尼罗罗非鱼钙结合蛋白S100P基因的克隆及功能分析
Cloning and functional gene analysis of calcium-binding protein S100P in Nile tilapia(Oreochromis niloticus)
李一 1吴雪莹 1钟勇 1庾娜 1黄瑜 1王蓓 1简纪常 1蔡佳1
作者信息
- 1. 广东海洋大学 水产学院,广东 湛江 524088;广东省水产动物病害防控与健康养殖重点实验室,广东 湛江 524088
- 折叠
摘要
为探究尼罗罗非鱼(Oreochromis niloticus)钙结合蛋白S100P 基因(On S100P)的功能,根据从罗非鱼头肾转录组获得的On S100P序列设计特异性引物,克隆得到On S100P,并对其推导的氨基酸序列进行生物信息学分析,利用荧光定量PCR技术,研究其在不同组织中的定位和表达水平及Poly(I∶C)刺激后该基因在各个组织中的表达模式,并表达与制备了重组蛋白,用于孵育尼罗罗非鱼头、肾、淋巴细胞后研究其对炎症因子转录的调控作用.结果表明:On S100P 基因CDS长度为 288 bp,可编码 96 个氨基酸,含有S100 蛋白家族的保守结构域;系统进化树结果分析表明,尼罗罗非鱼同斑马丽鱼(Maylandia zebra)的S100P具有较高的同源性;组织分布分析显示,On S100P在尼罗罗非鱼的不同组织中均有表达,其中以肌肉和鳃的表达量最高;经Poly(I∶C)刺激后,罗非鱼肠道、头肾和脑组织中On S100P表达水平显著升高且刺激 24h后达到高峰;制备的On S100P 重组蛋白(rOn S100P)可诱导肾淋巴细胞MIF、IL8、TNF-β及IL1-β的表达.研究表明,On S100P氨基酸序列具有S100 保守结构域,与其他S100 蛋白家族成员类似,可通过调节淋巴细胞炎症因子的表达影响鱼类的免疫应答.
Abstract
To explore the function of the calcium-binding protein S100P gene(On S100P)in Nile tilapia(Oreo-chromis niloticus),the On S100P gene was cloned through specific primers designed according to the On S100P se-quence obtained from the transcriptome data of tilapia head kidney.The tissue distribution and the expression pro-files of On S100P in various tissues of the Nile tilapia exposed to Poly(I∶C)stimulation were examined by real-time fluorescence quantitative PCR.The recombinant protein of On S100P was expressed and prepared,and was used to incubate Nile tilapia head kidney-derived lymphocytes for the determination of regulatory roles in inflamma-tory factors expression.The results showed that the length of the CDS of On S100P gene was 288 bp,encoding 96 a-mino acids and possessing a conserved S100 domain of S100 protein family.Phylogenetic tree analysis revealed that On S100P had the relative high homology with Maylandia zebra S100P.On S100P was expressed in different tissues of Nile tilapia,with the maximal levels in muscle and gill.After Poly(I∶C)stimulation,the expression levels of On S100P in the intestinal,head and kidney and brain of tilapia were significantly up-regulated,with the peak at 24 h post stimulation.The obtained On S100P recombinant protein(r On S100P)induced the expression of MIF,IL8,TNF-β and IL1-β in head kidney-derived lymphocytes.The findings indicated that On S100P had a conserved S100 domain,similar biological functions like other S100 protein family,and participated in the fish innate immunity by modulating the inflammatory factors expression of lymphocytes.
关键词
尼罗罗非鱼/钙结合蛋白S100P/重组蛋白/Poly(I∶C)/淋巴细胞Key words
Oreochromis niloticus/S100P/recombinant protein/Poly(I∶C)/lymphocyte引用本文复制引用
基金项目
国家自然科学基金(32002426)
国家自然科学基金(32073006)
湛江市科技计划项目(2022A01012)
湛江市科技计划项目(2022A01006)
农业农村部外来入侵水生生物防控重点实验室2023年度开放课题(20230302)
出版年
2024
NETL
NSTL
万方数据