生物钟相关基因在翘嘴鳜肝脏和心脏中的表达研究

发布时间：2018-08-03 15:50

【摘要】：生物钟是昼夜节律产生的物质基础,在生物体的新陈代谢、免疫调节和细胞周期等重要过程中起着关键作用。昼夜节律调节机制的研究,尤其是生物钟及其相关基因对各种生命过程的调节规律的研究,对揭示正常机体的节律性生理过程至关重要。然而迄今为止,维持生物钟运行的确切分子调控过程仍然不够清楚,并且存在较大的种属差异。目前在鱼类中,生物钟基因的研究对象主要为一些模式物种,其它养殖经济品种的相关研究还比较缺乏。本研究采用RACE-PCR方法对翘嘴鳜(Siniperca chuatsi) Clock (Circadian locomoter output cycles kaput)基因进行了克隆,并采用实时荧光定量PCR (qRT-PCR)技术检测了一系列生物钟相关基因与重要功能基因于LD光制(Light-Dark; 06:00-18:00为光照期,18：00-第二天06：00为黑暗期,模拟自然光照条件下约06：00天亮,18：00天黑的模式)下在翘嘴鳜肝脏与心脏组织中的表达水平,分析了各基因在翘嘴鳜肝脏和心脏内mRNA表达的昼夜节律性,以及翘嘴鳜肝脏和心脏内各生物钟基因之间及生物钟基因和功能基因之间表达的相关性。主要研究结果如下：(1)以翘嘴鳜白肌cDNA第一链为模板,克隆出翘嘴鳜Clock基因全长序列,GenBank登录编号为KT895225。获得的序列全长3698bp,包括5'端非翻译区(5'UTR)412bp,开放阅读框(ORF) 2697bp和3'端非翻译区(3'UTR) 589bp, ORF区共编码898个氨基酸。Clock蛋白分子量大小约为101.76kDa,等电点为7.14。(2)通过qRT-PCR技术,研究分析了13个生物钟基因Bmal1、Clock、Cry1、Cry3、 Cry-dash、Npas2、Npas4、Nrld1、Nr1d2、Per1、Per3、Rora、Tim口14个肝脏功能基因ApoA1、ApoB、ApoC1、Cpt1、Cyp、Dhcr7、EL、Fas、Hmgcr、Ldlr1、LPL Lxrα、Pparα、Pparβ在翘嘴鳜肝脏中的昼夜表达规律,以及各基因之间的相关性。13个生物钟基因中,Cry-dash、Npas4、Nrld1、Per1 和 Tim 5个基因在翘嘴鳜肝脏内具有显著的昼夜节律性；14个肝脏功能基因中,Cyp、EL、Hmgcr、Ldlr1、LPL、Lxra、 Pparα 和 Pparβ8个基因有显著的昼夜节律；上述具有昼夜节律的基因之间,生物钟基因Tim、Npas4、Nrld1口Per1,与8个肝脏功能基因中的1个或多个基因,在翘嘴鳜肝脏中的表达呈正相关关系。(3)采用qRT-PCR方法,研究分析了上述13个生物钟基因和12个心脏功能基因Acer2、Dusp28、Kcnh7、Mlf1、Mylk4、Raph1、Rhobtb2、Rhobtb3、Slco5A1、Socs3、 Socs5、Timp2在翘嘴鳜心脏中的昼夜表达规律,及各基因之间的相关性。13个生物钟基因中,Bmal1、Clock、Cry1、Npas2、Nrld1、Nrld2、Per3、Rorα 和 Tim 9个基因在翘嘴鳜心脏内都表现出明显的昼夜节律；12个心脏功能基因中,Dusp28、Raph1、 Rhobtb2、Rhobtb3、Slco5A1、Socs36个基因有昼夜节律显著性；上述具有显著昼夜节律的9个生物钟基因除Nrld1之外,其余8个基因和6个心脏功能基因中的1个或多个基因之间,在翘嘴鳜心脏中的表达具有相关性。本论文获得的结果为翘嘴鳜以及其它鱼类生物钟系统分子作用机制的深入研究提供了实验数据及理论依据。
[Abstract]:The biological clock is the material basis of the circadian rhythm, which plays a key role in the metabolism, immunoregulation and cell cycle of the organism. The study of the regulation mechanism of circadian rhythm, especially the regulation of the biological clock and its related genes to various life processes, is to reveal the rhythmic physiological process of the normal body. So far, the precise molecular regulation process for maintaining the biological clock is still not clear, and there are large species differences. In the fish, the research object of the biological clock gene is mainly some model species and the other breeding economic varieties are still lacking. The RACE-PCR method is used in this study. The Siniperca chuatsi Clock (Circadian Locomoter output cycles kaput) gene was cloned, and a series of biological clock related genes and important functional genes were detected by real time fluorescence quantitative PCR (qRT-PCR) technology. The expression level in the liver and heart tissues of Mandarin Mandarin, at 18:00, at 18:00, under the light conditions, and the expression level in the liver and heart tissues of Mandarin Mandarin, analyzed the circadian rhythms of mRNA expression in the liver and heart of the Mandarin Mandarin, as well as the expression of the genes and functional genes between the circadian clock genes and the circadian clock genes in the Mandarin liver and the heart. The main results are as follows: (1) the whole long sequence of Clock gene was cloned from the first chain of the white muscle cDNA of Mandarin Mandarin, and the full-length sequence of the Mandarin Clock gene was cloned. The sequence full length of 3698bp, including the 5'terminal non translation region (5'UTR) 412bp, the open reading frame (ORF) 2697bp and 3' end non translation region (3'UTR), was coded, and 898 were coded in the region. The molecular weight of amino acid.Clock protein is about 101.76kDa, and the isoelectric point is 7.14. (2). The 13 biological clock genes, Bmal1, Clock, Cry1, Cry3, Cry-dash, Npas2, Npas4, 14 liver function genes, are studied and analyzed. 5 genes of Cry-dash, Npas4, Nrld1, Per1 and Tim have significant circadian rhythms in the liver of Mandarin Mandarin, and the 5 genes of the 14 liver function genes, Cyp, EL, Hmgcr, Ldlr1, LPL, Lxra, Lxra, alpha and beta 8 genes have significant daytime Nocturnal rhythm; the above genes with circadian rhythms, clock genes Tim, Npas4, Nrld1 mouth Per1, and 1 or more genes of 8 liver function genes are positively related to the expression in the liver of Mandarin Mandarin. (3) the above 13 biological clock genes and 12 cardiac function genes, Acer2, Dusp28, Kcnh7, were studied by qRT-PCR. Mlf1, Mylk4, Raph1, Rhobtb2, Rhobtb3, Slco5A1, Socs3, Socs5, and Timp2 in the heart of Mandarin Mandarin, and the correlation between the genes and the.13 biological clock genes. In this case, Dusp28, Raph1, Rhobtb2, Rhobtb3, Slco5A1, and Socs36 genes have circadian rhythms; the above 9 clock genes with significant circadian rhythms except Nrld1, the other 8 genes and 1 or more genes of 6 cardiac function genes, are related to the expression in the heart of the Mandarin Mandarin. The results obtained in this paper It provides experimental data and theoretical basis for further research on molecular mechanism of biological clock system in mandarin fish and other fish.
【学位授予单位】：湖南农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S917.4

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