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研究生:蔣明蕙
研究生(外文):Ming-Hui Chiang
論文名稱:利用跳躍子突變探討Shewanellasp.TeC抗重金屬鎘與類金屬碲之特性
論文名稱(外文):Screening and characterization of cadmiumsensitive and reduced tellurite-reduction mutants ofShewanella sp. TeC by transposon mutagenesis
指導教授:簡志青
指導教授(外文):Chih-Ching Chien
學位類別:碩士
校院名稱:元智大學
系所名稱:生物科技與工程研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:65
中文關鍵詞:重金屬抗性亞碲酸鹽鎘離子Shewanella
外文關鍵詞:heavy metal resistancetelluritecadmiumShewanella
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本研究探討具有重金屬鎘與類金屬碲抗性之微生物Shewanella
sp. TeC 及用Tn5 轉位子插入基因之突變株Shewanella sp. A8 與
Shewanella sp. B21。本論文證實Shewanella sp. TeC 可在高濃度(3,000 μM)亞碲酸鹽環境中生長,並且具有還原亞碲酸鹽為元素態碲之能力;而Shewanella sp. A8 經由生長曲線測定、亞碲酸鹽還原量測定及含亞碲酸鹽培養盤等實驗顯示,被Tn5 轉位子插入之基因與還原亞碲酸鹽為元素態碲相關。Shewanella sp. B21 則是推測與亞碲酸鹽抗性相關之基因被破壞。利用分生實驗技術解序被Tn5 轉位子插入Shewanella sp. A8 之基因,經由定序及blastx 比對,得到與Pseudomonas fluorescens Pf0-1 之hflC 基因轉譯的HlfC 蛋白質有80%之同源性,同時於hflC 基因上游序列比對出與Pseudomonas entomophila L48 之hflK 基因轉譯的HlfK 蛋白質有80%之同源性;而被Tn5 轉位子插入Shewanella sp. B21 之基因轉譯後之序列則是比對到與Azotobacter vinelandii DJ 之metallopeptidase 蛋白質有46%之同源性。過去文獻顯示hflK 及hflC 基因具有相關性,未來將進一步探討此二基因在Shewanella sp. TeC 中,對還原能力所扮演的角色及釐清metallopeptidase 蛋白質與金屬抗性兩者之間的關聯。
Shewanella sp. TeC was isolated from soil contaminated with heavy metal and was able to tolerate high concentration of tellurite (up to 3,000μM) and cadmium. Black precipitates (presumably due to the production of tellurium from tellurite reduction) occurred in the growth
media of strain TeC. A cadmium sensitive strain B21 and a reduced tellurite-reduction strain A8 were obtained after screening mutants of Shewanella sp. TeC generated by transposon (Tn5) mutagenesis. Gene disrupted by Tn5 in strain A8 was identified as a homologue of hflC of
Pseudomonas fluorescens Pf0-1. In strain B21, a gene encode
homologue of metallopeptidase of Azotobacter vinelandii DJ was disrupted by Tn5. The possible roles of hflC homologue in tellurite reduction and metallopeptidase in heavy metals/metalloids resistance in Shewanella sp. TeC remain to be studied.
中文摘要................................................. I
Abstract................................................III
目錄......................................................V
圖表目錄................................................ IX
致謝................................................... XII
第一章 前言...............................................1
1.1 希萬氏菌(Shwanella sp.)介紹...........................1
1.2 微生物抵抗金屬離子衍生之作用機制......................3
1.2.1 通透性屏障隔離......................................4
1.2.2 金屬離子之主動運輸..................................5
1.2.3 金屬離子之蛋白質鍵結................................6
1.2.4 細胞外隔離..........................................6
1.2.5 酵素解毒作用........................................7
1.2.6 細胞標的對金屬敏感性之減低金屬對細胞毒性............8
1.3 微生物與金屬碲離子毒性之關係..........................9
1.3.1 金屬碲之毒性........................................9
1.4 微生物對於亞碲酸鹽之解毒作用..........................9
1.4.1 還原酵素之解毒作用.................................10

2.2.3 菌株培養條件及培養基配方...........................18
1.4.2 甲基轉移?之解毒作用...............................10
1.4.3 利用流出運輸之解毒作用.............................11
1.4.4 利用生物累積之解毒作用.............................11
1.5 研究目的.............................................12
第二章 材料與方法........................................13
2.1 實驗材料.............................................13
2.1.1 菌株與質體.........................................13
2.1.2 引子...............................................13
2.1.3 藥品與試劑.........................................14
2.1.4 試劑組.............................................15
2.1.5 酵素...............................................15
2.1.6 培養基.............................................16
2.1.7 抗生素.............................................16
2.1.8 DNA電泳試劑........................................16
2.1.9 實驗儀器...........................................16
2.2 實驗方法.............................................18
2.2.1 實驗菌株Shewanella sp. TeC之來源...................18
2.2.2 實驗菌株Shewanella sp. A8 及Shewanella sp. B21 之來源
.........................................................18

第三章
2.2.4 實驗菌株移除亞碲酸鹽特性之觀察.....................19
2.2.5 微生物生長曲線(growth curve)之測定.................19
2.2.6 亞碲酸鹽之定量.....................................20
2.2.7 菌株染色體DNA之純化(genomic DNA extraction)........20
2.2.8 以聚合?連鎖反應(Polymerase chain reaction, PCR)進行
Tn5 轉位子之確認.........................................21
2.2.9 瓊脂凝膠電泳分析(eletrophoresis) ..................22
2.2.10 菌株染色體DNA之限制?截切(restriction enzyme
digestion)...............................................22
2.2.11 以接合?進行DNA片段自體黏合(self ligation) .......22
2.2.12 倒轉聚合?連鎖反應(Inverse Polymerase chain reaction,Inverse PCR) ...................................23
2.2.13 目標DNA片段之膠體萃取(gel extraction) ............23
2.2.14 TA黏合(TA ligation) ..............................24
2.2.15 熱休克反應(heat shock)............................24
2.2.16 質體之純化(plasmid extraction) ...................25
實驗結果與討論...........................................27
3.1 實驗菌株移除亞碲酸鹽特性之觀察.......................27
3.2 實驗菌株生長曲線之測定...............................27
3.3 利用亞碲酸鹽定量測量實驗菌株之還原能力...............28


3.4 Tn5 轉位子插入之基因解序.............................30
3.5 hflK、hflC基因與還原亞碲酸鹽能力關係之討論...........32
3.6 metallopeptidase蛋白質與金屬抗性關係之討論...........33
第四章 結論與展望........................................59
參考文獻.................................................60
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