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24 h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。
To explore the function of the calcium-binding protein S100P gene(On S100P)in Nile tilapia(Oreochromis niloticus),the On S100P gene was cloned through specific primers designed according to the On S100P sequence obtained from the transcriptome data of tilapia head kidney.The tissue distribution and the expression profiles 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 inflammatory factors expression.The results showed that the length of the CDS of On S100P gene was 288 bp,encoding 96 amino 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-βand IL1-βin 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.
作者
李一
吴雪莹
钟勇
庾娜
黄瑜
王蓓
简纪常
蔡佳
LI Yi;WU Xueying;ZHONG Yong;YU Na;HUANG Yu;WANG Bei;JIAN Jichang;CAI Jia(College of Fisheries,Guangdong Ocean University,Zhanjiang 524088,China;Guangdong Provincial Key Laboratory of Aquatic Animal Disease Control and Healthy Culture,Zhanjiang 524088,China)
出处
《大连海洋大学学报》
CAS
CSCD
北大核心
2024年第4期613-621,共9页
Journal of Dalian Ocean University
基金
国家自然科学基金(32002426,32073006)
湛江市科技计划项目(2022A01012,2022A01006)
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)。
