臺灣博碩士論文加值系統

Xanthomonas campestris pv. campestris (Xcc)為革蘭氏陰性嗜氧菌，感染十字花科作物造成十字花科黑腐病。細菌細胞內表現 polyphosphate kinase ( PPK ) 的ppk基因，可調控細菌細胞內的 inorganic polyphosphate（polyP）之形成。Poly P是由數十個到數百個磷酸根殘基，經由ATP水解，然後再藉由高能的磷酐鍵所結合成的一鏈狀聚合物。 在許多的微生物、菌類、植物及動物細胞中都可發現poly P的存在。目前所知，poly P在細菌細胞之生長期間的生理上適應及對環境的抗性是必需的。
在本實驗中為了探討 ppk基因在Xcc中，對鞭毛的活動、致病因子的致病性、生長的情形、以及對植物致病性的胞外酵素在Xcc中所扮演的角色。利用insertional mutation的方法，將 ppk 基因突變為無效基因，則將造成Xcc的致病性及病徵減緩。此外 在胞外多醣 ( EPS ) 及鞭毛運動性 (flagellum motility ) 中，野生株與突變株有些差異但並不顯著。Xc17中ppk基因缺失下對環境的抗性方面，在酸性環境中生長的情形，野生株與突變株在pH5時都能正常生長，但是都無法生長於低於pH4的環境。而在營養較豐富的環境中培養，野生株與突變株的生長並無差別，但在較貧乏的環境中培養，突變株的stationary phase則需要較長的時間適應。此外 野生株與突變株在活菌數的方面，在兩者生長到stationary phase 後所做的測試，也沒有明顯的不同。由此結果來推測可能是Xcc的基因體中，並沒有rpoS相似功能的基因，而使得突變株在較貧乏的環境中需要較長的時間來適應培養環境的改變。

Xanthomonas campestris pv. campestris (Xcc) is an important plant pathogen which causes black rot disease in cruciferous plants. The ppk gene encodes a polyphosphate kinase (PPK) responsible for the synthesis of inorganic polyphosphate (poly P). Poly P is a polymer of tens to hundreds of orthophosphate (Pi) residues linked by high-energy phosphoanhydride bonds , as in ATP. Poly P is ubiquitous , having been found in all organisms (archaea, bacteria, fungi, plants, insects, and animal cell). It is well known that poly P plays a role in the physiological adaptation of microbial cells during growth and development and is required for the adaptation to stress.
Xcc ppk mutant is motile and is normal in the secretion of virulence factors, such as exopolysaccharide and exocellular enzymes which are essential for Xcc to invade and establish infections in host cells , production. Neither ppk mutants nor their parental strains can survive after a prolonged incubation at stationary phase. Both ppk mutants and wild-type strains are sensitive to acidified media (pH below 4.0). No homolog of rpoS is present in Xcc genome , that might explain the fragility of Xcc in stringency. In rich medium, ppk mutants grow as rapid as their parental strains. Nevertheless, ppk mutants present a serious delay in growth in poor medium especially after a downshift of nutrition. This suggests that ppk mutants need more time to adapt to an environmental change.

英文摘要 3
中文摘要 4
縮寫字對照表 5
壹、前言 8
貳、材料 15
参、實驗方法 19
ㄧ、細菌之培養與保存 19
二、點測試法 (Spot test) 19
三、DNA的製備 19
四、聚合酶連鎖反應 (Polymerase Chain Reaction, PCR) 21
五、洋菜膠體電泳分析 (agarose gel electrophoresis) 21
六、質體之選殖 21
七、轉形作用 23
八、致病性的測試 24
九、生長曲線的測試 25
十、胞外多糖量之測定 26
十ㄧ、菌體蛋白質量之測試 26
十二、胞外酵素之測定 27
十三、運動性 (Motility) 之測試 28
十四、活菌數 (Viability) 之測定 28
肆、結果 29
ㄧ、Xc17及P20H之ppk突變株的取得 29
二、野生株與突變株之特性分析 30
三、利用互補株來分析ppk對生長之影響 37
伍、討論 39
陸、参考文獻 43
柒、表 48
捌、圖 54

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