藉由轉殖∆6和∆5去飽合酶改善吳郭魚n-3多元不飽和脂肪酸合成並且調控膜之流動性增強其抗寒能力

Improvement of n-3 PUFA biosynthesis by transgenic Δ6-desaturase and Δ5-desaturase in tilapia can enhance cold tolerance by maintaining membrane fluidity

林正偉(Cheng-Wei Lin)

指導教授 ： 吳金洌

共同指導教授 ： 陳彥榮(Edward Chern)

國立臺灣大學/生命科學院/生化科技學系/碩士(2018年)

https://doi.org/10.6342/NTU201803195

摘要

深海魚油中的n-3多元不飽和脂肪酸已經被大量的利用在人類的營養食品與水產養殖，魚油中的EPA與DHA已經被證實會影響人類的心血管疾病、神經調控和免疫功能；在水養殖方面可以改善魚的孵化成長與抗寒能力。但是隨著漁業資源的耗竭導致魚油取得不易價格也不斷上升，因此全世界都正在開發可以解決魚油資源枯竭的問題。目前較常見的方法是利用基因轉殖的方式去提高EPA與DHA的合成，的確在哺乳類有一定的成果，但是在水產養殖魚類這方面的研究還是非常稀少。吳郭魚是目前一種全世界新興的養殖魚種，在2016年已經達到4,220,000噸的產量，但是其EPA與DHA的含量卻是只有養殖鮭魚的二十分之一，因此吳郭魚非常適合做為提升n-3多元不飽和脂肪酸的合成工廠。在近幾年氣候變遷，侵害養殖漁業的寒流不斷增強，導致漁民不小的損失，在提升吳郭魚EPA與DHA的同時，也可以增強其抗寒能力。但過去多數關於n-3多元不飽和脂肪酸的研究只侷限在膜的流動性又或者是冷壓力下單一組織總觀的機制調控。本研究希望藉由確認轉殖大西洋鮭魚的∆6與∆5去飽合酶基因之吳郭魚脂肪酸組成藉由mRNA與蛋白質表現確定轉基因吳郭魚的成功。並且以氣相色譜質譜分析脂肪酸組成，結果呈現雙轉魚的EPA與DHA各自在肝臟提升了91%與59%。在模擬台灣寒流的降溫模式實驗中，雙轉魚與單轉魚維持在寒冷下身體的平衡能力都比野生種提升了約一倍，並且在生存率方面雙轉魚更提升了1.1倍。腦做為遇到冷壓力的第一級反應器官，藉由次世代定序的分析本研究發現關於腦的膜上訊號與滲透壓都是被顯著調控，並且膜上磷脂質的雙鍵指數也是顯著上升影響膜的流動性。結論證實藉由改善吳郭魚的n-3多元不飽和脂肪酸合成，調控膜上磷脂質並影響膜的流動性，維持膜上訊號與滲透壓的狀態導致轉基因魚有更好的抗寒能力。

關鍵字

多元不飽和脂肪酸 ； 抗寒 ； 吳郭魚 ； 膜的流動性 ； 去飽合酶

並列摘要

The n-3 polyunsaturated fatty acids in deep sea fish oil have been heavily utilized in human nutritious foods and aquaculture. EPA and DHA in fish oil have been shown to affect human cardiovascular disease, neuromodulation and immune function; Farming can improve the hatching growth and cold resistance in fish. However, along with the depletion of fishery resources, the price of fish oil is increasing, so the world is solving the problem of depletion of fish oil resources. At present, the common method is to use genetic engineering to improve the synthesis of EPA and DHA. It does have some results in mammals, but research on aquaculture fish is still very rare. Tilapia is a developing of farmed fish species in the world. It has reached 4,220,000 tons in 2016, but its EPA and DHA content is only one tenth of that of farmed carp. Therefore, tilapia is very suitable for a synthetic factory that upgrades n-3 polyunsaturated fatty acids. In recent years, the climate change has intensified, and the cold surge of the aquaculture fishery has been continuously enhanced, resulting in many lose for the fishermen. While raising the EPA and DHA of the tilapia, it can also enhance its cold resistance. However, most of the research on n-3 polyunsaturated fatty acids in the past was limited to the membrane fluidity or the mechanism of single tissue observation under cold pressure. In this study, the success of transgenic tilapia was determined by mRNA and protein expression level and confirmed the fatty acid composition by gas chromatography mass spectrometry. EPA and DHA in double transgenic tilapia each grew 91% and 59% in the liver. In the experiment of simulating the cooling mode of the cold surge in Taiwan, the ability of the double transgenic and the single transgenic tilapia to maintain the equilibrium of body under cold shock was about double that of the wild type, and the double transgenic tilapia in the survival rate was increased by 1.1 times. As the first-stage reaction organ that encounters cold stress, the brain was significantly regulated according to the next generation of sequence analysis. The gene of membrane signal and osmotic pressure on the brain are also regulated, and the phospholipid double bond index on the membrane is also significant rise affects the fluidity of the membrane. Conclusion, it is confirmed that improved the synthesis of n-3 polyunsaturated fatty acids in tilapia, regulated the phospholipids on the membrane and affected the fluidity of the membrane, maintained the signal on the membrane and the osmotic pressure lead to better cold tolerance of the transgenic fish.

並列關鍵字

Polyunsaturated fatty acid ； Cold tolerance ； Tilapia ； Membrane fluidity ； Desaturase

參考文獻

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