- 學位論文
芝麻種子發芽時期 cDNA 庫之構築與脂肪酶基因之選殖
Construction of cDNA Libraries from Germinating Sesame Seeds and Cloning of Lipase Genes
摘要
脂肪酶（Lipase; EC 3.1.1.3）可水解三酸甘油脂形成甘油及脂肪酸，可廣泛地應用於脂肪、食品、清潔劑工業及生技產業上。芝麻（Sesamun indicum L.）屬於胡麻科，是目前世界上所栽種的重要油料作物之一；已知芝麻種子含油量超過 50%，且以三酸甘油酯的形式儲存能量，可提供作為種子發芽及幼苗生長所需之養份及能量來源。因芝麻種子在發芽兩天後約有超過 50% 的油脂被耗盡，故推測其參與水解油脂以產生能量的酵素-脂肪酶在此一時期應具極高的活性。本研究的目的即在於構築芝麻種子發芽時期的 cDNA library，選殖參與水解油脂的 lipase 基因以進行功能性基因表現。分別取各發芽時間點的芝麻種子粗萃液分析其脂肪酶酵素活性，發現在發芽 16小時後的lipase 酵素活性可達到最高，之後則隨著時間而下降。利用根據已知序列所設計的植物 lipase degenerate primers，以 PCR 偵測 lipase 基因於各發芽時間點的表現並選殖其 cDNA；結果發現可於發芽8小時的 cDNA library中篩選到三個 lipase/esterase 基因，序列比對的結果顯示它們應是三個不同的脂解酶基因。因此，目前已構築好芝麻發芽8小時及12小時之 cDNA library，以供選殖這三個脂解酶的 cDNA 全長序列，再分析這三個脂肪酶基因的表現。
並列摘要
Lipase (triacylglycerol acylhydrolase; EC 3. 1. 1. 3), an enzyme which catalyzes the hydrolysis of triacylglycerol to glycerol and fatty acids, has been widely used in fat, food ingredients, detergents and biotechnology industries. Sesame（Sesamun indicum L.）, belongs to the Pedaliaceae family, is one of most important oilseed crops in the world. Sesame seed possesses more than 50% oil content in dry weight. The storage triacylglycerols (TAGs) is used as energy source for germination and subsequent post-germinative growth. Because more than 50% of oil in sesame seed is exhausted after sprouting for two days, it is suggested that the lipase exhibits highest activity during this stage. The purpose of this study is to construct cDNA libraries from germinating sesame seeds and to clone the lipase genes for functional analysis of gene expression. Sesame seed extracts from different time points after imbibition are subjected for assaying lipase activity. It is found that lipase activity increases after imbibition and the maximum activity is detected at 16 hours after imbibition. Detection of lipase gene expression and cloning of partial cDNAs at different time points are conducted by PCR cloning by the use of designed plant lipase degenerate primers. Three partial lipase/esterase cDNA sequences are cloned, and the result of sequence alignment shows that they may be different lipase/esterase genes. Therefore, cDNA libraries of 8 and 12 hours after imbibition are constructed and used to clone full-length lipase/esterase cDNA sequences. Expression analysis of three lipase genes could be further studied.
參考文獻
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