臺灣博碩士論文加值系統

核醣核酸開關(Riboswitch) 為細菌體內感應並配對小分子代謝物來調節基因表現的RNA結構，其組成包含了和小分子代謝物配對結合的核醣核酸適體(aptamer)及下游的基因表現平台(expression platform)，當小分子代謝物與核醣核酸適體結合後會影響下游基因表現平台，並對基因的表現進行調控。細胞內SAM/SAH濃度的消長被報導與數種真核生物(含酵母菌)的壽命延長有關。由於過去文獻指出在真核細胞內沒有發現SAH核醣核酸開關的存在，因此本篇研究中嘗試使用酵母菌，作為在真核生物中利用SAH核醣核酸開關快速偵測細胞內SAH濃度變化的典範系統。-1計畫性核醣體轉譯軌道移轉(-1 programed ribosomal frameshifting, -1 PRF)為另一種調控基因表現機制，當一段mRNA序列上帶有滑動序列、間隔序列、下游重新編碼刺激元件，而核醣體在編碼過程中閱讀到此段序列，核醣體往mRNA的5’端滑動一個核苷酸，此時核醣體閱讀的mRNA解碼序列稱為-1 frame。利用綠色螢光報導基因結合過去實驗室所設計出的SAH核醣核酸開關作為-1 PRF的刺激元件，並藉-1 PRF效率變化來偵測酵母菌內源性SAH濃度之變化表現。結果發現SAH核醣核酸開關可在酵母菌內刺激-1 PRF的表現。接著在培養環境中額外加入SAH並觀察酵母菌內-1 PRF的表現，發現加入SAH濃度越高酵母菌內-1 PRF的表現就越強。除此之外，過去研究指出剔除掉與酵母菌內合成磷脂質有關的基因，可使得酵母菌內源性SAM/SAH的濃度消長，因此再進一步使用剔除與磷脂質合成相關基因Opi1與Cho2後的酵母菌突變株並將綠色螢光報導基因轉殖入內，以觀察突變株內源性SAH濃度變化對於-1 PRF的影響。剔除Opi1基因可使酵母菌內源性的SAH濃度提升，而在螢光顯微鏡下的確可以看到螢光的表現；剔除Cho2基因造成酵母菌內源性的SAM累積，而在螢光顯微鏡下看到的螢光表現比剔除Opi1基因不明顯，以西方墨點法來比較跟在顯微鏡下看到的螢光表現結果一致，表示酵母菌內源性的SAH濃度變化足以使在內的SAH核醣核酸開關調控-1 PRF的發生。

Riboswitches are RNA structures that bind and respond to small molecular metabolites to regulate gene expression in bacteria. A riboswitch consists of aptamer and gene expression platform. When a specific metabolite binds with the matched aptamer, it will affect the gene expression platform and regulate expression of gene. The variation of the endogenous SAM/SAH concentration has been reported to affect longevity in several eukaryotes, including yeast. However, the SAH riboswitch was not found in eukaryotes. Therefore, this study attempts to use yeast as the model system to detect the change of the SAH concentration by an engineering bacteria riboswitch. The -1 programed ribosomal frameshifting (-1 PRF) is one of the gene regulation platform with potential of riboswitch regulation. To trigger -1 PRF, three elements in the mRNA sequence are required: the slippery sequence, the spacer, and the simulator. When a ribosome encounters these three elements, the ribosome will move one nucleotide toward 5’ end of the mRNA. Under this situation, the reading frame was shifted to -1 frame.
An SAH riboswitch was designed as a -1 PRF simulator, and inserted into green fluorescent protein (GFP) reporter to detect the change of endogenous SAH concentration in yeast. The result showed that the SAH riboswitch could stimulate -1 PRF in yeast. Furthermore, addition of SAH into the medium enhances -1 PRF in yeast, suggesting a SAH-dependent -1 PRF stimulation. As knocking out the Opi1 and Cho2 that related to the production of phospholipid would lead to the endogenous SAH concentration variation, the SAH dependent -1 PRF reporter was transformed into both mutation strains to detect the SAH variation by -1PRF activity. The result showed that green fluorescence could be detected, and is consistent with the result of western blot. Therefore the change of endogenous SAH concentration can be detected by SAH riboswitch- mediated -1 PRF in yeast.

目錄
中文摘要............................................................................................................. i

Abstract............................................................................................................. ii

目錄.................................................................................................................... iv

圖表目錄..........................................................................................................viii

