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研究生:魏梓傑
研究生(外文):Wei, Zih-Jie
論文名稱:以魚類生殖細胞專一性 Piwi1 啟動子建立基因轉殖魚誘導式不孕技術
論文名稱(外文):Establishment of Inducible Infertility Technology for Transgenic Fish by Teleost Germline-Specific Piwi1 Promoter
指導教授:龔紘毅
指導教授(外文):Gong, Hong-Yi
口試委員:陳志毅胡紹揚吳貫忠龔紘毅
口試委員(外文):Chen, Jyh-YihHu, Shao-YangWu, Guan-ChungGong, Hong-Yi
口試日期:2016-01-25
學位類別:碩士
校院名稱:國立臺灣海洋大學
系所名稱:水產養殖學系
學門:農業科學學門
學類:漁業學類
論文種類:學術論文
論文出版年:2016
畢業學年度:104
語文別:中文
論文頁數:49
中文關鍵詞:基因轉殖魚Piwi1 啟動子生殖細胞專一性誘導性不孕
外文關鍵詞:transgenic fishPiwi1 promotergermline specificinducible infertility
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Piwi (P-element induced wimpy testis) 為專一性表現在生殖細胞之基因,扮演調控生殖腺發育及成熟之重要角色。Piwi最早發現於果蠅,而在斑馬魚中有兩個Piwi的同源基因,Piwi1/Ziwi和Piwi2/Zili ,其功能在演化上具有保守性和專一性。誘發性毒殺蛋白 Nitroreductase (NTR) 為一種硝基還原酶,藉由轉化基質 Metronidazole (Mtz)為細胞毒素,促使細胞產生細胞凋亡。故本實驗利用生殖細胞專一性Piwi1啟動子表現誘發性毒殺蛋白NTR來破壞生殖腺發育,以發展基因轉殖魚之不孕控制技術。選殖出斑馬魚Ziwi基因5 kb長度之生殖細胞專一性啟動子序列,搭配 NTR/Mtz 誘殺系統,成功建立 Tg(Ziwi:NTR-EGFP) 基因轉殖品系斑馬魚。將此基因轉殖斑馬魚透過5mM Mtz的浸泡處理飼養達一個月以期達到專一性殺死生殖細胞之目的。我們發現 Ziwi 5 kb 啟動子確實能夠驅動外源 NTR-EGFP 癒合基因專一性表現在生殖腺細胞中,且在受精後 12 小時即可以RT-PCR被偵測到。不論基因轉殖雄魚或雌魚其EGFP螢光訊號則要等到孵化後 25 至30 天才能以螢光顯微鏡偵測到。將一個月大基因轉殖斑馬魚經過 5 mM Mtz 基質處理一個月後之轉殖斑馬魚,偵測到其 NTR-EGFP 基因表現量有下降,螢光訊號的消失,促細胞凋亡基因 Bak-1 和 Bok-1 表現量增加,均顯示 NTR/ MTZ 誘殺系統為有效的。但在組織切片觀察及 TUNEL 染色上結果顯示毒殺效果不明顯,可能原因為本實驗使用之Mtz純度不夠與最適濃度可能需要改進。即使目前此技術無法達到 100% 不孕,未來也可透過Piwi1啟動子結合其他誘導性毒殺系統,如 Cre/loxP 或 Gal4/UAS 等系統建立基因轉殖魚的誘導式不孕技術。並期望此技術未來能應用於臺灣螢光觀賞魚產業,以解決基改螢光觀賞魚販售的限制,包括外源基因是否透過基因流而影響到野外族群基因庫之基改魚類對環境及生態之安全性疑慮,及優良品系外流導致市場價格崩盤的問題。
Piwi (P-element induced wimpy testis) is specifically expressed in germ cells and plays an important role in the control of germline development and maturation. It was originally found in Drosophila. There are two homologous genes Piwi1/Ziwi and Piwi2/Zili in zebrafish with conserved function and specificity in evolution. Inducible toxic protein Nitroreductase (NTR) can convert Metronidazole (Mtz) into a cytotoxic compound to induce cell apoptosis. In this study, we developed the infertility control technology of transgenic fish by using the germline-specific Piwi1 promoter to express the inducible toxin protein NTR that can destroy the development of germ cells in gonad. The 5kb germline-specific promoter of zebrafish Piwi1/Ziwi gene was cloned and combined with the NTR/Mtz sysment to establish transgenic zebrafish line Tg(Ziwi:NTR-EGFP). To achieve the purpose of germ cell-specific killing, transgenic fish with Ziwi:NTR-EGFP transgene were treated and reared in 5mM Mtz freshwater for one month. We found Ziwi 5 kb promoter indeed expressed