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草鱼褪黑激素受体Mtnr1基因克隆及组织表达分析
胡伟1,2, 吴慧2, 袁汉文1, 郜卫华1, 陈敦学3, 郭利伟1, 许巧情1
1.长江大学 湿地生态与农业利用教育部工程研究中心 湖北 荆州 434023;2.湖南师范大学 省部共建淡水鱼类发育生物国家重点实验室 湖南 长沙 410081;3.贵州大学动物科学学院 贵州 贵阳 550025
摘要:
为了探究褪黑激素受体(melatonin receptor, Mtnr1)基因在草鱼(Ctenopharyngodon idella)中的表达模式和功能,采用RACE技术从草鱼组织中克隆获得Mtnr1基因的6个亚型(Mtnr1Aa、Mtnr1Ab、Mtnr1Alike、Mtnr1Ba、Mtnr1Bb和Mtnr1C)的cDNA序列,采用ProtParam、TMpred、NetPhos、NetNGlyc、Singal4.1及Phyre2等在线软件对Mtnr1基因的6种亚型进行生物信息学分析。同时,通过实时荧光定量PCR检测6种亚型在不同组织中的表达。结果显示,Mtnr1基因6个亚型cDNA全长分别为2045、2036、2031、2799、2535和2477 bp,分别编码350、351、344、356、347和361个氨基酸。6种亚型均由7个跨膜结构域、NRY结构域、CYICHS结构域和NAXXY结构域及G蛋白偶联受体结合位点组成,同时含有多个磷酸化位点和糖基化位点。Mtnr1基因6个亚型蛋白均由20种氨基酸组成,均属于稳定的亲水性蛋白。氨基酸序列比较分析发现,6种亚型与其他鱼对应的亚型同源性很高,分别为93.1%~99.4%、84.8%~95.2%、82.8%~96.8%、90.1%~97.5%、79.7%~98.3%和90.6%~95.6%。邻接法构建系统进化树显示,6种亚型均与鲤科(Cyprinidae)鱼类聚为一支,与鲤鱼(Cyprinus carpio)、鲫鱼(Carassius auratus)和斑马鱼(Danio rerio)亲缘关系最近。此外,6种亚型mRNA在草鱼肝脏、心脏、鳃、脑、肌肉、前肠、中肠、后肠和肾脏9个组织中均有表达,其中,Mtnr1Aa和Mtnr1Ba基因分别在脑和肾脏中表达量最高,表明Mtnr1Aa和Mtnr1Ba可能在草鱼的神经调节和免疫反应中发挥重要作用。
关键词:  褪黑激素受体  草鱼  分子克隆  组织表达
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Molecular Cloning and Expression Analysis of the Melatonin Receptor Gene in Grass Carp (Ctenopharyngodon idella)
HU Wei1,2, WU Hui2, YUAN Hanwen1, GAO Weihua1, CHEN Dunxue3, GUO Liwei1, XU Qiaoqing1
1.Engineering Research Center of Ecology and Agricultural Use of Wetland, Ministry of Education, Yangtze University, Jingzhou, Hubei 434023, China;2.State Key Laboratory of Developmental Biology of Freshwater Fish, School of Life Sciences, Hunan Normal University, Changsha, Hu’nan 410081, China;3.School of Animal Science, Guizhou University, Guiyang, Guizhou 550025, China
Abstract:
In the present study, six isoforms of melatonin receptor 1 (Mtnr1) were cloned from grass carp (Ctenopharyngodon idella) using RACE technology and bioinformatic analysis. The distribution pattern of the six paralogs in different tissues was detected using real-time quantitative polymerase chain reaction (RT-qPCR). The full-length cDNAs of the six isoforms (Mtnr1Aa, Mtnr1Ab, Mtnr1Alike, Mtnr1Ba, Mtnr1Bb, and Mtnr1C) were 2045, 2036, 2031, 2799, 2535, and 2477 bp, with open reading frames (ORF) of 1053, 1056, 1035, 1071, 1044, and 1086 bp, encoding 350, 351, 344, 356, 347, and 361 amino acids, respectively. All of the proteins contain 7 transmembrane domains (TM), an NRY motif, a CYICHS motif, a NAXXY motif, conserved residues interacting with G-proteins in the TM3, asparagine-linked glycosylation sites, and several phosphorylation sites. The six Mtnr1 proteins are composed of 20 amino acids and are stable hydrophobic proteins. Protein sequence identity analysis showed that the six Mtnr1 isoforms share 93.1%-99.4%, 84.8%-95.2%, 82.8%-96.8%, 90.1%-97.5%, 79.7%-98.3%, and 90.6%-95.6% identities with their respective counterparts in other fishes. Phylogenetic analysis revealed that six grass carp Mtnr1 paralogs were clustered into the Cyprinidae clade, sharing a close relationship with those of Danio rerio, Cyprinus carpio, and Carassius auratus. In addition, the expression of the six Mtnr1 isoform genes was detected in the liver, heart, gill, brain, muscle, forgut, midgut, hindgut, and kidney tissues. Results showed that Mtnr1Aa and Mtnr1Ba were highly expressed in the brain and kidney, suggesting that they might play a vital role in nervous system regulation and the immune response in grass carp, respectively.
Key words:  Melatonin receptor  Ctenopharyngodon idella  Gene cloning  Tissue expression