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研究生:陳梵帆
研究生(外文):Fan-Fan Chen
論文名稱:新型砷抗性轉位子TnARS1之砷抗性操縱組的基因功能及調控特性分析
論文名稱(外文):Analysis of gene function and regulative feature of ars-operons from a novel arsenic resistance transposon TnARS1
指導教授:黃介辰
學位類別:碩士
校院名稱:國立中興大學
系所名稱:生命科學系所
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:2010
畢業學年度:98
語文別:中文
論文頁數:57
中文關鍵詞:砷抗性操縱組
外文關鍵詞:arsenicarsenic resistanceoperon
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  本實驗針對巢居於Bacillus species MB24汞抗性轉位子TnMARS1內之砷抗性轉位子TnARS1的序列進行了分析。其中與砷抗性相關的基因群可分為由兩個調控蛋白所分別帶領的兩組操作組:arsR1-orf3-orf4-orf5與arsR2-arsB-arsC-arsD-arsA-orf11-orf12,當中第二群操作組含有典型的五基因型砷抗性操作組(arsR-B-C-D-A)。針對這基因群中無法特定功能的五個基因進行分析的結果,orf3與orf4雖然彼此在胺基酸序列上只有22%的一致性,但是在保留性序列的分析中都顯示可能為thioredoxin reductase。這結果顯示此兩個蛋白可能參與五價砷還原&;#37238;ArsC的氧化還原反應鏈中。orf5及orf11在序列分析的結果中判斷分別為N-acetyltransferase及HesB-like domain protein,但過去並沒有此兩種蛋白與生物砷抗性具有相關性的研究報導。orf12的胺基酸序列中帶有一段HC(X)5R的序列,而此序列被認為是真核生物中五價砷還原&;#37238;Arr2p的活性中心。
  為了了解此兩組操縱組的相互關係及調控機轉,本研究針對兩調控基因進行了較深入的研究。從前人針對Bacillus subtilis str. 168及Escherichia coli質體R773的研究中可以知道砷抗性操縱組中的調控蛋白ArsR之辨識操作子序列為一段反重複序列,且在其中帶有一段[TCAT…TTTG]的序列為ArsR的結合位置。本實驗中針對arsR1基因的上游序列進行分析也發現一段反重複序列且帶有與上述結合序列僅相差一個核&;#33527;酸的[TCAA…TTTG]序列。另外在orf3基因的上游發現一段與arsR1上游之反重複序列完全一致的序列,推測可能為另一個ArsR的辨識序列。相反地,在針對arsR2基因的上游序列進行分析的結果並沒有發現可能為ArsR辨識位置的序列。而在5’-RACE實驗中分別得到了位於arsR1基因與arsR2基因上游的RNA轉錄起始點,且所得到的位置與預測的啟動子-35/-10 box距離相符。然而在針對orf3基因上游序列同樣進行5’-RACE實驗卻無法得到任何結果,指出orf3上游可能實際上並不存在轉錄起始點。
  以real-time RT-PCR針對上述三段可能的操作子序列進行三價砷的RNA誘導實驗,發現arsR1及orf3具有相似的表現情形,這可能致因於上述的完全一致之ArsR辨識序列或著如同5’-RACE實驗的結果一樣意味著arsR1及orf3實際上確實如原先預測般屬於同一個轉錄單位。另外,arsR2相較於前述兩個基因在較低的三價砷濃度下即有誘導表現的情形,顯示其所帶領的第二群操作組也會在較低的砷濃度環境下優先進行表現。以表現測試載體進行固態培養基的平板測試,結果也顯示arsR2前的[操作子/啟動子]在含有三價砷的環境相較於arsR1前的[操作子/啟動子]具有更明顯的誘導表現情形。由此推測第一群操作組的基因在Bacillus species MB24的砷抗性系統中可能並非主要的抗性提供者,而是在較高的砷濃度環境下輔助第二群操作組的表現蛋白以提高砷抗性的能力。


