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系統識別號 U0002-1707200815563100
中文論文名稱 合成致死對啤酒酵母菌JHD1功能相關基因之篩選與分析
英文論文名稱 Synthetic Lethal Analysis of JHD1 from Saccharomyces cerevisiae
校院名稱 淡江大學
系所名稱(中) 生命科學研究所碩士班
系所名稱(英) Graduate Institute of Life Sciences
學年度 96
學期 2
出版年 97
研究生中文姓名 林俊良
研究生英文姓名 Chun-Liang Lin
學號 695180447
學位類別 碩士
語文別 中文
口試日期 2008-07-17
論文頁數 46頁
口試委員 指導教授-陳銘凱
委員-林賜恩
委員-鄧述諄
中文關鍵字 合成致死  JHD1  突變 
英文關鍵字 Synthetic Lethal  JHD1  Mutagenesis 
學科別分類 學科別醫學與生命科學生物學
中文摘要 合成致死篩選是藉由兩個基因間交互作用的一種很有效的基因篩選法。原則上,經此種方式可篩選出合成致死性基因。合成致死即在篩選需仰賴興趣基因方可存活的菌株,當中使用菌落顏色分析的技術以視覺確認符合需求的突變株。

合成致死篩選必須遵照以下四個步驟 :
一.感興趣的基因功能需要在實驗菌株中被阻斷,此實驗菌株的ade2 ade3/ade8需無法作用,菌株最終呈現白色。
二.將含有野生型的感興趣基因與ADE3/ADE8的重組質體,導入步驟一的實驗菌株中。導入後的菌株會有紅白相間的扇形菌落產生,而白色菌落表示所導入的重組質體已遺失。
三.將步驟二的菌株執行隨機性突變,所要的突變株需仰賴重組質體上才得以存活,並呈現純紅色的菌落。
四.篩選出的合成致死菌株將會導入基因庫質體,如果突變株上的基因庫質體含有與原本的重組質體互補的DNA,則細胞將不需要在長期的依賴重組質體上的感興趣基因,就會再次呈現出紅白的扇形菌落。

最後,可從篩選出的互補性質體定序出其上所帶的合成致死基因。
英文摘要 The synthetic lethal screen is a powerful genetic screen that relies on finding the secondary molecular targets. In principle, this screen can identify any mutant gene which causes cell death with a nonlethal “primary” mutation. The synthetic lethal screen is a method of isolating novel mutants whose survival is dependent on the gene of interest. Combining the colony-color assay, this method will offer a means to visually detect a mutant that depends on a plasmid for survival.

The synthetic lethal screen must be carry out in the following four processes :
(i) The gene function of interest should be disrupted in a strain containing ade2 ade3/ade8 mutations, which result in a white phenotype.
(ii) Combining the wild-type gene of interest and ADE3/ADE8 in a plasmid which is then transformed into the strain of step (i). The cells will produce red-white sector, and the white colonies have lost the plasmid.
(iii) A mutagenesis will be performed to introduce random mutations into the strain genome. The mutant must dependents on the gene of interest from plasmid of step (ii) for survival and produce red solid colonies.
(iv) The synthetic lethal(SL) colonies are transformed with a library. Then the mutants containing complementing DNA are no longer dependent on the gene of interest, and the cells with red-white sectoring are identified.

Finally,the synthetic lethal gene can be sequencd from the complement plasmid.
論文目次 目錄

實驗簡介篇............................................... 1
第一章 序論............................................. 2
1.1 酵母菌簡述.......................................... 2
1.2 去甲基化酶的概論.................................... 2
1.3 合成致死介紹........................................ 3
1.4 研究動機與目的...................................... 3

實驗方法材料篇........................................... 4
第一章 基因剔除(Knockout)............................... 5
1.1 基因剔除片段的擴增.................................. 5
1.1-1質體pMPY-3xMyc的製備............................ 5
1.1-2引子的設計...................................... 6
1.1-3 聚合酶連鎖反應(PCR)............................ 6
1.2 PCR產物的確認與回收................................. 7
1.2-1瓊脂凝膠電泳.................................... 7
1.2-2 純化回收....................................... 8
1.3 基因剔除片段的導入與正確菌株的篩選.................. 8
1.3-1 製備酵母菌勝任細胞............................. 9
1.3-2用電穿孔法進行勝任細胞的轉型.................... 9
1.3-3菌株的篩選及確認................................ 9

