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研究生:邱佳恩
研究生(外文):CHIU,CHIA-EN
論文名稱:探討大腸桿菌表現泛酸激酶對白藜蘆醇產量之影響
論文名稱(外文):Improving resveratrol biosynthesis in Escherichia coli via pantothenate kinase expressed
指導教授:簡良榮
指導教授(外文):CHIEN,LIANG-JUNG
口試委員:謝欣如簡良榮吳弦聰
口試委員(外文):HSIEH,HSIN-JUCHIEN,LIANG-JUNGWu,HSIEN-TSUNG
口試日期:2022-08-25
學位類別:碩士
校院名稱:明志科技大學
系所名稱:化學工程系碩士班
學門:工程學門
學類:化學工程學類
論文種類:學術論文
論文出版年:2022
畢業學年度:110
語文別:中文
論文頁數:40
中文關鍵詞:白藜蘆醇泛酸激酶生物合成代謝工程
外文關鍵詞:ResveratrolPantothenate kinaseBiosynthesisMetabolic engineering
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白藜蘆醇是非黃酮類的多酚化合物,屬於芪類化合物的植物抗毒素、抗氧化劑,具有抗腫瘤、防治心血管疾病、抗菌、抗病毒、保肝等藥理作用,在防治疾病及保健美容等方面越來越引起人們的注意,是很有潜力且具有廣闊的市場前景的天然藥物。目前白藜蘆醇主要從植物中提取,但植物成分含量和提取效率較低。化學合成法生產白藜蘆醇,其生產過程易造成環境污染,近年來,生物合成法的快速發展已轉向利用微生物合成白藜蘆醇。
本研究利用基因工程技術,將基因 Stilbene synthases(Vitis vinifera)、 4-coumaroyl CoA ligase(Arabidopsis thaliana)、pantothenate kinase(Aspergillus nidulans) 分別建構在pET30b(+)及pACYC-184質體上,進行基因編碼優化並構築表現於大腸桿菌中。研究發現採用誘導培養基LB+ Sorbitol對於蛋白質可溶性有所助益,以及置換M9培養基方式進行生產轉化較利於白藜蘆醇生產。

Resveratrol, a phytoalexin is a natural secondary metabolite,biosynthesized by plants and predominantly found in grapes,peanuts, and blueberries. In recent years, the rapid development of biosynthesis has turned to the use of microbial synthesis of resveratrol.
In this study, using genetic engineering technology, the genes Stilbene synthases (Vitis vinifera), 4-coumaroyl CoA ligase (Arabidopsis thaliana), pantothenate kinase (Aspergillus nidulans) were constructed on pET30b(+) and pACYC-184 plastids, respectively, for gene encoding. Optimized and constructed for expression in E.coli. The study found that the induction medium LBS (LB+0.6M Sorbitol) was helpful for protein solubility, and the replacement of M9 medium for production transformation was more conducive to the production of resveratrol.

目錄
口試委員審定書 i
誌謝 ii
中文摘要 iii
Abstract iv
目錄 v
圖目錄 vii
表目錄 viii
第一章 緒論 1
第二章 文獻回顧 2
2.1 白藜蘆醇 2
2.2 白藜蘆醇的製程 3
2.2.1 植物萃取法 3
2.2.2 化學合成法 3
2.2.3 生物合成法 6
第三章 研究方法 12
第四章 實驗方法與步驟 13
4.1 實驗材料 13
4.2 E.coli 基因表現系統建構 15
4.2.1 聚合酶連鎖反應(PCR) 15
4.2.2 DNA電泳分析 17
4.2.3 PCR 產物純化 18
4.2.4 限制酶切割 18
4.2.5 接合反應(Ligation reaction) 19
4.2.6 基因轉殖篩選 20
4.3 蛋白質分析 22
4.3.1 SDS-PAGE 22
4.4 菌株培養 24
4.5 定量測定 27
第五章 實驗結果與討論 30
5.1 建構載體 30
5.1.1 蛋白質表現分析 31
5.2 PanK基因表現對白藜蘆醇產量之影響 32
5.3 轉化培養基對白藜蘆醇產量之影響 33
5.4 誘導時機對白藜蘆醇產量之影響 34
第六章 結論 36
參考文獻 37

圖目錄
圖 1.1健康食品主要13項保健功效 1
圖 2.1順/反白藜蘆醇之結構式 2
圖 2.2 Wittig reaction 4
圖 2.3 Perkin reaction 4
圖 2.4 Hech reaction 5
圖 2.5白藜蘆醇植物代謝途徑 8
圖 3.1 設計之載體 12
圖 4.1白藜蘆醇 標準品分析圖 28
圖 4.2香豆酸 標準品分析圖 28
圖 4.3白藜蘆醇檢量線 29
圖 4.4 香豆酸檢量線 29
圖 5.1 目標基因(STS、4CL、Pank)之 PCR 產物結果 30
圖 5.2 pET30b(+)建構STS與4CL基因篩選分析 31
圖 5.3 pACYC-184建構Pank基因篩選分析 31
圖 5.4 不同誘導時間之SDS-PAGE分析圖 32
圖 5.5 PanK基因表現對白藜蘆醇產量之影響 33
圖 5.6 轉化培養基對白藜蘆醇生產的影響。 34
圖 5.7 誘導時機對白藜蘆醇產量的影響 35

表目錄
表 2.1 三種製成優缺點比較 6
表 2.2 不同表達系統白藜蘆醇產率比較 9
表 4.1 Vector 設計之限制酶切點 13
表 4.2基因引子序列 14
表 4.3聚合酶連鎖反應所需溶液及體積 15
表 4.4聚合酶連鎖反應時間 16
表 4.5限制酶切割反應所需溶液及體積 18
表 4.6接合反應所需溶液及體積 19
表 4.7菌落 PCR 所需溶液及體積 21
表 4.8 菌落 PCR 反應所需時間 21
表 4.9 蛋白質電泳溶液配方 23
表 4.10抗生素及誘導劑配方 24
表 4.11 培養基配方 25
表 4.12 M9培養基配方 26


7參考文獻
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