請用此 Handle URI 來引用此文件:
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/23969
標題: | 生物技術在海洋水產養殖生物之研究與應用 Studying and application of biotechnology in marine aquacultural species |
作者: | Hsin-Liang Chen 陳信良 |
指導教授: | 蔡懷楨 |
關鍵字: | 生物技術,精子,九孔,小球藻,電破,生長激素,水產養殖, sperm,abalone,Nannochlorpsis oculata,gene transfer,growth hormone, |
出版年 : | 2006 |
學位: | 博士 |
摘要: | 做為經濟應用或是基礎研究的前提下,基因轉殖已經發展成為一個好用的技術工具,用以進行水產生物的特質改良,包括食用的魚介貝類、微細藻或是大型藻等。
我們注入了總體積10 μl濃度為10 μg/μl的線性化質體pOBA-YPGHc,至臺灣小鮑魚 (Haliotis diversicolor supertexta) 的精巢內,注入質體的構築是包含了日本稻田魚(medaka) β-actin基因的啟動子,驅動表現黃鰭鯛生長激素 (yellowfin growth hormone) 的cDNA片段。經過人工的配子誘導、授精與培育後,利用PCR (polymerase chain reaction) 檢測了在授精後九小時後 (larvae)、三週大 (juvrnile) 以及一年大的檢體,計算後得到外源基因的轉殖率分別為90%、92.5%與60%。其中也發現有約20%的成熟個體,其生殖巢的配子可以檢測到外源基因的存在。從PCR檢測得到正訊號反應的檢體,再藉由genomic Sothern blot的檢測,確認了外源性基因戡入這些檢體的基因體 (genome) 內。接著經由測量殼長與肉重,評估黃鰭鯛外源基因是否能夠促進臺灣小鮑魚的生長,從統計分析的結果發現基因轉殖臺灣小鮑魚,殼長與肉重的增長上與未經處理的對照組別有明顯差異(P<0.05)。總結來說,利用直接注射精巢的方法,產生基因轉殖臺灣小鮑魚品系,是一種簡單、大量的,且傷害性最小又非病毒感染式的有效率基因轉殖法。 我們也利用了電穿孔法 (electroporation) 將濃度為20μg/μl的線性化質體phr-YPGHc,導入到已製備完成之擬球藻 (Nannochloropsis oculata) 的原生質體 (protoplast) 中,進行擬球藻的基因轉殖工作。導入質體的構築是包含了衣藻 (Chlamydomonas reinhartii) 的Hsp70A與RBCS2基因之雙啟動子,驅動表現黃鰭鯛生長激素 (yellowfin growth hormone) 的cDNA片段。經過連續的繼代培育與檢測後,有四株轉殖藻株至少在50代以前,皆可以檢測出外源基因的存在,而且也確認了外源性基因戡入這四株的基因體 (genome) 內。將此四株轉殖藻株經過熱處理誘發後,分析並偵測黃鰭鯛生長激素的表現,並且測量從四株之50 ml的藻水中,可獲得的黃鰭鯛生長激素表現量由0.42 μg/ml到0.27 μg/ml。接著藉由豐年蝦 (Artemia franciscana) 代為消化藻纇6小時後,再投餵給紅色吳郭魚幼魚 (Oreochromis niloticus X O. mossambicus) 食用作為生物活性的測試。餵食四星期後,測量各組魚隻的體長與體重,做為評估生長促進的依據,並且從統計分析的結果發現,有餵食事先攝取轉殖藻之豐年蝦的幼魚,體長與體重的增加上與攝取非轉殖藻之豐年蝦的的幼魚別有明顯差異(P<0.01),在增重率上分別為239%和104%,在增長率上分別為217%和91%。因此,擬球藻可以表現有功能的黃鰭鯛生長激素,而且我們也推論擬球藻可能也是一種相當好用的生物反應器 (bioreactor) 材料。 Gene delivery has become a well used technique both for economic and research purpose and been applied in aquactic organisms, such as fish, shellfish, microalgae, and macroalgae. We microinjected the linearized plasmid pOBA-YPGHc (10 μg/μl), containing the yellowfin porgy growth hormone cDNA driven by the medaka β-actin promoter, into the testis of small abalone (Haliotis diversicolor supertexta) at a total volume of 10 μl. After polymerase chain reaction (PCR) -Southern blot detection, the gene-transfer efficiencies in larvae, juveniles, and 1-year-old adults of F1 were 90%, 92.5%, and 60%, respectively. Around 20% of F1 mature abalone contained the transgene in their gonads. Genomic Southern analysis of abalone that were PCR-Southern-positive revealed that the transgene was integrated into the genome. The average shell lengths and body weights of abalone in the gene-transfer group were significantly greater than those of the control group. We conclude that direct testis injection is a simple, high-throughput, minimally invasive, non-viral based, and efficient approach for producing transgenic line of abalone. This technique has great potential for application on many commercially important shellfish species. Plasmid phr-YPGHc, containing the fish growth hormone (GH) cDNA driven by a heat shock protein 70A promoter and a ribulose bisphospate carboxylase small subunit 2 promoter, was transferred into the protoplast of marine microalga, Nannochloropsis oculata, by electroporation. Four transgenic clones, which the transferred phr-YPGHc was integrated into genome and stably existed at least until the 50th generation, were obtained. When we treated these transgenic microalgae by heat shock, the heterologous fish GH was produced in a amount of 0.42 to 0.27 μg /ml from the 50 ml of medium. We incubated artemia with the wild-type and transgenic N. oculata for 6 hr and then fed these microalgae-treated artemia to red-tilapia larvae. After 4-week feeding, the growth of larvae that were fed artemia incubated with transgenic microalgae was greater than that of larvae that were fed artemia incubated with non-transgenic microalgae: 239% vs 104% in weight gain, and 217% vs 91 % in body length increase, respectively. Therefore, the N. oculata enables to produce functional GH and we propose it might be an excellent bioreactor material. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/23969 |
全文授權: | 未授權 |
顯示於系所單位: | 海洋研究所 |
文件中的檔案:
檔案 | 大小 | 格式 | |
---|---|---|---|
ntu-95-1.pdf 目前未授權公開取用 | 2.75 MB | Adobe PDF |
系統中的文件,除了特別指名其著作權條款之外,均受到著作權保護,並且保留所有的權利。