Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/20907
DC FieldValueLanguage
dc.contributor.advisor曾義雄zh_TW
dc.contributor.author方鈺森zh_TW
dc.date2000zh_TW
dc.date.accessioned2014-06-06T07:14:49Z-
dc.date.available2014-06-06T07:14:49Z-
dc.identifier.urihttp://hdl.handle.net/11455/20907-
dc.description.abstractfXo, isolated in our laboratory, is a filamentous bacteriophage specifically infecting Xanthomonas oryzae pv. oryzae, a gram-negative pathogen causing bacterial blight in rice. Similar to other filamentous phages, fXo possesses a circular, single-stranded DNA genome (7.6 kb), replicates using a replicative form (RF) as an intermediate, and propagates without lysis of the host cells. The nucleotide sequence of fXo has previously been determined for intergenic region (IR), genes III, VIII and VI (encoding capsid proteins pIII, pVIII and pVI, respectively) and genes I and XI (encoding pI and pXI, respectively, that are presumably required for morphogenesis). In order to understand the genome organization, the rest regions of the fXo RF DNA were sequenced and analyzed. Putting all the sequences determined together, a total of 7,613 bp was obtained. Open reading frame (ORF) analysis indicated the presence of 10 putative genes. These genes are arranged in the same order as that in Ff phages (f1, M13 and fd), IR-gII/X-gV-gVII-gIX-gVIII-gIII-gVI-gI/XI. In this study, the origin for fXo replication was located in a 1.5-kb MluI DNA fragment within the coding region of gII, the gene encoding the replication initiation protein. This 1.5-kb DNA fragment was inserted into the HindIII-SmaI sites of pUC19G to generate plasmid pGII1561G. After electroporation, pGII1561G was found to be maintained in X. oryzae pv. pryzae; however, no deletion clones containing an insert smaller than that of pGII1561G were maintained in complementation tests with the gII being provided in trans.en_US
dc.description.abstractfXo 係自本實驗室分離的一種線狀噬菌體,可專一感染革蘭氏陰性菌 Xanthomonas oryzae pv. oryzae,此菌可導致水稻白葉枯病。 與一般線狀噬菌體類似,fXo 具有環狀單股 DNA 基因體 (7.6 kb),以雙股複製形 DNA (RF DNA) 為其複製的中間產物,並在增殖的過程中不會溶裂宿主細胞。 本實驗室先前已完成 fXo-基因間區 (intergenic region, IR)、 gIII、 gVIII 及 gVI (分別主導 pIII、pVIII 及 pVI 這三個外套蛋白),與 gI、gXI (分別主導 pI 及 pXI,這兩者可能為 morphogenesis 所需的蛋白) 之定序工作。 為了進一步了解 fXo 整個基因體上各基因的排列情形,本研究繼續完成 fXo 基因體核酸的定序與分析。 綜合本研究以及前人研究所定出之 DNA 片段序列,定出 fXo 基因體 DNA 全長為 7,613 nt。 分析其 open reading frame (ORF) 的結果顯示含有10個可能的基因,與 Ff 類線狀噬菌體 (f1,M13 及 fd) 一樣,這些基因在基因體上的排列順序,依次為 IR-gII/X-gV-gVII-gIX-gVIII-gIII-gVI-gI/XI 。 在本研究中,fXo 的複製起始點 (replication origin) 定位在 1.5-kb MluI DNA 片段上,此 DNA 片段包含 fXo 的 gII ,主導複製起始蛋白 (replication initiation protein)。 將此 1.5-kb 的 DNA 片段選殖在 pUC19G 之 HindIII-SmaI 位置上,得到質體 pGII1561G,經由電孔法的方式送入 X. oryzae pv. oryzae 後,發現此質體可存活。 然而以 in trans 的方式提供 gII 功能,並無法得到小於 1.5-kb DNA 片段的刪減質體在菌體中存活。zh_TW
dc.description.tableofcontents目錄 縮寫字對照表1 中文摘要2 英文摘要3 前言4 材料與方法9 I. 實驗材料9 一、菌種、噬菌體與質體9 二、藥品、酵素及放射性同位素9 三、培養基9 四、抗生素10 五、試劑10 II. 實驗方法16 一、小量質體 DNA 的抽取16 二、大量質體 DNA 的抽取16 三、噬菌體 fXo RF DNA 之抽取17 四、噬菌體單股 DNA 之抽取17 五、質體 DNA 快速篩選法(rapid screening)18 六、質體 DNA 之回收18 七、勝任細胞之製作(competent cell)19 八、E. coli 之轉型作用(transformation)19 九、細胞轉染作用(transfection)20 十、核酸定序反應20 十一、DNA 定序膠體電泳(DNA Sequence gel electrophoresis)20 十二、fXo single-stranded DNA binding protein(SSB)的分離與純化21 十三、SDS-聚丙烯胺膠體電泳22 十四、膠體阻滯分析(Gel retardation assay)22 結果與討論23 一、fXo 基因體之分段選殖、定序及序列分析23 二、fXo EcoRI-HindIII 之 1.7-kb DNA 片段上各個 ORFs 所轉譯 蛋白胺基酸序列與 fLf 及 fXv 之比較24 三、fXo RF DNA 自主複製片段之選殖27 四、fXo 複製起始區之定位 28 五、fXo 基因 V 互補 fLf 基因 V 突變株之測試 29 參考文獻31 圖表37 附錄68zh_TW
dc.language.isoen_USzh_TW
dc.publisher分子生物學研究所zh_TW
dc.subject水稻白葉枯病菌zh_TW
dc.subject複製起始蛋白zh_TW
dc.subject線狀噬菌體zh_TW
dc.subject基因體核酸的定序zh_TW
dc.subject電孔法zh_TW
dc.subject革蘭氏陰性菌zh_TW
dc.titleSequence Analysis of the Filamentous phage fXo of Xanthomonas oryzae pv. oryzaeen_US
dc.title水稻白葉枯病病原菌線狀噬菌體 fXo 基因體之定序與分析zh_TW
dc.typeThesis and Dissertationzh_TW
item.openairecristypehttp://purl.org/coar/resource_type/c_18cf-
item.openairetypeThesis and Dissertation-
item.cerifentitytypePublications-
item.fulltextno fulltext-
item.languageiso639-1en_US-
item.grantfulltextnone-
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