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Please use this identifier to cite or link to this item: http://ntour.ntou.edu.tw:8080/ir/handle/987654321/53548

Title: 建立fat-1基因轉殖魚以增進omega-3多元不飽和脂肪酸合成及其抗發炎分子機制研究
Establishment of fat-1 transgenic fish to enhance omega-3 PUFAs synthesis and to study their molecular mechanism on anti-inflammation
Authors: Lin, Shi-Ting
Contributors: NTOU:Department of Aquaculture
Keywords: omega-3 多元不飽和脂肪酸;omega-3 去飽和酶;基因轉殖魚類;發炎反應
omega-3 PUFA;omega-3 desaturase;transgenic fish;inflammatory
Date: 2019
Issue Date: 2020-07-03T08:30:21Z
Abstract: 研究證實秀麗隱桿線蟲 fat-1 基因產物是 omega-3 desaturase 基因,能將 omega-6 PUFAs 轉換成 omega-3 PUFAs,並且已運用 fat-1 基因在小鼠和斑馬魚皆有提升 omega-3 PUFAs 的含量。由於 LC-PUFA 主要進行合成的位置在肝臟、脾臟和腸道,因此本實驗藉由建立肝臟專一性或腸道專一性表現魚類密碼子優化 fat-1 omega-3 desaturase 和螢光蛋白 AcGFP 基因轉殖魚,探討全身脂肪酸 omega-3 PUFAs 含量是否提升。我們同時也檢視了 fat-1 基因轉殖魚的脂肪酸代謝調節,並進一步探討內源性產生 omega-3 PUFAs 對發炎相關基因的影響。第一部分已建立肝臟專一性 Drlfabp 2.9 kb 啟動子表現 fat-1 基因轉殖斑馬魚品系。本實驗結果顯示肝臟專一性表現 fat-1 基因轉殖斑馬魚具有提升全身 omega-3 PUFAs 比例,比對照組提升了4 倍(23.85±2.70 % vs. 5.63±4.51 %)。屬於 omega-3 PUFAs 中的 ALA(18:3n-3)、EPA(20:5n-3)、DPA(22:5n-3)、DHA(22:6n-3)都有提升的趨勢,特別是 DHA 顯著增加 3 倍(15.70±4.10% vs. 5.42±4.86 %)。而 EPA 在對照組中沒有偵測到含量,fat-1 基因轉殖斑馬魚則有 2.49±1.46 % 的含量。在脂肪酸代謝相關的轉錄活性中,主要傾向於脂肪酸氧化過程,如 ppara、acox3 和 cyp4t8 皆有被活化。此外調控促發炎因子的相關基因如 IL-1β、IL-6、IL-12α、IFN-γ、TNF-a 和 NF-κB,在肝臟專一性表現 fat-1 基因轉殖斑馬魚中轉錄活性皆顯著被抑制。綜合以上結果表明,肝臟專一性表現 fat-1 基因轉殖斑馬魚在餵食 omega-6 PUFAs 飼料,除了能有效合成 DHA、EPA,還能降低促發炎因子的表現。第二部分在吳郭魚以腸道專一性表現的 fabp2基因 2.5 kb 啟動子轉錄因子結合位點預測出兩個 Enhancers 區域。包含 Enhancer 1 的 Onfabp2 1.5 kb 啟動子幾乎不具有活性,反之包含 Enhancer 1&2 的 Onfabp2 2.5 kb 啟動子在斑馬魚及吳郭魚均具有活性。根據活體內啟動子活性測試結果,將分別建立斑馬魚 Drfabp2 1.0 kb 啟動子及 Onfabp2 2.5 kb 啟動子表現 fat-1 與 AcGFP 之基因轉殖吳郭魚。進一步也將繼續探討內源性產生 omega-3 PUFAs 對腸道表現 fat-1 基因轉殖吳郭魚其發炎相關基因表現及抗病源菌之影響。
The fat-1 gene encoding the omega-3 desaturase was identified in C. elegan and was proved to be able to convert omega-6 PUFAs to omega-3 PUFAs in transgenic mice and zebrafish. Because the fatty acid synthesis is mainly in the liver, spleen and intestine, we would like to establish the transgenic fish expressing fat-1 in the liver or intestine. By establishment of a transgenic fish with liver-specific or intestine-specific expression of fish-codon optimized fat-1 and AcGFP, we investigated whether the omega-3 PUFAs in whole-body could be increased. We focused on the fatty acid metabolism regulation in transgenic fat-1 transgenic fish, and further explored the changes in endogenously produced omega-3 PUFAs on inflammation-related genes. Firstly, the results showed that the liver-specific expression of fat-1 transgenic zebrafish driven by Drlfabp 2.9 kb promoter increased the omega-3 PUFAs contents to 4-fold (23.85±2.70 % vs. 5.63±4.51 %). Omega-3 PUFAs including LA(18:3n-3), EPA(20:5n-3), DPA(22:5n-3)and DHA(22:6n-3) had an increasing trend in liver-specific fat-1 transgenic zebrafish, especially DHA was significantly increased to 3-fold (15.70±4.10 % vs. 5.42±4.86 %) compared with control transgenic zebrafish with AcGFP expression. EPA was not detected in the control group, and the liver-specific fat-1 transgenic zebrafish with the contents of 2.49±1.46%. The fatty acid metabolism was toward fatty acid oxidation, because the expression of ppar-alpha, acox3 and cyp4t8 were activated. In addition, IL-1β, IL-6, IL-12α, IFN-γ and TNF-a, which regulate the genes involved in the promotion of inflammation, are significantly inhibited in the liver-specific expression of fat-1 transgenic zebrafish. In summary, the liver-specific expression of fat-1 in transgenic zebrafish enhanced the EPA and DHA, and inhibited pro-inflammatory cytokines and downstream genes after fed with homemade omega-6 PUFA feed. Secondly, predicted transcription factor binding sites of the intestine-specific fabp2 promoter revealed two enhancer regions in fabp2 2.5 kb promoter of Nile tilapia. The 1.5 kb fabp2 promoter containing enhancer 1 had little promoter activity whereas 2.5 kb fabp2 promoter containing enhancer 1 & 2 were active in both zebrafish and tilapia. Based on the in vivo promoter activity, we are establishing intestine-specific transgenic tilapia driven by either zebrafish Drfabp2 1.0 kb promoter or tilapia Onfabp2 2.5 kb promoter to express fat-1 and AcGFP. Furthermore, we will also explore the effects of endogenously produced omega-3 PUFAs on inflammation-related genes and protection against bacterial pathogens in transgenic tilapia.
URI: http://ethesys.lib.ntou.edu.tw/cgi-bin/gs32/gsweb.cgi?o=dstdcdr&s=G0010633008.id
Appears in Collections:[水產養殖學系] 博碩士論文

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