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研究生:王鈺君
研究生(外文):Yu-Jun Wang
論文名稱:建立海鱺鰭細胞株及分子選殖海鱺脂肪酸Δ6去飽和酶與延長酶基因
論文名稱(外文):Establishment of fin cell line, molecular cloning of fatty acid Δ6 desaturase, and elongase genes from cobia
指導教授:陳昭德陳昭德引用關係鄭達智鄭達智引用關係
指導教授(外文):Jau-Der ChenTa-Chih Cheng
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:2006
畢業學年度:94
語文別:中文
論文頁數:95
中文關鍵詞:海鱺脂肪酸Δ6去飽和酶延長酶脂肪酸
外文關鍵詞:cobiadelta6 desaturaseelongasefatty acid
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海鱺(Rachycentron canadum)為新興箱網養殖魚種,具有高經濟價值與廣大的市場。但飼料中需使用高量的魚油以滿足其對多元不飽和脂肪酸的需求。降低魚油使用量除了以植物油替代部分魚油外,研究多元不飽和脂肪酸合成路徑及相關酶的基因是必要的。因此本論文1)已建立沒有受到微生物污染且不需使用抗生素之鰭細胞株,其培養條件為用以含10% FBS之L-15培養液於28℃培養,同時已建立了電穿孔基因轉殖條件為使用BTX 830電穿孔儀、4 mm電穿孔槽、生理食鹽水為緩衝液及425伏特進行電穿孔。冷凍保存時使用含10%之DMSO之FBS為抗凍劑,該細胞株將成為從事海鱺離體研究之必備平台。2)已選殖出脂肪酸Δ6去飽和酶及延長酶基因序列,包括啟動子、cDNA、5’及3’UTR。啟動子序列含有固醇調控特有序列(sterol regulatory element)。cDNA序列與其他魚種相似性高且演譯出之胺基酸亦與其他生物一樣仍保有重要結構區。兩個基因mRNA在各組織都有表現只是程度不同,但都以腦為最高。3)Δ6去飽和酶及延長酶基因重組質體已被轉入鰭細胞株,兩者重組蛋白基因都能被轉譯成為重組蛋白,當Δ6去飽和酶基因與綠色螢光蛋白基因融合後,其重組蛋白分布在細胞質特定區域,而延長酶基因表現以西方點墨法偵測其蛋白質的表現時發現重組蛋白呈現dimer。上述重組蛋白是否具有生理功能,有待進一步探討。本論文成果有助於研究海鱺多元不飽和脂肪酸之合成機制,並為創造出低或不需使用魚油之新品系海鱺目標踏出第一步。
Cobia is a new species with high economical value and potential international market. However, high percentage of fish oil is added in the formulated feeds to meet the requirement of polyunsaturated fatty acid for cobia becomes a barrier for the development of cobia culture. In addition to use vegetable oil to substitute fish oil partly, researches on pathways for synthesis of polyunsaturated fatty acid and genes encoding enzymes catalyzing each step are necessary. Therefore, 1) a cobia fin cell line is established without bacteria, fungi and mycoplasma contamination. The cell line is cultured in L-15 medium containing 10 % FBS at 28 oC and foreign genes can be transferred into cells using BTX 830 electroporator using 4 mm cuvette containing PBS at condition of one pulse of 450 voltage. Cells can be cryopreserved in FBS containing 10% DMSO. 2) Promoters, cDNA, 5’ and 3’ UTR sequences of delta 6 desaturase and elongase genes are cloned. Both promoters contain sterol regulatory elements. The cDNA sequences and amino acid sequence of both genes are highly conserved among fishes. Variable amounts of both genes mRNA are detected in tissues tests but highest amounts of both genes mRNA are detected in brain. 3) The plasmids containing either delta 6 desaturase or elongase cDNA are delivered into cobia fin cell line and recombinant proteins are expressed. Most of fusion proteins of delta 6 desaturase and green fluorescent protein are located at specific location in cytoplasm. The dimers of elongase recombinant protein are detected using Western blot. As regard to the biological function of these recombinant proteins remain to be evaluated.
目錄

