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研究生:李京儒
研究生(外文):Chin-Ju Lee
論文名稱:探討鏈黴菌線形質體SCP1上末端連結蛋白與末端蛋白中影響末端複製的胺基酸
論文名稱(外文):The amino acid residues of Tac and Tpc that affect the end replication of Streptomyces SCP1 linear plasmid
指導教授:楊千金
指導教授(外文):Chien-Chin Yang
學位類別:碩士
校院名稱:中原大學
系所名稱:化學研究所
學門:自然科學學門
學類:化學學類
論文種類:學術論文
論文出版年:2018
畢業學年度:106
語文別:中文
論文頁數:73
中文關鍵詞:鏈黴菌SCP1端粒連結蛋白末端蛋白
外文關鍵詞:streptomyceSCP1terminal associated proteinterminal protein
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鏈黴菌的染色體與部分質體呈線形。DNA複製時由中央的複製起點開始向兩側進行,當複製到末端時會在3’端留下單股缺口,端粒連結蛋白(Tap)會與缺口結合,牽引末端蛋白(TPs)作為引子,完成末端缺口的補齊。在此,Tap扮演著DNA聚合酶的角色;末端蛋白Tpg則作為引子。
少數鏈黴菌質體如S. coelicolor的SCP1端粒序列及末端蛋白和大多數鏈黴菌有所差異,SCP1末端蛋白(Tpc)基因上游具有端粒連結蛋白(Tac)基因,與Tap及Tpg系統相似,因此也可能以相同機制進行末端的修復。我們將Tac、Tap、用蛋白質做引子的Φ29 DNA聚合酶以及具有DNA聚合酶同源性的E. coli Pol II的胺基酸序列做比對,發現有三處位置的天門冬胺酸是四種蛋白共有,其中Asp398、Asp670對應到Φ29 DNA聚合酶和E. coli Pol II與鎂離子結合的位置;在Φ29 DNA聚合酶上的YCDTDS基序(motif)與其活性有關,其中的Thr具有高度保守性。本次研究目標之一是測試Tac與金屬離子結合的天門冬胺酸與YCDTDS基序中的蘇胺酸,將Asp398與Asp670突變成無法與二價金屬離子結合的天門冬醯胺或丙胺酸;Thr669突變成丙胺酸,再以鏈黴菌體內實驗測試,由於鏈黴菌要維持線形質體或染色體複製必須依靠Tac,若Tac失去活性則無法得到線形質體,pLUS894是一個能在鏈黴菌中維持線形複製的迷你質體,我們將三個突變後分別換入pLUS894中的tac,用限制酶AseI線形化後送到鏈黴菌內,抽取單株的質體,結果無法得到質體。
Tpc對鏈黴菌維持線形質體或染色體也極為重要,其主要的功能為引子,可以鍵結在DNA上讓Tac順利進行複製。先前實驗顯示,在Tap與Tpg系統中,Tpg是利用親水性高的第114個蘇胺酸和DNA鍵結。我們利用放射性磷的dG*作為標記,Tpc為引子,Tac為DNA聚合酶進行體外補齊實驗,再用二維TLC片推測Tpc可能利用絲胺酸與DNA鍵結,Tpc總共有13個絲胺酸,將胺基酸序列做親水性分析,結果Ser206為Tpc上較為親水的絲胺酸。本研究第二個目標為定位出Tpc與DNA鍵結的絲胺酸,先將Ser206突變成丙胺酸,再送入鏈黴菌做體內實驗,結果無法得到線形質體;之後將突變成蘇胺酸、酪胺酸以及具有咪唑的組胺酸,測試絲胺酸可否被以上胺基酸取代;另外我們也測試Ser178,將其突變成丙胺酸測試是否會有影響,若Tpc無法鍵結到DNA上則無法維持線形複製,將突變送入鏈黴菌的結果為Ser178換成丙胺酸無法得到線形質體,而Ser206換成上述三種胺基酸仍可以得到線形質體,但換成丙胺酸則無法得到線形質體,初步結果顯示Tpc的Ser206可能不是與DNA鍵結的位置,而丙胺酸的突變可能因為其含有His-tag而失去活性;Ser178還需要做其他突變進行更明確的判斷是否是Tpc與DNA結合的位置。
Some plasmids and chromosomes of Streptomyces are linear. The replication of streptomyces proceeds bidirectionally from central origin to the end of DNA, and leaves a 3’ overhang. Next, terminal asscoiated protein (Tap) links to the 3’ overhang and recruit terminal protein (Tpg) to repair the 3'' gap. In this case, Tap serves as a DNA polymerase, and Tpg serves as a primer. Some plasmids, like SCP1, have different telomeric DNA sequence that can’t align with others. The terminal asscoiated protein of SCP1 named Tac, and terminal protein named Tpc. The tac (gene of Tac) is on the upstream of tpc (gene of Tpc). This characteristic is similar to Tap-Tpg system, and therefore they may have the same mechanism of end-patching.

