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研究生:姚培莉
研究生(外文):Yao, Pei-Li
論文名稱:甘藷塊根Sporamin蛋白質定點突變對胰蛋白脢抑制因子活性之影響
論文名稱(外文):Effect of Structural Modification on Trypsin Inhibitory Activity of Sweet Potato Sporamin by Site-Directed Mutagenesis
指導教授:葉開溫葉開溫引用關係
指導教授(外文):Yeh, Kai-Wun
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:植物學研究所
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:1999
畢業學年度:88
語文別:英文
論文頁數:90
中文關鍵詞:甘藷甘藷塊根胰蛋白脢抑制因子定點突變三級結構模擬
外文關鍵詞:sweet potatosweet potato tubersporamintrypsin inhibitorsite-directed mutagenesisthree-dimensional modeling
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Sporamin為甘藷塊根中含量最豐富的儲藏性蛋白質,我們將所篩選出的sporamin cDNA 構築於表現載體 pGEX2T,並轉殖至大腸桿菌 (Escherichia coli) XL1-Blue 中表現,證實其具有胰蛋白脢抑制因子 (Trypsin inhibitor) 活性。經由初步分析胺基酸一級排列 (Sequence alignment)、二級結構推演 (Secondary structure prediction),並比對資料庫 (Protein data bank, PDB) 的結果,模擬 sporamin 的三級結構應與 Erythrina caffra 所含的 ETI 結構相近,因而推測出與活性有關的胺基酸位置所在,進一步以定點突變 (Site-directed mutagenesis) 的方式,分析突變對於 sporamin 活性的影響。發現 Asp70Val、Glu72Arg 及 Ser73Ile 突變株均喪失胰蛋白脢抑制因子活性,而 Ala 69Ser 則仍維持活性,乃確定甘藷塊根sporamin 的活性區域為 72Arg-73Ser;此結果與先前所模擬出的三級結構相符。此外,由於 sporamin 的結構中含有兩對雙硫鍵,乃設計將 45Cys 置換為 Leu以打破雙硫鍵,結果發現此突變株喪失了胰蛋白脢抑制因子活性。所以,維持 sporamin 結構的完整性,對於胰蛋白脢抑制因子活性的存在是有必要的。確定 sporamin 之活性區所在後,回歸三級結構的預測,便可重新定出 sporamin 完整的蛋白質三級結構圖。

In our earlier studies, we screened four sporamin cDNA clones from sweet potato tubers and demonstrated their trypsin inhibitory (TI) activities as well as insect resistance. However, we could not identify the precise TI activity domain of our clones by amino acid sequence alignment with other TIs or three-dimensional (3D) modeling. In this study, we identified amino acid residues that were important for the sporamin TI activity by site-directed mutagenesis. Modification on three amino acid residues, namely Asp70Val, Glu72Arg, and Ser73Ile, resulted in a complete loss of TI activity. In contrast, modification of Ala69Ser retained the TI activity. Therefore, we concluded that the TI activity domain of the sweet potato sporamin resided in 69Asp-73Ser. Results using 3D modeling also supported our conclusion. We also identified two disulfide bonds within sporamin. When one of the cysteine residues, Cys45, was mutated to leucine, the TI activity was lost. This indicated that disulfide bonding and the integrity of the structure of sporamin were important factors contributing to the TI activity.

中文摘要-----------------------------------------------------1
Abstract--------------------------------------------------------2
Introduction--------------------------------------------------3
Materials and Methods-----------------------------------13
Results---------------------------------------------------------37
Discussion----------------------------------------------------67
References----------------------------------------------------77
Appendix-----------------------------------------------------83

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