誌謝 i
中文摘要 ii
Abstract iii
表目錄 x
圖目錄 iv
1. 動機與目的 1
1.1. 動機 1
1.2. 目的 2
2. 前言 2
2.1. 海鱺 2
2.2. 魚類細胞株 3
2.3. 脂肪酸 4
2.4. 多元不飽和脂肪酸的合成 4
2.5. 魚類多元不飽和脂肪酸的需求與來源 6
2.6. 魚類脂肪酸去飽和酶及延長酶 8
3. 材料方法 8
3.1. 實驗魚種 9
3.2. 海鱺鰭細胞培養 9
3.2.1. 細胞分離及初代培養 9
3.2.2. 細胞之繼代培養(Subculture)與冷凍保存 9
3.2.3. 溫度及胎牛血清濃度對鰭細胞生長之影響 10
3.2.4. 微生物(細菌、真菌及黴漿菌) 汙染偵測 10
3.2.5. 染色體端點酶活性之偵測 11
3.3. 海鱺脂肪酸Δ6去飽和酶及延長酶基因選殖 11
3.3.1. 脂肪酸Δ6去飽和酶及延長酶部分(partial)片段基因序列選殖 11
3.3.1.1. 肝臟total RNA抽取 11
3.3.1.2. RNA電泳(Electrophoresis) 12
3.3.1.3. 反轉錄作用(Reverse Transcription) 12
3.3.1.4. 引子的設計 12
3.3.1.5. 聚合酶連鎖反應(PCR) 13
3.3.1.6. 電泳膠體之製備及電泳分析 13
3.3.1.7. 定序載體的構築及保存 14
3.3.1.7.1. DNA分子萃取 14
3.3.1.7.2. 接合作用(Ligation) 14
3.3.1.7.3. 轉形作用(Transfermation) 14
3.3.1.7.4. 重組DNA檢定 15
3.3.1.7.5. 定序及保存 15
3.3.2. Rapid amplification of the cDNA ends-polymerase chain reaction (RACE-PCR) 15
3.3.2.1. mRNA 純化 16
3.3.2.2. 合成5’及3’RACE cDNA 16
3.3.2.3. 聚合酶連鎖反應(PCR) 17
3.3.2.3.1. 第一次聚合酶連鎖反應(First PCR) 17
3.3.2.3.2. 第二次聚合酶連鎖反應(Second PCR) 17
3.3.3. 定序載體的構築及保存 17
3.3.4. 序列分析 18
3.3.5. 脂肪酸Δ 6去飽和及延長基因(cDNA)全長的選殖 18
3.3.5.1. 聚合酶連鎖反應(PCR) 18
3.3.5.1.1. 第一次聚合酶連鎖反應(First PCR) 18
3.3.5.1.2. 第二次聚合酶連鎖反應(Second PCR) 18
3.3.5.2. 載體構築 19
3.3.6. 脂肪酸Δ 6去飽和酶及延長酶啟動子核苷酸序列基因選殖 20
3.3.6.1. Genomic DNA 抽取 20
3.3.6.2. 脂肪酸Δ 6去飽和及延長基因5’端上游序列選殖 20
3.3.6.2.1. DNA 製備 21
3.3.6.2.2. 引子設計 21
3.3.6.2.3. 聚合酶連鎖反應(PCR) 21
3.3.6.2.3.1. 第一次聚合酶連鎖反應 22
3.3.6.2.3.2. 第二次聚合酶連鎖反應 22
3.4. 海鱺脂肪酸Δ6去飽和酶及延長酶基因mRNA在各種組織之表現 22
3.4.1. 各組織total RNA 之抽取 22
3.4.2. 反轉錄聚合酶連鎖反應(Reverse Transcription-PCR, RT-PCR) 23
3.4.2.1. 引子設計 23
3.4.2.2. 聚合酶連鎖反應(PCR) 23
3.4.3. Real time PCR分析 24
3.5. 基因轉殖海鱺鰭細胞株之特性分析 24
3.5.1. 電穿孔基因轉殖(Electroporation) 24
3.5.1.1. 最佳電穿孔條件 25
3.5.1.2. 最佳基因表現時間 25
3.5.2. 大量質體的抽取 25
3.5.3. 基因轉殖構築載體分析 26
3.5.3.1. 觀察脂肪酸Δ6 去飽和酶螢光融合蛋白 26
3.5.3.2. 西方點墨法(Western blotting) 27
3.5.3.2.1. 10% SDS-PAGE 膠體製備 27
3.5.3.2.2. SDS-PAGE 凝膠電泳 27
3.5.3.2.3. 西方點墨法(Western Blotting) 27
3.6. 統計分析 28
4. 結果 28
4.1. 海鱺鰭細胞繼代培養 29
4.2. 分子選殖海鱺Δ6 去飽和酶及延長酶基因cDNA全長與比對 30
4.2.1. 脂肪酸Δ6 去飽和酶基因 30
4.2.2. 脂肪酸延長酶基因cDNA 31
4.2.3. 海鱺脂肪酸Δ 6去飽和酶及延長酶啟動子序列的
選殖 32
4.3. 海鱺Δ6 去飽和酶及延長酶基因在各組織的表現 32
4.3.1. 利用反轉錄-聚合酶連鎖反應(RT-PCR)分析 32
4.3.2. 利用Real time PCR分析 33
4.4. Δ6去飽和酶及延長酶cDNA基因轉殖入海鱺鰭細胞株後其特性研究 33
4.4.1. 海鱱鰭細胞之最佳電穿孔基因轉殖條件 33
4.4.2. 螢光基因蛋白在鰭細胞株表現之最佳觀察時間 34
4.4.2.1. 海鱺Δ6 去飽和酶重組蛋白在海鱺鰭細胞內表現
特性 34
5. 討論 35
5.1. 細胞株之討論 35
5.2. 脂肪酸Δ6去飽和酶及延長酶之選殖 36
5.3. Δ6 去飽和酶及延長酶基因轉殖入鰭細胞後之特性分析 39
6. 結論 40
6.1. 建立海鱺細胞及其基因轉殖條件 40
6.2. 選殖海鱺脂肪酸Δ6去飽和酶及延長酶基因 40
6.3. 研究脂肪酸Δ6去飽和酶及延長酶cDNA基因在所建立之鰭細胞株內之特性 40
7. 參考文獻 41