第二章 JHD1功能補回.................................... 11
2.1 表現片段的擴增..................................... 11
2.1-1 基因體DNA的製備............................... 11
2.1-2 引子的決定.................................... 11
2.1-3 PCR........................................... 12
2.2 PCR產物的確認...................................... 13
2.2-1 瓊脂凝膠電泳.................................. 13
2.2-2 純化回收...................................... 13
2.3 TA Cloning......................................... 13
2.3-1 進行TA連接.................................... 13
2.3-2 大腸桿菌勝任細胞(DH5α)的製備.................. 14
2.3-3 勝任細胞轉型及藍白篩選........................ 15
2.4 從轉型後的Clone確認目標片段的存在及定序............ 16
2.4-1 Clone的挑選及培養............................. 16
2.4-2 質體的萃取.................................... 16
2.4-3 限制酶的切割確認:EcoR I切割.................. 17
2.4-4 定序目標片段.................................. 17
2.5 表現載體的製備..................................... 17
2.5-1 限制酶切割-double digests method.............. 17
2.5-2 重組質體的建構................................ 19
2.5-3 重組質體的確認................................ 20
2.6 表現質體(pr79J)的導入與轉型後的確認................ 20
2.6-1 製備酵母菌勝任細胞............................ 20
2.6-2 用電穿孔法進行勝任細胞的轉型.................. 21
2.6-3 菌株的篩選及確認.............................. 21

第三章 隨機性突變...................................... 22
3.1 EMS突變............................................ 22
3.2 突變後的培養....................................... 22
3.3 突變株的篩選與確認................................. 23
3.3-1 轉盤測試...................................... 23
3.3-2 測試質體p315J的建構........................... 23
3.3-3 p315J的導入取代測試........................... 23

第四章 互補性基因庫篩選................................ 24
4.1 pRS425 base基因庫的導入............................ 24
4.1-1 製備酵母菌勝任細胞............................ 24
4.1-2 以電穿孔法進行勝任細胞的轉型.................. 24
4.2 JHD1功能互補性菌株的篩選........................... 24

實驗結果討論篇.......................................... 26
第一章 JHD1的功能破壞.................................. 27
第二章 突變株的選擇.................................... 28
第三章 互補基因庫質體的篩選............................ 30
第四章 結論............................................ 31

附錄1 實驗使用的序列比對............................... 35
附錄2 double digest相關資料............................ 38
附錄3 實驗引子與相關配方............................... 39
附錄4 實驗使用的培養基/液.............................. 41
附錄5 使用的DNA marker................................. 42
附錄6 儀器設備......................................... 43


實驗圖表目錄
圖 1-0 : JHD1 knockout fragment (1.63kb) from PCR........ 7
圖 1-1 : CHY125於SC-Ura medium的測試圖.................. 33
圖 1-2 : JHD1 knockout fragment導入後於SC-Ura medium的生長情況.................................................... 33
圖 1-3 : 同圖1-2將相同的細胞型態,挑出轉至相同的培養基.. 33
圖 1-4 : 將PCR檢測後符合的重組株(A/D/F)轉塗於5-FOA上的生長結果.................................................... 33
圖 1-5 : 將圖1-4有生長的菌落,隨機挑選回塗SC-Ura medium的測試圖.................................................... 33
圖 1-6 : 重組株的URA3 pop-out前後的PCR檢測結果.......... 10
圖 2-0 : 以PCR擴增wild-JHD1 fragment的結果圖............ 13
圖 2-1 : 以EcoR I確認N1質體的測試圖..................... 17
圖 2-2 : 以double digest方式處理N1質體與pYT79的結果圖... 18
圖 2-3 : 以double digest方式確認pr79J的測試圖........... 20
圖 2-4 : 以EcoR I與EcoR V確認pr79J的測試圖.............. 20
圖 3-0 : 呈現穩定紅色狀態的突變株....................... 34
圖 3-1 : 圖 3-0的突變株塗於5-FOA medium的測試圖......... 34
圖 3-2 : p315J導入mPCW3001後於SC-Leu-Ade* medium的取代測試圖...................................................... 34
圖 3-2-1 : 圖 3-2的白色菌落轉回YPD-Ade* medium的結果圖.. 34
圖 3-3 : pr79J lost rate極低的突變株.................... 34
圖 4-0 : PCW1 ~ PCW4001於YPD-Ade* medium上的細胞狀態圖.. 35
圖 4-1 : p315CK導回mPCW3001後於YPD-Ade* medium上的顏色變化圖...................................................... 35
圖 4-2 : p315CK導回mPCW3001後於SC-Leu-Ade* medium上的顏色變化圖.................................................... 35
圖 4-3 : PCW4001塗於SC-Leu-Ade* medium,置放4℃的顏色變化圖...................................................... 35

表一 : Ura3 marker存在於否的PCR檢測結果................. 10
表二 : TA連結的成分比例................................. 14
表三 : double digests操作流程........................... 18
表四 : pr79J連結的成分比例.............................. 19
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