【摘要】：生物鐘是晝夜節(jié)律產(chǎn)生的物質(zhì)基礎(chǔ),在生物體的新陳代謝、免疫調(diào)節(jié)和細胞周期等重要過程中起著關(guān)鍵作用。晝夜節(jié)律調(diào)節(jié)機制的研究,尤其是生物鐘及其相關(guān)基因?qū)Ω鞣N生命過程的調(diào)節(jié)規(guī)律的研究,對揭示正常機體的節(jié)律性生理過程至關(guān)重要。然而迄今為止,維持生物鐘運行的確切分子調(diào)控過程仍然不夠清楚,并且存在較大的種屬差異。目前在魚類中,生物鐘基因的研究對象主要為一些模式物種,其它養(yǎng)殖經(jīng)濟品種的相關(guān)研究還比較缺乏。本研究采用RACE-PCR方法對翹嘴鱖(Siniperca chuatsi) Clock (Circadian locomoter output cycles kaput)基因進行了克隆,并采用實時熒光定量PCR (qRT-PCR)技術(shù)檢測了一系列生物鐘相關(guān)基因與重要功能基因于LD光制(Light-Dark; 06:00-18:00為光照期,18：00-第二天06：00為黑暗期,模擬自然光照條件下約06：00天亮,18：00天黑的模式)下在翹嘴鱖肝臟與心臟組織中的表達水平,分析了各基因在翹嘴鱖肝臟和心臟內(nèi)mRNA表達的晝夜節(jié)律性,以及翹嘴鱖肝臟和心臟內(nèi)各生物鐘基因之間及生物鐘基因和功能基因之間表達的相關(guān)性。主要研究結(jié)果如下：(1)以翹嘴鱖白肌cDNA第一鏈為模板,克隆出翹嘴鱖Clock基因全長序列,GenBank登錄編號為KT895225。獲得的序列全長3698bp,包括5'端非翻譯區(qū)(5'UTR)412bp,開放閱讀框(ORF) 2697bp和3'端非翻譯區(qū)(3'UTR) 589bp, ORF區(qū)共編碼898個氨基酸。Clock蛋白分子量大小約為101.76kDa,等電點為7.14。(2)通過qRT-PCR技術(shù),研究分析了13個生物鐘基因Bmal1、Clock、Cry1、Cry3、 Cry-dash、Npas2、Npas4、Nrld1、Nr1d2、Per1、Per3、Rora、Tim口14個肝臟功能基因ApoA1、ApoB、ApoC1、Cpt1、Cyp、Dhcr7、EL、Fas、Hmgcr、Ldlr1、LPL Lxrα、Pparα、Pparβ在翹嘴鱖肝臟中的晝夜表達規(guī)律,以及各基因之間的相關(guān)性。13個生物鐘基因中,Cry-dash、Npas4、Nrld1、Per1 和 Tim 5個基因在翹嘴鱖肝臟內(nèi)具有顯著的晝夜節(jié)律性；14個肝臟功能基因中,Cyp、EL、Hmgcr、Ldlr1、LPL、Lxra、 Pparα 和 Pparβ8個基因有顯著的晝夜節(jié)律；上述具有晝夜節(jié)律的基因之間,生物鐘基因Tim、Npas4、Nrld1口Per1,與8個肝臟功能基因中的1個或多個基因,在翹嘴鱖肝臟中的表達呈正相關(guān)關(guān)系。(3)采用qRT-PCR方法,研究分析了上述13個生物鐘基因和12個心臟功能基因Acer2、Dusp28、Kcnh7、Mlf1、Mylk4、Raph1、Rhobtb2、Rhobtb3、Slco5A1、Socs3、 Socs5、Timp2在翹嘴鱖心臟中的晝夜表達規(guī)律,及各基因之間的相關(guān)性。13個生物鐘基因中,Bmal1、Clock、Cry1、Npas2、Nrld1、Nrld2、Per3、Rorα 和 Tim 9個基因在翹嘴鱖心臟內(nèi)都表現(xiàn)出明顯的晝夜節(jié)律；12個心臟功能基因中,Dusp28、Raph1、 Rhobtb2、Rhobtb3、Slco5A1、Socs36個基因有晝夜節(jié)律顯著性；上述具有顯著晝夜節(jié)律的9個生物鐘基因除Nrld1之外,其余8個基因和6個心臟功能基因中的1個或多個基因之間,在翹嘴鱖心臟中的表達具有相關(guān)性。本論文獲得的結(jié)果為翹嘴鱖以及其它魚類生物鐘系統(tǒng)分子作用機制的深入研究提供了實驗數(shù)據(jù)及理論依據(jù)。
[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.
【學位授予單位】：湖南農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：S917.4

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本文編號：2162219