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研究生:陳信良
研究生(外文):Chen Hsin-Liang
論文名稱:魚類生長激素基因在水產養殖上的應用
論文名稱(外文):The application of fish growth hormone gene on aquaculture
指導教授:蔡懷楨蔡懷楨引用關係許建宗許建宗引用關係
指導教授(外文):Tsai Huai-TenHsu Chien-chung
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:海洋研究所
學門:自然科學學門
學類:海洋科學學類
論文種類:學術論文
論文出版年:1999
畢業學年度:88
語文別:中文
論文頁數:96
中文關鍵詞:重組魚類生長基素精子載體九孔
外文關鍵詞:recombinant fish growth hormonesperm-mediationabalone
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由於人口暴增而對水產動物蛋白質的需求日漸迫切,於是不斷提昇遠、近洋漁業的漁獲量,但是卻因過度捕撈,不僅漁穫的相對捕獲量與努力量減少了,並且也對海洋漁業造成資源枯竭的危機。為了能使資源可以有效及永續的利用,並解決海洋資源枯竭的危機,而發展了內陸的淡、海水養殖方式。不過內陸的養殖,卻因為水資源利用有限,而且養殖空間不足,造成養殖生物體型小,且成長速率慢導致養成期長。因此,為解決此項問題,本實驗擬應用水產生物技術的方法,來達到促進魚、貝類成長較快的目標。
天然的生長激素 (growth hormone﹐GH) ,是由腦下垂體萃取而得,來源相當不易;若以基因工程的技術,即可經由原核系統之大腸桿菌 (Escherichia coli) 及真核系統之酵母菌 (Pichia pastoris) 大量生產recombinant GH (rGH) ,然後再應用在水產養殖上。培養100 ml含有黃鰭鯛 (Acanthopagrus latus) GH cDNA 表現載體的E. coli菌液,在菌體打破後,以離心法沈降,得到約87.36±0.28 mg的內涵體,再以6 M Guanidine HCl溶解後,經過透析純化出34.04±0.17 mg rGH。純化後的rGH以每三天一次浸泡單性吳郭魚 (Oreochromis niloticus x O. aureus) 魚苗30分鐘,18天後,結果顯示以浸泡500μg/liter的效果最好,實驗組的平均增重率比對照組的高出298%,平均體重也高出約75%。另外,在酵母菌方面則是將已帶有ypGH cDNA質體 (pSJ04) 之酵母 (BC01) ,以100%甲醇誘發轉形酵母菌株製造rGH,經SDS-PAGE發現在分子量約22 kDa處,有一對照組所無之蛋白質條紋出現;再進一步以rabbit anti-ypGH polyclonal antibody進行Western blot分析,結果為免疫正反應。從800 ml菌體培養液中,經誘發72小時後,以ELISA測定轉形酵母菌其萃取液中含rGH的含量約為0.421±0.015 μg/ml。大量培養後的轉形酵母菌經冷凍乾燥,依比例添加於飼料中,餵食歐洲鰻 (Anguilla anguilla) 鰻苗與馴化成海水的單性吳郭魚魚苗,結果顯示在飼料中添加含0.32%之rGH的酵母菌,對促進鰻苗生長最為明顯 (P<0.01) ,實驗組鰻魚魚苗的增重率,在42天後比平均對照組的高出139%,平均體重也高出約57%;實驗組的海水馴化吳郭魚魚苗,在28天後的平均增重率比對照組的高出69%,平均體重也高出約36%。
此外本實驗也利用基因轉殖方式來改良貝類品種。將含有魚類生長激素的外源性基因,以電破法導入九孔的精子內,藉由精子為載體攜入卵中,經過半年的蓄養,各組逢機選取一隻九孔,萃取其腹足、生殖腺與內臟組織的genomic DNA後,以PCR檢測外源性基因的存在。結果發現凡電破含有外源性基因之實驗組的九孔,至少有一組織顯示有PCR之正反應。至於檢測蓄養一年十隻經8 kV電破含200μg/ml外源性基因處理的九孔中,發現有一隻九孔在腹足有正反應。在成長方面該組實驗組的九孔與未經處理的九孔比較,在平均體長上多54%、平均體寬多46%、平均體重多63%,不過,這初步結果,還有待更進一步的實驗證明之。
由本實驗證實了利用微生物可以大量生產具有生物活性的rGH,並且也提供了利用浸泡與餵食rGH的方式,皆可達到促進魚苗成長的效果,此外也證明了利用電破法進行基因轉殖,基因確實能被攜入個體內,且經過一年仍然存在,因此未來可更廣泛地的應用rGH來促進養殖生物的生長,或以電破法導入能增進生長、抗逆境或抗病等基因而得到新的優勢品種,以求幫助提高養殖業的競爭力。
We apply genetic engineering and gene transferring technologies on aquaculture. By using genetic engineering technology, large amount of recombinant growth hormone (rGH) can be obtained from both prokaryotic and eukaryotic expression systems. In prokaryotic system, 34.04 ± 0.17 mg of rGH was obtained out of 100 ml Escherichia coli culture containing yellowfin porgy GH (ypGH) cDNA. The resultant rGH was used to immerse fries of tilapia (Oreochromis niloticus X O. aures). After 18 days of experiment, results showed that immersion with 500 mg rGH/l promoted fries growth most prominently. Compared with control group, the weight gain rate and average body weight were 298 % and 75 % greater, respectively.
In eukaryotic system, yeast (Pichia pastoris) transformant containing ypGH cDNA was cultured and induced for 72 hr to produce rGH. The transformed yeast produced rGH at the concentration of 0.421 ± 0.015 μg/ml based on ELISA measurement. The transformed yeast harvested from large-scale preparation was frozen-dried, added with various proportions to the feed powder as a supplement. After feeding the fries of European eel (Anguilla anguilla) for 42 days and seawater-adapted tilapia for 28 days, results showed that group of feeding 0.32 % transformed yeasts containing rGH enhanced most prominently the growth of fish fry (P<0.01). Compared with control group, the weight gain rate and average body weight of European eel fries were 139 % and 57 % greater, respectively. For seawater-adapted tilapia fries were 69 % and 36 % greater, respectively.
We also used electroporated-sperm mediation to transfer foreign DNA fragment that contained salmon GH cDNA into small abalone (Haliotis divorsicolor suportexta) . After 180 days post-fertilization, abalone were randomly chosen from each groups and extracted their genomic DNAs for PCR analysis. Results showed that at least one of tissues of abalone which electroported with foreign gene contained positive band. PCR and Southern blot analysis also demonstrated that one-year-old abalone derived from electroporated sperm at 8kV in the presence of 200 μg/ml DNA showed the positive signal. Compared with the untreated group, the average body length、body width and body weight were 54 %、46 % and 63 % higher, respectively.
The experiments indicate that using microorganisms to product large amount of biologically active rGH is achieved. rGH enhances the growth rate of fries by both immersing and feeding approaches. Besides, we also confirm that the foreign gene is transferred into abalone embryos by elcetroporated-sperm mediation. And, the foreign gene still exist in one-year-old transgenic abalone.
目錄……………………………………………………………Ⅰ
中文摘要………………………………………………………Ⅱ
英文摘要………………………………………………………Ⅳ
前言……………………………………………………………1
材料與方法……………………………………………………10
結果……………………………………………………………35
討論……………………………………………………………43
參考文獻………………………………………………………53
圖表……………………………………………………………65
附圖……………………………………………………………85
附錄……………………………………………………………88
附表……………………………………………………………91
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