2A,PfIRF2B and PfIRF9 were detected at 24h.In spleen,PfIRF5 expression reached a peak at 3h,and the expression of PfIRP1,PfIRF2A and PfIRF6 reached maximum at 6h.PfIRF9 and PfIRF4B were induced drastically at 12h and 24h,respectively.The expression of PfIRF2B,PJIRF3 and PJIRF10 were the highest at 48h,while those of PfIRF4A,PfIRF4B,PfIRF7 and PfIRF8 were the highest at 72h.In the head kidney treated with LPS,up-regulation of PfIRF2B,PfIRF5,PfIRF7,PfIRF9 and PfIRF10 were evident at 6h.The expression of PfIRF2A,PfIRF3,PfIRF4B5 PfIRF4C and PJIRF6 reached a peak at 6h.In spleen,the highest levels of PfIRF4A and PJIRF8 were found at 6h.The expression of PfIRFl,PfIRF2B,PfIRF3,PfIRF4B,PfIRF4C,PfIRF6 and PfIRF7 reached a peak at 12h.Significant induction of other IRFs were observed at 24h.In the head kidney infected with E.ictaluri,the expression of PfIRF6,PfIRF5 and PfIRF4B were detected at 6h,12h,and 48h,respectively.Most of the IRF genes(i.e.PfIRF1,PfIRF2A,PfIRF2B,PfIRF3,PfIRF4A,PfIRF4C,PfIRF6,PfIRF7 and PfIRF9)were induced drastically at 72h.PfIRF10 was also induced,with the highest expression at 96h.In spleen,PfIRF4B,PfIRF4A and PfIRF5 were significantly upregulated at 6h,12h,and 48h,respectively.The highest induction of PfIRF2B and PJIRF4C were found at 24h.The levels of PfIRF1,PfIRF2A,PfIRF3 and PfIRF6 reached a peak at 72h,and those of PfIRF7,PfIRF8,PfIRF9 and PfIRF 10 reached a peak at 96h.4.Functional analysis of PfIRF3,PfIRF7 and PfIRF9To investigate the localization of PfIRF3,PfIRF7 and PfIRF9,three expression plasmids were constructed and transfected into EPC cells,namely pEGFP-PfIRF3,pEGFP-PfIRF7 and pDsRed-PfIRF9.At 24h post transfection,GFP fluorescence was detected in the cytoplasms of cells transfected with pEGFP-PfIRF3,suggesting an intracellular localization of PfIRF3.The GFP signal was observed throughout the cells transfected with pEGFP-PfIRF7,whilst the DsRed signal was found in the nucleus of pDsRed-PfIRF9 expressing cells.Furthermore,the cellular trafficking of three transcription factors(PfSTATIA,PfSTAT1B and PfSTAT2)were studied when overexpressed alone or in combinations with PfIRF9.PfSTAT1 A resided around the nucleus,PfSTAT1B distributed in the cytoplasm and nucleus,and PfSTAT2 localized in the cytoplasm.However,when co-expressed with PfIRF9,PfSTAT2 accumulated in the nucleus and colocalized with PfIRF9.Individual PfIRF3,PfIRF7 and PfIRF9 constructs with a GFP tag were transfected into EPC cells.Cells were harvested at 48h post transfection and qPCR was performed to assess the expression level of IFN.As a result,significant induction of IFN was observed for overexpressed PfIRF33 PfIRF7 and PfIRF9,when compared to cells transfected with the plasmid control. |
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