河川沙塘鳢TLR2基因的克隆及表达分析  

Clone and Expression Analysis of TLR2 Gene from Odontobutis potamophila

作　　者：于兴达 王佩佩[1] 汪亚媛[1] 贾秀琪 张国松[1] 尹绍武[1] 陈树桥 周国勤 

机构地区：[1]南京师范大学,生命科学学院,江苏省生物多样性与生物技术重点实验室,南京210023 [2]南京市水产科学研究所,南京210036

出　　处：《基因组学与应用生物学》2016年第2期285-293,共9页Genomics and Applied Biology

基　　金：江苏省科技支撑计划(农业)项目(BE2013441);“江苏省六大人才高峰”高层次人才项目(2012-NY-032);南京师范大学科技成果转化基金项目(2013-02);江苏省2015年度普通高校研究生实践创新计划项目(SJLX15_0300)共同资助

摘　　要：本研究以河川沙塘鳢（Odontobutis potamophila）为研究对象,采用RACE、实时荧光定量PCR等技术,首次对该鱼的TLR2基因进行了克隆和表达模式分析。主要研究结果如下：TLR2基因的c DNA全长序列为3 473 bp,包括2 631 bp开放阅读框（ORF）、75 bp的5＇UTR区和767 bp的3＇UTR区,并且具有脊椎动物典型的加尾信号AATAAA和26 bp的poly A尾巴,推测该序列编码876个氨基酸。蛋白结构预测表明TLR2蛋白的TIR结构域和LRR基序符合TLR家族的共同特征,其蛋白分子也存在多个功能位点。系统树中河川沙塘鳢与所有鱼类聚类为独立的分支,与鲈形目亲缘关系最近。应用RT-PCR法检测健康河川沙塘鳢鱼体中TLR2基因的m RNA组织表达差异情况。结果显示,TLR2基因在检测的12种组织中均有表达,在肝、肾、肠、血液和胃中都有较高的表达水平。人工感染嗜水气单胞菌后,对河川沙塘鳢3种主要免疫组织（脾、肾、肝）中进行不同时段表达量变化的检测,结果表明,TLR2基因在脾中感染后4 h表达水平发生下降,随后的24~48 h又上升,48 h时达到顶峰,72~96 h维持在略低于24 h水平上。在肾中,感染后表达水平持续上升,且24~96 h间保持相对稳定的表达水平。在肝组织中,感染后一直呈现上升趋势,72 h时达到顶峰,其他时段表达水平相差不大。可见TLR2在河川沙塘鳢抵御外源微生物侵染的先天免疫中发挥重要作用。With Odontobutis potamophila as research object, rapid amplification of cDNA ends （RACE） approaches and quantitative real-time PCR were used to obtain the full length of TLR2 cDNA and to analysis the expression pattern for the first time. The main results were shown as follows： sequence analysis revealed that the full length of TLR2 cDNA were 3 473 bp. And the putative length of open reading frame （ORF） in the gene was predicted as 2 631 bp that encoded 876 amino acids. The length of the 5＇ UTR of the gene was 75 bp, and the length of 3＇ UTR was 767 bp. Each 3＇ UTR in the gene possessed a poly adenylation signal sequences of AAT AAA, and a polyA tails of 26 bp which was the vertebrate typical tailing signal. TLR2 protein of O. potamophila had LRR motifs and TIR domain structure. This was an important characteristic of TLR family. In the TLR2 protein molecule, there also were many functional sites. Phylogenetic trees showed that Odontobutis potamophila was an independent branch and the gene in O. potamophila had closed genetic relationship with Perciformes. Quantitative real-time PCR was used to quantify the mRNA tissue expression differences expression of TLR2 gene in healthy O. potamophila. The results showed that TLR2 gene of 12 tissue were expressed and TLR2 expressed highly in liver, kidney, intestine, blood and stomach. The mRNA expression levels of TLR2 genes in liver, spleen and kidney of O. potamophila were quantified after A eromonas hydrophila infection. The results showed that in the spleen, the expression quantities dropped at 4 hours after injection, followed by another rise at 24-48 hours and reached peak after 48 hours, 72-96 hours maintained at a level slightly lower than 24 hours. In the kidney, the expression levels of infection continue to rise, and the expression level between 24-96 hours remained relatively stable. In liver tissue, it had been showing an upward trend after infection, peaked at 72 hours, the expression levels of other times had no big difference. The results sh

关 键 词：河川沙塘鳢 TLR2 RT-PCR 表达分析 

分 类 号：S917.4[农业科学—水产科学]