臺灣博碩士論文加值系統

Xanthomonas campestris pv. campestris（Xcc）為黑腐病菌屬革蘭氏陰性菌能感染十字花科植物。從文獻得知 FliF 蛋白可構成第三類型分泌系統，為控制 Xcc 鞭毛蛋白生成重要角色。
本實驗目的在於探討 Xcc 中 fliF 基因之功能性，利用同源重組技術在 fliF 基因中插入一段外來抗藥基因，破壞原本基因之功能，得到 fliF 突變株。實驗數據顯示 : fliF 基因突變後與 Xc17 相比：（1）fliF 運動性降低 ;（2）fliF 基因被破壞後不影響致病能力；（3）在西方點墨法的結果顯示 fliF 突變株蛋白表現與 fleQ、rpoN1、rpoN12 和 fliA 突變株等的蛋白表現無顯著差異而在與 fliC 突變株蛋白表現有顯著差異；（4）藉由酵母雙雜交實驗測試 rsbR、rsbS、rsbT間蛋白交互關西與 fliF 基因與其他 Xcc 基因是否有蛋白之間交互作用。其結果顯示 : FliF與 FlhF 有蛋白交互作用。

Xanthomonas campestris pv. campestris (Xcc) is a gram-negative bacterium. It can infect cruciferous plants and cause black rot disease. Xcc secrets various type of enzymes and polysaccharides which are essential for the bacterial virulence. Previous data from our lab have indicated that Xcc has a single polar flagellum. Biogenesis of the flagellum is regulated by the three-tier regulation of sigma factors RpoD, RpoN and FilA. A basal body composed of a flagellar type-III secretion system is essential for the secretion of the flagellin protein FliC. In other Gram-negative bacteria, FliF protein is the first element constituting the type III secretion system. Homolog of fliF was identified in the complete sequenced genome of Xcc. The aim of this study is to characterize the biological function of fliF gene in Xcc. Mutant of fliF was constructed by insertional mutagenesis. Phenotypes of the fliF mutant were analyzed by motility assay and pathogenic analysis. Regulatory function of fliF was analyzed by western blot. Interactions between FliF and other flagellar proteins were studied with the yeast-two hybrid system. The results demonstrated that 1) FliF is essential for the motility; 2) FliF is not involved in the pathogenicity; 3) FliF positively regulates the production of FliC, and 4) FliF interacts with FlhF protein.

目錄
縮寫字母對照表 1
Abstract 4
摘要 5
壹、前言 6
貳、文獻回顧 9
一、鞭毛細菌的型態與3D結構： 9
二、同型菌株之分析 10
1. Salmonella typhimurium 10
2. Pseudomonas aeruginosa 11
參、材料與方法 12
一、材料 12
（一）實驗相關材料來源 12
（二）藥品 12
（三）儀器 12
（四）培養基 13
二、方法 18
（一）細菌之培養與保存 18
（二）質體 DNA 之抽取 18
（三）聚合酶鎖反應（Polymerase chain reaction、PCR） 19
（四）洋菜膠體電泳分析（Agarose gel electrophoresis） 20
（五）質體的選殖（Cloning） 20
（六）勝任細胞之製備（Competent cell） 22
（七）轉形作用（Transformation） 22
（八）溶菌斑測試法（Spot test） 23
（九）運動性測試（Motility assay） 24
（十）複製噬菌體 (Amplification of bacteriophage) 24
（十一）噬菌體濃度測試 (Phage titer assay) 25
（十二）病原性測試（Pathogenecity） 25
（十三）蛋白質凝膠電泳分析（SDS-PAGE） 26
（十四）西方墨點法（Western blot） 27
（十五）酵母菌勝任細胞之製備（Competent cell） 28
（十六）酵母菌轉形作用（Yeast transformation） 28
（十七）酵母菌活性濾膜分析法（Colony-lift filter assay） 29
肆、結果討論 30
一、探討 Xcc 中 fliF 基因的特性分析 30
（一）fliF 基因之分析 30
（二）fliF 突變株之構築 30
（三）pBBD1 質體之構築 31
（四）pBBD2 質體之構築 31
二、FliF 突變株對噬菌體之敏感性測試 32
三、運動性分析 32
四、病原性分析 32
五、fliF 基因是否為其他鞭毛基因所需 32
（一）FleQ 蛋白偵測 33
（二）RpoN1 蛋白偵測 33
（三）RpoN2 蛋白偵測 33
（四）FliA 蛋白偵測 34
（五）FliC 蛋白偵測 34
六、蛋白與蛋白交互作用分析 35
（一）酵母菌雙雜交系統 (Yeast two-hybrid system,YTH) 35
（二）探討 Xcc 中 rsbR、rsbS 和 rsbT 基因的特性分析 36
（三）濾膜呈色分析 (Colony filter lift assay) 結果 : 40
七、總結 47
表1、實驗研究所使用之菌株 48
表2、實驗研究所使用之載體與質體 57
表3、實驗研究所使用之噬菌體 60
表4、Xc17 系列致病性測試感染次數共16次之統計分析 61
表5、利用西方點墨法偵測 Xcc 中部分鞭毛蛋白 FleQ、RpoN1、RpoN2、FliA和FliC與FliF和其他突變株間的關係 62
圖1、pBBD1 (AD) 之構築 63
圖2、fliF 突變株之構築 64
圖3、pBBD1 (DBD) 之構築 65
圖4、溶菌斑測試 (spot test) 66
圖5、pBBD2 (DBD) 之構築 67
圖6、pBBD2 (AD) 之構築 68
圖7、fliF突變株比較 Xc17 運動性測試 69
圖8、Xc17系列致病性測試 70
圖9、Western blot using anti-FleQ anti serum 71
圖10、Western blot using anti-RpoN1 anti serum 72
圖11、Western blot using anti-RpoN2 anti serum 73
圖12、Western blot using anti-FliA anti serum 74
圖13、Western blot using anti-FliC anti serum 75
圖14、pBBrsbR (DBD) 質體之構築 76
圖15、構築 rsbR、rsbS、rsbT、D1、D2 基因至 pAS2-1 與 pACT2 載體 77
圖16、pBBrsbR (AD) 質體之構築 78
圖17、pBBrsbS (DBD) 質體之構築 79
圖18、pBBrsbS (AD) 質體之構築 80
圖19、pBBrsbT (DBD) 之構築 81
圖20、pBBrsbT (AD) 之構築 82
圖21、酵母菌雙雜交濾膜分析 83
圖22、酵母菌雙雜交濾膜分析 84
圖23、酵母菌雙雜交濾膜分析 85
圖24、酵母菌雙雜交濾膜分析 86
圖25、酵母菌雙雜交濾膜分析 87
圖26、酵母菌雙雜交濾膜分析 88
圖27、酵母菌雙雜交濾膜分析 89
圖28、酵母菌雙雜交濾膜分析 90
圖29、酵母菌雙雜交濾膜分析 91
圖30、酵母菌雙雜交濾膜分析 92
圖31、酵母菌雙雜交濾膜分析 93
圖32、酵母菌雙雜交濾膜分析 94
圖33、酵母菌雙雜交濾膜分析 95
圖34、酵母菌雙雜交濾膜分析 96
圖35、酵母菌雙雜交濾膜分析 97
圖36、酵母菌雙雜交濾膜分析 98
圖37、Xcc 鞭毛合成構照圖 99
圖38、鞭毛調控關係圖 100
附錄一、實驗研究所設計之引子 101
附錄二、胺基酸縮寫表 102
柒、參考文獻 103

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