臺灣博碩士論文加值系統

促性腺素釋放激素(Gonadotropin Releasing Hormone ,GnRH)是調控脊椎動物生殖作用重要的胜肽分子。近年來在鱸形目魚種已證實，腦中存在三種不同形式的GnRH，分別是sea bream form GnRH (sbGnRH 或 GnRH1) chicken GnRH II form (cGnRH-II或GnRH2) 和 salmon form GnRH (sGnRH或GnRH3)。本實驗藉由選殖並定序得到黃錫鯛三種GnRH的cDNA全長以及包含5’flanking regions的genomic DNA，在三種GnRH的基因構造上發現具有高度的保守性，皆由4個表現子(exon)及3個插入子(intron)所組成，exon I 轉錄成5’端非轉譯區(5’ untranslated region ,5’UTR)；exon II 轉錄成訊號序列(signal peptide)、GnRH胜肽、蛋白水解酶辨識的切位 (Gly-Lys-Arg, GKR)、GnRH-associated peptide的胺基端(N-terminus)；exon III 轉錄成GnRH-associated peptide 的中段區域；exon IV 轉錄成GnRH-associated peptide的羧基端(C-terminus)和3’端非轉譯區，本研究利用genome walking的方法，得到2.6 kb sbGnRH、1.9 kb cGnRH-II及3.2 kb sGnRH啟動子區域，再利用genomatix資料庫的程式，搜尋到許多相關轉錄因子（transcription factor）結合位置，例如Brn-2、Estrogen response element (ERE)、progesterone response element (PRE)、octamer-binding factor 1(Oct-1)、GATA binding factor 1 (GATA-1)。本研究以斑馬魚為模式魚種，進行基因轉殖及啟動子活性的分析，從螢光蛋白(fluorescent protein)表現的情形和位置，實驗結果發現黃錫鯛的sbGnRH啟動子無法在斑馬魚胚胎時期表現，而cGnRH-II啟動子則在受精後34小時開始表現在斑馬魚的脊椎上，而sGnRH啟動子則可專一性的表現在斑馬魚olfactory bulb-terminal nerve region (OB-TN)以及the ventral telencephalon-proptic area (VT-POA)，選殖出GnRH啟動子序列不僅可以初步了解這個基因的調節機制，輔以適當的基因轉殖技術，對於發展出使GnRH基因失去功能導致不孕的技術，提供一個良好的契機。

Gonadotropin-releasing hormone (GnRH), which is an essential decapeptide, plays an important role in the control of reproductive function in vertebrates. Three forms of GnRH had been demonstrated in the brains of perciforms including, sea bream GnRH (sbGnRH or GnRH1), chicken GnRHII (cGnRH-II or GnRH2), and salmon GnRH (sGnRH or GnRH3). In this study, complementary as well as genomic DNA, encoding the three forms of GnRH from silver seabream (Sparus sarba), were cloned and sequenced. The genomic structure of GnRH is highly conserved, with 4 exons and 3 introns. The 5’ untranslated region (5’UTR) is encoded by exon I, whereas the signal peptide, GnRH decapeptide, proteolytic cleavage site (Gly-Lys-Arg) and the N-terminus of the GnRH associated peptide (GAP) are encoded by exon II. The exon III encodes the mid region of GAP and exon IV encodes C-terminus of GAP and 3’UTR. Using genome walking strategy, we cloned the promoter regions of sbGnRH (2.6 kb), cGnRH-II (1.9 kb), and sGnRH (3.2 kb) and identified several putative DNA-binding sites of transcription factors such as, Brn-2, progesterone response element (PRE), octamer-binding factor 1 (Oct-1), and GATA binding factor 1 (GATA-1). Furthermore, functional activities of these three promoters were analyzed using a reporter gene in zebrafish (Danio rerio). The expression patterns of sGnRH promoter shown significant specifity of GFP in olfactory bulb-terminal nerve region (OB-TN) and ventral telencephalon-proptic area (VT-POA). However, sbGnRH promoter showed no significant expression and a nonspecific expression was produced by cGnRH-II promoter. These results suggest that cloning of the upstream regulatory region of this gene would provide an opportunity to demonstrate its role in silver seabream and possibility of attempting to ‘knock down’ the expression of this gene to develop a sterile transgenic fish.

目 錄
中文摘要 I
英文摘要 III
目錄 V
圖表目錄 VII
第一章、前言 1
ㄧ、黃錫鯛之簡介 2
二、魚類生殖內分泌系統概論 3
三、促性腺激素釋放素(Gonadotropin Releasing Hormone ,GnRH)
1.GnRH基因的發現和命名 4
2.GnRH的基因結構和生物體內的分布 7
3.GnRH的功能 10
4.GnRH在硬骨魚類的功能 12
第二章、實驗流程 14
第三章、實驗材料
一、生物材料 15
二、儀器及器材 16
三、反應試劑 16
第四章、實驗方法
一、選殖黃錫鯛(Sparus sarba)促性腺素釋放激素cDNA序列 23
二、選殖黃錫鯛(Sparus sarba)促性腺素釋放激素啟動子(promoter)之基因體序列 30
三、構築黃錫鯛促性腺素釋放激素啟動子螢光蛋白表現質體 32
第五章、實驗結果
ㄧ、黃錫鯛GnRH基因之選殖 36
二、黃錫鯛GnRH基因比對結果 37
三、GnRH基因在黃錫鯛不同器官表現情形 38
四、黃錫鯛GnRH基因啟動子之選殖 38
五、GnRH基因啟動子在斑馬魚斑馬魚胚胎發育時期表現之情形 39
第六章、討論 41
ㄧ、黃錫鯛GnRH基因和啟動子之選殖 42
二、GnRH基因在黃錫鯛不同器官表現情形 42
三、黃錫鯛GnRH基因啟動子在斑馬魚胚胎發育時期表現之情形 43
第七章、參考文獻 46
圖表

沈世傑，1993，台灣魚類誌，國立台灣大學動物學系，台北市。
陳兼善、于名振，1984，台灣脊椎動物誌，台灣商務印書館，台北市。
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