臺灣博碩士論文加值系統

Xanthomonas campestris pv. campestris (Xcc) 為十字花科黑腐病之病原菌。 此菌所產生的胞外酵素 (包括 alpha-澱粉酶、 纖維素酶、 蛋白酶和果膠酶) 及胞外黏多醣 (exopolysaccharide， 簡稱 EPS) 為致病力所需。 由目前研究已知， 這些致病因子 （virulence factors） 的產生會受到 Clp （cyclic AMP receptor protein-like protein） 及 rpf (regulation of pathogenicity factors) 基因群所調控。 其中， rpfF 為 DSF （diffusible signal factor，為訊號傳遞之小分子） 合成所必需， 目前研究顯示， DSF 會參與細胞間的訊息傳遞， 藉由傳遞過程間接調控致病因子的合成。 先前， 實驗室得到一 rpfF 突變株， P20H(rpfF::Gm)， 意外的發現， 以噬菌體 fLf 進行點測試法 (spot test) 時， 此突變株出現較野生株更澄清的溶菌斑。 為了探討 rpfF/DSF system 在線狀噬菌體 fLf 感染過程的作用， 利用跳躍子 (mini-Tn5) 致突變法來誘變 P20H(rpfF::Gm)， 得到 rpfF 與 clp 雙基因突變株 M67(rpfF::Gm;clp::Tc)。 此突變株的溶菌斑回復成與野生株相同的表現型， 於是進行一系列實驗， 以探討 RpfF/DSF system 與 Clp 如何共同參與 fLf 感染過程， 而得到以下的結果： 1) RpfF/DSF system 對於 fLf 自然感染及吸附作用具有負向調控的作用， 而 Clp 具有正向調控的作用； 2) rpfF/DSF system 對於 pilA1 的轉錄具有負向調控的作用， 而 Clp 具有正向調控的作用； 3) RpfF/DSF system 對 clp 轉錄作用似乎具有負向調控作用； 4) RpfF/DSF system 及 Clp 並不是透過調控

Xanthomonas campestris pv. campestris (Xcc) is the causal agent of black rot in cruciferous plants. Xcc produces a range of extracellular enzymes (including alpha-amylase, cellulases proteases and pectinases) and an exopolysaccharide (EPS), which are collectively essential for pathogenesis. Previous studies have shown that production of these virulence factors is positively regulated by Clp (cyclic AMP receptor protein–like protein) and rpf gene cluster (for regulation of pathogenicity factors). Synthesis of diffusible signal factor (DSF) is completely dependent on RpfF. As well as being implicated in the perception of DSF and signal transduction leading to virulence factors synthesis. P20H(rpfF::Gm), a mutant derived from Xcc P20H, which plaque is more clear than wild-type when filamentous phage (fLf) infected., transposon mutagenesis was used in this study with mini-Tn5 to induce P20H(rpfF::Gm), to obtain rpfF and clp double mutant, M67(rpfF::Gm;clp::Tc), while plaque return to as clear as wild-type when filamentous phage (fLf) infected. The experiments were designed for understanding how RpfF/DSF system and Clp influence the

目錄
中文摘要 1
英文摘要 3
縮寫表 4
第一章 前言 6
第二章 前人研究 11
第三章 材料與方法 19
一、 菌種、 噬菌體及質體 20
二、 試劑、 藥品及酵素 20
三、 DNA 的製備 20
1. 質體 DNA (plasmid DNA) 製備 20
2. 染色體 (chromosomal DNA) 製備 21
3. 洋菜凝膠電泳分析 (agarose gel electrophoresis) 21
4. 限制酶切割分析 (Restriction enzyme digestion) 22
5. DNA 片段回收 22
6. DNA 黏接試驗 (ligation) 23
四、 DNA 之轉型實驗(DNA transformation) 23
1. 大腸桿菌 (E. coli) 之轉型實驗 23
2. X. campestris pv. campestris 之轉形實驗 24
五、 噬菌體培養與操作 25
1. 線狀噬菌體

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