臺灣博碩士論文加值系統

十字花科蔬菜黑腐病的病原菌 Xanthomonas campestris pv. campestris 為革蘭氏陰性菌， 在十字花科蔬菜的生產上造成嚴重損害。 此菌會分泌大量之胞外多醣 (exopolysaccharide， 簡稱 EPS)， 在工業上此胞外多醣稱為 xanthan gum， 當作穩定劑、 增稠劑、 乳化劑及懸浮劑等， 是目前廣泛使用的細菌膠。 因此， xanthan gum 的合成極具研究價值。
本實驗室研究 X. campestris pv. campestris 之 xanthan gum 合成基因多年， 曾以 Tn5(pfm)CmKm 跳躍子致變的方式選殖到一個 eps8 基因。 深入分析後得知， eps8 基因即為 clp 基因， 其所主導的蛋白產物與 Escherichia coli 的 cyclic AMP receptor protein (CRP) 相似， 而 CRP 被發現具有參與調控轉錄作用的功能。 此外， 曾有報導指出破壞 X. campestris 的 clp 基因將使其 cellulase 的產量減少 85%， 此現象與本實驗室的發現具有類似的情形， 這也暗示著 X. campestris 的 engXCA 基因可能具有 Clp-dependent promoter。 所以， 本研究便針對 engXCA 基因之轉錄作用遭受調控之情形加以探討。
為了解 Xc17 的 engXCA 基因之轉錄作用遭受調控之情形， 本研究利用 PCR 的方式擴增出推測之 engXCA 基因啟動子片段， 並將其選殖於含有 lacZ 報導基因之無啟動子泛寄主載體 pFY13-9。 將所得質體分別送入 Xc17 野生株及 clp 基因突變株 AU56E 之內， 藉由觀察 lacZ 報導基因之作用情形來了解 engXCA 基因啟動子所表現之活性。 分析結果得知， engXCA 基因啟動子在 clp 基因突變株 AU56E 之內幾乎無明顯活性， 顯示 Clp 確實調控 engXCA 基因轉錄作用。 另一方面， 藉由插入 xylE 報導基因之方式觀察 engXCA 基因之啟動子活性， 並利用 mini-Tn5-Tc 跳躍子針對 engXCA 基因突變株進行二次突變， 篩選 engXCA 基因啟動子強度下降之二次突變株。 因此， 所得到之突變株遭受致變位置之基因， 便可能具有調控 engXCA 基因轉錄作用之功能。 接著， 分析篩選到之兩突變株， 發現遭受破壞位置之基因分別與 Xylella fastidiosa 的 ppsA 基因 (86%) 及 gpdA 基因 (76%) 最為相似， 此兩基因所主導之蛋白產物分別為 phosphoenolpyruvate synthase 及 glycerol-3-phosphate dehydrogenase。 然而， 還需進行更深入之實驗才能了解此兩基因與 engXCA 基因之轉錄調控之間的關係。 另外， 病原性測試結果顯示 engXCA 突變株仍具病原性， 顯示 engXCA 基因所主導之 cellulase 蛋白在 Xc17 病原性之貢獻上並非扮演非常重要之角色； 而 gpdA 基因突變株則不具病原性， 推測可能是因為突變株之能量代謝過程受阻所導致。

The Gram-negative plant pathogenic Xanthomonas campestris pv. campestris causes black rot in crucifers, which results in large damage in agriculture. X. campestris. pv. campestris secretes a large mount of exopolysaccharide (EPS), xanthan gum, which is widely used as a stabilizing, viscosifying, emulsifying, thickening, and suspending agent. Therefore, it is of importance to study the biosynthesis of xanthan gum. Previously, by using Tn5(pfm)CmKm transposon mutagenesis, the gene in locus eps8 involved in xanthan synthesis and pathogenicity was cloned, sequenced and found to encode Clp, homologous to the global transcription factor CRP (cyclic AMP receptor protein) in Escherichia coli. It was reported that in clp mutant of X. campestris pv. campestris, cellulase production is decreases by 85% compared to its wild-type cells. Therefore, it was interesting to know whether transcription of engXCA gene is regulated by Clp.
In this study, two fragments in the upstream region of engXCA were amplified by PCR and cloned into the promoter-less promoter-probing vector pFY13-9 with lacZ as the reporter gene. The resultant plasmids were introduced into the wild type Xc17 and the clp mutant AU56E, followed by measuring the b-galactosidase activities. It was found that expression of the engXCA promoter was much lower in AU56E than Xc17, ranging from 22 to 40 versus 1793 to 4166 Miller Units. These data indicate that Clp is essential for the transcription of engXCA gene.
It was also interesting to understand whether proteins other than Clp are involved in the regulation of engXCA expression. To achieve this end, the promoter-less xylE reporter gene (encoding catechol 2,3-dioxygenase) was inserted into engXCA gene of Xc17 resulting in mutant HC117(engXCA::xylE-GmW), a strain deficient in cellulase and showing XylE activity. HC117(engXCA::xylE-GmW) was then used as the recipient for Mini-Tn5-Tc mutagenesis for the selection of mutants that exhibited lower or none XylE activity, with the rational that mutation of a gene required for the transcription of engXCA would turn off the xylE expression in HC117(engXCA::xylE-GmW). Six such mutants were obtained and two of them were further studied. Sequence analysis of the responsible DNA regions from these two mutants revealed that one has a mutated ppsA gene (coding for phosphoenolpyruvate synthase) and the other has a mutated gpdA gene (coding for glycerol-3-phosphate dehydrogenase). Although further studies are needed to assure the involvement of these genes in transcription of the engXCA, the pathogenicity tests have indicated that gpdA gene is required for pathogenicity.