We align the amino acid sequence of Tac, Tap, Φ29 DNA polymerase, and E.coli DNA Pol II. The alignmet showed that there are three conservative aspartic acid residues. Two of them have been identified that are the Mg2+ binding site of Φ29 DNA polymerase and E.coli DNA pol II[1,29], and the corresponding positions in Tac are Asp398 and Asp670.The essentialty of these two residues was examined in vivo by mutating the corresponding positions in tac gene in plasmid pLUS892L, a mini linear SCP1 plasmid. The mutants of them named TacD398Q and TacD670A.The results showed that there had no linear plasmids formed. Additionally, Φ29 DNA polymerase has a motif (YCDTDS) to do with protein-primed and polymeration. The threonine is important for its activity. The essentialty of Thr669 of Tac was also tested. The in vivo results suggested that it might be critical.

The essential aminio acid residues on Tpc were also examined. We use phosphoamino acid analysis (PAA) to determine that Tpc used serine to bond to the end of DNA. There are thirteen serines in Tpc, we first check Ser206 because of its hydrophilicity. It was mutated to alanine, and named TpcS206A. The in vivo result suggested that Tpc might use Ser206 to bond to DNA end. Further, Ser206 was mutated to histidine, threonine and tyrosine. The in vivo results showed that Ser206 can be replace by those amino acid. The result of TpcS206A might be influence by his-tag, hence there had no linear plasmid formed. We also picked Ser178 to mutate to alanine then had in vivo testing. The result identicated that Ser178 can’t get any linear plasmid. There was an unecpected mutation caused by polymerase chain reaction (PCR), the His135 of Tpc mutated to tyrosine. The in vivo result that His135 might also be critical.
目錄
摘要 I
Abstract II
目錄 III
圖目錄 V
表目錄 VII
第一章 序論 1
1.1鏈黴菌簡介 1
1.2鏈黴菌線形染色體複製 2
1.3鏈黴菌的末端序列 3
1.4 Φ29 DNA聚合酶 5
1.5染色體端粒的不穩定性 6
1.6論文研究內容 7
第二章 材料與實驗方法 11
2.1儀器 11
2.2藥品 11
2.3試劑 12
1.培養基與培養液 12
2. Buffer 12
3. Antibiotic 13
2.4菌種與質體 14
1.菌種目錄 14
2.質體目錄 14
2.5本實驗使用的引子 16
1.構築Tac D398Q pet28a 16
2.構築Tac D670A pet28a 16
3.構築792 Tpc-S206A 16
4.構築793-4 17
5.構築793-4 Tac-T669A 17
6.構築793-4 Tpc-S206T(與Tpc-E208Q共用) 17
7.構築793-4 Tpc-E208Q(與Tpc-S206T共用) 17
8.構築793-4 Tpc-S206H 17
9.構築793-4 Tpc-S206Y 18
10.構築793-4 Tpc-S178A 18
2.6實驗方法 18
1.鏈黴菌原生質體製備 [28] 18
2.鏈黴菌轉型 [28] 19
3.鏈黴菌質體純化 [28] 19
4.E.coli質體純化 (Novelgene Plasmid extraction kit) 19
5.E.coli勝任細胞製備 [16] 20
6.E.coli轉型 [16] 20
7.菌種儲存 20
8.蛋白質表現 20
9.超音波震盪破菌 21
10.PCR反應 21
11.In-Fusion 23
第三章 實驗結果 24
3.1 SCP1端粒連結蛋白活性的分析 24
1.Tac-D398Q-pet28a質體構築 24
2.Tac-D670A-pet28a質體構築 26
3.Tac D398Q與Tac D670A蛋白質表現 28
