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研究生:張楷煒
研究生(外文):Kai-Wei Chang
論文名稱:十字花科黑腐病菌UDP-葡萄糖去氫基因及其鄰近有關醣類代謝基因之分析
論文名稱(外文):Characterization of udgH and the Flanking Genes Involved in Carbohydrate Metabolism in Xanthomonas campestris pv. campestris
指導教授:曾義雄曾義雄引用關係
指導教授(外文):Yi—Hsiung Tseng
學位類別:碩士
校院名稱:國立中興大學
系所名稱:分子生物學研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2001
畢業學年度:89
語文別:中文
論文頁數:118
中文關鍵詞:十字花科黑腐病菌UDP-葡萄糖去氫基因
外文關鍵詞:Xanthomonas campestris pv. campestrisudgH
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十字花科黑腐病菌 (Xanthomonas campestris pv. campestris) 為桿狀有單極單鞭毛革蘭氏陰性的好氣性植物病原菌, 核酸所含鹼基 G + C 量比率高達 64%。 X. campestris pv. campestris 所分泌的黃原膠 (xanthan gum), 與本菌之致病能力有關。 黃原膠之合成包括許多步驟, 本實驗室近來也找到與其生合成有關的基因: 為 eps1、 eps2、 eps3、 eps4、 eps5、 eps6、 eps7 及 eps8。 其中 eps3 帶有 UDP-glucose dehydrogenase 基因 (udgH)。
為了了解 X. campestris pv. campestris 17 的 UDP-glucose dehydrogenase 基因 (udgH) 基因上下游是否有其他相關基因, 首先利用 chromosome walking 的方式, 選殖出其鄰近 DNA 片段, 再加以定序及分析各基因突變對醣類代謝與致病性的影響, 以及啟動子活性等。 chromosome walking 之後, 得到以 pOK12 為載體而帶有udgH 基因上下游 DNA 片段的質體 pCKW-1 與 pCKW-2。 經定序分析, 找到九個 ORF, 分別為: gntR 基因, nt 441 到 nt 79; glutathione peroxidase 基因 (gpxX), nt 801 到 nt 1,385; peptidyl-prolyl cis-trans isomerase 基因 (ppiA), nt 1,546 到 nt 2,439; UDP-glucose dehydrogenase 基因 (udgH), nt 2,464 到 nt 3,885; ORF225, nt 4,050 到 nt 4,727; ORF278, nt 4,981 到 nt 5,817; PckR 基因 (pckR), nt 6,134 到 nt 7,171; glucose/galactose transporter 基因 (gluP), nt 7,185 到 nt 8,495; fructokinase 基因 (pfkB), nt 8,492 到 nt 9,475。以 insertion mutation 方法分別構築突變株, 進行醣類代謝、 啟動子活性及致病性分析。 udgH 基因之缺失會導致菌落外觀之改變, 較野生株乾而無濕黏狀, 並失去致病力; pfkB 基因之缺失會導致菌體生長較快, 其致病力亦增加; 而在 ppiA 基因、 pckR 基因及 gluP 基因之缺失上, 並不會影響菌落外觀與致病能力, 於生長方面亦影響不大。
在醣類代謝方面, 各突變株與野生株並無顯著的差異, 而各菌株對醣利用之情形均為 sucrose 較 glucose 好, 依次則為 fructose、 maltose、 xylose、 galactose 及 lactose。 在啟動子活性方面 udgH 基因之啟動子活性較其他四個基因強, 而菌株生長狀況較佳者各該基因啟動子活性亦表現較高。

Xanthomonas campestris pv. campestris is a gram-negative, rod-shaped and monotrichously flagellated plant pathogenic bacterium. The chromosome of X. campestris pv. campestris has a G + C content of 64%. It produces copious amounts of xanthan gum which has been implicated to associate with the virulence of the bacterium. Biosynthesis of xanthan gum involves multiple steps and a multitude of enzymes. Rescently, our laboratory has localized the genes involved in xanthan synthesis at eight loci on the X. campestris pv. campestris 17 (Xc17) chromosome map. Thes loci are designated as eps1, eps2, eps3, eps4, eps5, eps6, eps7 and eps8, respectively. Among them, eps3 contains the gene encoding UDP-glucose dehydrogenase gene (udgH). In this study, I employed a chromosome walking strategy to clone the DNA fragments flanking the udgH gene from the (Xc17) chromosome. Two clones, pCKW-1 and pCKW-2, carrying the upstream and the downstream DNA fragment, respectively, were thus obtained. Sequence analysis revealed 10,200 bp containing nine ORFs : gntR gene, nt 441 to nt 79; glutathione peroxidase gene (gpxX), nt 801 to nt 1,385; peptidyl-prolyl cis-trans isomerase (PPIase) gene (ppiA), nt 1,546 to nt 2,439; UDP-glucose dehydrogenase gene (udgH); nt 2,464 to nt 3,885; ORF225, nt 4,050 to nt 4,727; ORF278, nt 4,981 to nt 5,817; PckR gene (pckR), nt 6,134 to nt 7,171; glucose/galactose transporter gene (gluP), nt 7,185 to nt 8,495; fructokinase gene (pfkB), nt 8,492 to nt 9,475. Mutants of these genes were constructed by insertional mutagenesis. Mutation of udgH gene resulted in a non-mucoid phenotype and a loss of pathogenicity; the mutation of pfkB gene caused higher rates of growth and increased virulence; the mutation in ppiA, pckR and gluP gene did not affect the colony morphology, pathogenicity and growth rate.
No differences were found in sugar utilization between the mutant and the wild-type strains. Sucrose can support better growth than dose glucose, and the oder of efficiency in sugar utilization by X campestris pv. campestris is fructose> maltose> xylose> galactose> lactose. The udgH gene promoter is stronger than that of the other four genes, and levels of promoter activity are always higher when cells are cultured rich medium which can support higher growth rates.

中文摘要i
英文摘要ii
壹、前言1
貳、材料4
參、實驗方法6
一、細菌之培養6
二、噬菌體 fLf 與 fL7 之增殖 (amplification)6
三、DNA 之製備及操作6
四、DNA之操作與質體之構築 (cloning)7
五、凝膠體電泳製備與分析 (gel electrophoresis)8
六、DNA片段回收8
七、DNA定序反應(DNA sequencing)9
八、重組 DNA 之轉形作用 (transformation)10
九、DNA 之快速篩檢法11
十、南方墨點雜交法 (Southern blot hybridization)11
十一、溶菌斑測試 (spot test)12
十二、生長曲線之測定12
十三、病原性測試13
十四、啟動子之活性分析 (C23O 活性分析)13
十五、Xanthan polysaccharide (xanthan gum)含量測試13
十六、Computer analysis13
肆、結果與討論14
一、UDP-glucose dehydrogenase基因上、下游 DNA 定序分析14
二、SD701 片段鄰近區域的定序分析14
三、DNA 序列分析17
四、蛋白質電腦分析20
五、不同菌種之基因組織 (genome organization)22
六、突變株之分析23
七、野生株 Xc17 與各突變株在不同培養基之生長情形28
八、基因的表現分析31
九、病原性測試分析32
伍、參考文獻33
陸、圖39
柒、表101
捌、DNA sequence and amino acid sequence114

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