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Please use this identifier to cite or link to this item: http://ntour.ntou.edu.tw:8080/ir/handle/987654321/16803

Title: 點帶石斑 (Epinephelus coioides) 生長素釋質 (ghrelin) 基因之分子選殖及特性分析
Molecular cloning and characterization of ghrelin gene in orange-spotted grouper ( Epinephelus coioides )
Authors: Shih-Tsung Huang
Contributors: NTOU:Department of Aquaculture
Keywords: 生長素釋質;促生長素分泌素;攝食;分子選殖
ghrelin;GHS;feeding;molecular cloning
Date: 2006
Issue Date: 2011-06-30T08:42:26Z
Abstract: 生長素釋質 (Ghrelin) 為一種內生型促生長荷爾蒙分泌素 (growth hormone secretagogue, GHS),能促進GH 由腦下垂體分泌,近來年證實ghrelin 也與攝食行為有關。本論文目標是研究點帶石斑 (Epinephelus coioides) 生長、攝食調控因子 ─ ghrelin 基因之分子選殖及其特性分析。根據已知脊椎動物ghrelin 之胺基酸及核酸序列設計退化性引子,以點帶石斑胃組織之cDNA 使用RT-PCR及RACE等方法選殖出ghrelin基因cDNA 序列,其全長為1485 個核苷酸,可轉譯出102 個胺基酸序列。本研究亦選殖出Ghrelin 基因體DNA 為3257 個核苷酸,ghrelin基因結構由四個編碼子及三個插入子所構成。經由Real-time PCR 分析結果顯示點帶石斑ghrelin 基因廣泛表現於各組織中,且以胃表現量最高。在攝食前胃中ghrelin基因及腦中NPY及GH基因表現量逐漸上升,並於攝食後下降。飢餓使ghrelin 基因表現量上升,此與GH 及NPY 基因表現情形相類似。人工合成的人類ghrelin及八碳、十碳修飾的石斑ghrelin皆能促進體外培養腦組織中之GH 及NPY 基因表現。內腦腔 (I.C.V.) 注射八碳修飾之石班ghrelin 可促進腦中GH 及NPY 基因表現。於石斑飢餓時注射葡萄糖,造成胃中ghrelin 基因表現量下降。高脂肪飼料能顯著的降低ghrelin 基因表現量。使用飼料及棉花進行胃部填充能使ghrelin 基因表現量下降。具厭食症的石斑魚其胃中ghrelin 基因表現量顯著的高於正常攝食之石斑。魚類運輸撈捕時所造成的緊迫使ghrelin 基因表現量降低,低溫處理則不會影響ghrelin 表現量。誘引劑可增加ghrelin 基因表現量,推測ghrelin 基因表現量可能受到嗅覺的影響。
Ghrelin an endogeneous growth hormone secretagogues (GHSs), that stimulating growth hormone secretion from pituitary. In recent study show that ghrelin is related in appetite. In this study, we have cloned and characterization of the full-length cDNA of ghrelin gene from orange-spotted grouper (Epinephelus coioides). We base on the ghrelin sequence of vertebrate animals to design degenerate primers. Use the cDNA of stomach tissue to clone the ghrelin cDNA sequence by RT-PCR and RACE methods. Grouper full-length ghrelin cDNA was 1485 bp in length and deduced 102 amino acids. The length of ghrelin genomic DNA was 3257 bp, and the genomic DNA structure was composed of four exons and three introns. Real-time quantitative PCR analysis showed ghrelin gene distribution in every tissues, and dominant expressed in stomach. Grouper maintained on a scheduled feeding regimen show that ghrelin, GH and NPY mRNA level increase before the feeding time, and the level rapid decrease after the feeding time. Food deprivation cause the ghrelin, GH and NPY mRNA level increase. The synthetic humen ghrelin and the grouper ghrelin that was modified by octanoic acid and decanoic acid all cause the GH and NPY mRNA level increase in vitro. Brain intracerebroventricular (i.c.v) injection of synthetic grouper ghrelin showed the stimulation of GH and NPY mRNA level. Glucose intraperitoneally (IP) injection decreased the ghrelin mRNA level. Use feed and cotton to fill the stomach showed that ghrelin gene expression was significantly decreased. Higher ghrelin mRNA was found in anorexia grouper. To handle fish would cause stress that decreased the ghrelin mRNA level in grouper. Attractant increased ghrelin mRNA expression, showed that ghrelin might related to olfaction.
URI: http://ethesys.lib.ntou.edu.tw/cdrfb3/record/#G0M94330011
Appears in Collections:[水產養殖學系] 博碩士論文

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