| Blunt snout bream(Megalobrama amblycephala)is one of the important herbivorous economic fishes in our country.In recent years,with the increase of aquaculture density and the deterioration of environment,many diseases have broken out in the culture of M.amblycephala,which not only caused huge economic losses,but also posed a threat to human health.As a serine/threonine kinase,Interleukin(IL)-1 receptor-associated kinase 4(IRAK4)plays a critical role in the TLR/IL-1R pathway of innate immune.So far,only a few homologues of IRAK4 in teleost fishes,such as large yellow croaker,grass carp,spotted grouper and zebrafish,have been identified,while the structure and function of IRAK4 in M.amblycephala(MaIRAK4)has not been studied.In this study,we cloned and identified the MaIRAK4 gene and preliminarily explored its functions,which will contribute to enrich the relevant theoretical knowledge of fish IRAK4 s and deepen the understanding of innate immune defense mechanism of M.amblycephala.Therefore,it could provide new approaches for the treatment of pathogen-related diseases.Methods: First,we extracted total RNA from the tissues and then reverse transcribed into c DNA.The primers were designed based on the results of high-throughput sequencing and multiple alignment.The open reading frame(ORF)region of MaIRAK4 was amplified by PCR using spleen c DNA as template,and then the bioinformatics analysis was carried out.Secondly,q RT-PCR was conducted to detect the expression patterns of MaIRAK4 in normal tissues and LPS stimulation or Aeromonas hydrophila infection at different times.Then,we investigated the regulation of MaIRAK4 on downstream NF-κB and pro-inflammatory cytokines by dual-luciferase reporter assay and q RT-PCR,respectively.Finally,the interaction between MaIRAK4 and MaMy D88 was studied by subcellular localization and co-immunoprecipitation to further deepen the recognize of the function of MaIRAK4.The results obtained are as follows:1.The ORF region of MaIRAK4 was 1422 bp,encoding 473 amino acids.SMART analysis showed that MaIRAK4 contains two characteristic domains: death domain(DD)and kinase domain(S＿TKc).Sequence similarity analysis and multiple alignment results revealed that the identity of MaIRAK4 sequence with other fishes IRAK4 were more than 56%,and the identity with grass carp was the highest(97.67%).In phylogenetic trees,MaIRAK4 clustered with grass carp firstly and then with other fishes.2.The expression analysis showed that MaIRAK4 was expressed in all examined tissues,with the highest expression level in liver and spleen and the lowest expression level in muscle.The expression of MaIRAK4 significantly increased in spleen and head-kidney after LPS stimulation,and also significantly up-regulated in spleen,head-kidney and liver after Aeromonas hydrophila infection.These results suggest that MaIRAK4 may be involved in the innate immunity of M.amblycephala.3.Dual-luciferase reporter assay showed that MaIRAK4 could slightly induce NF-κB activity.However,when MaIRAK4 were co-transfected with MaMy D88,the NF-κB activity was significantly increased compared with the group transfected with MaIRAK4 alone,while the activity of NF-κB was decreased compared with the group transfected with MaMy D88 alone.These results suggested that MaIRAK4 could obviously promote the NF-κB activity via MaMy D88.Si RNA knockdown assay showed that when MaIRAK4 was knocked down,the expression levels of downstream proinflammatory factors(IL-6,IL-8,IL-1β and TNF-α)were also decreased.These results suggested that MaIRAK4 might be involved in the innate immune response by regulating the NF-κB signal to mediate the expression of downstream inflammatory factors to inhibit A.hydrophila infection.4.In view of the interaction between IRAK4 and My D88 in TLR/IL-1R signaling pathway,we verified by subcellular localization and coimmunoprecipitation assay.The results showed that MaIRAK4 and MaMy D88 might interact through DD domain to form a complex,which transmits downstream signaling. |
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