Cloning and functional analysis of S100A16 gene from Nile tilapia

【Objective】The purpose of the study was to study the structural characteristics, phylogenetic relationship and tissue expression distribution of Nile tilapia(Oreochromis niloticus) calcium binding protein S100A16, to further analyze the role of S100A16 in antimicrobial immunity, and to provide a reference for further study of the function of S100A16 gene in fish immune defense. 【Method】Specific primers were designed according to the sequence of S100A16gene to clone the S100A16 gene of Nile tilapia(On-S100A16). The prokaryotic expression vector was constructed and the amino acid sequence of On-S100A16 was analyzed bioinformatically by using SMART 4.0,DNAMAN 9.0 and MEGA 6.0 software. Real-time fluorescence quantitative PCR was used to detect the tissue distribution of On-S100A16 in Nile tilapia and the expression changes in different tissues of Nile tilapia after Streptococcus agalactiae infection. The fusion protein was prepared and used to incubate Nile tilapia head kidney lymphocytes for detection of inflammatory related factor genes expression after incubation. 【Result】The ON-S100A16 gene obtained by cloning had a coding region of 276 bp in length, encoding 91 amino acid residues,containing S100 conserved domain. On-S100A16 amino acid sequences had high similarity to S100A16 amino acid sequences of other fishes,such as Maylandia zebra,Cyprinodon variegatus,Micropterus salmoides and so on. Phylogenetic analysis showed that On-S100A16 was most closely related to S100A16 of M. zebra. On-S100A16 gene was widely expressed in all tissues of Nile tilapia,and the relative expressions of OnS100A16 were the highest in liver tissue. After S. agalactiae infection,the expressions of On-S100A16 gene in brain, intestine and spleen tissues of Nile tilapia were up-regulated, and all of them reached the peak 24 h after infection. The recombinant protein S100A16 was successfully prepared and used to incubate head kidney lymphocytes. Real-time fluorescence quantitative PCR results showed that the relative expressions of anti-inflammatory factor genes IL-4 and TLR1 were extremely significantly up-regulated(P<0.01, the same below), while the relative expressions of pro-inflammatory factors gene IL-6 and TNF-α were extremely significantly down-regulated. 【Conclusion】The amino acid sequence of OnS100A16 contains the typical S100 structural domain of the S100 protein family, which may have similar biological functions to other S100 proteins. On-S100A16 is involved in the antimicrobial immune response of Nile tilapia by regulating lymphocyte activity.