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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 楊健志(Chien-Chih Yang) | |
| dc.contributor.author | Pei-Chung Wang | en |
| dc.contributor.author | 王培仲 | zh_TW |
| dc.date.accessioned | 2021-06-08T05:11:50Z | - |
| dc.date.copyright | 2006-07-28 | |
| dc.date.issued | 2006 | |
| dc.date.submitted | 2006-07-21 | |
| dc.identifier.citation | 黃玟寧. (2004). 紅麴中 Monacolin K 生合成相關基因選殖與分析. 國立臺灣大學微生物與生化學研究所碩士論文.
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Recent developments towards the heterologous expression of complex bacterial natural product biosynthetic pathways. Curr Opin Biotechnol 16, 594-606. Wicks, R.J. (1986). RNA molecular weight determination by agarose gel electrophoresis using formaldehyde as denaturant: comparison of RNA and DNA molecular weight markers. Int J Biochem 18, 277-278. Wilfinger, W.W., Mackey, K., and Chomczynski, P. (1997). Effect of pH and ionic strength on the spectrophotometric assessment of nucleic acid purity. Biotechniques 22, 474-476, 478-481. Yadav, G., Gokhale, R.S., and Mohanty, D. (2003). Computational approach for prediction of domain organization and substrate specificity of modular polyketide synthases. J Mol Biol 328, 335-363. Yamamoto, K.R., Alberts, B.M., Benzinger, R., Lawhorne, L., and Treiber, G. (1970). Rapid bacteriophage sedimentation in the presence of polyethylene glycol and its application to large-scale virus purification. Virology 40, 734-744. | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/23862 | - |
| dc.description.abstract | Polyketides 為微生物或真菌所產生結構多樣的天然化合物，它們往往具有許多生理活性，例如抗生素，毒素，香味及色素分子，在許多的植物中亦可發現。Polyketides 是由 polyketide synthases (PKSs) 所合成，它在演化上和 fatty acid synthase (FAS) 類似，但是催化情況更加複雜，才能合成結構複雜的二級代謝物。monacolin K (lovastatin) 為一種真菌合成的 polyketide 類化合物，它是膽固醇合成的關鍵酵素 HMG-CoA reductase 的抑制劑。最初在 Aspergillus terreus 中發現，後來在中國傳統醱酵菌種 Monascus spp. 中亦可發現。A. terreus 中的 lovastatin 合成酵素的基因序列已被發現 (lovB, lovF)，分別可轉譯出 lovastatin nonaketide synthase (LNKS) 及 lovastatin diketide synthase (LDKS)，此二蛋白質合成 lovastatin 之主體結構，另外需要其他酵素之協助才能合成完整的 lovastatin。先前的研究建立了紅麴染色體基因組庫 (黃, 2004)，並且篩選出可能含有 LNKS 或 LDKS 基因之正反應株 pDASHB3。它的全長約有 17kb，以限制酶作用為五個片段後個別分析，並利用不同的引子對可將其完整序列拼湊出。其中內含一可能之 PKS 基因 (putPKS)，一個可能的 ORF，一個蛋白質功能可能為 cytochrome P450 monooxygenase 之 mLovA 基因及功能可能為 hydoxylase 活性之 mlovX。根據 pDASHB3 已知基因序列設計專一性引子針對紅麴菌進行 RT-PCR 可放大出部分 putPKS 序列及輔助基因 mlovA。本論文另外測定了紅麴菌液態培養之 monacolin K 生合成曲線，並參考建構了可利用酵母菌雙雜合實驗研究蛋白質交互作用的紅麴菌 cDNA 庫。我們亦建構了輔助蛋白質 MLovA 的蛋白質表現系統，並且成功將原態 MLovA 及突變 MLovA 蛋白質進行初步表現。 | zh_TW |
