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研究生:郭子瑋
研究生(外文):Tzu-wei Kuo
論文名稱:運用突變的pheS基因做為反向篩選標記進行馬紅球菌基因體中kstD基因的剔除
論文名稱(外文):Using mutant pheS gene as the counter-selection marker to delete the kstD genes in Rhodococcus equi
指導教授:孟孟孝
指導教授(外文):Meng-Hsiao Meng
口試委員:李重義楊昭順
口試委員(外文):Chung-Yi LeeChao-Shun Yang
口試日期:2017-07-25
學位類別:碩士
校院名稱:國立中興大學
系所名稱:生物科技學研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2017
畢業學年度:105
語文別:中文
論文頁數:54
中文關鍵詞:反向篩選標記馬紅球菌
外文關鍵詞:counter-selection marker:Rhodococcus equipheSkstD
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4-雄烯二酮 (4-Androstene-3,17-dione, AD)在固醇藥物 工業的製程上 是一個重 要的前驅物,能經由一系列化學合成來形不同這類廣泛應用於醫藥產業,例如消炎、避孕等。在自然界中部分微生物具有降解固醇的能力,而 4-雄烯二酮為降解路徑之中間物,因此以微生來產 4-雄烯二 酮具有相當高的潛力。本實驗研究主角馬紅球菌 (Rhodococcus equi)屬於 革蘭 氏陽性菌 。3-ketosteroid Δ1-dehydrogenase (kstD)在固醇代謝的路徑上扮演 在固醇代謝的路徑上扮演 , 將 4-雄烯二酮催化成 1,4-雄二烯酮 (1,4-Androstadiene-3,17-dione , ADD) 的角 色。根據基因組資訊預測馬紅球菌存在著五個不同的 kstD基因,編碼分別 是 Req_06770、Req_10320、Req_13080、Req_15820、Req_30200。目前對於這 。目前對於這 些基因在固醇代謝路徑中的角色仍缺實驗證明 。因此本實驗先建立一套適合馬 紅球菌的非標記性基因剔除方法,逐一 在實驗室的 馬紅球菌 UV1(R. equi UV1)菌株上 進行個別 kstD基因剔除。目前已篩選到四株菌, 基因標號 分別是 Req_10320、Req_13080、Req_15820與 Req_06770。之後 針對基因剔除菌株, 再以薄層分析 (Thin layer chromatography)觀察 4-雄烯二酮累積情況。結果發現 這四株 kstD剔除菌株皆未能在培養基中累積 4-雄烯二酮。我們推測主要負責 4-雄烯二酮催化的基因尚未被成功剔除,抑或是這五個 KSTD蛋白皆具有轉換 4-雄烯二酮成 1,4-雄二烯酮的功能,導致被代謝而無法順利累積。 我們將於馬紅球菌中持續剔除 Req_30200或於同一株菌中累積多個剔除基因, 或於同一株菌中累積多個剔除基因, 以得到能累積 4-雄烯二酮的菌株。
4-Androstene-3,17-dione (AD), an important precursor in steroids industry, can be transformed into a variety of steroids via a series of chemical synthesis. These steroids have been widely used as anti-inflammatory drugs, immunosuppressant, and contraceptives etc. In nature, some microorganisms are able to degrade sterol; therefore, utilization of these microorganisms to accumulate AD is considerably potential. Rhodococcus equi, a gram-positive bacterium, use the 3-ketosteroid Δ1-dehydrogenase (kstD) to converse AD into 1,4-androstadiene-3,17-dione (ADD) in steroid metabolism. According to the genomic information of R. equi, there are probably five kstD paralogs, which include Req_06770, Req_10320, Req_13080, Req_15820, and Req_30200, in R. equi. Nonetheless, the role of these genes in the sterol pathway is still unclear. In this study, an unmarked gene knockout method applicable to R. equi was established to delete the putative kstD genes and the resulting effects on AD accumulation in the culture medium were assayed by thin layer chromatography (TLC). So far Req_10320, Req_13080, and Req_15820、Req_06770 have been deleted respectively. Unfortunately, none of the four kstD-deleted strains could accumulate AD. We presume either the major gene responsible for AD to ADD conversion has not been deleted or all the KSTD proteins are involved in AD catabolism so that single kstD deletion cannot result in AD accumulation. In the future, we will continue to delete Req_30200 and create mutant strains with multiple kstD gene deletions in order to obtain the ideal strain for AD production.
中文摘要........................... i
Abstract........................... ii
目錄............................... iii
表圖目錄........................... iv
第一章 緒論
第一節 固醇類化合物...................... 1
第二節 類固醇藥物的製造與合成............. 4
第三節 馬紅球菌與其膽固醇降解的代謝途徑.... 8
第二章 研究目的
第一節 先前研究......................... 11
第二節 篩選策略......................... 12
第三節 研究目標......................... 13
第三章 材料與方法
第一節 實驗材料......................... 15
第二節 實驗方法......................... 18
第四章 實驗結果......................... 24
第五章 討論............................ 48
參考文獻................................. 51
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