臺灣博碩士論文加值系統

摘要

芳香�� (aromatase) 屬於細胞色素 P450 家族的成員之一，由 cyp19 基因所轉錄轉譯出來的蛋白質產物，能催化雄激素成為雌激素。多數硬骨魚類以cyp19 基因在生殖腺發育期間，各組織的表現模式與功能性的不同可被分為兩型，且兩個基因位於不同染色體上。cyp19a 主要表現於卵巢；cyp19b 主要表現於腦與腦下垂體中。本實驗利用 RT-PCR、5’RACE、3’ RACE 技術選殖出黃錫鯛 cyp19a 基因與 cyp19b 基因之 cDNA 序列全長。其中黃錫鯛 cyp19a 基因 cDNA 序列全長包含 1842 個核�˙纂A可轉譯 (deduced) 成519 個胺基酸；黃錫鯛 cyp19b 基因 cDNA 序列全長為 1918 個核�˙纂A可轉譯成 500 個胺基酸。黃錫鯛 CYP19 蛋白質3-D 結構包含N 端膜定位區域 (membrane-targeting segment)、膜轉移螺旋區域 (transmembrane helix domain)、I螺旋 (I-helix)、類固醇結合螺旋 (steroid binding helix; ozol’s peptide region) 與血基質結合螺旋 (heme-binding helix) 等保守區域。以即時定量-聚合�○s鎖反應 (Real-time quantitative PCR) 分析點黃錫鯛 cyp19a 與 cyp19b 基因各組織間表現情形。結果顯示黃錫鯛 cyp19a 在生殖腺組織表現量最高。雌魚的卵巢表現量又高於雄魚的精巢。黃錫鯛 cyp19b在腦組織表現量最高，而雄魚腦表現量高於雌魚的腦。以芳香�“磻蹌� (aromatase inhibitor, formastane) 餵食斑馬魚、吳郭魚，造成魚體雄性化且生殖腺發育與卵細胞發育及精子生成 (spermatogenesis) 皆受到明顯抑制，且抑制程度與芳香�“磻蹌紋窄q濃度成正比。其中少數魚的生殖腺內還同時存在精細胞與卵細胞，即所謂兩性魚 (intersex)。

Abstract


P450 aromatase (P450arom, CYP19), a cyp19 gene product, is an important enzyme in the steroidogenic pathway and belongs to the cytochrome P450 superfamily. Aromatase is responsible for the conversion of androgen into estrogen and is found throughout the vertebrate phylum. Most teleosts have two isoforms of the cyp19 gene, ovary type and brain type, termed cyp19a and cyp19b. In the present study, the reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA end (RACE) were applied to clone the silver sea bream (Rhabdosargus sabar) cyp19a and cyp19b genes. Full length cDNA of silver sea bream cyp19a is 1842 bp, containing 50 bp in 5’-untranslation region, 232 bp in 3’-untranslation region and 1560 bp in coding region which can be deduced 519 amino acids. The full length cDNA of silver sea bream cyp19b is 1916 bp, containing 276 bp in 5’-untranslation region, 137 bp in 3’-untranslation region and 1503 bp in coding region which can be deduced 500 amino acids. The 3D structure of CYP19a is comprised of membrane-targeting segment, transmembrane helix, I-helix, steroid binding helix (Ozol’s peptide region) and heme-binding helix. Comparison of the deduced amino acid sequence of silver sea bream cyp19a to that of the cyp19b revealed only a 59% similarity. The real-time quantitative PCR was performed to study tissue-specific expression of both cyp19a and cyp19b gene in adult silver sea bream. Cyp19a was mainly expressed in silver sea bream gonads, both ovary and testis, but was also found at low levels in the kidney. In contrast, cyp19b was expressed at higher levels in brain of silver sea bream than that of female silver sea bream. Zebrafish and tilapia during the period of gonadal differentiation were treated with the non-steroidal aromatase inhibitor (formestane) causing masculinization. The gonad development, oocyte growth and spermatogenesis is suppressed with aromatase inhibitor in a dose-dependent manner.

