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研究生(外文):Cheng-Hung Tsai
論文名稱(外文):The Role of Loop 78 in the Primer-Independent Amylose Synthesizing Activity of Plastidial Starch Phosphorylase from Sweet Potato Roots
外文關鍵詞:starch phosphorylasePho1L78primer-independentPI
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甘藷塊根中的質體型澱粉磷解酶 (L-form starch phosphorylase, Pho1) 具有一段78個胺基酸的序列,稱為L78。當L78存在時,Pho1可以在不含短鏈寡糖作為引子的狀態之下進行直鏈醣的合成,意即具有不需醣引子澱粉合成活性 (primer-independent activity, PI activity)。在前人的研究中,已知Pho1透過pyridoxal phosphate (PLP) 與第一個Glc-1-P結合,而L78上有三組Glu-Lys,可能組成Glc-1-P第二結合位。在本研究中以實驗室原有之L78表現蛋白為模板,製備6株在這三組Glu或Lys單點突變的L78 mutant,並將其添加回L78完整斷裂的Pho1*,發現Pho1可能主要透過Lys529與Glc-1-P結合。另外,在Pho1之L78的功能相關的研究方面,幾乎都會使用中央斷裂型Pho1 (Pho1*) 來觀察L78斷裂前後對其活性的影響。先前Pho1*主要是採用自然斷裂法來製備,但是缺點是花費時間久又無法調控。本論文嘗試以三種方法來促進Pho1的斷裂,以期能發展出中央斷裂型Pho1的標準化製備法。但三種方法皆無法成功的移除L78。未來可以構築具有人造蛋白酶切位的Pho1的表現載體,並使用特定的蛋白酶來移除L78。

L-form Starch phosphorylase (Pho1) from sweet potato contains a 78 amino acids residues insertion (Loop 78, L78) and may catalyze primer-independent activity (PI activity) to synthesize starch. Pho1 may lose its PI activity completely when L78 is degraded, and the addition of extrinsic expressed L78 rescues the PI activity of the degraded Pho1. Previous studies have indicated that the first Glc-1-P may be bounded to the active site of Pho1 via pyridoxal phosphate (PLP), while the second Glc-1-P binding pocket may compose of 3 pairs of Glu-Lys repeats including Glu528-Lys529, Glu534-Lys535 and Glu540-Lys541 in L78. In this study, we constructed expression vector for L78 as well as its mutants for Glu-Lys pairs and tested their effects on the PI activity. The results showed that mutation at Lys529 may block the PI activity, demonstrating that Lys529 could be a molecular determinant for the second Glc-1-P binding site. On the other hand, the studies for the role of L78 involve in the preparation of L78-removed Pho1 (Pho1*). Previous methods for Pho1* preparation are usually time-costing, and the quality of Pho1* lacks for consistency. This study used trypsin, heat treatment and proteasome to accelerate degradation of Pho1, respectively. But neither of them removed L78 successfully. In the future, we will construct Pho1 expression vector with artificial protease cutting site and use designated protease to remove L78.

iii 中文摘要
iv Abstract
1 第一章 緒論
1 1.1 植物之澱粉代謝
2 1.2 澱粉磷解酶
3 1.2.1 澱粉磷解酶之生理功能
3 1.2.2 L型澱粉磷解酶之胺基酸序列特性
5 1.2.3 不需醣引子澱粉合成活性
7 1.3 L型澱粉磷解酶之調控機制
8 1.3.1 L78可能具有調控功能
9 1.3.2 L型澱粉磷解酶可受到磷酸化修飾
10 1.3.3 20S proteasome 降解路徑
11 1.4 研究動機
14 第二章 材料與方法
14 2.1 一般分析法
14 2.1.1 蛋白質定量
14 2.1.2 Pho1活性分析 (添加醣引子)
15 2.1.3 Pho1活性分析 (不添加醣引子)
15 2.1.4 電泳分析及CBR染色
16 2.1.5 活性染色法
16 2.1.6 免疫染色法
17 2.2 甘藷塊根澱粉磷解酶的製備
17 2.2.1 實驗材料
17 2.2.2 甘藷塊根完整型澱粉磷解酶 (Pho1) 之純化
18 2.2.3 甘藷塊根中央斷裂型澱粉磷解酶 (Pho1*) 之製備
18 2.2.4 37˚C處理加速Pho1降解試驗
18 2.2.5 Trypsin處理加速Pho1降解試驗
19 2.2.6 20S proteasome deletion mutant (DM) 處理加速Pho1降解試驗
19 2.3 E. coli表現系統之蛋白質表現
19 2.3.1 E. coli表現系統之載體構築
19 2.3.2 點突變技術 (site-direct mutagenesis)
20 2.3.3 L78重組蛋白質於E. coli之誘導表現與純化
21 2.3.4 20S proteasome deletion mutant (DM) 重組蛋白質於E. coli之誘導表現與純化
24 第三章 結果與討論
24 3.1 中央斷裂型澱粉磷解酶之標準化製備
26 3.1.1 37˚C處理加速Pho1降解
29 3.1.2 Trypsin處理加速Pho1降解
33 3.1.3 20S proteasome deletion mutant (DM) 處理加速Pho1降解
36 3.2 表現載體之建立與蛋白質表現
36 3.2.1 Wild-type L78表現載體之重新構築
40 3.2.2 20S proteasome deletion mutant (DM) 表現載體之重新構築
43 3.3 不需醣引子合成活性之Glc-1-P結合位
48 第四章 總結
48 4.1 Lys529在PI activity中可能做為主要與Glc-1-P結合的位點
48 4.2 未來展望
49 參考文獻
52 問答錄

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