Transcriptional Expression Patterns Of Grass Carp (Ctenopharyngodon Idella) Immune-related Gene TAK1 And Its Effect On The Activity Of NF-?B Promoter

TAK1 is a member of Ser/Thr protein kinase family.It has been confirmed that mammalian TAK1 and TAK1-binding protein 1(TAB1)are crucial upstream mediators in the NF-?B(Nuclear Factor kappa-B,NF-?B)signaling pathway.The interaction between TAK1 and TAB1 participates in many important physiological and pathological processes,such as cell differentiation,apoptosis and immunoregulation,through activating the NF-?B promoter and then triggering its downstream cascade reaction.Although the homologs of TAK1 gene and NF-?B signaling pathway in grass carp(Ctenopharyrgodon idellus)have been identified,it is still unclear whether Ci TAK1 protein alone has the effect on the Ci NF-?B activation.In our previous studies,we found that the targeted DNA vaccine of Vibrio mimicus could elicit a significant intestinal mucosal immune response in grass carp.And the Ci TAK1 protein in the intestine of the immunized grass carp was significantly upregulated and enriched in the Ci NF-?B signaling pathway.From these results,we guess that the Ci NF-?B signaling pathway activated by Ci TAK1 protein involved in regulating the above intestinal mucosal immune enhancement.Accordingly,Ci TAK1 protein was used as a research object in the study.The transcriptional expression patterns of Ci TAK1 gene in different tissues of healthy grass carp and C.Idella kidney(CIK)cells before and after immune stimulation were analyzed.And subcellular localization of Ci TAK1 protein and its colocalization with Ci TAB1 protein were determined.On the basis,the effect of the Ci TAK1 protein and the interaction between Ci TAK1 and Ci TAB1 proteins on Ci NF-?B promoter activation were further investigated by using CIK cell model.These findings lay a foundation for thorough studies on the Ci TAK1 protein regulatory role in the Ci NF-?B signaling pathway.The primary research contents and results were summarized as follows:1)Analysis of transcriptional expression patterns of Ci TAK1 gene.Here,the total RNA in healthy grass carp tissues was extracted by using TRNzol-A~+total RNA extracting reagent,and then being reverse transcribed into c DNA.The transcriptional expression profiles of Ci TAK1 gene in nine different tissues were firstly detected by RT-q PCR using c DNA as a templet,and we found that Ci TAK1gene was ubiquitous in all examined tissues.Subsequently,lipopolysaccharide(LPS),tumor necrosis factor-?(TNF-?)and vaccine antigen of Vibrio mimicus(named OVepis)in different concentrations were used to stimulated CIK cells.The temporal transcriptional expression levels of Ci TAK1 gene in CIK cells before and after immunostimulation were detected by RT-q PCR,either.The results showed that all tested immunostimulants could significantly upregulate the transcription of Ci TAK1gene,but the up-regulated levels varied according to the different immunostimulants and their concentrations.2)Analysis of subcellular localization of Ci TAK1 protein and its co-localization with TAB1 protein.Here,the secondary structure domain and subcellular localization of Ci TAK1 protein were firstly predicted using online analysis software.Three eukaryotic expression recombinant plasmids with fluorescence labels(pm Cherry-C1-Ci TAK1,p EGFP-C1-Ci TAK1 and pm Cherry-C1-Ci TAB1)were then constructed,respectively.Finally,the resulting recombinant plasmids were transfected individually or co-transfected into CIK cells.After transfection,cells were fixed with polyformaldehyde,followed by nucleus stain with DAPI and fluorescence observation under a confocal microscopy(1200×)or a fluorescence microscopy(400×).The results showed that the CIK cells transfected with pm Cherry-C1-Ci TAK1 alone emitted red fluorescence in the cytoplasm.While in the CIK cells co-transfected with p EGFP-C1-Ci TAK1 and pm Cherry-C1-Ci TAB1,green fluorescence(EGFP-Ci TAK1)overlapped with red fluorescence(m Cherry-Ci TAB1)to form a orange fluorescence in the cytoplasm.These results suggested that Ci TAK1 is a cytoplasmic protein and can co-localize with Ci TAB1 protein in the cytoplasm of CIK cells.3)Effect of the Ci TAK1 over-expression on the Ci NF-?B promoter activity.In the paper,the reporter gene vector(p GL3-basic-Ci NF-?B1)and overexpression vectors(p CMV-Myc-Ci TAK1,p CMV-Myc-Ci TAB1)were firstly constructed,respectively.The expressions of these overexpression vectors were then detected in the transfected CIK cells by western blotting.Finally,a dual-luciferase reporter assay was performed to detect the Ci NF-?B promoter activity in CIK cells stimulated or not with immunostimulation after Ci TAK1 and/or Ci TAB1 transfection.Western blotting showed that Ci TAK1 and Ci TAB1 proteins could express well in the CIK cells after Ci TAK1 and/or Ci TAB1 transfection.The results of dual-luciferase reporter assays showed that both the overexpression of Ci TAK1 protein and co-overexpression of Ci TAK1 and Ci TAB1 could all significantly upregulate the Ci NF-?B promoter activity,and the promoter activity was higher in the latter compared with the former,while the promoter activity was significantly downregulated when overexpressing Ci TAB1 alone.Meanwhile,it was found that immunostimulation could also promote the Ci NF-?B1 promoter activation,especially when the two proteins were co-overexpressed.However,the Ci NF-?B1 promoter activity had no significant change when overexpressing Ci TAK1 alone,and downregulated when overexpressing Ci TAB1 alone.In conclusion,the Ci TAK1 gene were ubiquitously expressed in grass carp tissues.The transcriptional expression level of Ci TAK1 could be significantly upregulated by different immunostimulants such as Vibrio mimicus vaccine antigen.Ci TAK1 is a cytoplasmic protein and can co-localize with Ci TAB1 protein in the cytoplasm.The overexpression of Ci TAK1 alone and co-overexpression of Ci TAK1and Ci TAB1,as well as the vaccine antigen of Vibrio mimicus could all promote the Ci NF-?B1 promoter activation.And there was a synergistic activation between the co-overexpression and vaccine antigen stimulation.Our results lay a good foundation for further exploring the regulatory role that Ci TAK1 protein plays in the Ci NF-?B1signaling pathway.