臺灣博碩士論文加值系統

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 量為 60%以上。 X. campestris pv. campestris 分泌大量的胞外多醣，使得菌落外觀呈現黃色黏濕狀，與本菌之致病能力有關。 在工業上稱此胞外多醣為 xanthan gum，可利用於食品加工，石油鑽探等農業與工業上。 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。 在 Escherichia coli (E. coli) 的研究中，lon基因之突變會導致 E. coli 的 capsular polysaccharide 增加，而使得外觀呈現濕粘狀，另外對紫外光耐受性降低，在受射線如紫外光、X 光照射後，細胞會因抑制細胞分裂蛋白累積，無法形成隔壁，使得細胞長度增加，呈現極長的細胞外觀。
為了想了解 X. campestris pv. campestris 的 lon 基因否有也影響胞外多醣的合成， 首先利用帶有 lon 基因起始點之 DNA 片段利用 homologous recombination 的方式，插入野生株 Xc17 的 lon 基因中，再以酵素切割及自我黏合，選殖出帶完整 lon基因的 DNA 片段，並進行定序及分析基因突變對胞外多醣、致病性與紫外光耐受性的影響。 經過上述方法，得到以一個以 pOK12 為載體而帶有 lon基因全長片段的質體 pOKRV。 經定序分析， 預測有兩個 ORF， 分別為：(1) nt 721 到 nt 3189 主導一包含 823 個氨基酸，分子量為 90,631.27 的蛋白質產物，名為 ATP-dependent protease La，基因名稱為 lon。 此基因序列與 E. coli與 X. fastidiosa 的 lon gene 有 65% 以上的相似性，並帶有 lon 基因所應有的 ATP/GTP binding motif 與ATP-dependent serine protease active site。 (2) 由nt 3307 到 nt 3680 可主導一包含 90 個氨基酸，分子量為 9327.13 的蛋白質產物，名為 Histone-like DNA binding protein，此基因名稱為 hupb。 此基因序列與 X. fastidiosa 與 E. coli 的 hupb gene 有 70% 以上的同質性，並帶有 hupb gene 所具有的 Bacterial histone-like DNA-binding proteins signature motif。 利用 double cross 的方法構築 lon 基因的突變株，進行胞外多醣、SOS response 及致病性分析。 lon 基因突變株，其菌落外觀並無改變，其紫外光感受性並無變化，於生長方面亦無影響。

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 more then 60%. 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. Recently, our laboratory has localized the genes involved in xanthan synthesis at eight loci on the X. campestris pv. campestris 17 (Xc17) chromosome map. In E. coli, lon gene mutations made cell mucoid, UV sensitive and long form cell.
In this study, I employed a homologous recombination strategy to clone the of lon gene from the Xc17 chromosome . pOKRV carrying the DNA fragment of complete lon gene as thus obtained. Sequence analysis revealed 3955 bp containing two ORFs. The lon gene, nt 721 to nt 3189; encodes a protein that has 823 amino acids with a molecule weight of 90,631.27. The Lon protease deduced from the Xc17 possesses 65% similarity to that of E. coli and X. fastidiosa. Two motifs include the gene are ATP/GTP binding motif and ATP-dependent serine protease active site. The hupb, nt 3307 to 3680; encodes a protein 90 amino acids with a molecule weight of 9327.13, whose product is named Histone-like DNA binding protein. The sequence similarity of hupb is more then 65% with E. coli and P. fluorescens. A motif, the bacterial histone-like DNA-binding proteins signature motif, is present in the deduced HupB protein. Mutants of lon were constructed by insertional mutagenesis. No differences were found in colony morphology, pathogenicity, growth rate and UV-sensitivity between the mutant and the wild type cells

中文摘要
英文摘要
壹、前言
貳、材料參、實驗方法
一、細菌之培養
二、噬菌體 fLf 與 fL7 之增殖 (amplification)
三、小量質體 DNA、 fLf RF DNA之製備
四、染色體 DNA 之抽取
五、噬菌體單股 DNA 之抽取8六、DNA之操作與質體之構築 (cloning)
七、凝膠體電泳製備與分析 (gel electrophoresis)
八、DNA片段回收
九、DNA定序反應(DNA sequencing)
十、重組 DNA 之轉形作用 (transformation)
十一、轉形菌落之鑑定DNA 之快速篩檢法
十二、南方墨點雜交法 (Southern blot hybridization)
十三、溶菌斑測試 (spot test)
十四、生長曲線之測定
十五、病原性測試
十六、exo-polysaccharide (EPS)含量測
十七、紫外光敏感性測試
十八、聚合?A連鎖反應(Polymerase Chain Reaction, PCR)
十九、Computer analysis
肆、結果與討論
一、lon 基因上、下游DNA 定序分析
二、DNA 序列分析
三、蛋白質電腦分析
五、不同菌種之基因組織 (genome organization)
六、突變株之分析
七、野生株 Xc17 與各突變株在不同培養基之生長情形
八、病原性測試分析
伍、圖
陸、表
柒、附錄
DNA sequence
Sequence alignment by ClustalX1.81
捌、參考資料

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