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標題: Study of infection of Xanthomonas campestris pv. campestris by filamentous phage phiLf
Xanthomonas campestris pv. campestris 對線狀噬菌體 phiLf 敏感性之研究
作者: 呂紹慈
Shao-Tzu Lu
關鍵字: 線狀噬菌體 phiLf;filamentous phage phiLf
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Filamentous phage φLf specifically infects Xanthomonas campestris pv. campestris (Xcc). It has a single-stranded circular DNA with a genome site of 6,008 nucleotides. The φLf viral strand encodes nine genes organized into the order of gII-gX-gV-gVII-gIX-gVIII- gIII-gVI-gI, with gII, gX and gV shown to be required for replication, gVII, gIX, gVIII, gIII, and gVI encoding coat proteins, and gI responsible for assembly and morphogenesis. φLf-homologous region (fhr) is present on the chromosome of Xcc strain Xc17, next to the dif (deletion induced filamentation) site that is the end of chromosome replication. This region has been shown to site specific integration between dif site (attB) and the dif-homologous attP site in φLf, and homologous recombination via the fhr region outside dif. φLf can infect Xcc strain P20H, but can't infect Xc17. Xc17fhrΔ8361 was constructed from Xc17 containing gVIII, gIII, gVI and gI of fhr region replaced by Gmr cartridge.φLf can infect Xc17fhrΔ8361. The purpose of this study was to understand why φLf can't infect Xc17 ? Two experimental strategies were used for study: (I) Xc17 mutants with Gmr cartridge replaced the gVIII,gIII,gVI or gI gene in the fhr region were contracted and infected by φLf. (II) Xc17fhrΔ8361 transformants complemented with DNA fragments carrying Xc17fhr gVIII,gIII,gVI and gI were obtained and infected by φLf. Results showed that mutation of gVIII,gIII,gVI or gI gene in the fhr region of Xc17 causes no effects on φLf infection. Adsorption test results appeared that Xc17 mutants is no significant difference by φLf. Therefore, mutation in the gVIII~gIII, gVIII~gVI,gVIII~gI and gVIII~gI gene of Xc17 causes no effects on φLf infection. PCR amplification analysis results, showed that fhr region is absent in the genome of Xc17fhrΔ8361 and P20H. Based on the results, φLf infection isn't associated with gVIII,gIII,gVI,gI gene but pilA1. Expression levels of pilA1 are various among Xc17, Xc17fhrΔ8361 and P20H by real-time PCR. Higher expression level of pilA1 in P20H, φLf can efficiency infect host, and lower expression level of pilA1 in Xc17fhrΔ8361, φLf also can efficiency infect host, but lower expression level of pilA1 in Xc17, φLf can't infect host. Based on the results, presumably there is other factor more influence than pilA1 efficiency when φLf infected host.

φLf 是十字花科蔬菜黑腐病菌 Xanthomonas campestris pv. campestris (Xcc) 之線狀噬菌體,其基因體為單股環狀 DNA,包含 6,008 個核苷酸。φLf 的基因排列與 E. coli Ff 噬菌體相似,順序為 gII - gX - gV - gVII - gIX - gVIII - gIII - gVI - gI。gII、gX 和 gV 與 DNA 複製有關,gVII、gIX、gVIII、gIII 和 gVI 主導噬菌體之外套蛋白,而 gI 產物則負責噬菌體之裝配與釋放。Xcc strain 17 (簡稱 Xc17) 的染色體複製終點 dif (deletion induced filamentation) site,也是 attB site,鄰近於 φLf–homologous region (fhr) 的右邊界。φLf 上的 attP site (位於 gI 下游) 可與宿主上的 attB 接觸,進行 site-specific integration,而 dif site 外圍之 fhr 區域則可供作進行 homologous recombination。φLf 可感染 Xcc strain P20H,但無法感染 Xc17;然而,前人將 Xc17 染色體上 fhr 區域中包含 gVIII、gIII、gVI和gI 的 DNA 片段以 Gmr 取代後,突變株即可被 φLf 感染,此突變株被命名為 Xc17fhrΔ8361。本研究目的為了解 Xc17 無法被 φLf 感染的原因。使用實驗策略如下:以插入突變法分別破壞 Xc17 染色體上 fhr 區域中的 gVIII、gIII、gVI 和 gI 基因,以了解哪個基因產物與 φLf 之感染有關。以及將 gVIII、gIII、gVI 和 gI 基因分別構築於質體上,轉殖送入 Xc17fhrΔ8361 突變株中,測試 φLf 的釋放情形。目前發現 φLf 無法感染上述個別基因被破壞的 Xc17 突變株。吸附率測試結果發現,相較於 Xc17,φLf 對於這些突變株的吸附能力亦無明顯差異。將 gVIII、gIII、gVI 和 gI 基因分別構築於質體上,再轉殖送入 Xc17fhrΔ8361 突變株後,以 spot test 和 plaque assay 測試,發現 φLf 依然能感染轉殖株,且轉殖株與 Xc17fhrΔ8361 釋出的噬菌體 titer 並無明顯差異。又構築 gVIII~gVI、gVIII~gI (Xc17fhrΔ8361L-1,gVIII、gIII、gVI 和 gI 基因全部被 Gmr 取代) 和 gVIII~gI (Xc17fhrΔ8361L-2,gVIII、gIII、gVI 和 gI 基因被 Gmr 取代,gVIII 基因部分保留) 刪除之突變株,發現 φLf 依然無法感染這些突變株。在實驗過程中發現,Xc17fhrΔ8361 與 Xc11 的突變株 P20H 的 EPS 含量和移動能力相似。於是,進一步利用 PCR 增幅分析 Xc17fhrΔ8361 與 P20H 在 fhr 區域以及 dif1 與 dif2 之相似性,發現 Xc17fhrΔ8361 與 P20H 都不具有 fhr 區域。由以上實驗中得知前人構築的 Xc17fhrΔ8361 基因體圖譜似乎與預期的不同,且 φLf 能感染宿主之原因與 fhr 區域的 gVIII、gIII、gVI 和 gI 基因之存在與否無關。已知 pilA1 與 φLf 的感染有關,因此利用 real-time PCR 檢測 Xc17、Xc17fhrΔ8361 與 P20H 之 pilA1 RNA 表現量。結果顯示菌株中 pilA1 RNA 量高低與 φLf 感染率無絕對相關。推測除了 pilA1 外,尚有其它因子能影響 φLf 是否會感染宿主。
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