Cloning and functional analysis of S100A16 gene from Nile tilapia

Abstract

[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 S100A16 gene 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 TNFα 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.

Abstract

摘要：【目的】 研究尼罗罗非鱼 ( Oreochromis niloticus) 钙结合蛋白S100A16的结构特征、系统进化关系及组织表 达分布情况等, 进一步分析S100A16在抗菌免疫中的作用, 为深入研究 S100A16基因在鱼类免疫防御中的功能提供参 考依据。 【方法】根据 S100A16基因序列设计特异性引物克隆尼罗罗非鱼 S100A16基因 ( On-S100A16), 构建原核表达载 体并采用SMART 4.0、DNAMAN 9.0及MEGA 6.0等在线软件对On-S100A16氨基酸序列进行生物信息学分析。通过 实时荧光定量PCR检测 On-S100A16在尼罗罗非鱼各组织中的分布情况及无乳链球菌感染后在尼罗罗非鱼各组织中 的表达变化, 并制备融合蛋白用于孵育尼罗罗非鱼头肾淋巴细胞后, 检测炎症相关因子基因的表达情况。 【结果】克隆 获得的 On-S100A16基因编码区长度为276 bp, 共编码91个氨基酸残基, 含有S100保守结构域。On-S100A16氨基酸序 列与斑马拟丽鱼、杂色鳉、大口黑鲈等其他鱼类的S100A16氨基酸序列具有较高的相似度, 系统发育进化分析也显示 On-S100A16与斑马拟丽鱼S100A16的亲缘关系最近。 On-S100A16基因在尼罗罗非鱼的各组织中广泛表达, 在肝脏 组织中的相对表达量最高; 经无乳链球菌感染后, On-S100A16基因在尼罗罗非鱼脑、肠道和脾脏组织中的表达量呈 上调趋势, 且均在感染24 h后达峰值。成功制备融合蛋白S100A16后用于孵育头肾淋巴细胞, 实时荧光定量PCR检测 结果表明, 抗炎因子基因 IL-4和 TLR1的相对表达量极显著上调 ( P<0.01, 下同), 促炎因子基因 IL-6和 TNF-α的相对表 达量则极显著下调。 【结论】On-S100A16氨基酸序列含有S100蛋白家族典型的S100结构域, 其可能具有与其他S100蛋 白相似的生物学功能。On-S100A16通过调控淋巴细胞活性来参与尼罗罗非鱼的抗菌免疫反应。