中文摘要 1
英文摘要 2
縮寫字對照表 3
前言 5
實驗材料與方法 12
一. 菌種及質體。 12
二. 藥品、 酵素及放射性同位素。 12
三. 培養基。 12
1. 液體培養基。 12
2. 固體培養基。 12
四. 試劑與緩衝溶液。 13
1. 抗生素濃度。 13
2. 抽取質體試劑。 13
3. 抽取染色體之試劑。 13
4. 一般電泳試劑。 13
5. 南方轉漬法反應試劑。 13
6. 製備探針所需之試劑。 14
7. Catechol 2,3-dioxygenase (C23O) 試驗所需之試劑。 14
五. 細菌之培養與保存。 14
六. DNA 之製備。 14
1. 小量質體 DNA 之抽取。 14
2. 染色體 DNA 抽取法。 15
七. 洋菜膠體電泳分析 (Agarose gel electrophoresis)。 16
八. 質體之選殖 (Cloning)。 16
1. 限制的切割分析。 16
2. DNA 回收。 16
3. DNA 的補齊 (Fill-in)。 17
4. DNA 的黏接 (Ligation)。 17
九. 質體 DNA 之快速篩選法 (Rapid screen)。 17
十. 轉形作用 (Transformation)。 18
1. E. coli 之轉形。 18
2. X. campestris 之轉形。 18
十一. 聚合媒鏈鎖反應 (Polymerase Chain Reaction, PCR)。 19
十二. 南方轉漬法 (Southern hybridization)。 19
十三. Catechol 2,3-dioxygenase (C23O) test。 20
十四. 分泌型酵素 cellulase 活性測試。 21
十五. 接合作用 (Conjugation)。 21
十六. 點測試法 (Spot test)。 22
十七. 病原性測試 (Pathogenicity assay)。 22
十八. 生長曲線測定 (Growth curve)。 22
十九. b-galactosidase 之活性測試。 23
實驗結果 24
一. engXCA 基因啟動子在 Xc17 之 clp 基因突變株之表現情形。 24
1. engXCA 基因啟動子之選殖與質體之構築。 24
2. 轉型株之篩選。 24
3. engXCA 基因啟動子之表現情形。 25
二. Xc17 野生株之 engXCA 基因突變株之篩選。 26
1. 構築質體 pOCEL。 26
2. 以 pOCEL 質體為基礎， 構築質體 pOCELGT。 26
3. xylE-Gmr cartridge 之 insertional mutation。 27
4. 轉型株的南方轉漬法 (Southern blotting) 分析。 27
三. Xc17 之 engXCA 基因突變株 HC117(engXCA::xylE-GmW) 之特性分析。 28
1. Cellulase activity 定性分析。 28
2. Catechol 2,3-dioxygenase (C23O) activity 定性分析。 28
3. C23O activity 定量分析。 29
4. 生長曲線測試。 29
四. engXCA 突變株 HC117(engXCA::xylE-GmW) 的二次突變株篩選。 30
1. 進行 transposon mutagenesis 獲得二次突變株。 30
2. 二次突變株之初步篩選。 30
3. 二次突變株的次級篩選。 31
4. 二次突變株的三級篩選。 31
五. 遭受 mini-Tn5-Tc 跳躍子插壞位置之基因序列特性分析。 33
1. tetracycline 抗藥基因之重組交換。 33
2. 基因片段之選殖與分析。 33
3. 進行 sequence分析與網路比對。 34
六. ppsA 完整基因之選殖。 34
1. 以部份基因進行基因同質性重組交換。 34
2. 轉型株的 Southern blotting 分析。 35
3. 完整基因之選殖。 35
七. gpdA 突變株之篩選。 36
1. 質體 pOSP 之構築。 36
2. 基因間同質性重組交換。 36
3. 進行轉型株的 Southern blotting 分析。 36
八. 病原性測試 (Pathogenicity assay)。 37
討論 38
參考文獻 46
圖表 56

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