Erwinia. carotovora subsp. carotovora是一種會造成許多植物根莖部腐爛的病原菌。大多作物常因感染此病原菌而失去經濟價值，但是到目前為止仍沒方法可以有效抑制此菌種造成的損害。由於此菌種會生產一至數種細菌素以抑制其他親屬相近的菌種生長，而被當作是一種有效的微生物農藥使用，但其細菌素相關研究仍然十分不足。
TH22-6 是一株被transposon Tn5 阻斷其低分子量細菌素生產相關基因的Erwinia. carotovora subsp. carotovora 野生株H-rif-8-6 的突變株。我們利用TAIL-PCR 的方法增幅並定序transposon Tn5 左右兩端向外延伸的DNA片段。此序列經過美國國家衛生研究院醫學圖書館的資料庫(NCBI)比對胺基酸序列(BLAST)，結果發現在此DNA片段與第三類型分泌系統的蛋白胺基酸序列有相當高的同質性。因此我們認為被Tn5 阻斷的基因為一種類似第三類分泌系統蛋白的基因。根據此比對結果我們推測Erwinia. carotovora subsp. carotovora可能經由類似第三類分泌系統的機制運送低分子量細菌素。
第三類分泌系統主要功能為分泌由病原菌產生的毒性蛋白，目前文獻指出其作用對象為動物或植物細胞。有趣的是在本文，我們卻發現低分子量細菌素可藉由類似第三類型分泌系統的機制分泌並感染其他菌種。希望此結果能幫助我們更加了解Erwinia carotovora subsp. carotovora 產生的低分子量細菌素其分泌系統作用的機制。Erwinia. carotovora subsp. carotovora is a phytopathogenic bacterium responsible for the soft-rot disease of many plants. It will decompose the crops, but there is no efficiently approach to bate the damages caused by the bacterium. Low-molecular-weight bacteriocin is an antibacterial substance which could be produced by Erwinia. carotovora subsp. carotovora, and inhibit the related strain growth. The bacteriocin could be used as effectively biopesticides, but the realtived study is still not enough.
TH22-6 is a Transposon Tn5 insertional mutant strain from Erwinia. carotovora subsp. carotovora, it could not generate low-molecular-weight bacteriocin. We used TAIL-PCR method to amplify and analyze the DNA sequence of the insert end from transposon Tn5. The DNA sequence was compared with the BLAST of the National Center for Biotechnology Information server, then the result show that has high homology with the genes of secretary protein in bacterium, especially the type Ⅲ secretion system. According to the comparison, this result suggest the low-molecular- weight bacteriocin may be transport by the type Ⅲ-like secretion system.
The function of type Ⅲ secretion system is usually the infection to the animal or the plant cell. In this study, it is interesting to find the relationship between low-molecular-weight bacteriocin and the type Ⅲ-like secretion system. The result may helpfully let we know the mechanism of low-molecular-weight bacteriocin and secretion system in Erwinia. carotovora subsp. carotovora.目次
第一章 緒論 …………………………………….………………………….....1
第二章 利用Transposon Tn5 阻斷E.C.C. 的低分子量細菌素生產相關基因
一. 目的……………………………………………………………..….8
二. 材料與方法……………………………………………………..….8
三. 結果……………………………………………………………….10
第三章 利用TAIL-PCR 的方法增幅並定序被Transposon Tn5阻斷的基因
一. 目的…………………………………………………………….…12
二. 材料與方法……………………………………………………….12
三. 結果……………………………………………………………….15
第四章 Erwinia. carotovora subsp. carotovora 低分子量細菌素分泌蛋白基因(BSP)的選殖
一. 目的…………………………………………………………….…21
二. 材料與方法……………………………………………………….21
三. 結果……………………………………………………………….24
第五章 BSP蛋白基因表現質體的構築與低分子量細菌素恢復實驗
一. 目的…………………………………………………………….…32
二. 材料與方法……………………………………………………….32
三. 結果……………………………………………………………….34
第六章 結論 ………………………………………………………………... 44
附錄………………………………………………………………………………55
參考文獻…………………………………………………………………………6
