臺灣博碩士論文加值系統

二苯乙烯合成酶（STS，EC2.3.1.95），為白藜蘆醇的生物合成中的關鍵酵素，目前已知白藜蘆醇具有殺真菌劑，抗氧化劑，抗血小板聚集作用，和抗炎活性。研究顯示，經二苯乙烯合成酶生產的生產白藜蘆醇化合物主要成分為植物抗毒素，具防禦植物性真菌病原體之反應，是藥物學上具有重要性的高生物活性物質。白藜蘆醇衍生物自苯基丙酸路徑，已知對人體健康有正向影響，已被證實有助於治療阿爾茨海默氏症、糖尿病、冠狀動脈心臟疾病。本研究目的為使用微生物的生物系統去生產有價值的分子，利用生產白藜蘆醇的工程菌，可作為大量生產的一種重要方法。利用微生物的系統去生產白藜蘆醇以降低生產的成本，並且還可防止過度採集和天然資源的破壞。然而二苯乙烯路徑並不存在於細菌或酵母中，因此需要引入整個具功能性的途徑；且要考量到整個生產過程中所引入來自二苯乙烯路徑中的一個或兩個基因以及添加的前驅物，可能會明顯的限制獲得代謝物所需要的成本和效果。以野外生長的葡萄屬植物 DNA 為模板，設計二苯乙烯合成酶基因特異性引子並利用 PCR 選殖二苯乙烯合成酶基因的全長片段。共選殖到七個二苯乙烯合成酶基因，分別是 STS1, STS16, STS43, STS46, STS4, STS51 及 STS58。序列分析顯示這些二苯乙烯合成酶基因的全長片段皆包含了兩個 exons 以及一個 intron。二苯乙烯合成酶基因編碼區域的長度有 1,179 個核苷酸，將其選殖至具有 NcoI和XhoI限制酶切位的表達載體 pET28a 上。重組STS58蛋白質之分子量大小為 43 kDa，在大腸桿菌 BL21（DE3） 細胞中，藉由異丙基-β-D-硫代半乳糖苷 （IPTG） 之誘導可使重組STS58蛋白質大量表達；但本實驗中重組STS58蛋白質由於不正確的蛋白質折疊和轉譯後修飾，因此形成不溶性的聚集體。在未來，研究將集中於重組STS58 蛋白質在真核表現系統中的表達。

Stilbene synthase (STS, EC 2.3.1.95), a key enzyme in resveratrol (trans-3, 5, 40-trihydroxy-stillbene) biosynthesis, and resveratrol are known to have antifungal, antioxidant, anti-platelet aggregation, and anti-inflammatory activity. Recent studies showed that stilbene synthase could lead to the production of resveratrol compounds, which were major components of the phytoalexin response against fungal pathogens of plant and were highly bioactive substances of pharmaceutical interest. Resveratrol, derived from the phenylpropanoid pathway, is also known for its positive effect on human health. Resveratrol has been suggested for the treatment of Alzheimer’s disease, diabetic, and coronary heart disease. The objectives of this study are to use biological systems for the production of numerous valuable molecules in microorganisms. Engineering bacteria for resveratrol production might thus represent a valuable means for its production in large quantities. The microorganism production of resveratrol would lower the cost, and also prevent intensive cutting and decimation of the natural sources. However, as the stilbene pathway does not exist in bacteria, or in yeast, the entire functional pathway needs to be introduced. Thus, the introduction of only one or two genes from the stilbene pathway and supplementing the cells with a precursor might significantly limit the cost and effort required to obtain the desired metabolite, and should be taken into account when considering the entire production process. Isolation of genome DNA from wild-growing grape Vitis sp. and design of gene-specific primers of the grape stilbene synthases are used for the cloning the full-length stilbene synthases gene by PCR. Seven STS genes, including STS1, STS16, STS43, STS46, STS4, STS51 and STS58, respectively. Sequence analysis indicated that these full-length STS genes all contained two exons and one intron. The coding regions of STS genes are 1,179 nucleotides in length, and they are introduced into expression vector pET28a at NcoI and XhoI restriction sites. The recombinant STS58 proteins, with a molecular weight of 43 kDa, are highly expressed in Escherichia coliBL21(DE3) cells by isopropyl-β-D-thiogalactoside (IPTG) induction, but they are present as insoluble aggregates due to inappropriate protein folding and post-translational modification. In the future, studies have been focused on eukaryotic expression of the recombinant STS58 proteins.