the foreign NTR-EGFP fusion gene in germ cells and can be detected in 12 hpf by RT-PCR. However, the EGFP fluorescent signals were detected until 25-30 dpf in both transgenic male and female fish by fluorescent microscopy. Transgenic zebrafish treated with 5 mM Mtz could induce the down-regulation of NTR-EGFP expression level, disappearance of the fluorescent signal and up-regulation of the proapoptotic Bax-1 and Bok-1 genes. These results reveal that NTR/Mtz system in transgenic zebrafish was workable. However, the H&E and TUNEL staining showed the toxic effect were not obvious. It might be caused from the Mtz purity and optimum concentration used in this study. Even though this technology can not reach 100% infertility, the Piwi1 promoter can be combind with other system, like Cre/loxP or Gal4/UAS to establish other inducible infertility technology for transgenic fish. We expect this technology will apply to fluorescent ornamental fish industry in Taiwan to solve the restrictions on sale of transgenic fluorescent ornamental fish, including the possible impact on wild-type gene pool by geneflow for the issue of safety of environment and ecology, and the problem of outflow of good strain to lead to the market price collapse.
謝辭 I
摘 要 III
Abstract IV
目 錄 V
圖目錄 VIII
壹、前言 1
一、 Piwi 簡介 1
二、 Ziwi 基因的啟動子 1
三、 現有的不孕技術 2
四、基因轉殖魚誘導性不孕技術 3
五、基因轉殖技術 (Transgenesis) 6
六、 研究動機及目的 7
貳、實驗材料及方法 8
一、實驗材料 8
1. 實驗動物 8
2. 實驗質體 8
3. 實驗菌株 8
4. 培養基 8
5. 化學藥品 8
6. 酵素及生物反應試劑套組 9
7. 實驗器材與儀器 9
二、實驗方法 10
1. 斑馬魚 genomic DNA萃取 10
2. 聚合酶鏈鎖反應 (Polymerase Chain Reaction, PCR) 10
3. DNA 瓊脂膠體電泳 (DNA agarose gel electorphoresis) 10
4. 膠體萃取 (Gel extraction) 11
5. 接合作用 (Ligation) 11
6. 轉型作用與藍白篩 (Transformation and Blue/White screening) 11
7. 小量質體 DNA 之備製 (Extraction of mini plasmid DNA) 12
8. 限制酵素剪切 (Restriction enzyme digestion) 12
9. 核酸定序 (Sequencing) 12
10. 中量質體 DNA 之備置 (Extraction of midi plasmid DNA) 12
11. Total RNA之萃取 (Extraction of Total RNA) 13
12. Total RNA之純化 (Purification of Total RNA) 13
13. 反轉錄作用 (Reverse transcription, RT) 14
14. 即時定量聚合酶鏈鎖反應 (Real-time Quantitative PCR, Q-PCR) 14
15. 建立基因轉殖斑馬魚 14
16. Metronidazole 基質浸泡處理 17
17. 石蠟切片 17
18. TUNEL Cell Death Assay 17
19.統計分析 17
參、結果 19
一、 以 Tol2 轉位子系統建立 Ziwi 啟動子表現誘殺蛋白基因 NTR-EGFP 之基因轉殖斑馬魚 19
二、外源性及內生性基因偵測 19
三、 浸泡 5 mM Mtz 基質進行誘導專一性毒殺生殖細胞 20
四、 H&E 染色及組織切片觀察 20
五、 TUNEL Cell Death Assay 21
肆、討論 23
一、 以 Tol2 轉位子系統建立 Ziwi 啟動子表現誘殺蛋白基因 NTR-EGFP 之基因轉植斑馬魚 23
二、外源性及內生性基因偵測 24
三、 浸泡 5 mM Mtz 基質進行誘導專一性毒殺生殖細胞 24
四、 H&E 染色及組織切片觀察 26
五、 TUNEL Cell Death Assay 27
伍、結論 29
陸、參考文獻 30

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