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研究生:黃曉雲
研究生(外文):Huang Hsiao-Yun
論文名稱:以GH1基因序列設計之探針將台灣櫻花鉤吻鮭從其他亞種的鉤吻鮭屬中區分出來及開發快速又經濟的鮭魚卵DNA抽取技術
論文名稱(外文):A novel growth hormone 1 gene-derived probe for Oncorhynchus masou formosanus distinguishing from the Oncorhynchus subspecies and development of the rapid and economic DNA extraction from a single salmon egg
指導教授:楊景雍楊景雍引用關係張學偉
指導教授(外文):Yang Jing-IongChang Hsueh-Wei
學位類別:碩士
校院名稱:國立高雄海洋科技大學
系所名稱:水產食品科學研究所
學門:農業科學學門
學類:食品科學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:78
外文關鍵詞:BiomarkerTaiwan salmonGrowth hormone gene 1Oncorhynchus
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第一部分:以分子生物學進行物種鑑定的研究方面,在傳統上普遍使用粒線體16S rRNA 為標的。然而,粒線體粒線體16S rRNA難以作為鉤吻鮭屬魚種(Genus Oncorhynchus)間的親源鑑定依據。本研究的目的在於尋找出可區別台灣櫻花鉤吻鮭(Oncorhynchus masou formosanus)與其他鉤吻鮭屬鮭魚之生物標誌,並發展出一套快速鑑定套組。藉由第一型生長賀爾蒙基因(growth hormone 1 gene,GH1)的intron 3 DNA片段進行聚合酶連鎖反應(Polymerase chain reaction, PCR)。將11種鉤吻鮭屬鮭魚的GH1基因intron 3的序列排比後,設計出一個新的PCR引子,可將台灣櫻花鉤吻鮭與其他三種櫻鮭亞種及其餘鉤吻鮭屬鮭魚區別。這個技術可迅速的區別台灣原生種櫻花鉤吻鮭與其他櫻鮭亞種,將可有效的防範外來種入侵。
第二部份:開發快速又經濟的鮭魚卵DNA抽取技術以適用於即時定量聚合酶連鎖反應
本實驗目的在於發展兩種新的單顆鮭魚卵DNA抽取方法,並與商業組織DNA抽取套組(Qiagen)及Chelex100萃取法進行DNA抽取效率之比較。就單顆鮭魚卵的DNA量(O.D. 260 nm)而言:Chelex100萃取法>過濾離心法>稀釋法>Qiagen組織DNA抽取套組。然而以DNA的品質(O.D. 260nm/280nm)看來,則為:過濾離心法>稀釋法>Qiagen組織DNA抽取套組>Chelex100萃取法,比值分別為,1.79±0.12, 1.49±0.30, 1.57±0.82, and 0.75±0.09 (n = 7)。然後我們再以不同稀釋倍數的DNA,利用粒線體12S rRNA基因所設計的不同長度產物之引子去進行real-time PCR。以過濾離心法及稀釋法所抽取的DNA,他們運用於PCR的擴增反應時,最好的DNA稀釋程度可至102及103倍。雖然過濾離心法及稀釋法的DNA抽取效率不比Qiagen組織DNA抽取套組和Chelex100萃取法來的好,但是過濾離心法及稀釋法所需的時間分別為10分鐘及7分鐘,比起另外兩種方法,著實更為省時。而且以過濾離心法及稀釋法所抽取的DNA量,至少可進行500及5000次的PCR反應,並可得到可接受之結果。況且,我們的目的在於讓這兩種相當簡單又符合成本效應的方法得以實際運用於單顆鮭魚卵的DNA抽取。
Part 1:A novel growth hormone 1 gene-derived probe for Oncorhynchus masou formosanus distinguished from the Oncorhynchus subspecies
Traditional mitochondrial 16S rRNA is commonly used in many species identification studies. However, it is difficult to apply to the phylogenetic studies among the Oncorhynchus species, which is a crucial need for management purposes for Oncorhynchus masou formosanus, Taiwan salmon. In this study, we have developed an improved species identification method for Taiwan salmon distinguished with other Oncorhynchus species tested by exploiting PCR for growth hormone (GH) 1 gene. By comparing DNA sequences for GH1 from 11 species of Oncorhynchus species we designed novel PCR primers that exploit differences between Taiwan salmon and other Oncorhynchus subspecies. Therefore, the technique is an important tool in the management of populations of the endangered land-locked Taiwan salmon preventing from their possible hybrids with other Oncorhynchus masou subspecies once tested.