3.2 tac突變質體構築 29
1. 構築892 Tac-D398Q與892 Tac-D670A與體內實驗 29
2. 構築793-4與894 32
3. 894 Tac-T669A質體構築與體內實驗 34
4. 894 Tac-D398Q與894 Tac-D670A質體構築與體內實驗 38
3.3 tpc突變質體構築 41
1.構築892 Tpc-S206A (89201)與體內實驗 41
2. Tpc-S206T、Tpc-E208Q質體構築 46
3. Tpc-S206H質體構築 49
4. Tpc-S206Y質體構築 51
5. Tpc-S178A質體構築 53
3.4測試tpc突變質體對鏈黴菌線型複製的影響 55
1. 突變tpc上絲胺酸對鏈黴菌線型質體複製的影響 55
第四章 結論 59
參考文獻 61
附錄 64
Appendix 1. 792-KN pRSETA質體構築 64
Appendix 2. 質體893 65
圖目錄
圖1.1:鏈黴菌的生長史 1
圖1.2:鏈黴菌線型質體複製機制 2
圖1.3:鏈黴菌3’末端單股167 bp 可能形成的二級結構 3
圖1.4:鏈黴菌染色體末端示意圖 4
圖1.5:各種鏈黴菌染色體或質體之末端序列比對 5
圖1.6:S. lividans ZX7 線形染色體端點刪除與環化 6
圖1.7:TapC、TaC、E.coli DNA polymerase II與 29 DNA polymerase胺基酸序列比對 9
圖1.8:Tpc中胺基酸的親疏水性 10
圖3.1.1:tac與tac-D398Q突變處的DNA序列比較和定序結果 24
圖3.1.2:Tac D398Q pet28a質體構築流程 25
圖3.1.3:tac與tac-D670A突變處的DNA序列比較和定序結果 26
圖3.1.4:Tac D670A pet28a質體構築流程 27
圖3.1.5:Tac His pet28a、Tac D398Q、Tac D670A BL21*表現3小時結果 28
圖3.1.6:Tac His pet28a、Tac D398Q、Tac D670A超音波破菌結果
28
圖3.2.1:892 Tac-D398Q與892 Tac-D670A質體構築流程 30
圖3.2.2:892 Tac-D398Q洋菜膠電泳圖 31
圖3.2.3:Tac D670A洋菜膠電泳圖 31
圖3.2.4:質體793-4與894構築流程 33
圖3.2.5:tac與tac-T669A突變處的DNA序列比較和定序結果 34
圖3.2.6:Tac-T669A構築流程圖 36
圖3.2.7:894與Tac T669A洋菜膠電泳圖 37
圖3.2.8:894與Tac T669A質體純化與酵素檢驗電泳圖 37
圖3.2.9:894 Tac D398Q與894 Tac D670A構築流程 39
圖3.2.10:894與Tac D670A洋菜膠電泳圖 40
圖3.2.11:894與Tac D670A質體純化與酵素檢驗電泳圖 40
圖3.3.1:tpc、 tpc-S206A突變處的DNA序列比較和定序結果 41
圖3.3.2:質體89201構築流程 43
圖3.3.3:89201洋菜膠電泳圖 44
圖3.3.4:89201切SacI洋菜膠電泳圖 44
圖3.3.5:線形892質體切SacI示意圖 44
圖3.3.6:線形89201質體切SacI示意圖 45
圖3.3.7:環形89201質體切SacI示意圖 45
圖3.3.8:tpc與tpc-S206T突變處的DNA序列比較和定序結果 46
圖3.3.9:tpc與tpc-E208Q突變處的DNA序列比較和定序結果 47
圖3.3.10:Tpc-S206T與Tpc-E208Q構築流程圖 48
圖3.3.11:tpc與tpc-S206H突變處的DNA序列比較 49
圖3.3.12:Tpc-S206H質體構築流程 50
圖3.3.13:tpc與tpc-S206Y突變處的DNA序列比較 51
圖3.3.14:Tpc-S206Y質體構築流程圖 52
圖3.3.15:tpc與tpc-S178A突變處的DNA序列比較 53
圖3.3.16:Tpc-S178A質體構築流程圖 54
圖3.4.1:Tpc S206Y與Tpc E208Q 1~5號100 volt洋菜膠電泳圖 55
圖3.4.2:Tpc S206Y與Tpc E208Q 1~5號50 volt洋菜膠電泳圖 56
圖3.4.3:Tpc S206H與Tpc E208Q 6~10號100 volt洋菜膠電泳圖 56
圖3.4.4:Tpc S206H與Tpc E208Q 6~10號50 volt洋菜膠電泳圖 56
圖3.4.5:894與Tpc S206T 100 volt洋菜膠電泳圖 57
圖3.4.6:894與Tpc S206T 50 volt洋菜膠電泳圖 57
圖3.4.7:894與Tpc S178A洋菜膠電泳圖 57
圖3.4.8:Tpc突變株與894切SacI檢驗 58
圖3.4.9:線形化與環形化的894質體 58
圖A1.1:792-KN pRSETA構築流程 64
圖A2.1:tpc與 tpc-H135Y的DNA序列比較 65
圖A2.2:894與893洋菜膠電泳圖 66
圖A2.3:894與893質體純化電泳圖 66
表目錄
表2.1:使用菌種目錄 14
表2.2:使用質體目錄 16
表2.3:Super-Run Taq DNA Polymerase反應試劑及條件 21
表2.4:Super-Run Taq DNA Polymerase反應溫度及時間 22
表2.5:kapa Hifi PCR kit反應試劑及條件 22
表2.6:kapa Hifi PCR kit反應溫度及時間 22
表3.2.1:894與Tac T669A的thior轉型株數量 35
表3.2.2:突變的Tac質體與894的thior轉型株數量 38
表3.4.1:突變的Tpc質體與894轉型至鏈黴菌MRO4的轉型株總量 55
表A2.1:Tpc H135Y與894轉型至鏈黴菌MRO4的轉型株數量 65
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