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研究生:蘇威仁
論文名稱:斑馬魚表皮型組織專一性啟動子之功能分析
論文名稱(外文):Functional analysis of epidermal tissue-specific promoters from zebrafish (Danio rerio)
指導教授:耿全福
學位類別:碩士
校院名稱:國立彰化師範大學
系所名稱:生物技術研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2007
畢業學年度:95
語文別:中文
論文頁數:132
中文關鍵詞:表皮型組織專一性啟動子十二脂氧酵素黏附蛋白角蛋白酪胺酸酶晶體蛋白
外文關鍵詞:epidermal tissue-specific promoter12-lipoxygenasecadherin 1keratintyrosinasecrystalline beta B1
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為了建立斑馬魚魚鰭、體表及眼睛等組織特異性轉基因斑馬魚,所以我們篩選了斑馬魚的表皮型及眼睛組織專一性啟動子,並選殖了九種斑馬魚基因啟動子分別包括4.0kb的角蛋白4(keratin 4)啟動子、1.5kb的角蛋白5(keratin 5)啟動子、1.5kb的角蛋白(krtt1c6)啟動子、1.5kb的角蛋白15(keratin 15)啟動子、1.3kb的角蛋白17(keratin 17)、2.0kb的十二脂氧酵素(12-lipoxygenase)啟動子、1.0kb的黏附蛋白(cadherin 1)啟動子、600bp的晶體蛋白(β crystallin B1)啟動子及1.5kb的酪胺酸酶(tyrosinase)啟動子並進一步分析被選殖出啟動子之活性,其中我們發現keratin 4啟動子具有明顯的表皮組織專一性表現的能力。利用serial deletion分析將keratin 4啟動子分為-8bp/+97bp、-41bp/+97bp、-205bp/+97bp、-566bp/+97bp、-1061bp/+97bp、-2241bp/+97bp、-3211bp/+97bp等片段,分別構築至pEGFP-1載體上,再利用顯微注射方式分別送入斑馬魚胚胎中,其中zkrt4-(-1061bp/+97bp)-EGFP及zkrt4-(-566bp/+97bp)-EGFP均可以啟動綠螢光蛋白主要表現在頭部、表皮組織、鱗片、胸鰭、尾鰭等位置,而zkrt4-(-205bp/+97bp)-EGFP啟動子的綠螢光蛋白表現位置卻只在部份表皮組織,失去魚鰭專一性表現能力。zkrt4-(-41bp/+97bp)-EGFP及zkrt4-(-8bp/+97bp)-EGFP啟動子的綠螢光蛋白幾乎喪失表現能力。在-566bp/-205bp區域可能負責胸鰭組織專一性表現,而-205bp/-41bp負責表皮組織專一性表現。接著利用區間刪除分析,結果發現-123bp/-107bp之間EKLF為重要的角蛋白4啟動子調控的轉錄因子之一,利用Luciferase定量分析啟動子發現-1061bp/-566bp可能為enhancer element或co-activator,而EKLF可以使其活性增加10倍。我們利用crybb1-600bp-EGFP可啟動綠螢光蛋白在斑馬魚眼睛晶體部位明顯表現。除此之外,keratin 5、krtt1c6及aLOX12都是很好的組織專一性表現啟動子,未來將建立斑馬魚魚鰭、體表及眼睛等組織特異性轉基因斑馬魚。

關鍵字:表皮型組織專一性啟動子、十二脂氧酵素、黏附蛋白、角蛋白、酪胺酸酶、晶體蛋白
In order to character the epidermal tissue-specific promoters of zebrafish, we have cloned nine zebrafish promoters that include 12-lipoxygenase (2.0kb), cadherin 1 (1.0kb), beta crystalline B1 (0.6kb), tyrosinase (1.5kb), keratin 4 (4.0kb), keratin 5 (1.5kb), krtt1c6 (1.5kb), keratin 15 (1.5kb) and keratin 17 (1.3kb) promoter region. We found keratin 4 promoter can drive EGFP expression in epidermal tissues, especial on fin. We divided the keratin 4 promoter into -8bp/+97bp, -41bp/+97bp, -205bp/+97bp, -566bp/+97bp, -1061bp/+97bp, -2241bp/+97bp, and -3211bp/+97bp fragments, then fused with EGFP for promoter activity assay in zebrafish embryo. We found that both zkrt4-(-1061bp/+97bp)-EGFP and zkrt4-(-566bp/+97bp)-EGFP were expressed in head, scales, pectoral fin, and cauldal fin. On the other hand, the zkrt4-(-205bp/+97bp)-EGFP only expressed in the part of epidermal and loss of fin specific expression property. The zkr4t-(-8bp/+97bp)-EGFP was not driven EGFP. That -566bp/-205bp probably response to fin tissues specific expression. We also found -123bp to -107bp transcription factor EKLF was important for keratin 4 promoter activity. The same results were confirmed by luciferase reporter assays. To screen other gene promoters, we found that keratin 5, krtt1c6 and aLOX12 were as the good candidates for epidermal-tissue and beta crystalline B1 can drive EGFP specific expression in zebrafish lens.

