本論文分為兩部分，分別為甘藷穀胱甘胺酸還原?之基因選殖、表現及酵素特性分析，另外，也針對樟芝穀胱甘胺酸還原?進行基因選殖。
本實驗利用聚合?連鎖反應，自臺農 57 號甘藷 (Ipomoea batatas [L.] Lam) 塊根中，選殖出 glutathione reductase (GR) 之 cDNA，基因全長 2062 bp，轉譯區包含 1482 bp，可轉譯出 494 個胺基酸，分子量為 54.7 kDa。在以往的研究發現，GR 胺基酸序列會包含兩個高保留的半胱胺酸，IbGR (Cys 70 與 75) 符合此特性。
IbGR以pYEX-S1作為表現載體，S. cerevisiae CK3 作為表現宿主，可表現出大量的重組蛋白質，而所表現的重組蛋白質，可利用含鎳螯合物凝膠之親和性管柱來純化。
從 IbGR 的酵素動力學分析，得到 IbGR 對 GSSG 的 Km 值為0.114 mM，而對於 NADPH 的 Km 值為 0.056 mM。在特性分析上，IbGR 經 70 ℃ 加熱 2 分鐘，活性仍保有 79 %，其半衰期為 3.3 分鐘；經二價金屬離子處理，活性影響分別為硫酸銅使活性完全喪失，硫酸鋅殘存 42 % 活性，氯化鈣不受影響；經不同 pH 值的緩衝液處理，顯示 pH 2.0∼4.0 會讓 IbGR 的活性完全喪失，而在 pH 6.0∼11.0 的範圍內，活性均很安定；經不同濃度的 imidazole 處理，IbGR 的活性均很安定；經 chymotrypsin 與 trypsin 在 37 ℃ 下反應 40 分鐘，活性仍保有 76 % 和 73 %。
另外，本實驗利用聚合?連鎖反應，自牛樟芝 (Taiwanofungus camphoratus) 子實體中，選殖出 GR 部分 cDNA 序列，長度為 396 bp。

The purpose of this thesis falls into two parts. We study an antioxidant enzyme which is glutathione reductase (GR) from Ipomoea batatas and Taiwanofungus camphoratus.
A full length cDNA of 2062 bp encoding a putative GR from Ipomoea batatas was cloned by polymerase chain reaction. It contains an open reading frame of 1482 bp which encodes a protein of 494 amino acid residues with a predicted molecular mass of 54.7 kDa. The two residues coordinating active site Cys 70, Cys 75 are conserved as they are present in all reported GR sequences.
To further characterize the IbGR, the coding region was subcloned into an expression vector pYEX-S1 and transformed into yeast S. cerevisiae CK3. The expression of the IbGR was purified by nickel chelating sepharose superflow column.
The Michaelis constant (Km) values for GSSG and NADPH were 0.114 mM and 0.056 mM for the IbGR. The protein's half-life of deactivation at 70 °C was 3.3 min. The enzyme activity was inhibited by Cu2+ and Zn2+, but not by Ca2+. The enzyme activity was stable in a broad pH range from 6.0 to 11.0, but had little or no activity at pH 4.0 or lower. Addition of imidazole up to 0.8 M had no significant effect on the activity. Last, the native enzyme was resistant to digestion by chymotrypsin or trypsin.
The other part of this thesis was cloned a partial cDNA of 396 bp from Taiwanofungus camphoratus by polymerase chain reaction.

中文摘要..................................................Ⅰ
英文摘要..................................................Ⅲ
縮寫表.....................................................V
壹、 前言..................................................1
一、 甘藷.................................................1
二、 樟芝.................................................3
三、 自由基...............................................5
四、 植物的抗氧化機制....................................11
五、 穀胱甘胺酸還原?....................................15
六、 研究動機............................................17
貳、 實驗材料.............................................26
一、 材料................................................26
二、 載體................................................26
三、 宿主................................................26
四、 培養基..............................................27
五、 實驗耗材............................................28
六、 儀器設備............................................28
七、 實驗藥品............................................29
八、 引子................................................32
參、 實驗方法..............................................33
一、 mRNA萃取...........................................33
二、 cDNA合成...........................................34
三、 甘藷與樟芝穀胱甘胺酸還原?之基因選殖.................35
四、 甘藷穀胱甘胺酸還原?之蛋白質表現與純化...............40
五、 甘藷穀胱甘胺酸還原?之活性測定.......................47
六、 甘藷穀胱甘胺酸還原?之特性分析.......................48
肆、 結果..................................................51
實驗一 甘藷穀胱甘胺酸還原?
一、 甘藷穀胱甘胺酸還原?之基因選殖.......................51
二、 甘藷穀胱甘胺酸還原?之蛋白質表現與純化...............53
三、 甘藷穀胱甘胺酸還原?之酵素動力學.....................55
四、 甘藷穀胱甘胺酸還原?之特性分析.......................56
實驗二 樟芝穀胱甘胺酸還原?
一、 樟芝穀胱甘胺酸還原?之基因選殖.......................60
伍、 討論..................................................73
陸、 參考文獻..............................................78
附錄......................................................87

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