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研究生:黃玟寧
研究生(外文):Wen-Nin Huang
論文名稱:紅麴中MonacolinK生合成相關基因選殖與分析
論文名稱(外文):Cloning and analysis of the monacolin K biosynthesis related genes in monascus purpureus BCRC 31615
指導教授:楊健志
指導教授(外文):Chien-Chih Yang
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:微生物與生化學研究所
學門:生命科學學門
學類:微生物學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
論文頁數:106
中文關鍵詞:Monacolin KPolyketide 合成脢紅麴
外文關鍵詞:Monacolin KPolyketide synthaseMonascus
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Polyketide 為自然之結構多樣性化合物,是微生物所產生之二次代謝物,大部分由真菌及絲狀細菌所產生,具有釵h的生理活性,包括抗生素或真菌毒素。其負責合成之酵素 polyketide synthase (PKS) 十分類似脂肪酸合成酶 (fatty acid synthase, FAS)。PKS 與 FAS 不同的是其可允雩鼮L或增加某些還原及脫水反應,而可產生釵h結構多樣性之化合物。Monacolin K (lovastatin) 為一種 polyketide,是膽固醇生合成之關鍵酵素 HMG-CoA reductase 的抑制劑,而 Aspergillus terreus 及 Monascus spp. 為自然界中可生產 monacolin K 之兩種真菌。在 A. terreus 中,lovastatin 主要由兩個 polyketide synthase (PKS) 所合成,分別為 lovastatin nonaketide synthase (LNKS) 及 lovastatin diketide synthase (LDKS),負責合成 lovastatin 之主體結構,此外,還需要其他輔助酵素的作用,例如 LovA、LovC 及 LovD,才能合成完整之 lovastatin。為了瞭解 monacolin K 在紅麴 (M. purpureus) 中的生合成機制,必須要得到參與其合成之 PKS 基因。本論文建立紅麴基因組庫,並利用 PCR 製備 KS domain 與 AT domain 專一性探針,篩選紅麴之基因組庫,得到一含有 15-18 kb 插入片段之選殖體 p3,將其以 NotI 及 XhoI 切割成五個片段,分別得到五個次選殖體。目前四個次選殖體已完成定序工作,共約 8.9 kb (pDASHB5),內含一可能之 PKS 基因,及一可能的 ORF。此 PKS 基因前半部與 lovB (LNKS) 基因具同源性,而後半部則與 lovF (LDKS) 具同源性,且轉譯後之胺基酸序列也獲得相同的結果。根據 domain 預測結果,此可能之 PKS 具有 KS、(AT)、ER、KR 與 ACP domain,排列方式與一般 PKS 無異,但比起 LDKS 及 LNKS 則缺乏了 DH 及 MT domain。另外,pDASHB5 也含有一可能之 ORF,與 lovastatin 生合成基因 cluster 中的 ORF12 具相似性,但轉譯後之胺基酸序列則完全不同,為一未知弁鄋熙J白質。依據所得之部分序列設計三條胜肽 (KS15p、KS25p 與 AT25p),用以免疫小白鼠,以獲得抗體,其中 KS25p 所得到的多株抗體可以專一性辨認胜肽本身,但經由西方轉印分析卻無法辨識紅麴全蛋白質粗抽液中大於 200 kD 的蛋白質。

目錄....................................................................................................................I
中文摘要...........................................................................................................IV
Abstract.............................................................................................................V
縮寫表..............................................................................................................VI

第一章 緒論.................................................................................1

1.1 Polyketide 簡介及其重要性.......................................................................1
1.2 Polyketide 緣起與定義..............................................................................3
1.3 Polyketide 之生合成..................................................................................5
1.3.1 Polyketide 與脂肪酸 (Fatty acid) 之生合成反應 ……………….…5
1.3.2 Polyketide 合成酶 (Polyketide synthase, PKS) ……………….......7
1.4 Monacolin K 生合成 ……………………………………………………..….11
1.4.1 Monacolin K 之結構與弁?…………………………………….......11
1.4.2 Monacolin K 與 ML-236B 生合成相關酵素與基因 ………..........12
1.4.3 Monacolin K 之生合成路徑與反應 ………………………………...16
1.5 紅麴菌 ………………………………………………………………….…...…18
1.6 研究動機、目的與方向 …………………………………………………........21

