日本虎斑猛水蚤之基因表現與功能性基因庫篩選

Gene expression and function genomic library screening of copepod Tigriopus japonicus

潘國文(Guo-Wen Pan)

指導教授 ： 張繼堯

國立臺灣大學/生命科學院/漁業科學研究所/碩士(2009年)

https://doi.org/10.6342/NTU.2009.10369

摘要

橈足類也許是地球上數量最多的多細胞生物，超過11,500個種類比陸地上的昆蟲數量還多。此成功演化的原因主要歸功於其高生殖率及快速的胚胎發生。而海洋橈足類也成為自然界孕育魚苗最佳的餌料生物。橈足類因為體型小富含高度長鏈不飽和脂肪酸，有潛力發展成為魚苗初期發育最佳的餌料生物。本研究對象日本虎斑猛水蚤對環境適應力強，且為雜食性，容易培養，因此有潛力發展為大量培養的魚苗生物餌料種類之一。關於日本虎斑猛水蚤生長發育及營養分析的研究不少，但基因層次上的相關研究並不多。本研究利用餵食日本虎斑猛水蚤不同的食物，並探討其生長情形與體內脂肪酸組成，再構築日本虎斑猛水蚤之cDNA基因庫，來探討其營養代謝合成相關基因和發育生長相關基因群之表現。本實驗以不同食物來培養日本虎斑猛水蚤14天，並觀察族群變化。於第14天收集蟲體分析脂肪酸組成，發現餵食周氏扁藻的蟲體，密度為每公升302.5隻，DHA和EPA分別佔總脂肪酸的1.56%與1.58%。餵食發酵液的蟲體，密度為每公升390隻，DHA和EPA分別佔總脂肪酸的5.53%與8.27%。餵食酵母的蟲體，密度為每公升392.5隻，DHA和EPA分別佔總脂肪酸的7.57%與11.57%。餵食奶粉的蟲體，密度為每公升1,217.5隻，DHA和EPA分別佔總脂肪酸4.24%與6.12%。以產量和食物取得便利性而言，奶粉為培養日本虎斑猛水蚤之最佳食物來源。本實驗利用cDNA 微陣列晶片經交叉比對後篩選出142個差異性表現基因，經定序比對分析，其中86個比對到相似序列基因，11個為功能不明的基因序列，扣除重複後共得72個促進表現基因。這些促進表現基因包括轉譯因子、呼吸鏈、結構、信息傳遞、發育、代謝、生長因子、運輸蛋白和調控蛋白等。此外，我們也選殖出Delta-9-desaturase基因，此將有助於日本虎斑猛水蚤在脂質代謝方面的相關研究。

關鍵字

虎斑猛水蚤 ； 不飽和脂肪酸

並列摘要

Copepods may be the most abundant multi-cellular organism in the world, which contains more than 11,500 species that more than that of insects. Its evolutionary success contributes to its high reproduction rate and rapid embryonic development, and the oceanic copepods naturally become the best living feed for fish fry in the nature. Copepods has the potential to become the best living feed for fish fry early development because of its small size and rich in long chain unsaturated fatty acid. The Tigriopus japonicus in this study has great adaptability to environment, polyphagia and easy to culture, so has the potential to develop as one of the fish fry living feed species that can be mass produced. Although there are quite a few studies on growth development and nutritional analysis of Tigriopus japonicus, however, the study on genetic level is still quite limited. In this study, we fed Tigriopus japonicus with different food to investigate the growth situation and fatty acid composition. The cDNA library of Tigriopus japonicus was further constructed to discuss the expression of nutritional metabolism related genes and development related genes. Tigriopus japonicus were fed with different food for 14 days and the population changes were observed. The fatty acid composition analysis on the 14th day showed that the density of Tigriopus japonicus fed with Tetraselmis chui was 302.5/L, and the DHA and EPA was 1.56% and 1.58% of total fatty acid respectively. The density of Tigriopus japonicus fed with fermented liquid was 390/L, and the DHA and EPA was 5.53% and 8.27% of total fatty acid respectively. The density of Tigriopus japonicus fed with yeast was 392.5/L, and the DHA and EPA was 7.57% and 11.57% of total fatty acid respectively. The density of Tigriopus japonicus fed with milk powder was 1217.5/L, and the DHA and EPA was 4.24% and 6.12% of total fatty acid respectively. From the production amount and convenience point of view, milk powder is the best food source of Tigriopus japonicus culture. In this study, we screened and obtained 142 up-regulated genes from cDNA library. After blasting, 86 of which can find the homologous genes, 11 of which are unknown function. The up-regulated genes include transcription factors, respiratory chain, structure, signal transduction, development, metabolism, growth factors, transportation proteins and regulatory proteins. Besides, we also subcloned Delta-9-desaturasae gene, which might be helpful on the research of Tigriopus japonicus on lipid metabolism.

並列關鍵字

Copepoda ； Tigriopus japonicus ； DHA ； EPA

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