一、 實驗背景介紹........................................................................................ 1
1.1. 核醣核酸開關(Riboswitch) ................................................................... 1
1.1.1. 核醣核酸開關之構造及調控功能.................................................. 1
1.1.2. 核醣核酸開關的分類...................................................................... 4
1.2. 生物體內的甲基化及S-腺苷甲硫胺酸(SAM)代謝循環與其生理功能............................................................................................................. 5
1.2.1. 生物體內的SAM代謝循環............................................................ 6
1.2.2. SAM及SAH代謝循環與生物體壽命的關聯............................... 8
1.2.3. SAM及SAH代謝循環與磷脂質生成之關係............................. 10
1.3. 計畫性核醣體轉譯軌道移轉(programed ribosomal frameshifting, PRF) ...................................................................................................... 11
1.3.1. -1計畫性核醣體轉譯軌道移轉(-1 programed ribosomal frameshifting, -1 PRF) .................................................................. 11
1.3.2. S-腺苷高半胱氨酸核醣核酸開關(S-adenosylhomocysteine riboswitch)於細菌基因表現之調控............................................. 12
1.3.3. SAH核醣核酸開關結合-1 PRF於真核細胞之運用................... 14
二、 動機......................................................................................................... 15
三、 材料......................................................................................................... 16
3.1. 質體....................................................................................................... 16
3.2. 本實驗使用之酵母菌菌株................................................................... 17
3.3. 本實驗所使用之試劑及藥品............................................................... 18
3.3.1. 藥品縮寫列表................................................................................ 18
3.3.2. 試劑配方........................................................................................ 18
四、 實驗方法............................................................................................... 19
4.1. 質體構築............................................................................................... 19
4.1.1. 聚合酶連鎖反應(polymerase chain reaction, PCR) .................... 19
4.1.2. 接合反應( ligation) ....................................................................... 19
4.1.3. 大腸桿菌(DH5α)勝任細胞製備................................................... 20
4.1.4. 大腸桿菌轉型(E. coli transformation) ......................................... 20
4.1.5. 菌落選殖........................................................................................ 20
4.1.6. 質體DNA萃取.............................................................................. 21
4.2. 酵母菌相關實驗前置作業................................................................... 22
4.2.1. 酵母菌菌株W303與BY4741勝任細胞製備.............................. 22
4.2.2. 酵母菌轉型(yeast transformation) ............................................... 22
4.3. 酵母菌螢光偵測觀察........................................................................... 23
4.4. 西方墨點法(Western blot) ................................................................... 23
4.4.1. 酵母菌裂解液收集(鹼裂解法) .................................................... 23
4.4.2. 測量酵母菌裂解液中蛋白質濃度................................................ 23
4.4.3. 聚丙烯醯胺凝膠電泳(SDS polyacrylamide-gel-electrophoresis, SDS-PAGE) ................................................................................... 23
4.4.4. 轉漬(Transfer) ............................................................................... 24
4.4.5. 阻攔(Blocking) ............................................................................. 24
4.4.6. 免疫墨點(Immunoblot) ................................................................ 24
4.4.7. 轉漬訊號偵測................................................................................ 24
4.5. 在外界培養環境中加入SAH不同濃度的SAH.................................. 25
4.5.1. 配製含有SAH之minimal SD DO-Ura液態培養液.................... 25
4.5.2. 於實驗培養環境加入SAH之時機與後續處理步驟................... 25
4.6. 酵母菌實驗詳細步驟流程............................................................................26
4.6.1. Yeast transformation.......................................................................26
4.6.2. Yeast strain W303 & BY4741 competent cell 製備......................26
4.6.3. Yeast western blot...........................................................................27
五、 實驗結果............................................................................................... 31
5.1. 以帶有SAHpk-s7之綠色螢光報導基因檢測W303酵母菌內發生-1 PRF的可行性........................................................................................ 31
5.1.1. 帶有SAHpk-s7之報導基因在W303酵母菌內發生-1 PRF........ 31
5.2. 檢測SAH riboswitch不同長度的間隔序列在酵母菌系統中之-1 PRF表現....................................................................................................... 35
5.2.1. 以點突變方式將SAHpk-s7之間隔序列縮減為SAHpk-s6........ 35
5.2.2. 以帶有不同長度之間隔序列SAH riboswitch之綠色螢光報導子檢測W303酵母菌-1 PRF表現差異............................................. 37
5.3. 於培養環境中外加SAH並觀察-1 PRF的變化.................................. 40
5.3.1. 以帶有SAHpk-s7之螢光報導子檢測於培養環境中外加SAH可以在W303酵母菌內引起-1 PRF的變化..................................... 40
5.4. 嘗試其他實驗常用的酵母菌種類並檢測其-1 PRF的可行性........... 43

5.4.1. 檢測帶有SAH riboswitch之綠色螢光報導子影響在BY4741酵母菌內發生-1 PRF的可行性........................................................ 43
5.4.2. 檢測BY4741剔除Opi1以及Cho2基因後其-1 PRF的影響...... 46
六、 討論......................................................................................................... 51
6.1. SAH riboswitch在酵母菌內調控-1 PRF的可能性............................. 51
6.2. 外界環境中SAH的濃度變化對於酵母菌中SAH riboswitch調控-1 PRF的系統影響關聯............................................................................ 51
6.3. SAH riboswitch調控-1 PRF對於在BY4741酵母菌菌株的突變株的影響.................................................................................................... 52
七、 參考文獻............................................................................................... 53
八、 附錄......................................................................................................... 55
附錄一、試劑配方.......................................................................... 55
8.1. E. coli培養液配方................................................................................ 55
8.2. 酵母菌培養液配方............................................................................... 55
8.3. Western blot試劑配方.......................................................................... 56
8.4. ECL冷光呈色試劑配方....................................................................... 57
附錄二、藥品來源.......................................................................... 58

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