Key word: epidermal tissue-specific promoter、12-lipoxygenase、cadherin 1、keratin、tyrosinase、crystalline beta B1
第一章、前言 ………………………………………………………1
第一節、基因轉殖技術與基因轉殖動物發展 …………………1
第二節、基因轉殖魚動物模式發展 ……………………………4
第三節、斑馬魚與基因轉殖斑馬魚動物模式 …………………6
第四節、基因轉殖螢光魚應用 …………………………………8
第五節、研究目的 ……………………………………………17
第二章、實驗材料 …………………………………………………18
第三章、實驗方法 …………………………………………………25
3.1 斑馬魚飼養與交配產卵 ……………………………………25
3.2 斑馬魚的全量DNA抽取 …………………………………25
3.3 斑馬魚的表皮型組織專一性啟動子之選殖 ……………26
3.4 斑馬魚表皮型組織專一性啟動子之功能分析 …………34
3.5 抽取中量質體 …………………………………………38
3.6 基因轉殖 ………………………………………………39
3.7 斑馬魚表皮型組織專一性啟動子強度分析 …………40
第四章、結果 ……………………………………………………42
壹、斑馬魚之角蛋白4 (keratin 4)啟動子功能分析 …………42
第一節、角蛋白4(keratin 4)啟動子基因選殖 ……………42
第二節、角蛋白4(keratin 4)啟動子啟動綠螢光蛋白表現分析 …42
第三節、角蛋白4 (keratin 4)啟動子系列刪除後各片段之綠螢光表現分析 …………………………………………………43
第四節、角蛋白4 (keratin 4)啟動子不同組織專一性調控區域之綠螢光表現分析 …………………………………………44
第五節、角蛋白4(keratin 4)近端啟動子之轉錄因子預測及綠螢光表現分析 ………………………………………………46
第六節、角蛋白4 (keratin 4)啟動子系列刪除後各片段表現能力定量研究 ……………………………………………………47
第七節、角蛋白4 (keratin 4)啟動子不同調控區域之表現能力定量研究 ………………………………………………………48
貳、斑馬魚之角蛋白5 (keratin 5)啟動子基因選殖與綠螢光蛋白表現分析 ………………………………………………………………50
參、斑馬魚之角蛋白(krtt1c6)啟動子基因選殖與綠螢光蛋白表現分析 …………………………………………………………………50
肆、斑馬魚之角蛋白15 (keratin 15)啟動子基因選殖與綠螢光蛋白表現分析 ……………………………………………………………51
伍、斑馬魚之角蛋白17 (keratin 17)啟動子基因選殖與綠螢光蛋白表現分析 ……………………………………………………………52
陸、斑馬魚之酪氨酸酶(tyrosinase)啟動子基因選殖與綠螢光蛋白表現分析 ……………………………………………………………52
柒、斑馬魚之黏附蛋白1(cadherin 1)啟動子基因選殖與綠螢光蛋白表現分析 …………………………………………………………53
捌、斑馬魚之十二脂氧酵素(12-lipoxygenase)啟動子基因選殖與綠螢光蛋白表現分析 …………………………………………………54
玖、斑馬魚之晶體蛋白(β crystallin B1)啟動子基因選殖與綠螢光蛋白表現分析 ………………………………………………………54
第五章、討論 …………………………………………………………56
壹、斑馬魚之角蛋白4 (keratin 4)啟動子功能分析 ………………56
第一節、角蛋白4(keratin 4)啟動子基因選殖與序列分析 ……56
第二節、角蛋白4(keratin 4)啟動子啟動綠螢光蛋白表現與活性分析 …………………………………………………56
貳、斑馬魚體表組織專一性啟動子選殖與特性分析 ……………60
參、基因轉殖螢光魚觀賞魚對於GMO問題的探討 ……………62
肆、未來展望 ………………………………………………………64
參考文獻 ……………………………………………………………65
圖表與附錄 ………………………………………………………75
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