| dc.description.abstract | Polyketides are structure-diversified natural products, which are produced by microorganisms, fungi, and also by some plants. They display a wealth of physiologically activites, including antibiotic, toxin, aroma and pigments. Polyketides are produced by polyketide synthases (PKSs) which are evolutionarily related to fatty acid synthase (FAS), but much more sophicated to synthesize complicated secondary metabolites. monacolin K (lovastatin), a polyketide from fungus, is an inhibitor of HMG-CoA reductase, which is a key enzyme in the biosynthesis pathway of cholesterol. They are found in Aspergillus terreus, and also found in fungi Monascus spp., which is applied to Chinese traditional fermentation. At least two lovastatin biosynthesis genes from A. terreus have been cloned (lovB, lovF), and they encode lovastatin nonaketide synthase (LNKS) and lovastatin diketide synthase (LDKS). LNKS and LDKS together are responsible for the production of the core structure of lovastatin, while other enzymes are required to produce the full-functional lovastatin. In previous study we constructed the Monascus genomic library (Huang, 2004), and isolated a positive clone that contained putative LNKS or LDKS. The 17kb insert in the positive clone, pDASHB3, was digested to 5 fragments and subcloned. The gene arrangements were determined by using different primer sets. The genes contained in this clone were predicted to have a putative PKS gene, an unclassified ORF, a putative cytochrome P450 monooxygenase MLovA, and a putative hydroxylase gene mlovX. According to the genomic sequences ontained from pDASHB3, we designed several specific primers to amplify the partial putative Moncolin K biosynthesis gene putPKS and an auxiliary protein mlovA from Monascus by RT-PCR. In addition, we measured the production curve of monacolin K in Monascus broth culture, and construced a Monascus cDNA library which can be used to fish interacting proteins of PKSs using yeast-two hybrid techniques. We also construced the protein expression system of a putative auxiliary protein MLovA, and heterologous protein expression of MLovA was performed. | en |
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| dc.description.tableofcontents | 目錄 I
中文摘要 IV Abstract V 縮寫表 VI 第一章緒論 1 1.1 二次代謝物的定義及其功能角色 1 1.2 Polyketide synthase 的簡介及近代研究發展 4 1.3 Polyketide synthase 之分類 7 1.4 脂肪酸 (fatty acid) 生合成反應 9 1.5 Monacolin K 生合成 10 1.5.1 Monacolin K 簡介 10 1.5.2 Lovastatin 生合成相關基因 12 1.6 Polyketide synthase 在組合化學 (combinatorial chemistry) 上之發展 15 1.7 紅麴菌 (Monascus spp.) 19 1.7.1 紅麴菌之簡介及特性 19 1.7.2 紅麴菌之培養 20 1.7.3 紅麴在食品工業及保健食品上之應用 20 1.8 研究動機及目的 21 第二章 材料與方法 24 2.1 實驗菌種 24 2.1.1 紅麴菌 (Monascus spp.) 24 2.1.1.1 固態培養及菌種保存 24 2.1.1.2 液態培養 24 2.1.2 載體 (Vectors) 24 2.1.3 