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研究生:楊孟學
研究生(外文):Meng-Hsueh Yang
論文名稱:枯草桿菌EzrA蛋白之截切對分裂細胞間細胞膜形成之影響
論文名稱(外文):The effects of EzrA truncation on membrane formation between the dividing cells
指導教授:張邦彥張邦彥引用關係
指導教授(外文):Ban-Yang Chang
學位類別:碩士
校院名稱:國立中興大學
系所名稱:生物化學研究所
學門:生命科學學門
學類:生物化學學類
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:45
中文關鍵詞:枯草桿菌分裂細胞細胞膜
外文關鍵詞:Bacillus subtilisEzrAdividing cellscell membrane
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在Bacillus subtilis細胞分裂週期中,一種類似真核生物tubulin的FtsZ蛋白會在細胞中央組裝成一環狀構造- Z-ring。接著,參與細胞分裂有關的蛋白會被招募至Z-ring處,其中有些蛋白對Z-ring的形成具有正向或負向的調控功能。EzrA為參與細胞分裂的蛋白之ㄧ,它會抑制FtsZ在細胞兩端不正常的聚合,讓Z-ring只在細胞中央形成;此為EzrA對FtsZ聚合的負向調控。根據研究,不論ezrA基因之刪除、EzrA C端QNR patch之刪除或突變、或降低細胞中EzrA含量,都會使細胞長度增加。因此,一般認為EzrA除了負向調控FtsZ單體之聚合外,也能正向調控與Z-ring特性有關的細胞分裂。本研究乃欲探討EzrA是透過其蛋白上那一個區域的胺基酸和FtsZ單體交互作用,發揮其抑制FtsZ聚合的負向調控功能、或透過那一個區域的胺基酸 (除了QNR patch) 和Z-ring交互作用,發揮其正向調節功能。本研究將ezrA進行一系列刪除突變,並進行突變菌體形態的觀察,發現除了協助EzrA座落至Z-ring的QNR patch (505-511) 外,位於EzrA C端511-562這段胺基酸序列,可能亦具有正向調控功能。另外,本研究中各種ezrA刪除突變都會使分裂細胞及染色體間之膜狀構造形成受到延遲。不過,這種延遲是否因為ezrA突變而所造成的Z-ring內縮缺失,則需進一步探討。

At initiation of cell division, FtsZ, a tubulin-like protein, will polymerize into a Z-ring structure at the mid-cell. Z-ring provides a cytoskeletal scaffold that recruits other division proteins and some of which possess positive or negative regulation for Z-ring formation. EzrA is one of the division proteins and has a negative role for FtsZ polymerization; it inhibits aberrant Z-rings from assembling in cell poles and thus cytokinesis is restricted to mid-cell. In addition to the negative role, several studies have been proposed that EzrA also has a positive role in cytokinesis. Those studies found that either the null mutation or the conserved QNR patch mutation in ezrA or deficiency in EzrA, all these mutations make the cell length of mutant cells longer than that of wild type. According to the above evidences, EzrA is thought that it could maintain the dynamic nature of medial Z-ring, thereby renders Z-ring sensitive to coordinate between cell growth and cell division. The aim of this study is to understand which amino acid residues of EzrA are able to interact with FtsZ, to either inhibit its polymerization or to maintain its assembly dynamics with Z-ring. We make use of a serial EzrA deletion mutation for analysis and observe the phenotype of these mutants. We discovered that 52 amino acid residues (511-562) on C terminal of EzrA might have regulated for the dynamic nature of Z-ring except known QNR patch (505-511). Besides, all ezrA mutations results in delay of membrane formation between the dividing cells and chromosomes. However, where this delay is a result of defective Z-ring constriction by ezrA mutation remains to be answered.

摘要 i
Abstract ii
前言 1
材料與方法 7
一、試藥來源 7
二、菌株、質體 7
三、實驗方法 7
1. Truncated ezrA基因之構築 7
2. pMS質體之構築 7
3. pMS-X質體之構築 8
4. 嵌入型菌株DB2X之構築 8
5. 表現EzrA蛋白載體之構築 8
6. 嵌入型菌株DB2X/pEAX之構築 9
7. 嵌入型菌株DB2X、DB2X/pEAX、ezrA啟動子遭破壞突變株DB2003與野生型菌株DB2菌體形態差異之分析 9
8.嵌入型菌株DB2X、DB2X/pEAX內EzrA蛋白含量之分析 10
9. 嵌入刪除突變型ezrA對菌株細胞膜內凹之影響 11
結果 12
一、嵌入刪除突變型ezrA對B. subtilis細胞長度之影響 12
二、嵌入型菌株與野生型菌株內EzrA蛋白含量之分析 13
三、嵌入型菌株內EzrA蛋白含量之分析 14
四、嵌入型菌株內EzrA蛋白增加對菌體長度之影響 14
五、嵌入刪除突變型ezrA對分裂細胞間的細胞膜形成之影響 15
討論 17
附圖 19
圖一、一系列EzrA刪除區域 19
圖二、pMS質體之構築 20
圖三、pMS-X質體之構築 21
圖四、嵌入型菌株DB2X之構築 22
圖五、嵌入刪除突變型ezrA對B. subtilis細胞長度之影響 23
圖六、野生型菌株DB2與各嵌入型菌株DB2X在指數生長中期與生長靜止期不同長度範圍之分布 24
圖七、嵌入型菌株與野生型菌株內EzrA蛋白含量之分析 25
圖八、pEA-X質體之構築 26
圖九、嵌入型菌株內EzrA蛋白含量之分析 27
圖十、嵌入型菌株內EzrA蛋白增加對菌體長度之影響 28
圖十二、嵌入刪除突變型ezrA對分裂細胞間的細胞膜形成之影響 30
表格 31
表一、野生型菌株DB2與各嵌入型菌株DB2X在指數生長中期與生長靜止期不同長度範圍之分布 31
表二、嵌入型菌株DB2X、DB2X/pEAX在指數生長中期不同長度範圍之分布 33
表三、菌株 34
表四、質體 35
表五、Primers used in this study 36
附錄 38
附錄一、Bacillus subtilis 細胞分裂周期 38
參考文獻 40



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