臺灣博碩士論文加值系統

Pectobacterium carotovorum subsp. carotovorum為腸道菌科的革蘭氏陰性菌，此菌株偏好寄生於植物，由於會產生果膠酶，因此常造成十字花科作物根莖部腐爛之疾病，導致經濟作物損傷。至目前為止，尚沒有一個有效的方法防治此菌株所產生的疾病，農用化學物質是目前經常採用的抗菌藥劑，但其效用並沒有針對性，可能造成使用地區無法避免的環境污染。
細菌素是一種由細菌分泌的胞外蛋白性化合物，此物質具有抗生活性，能夠抑制或殺死與生產菌株有相近親緣關係的菌種，由於細菌素所抑制菌株的種類相當有限，且有高度特異性，並對環境低毒害等優點，可以拿來製作成生物性抗菌試劑，有效防疫Pectobacterium所造成的經濟損失。
鞭毛系統是一超過20種不同蛋白所組成的旋轉裝置，目的是使細胞在液體中移動。在本研究室的先前研究中，發現低分子量的細菌素Carocin，其主要是經由第三類分泌系統中的鞭毛系統分泌至細胞外。此外，從先前的研究中得知，在Pcc菌種內的Carocin表現相關機制裡，DGC與CRP等調控因子扮演了重要的角色。
在本研究中，我們藉由RT-PCR方法分析H-rif-8-6及3F-3菌株的多株突變株，當調控因子crp基因被阻斷，其會連動使運輸胞器的結構蛋白失去正常運作的能力，證實兩者會影響相互間的正常表現，從而發現彼此間的交互關係。

Pectobacterium carotovorum subsp. carotovorum is a Gram-negative, phytopara-sitic enterobacterium responsible for the soft-rot disease of the family Brassicaceae by producing extracellular pectinases. It causes greatly economic losses every year. So far, there is no efficient method, either chemical or otherwise, which has been found to con-trol this worldwide disease. Agrochemicals are generally used against this disease, but they cause the environmental pollutions.
Bacteriocins are antimicrobial and extracellular toxic proteins, which are produced by some bacteria and kill closely related bacteria. However, these proteinaceous toxins are highly specific and low environmental contaminations. They can be exploited as an-timicrobial agents and are efficiently preventing the pathogen that causes the economic damage.
The flagellum secretion system consisting of more than twenty different proteins is a rotary device for motility in liquid. From our previous studies, low-molecular-weight bacteriocin, Carocin, had already been confirmed and reported that it was exported to extracellular through the bacterial flagellum type III secretion system. In addition, from previous research, the regulation factors, DGC and CRP, play an important role in ex-pression mechanism of Carocin in Pcc.
In this study, we use RT-PCR method to analysis the mutants of strains, H-rif-8-6 and 3F-3. When the regulation factor, the crp gene, are interrupted, the flagellum stru-ture proteins can not be express normally. They are identified to be influenced each oth-ers’ normal expression. And then, we can know their cooperation mechanism.

名詞縮寫表 i
摘要 ii
Abstrate iii
目次 iv
表目次 vi
圖目次 vii

第一章 序論 1
一、 細菌性軟腐病 (Bacterial soft-rot disease) 1
二、 細菌素 (Bacteriocin) 2
三、 細菌素的命名與分類 3
四、 分泌系統 3
五、 第三類分泌系統 (Type III secretion system, T3SS) 4
六、 細菌素基因的調控機制 5
七、 研究目的 6
第二章 材料與藥品 12
一、 實驗使用之菌株、質體及引子 12
二、 各類試劑 15
第三章 實驗方法 21
一、 Genomic DNA製備 21
二、 PCR 21
三、 洋菜膠體配製與DNA電泳分析 22
四、 DNA純化回收 22
五、 TA-cloning 22
六、 Competent cell製備 (Hanahan法) 23
七、 Transformation (Hanahan法) 23
八、 小量plasmid DNA製備 24
九、 大量plasmid DNA製備 24
十、 DNA digestion with restriction endonuclease 25
十一、 去蛋白處理 25
十二、 DNA ligation 25
十三、 SLIM 26
十四、 蛋白大量誘導表現 26
十五、 SDS-PAGE製備與蛋白質電泳分析 27
十六、 RNA製備 27
十七、 甲醛洋菜膠體配製與RNA電泳分析 28
十八、 RT-PCR 29
第四章 結果 30
一、 構築ysaT基因重組載體 30
二、 構築fliG基因重組載體 31
三、 誘導融合蛋白大量表現 31
四、 ysaT基因的mRNA表現 32
五、 Pcc突變株的RNA轉錄表現 32
第五章 討論 34
參考文獻 63

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