臺灣博碩士論文加值系統

Bacillus subtilis DB1001是一對溫度敏感的σA突變株，其σA蛋白在與啟動子 -10 DNA的結合區域上，具有Ala-197-Pro的取代突變。不同於野生型B. subtilis DB2的短桿狀，B. subtilis DB1001在37℃下具有長絲狀的生長形態。雖然B. subtilis DB1001在生長過程中，細胞間仍會形成隔膜，但是隔膜中細胞壁的內凹，卻非常緩慢。只有在營養生長末期，它才有明顯細胞分裂的現象，推測此可能與B. subtilis DB1001細胞內分解細胞壁的自溶素的延遲生產有關。由於只有調控自溶素表現的sigD基因，能夠補救B. subtilis DB1001的長絲狀生長缺失，因此我們相信B. subtilis DB1001細胞分裂延遲的原因，可能和其細胞內σD蛋白的含量或σD蛋白的轉錄活性不足有關。本研究乃在澄清上述兩種可能性，並希望藉此瞭解B. subtilis細胞中sigD基因表現之調控。
利用西方墨點法分析，探討B. subtilis DB1001中σD及FlgM蛋白的含量，發現它的σD蛋白的含量似乎較野生型者來的少；FlgM蛋白含量在兩者之間，則沒有明顯的差異。利用β-galactosidase activity分析控制sigD operon的各個啟動子 (PD-3、PA、PsigD) 活性，發現B. subtilis DB1001的雙重啟動子（tandem promoters；PD-3及PA) 活性遠較野生型者低，但是各別的PD-3及PA啟動子活性，則差異不大。這種結果顯示B. subtilis DB1001細胞分裂延遲的原因，應該是sigD mRNA表現量不足，造成細胞內σD蛋白含量不足所致。另外，B. subtilis sigD的表現，可能會受到一種特殊的不同σ-RNA聚合酶之轉錄協同作用所調控。

Bacillus subtilis DB1001 is a temperature-sensitive (Ts) sigA mutant with an Ala-197-Pro substitution in the promoter -10 binding region of the σA factor. Different from the small rod shape of the wild-type (Wt) sigA strain, B. subtilis DB2, the Ts sigA strain assumes a filamentous growth phenotype at 37°C. Although the septum between two sister cells of B. subtilis DB1001 generates normally during vegetative growth, invagination of septum was fairly slow. Not until stationary phase does the cell division become significant, suggesting that the filamentous phenotype of B. subtilis DB1001 is ascribed to the autolysis delay of cell wall. Since only sigD, which controls the expression of some of the atutlysin genes, can rescue the filamentous growth phenotype of B. subtilis DB1001, it is believed that the division delay of B. subtilis DB1001 is attributed to insufficient content or transcription activity of the σD. This study is aimed to distinguish between these two possibilities and to understand the regulation of B. subtilis sigD expression.
Western blot analyses of the contents of σD and FlgM in B. subtilis revealed that the content of σD in B. subtilis DB1001 is much lower than that found in the Wt sigA strain; however, there is no significant difference in the contents of FlgM between the two sigA strains. The β-galactosidase activity assays revealed that the activity of the tandem operon promoters (PD-3 and PA) of the sigD in B. subtilis DB2 is 10-fold higher than that found in B. subtilis DB1001. However, the activity difference became insignificant as the tandem promoters were separated. Taken together, these results indicated that the delay of B. subtilis DB1001 cell division must be due to the insufficient expression of sigD mRNA and thus the insufficient content of σD. More importantly, these results implicated that the expression of B. subtilis sigD operon is under the control of a novel mechanism which involves a synergistic effect of two different σ-RNA polymerase holoenzymes.

前言………………………………………………………………………1
材料與方法 …………………………………………………………… 8
一、試藥來源……………………………………………………………8
二、菌株、質體…………………………………………………………8
三、實驗方法……………………………………………………………8
1. pET21d-flgM質體之構築……………………………………………8
2. pT7-5-sigD質體之構築…………………………………………… 8
3. 野生型σD蛋白及FlgM蛋白之大量生產及抗體製備……………… 9
4. 不同生長時期中B. subtilis DB2、DB1001細胞內σA蛋白、σD蛋白及FlgM蛋白的含量比較………………………………………………9
5. 枯草桿菌sigD基因缺陷突變株之構築……………………………10
6. pCoiZA-PD-3、PA、PSigD、PD-3A、PD-3ex、PAex 質體之構築……………………………………………………………………… 11
7. 分析B. subtilis DB2、DB1001中控制sigD operon基因之起動子DNA表現的情形…………………………………………………………11
8. 野生株B. subtilis DB2及突變株B. subtilis DB1001 sigD mRNA表現之分析…………………………………………………………… 12
9. pCT24-PD-3、PA、PSigD、PD-3A、PD-3ex、PAex質體之構築 13
10. 蛋白質之定量…………………………………………………… 13
11. σ蛋白之重組RNA聚合酶的多循環體外轉錄活性（in vitro multiple cycle transcription activity）分析…………………13
結果 ……………………………………………………………………15
一、σD及FlgM蛋白之大量生產及抗體製備………………………… 15
二、不同生長時期中B. subtilis DB2、DB1001細胞內σA、σD及FlgM蛋白的含量比較 …………………………………………………… 15
三、sigD 破壞突變株之構築…………………………………………17
四、B. subtilis DB2、DB1001中控制sigD operon基因之起動子DNA表現之情形…………………………………………………………… 17
五、B. subtilis DB2及B. subtilis DB1001中 sigD mRNA含量之分析……………………………………………………………………… 19
六、不同RNA聚合酶轉錄協同作用之分析……………………………19
討論及結論 ……………………………………………………………20
附圖及說明…………………………………………………………… 23
表格 ……………………………………………………………………39
參考文獻 ………………………………………………………………43

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