臺灣博碩士論文加值系統

真核生物利用細胞計畫性死亡 (programmed cell death, PCD) 機制控制細胞自殺與消滅受損或不需要的細胞。近年來發現在原核生物中亦有 PCD 機制，例如 Xanthomonas axonopodis pv. glycines (Xag) 的 rapid cell death (RCD) 現象。先前研究發現將 Xag 培養在 LB medium 中48小時後會有 RCD 現象，而在 starch medium 中則否。為探討造成 Xanthomonas RCD 現象之機制，本研究中首先比較 Xag 與 Xanthomonas campestris pv. campestris (Xcc) 在不同培養時期之生長曲線與存活率。Xag 有出現RCD 現象，Xc17則無。然後利用 EZ-Tn5™ <R6Kγori/KAN-2>Tnp Transposome™ kit 隨機突變 Xag基因體，期能找出導致 RCD 現象減弱或消失的相關基因。初步得到3個疑似 RCD 減弱的突變株，但進一步實驗發現其生長與存活情形與野生株相似。前人研究中發現在 LB中添加硫酸亞鐵會影響RCD現象，本研究發現培養基中超過一定濃度的硫酸亞鐵會抑制細菌生長；但低濃度之硫酸亞鐵會使 Xag 生長延遲與減緩RCD。依據 microarray 結果與研究分析，選擇與 catalase 活性及鐵離子吸收相關的 catB、fhuA、iroN 等基因進行突變，並探討其與 RCD 及病原性之相關性。結果顯示，XagcatB 與 XagiroN 之 RCD 現象與野生株相似。Xag 與 Xc17之 iroN 與 fhuA 基因產物可能與鐵離子吸收能力有關；然而在鐵離子缺乏的環境下，XagiroN、XagfhuA、Xc17iroN、Xc17fhuA突變株生長並不會受到影響。XagcatB突變株的 catalase 活性降低，但細菌的 catalase活性高低似乎與 RCD 無相關。測試catB、fhuA、iroN 等突變株的胞外酵素活性後，結果顯示突變株與野生株差異不明顯。進行Xag病原性測試時發現Xag 的 catB、mopB、fhuA突變株的病原性減弱。

Many eukaryotic organisms practice programmed cell death (PCD) to eliminate damaged or abandoned cells. Recently, some studies suggest that some prokaryotes also possess PCD mechanism. For an example, Xanthomonas axonopodis pv. glycines (Xag) undergoes rapid cell death (RCD) when grown in LB medium for 48 hr but not in starch medium. In order to study the mechanism of RCD, Xag and the RCD-deficient Xanthomonas campestris pv. campestris (Xcc) were compared and no significant differences in growth and survival rates were observed. Using random transposon EZ-Tn5 for mutagenesis, three Xag mutants that initially showed less or no RCD were isolated. However, consistent RCD was no longer observed in further experiments. In LB medium, FeSO4 at low concentration was found to slow down RCD of Xcg, while the cell growth was inhibited at high concentrations. This is in agreement with the result of a previous study showing that FeSO4 can reduce RCD. To further study the Xcg RCD, several mutants were isolated based on the microarray data. Analysis on these mutants revealed that 1) XagcatB, a catalase mutant, has a catalase level which was 50% of that in wild type, but no significant change in RCD was observed, 2) growth of XagiroN, XagfhuA, Xc17iroN, and Xc17fhuA was not affected by iron deficiency in the medium, although it has been shown that FhuA and IroN play some roles in ferric uptake in Xag and Xc, 3) levels of extracellular enzymes were not altered in mutants catB, fhuA, and iroN,and 4) Xag mutants catB, mopB, and fhuA exhibited reduced virulence in causing pustule disease in soybean.