摘要 I
Abstract III
致謝 V
目錄 VII
圖目錄 XIV
表目錄 XVII
第一章 前言 1
第二章 文獻回顧 5
2.1 野生山葡萄 5
2.1.1 山葡萄之分類地位 5
2.1.2 漢氏山葡萄之型態特徵、分佈及其用途 6
2.1.3 細本山葡萄之型態特徵、分佈及其用途 6
2.2 植物基因轉殖在醫藥上之應用 8
2.3 植物之二次代謝產物 9
2.4 中草藥ITS序列資料庫 11
2.5 白藜蘆醇 14
2.5.1 白藜蘆醇之來源 14
2.5.2 白藜蘆醇之化學性質 16
2.5.3 白藜蘆醇之生合成路徑 16
2.5.4 國內外白藜蘆醇的研究概況及發展 19
2.5.5 白藜蘆醇的生理功能 19
2.6 反式白藜蘆醇及其衍生物的分析方法 23
2.6.1 紫外光光度法 23
2.6.2 薄層層析-紫外分光光度法 24
2.6.3 螢光光度法 24
2.6.4 高壓液相色層分析法 25
2.6.5 氣相層析質譜分析儀 25
2.6.6 毛細管電泳法 25
2.6.7 非親水性毛細管電泳法 26
2.6.8 高錳酸鉀褪色分光光度法 26
2.7 原核表現系統 28
第三章 材料與方法 31
3.1實驗材料 31
3.1.1 藥品 31
3.1.2 實驗儀器 31
3.1.3 載體 32
3.1.4 DNA 分析用緩衝溶液 32
3.1.5 聚合酶連鎖反應（polymerase chain reaction; PCR）之引子 33
3.1.6 酵素 34
3.2 實驗架構 35
3.3 實驗方法 35
3.4 白藜蘆醇合成酶基因選殖 36
3.4.1 Genomic DNA 之萃取 36
3.4.2 設計引子 36
3.4.3 聚合酶連鎖反應之擴增 STS 基因 37
3.4.4 PCR 產物鑑定 37
3.4.5 DNA 切膠純化 37
3.4.6 yT＆A Cloning 38
3.4.7 接合反應 (ligation) 38
3.4.8 轉型 (transformation) 39
3.4.9 plasmid DNA 小量萃取 40
3.4.10 限制酶確定重組基因 41
3.3.11 核酸定序 42
3.3.12 白藜蘆醇合成酶基因之序列分析 42
3.3.13 白藜蘆醇合成酶基因之 intron 序列分析 43
3.3.14 白藜蘆醇合成酶序列比對與分析 43
3.5 白藜蘆醇合成酶基因之exon1st 以及 exon2nd 個別 DNA 片段選殖 43
3.5.1 引子設計 44
3.5.2 聚核酶連鎖反應之擴增 STS genes 之exon1st 以及 exon2nd 個別 DNA 片段 45
3.5.3 STS genes 之exon1st 以及 exon2nd接合反應 (ligation) 47
3.5.4 DNA Clean-up 47
3.5.5 聚核酶連鎖反應之擴增 STS genes 之exon1st-exon2nd DNA 片段 48
3.5.6 PCR 產物鑑定 48
3.5.7 DNA 切膠純化 48
3.5.8 yT＆A Cloning 49
3.5.9 接合反應 49
3.5.10 轉型 (transformation) 50
3.5.11 增殖含選殖片段之菌落 50
3.5.12 plasmid DNA 小量萃取 50
3.5.13限制酶確定重組基因 50
3.5.14 核酸定序 52
3.5.15 白藜蘆醇合成酶基因之序列分析 52
3.5.16 生物資訊分析方法 52
3.6 白藜蘆醇合成酶基因 (STS58) 選殖至原核表達載體 54
3.6.1 選殖載體之製備與純化 54
3.6.2 STS58 基因與選殖載體之接合作用 54
3.6.3 轉型 55
3.6.4 限制酶確認重組基因 55
3.6.5 核酸定序 55
3.7 原核系統進行白藜蘆醇合成酶表達 56
3.7.1 重組載體轉型至蛋白質表達菌株 56
3.7.2 白藜蘆醇合成酶 STS58 基因之誘導 56
3.7.3 最佳化白藜蘆醇合成酶誘導條件測試 57
第四章 結果與討論 60
4.1 白藜蘆醇合成酶 STS 基因選殖 60
4.1.1 葡萄屬植物 Genomic DNA 之萃取 60
4.1.2 葡萄屬植物 ITS 序列分析結果 60
4.1.3 聚合酶連鎖反應之擴增 STS genes 基因 60
4.1.4 白藜蘆醇合成酶基因選殖至 yT&;A 載體 60
4.2. 葡萄屬植物白藜蘆醇合成酶基因之序列分析 (含intron) 66
4.2.1 白藜蘆醇基因含 intron 之核苷酸序列片段 67
4.2.2 白藜蘆醇基因之 intron 序列比對 67
4.2.3 白藜蘆醇基因扣除 intron 之核苷酸序列片段 68
4.3 白藜蘆醇基因核苷酸序列比對 68
4.4 白藜蘆醇合成酶胺基酸序列比對與分析 68
4.5 STS58之exon1st-exon2nd 選殖至 yT&;A 載體 92
4.5.1 白藜蘆醇合成酶基因之exon1st 以及 exon2nd 個別 DNA 片段選殖 92
4.5.2聚核酶連鎖反應之擴增 STS58 之exon1st-exon2nd DNA 片段 92
4.5.3 STS58之exon1st-exon2nd 選殖至 yT&;A 載體 92
4.6 白藜蘆醇合成酶基因 STS58 核苷酸序列比對 98
4.7 原核表達之白藜蘆醇合成酶基因選殖與表達 101
4.7.1白藜蘆醇合成酶基因選殖至 pET28a 載體 101
4.7.2 確認白藜蘆醇合成酶基因選殖至 pET28a 載體 101
4.7.3 白藜蘆醇合成酶基因最佳化表現條件測定 105
4.7.3.1 白藜蘆醇合成酶基因於不同時間點下之重組蛋白質表達 105
4.7.3.2 白藜蘆醇合成酶基因於不同溫度下之重組蛋白質表達...........................................................................................113
4.7.3.3 白藜蘆醇合成酶基因於不同濃度 IPTG 下之重組蛋白質表達..........................................................................116
4.8 白藜蘆醇合成酶 3D 結構預測 119
第五章 結論 124
參考文獻 126
附錄 137
作者簡介 139

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