臺灣博碩士論文加值系統

柑橘潰瘍病菌具有二個 lexA 基因，分別合成 LexA1 和 LexA2 蛋白，推測具有抑制蛋白 (repressor) 的功能，調控許多與 DNA 修補相關之基因。分析二 LexA 蛋白胺基酸序列，發現負責與 DNA 結合之相關序列 (α domain) 並不相似，推論二蛋白應與不同之 DNA 片段結合。為了得到進一步的證實，遂利用 Electrophoretic mobility shift assay (EMSA) 分析，結果顯示 LexA1 蛋白可與 lexA1 啟動子 (-40 ~ +155，以轉譯始點為 +1) 結合，卻無法與 lexA2 啟動子 (-71 ~ +134，以轉譯始點為 +1) 結合; 而 LexA2 蛋白則與之相反，只能與此 lexA2 啟動子結合。LexA1 蛋白作用於 lexA1 啟動子之所在，已知為一顛倒重覆序列: 5’-TTAGTAGTAATACTACTAA-3’，為了得知 LexA2 蛋白作用之所在，便將 lexA2 啟動子之縮短片段與 LexA2 蛋白進行 EMSA 分析，結果發現一不同於 LexA1 蛋白結合位置 (TTAG-N8~11-CTAA) 之序列，5’-GGTGTACAAATGTACACC-3’，為其作用所在。為了解此序列之對稱性和序列內核苷酸之必要性，也以 EMSA 測定突變序列與 LexA2 蛋白之作用，目前數據顯示 LexA2 蛋白結合之必要序列，可能為 TGTN8~10ACA。在 lexA2 基因轉譯始點 ATG 上游 115 bp 處，發現一相似於 LexA1 蛋白結合位置 (TTAG-N8~11-CTAA) 之序列: 5’-TTAGTACTAAAGTTATAA-3’，藉由分析發現 LexA1 蛋白可與之結合，但親合性不若 lexA1 啟動子上之LexA1 蛋白結合位置好。此外，已知 LexA1 蛋白可作用於 recA 啟動子，為了知道 LexA2 蛋白是否也可與之作用，便進行 EMSA 分析，結果發現 LexA2 蛋白亦可作用於 recA 啟動子上。

There are two lexA genes in Xanthomonas axonopodis pv. citri. LexA1 and LexA2 proteins, which are supposed to function as a repressor and modulate many DNA repair genes. By analyzing two sequences of LexA proteins, we discover they are not the same as the relative sequences (α domain) that bind to DNA. Therefore, we assume that the two proteins bind to different DNA sequences. In order to get further evidence, Electrophoretic mobility shift assay (EMSA) is adopted. The result shows that LexA1 protein can bind to lexA1 promoter (nucleotides -40 to +155, relative to the translation start site, +1),rather than to lexA2 promoter (nucleotides -71 to +134, relative to the translation start site, +1); on the contrast, LexA2 protein functions reversely, only bind to lexA2 promoter. LexA1 protein functions on where lexA1 promoter locates, which is known as an inverted repeat, 5’-TTAGTAGTAATACTACTAA-3’. In order to know where LexA2 protein functions, we analyze the shorten sequence of lexA2 promoter with LexA2 protein by EMSA. The result indicates that LexA2 protein binds to another sequence, 5’-GGTGTACAAATGTACACC-3’, which is different from the original one (TTAG-N8~11-CTAA). To get a better understanding of the symmetry of the sequence and the necessity of nucleotides in the sequence, EMSA is performed to determine the function of mutated sequence with the LexA2 protein. The data shows that the necessary sequence to which LexA2 protein binds probably is TGTN8~10ACA. We discover that, at the place where 115 bp upstream of the translation start site of the lexA2 gene, a specific sequence (5’-TTAGTACTAAAGTTATAA-3’), which is similar to the sequence (TTAG-N8~11-CTAA), to which LexA1 protein binds. We find that LexA1 protein can bind to this specific sequence, but the affinity is not as well as to the LexA1 protein on lexA1 promoter. Besides LexA1 protein can function on recA promoter, EMSA analysis is conducted to know if it works with LexA2. The result demonstrates LexA2 protein can also function on recA promoter.

目 錄

頁
目 錄 I
圖目錄 III
表目錄 IV
摘要 1
Abstract 2
前言 3
材料與方法 9
一. 實驗材料 9
1. 菌種與質體及引子 9
2. 實驗藥品 13
3. 培養基與抗生素 14
4. 緩衝液及溶液 14
5. 儀器設備 19
二. 實驗方法 20
1. 細菌之培養與保存 20
2. 質體DNA之抽取 20
3. DNA之限制酶作用 　 21
4. 洋菜膠電泳法 21
5. 聚合酶鏈反應 22
6. DNA片段之回收 22
7. 大腸桿菌之轉形作用 23
8. 蛋白質之純化 23
9. 蛋白質濃度之測定 24
10. 細胞萃取物之製備 25
11. SDS-Polyacrylamide凝膠電泳分析 25
12. Electrophoretic mobility shift analysis 26
結果 28
一. LexA 兩蛋白胺基酸序列之比對 28
二. LexA 蛋白之純化 30
三. LexA 蛋白與 lexA 啟動子片段之結合 32
1. LexA 蛋白與啟動子之結合情形 32
2. LexA 蛋白結合於啟動子之親和性 33
四. LexA2 蛋白在 lexA2 啟動子結合位置之確認 33
1. LexA2 蛋白之結合位置 33
2. LexA2 蛋白與 lexA2 突變啟動子之結合 36
五. LexA1 蛋白與 lexA2 基因上游區域之結合 38
1. LexA1 蛋白與啟動子之結合情形 38
2. LexA1 蛋白結合於啟動子之親和性 44
六. LexA2 蛋白與 lexA1、recA 啟動子之結合 48
1. LexA2 蛋白與 lexA1 啟動子之結合情形 48
2. LexA2 蛋白與 recA 啟動子之結合情形 48
討 論 53
參考文獻 58

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楊源昌 (2000) 柑橘潰瘍病菌參與DNA修補基因之結構與特性分析。國防醫學院生命科學研究所博士論文

徐千琇 (2002) 柑橘潰瘍病菌 LexA 蛋白結合位置之確定。輔仁大學生物研究所碩士論文

蘇宿睿 (2003) 柑橘潰瘍病菌 lexA2 基因對 SOS DNA 修補系統的調控。輔仁大學生命科學研究所碩士論文