臺灣博碩士論文加值系統

生質柴油是一種新興的替代能源，能夠取代石化柴油的燃料，其原料可來自含有高含量三酸甘油脂的植物油或廢棄生物質，藉由轉酯化反應產生單烷基酯長鏈脂肪酸與短鏈醇，如脂肪酸甲基酯。在最近幾年應用基因工程微藻生產生質柴油受到許多注目。脂肪酸合成是先將acetyl-CoA轉變成malonyl-CoA，這個反應由acetyl-CoA carboxylase (ACCase)的催化。因此ACCase往往被視為脂肪酸合成途徑中的關鍵酵素。根據這論點，我們希望增加ACCase的表現以達到增加細胞內脂質的含量，進一部增加生質柴油的量產。藍綠菌 (Synechocystis sp. PCC 6803)屬於藍綠菌的一種，因為其易於培養的生長特性，成為生質能源的首選。然而，現今沒有適用於藍綠藻的穿梭載體可用；因此本論文主要包括兩大方向：(1) 構築ACCase至表現載體中。(2) 設計和建構大腸桿菌與藍綠菌適合的穿梭載體，以利將來用於基因選殖和功能分析。研究結果顯示在大量表現ACCase時，細胞的脂質組成有所改變，改變的差異可以當作是否採用增加細胞ACCase來增加細胞內的脂質作為生質柴油研發的策略。而穿梭載體部分，我們將構築好的穿梭載體送入藍綠菌中，觀察穿梭載體是否能保留在菌體內，同時我們也測試出藍綠菌對ampicillin耐受性。

Biodiesel is an alternative energy source and a substitute for petrochemical diesel fuels. Transferification of triacylglycerols from plant oil or waste biomass yields monoalky esters of long-chain fatty acids with short chain alcohol such as fatty acid methyl esters. More attention has recently focused on the application of genetically engineered microalgae in the production of biodiesel. The reaction of acetyl-CoA to malonly-CoA is catalyzed by acetyl-CoA carboxylase (ACCase). ACCase is often regarded as the first committed step of the fatty acid synthetic pathway. My aim is to increase the lipid content of the cell through enhancing the expression of ACCase. Cyanobacterium Synechocystis sp. PCC 6803 (Synechocystis) is one of the best-characterized microalgae species and, given its robust growth characteristic, becomes the organism of choice for applications. Detailed studies on techniques for genetic manipulation of this species are available. Unfortunately, no suitable vector for cyanobacteria is courrently available. Therefore, the thesis has two main aims: (1) cloning ACCase on expression vector. (2) constructing a E.coli / Synechocystis suitable shuttle vector for efficient gene cloning and functional assays. My results show that overexpression of ACCase increases the lipid content of the cells. In addition, the shuttle vector constructed was transformed into Synechocystis for testing. As it turned out, it conferred antibiotic resistance to the cell and could be recovered as a plasmid form.

中文摘要 i
ABSTRACT ii
誌 謝 iii
目 錄 iv
圖 目 錄 vii
表 目 錄 viii
第一章 緒論 1
1.1 生質能源的簡介 1
1.1.1 何謂生質能源 1
1.1.2 生質能源的演進 2
1.2 生質柴油的簡介 3
1.2.1 生質柴油的介紹 3
1.1.2 生質柴油的製程 4
1.3 Acetyl-CoA carboxylase (ACCase)的簡介 6
1.3.1 ACCase的生化特性 6
1.3.2原核ACCase的介紹 7
1.3.3真核 ACCase的介紹 8
1.4藍綠菌Synechocystis sp. PCC 6803的簡介 10
1.5 實驗目的 12
第二章 材料與方法 14
2.1菌株、載體及培養基 14
2.2大腸桿菌勝任細胞的製備與轉型作用 15
2.2.1大腸桿菌勝任細胞的製備 16
2.2.2大腸桿菌勝任細胞的轉型作用 17
2.3酵母菌勝任細胞的製備與轉型作用 17
2.3.1酵母菌勝任細胞的製備 17
2.3.2酵母菌勝任細胞的轉型作用 18
2.5建構穿梭載體 19
2.6質體之選殖 20
2.7蛋白質製備 21
2.8 SDS-PAGE之蛋白質分子量分析 22
2.9西方點墨法 22
2.10細胞內脂質的測定 24
第三章 結果 25
3.1測試穿梭載體的功能 25
3.2 ACCase的表現 27
3.3 ACCase送入細胞後，胞內脂質含量的改變 28
第四章 討論 30
參考文獻 33

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