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研究生:謝承陸
研究生(外文):Cheng-Lu Hsieh
論文名稱:高溫嗜酸菌 Alicyclobacillus acidocaldarius木糖操縱組調控因子之研究
論文名稱(外文):Xylose operon transcriptional regulator in Alicyclobacillus acidocaldarius 49-1
指導教授:曾惠中曾惠中引用關係
指導教授(外文):Huei-Chung Tseng
學位類別:碩士
校院名稱:東吳大學
系所名稱:微生物學系
學門:生命科學學門
學類:微生物學類
論文種類:學術論文
論文出版年:2011
畢業學年度:99
語文別:中文
論文頁數:132
中文關鍵詞:Alicyclobacillus acidocaldarius木糖調控蛋白黑色素操縱組
外文關鍵詞:Alicyclobacillus acidocaldariusxylose repressormelC operon
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  Alicyclobacillus acidocaldarius屬產胞革蘭氏陽性桿菌,為異營性腐生菌,具有嗜酸嗜熱特性,並可利用多種單醣或多醣作為唯一碳源。本實驗室已成功選殖A. acidocaldarius 49-1之木糖操縱組基因,全長約11 kb,排列順序為xylT-xylA-xylB,此種基因排列順序與其他菌屬明顯不同,且其中並未發現木糖調控基因。本研究首先重新建構木糖啟動子至低複製數載體上,並以黑色素操縱組作為報導基因,自A. acidocaldarius基因庫進行木糖調控蛋白基因的篩選,同時自基因組中設計專一性引子直接選殖。結果顯示大約有33%的轉型株皆成功接上A. acidocaldarius 49-1染色體片段,然而於CUTY固體培養基上依舊呈現黑色,推測這些轉型株中所含有的3~5 kb片段皆不含有xylR基因。另外根據A. acidocaldarius基因組序列,以專一性引子自A. acidocaldarius 49-1選殖一調控蛋白家族相關的基因,且經由反轉錄聚合酵素鏈反應證實該基因擁有獨立的啟動子,並經由保守性區域與親緣比對之鄰接法,推斷為木糖調控蛋白。隨後將含有木糖調控基因之質體與含有木糖啟動子的質體同時送入大腸桿菌表現,培養於30℃下24小時,可觀察到報導基因產生的黑色素明顯的被抑制,而添加木糖至培養基時,則抑制現象明顯被減少。另一方面,藉由反轉錄聚合酵素鏈反應結果間接推測,參與木糖操縱組之啟動子位於orf 771上游,同時開啟下游orf 771至木酮糖激酶基因共四個基因之表現。本研究確實成功選殖A. acidocaldarius的木糖調控蛋白基因。另外,仍需進一步確認木糖操縱組上調控蛋白結合位置以及是否同樣受到碳代謝物抑制物所共同調控。
Alicyclobacillus acidocaldarius is a thermoacidophilic, spore-forming, Gram-positive saprophytic bacteria, and can utilize various monosaccharide or polysaccharide as sole carbon and energy source. Recently, our lab had cloned the whole xyl operon from A. acidocaldarius 49-1 strain. The operon contains xylT, xylA and xylB, and the arrangement is different from other known xyl operon, no regulator gene found in neighboring area. The purpose of this work was to search the xylR of the A. acidocaldarius 49-1. First, we inserted xylA promoter to low-copy number plasmid and used the melC operon as reporter. Second, we constructed a genomic library containing 3~5 kb DNA fragments and designed specific primers from A. acidocaldarius 104-IAT genomic sequence. The results showed that about 33% of the transformants were containing A. acidocaldarius 49-1 3~5 kb genomic fragment, however, transformants were still producing melanin on CUTY plate, suggested that neither clones contained xylR gene. Furthermore, we successful cloned a gene of the family of regulatory protein of A. acidocaldarius, and confirmed the regulatory gene has an independent promoter by reverse transcription polymerase chain reaction, and suggested this regulatory protein was xylose repressor by conserved region analysis and neighbor-joining method. The repression activity of the gene of regulatory protein was examined by expressed in E. coli that had been transformed a plasmid carrying xylA promoter. After incubating at 30℃ for 24 hours on CUTY plate, the result showed that melanin formation was significantly decreased. While adding xylose to the medium, the repression was significantly decreased. On the other hand, xylose promoter located upstream of orf 771 was shown by reverse transcription polymerase chain reaction, it suggested that the transcription began from orf 771 to xylulose kinase gene in vivo. In conclusion, we successfully cloned the A. acidocaldarius 49-1 xylose regulatory gene. The regulatory protein binding site and phenomenon of catabolite repression still need further study.
中文摘要.................................................I
英文摘要...............................................III
目錄.....................................................V
圖次....................................................IX
表次....................................................XI
第一章 前言............................................1
第一節 Alicyclobacillus acidocaldarius.................1
第二節 植物細胞壁之組成................................2
第三節 木糖異構酶......................................5
第四節 木糖操縱組......................................7
第五節 木糖調控蛋白...................................10
第六節 A. acidocaldarius 49-1基因的選殖...............12
第七節 報導基因melC operon............................16
第八節 研究目的、策略與架構...........................17
第二章 材料與方法.....................................20
第一節 材料...........................................20
一、 菌株與質體.....................................20
二、 引子...........................................20
三、 藥品與器材.....................................20
四、 培養基配方.....................................22
五、 試劑配方.......................................25
第二節 方法...........................................30
一、 製備A. acidocaldarius 49-1菌株之染色體DNA......30
二、 製備大腸桿菌質體DNA............................31
三、 限制酶反應.....................................33
四、 聚合酶鏈反應...................................33
五、 重新建構PxylA-melC operon片段之選殖株..........34
六、 洋菜醣膠體電泳.................................34
七、 製備部份切割之A. acidocaldarius 49-1染色體DNA片段 ...............................................35
八、 DNA片段之回收..................................36
九、 黏接反應.......................................37
十、 勝任細胞的製備.................................37
十一、 大腸桿菌之轉型.................................39
十二、 製備大腸桿菌RNA................................40
十三、 反轉錄聚合酶鏈反應.............................41
十四、 細胞萃取物的製備...............................42
十五、 蛋白質濃度的測定...............................42
十六、 酪胺酸酶活性測定...............................43
第三章 結果...........................................44
第一節 重新建構含黑色素操縱組為報導基因之選殖株.......44
第二節 建構A. acidocaldarius 49-1的基因組資料庫.......44
第三節 A. acidocaldarius 49-1木糖調控蛋白基因選殖.....46
第四節 木糖調控蛋白基因序列比對與分析.................47
第五節 木糖啟動子活性分析.............................49
第六節 木糖啟動子位置.................................52
第四章 討論...........................................55
第一節 成功建構含黑色素操縱組為報導基因的低複製數載體.55
第二節 A. acidocaldarius 49-1 3~5 kb基因組資料庫之建構 ...............................................56
第三節 以反向遺傳研究法選殖A. acidocaldarius 49-1木糖調控蛋白 ...............................................58
第四節 木糖基因開啟表現與受到抑制.....................60
第五節 木糖啟動子區域與其於基因組上可能的位置.........62
參考文獻................................................65
圖......................................................81
表.....................................................105
附錄...................................................111
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