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研究生:徐千琇
研究生(外文):Chien-Hsiu Hsu
論文名稱:柑橘潰瘍病菌LexA蛋白結合位置之確定
論文名稱(外文):Identification of the consensue sequence of LexA binding site in Xanthomonas campestris pv. citri
指導教授:楊美桂楊美桂引用關係
指導教授(外文):Mei-Kwei Yang
學位類別:碩士
校院名稱:輔仁大學
系所名稱:生物學系
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
論文頁數:82
中文關鍵詞:柑橘潰瘍病菌LexA蛋白
外文關鍵詞:Xanthomonas campestris pathovarlexA-recA-recXLexA repressorSOS boxEMSALexA binding site
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摘要
與DNA修補有關的lexA、recA與recX基因,在柑橘潰瘍病菌 (Xanthomonas campestris pv. citri) 呈lexA- recA- recX連續排列的特殊結構,同時也存在於十字花科黑腐病菌( X. c. pv. campestris ) 和水稻白葉枯病菌( X. oryzea pv. oryzae )等不同黃原桿菌中。藉由Electrophoretic mobility shift analysis (EMSA)分析,顯示X. c. pv citri的 LexA蛋白也可與X. c. pv. campestris 與 X. o. pv. oryzae之lexA與recA啟動子作用,作用後會抑制其表現,表示柑橘潰瘍病菌的LexA蛋白可跨過病原小種的界限,作用於不同的黃原桿菌。分析lexA基因之啟動子,發現一顛倒重複序列:5’-TTAGTAGTAATACTACTAA-3’,為LexA蛋白作用之所在。以定點突變法證實此對稱結構為LexA結合所必需。若將突變啟動子接上luxAB基因,並進行轉錄分析,發現lexA啟動子轉錄加強,表示LexA確定無法與突變的啟動子作用。此突變啟動子的轉錄,可促進LexA之表現量明顯增加,RecA則不易形成。表示lexA啟動子的突變,影響LexA蛋白的結合,lexA方得以啟動。recA與recX啟動子也有類似但不完全相同的序列,可為LexA蛋白所附著,但作用較弱,故知此一顛倒重複序列為LexA蛋白作用之重要序列。
Abstract
A Xanthomonas campestris gene cluster consisted of lexA, recA and recX genes was identified and characterized. Electrophoretic mobility shift analysis indicated that the LexA protein of X. c. pv. citri bind to the lexA and recA promoters of X. c. pv. campestris and X. oryzae. pv. oryzae and repress the Xanthomonas campestris recA gene, indicating that the X. c. pv. citri LexA protein was functional in different Xanthomonas campestris pathovars. It was confirmed that a symmetrical sequence of TTAGTAGTAATACTACTAA within the lexA promoter region is essential for the LexA protein binding by site-directed mutagenesis and electrophoretic mobility shift analysis. A lexA mutated promoter increased in the transcriptional and translational level, due to loss of the binding ability of the LexA protein. However, the LexA protein was able to bind to the similar sequences of the recA and recX promoters. It was suggested that the consensus sequence of LexA binding in X. c. pv. citri is TTAGTAGTAATACTACTAA.
目 錄
中文摘要 1
英文摘要 2
前言 3
材料與方法 8
一. 實驗材料 8
二. 實驗方法 21
1. 染色體DNA之抽取 21
2. 質體DNA之抽取 21
3. 重組質體之構築   22
4. 洋菜膠電泳法 23
5. 聚合酶鏈反應 23
6. DNA片段之回收 23
7. 大腸桿菌之轉形作用 24
8. 柑橘潰瘍病菌之轉形作用 25
9. DNA探針之製備 25
10. 南氏雜交反應 26
11. 蛋白質之純化 27
12. 蛋白質濃度之測定 28
13. 抗體之製作 29
14. 細胞萃取物之製備 29
15. SDS-Polyacrylamide凝膠電泳分析 29
16. 西方墨點法 30
17. 細胞冷光酵素活性之測定 31
18. Electrophoretic mobility shift analysis 31
19. 菌落雜交法 31
結果
一. 不同黃原桿菌lexA、recA、recX基因之選殖
1. 水稻白葉枯病菌
(1) recA基因之選殖 33
(2) pAP41之限制酶圖譜 33
(3) 序列分析與比對 36
2. 十字花科黑腐病菌
(1) recA基因之選殖 36
(2) pAP60之限制酶圖譜 39
(3) 序列分析與比對 39
二. 不同黃原桿菌lexA、recA、recX基因之序列比對
1. lexA基因 44
2. recA基因 44
3. recX基因 45
三. 柑橘潰瘍病菌LexA蛋白之作用
1. 三種黃原桿菌lexA、recA與recX啟動子序列分析 47
2. 三種黃原桿菌之啟動子與 LexA蛋白結合測定 47
(1) lexA啟動子 47
(2) recA啟動子 48
3. LexA調控十字花科黑腐病菌recA的表現 52
四. LexA蛋白結合位置之確認
(一) lexA啟動子之突變分析 55
1. lexA突變啟動子之結合測定 55
2. lexA突變啟動子之轉錄活性 58
(1) 重組質體之構築 58
(2) 生長穩定性之測定 60
(3) 冷光值之測定 60
3. lexA突變啟動子之LexA蛋白表現 64
(1) 構築含lexA表現載體 64
(2) 分析LexA之表現量 65
(二) recA啟動子之突變分析 68
(三) recX啟動子之突變分析 68
討 論 74
參考文獻 77
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