大腸桿菌 (Escherichia coli) 25 2.2 實驗藥品 26 2.2.1 培養基 26 2.3 實驗儀器 27 2.4 實驗方法 28 2.4.1 DNA 之純化與分析 28 2.4.1.1 紅麴菌基因組 DNA 之抽取 28 2.4.1.2 噬菌體 DNA 之抽取 29 2.4.1.3 質體 DNA 之抽取 30 2.4.1.4 DNA 片段之純化 32 2.4.1.5 DNA 洋菜膠體電泳分析 33 2.4.2 RNA 之純化與分析 33 2.4.2.1 紅麴菌 Total RNA 之抽取 33 2.4.2.2 mRNA 之純化 34 2.4.2.3 甲醛洋菜膠體電泳 34 2.4.3 紅麴菌 cDNA 庫之建構 35 2.4.3.1 單股 cDNA 合成 36 2.4.3.2 雙股 cDNA 合成 36 2.4.3.3 補平 cDNA 尾端序列 36 2.4.3.4 cDNA 片段之修飾與純化 37 2.4.3.5 載體接合反應與體外噬菌體包裝 (In vitro packaging reaction) 37 2.4.3.6 價數 (Titer) 測定及 cDNA 庫之放大 38 2.4.4 紅麴菌 monacolin K 生合成相關基因序列之選殖及建構 39 2.4.4.1 探針設計 39 2.4.4.2 確認正反應選殖體片段拼接順序 40 2.4.4.3 反轉錄酶-聚合酶連鎖反應 (Reverse Transcription-Polymerase Chain Reaction, RT-PCR) 41 2.4.4.4 接合反應 41 2.4.4.5 質體 DNA 轉形 42 2.4.5 序列點突變實驗 (Point Mutation Experiment) 43 2.4.6 紅麴菌 cDNA 基因組庫之篩選 44 2.4.6.1 探針製備 44 2.4.6.2 噬菌體之培養 45 2.4.6.3 基因組庫之轉印 45 2.4.6.4 雜合反應 45 2.4.6.5 挑選正反應噬菌體 46 2.4.6.6 正反應株 DNA 片段之選殖 47 2.4.7 重組蛋白質之建構、抽取及電泳分析 47 2.4.7.1 重組蛋白質載體之建構 47 2.4.7.2 最佳蛋白質表現條件之探討 48 2.4.7.3 SDS 膠體電泳 48 2.4.7.4 蛋白質染色 49 2.4.7.5 蛋白質電泳轉印及酵素免疫染色法 50 2.5 Monacolin K 之萃取及分析 50 2.5.1 紅麴菌二級代謝物 monacolin K 萃取 50 2.5.2 高壓液相層析法 (High Performance Liquid Chromatography HPLC) 51 2.6 序列分析 51 第三章 結果與討論 52 3.1 基因組庫正反應株之序列分析 52 3.1.1 pBSBf8 53 3.1.2 基因組序列片段拼接 53 3.2 Monacolin K 合成之測定 54 3.2.1 Monacolin K 標準品及樣品之 HPLC 圖譜 55 3.2.2 Monacolin K 生合成曲線 55 3.3 紅麴菌 cDNA 庫之建構 55 3.3.1 RNA 之抽取及 poly ( A )+ RNA 之純化 55 3.3.2 cDNA library 之建構 56 3.4 紅麴菌 monacolin K 生合成相關基因 cDNA 之選殖 57 3.4.1 cDNA 庫之篩選 57 3.4.2 以 RT-PCR 進行紅麴菌 monacolin K 合成酶 cDNA 之分析 57 3.4.2.1 反轉錄作用 ( Reverse transcription reaction ) 58 3.4.2.2 putPKS cDNA 序列之部分片段選殖及比對分析 58 3.4.2.3 mlovA 基因序列之選殖及比對分析 61 3.4.2.4 mlovX 基因序列之選殖及比對分析 62 3.5 mlovA 相關蛋白質之表現 63 3.5.1 重組蛋白質載體建構 63 3.5.2 最適蛋白質誘導條件探討 63 第四章 結論與展望 65 參考文獻 68 圖與表 74 碩士論文口試問答摘要 107 | |
| dc.language.iso | zh-TW | |
| dc.subject | 基因組庫 | zh_TW |
| dc.subject | 紅麴菌 | zh_TW |
| dc.subject | monacolin K | en |
| dc.subject | cDNA library | en |
| dc.subject | monascus | en |
| dc.title | 紅麴菌中 Monacolin K 生合成相關基因 cDNA 之選殖與分析 | zh_TW |
| dc.title | Cloning and Analysis of the monacolin K cDNA encoding Biosynthesis Related Genes in Monascus purpureus BCRC 31615 | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 94-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 蘇仲卿,潘子明(Tzu-Ming Pan),袁國芳(Kuo-Fan Yuan),常怡雍 | |
| dc.subject.keyword | 紅麴菌,基因組庫, | zh_TW |
| dc.subject.keyword | monascus,cDNA library,monacolin K, | en |
| dc.relation.page | 111 | |
| dc.rights.note | 未授權 | |
| dc.date.accepted | 2006-07-22 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 微生物與生化學研究所 | zh_TW |
| 顯示於系所單位： | 微生物學科所 | |
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