三酸甘油酯(Triacylglycerol, TAG)係由Diacylglycerol acyltransferase(DGAT)將diacylglycerol (DAG)接上一脂肪酸組合而成，而脂肪酸由ACCase (Acetyl-CoA carboxylase)及FAS (Fatty acid synthase)合成。本研究以單胞藻(Chlamydomonas reinhardtii)薄細胞壁(cell-wall less)品系CC400探討合成TAG及脂肪酸相關基因，包含五個DGAT [DGAT1 (JGI 184281)、DGAT2 (JGI 400751)、 DGAT3 (JGI 285889)、DGAT4 (JGI 141301)及DGAT5 (JGI 190539)]與兩個FAS之subunit [三個 β ketoacyl-ACP reductase isoforms ( (JGI 153976)、(JGI 153976)及(JGI 194728)) 與兩個β ketoacyl-ACP synthase (KAS) isofroms ( (JGI 139619)和(JGI 205887)) ]及三個ACCase ( ACCα (NCBI XP_001696945.1)、ACCβ (NCBI XP_001703187.1)與ACC biotin carboxylase (NCBI XP_001702319.1)之mRNA量與缺氮誘導油脂累積之關係。以有機碳源(acetate)添加之有無觀察藻體油脂累積及mRNA量變化，在缺氮下CC400有油脂累積，同時CC400有DGAT3之mRNA量增加，而FAS中則無差異；缺氮之CC400 ACCα、ACCβ 及ACC biotin carboxylase mRNA量皆下降。結果顯示DGAT3為缺氮下油脂累積之必須。以上述結果為依據，以DGAT基因表現為調查缺氮且缺acetate之指標，發現在缺氮誘導之DGAT1、DGAT2及DGAT3 mRNA量在缺氮且缺acetate下會部分被抑制，而當培養基缺acetate 時則使DGAT4 mRNA 量上升。因此，本研究證實在C. reinhardtii 中acetate為缺氮下油脂累積及DGAT3 基因表現(mRNA量) 之必須。

Diacylglycerol acyltransferase (DGAT) is a key for the synthesis of triacylglycerol (TAG) from diacylglycerol in the unicellular green alga Chlamydomonas reinhardtii.Acetyl-CoA carboxylase (ACCase) and fatty acid synthase (FAS) are responsible for the synthesis of fatty acids. We found the TAG and fatty acid synthesis related genes in C. reinhardtii, including five DGAT (DGAT1 (JGI 184281), DGAT2 (JGI 400751), DGAT3 (JGI 285889), DGAT4 (JGI 141301), and DGAT5 (JGI 190539)), three β ketoacyl-ACP reductase isoforms ( (JGI 153976), (JGI 153976), and (JGI 194728)) and two β ketoacyl-ACP synthase isofroms ( (JGI 139619) and (JGI 205887)) for FAS, and ACC α (NCBI XP_001696945.1), ACC β (NCBI XP_001703187.1) and ACC biotin carboxylase ( NCBI XP_001702319.1)) for ACCase in C. reinhardtii. This investigation designed the primers of the above genes to determine whether acetate influences their mRNA expression levels in cell-wall less strain CC400 in the nitrogen starvation condition. The results showed that the absence of nitrogen in the medium triggered the lipid accumulation for the strains of CC400 in the condition of 50 μE light. DGAT3 mRNA levels were increased by nitrogen starvation. For the FAS genes, in the strain of CC400 showed no increased mRNA levels upon exposure to nitrogen starvation. The mRNA levels of ACCα, ACC β and ACC biotin carboxylase were more or less decreased by nitrogen starvation in CC400 strains. Thus, the responses of DGAT gene expression to acetate supplement were checked. The absence of acetate from the medium partly inhibited the nitrogen starvation induced increases in lipid and DGAT3 mRNA levels, and the mRNA levels of DGAT1 and DGAT2 in the nitrogen starvation condition. However, DGAT4 mRNA levels were significantly induced by the absence of acetate from the medium. In conclusion, the present study demonstrate that acetate is required for the nitrogen starvation induced DGAT3 gene expression (mRNA levels) and lipid accumulation in C. reinhardtii.

中文摘要 ...................................................................................................................... ii
英文摘要 ..................................................................................................................... iii
目錄 ................................................................................................................................v
表目錄 ..........................................................................................................................vi
圖目錄 ........................................................................................................................ vii
附錄目錄 ................................................................................................................... viii
縮寫字對照 ...................................................................................................................x
一、前言 .......................................................................................................................1
二、材料方法 ...............................................................................................................9
三、藥品與儀器 .........................................................................................................16
壹、藥品 .................................................................................................................. 16
貳、儀器 .................................................................................................................. 22
参、藥品配方 .......................................................................................................... 23
四、結果 .....................................................................................................................25
五、討論 .....................................................................................................................31
六、參考文獻 .............................................................................................................33
七、表 .........................................................................................................................40
八、圖 .........................................................................................................................42
九、附錄 .....................................................................................................................56

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