The arsenic resistance transposon TnARS1, nested in the mercury resistance transposon TnMARS1 of Bacillus species MB24, was identified and two arsenic- resistance (ars) operons were found. The first operon (arsR1-orf3-orf4-orf5) contains an arsenic-dependent regulator followed by three unknown genes, and the second operon (arsR2-arsB-arsC-arsD-arsA-orf11-orf12) contains a complete five-gene type ars operon which is usually found in Gram-negative bacteria originated plasmid. Translated peptide sequences of orf3 and orf4 both do not have similarity with arsenic-resistance related proteins in the results of blastp. However, according to the conserved-domains, they both seem to have similarity with thioredoxin reductase, even there is only 22% of similarity between these two peptide sequences. This result indicates that these genes may involve with the thioredoxin reduction which is followed by the reduction of arsenate reductase (ArsC). Peptide sequences of orf5 and orf11 have high similarity to N-acetyltransferase and HesB-like domain protein, respectively, but the relationship between these two proteins and arsenic resistance mechanism is still need to be elucidated. There is a HC(X)5R sequence in the peptide sequence of orf12, which is determined to be a active site sequence of eukaryotic arsenate reductase Arr2p.
The binding sites of arsenic-dependent regulator ArsR in Bacillus subtilis str. 168 and Escherichia coli plasmid R773 both harbor a pair of inverted repeats, and the major binding sequence is [TCAT…TTTG]. We also found inverted repeats upstream the arsR1 gene and this sequence have a sequence [TCAA…TTTG] which have only one base pair difference with [TCAT…TTTG]. In addition, there is a completely identical sequence of this inverted repeat on the upstream sequence of orf3 gene, indicates there may be another ArsR1 binding site. In the other way, there is no ArsR binding side liked sequence upstream the arsR2 gene. We also found the RNA transcriptional starting point upstream the arsR1 and arsR2 genes by 5’-RACE, and the location of these starting points are both correspond with the predicted promoters location. However, We didn’t get any result from 5’-RACE experiment of the orf3 gene, indicates there is no promoter upstream the orf3.
The regulative activity of these three hypothetical regulator sequences was analyzed by real-time RT-PCR, and we found the transcriptional activity of arsR1 and orf3 is similar. This result may because of the completely identical regulator sequences or these two genes are included within the same one transcriptional unit. The arsR2 gene has higher transcriptional level then other two, indicated the second operon will be transcripted in lower arsenic concentration than the first operon. That means the first operon may be not the main role of arsenic-resistance system of Bacillus species MB24, but have ability to boost the resistance activity provided by the second operon.


摘要 i
Abstract iii
目次 v
表目次 viii
圖目次 ix
一、 導論 1
1-1 砷污染問題 1
1-2 砷的化學特性及毒性機制 1
1-3 生物的砷抗性機制 2
1-4 砷操縱組的調控 3
1-5 砷抗性轉位子TnARS1 4
1-6 研究動機 5
二、 材料與方法 6
2-1 菌種與質體 6
2-2 培養基 6
2-3 菌種培養與保存 6
2-3-1 液態培養 6
2-3-2 固態培養 6
2-4 DNA的萃取 6
2-4-1 Genomic DNA萃取 6
2-4-2 plasmid DNA萃取 7
2-5 RNA之萃取 7
2-6 DNA、RNA純度鑑定與定量分析 7
2-7 聚合酶鏈反應(Polymerase chain reaction, PCR) 7
2-7-1 聚合酶鏈反應 7
2-7-2 反轉錄聚合酶鏈反應(Reverse-Transcription PCR, RT-PCR) 8
2-7-3 即時偵測聚合酶鍊反應(real-time PCR) 8
2-7-4 偵測RNA表現量 8
2-8 洋菜膠體電泳(Agarose gel electrophoresis)分析及記錄 9
2-9 DNA片段回收及純化 9
2-9-1 純化溶液中的DNA 9
2-9-2 回收洋菜膠體內的DNA片段 10
2-10 DNA黏合反應 10
2-11 大腸菌(Escherichia coli)DH5α轉型 10
2-12 枯草桿菌(Bacillus subtilis) 168轉形 10
2-12-1 培養基配置 10
2-12-2 轉型實驗流程 11
2-13 砷抗性測試 11
2-14 5’-cDNA末端快速增幅(5’- Rapid Amplification of cDNA Ends, 5’-RACE) 11
2-15 蛋白質表現載體誘導表現測試 12
2-16 Tricine SDS-PAGE蛋白電泳 12
2-16-1 配置膠體 12
2-16-2 SDS-PAGE電泳 13
2-16-3 膠體染色 13
三、 結果與討論 14
3-1 TnARS1序列解析 14
3-2 操縱子(operator)序列解析 15
3-2-1 ArsR1辨識區域 15
3-2-2 ArsR2辨識區域 15
3-2-3 疑似ArsR1辨識區域 15
3-3 啟動子(promoter)序列解析 16
3-3-1 -10與-35 box 16
3-3-2 轉錄起始點 16
3-4 ArsR1與ArsR2調控測試 17
3-4-1 RNA表現調控測試 17
3-4-2 平板測試 17
3-5 確定operon1為同一轉錄序列 18
四、 結論及未來展望 19
4-1 兩組操縱組的關係 19
4-2 未知基因之解析與預測 19
4-3 ArsR1及ArsR2的調控表現 21
4-4 operon1是否為單一轉錄序列 21
4-5 總結 22
參考文獻 23
表 27
圖 34
附錄 51

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