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標題: | 線狀噬菌體DNA嵌入宿主染色體的研究 STUDIES ON THE INSERTION OF FILAMENTOUS PHAGE DNA INTO THE HOST CHROMOSOME |
作者: | 白美蜀 |
出版年 : | 1989 |
學位: | 碩士 |
摘要: | 根據本實驗室研究發現,在線狀噬菌體Xf的生活史中,會將基因體嵌入宿主水稻白葉枯病病原菌的染色體上。在Xf基因體中,與Xf嵌入有關的區域,已被選殖出來稱為Ins7;在水稻白葉枯病病原菌染色體上,與Ins7 DNA具有同質性的部分,也已被選殖出來,稱為Xo12。核酸序列分析的結果顯示Ins7與Xo12有274bp的相同序列。此外,在Ins7 DNA中的15bp序列5'-TATACATTATGCGAA-3',與Cf16-v1嵌入宿主(柑橘潰瘍病病原菌)染色體所需的核心序列完全相同。但是在Xo12中卻無此序列。為了瞭解Xf嵌入宿主染色體是經由15bp或是274bp,因此將迷你噬菌體f42(含有整個Ins7 DNA,亦即包括15bp的核心序列)與pUC18-Cm黏接,構築成f42-pUC18-Cm,作嵌入分析。又因為水稻白葉枯病病原菌有限制系統,操作不易,所以將f42-pUC18-Cm送入與其相近而沒有限制系統的柑橘潰瘍病病原菌XW47中。實驗結果顯示f42-pUC18-Cm嵌入XW47染色體上。由於XW47不含該274bp,故嵌入應是以此15bp為仲介。利用電腦分析Xo12的核酸序列後,推演出兩個ORFs,二者各有一個核糖體鍵結位置,可轉譯出64與106個胺基酸的多勝鏈,但是功能不明。已知f42-pUC18-Cm可嵌入XW47染色體之中,其是否能成為嵌入載體與穿梭載體,也加以討論。 The filamentous bacteriophage Xf contains a circular single-stranded DNA of 8 kilobases. After infection, the single-stranded DNA replicates to form double-stranded replicative form DNA (RF DNA). It was found that the phage genome could integrate into the chromosome of its host cells, Xanthomonas campestris pv oryzae, and exist as lysogenic state. DNA regions which are responsible for the insertion were found to locate within a BamHI-KpnI fragment of 865 bp fragment. This DNA fragment has been cloned into M13mp18 and called Ins7. A BamHI fragment (1.2 kb) of the host chromosomal DNA was found to have high homology with Ins7 probe under high stringent hybridization conditions. This 1.2 kb BamHI fragment was cloned into BamHI site of pBR322 and called Xo12. DNA sequences of Ins7 and Xo12 were determined by dideoxy sequencing method. The high homology region between Ins7 and Xo12 DNA was found in the 274 bp sequence. In a previous report, a core sequence of 15 bp (5'TATACATTATGCGAA3') in the integration site of Cf16-vl and its host system has been reported. This core sequence appeared only in Ins7 DNA but not in Xo12 sequence. To understand the integration mechanism of Xf phage, we constructed a shuttle vector f42-pUC18-Cm containing the DNA of a miniphage f42. The DNA of f42 is 3.5 kb, which contained the complete Ins7 sequence. Since the host bacteria X. oryzae have restriction systems, competent cells made of X. citri XW47 were chosen for the recipient cells of transformation analysis. The result showed that f42-pUC18-Cm molecule did integrate into XW47 chromosome. This indicates the integration is via core sequence (15 bp) in the X. citri system, since XW47 chromosome did not have the aforementioned 274 bp. Computer search of Xo12 DNA sequence, two ORFs with two ribosome binding sites could be found. The ORF-1 has 64 amino acids and the ORF-2 has 106 amino acids. The function of Xo12 DNA with 274 bp homology and the deduced two ORFs are not known. However, the shuttle vector properties of f42-pUC18-Cm could only be used for the integration cloning system. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/75692 |
全文授權: | 未授權 |
顯示於系所單位: | 植物科學研究所 |
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