謝辭…………………………………………………………………… i
摘要………………………………………………………………….... ii
目錄……………………………………………………………………. v
表目錄………………………………………………………..………... viii
圖目錄………………………………………………………..………... ix
壹、前言…………………………………………………………..…... 1
一、 魚類生殖內分泌系統………………..…………………….. 1
二、 性類固醇激素的生成與生理功能……………...…………. 2
三、 魚類性別決定與分化……………………………………… 4
四、 魚類生殖腺分化與發育…………………………………… 6
五、 細胞色素P450超級家族 (Cytochrome P450 superfamily).. 7
六、 芳香�� (Cytochrome P450 Aromatase)……………………. 8
七、 內分泌干擾物質 (Endocrine disruption compounds) 造成不孕之機制…………………………………………………
11
八、 基因轉殖水產生物對環境之影響與不孕技術之研究……. 12
九、 芳香�“磻蹌� (Aromatase Inhibitor, AI)………………….. 13
十、 本研究之目標………………………………………………. 15
貳、實驗材料…………………………………………………………. 17
一、 實驗生物………………………………………..…………... 17
1. 黃錫鯛 (Rhabdosargus sabar)………………………… 17
2. 斑馬魚 (Danio rerio)..………………………………… 17
3. 莫三鼻克吳郭魚 (Oreochromis mossambicus)……….. 17
二、 實驗菌株………………………………………..…………... 17
三、 實驗質體………………………………………..…………... 17
參、實驗方法…………………………………………………………. 18
1. 分子選殖黃錫鯛之cyp19a與cyp19b 基因cDNA序列…… 18
1-1 去TRIZOL reagent 抽取 total RNA..………………….. 18
1-2 RNA電泳分析……………………..……………………. 19
1-3 引子 (primer) 設計….…….............................................. 20
1-4 反轉錄�﹞狨� (Reverse Transcription)………………… 20
1-5 聚合�○s鎖反應 (PCR)………..……………………….. 21
1-6 膠體萃取 (Gel extraction)…............................................ 21
1-7 定序載體的構築與選殖………………………………… 22
1-7-1 製備勝任細胞-氯化鈣法………………………… 22
1-7-2 接合作用 (Ligation)……………………………... 22
1-7-3 轉型作用 (Transformation)……………………… 23
1-7-4 篩選 (Selection)………………………………….. 23
1-7-5 小量質體製備 (Mini-preparation)……………….. 23
1-7-6 質體 DNA 濃度和純度的計算…………………. 24
1-7-7 分析與定序 (Auto-sequence)……………………. 24
2. 黃錫鯛cyp19a與cyp19b 基因之cDNA全長之選殖…….. 25
2-1 採用 GeneRacerTM Kit (Invitrogen, USA) 選殖黃錫鯛 cyp19a 與黃錫鯛 cyp19b 之 cDNA 序列全長………
25
2-1-1 去除 RNA 5’ 端磷酸根…………………………. 25
2-1-2 純化去除磷酸根RNA……………………………. 25
2-1-3 去除 mRNA 5’端帽子結構 (5’Cap structure)…... 26
2-1-4 純化去除 mRNA 5’端帽子結構之 RNA……….. 26
2-1-5 接合寡 RNA (Oligo RNA) 片段………………... 26
2-1-6 純化接合寡RNA (Oligo RNA) 片段之RNA…… 26
2-1-7 第一股cDNA之合成 (First-strand cDNA synthesis)…………………………………………..
27
2-2 5’ RACE (Rapid amplification of 5’ cDNA ends)……….. 27
2-2-1 5’ UTR 之第一次聚合酵素連鎖反應 (First Polymerase Chain Reaction) 與巢氏聚合酵素連鎖反應 (Nest PCR)……………………………….

27
2-2-2 膠體萃取 (Gel extraction)……………………….. 28
2-2-3 定序載體的構築與選殖…………………………. 28
2-3 3’RACE (Rapid amplification of 3’ cDNA end )………... 29
2-3-1 3’ UTR之第一次聚合酵素連鎖反(First Polymerase Chain Reaction) 與巢氏聚合酵素連鎖反應 (Nest PCR)……………………………….

30
2-3-2 膠體萃取 (Gel extraction)……………………….. 30
2-3-3 定序載體的構築與選殖………………………….. 30
2-4 定序結果與分析………………………………………… 30
3. 黃錫鯛 cyp19a 基因及 cyp19b 基因在各組織間之表現情形…………………………………………………………...
30
3-1 實驗設計………………………………………………… 30
3-2 抽取黃錫鯛各組織之total RNA………………...……... 31
3-3 反轉錄聚合�○s鎖反應 (RT-PCR)…………………….. 31
3-4 即時定量聚合酵素鏈鎖反應 (real-time quantitative PCR)……………………………………………………...
31
3-4-1 專一性引子之選擇……………………………….. 32
3-4-2 絕對定量標準曲線 (standard curve) 之建立與樣品定量…………………………………………….
33
4. CYP19a蛋白質3D結構…………………………………….. 34
5. 利用 AI 餵食斑馬魚、吳郭魚之影響……………………… 34
5-1 實驗設計………………………………………………… 34
5-1-1 斑馬魚…………………………………………….. 34
5-1-2 吳郭魚…………………………………………….. 34
5-2 添加芳香�“磻蹌祖犒篘蝜}料製備…………………… 35
5-3 生殖腺發育狀況的觀察………………………………… 35
5-3-1 生殖腺外觀觀察與生殖腺成熟指數之分析…….. 35
5-3-2 卵徑之測量……………………………………….. 36
5-3-3 組織切片………………………………………….. 36
5-3-4 觀察生殖細胞發育狀況………………………….. 36
5-3-5 性別比…………………………………………….. 38
5-4 統計分析………………………………………………… 38
肆、結果………………………………………………………………. 39
一、 黃錫鯛 cyp19a 與 cyp19b 基因 (cDNA) 序列全長與比對結果………………………………………………………...
39
二、 黃錫鯛 CYP19a 蛋白質3D結構………………………….. 41
三、 黃錫鯛 cyp19a 與 cyp19b 基因在各組織間之表現情形... 42
四、 餵食芳香�“磻蹌� (Aromatase inhibitor) formestane對斑馬魚生殖腺之影響…………………………………………...
42
五、 餵食芳香�“磻蹌� (Aromatase inhibitor) formestane對吳郭魚生殖腺之影響…………………………………………...
43
伍、討論………………………………………………………………. 47
一、 黃錫鯛 cyp19a 與 cyp19b 基因 (cDNA) 序列全長與比對結果………………………………………………………...
47
二、 黃錫鯛 CYP19a 蛋白質3D結構…………………………... 48
三、 黃錫鯛 cyp19a 與 cyp19b 基因在各組織間之表現情形... 49
四、 餵食芳香�“磻蹌� (Aromatase inhibitor) formestane對斑馬魚生殖腺之影響…………………………………………...
50
五、 餵食芳香�“磻蹌� (Aromatase inhibitor) formestane對吳郭魚生殖腺之影響…………………………………………...
51
陸、參考文獻…………………………………………………………. 54
圖表……………………………………………………………………. 65
附錄……………………………………………………………………. 表一、 各物種cyp19 cDNA序列之同一性比較….................... 65
表二、 各物種cyp19 cDNA胺基酸序列之相似性比較............ 66
96

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