Cloning and expression characteristics analysis of IRF9 gene in GIFT Oreochromis niloticus

【Objective】 To clarify the structural characteristics and tissue expression patterns of the interferon regulatory factor 9 gene（OnIRF9） in GIFT Oreochromis niloticus, and its immune response after infection with Streptococcus agalactis and stimulation with Poly（I:C）, which provided theoretical basis for revealing the function mechanism of IRF9in immune response reaction of GIFT O. niloticus and enriching the immunological knowledge of IRF in fish. 【Method】 The cDNA sequence of the OnIRF9 gene was amplified by using RACE technology, bio-informatics analysis was performed by applying online softwares, such as ExPASy, SMART, TMHMM-2.0 and Euk-mPLoc 2.0, while real-time fluorescence quantitative PCR was used to detect the expression characterisitics of the OnIRF9 gene in healthy GIFT O. niloticus tissues and its expression changes after infection with S. agalactiae and stimulation with Poly（I:C）. 【Result】 The full-length cDNA sequence of OnIRF9 gene was 1828 bp, containing a 50 bp 5’-end non-coding region（5’-UTR）, a 485bp 3’-end non-coding region（UTR）, and a 1293 bp open reading frame（ORF）, encoding 430 amino acid residues.OnIRF9 protein had a molecular weight of 48.42 kD and a theoretical isoelectric point（pI） of 6.23 and contained two typical IRF domains of DBD and IAD, without signal peptides and transmembrane domains. The amino acid sequence of OnIRF9 shared 56.2%-95.7% similarity with IRF9 amino acid sequences from other teleosts fish species, had the highest similarity with IRF9 amino acid sequences of Astatotilapia calliptera（95.7%）.The phylogenetic tree constructed based on IRF9 amino acid sequence similarity showed that GIFT O. niloticus clustered with other reference fish species, particularly exhibiting the closest relationship with A. calliptera. The OnIRF9 gene was expressed in brain, blood, muscle, liver, skin, heart, spleen, head kidney, intestine and gill of healthy GIFT O. niloticus, with the highest relative expression level in the brain, which was significantly higher than other tissues（P＜0.05）, while the relative expression level in the gill was the lowest. After S. agalactiae infection and Poly（I:C） stimulation, the expression levels of OnIRF9 gene were extremely significantly up-regulated within 6 h in the spleen, head kidney and brain（P＜0.01）. However, the time at which the relative expression level reached its peak was not consistent, suggesting that the OnIRF9 gene might have complementary coordination in response to immune responses in various tissues. 【Conclusion】 OnIRF9 contains two typical domains of IRF（DBD and IAD）. Especially, the function of the DBD domain is relatively conserved in vertebrates.The OnIRF9 gene plays an important role in the central nervous system and is involved in the immune response reaction of GIFT O. niloticus in resisting pathogen invasion.