摘要 I
Abstract II
縮寫字對照表 III
前言 1
一、 Xanthomonas 1
二、 真核細胞凋亡機制 1
三、 X. axonopodis pv. glycines 的 rapid cell death (RCD)現象 2
四、 鐵離子與細菌生長之探討 3
五、 研究目的 3
實驗材料 4
一、 菌種、實驗用質體 4
二、 藥品 4
三、 酵素 4
四、 引子 4
五、 緩衝液與培養基 4
實驗方法 6
一、 菌種之培養與保存 6
二、 存活菌數測試 6
三、 生長曲線測試 6
四、 質體 DNA 之抽取 (BioKit) 6
五、 洋菜膠電泳法 (agarose gel electrophoresis) 7
六、 聚合酶連鎖反應 (Polymerase Chain Reaction, PCR) 7
七、 膠體 DNA 之回收 (Gel/PCR DNA Fragments Extraction Kit) 7
八、 限制酶酵素切割 (restriction enzyme digestion) 8
九、 DNA 之黏接反應 (ligation reaction) 8
十、 大腸桿菌勝任細胞 (Competent cell) 製備 8
十一、 轉型作用 (transformation) 9
十二、 電穿孔實驗 (electroporation) 9
十三、 catalase 活性測試 10
十四、 胞外酵素活性測試 10
1. cellulase 測試： 10
2. amylase 測試： 10
3. protease 測試： 11
十五、 鐵離子螯合劑 2,2''- dipyridyl對於 Xag 與 Xcc 野生株與突變株生長影響 11
十六、 鐵離子吸收輔助因子 siderophore 分泌量測試 11
十七、 Xag 病原性測試方法之建立 11
十八、 Xc17之病原性測試 11
十九、 突變株之構築 12
1. XagcatB 突變株之構築 12
2. XagiroN突變株之構築 12
3. Xc17 fhuA突變株之構築 13
4. Xc17iroN突變株之構築 13
結果與討論 14
一、 Xanthomonas 的RCD 現象之探討 14
二、 以transposon 逢機突變策略，探討與 Xag RCD 相關之基因 14
三、 測試不同濃度的FeSO4‧6H2O對於 Xag 生長之效應 15
四、 Xag catB、iroN 與 fhuA 等突變株之特性 16
1. XagcatB、XagiroN、Xc17iroN、Xc17fhuA 突變株之構築 17
2. XagcatB、XagiroN 的 RCD 現象之探討 17
3. XagiroN、XagfhuA、Xc17fhuA、Xc17iroN 的鐵離子吸收測試 17
4. XagiroN、XagfhuA、Xc17fhuA、Xc17iroN 於缺鐵環境中的生長測試 18
5. XagcatB 及其他突變株之 catalase 活性測試 19
6. XagcatB、XagiroN、XagfhuA、Xc17iroN、Xc17fhuA等基因突變株的胞外酵素 endoglucanase、protease、amylase 活性之探討 20
五、 Xag 與其突變株之病原性測試 20
六、 Xc17 與其突變株之病原性測試 21
參考文獻 23
表一、本實驗所使用之菌種 26
表二、本實驗中所使用之質體 28
表三、Xag 與 Xc17 的 catalase 表現之差異 29
表四、Xag catalase 基因 30
表五、Xag 野生株與突變株之病原性 31
圖一、Xag12609 與 Xc17 生長曲線與存活率。 32
圖二、Xag12609 生長曲線與存活率。 33
圖三、不同濃度的 FeSO4 對於 Xag 生長之效應。 34
圖四、FeSO4 對於 LB pH值與混濁程度的影響。 35
圖六、XagcatB 生長曲線與存活率。 37
圖七、XagiroN 生長曲線與存活率。 38
圖八、鐵離子吸收輔助因子 siderophore 分泌量測試。 39
圖九、在培養基中加入 2,2’-dipyridyl 之Xag與突變株 fhuA 及 iroN 生長曲線。 40
圖十、在培養基中加入 2,2’-dipyridyl 之 Xc17 與突變株 fhuA 及iroN生長曲線。 41
圖十一、catalase 活性測定。 42
圖十二、Xag 及 Xc17 野生株與突變株的胞外酵素Amylase、Protease 及 Endoglucanase 之活性測試。 43
圖十三、Xag野生株與突變株之病原性測試。 44
圖十四、Xc17野生株及 fhuA與iroN 突變株之病原性測試 45
附錄一、本實驗所使用之引子 50
附錄二、培養基緩衝溶液 51

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