臺灣博碩士論文加值系統

中文摘要
促進生長、改進飼料轉換率是水產養殖一直追求的目標，本研究的長期目標是使用大量基因轉殖技術來產生成長快速的高經濟海水魚類－黃錫鯛魚（Sparus sarba），以增進養殖產量。電破法處理之精子其攜帶外源DNA，經由受精作用，將外源DNA轉殖入海水魚的胚胎之中。在經由電破法處理精子後進行受精的魚苗中，約有56至70%魚苗的PCR分析為轉基因海水魚。由於以人工擠壓的方式進行收集配子（精子與卵），會造成種魚緊迫受傷、病原菌感染導致高死亡率，大部份的雌魚僅能進行擠卵採集一次，故為了避免擠精卵操作對種魚所造成的緊迫(stresses)與傷害，故需要有其它替代的基因轉殖方法，本研究利用微脂體lipospermine L-dioleoyl phosphatidylethanolamine（DOPE；Promega, USA）lipofection感染法，將外源DNA以liposome為載體（攜帶者）送入黃錫鯛的精巢之中，每隻雄魚感染所使用的劑量約為0.7 毫升之微脂體與DNA之混合液，約48小時生殖巢感染後雄種魚與正常雌魚進行交配與產卵，藉精巢lipofection感染與正常雌魚交配所得之受精卵孵化後之幼苗，約有17 % 到50 %經PCR分析結果推定為轉基因魚，藉由多重生殖巢感染處理的基因轉殖效率明顯有增加（58-90%）。在本研究GH促進生長的研究實驗中，攜帶鱒魚生長基因的轉基因黃錫鯛魚，其生長速度有增加；魚體之體組成結果顯示出，其脂質（lipids）含量有明顯降低；由RT-PCR分析得知，部份帶有rtGH1的轉基因魚會表現轉殖入之外源性基因；本研究結果顯示藉由這兩種大量基因轉殖的技術可以得到生長快速的黃錫鯛。

英文摘要
The objective of the present study is to produce fast growing sliver sea bream (Sparus sarba) for intensive aquaculture by employing various mass gene transfer technologies. We have transferred a "all-fish" growth hormone (GH) gene construct into silver sea bream embryos by electroporating the transgene construct into sperm. Ranging from 50 to 70% of the survived animals developed from eggs fertilized with electroporated sperm were found PCR positive. Striping gametes from sliver sea bream broods can cause severe stress to fish, frequently leading to serious mortality, and most female broods can only be striped once. To eliminate handling stresses in the brood fish, an alternative mass gene transfer method is developed. We employed lipid vesicles made of lipospermine L-dioleoyl phosphatidylethanolamine (DOPE) to deliver the GH transgene into testis. Each male fish received 0.7 ml of liposome/DNA mixture. Forty-eight hours after gonadal lipofection, treated male fish were mated to untreated females and fertilized eggs were collected every day. Ranging from 17% to 50% of the resulting larvae were tested positive for the transgene by PCR analysis. The percentage (58-90%) of PCR positive fry was significantly improved if multiple gonadal lipofection was carried out in the same male fish. Furthermore, preliminary analysis showed that a significant number of PCR positive GH transgenic silver sea bream can promote the sea bream growth. Body component analysis of transgenic fish indicated that the fat content of transgenic sea bream muscle was lower than the control sea bream in muscle tissue. Transgene expression also has been demonstrated in PCR positive fish by RT-PCR analysis. These results suggest that faster growing silver sea bream can be produced by various mass gene transfer technologies.

目錄
中文摘要 4
英文摘要 5
感謝 6
前言 7
材料 15
1、實驗動物---黃錫鯛（Sparus sarba） 15
2、質體與菌種 15
3、脂小體（DOPE） 16
方法 17
１、轉殖之DNA的製備 17
1-1、製備具勝任性的大腸桿菌細胞 17
1-2、轉形作用 17
1-3、選取 18
1-4、DNA的小量備製 18
1-5、瓊脂醣膠體電泳法 18
２、質體DNA分離純化與線性化 19
2-1、大量質體DNA之抽取法 19
2-2、DNA的線性化 20
３、種魚配子的取得與處理 20
3-1、黃錫鯛種魚的取得 20
3-2、種魚荷爾蒙催熟處理 20
3-3、配子的取得 20
3-4、精子濃度的觀察 21
3-5、卵質的觀察 21
3-6、精子活動力的觀察 21
４、利用電破法進行基因轉殖 22
4-1、電破液 22
4-2、電破法的處理 22
4-3、人工授精與洗卵 22
５、利用生殖腺脂小體感染法進行基因轉殖 23
5-1、脂小體感染液製配 23
5-2、生殖腺感染處理過程 23
5-3、自然受精與收卵 24
６、幼苗的孵化與飼育 24
6-1、受精卵的孵化 24
6-2、綠藻的培養 24
6-3、輪蟲的培養 25
6-4、豐年蝦之培養 26
6-5、幼苗的飼育 26
6-6、海上箱網的養殖 28
７、樣本分析 28
7-1、黃錫鯛魚的標幟 28
7-2、黃錫鯛genomic DNA的抽取 28
7-3、多聚合酵素連鎖反應（PCR）分析 29
7-4、DNA的回收 29
7-5、探針的製備法 30
7-6、南方轉換分析 31
7-7、南方氏浸漬法 32
7-8、RT-PCR分析 34
7-9、黃錫鯛幼魚體長與體重之測量與分析 37
7-10、轉基因黃錫鯛之體組成分析 37
結果 40
討論 44
參考文獻 53
附圖 65
附表 80
附錄 86

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