表目錄

表一、各物種脂肪酸Δ6去飽和酶cDNA轉譯區域(coding region)
序列之同一性(identity)比較 50
表二、各物種脂肪酸Δ6去飽和酶胺基酸序列之相似性(similarity)
比較 51
表三、各物種脂肪酸延長酶cDNA轉譯區域(coding region)序列之相同性(identity)比較。 52
表四、各物種脂肪酸延長酶胺基酸序列之相似性(similarity)比較 53
表五、 魚類脂肪酸去飽和酶基因cDNA在酵母菌S. cerevisiae表現時轉換受質成為產物的效率 54
表六、魚類脂肪酸延長酶cDNA在酵母菌S. cerevisiae表現時轉換受質成為產物的效率 55


圖目錄

頁碼
圖一、多元不飽和脂肪酸合成流程圖 56
圖二、海鱺鰭細胞繼代培養 57
圖三、溫度及胎牛血清濃度對鰭細胞生長之影響 58
圖四、微生物(細菌、真菌及黴漿菌)污染偵測 59
圖五、測試鰭細胞之染色體端點酶活性 60
圖六、海鱺脂肪酸Δ6 去飽和酶及延長酶基因部分片段(partial)
電泳圖 61
圖七、以GSP及UPM快速增幅海鱺脂肪酸Δ6去飽和酶基因
電泳圖 62
圖八、海鱺脂肪酸Δ6去飽和酶基因及延長酶cDNA基因全長之
電泳圖 63
圖九、各物種間脂肪酸Δ6 去飽和酶胺基酸序列之生物演化樹 64
圖十、以GSP及UPM快速增幅海鱺脂肪酸延長酶基因電泳圖 65
圖十一、各物種間脂肪酸延長酶胺基酸序列之生物演化樹 66
圖十二、 海鱺脂肪酸Δ6 去飽和酶及延長酶之啟動子核苷酸序列之
電泳圖 67
圖十三、海鱺β肌動蛋白3基因在各組織的mRNA表現 68
圖十四、(A )pcDNA3.0-HA yun及(B) pcDNA3.0-EGFP載體結構圖 69
圖十五、比較海鱱鰭細胞之最佳電穿孔基因轉殖條件 70
圖十六、探討海鱱轉殖鰭細胞之最佳表現時間 71
圖十七、pD6K-DNA3.0EGFP載體結構圖 72
圖十八、海鱺Δ6 去飽和酶螢光融合蛋白(EGFP fusion protein) 在其鰭細胞內之表現(1) 73
圖十九、海鱺Δ6 去飽和酶螢光融合蛋白(EGFP fusion protein) 在其鰭細胞內之表現(2) 74
圖二十、載體結構圖 75
圖二十一、以SDS-PAGE及Western Blotting分析海鱺鰭細胞轉殖其
Δ6 去飽和酶及延長酶基因的表現 76


附錄

附錄一、Genbank上各物種脂肪酸Δ6去飽和酶序列 77
附錄二、Genbank上各物種脂肪酸延長酶序列 78
附錄三、海鱺脂肪酸Δ6去飽和酶 Real time PCR 統計分析 79
附錄四、海鱺脂肪酸延長酶 Real time PCR 統計分析 80
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