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標題: Sequence Analysis of the Filamentous phage fXo of Xanthomonas oryzae pv. oryzae
水稻白葉枯病病原菌線狀噬菌體 fXo 基因體之定序與分析
作者: 方鈺森
關鍵字: 水稻白葉枯病菌;複製起始蛋白;線狀噬菌體;基因體核酸的定序;電孔法;革蘭氏陰性菌
出版社: 分子生物學研究所
fXo, 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.

fXo 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。
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