Cloning And Functional Study Of Zebrafish Natural Immunity Related Genes TAK1 And TANK

As an important model organism,zebrafish is widely used in the research of vertebrate development and hematopoiesis in biomedicine,and has also been applied in immunity in recent years.Transforming growth factor ?-activated kinase 1(TAK1),a member of the mitogen-activated protein kinase kinase(MAP3K)family.TAK1 plays a vital role in IL-1-mediated NF-?B,JNK,and p38 activation in human and mammals.The TRAF family member-associated NF-?B activator(TANK)is involved in the regulation of the nuclear factor-?B(NF-?B)signaling pathway.However,there are few reports in zebrafish about TAK1 and TANK in innate immunity.To explore the roles of zebrafish TAK1(Danio rerio TAK1,DrTAK1)and zebrafish TANK(Danio rerio TANK,DrTANK)in innate immune,TAK1 splice isomer and TANK gene cloned.The open reading frame(ORF)of DrTAK1 consists of 1 737 nucleotides and codes 578 amino acids,which includes a N-terminal Serine/Threonine protein kinases(S/TKc)and a C-terminal coiled-coil region.The DrTANK comprises 1 047 nucleotides and codes 349 amino acids.The predictions of DrTAK1 and DrTANK are 65 kDa and 39.27 kDa.The isoelectric point of DrTAK1 and DrTANK are 6.64 and 5.64.DrTAK1 migrated around 72 kDa and DrTANK migrated around 50 kDa in immunoblotting assay and were both of DrTAK1 and DrTANK were identified as a cytosolic protein by immunofluorescence staining.Luciferase reporter assay show that overexpression of DrTAK1 in EPC cells down-regulates the activity of IFN in EPC cells;however when co-transferred with zebrafish IRF7,the DrTAK1 can apparently promote the IFN-activation ability of IRF7.And the subsequent experiments have also verified that this phenomenon helps zebrafish resist viruses.DrTANK could not induce the activition of IFN in luciferase reporter assay in EPC cells.However,the IFN-activation was obviously enhanced by DrTANK when the cells were treated with poly(I: C)or grass carp reovirus(GCRV).The data in this study showed that DrTAK1 and DrTANK may play an important role in the zebrafish antiviral innate immune response,laying a foundation for the subsequent research on DrTAK1 and DrTANK functions.