Part 2:Rapid and economic DNA extraction from a single salmon egg for real-time PCR amplification
This subject is to develop two novel methods for DNA extraction from a single salmon fish egg compared with the commercial tissue kit (Qiagen) and Chelex 100-based method. The order of DNA amount (O.D. 260 nm) for one salmon egg is: Chelex 100 > filtration > dilution > tissue kit. DNA quality (O.D. 260nm/280nm) for the filtration, dilution, tissue kit, and Chelex 100 protocols is 1.79±0.12, 1.49±0.30, 1.57±0.82, and 0.75±0.09 (n = 7), respectively. These results suggest that O.D. 260 nm is biased to DNA measurement. Alternatively, using DNA templates with different dilutions, real-time PCR for the mitochondrial 12S rRNA gene was performed for different lengths of PCR amplicons. Filtration and dilution protocols yielded their best result in PCR amplification in dilutions within 102 and 103, respectively, although other methods are more sensitive. Fortunately, the operation time for the dilution and filtration protocols is 7 and 10 min and the proteinase K and Chelex 100 are not required. In estimation, at least 500 and 5,000 PCR tests are acceptable for the filtration and dilution protocols, respectively. Therefore, our proposed two methods are quite simple and economical for DNA extraction of a single salmon fish egg.
目錄....................................................................................................... i
圖表目錄............................................................................................... iv
Part 1
第一部分:以GH1基因序列設計之探針將台灣櫻花鉤吻鮭從其他亞種的鉤吻鮭屬中區分出來...........................................................
1
中文摘要............................................................................................... 2
英文摘要............................................................................................... 3
壹、 前言........................................................................................... 4
貳、 文獻探討................................................................................... 7
一、台灣櫻花鉤吻鮭簡介....................................................... 7
二、發現與命名過程............................................................... 8
三、保育工作的發展史........................................................... 10
四、台灣櫻花鉤吻鮭的核酸序列鑑定之研究....................... 10
参、 實驗材料與方法....................................................................... 13
一、實驗流程........................................................................... 13
二、實驗檢體及來源............................................................... 14
三、實驗器材........................................................................... 15
1. 共同器材.............................................................................. 15
2. 萃取鮭魚肌肉組織Total DNA............................................ 15
3. 分析特定基因之表現.......................................................... 15
4. 膠體電泳.............................................................................. 16
5. 膠體純化.............................................................................. 17
四、實驗步驟............................................................................ 17
1. 利用系統發生學的分析得到Oncorhynchus subspecies
之生長賀爾蒙1(growth hormone 1, GH1)序列................
17
2. 引子設計.............................................................................. 18
3. 萃取鮭魚肌肉組織Total DNA............................................ 18
4. 分析依GH1基因所設計的引子在實驗物種中的表現
是否如預期.........................................................................
19
5. DNA純化與定序................................................................. 20
肆、 實驗結果................................................................................... 22
一、由系統發生學得到Oncorhynchus subspecies的GH1
基因序列...........................................................................
22
二、序列排序並設計出對台灣櫻花鉤吻鮭具專一性的引
子.......................................................................................
23
三、以聚合酶連鎖反應(Polymerase chain reaction, PCR)
和膠體電泳去驗證台灣櫻花鉤吻鮭引子的專一性.......
24
伍、 討論........................................................................................... 26
陸、 結論........................................................................................... 28
Part 2
第二部份:開發快速又經濟的鮭魚卵DNA抽取技術以適用於即時定量聚合酶連鎖反應.......................................................................
29
中文摘要............................................................................................... 30
英文摘要............................................................................................... 31
壹、 前言............................................................................................ 32
貳、 文獻探討.................................................................................... 35
參、 實驗材料與方法........................................................................ 37
一、實驗流程............................................................................. 37
二、實驗檢體及來源................................................................. 38
三、實驗器材............................................................................. 38
1. 共同器材............................................................................... 38
2. 萃取組織Total DNA............................................................ 38
A. 過濾離心法.................................................................... 38
B. 稀釋法............................................................................ 39
C. 商業組織DNA抽取套組(Qiagen)............................... 39
D. Chelex100沉澱法........................................................... 39
3. DNA濃度測定...................................................................... 40
4. 定量分析特定基因之表現................................................... 40
5. 膠體電泳............................................................................... 41
四、實驗步驟............................................................................. 41
1. 萃取組織Total DNA............................................................ 41
A. 過濾離心法.................................................................... 42
B. 稀釋法............................................................................ 43
C. 商業組織DNA抽取套組(Qiagen)............................... 44
D. Chelex100沉澱法........................................................... 45
2. 以分光光度計 (GeneQuant pro, Califormia, USA)測定
不同方法抽取的DNA濃度.................................................
46
3. 12S rRNA的引子設計.......................................................... 46
4. 不同方法所萃取的DNA,以定量分析不同長度的特
定基因之表現........................................................................
46
肆、 實驗結果.................................................................................... 49

一、以分光光度計測DNA濃度並比較四種萃取法之
DNA抽取效率..................................................................

49
二、以即時定量聚合酶連鎖反應(Real time PCR)進行四種
DNA萃取法之效率比較..................................................
50
三、以膠體電泳再驗證real-time PCR之結果....................... 51
伍、 討論............................................................................................ 53
陸、 結論............................................................................................ 57
柒、 參考文獻.................................................................................... 58
捌、 圖表............................................................................................ 64
玖、 附錄
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