第二章 材料與方法………………..…………….…….…………....24

2.1 實驗材料 …………………………………………………………………...…..24
2.1.1 紅麴與培養條件 ……………………………………………………...24
2.1.1.1 固態培養 …………………………………………………...…....24
2.1.1.2 液態培養 …………………………………………………...…....24
2.1.2 載體 (Vectors) ……………………………………………………......25
2.1.3 大腸桿菌 (Escherichia coli) …………………………………………25
2.2 實驗藥品 …………………………………..……………………………….....25
2.3 實驗儀器 ………………………………………………………………….......25
2.4 實驗方法 …………………………………………………………………...…26
2.4.1 DNA 之抽取與分析 ……………………………………………….....26
2.4.1.1 紅麴基因組 DNA 之抽取 …………………………....…..........26
2.4.1.2 噬菌體 DNA 之抽取 …………………………………….......…27
2.4.1.3 質體 DNA 之抽取與分析 ………………………………......…27
2.4.1.4 DNA 洋菜膠體電泳分析 ………………………………...........29
2.4.1.5 DNA 片段之分離純化 ………………….……………….…......30
2.4.1.6 南方轉印法 (Southern blotting)............. ………………..…....31
2.4.1.7 DNA 墨點轉印 (DNA dot blotting).………………………........31
2.4.1.8 雜合反應 …………………………….……………………..…....31
2.4.2 紅麴基因組庫之建立 …………………....………………………..….32
2.4.2.1 基因組 DNA 以 Sau3AI 不完全切割之最適濃度測定 ……..32
2.4.2.2 大量 DNA 以 Sau3AI 不完全切割 ……...……………...…...32
2.4.2.3 基因組 DNA 片段與載體 (Lambda DASH II) 之接合反應....33
2.4.2.4 體外噬菌體包裝 (In vitro Packaging) …………………..….....33
2.4.2.5 價數測定 ………………………………………………….…......33
2.4.2.6 基因組庫之放大 …………………………………………….......34
2.4.3 紅麴 monacolin K 生合成相關基因部分序列選殖.........................34
2.4.3.1 部分序列之增殖 ……………………………………….…..........34
2.4.3.2 載體之處理 ……………………………………………….…......35
2.4.3.3 接合反應 (T-A cloning) ………………………………….…..... 35
2.4.3.4 質體 DNA 之轉形 ………………………………………......….35
2.4.3.5 菌種保存 ……………………………………………….….….....36
2.4.4 紅麴基因組庫之篩選 ……………………………………………...….36
2.4.4.1 探針設計與製備 ………………………………………..…….....36
2.4.4.2 塗佈... ……………………………………………………......…..36
2.4.4.3 基因組庫之轉印 …………………………………………..….....37
2.4.4.4 雜合反應 …………………………………………………......….37
2.4.4.5 挑選正反應噬菌體 ………………………………………...........37
2.4.4.6 正反應株 DNA 片段的選殖 …………………………….…......38
2.4.4.7 以 PCR 確認片段拼接順序及方向 ….....……………….........38
2.4.5 紅麴蛋白質抽取法與電泳分析 …………………………………..…..39
2.4.5.1 紅麴蛋白質粗抽取 ……………………………………….…......39
2.4.5.2 蛋白質定量 ……………………………………………….…......39
2.4.5.3 蛋白質電泳分析 ………………………………………….…......40
2.4.5.4 蛋白質染色 ……………………………………………….…......40
2.4.6 單株抗體的製備 ………………………………………………….......41
2.4.6.1 胜肽合成 ………………………………………………….…......41
2.4.6.2 胜肽定量 ………………………………………………….…......42
2.4.6.3 小白鼠免疫 ……………………………………………….…......43
2.4.6.4 細胞融合 ………………………………………………….…......43
2.4.7 免疫學方法 …………………………………………………...…..…..45
2.4.7.1 酵素連結免疫吸著分析法 (ELISA) …………………….….......45
2.4.7.2 蛋白質電泳轉印及酵素免疫染色法 ………………….…....…..46

第三章 結果與討論 ……….……………………..….……………..47

3.1 紅麴 monacolin K 生合成相關基因部分序列之選殖 ……………..........47
3.1.1 紅麴基因組 DNA 之抽取 …………………………………….....….47
3.1.2 部分序列之選殖 ……………………………………………….....….47
3.2 紅麴基因組庫的建構 ……………………………………………………......49
3.2.1 基因組 DNA 以 Sau3AI 不完全切割片段之製備 ………….……49
3.2.2 紅麴基因組庫的建構 ……………………………….…………....….49
3.3 紅麴 monacolin K 生合成相關基因的選殖 …………………………......50
3.3.1 基因組庫之篩選 …………………………………………….…....….50
3.3.2 正反應株的鑑定 ………………………………………………..…....50
3.3.3 序列分析 …………………………………………………….….…....51
3.3.3.1 pBSBf0 …..……………………………….……………….….....51
3.3.3.2 pBSBf2 …………………………………………………….........52
3.3.3.3 pBSBf1 …………………………………………………….…....53
3.3.3.4 pBSBf3 …………………………………………………….…....53
3.3.3.5 pBSBf8 ………………………………………………….……....54
3.3.3.6 序列拼接 …………………………………...………….…….....54
3.4 專一性抗體的製備與檢測 …………………………………………….........57
3.4.1 胜肽抗原製備 ………………………………………………….…....57
3.4.2 多株抗體的取得與單株抗體之製備 ………………………….…....58
3.4.3 紅麴粗蛋白質的抽取 ………………………………………….…....59
3.4.4 抗體專一性檢定 ……………………………………………….…....60

第四章 結論與未來展望…………………………..……...…….....62

參考文獻…………………………………………………………………..............65
圖與表……………………………………………………………………..............71
碩士論文口試問答摘要................................................................................102




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