The Effects Of Akirin1 And Akirin2 On Resistance To Aeromonas Hydrophila Infection In The Blunt Snout Bream(Megalobrama Amblycephala)

In just over a decade since Akirin was first identified in Drosophila melanogaster and Mus musculus in 2008,nuclear factor Akirin has been recognized closely associated with innate immune defense mechanisms,neural development,limb formation,myogenesis,and tumorigenesis in a variety of animals.In recent years,with the deterioration of water quality and high-density breeding,disease resistance of a unique freshwater fish Megalobrama amblycephala in China has declined,and the incidence rate has continued to rise.Bacterial septicemia caused by Aeromonas hydrophila has the highest mortality rate and is one of the most serious diseases in aquaculture.In the present study,we cloned the Akirin1 and Akirin2 genes in M.amblycephala,detected their expression patterns and immunoprotective effects,and analyzed the regulatory effects of Akirin1 and Akirin2 on NF-κB p50,NF-κB p65 and the antimicrobial peptides LEAP-1and LEAP-2 after overexpression / knockdown in L8824 cells.The interaction of Akirin1/ Akirin2 with NF-κB p50 / NF-κB p65 in vitro was further analyzed using GST pull-down assay,and the transcriptional regulation of LEAP-1 and LEAP-2 on NF-κB p65 was examined by dual-luciferase reporter assay.Therefore,exploring the role of Akirin1 and Akirin2 genes in resistance to bacterial infection and understanding the regulation mechanism of innate immune response of M.amblycephala is helpful to solve the problem of frequent fish diseases and provide an important reference for the development of efficient fish vaccines.The main results of the study are as follows:1.The Akirin1 ORF was 570 bp in length,encoding 189 amino acids,with a nuclear location signal(KRRRC)at the N-terminal 23-27.The Akirin2 ORF was 558 bp in length,encoding 185 amino acids with two nuclear localization signals,26-30(KRRRC)and 75-78(KRRH),respectively.Multiple sequence alignment analysis showed that Akirin had high similarity among different species.Phylogenetic analysis showed that Akirin of M.amblycephala was most closely related to those of C.idella,followed by D.rerio and P.promelas.2.It was found by q RT-PCR that Akirin1 and Akirin2 were expressed in ten tissues tested in healthy M.amblycephala.Akirin1 and Akirin2 expressed highly in the head kidney,spleen and liver,and lowly in the blood.After being infected with A.hydrophila,the expression levels of Akirin1,Akirin2,NF-κB p50,NF-κB p65,LEAP-1and LEAP-2 in the liver and spleen tissues of M.amblycephala were significantly up-regulated.These changes in gene expression indicated that they were involved in the immune response against A.hydrophila infection.The expression levels of Akirin1,Akirin2,NF-κB p50,NF-κB p65,LEAP-1 and LEAP-2 were up-regulated significantly after stimulation of primary hepatocytes and splenocytes of M.amblycephala by LPS and A.hydrophila,indicating that they can respond to the immune response against pathogens in different degrees.Akirin1 and Akirin2 were overexpressed or knocked down in L8824 cells,respectively,and the results showed that Akirin2 positively regulated expression of the NF-κB p50,NF-κB p65,LEAP-1 and LEAP-2 genes.3.Akirin1 and Akirin2 recombinant proteins with His tag were expressed in prokaryotic cells.Then,M.amblycephala was injected with the purified recombinant proteins intraperitoneally.After two days of protein injection,it was challenged with A.hydrophila,and the results showed that both Akirin1 and Akirin2 could improve the cumulative survival rate of M.amblycephala.The GST-tagged NF-κB p65 and the IPT and RHD domains of NF-κB p50 were expressed.The results of GST pull-down assay showed that Akirin1 did not bind to NF-κB p50 and NF-κB p65,while Akirin2 could bind to the IPT domain of NF-κB p50 in vitro.4.The promoter sequences of LEAP-1 and LEAP-2 were obtained by cloning method.Sequence analysis showed that there were transcription factor NF-κB p65 sites in their promoter sequences.Further site-mutations were performed on NF-κB p65 binding sites in the LEAP-1 and LEAP-2 promoters.Dual luciferase reporting assay showed that the activities of LEAP-1 and LEAP-2 promoters reduced after the mutation of NF-κB p65 binding site.