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研究生:陳彥君
研究生(外文):Yen-Chun Chen
論文名稱:Pectobacterium carotovorum subsp. carotovorum. 低分子量細菌素受環磷酸鳥苷與環磷酸腺苷受體蛋白調控作用之探討
論文名稱(外文):Low-molecular-weight Bacteriocin Gene Is Regulated by the c-di-GMP and Cyclic AMP Receptor Protein Signaling in Pectobacterium carotovorum subsp. carotovorum
指導教授:莊敦堯
口試委員:金德航溫育德
口試日期:2011-07-29
學位類別:碩士
校院名稱:國立中興大學
系所名稱:化學系所
學門:自然科學學門
學類:化學學類
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:52
中文關鍵詞:細菌素
外文關鍵詞:Pectobacterium
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Pectobacterium carotovorum subsp. carotovorum (Pcc) 屬於腸道菌科,它是一種會造成許多植物疾病的病源菌。大部分的 Pcc 會產生低分子量細菌素去抑制其他親源相近的菌種生長。目前文獻對於 Pcc 菌種中低分子量細菌素產生其調控機制主要是藉由 SOS 機制來進行調控細菌素基因的表現,但所參與的因子在各菌種內的報導並無一致。
在細菌內環磷酸鳥苷 (cyclic-di-GMP) 是做為二級訊號,由酵素鳥苷環化酶 (Diguanylate cyclase) 催化兩分子的 GTP 所形成的。cyclic-di-GMP 參與細菌細胞許多生理功能的調控,其中即有毒性蛋白的產生。環磷酸腺苷受體蛋白 (Cyclic AMP receptor protein) 會與環磷酸腺苷 (cAMP) 結合形成複合物做為轉錄因子,而結合至目標啟動子來調節基因,可以調控鞭毛的合成與毒性基因的表現。我們在實驗中發現,Diguanylate cyclase (dgc) 與 Cyclic AMP receptor protein (crp) 的突變皆造成細菌素無法產生。
在本篇研究中,我們利用同質互換的方式將 crp 基因阻斷。並利用 NCBI 的BLAST的程式比對胺基酸序列得知從 Pcc 中所獲得的 crp 與其他物種的 crp 基因有將近百分之百的同質性。我們發現在 CRP 突變株的轉錄實驗中細菌素 (Carocin) 基因是無法轉錄,並經由 in vivo 實驗分析後,在 CRP 突變株中的低分子量細菌素 (Carocin) 也無法表現。而由於 CRP 與 cAMP 結合後可以作為轉錄因子,能辨識特別的DNA序列或是結合至目標基因啟動子上,進而與 RNA 聚合酶作用進行轉錄調控,我們認為 CRP 可能結合在細菌素基因的啟動子位置進行調控作用。我們推測 DGC 所產生的二級訊號,可能與細菌素的生成或是分泌至細胞外的訊號傳遞有關。CRP-cAMP 做為轉錄因子,可能會結合在細菌素基因的啟動子。我們認為 DGC 與 CRP 皆在細菌素基因的表現扮演很重要的角色。


Pectobacterium carotovorum subsp. carotovorum (Pcc) is an enterobacterium that causes various plant diseases. Some of the Pcc strains produce low-molecular-weight bacteriocin (LMWB) to inhibit the related strain growth. The bacteriocin could be used as effectively biopesticides. However, little is known about the genetic regulation of bacteriocin production in Pectobacterium species. The bacteriocin expression was regulared by the SOS response、second messenger or transcription factor.
The bacterial second messenger, Cyclic-di-GMP, is produced from two molecules of GTP by diguanylate cyclase enzymes (DGC). As previous report, the cyclic-di-GMP cause the biofilm formation, motility of flagellum and virulence protein expression. The bacterial cyclic AMP receptor protein (CRP) that binds cAMP could act as transcription factor which mediates transcription activation related to the flagellum synthesis and toxin production. Strikingly, it was found that the dgc or crp mutants do not expressed bacteriocin in Pcc.
In this study, the crp mutant was acquired from homologues recombination method. After deduction of amino acid sequence, the Pcc homology of crp was similar to the other strain(100%). In RNA level, the crp mutant was absent in the expression of bacteriocin gene. Subsequently, the bacteriocin assay showed that bacteriocin would not express in the crp mutants. CRP function by binding, in the presence cAMP, to specific DNA site in or near target promoter and enhancing the ability of RNA polymerase to bind and initiate transcription. It suggested that CRP could be a regulator promoting the production of bacteriocin. As shown herein, the second messenger producer DGC and transcription factor CRP play an important role in bacteriocin expression.


中文摘要…………………………………………………………………………..i
英文摘要…………………………………………………………………………..ii
目次………………………………………………………………………………..iii
表目次……………………………………………………………………………..v
圖目次……………………………………………………………………………..vi
縮寫表……………………………………………………………………………..vii
正文
第一章 序論……………………………………………………………………….1
第二章 材料與藥品……………………………………………………………….5
第三章 實驗方法………………………………………………………………….7
一. 染色體DNA的分離……………………………………………………...7
二. 聚合酶鏈鎖反應……………………………………………………..........7
三. DNA回收…………………………………………………………………7
四. TA-cloning…………………………………………………………………8
五. 勝任細胞 (Competent cell) 製備與轉型作用 (Transformation) ………..8
六. 小量質體的分離…………………………………………………………...8
七. 電穿孔轉型作用 (Electroporation)………………………………………..8
八. 探針 (Probe) 之製備……………………………………………………....9
九. 南方轉漬法 (Southern blotting)…………………………………………...9
十. Thermal asymmetric interlaced PCR (TAIL-PCR)………………………....9
十一. RNA 的分離………………………………………………………..10
十二. 反轉錄聚合酶鏈反應 (Reverse transcription PCR)……………….10
十三. 載體的去磷酸化 (CIAP)…………………………………………...11
第四章 結果………………………………………………………………………..12
一. crp 基因之選殖……………………………………………………………12
二. crp 突變株之構築…………………………………………………………12
三. 基因剔除 (knock-out)……………………………………………………...12
四. crp 突變株初步篩選......................................................................................12
五. crp 突變株確認…………………………………………………………….13
六. TAIL PCR 實驗…………………………………………………………….13
七. crp 之 ORF (open reading frame)………………………………………….14
八. crp 基因 DNA 定序與分析…………………….......................................14
九. Reverse transcriptase PCR (RT-PCR) 實驗…………………………………15
十. crp 基因表現質體的構築與細菌素恢復實驗…………………………...15
第五章 討論………………………………………………………………………......17
參考文獻………………………………………………………………………………44


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