Sequence Analysis And Preliminary Verification Of Immunological Functions Of Fish Akirin Genes

The innate immune system is the first defense against the microorganisms in all metazoans. Akirinis a new member of innate immune system, and plays a role in the transcription of NF-κB-dependentgenes in the immune deficiency (Imd) pathway of Drosophila melanogaster. Akirins with diversenuclear functions related to metabolism have also been reported. The Akirin homologs from fishparasites like arthropods (i.e. ticks and red mites) and sea lice have self-protective effects, and havebeen developed into vaccines to improve host immunity. China is one of the top aquaculture countries.Study on fish Akirin can enrich the genetic resources and reveal its biological functions in the immumesystem, which is quite important for the aquaculture development.The cDNAs of three akirin2(csakirin2, cpakirin2and pdakirin2) and one akirin1(pdakirin1) werecloned from the transcriptomes of Cynoglossus semilaevis, Chiloscyllium punctatum, andParamisgurnus dabryanus by touchdown PCR, TAIL-PCR,3-RACE and5-RACE techniques.Deduced amino acid sequence analysis indicated that PdAkirin2contained only one N-terminal nuclearlocation signal (NLS), and three others had two NLSs. Sequence alignments and phylogenetic analysisdemonstated that deduced CsAkirin2and CpAkirin2were92%and77%identical to the Akirin2s ofMaylandia zebra and Latimeria chalumnae, respectively. PdAkirin1and PdAkirin2were closely relatedto Akirin1and Akirin2of Danio rerio with the identities of87%and91%, separately.pdakirin2was selected for sub-cellular localization analysis. Four gene fragments encoding formature PdAkirin2with and without the first NSL (pdakirin2-△NLS1), the second NSL(pdakirin2-△NLS2) and both NSLs (pdakirin2-△NLS1&2) were obtained by overlap PCR with specificprimers, subcloned into vector pDsRed2-C1, and transfected into zebrafish embryos cell line ZF4. Theresults showed that PdAkirin2was specifically located in the nucleus, single NLS-truncated mutantswere detected in both cytoplasm and nucleus, and double NLS-truncated mutant were diffuse in thecytoplasm.qRT-PCR was conducted to identify the expression profile of pdakirin2in different tissues ofChinese loach (skin, liver, brain, spleen, gill, sperm, oocyte, intestine, heart, muscle and kidney), itsspatio-temporal expression post bacterial chanllenge, and effects on the expression of transcriptionfactors NF-κB and cytokine (TNFα, IFN-α, IFN-γ, IL-4and IL-1β). pdakirin2was detected in all tissueswith the highest lever in kidney, moderate in sperm and muscle, and lowest in spleen and gill. Itstranscription in kidney, liver and gill was up-regulated in response to challenge with pathogenicAeromonas hydrophila. In the presence of A. hydrophila, poly I:C and LPS, the expression of pdakirin2in primary spleen cells were also stimulated. Consequently, its over-expression induced theup-regulation of most immune cytokines except for IFN-α and IFN-γ. This interaction (i.e. PdAkirin2and14-3-3β) was further confirmed by yeast two-hybrid (Y2H) assay.In summary, four akirin genes were cloned from three fish, and the subcelular location and immunity-related function of PdAkirin2were determined by using mutagenesis and immunologicaltechniques. This study provides theoretical basis to explore the defense mechanism, disease occurence,medicine development and potential application.