Preliminary Study On The Immune Function Of TAK1 And Its Binding Protein TAB1 In Blunt Snout Bream

Transforming growth factor-β activated kinase-1(TAK1)is an important upstream signaling molecule in the nuclear factor-κB(NF-κB)signaling pathway.It is activated and participates in various signaling pathways such as differentiation,apoptosis,and immune response.The fish septicemia caused by Aeromonas hydrophila is a century-old problem that has plagued the aquaculture industry for many years and needs to be solved urgently.More and more studies have shown that TAK1 plays an important role in innate immunity as an immune performance gene.Among them,the activation of TAK1 by TAB1 exhibits a complex regulatory pattern.However,the relevant role of TAK1 in Megalobrama amblycephal a(MaTAK1)and the effect of the interaction of TAK1 and TAB1 on the activation of the transcription factor NF-κB remain unclear.This study took the immune-related gene TAK1 as the entry point,hoping to clarify its molecular regulation mechanism in the innate immunity of Tuantou bream,to provide a new target for improving the innate immunity of Tuantou bream against infection,and to solve the antibiotics in the treatment of fish diseases.Dependency issues,offering new possibilities for healthy fish farming.Research methods: The TAK1 and TAB1 genes(MaTAK1 and MaTAB1)of the bream were cloned and characterized by transcriptome sequencing technology and PCR,and their effects in vitro and in vivo were analyzed by q TR-PCR.The expression profile of the reaction was used to verify the effect of MaTAK1 and MaTAB1 on NF-κB promoter activity by dual-luciferase reporter assay.Overexpression plasmids and siRNA were constructed to clarify the effect of MaTAK1 and MaTAB1 on inflammatory factors.By fusing reporter genes and Co-IP to explore the intracellular localization and in vitro interaction of MaTAK1 and MaTAB1.Findings: The ORF sequence of the MaTAK1 gene c DNA is 1626 bp in length,encoding 541 amino acids,the calculated molecular weight is60.8 k Da,and the predicted theoretical isoelectric point is 6.79.The ORF sequence of the MaTAB1 gene c DNA is 1497 bp in length,encoding 498 amino acids,the calculated molecular weight is 53.6k Da,and the predicted theoretical isoelectric point is 5.68.Multiple sequence alignment revealed that MaTAK1 and MaTAB1 are highly conserved among species.Phylogenetic analysis showed that MaTAK1 and MaTAB1 have high species homology and cluster together with other teleosts as new members of the TAK1 and TAB1 family.MaTAK1 and MaTAB1 were specifically expressed at different levels in all the examined tissues.After LPS stimulation and A.hydrophila infection,the expressions of MaTAK1 and MaTAB1 were significantly enhanced in different tissues and MAF cells.Overexpression of MaTAK1 or MaTAB1 alone could not enhance NF-κB promoter activity;after co-overexpression of MaTAK1 and MaTAB1,NF-κB promoter activity was significantly up-regulated.After overexpression of MaTAK1 and MaTAB1,the transcriptional expression levels of pro-inflammatory cytokines IL-1β,IL-6 and IL-8 were significantly up-regulated in MAF cells;after knockdown of MaTAK1,pro-inflammatory cytokines IL-1β,IL-6 The transcriptional expression levels of IL-8 and IL-8 were significantly down-regulated.MaTAK1 and MaTAB1 positively regulate the expression of intracellular inflammatory factors.Subcellular localization analysis showed that both MaTAK1 and MaTAB1 were cytoplasmic proteins.Co-immunoprecipitation and WB experiments showed that MaTAK1 could interact with MaTAB1 to form a complex in vitro.Taken together,the data from this study suggest that MaTAK1 can interact with MaTAB1 and regulate the NF-κB signaling pathway to produce pro-inflammatory factors and then mediate host innate immune responses against pathogen invasion.This study lays a solid theoretical foundation for solving the problem of antibiotic dependence in the treatment of fish diseases and establishing new therapies targeting bacterial infections in fish.