Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/21599
標題: Transcriptional analysis of pilBA2A1CD cluster in xanthomonas campestris pv. campestris
十字花科黑腐病菌 pilBA2A1CD 之轉錄分析
作者: 黃培瑄
關鍵字: Xanthomonas camperstris pv. campestris 17
clp
type IV pilus
pilA
pilB
pilC
pilD
Xanthomonas campestris pv. campestris
clp
type IV pilus
pilA
pilB
pilC
pilD
出版社: 分子生物學研究所
摘要: AU56E, a clp mutant derived from Xanthomonas campestris pv. campestris strain 17, synthesizes no significant amounts of xanthan gum (an exopolysaccharide). In addition, this mutant is resistant to filamentous phage fLf due to the failure in phage adsorption. Furthermore, studies carried out in our laboratory have indicated that 1) fLf infection is through attachment to the type IV pilus, the primary receptor, 2) pilRSBA1A2CD gene cluster is essential for the normal biogenesis of pilus, and 3) biogenesis of the type IV pilus is regulated by Clp, a global transcription factor. In order to understand the expression and regulation of genes pilA1, pilA2, pilB, pilC, and pilD, the promoter activities of pilA1, pilB and pilC were measured in two types of transcriptional fusions: one with the promoter-less xylE-GmW cartridge and the other with the promoter-less lacZ as the reporter. In the former assays, the cartridge was inserted into the coding regions of the genes to be studied while in the latter assays a promoter region was cloned upstream of the lacZ gene. The results of xylE-GmW reporter assays showed that pilA1 promoter is drastically reduced in clp mutant, indicating that it is positively regulated by Clp. No effects were observed in the expression of pilB and pilC promoters. In lacZ fusion assays, the result indicated that 1) neither growth of each strains nor the activities of the promoters were affected by fLf infection, 2) expression of the pilA1 and pilC promoter was higher in the wild-type Xc17 than those in AU56E, indicating that that transcription of pilA and pilC gene requires Clp, and 3) the promoter activities of pllA2, pilB and pilD were not affected by the mutation of clp gene. Primer extension analysis showed that the transcription start site of pilB is the T located at 15 nt upstream from the ATG start codon, and that of pilC is the G located at 21 nt upstream from the ATG start codon. In addition, Northern blot analysis results suggest that pilA1, pilA2 and pilC are transcribed from different promoters.
中文摘要 AU56E 是 Xanthomonas camperstris pv. campestris 17 (簡稱 Xc17) 胞外黏多醣合成基因之突變株,其突變位置位在 eps8 基因座 (locus)。 經由選殖及定序分析的結果發現 eps8 帶有 clp 基因。 AU56E 無法當作 indicator host 供給線狀噬菌體 fLf 感染,以產生溶菌斑 (plaque),但若將 fLf 的 RF DNA 以電孔法送入 AU56E,則仍能加以繁殖並釋放出正常的後代噬菌體顆粒。 這些結果顯示 AU56E 仍然具有正常讓 fLf 進行 DNA 複製、morphogenesis、包裝及釋放的功能。 因此推論 AU56E 所缺失者應是在於缺乏 phage receptor。 另外,根據研究結果推論 fLf 經由以 type IV pilus 為其 primary receptor 進行感染 X. camperstris pv. campestris,而此 pilus 之合成可能需要 Clp 的正調控。 為了了解 Clp 對一些已知與 pilus 合成有關基因表現的影響,故本研究將選殖一些已知與 pilus 合成有關的基因啟動子,如 pilA、pilB、pilC、pilD。 首先,以 insertion mutation 的方式,構築含有 xylE 報導基因之諸 pil 基因啟動子報導株,進行啟動子活性分析。 結果顯示,當 pilA1 基因帶有xylE-GmRW cartridge 時,其啟動子活性在 Xc17 菌體中較在 AU56E 菌體中高。 而 pilB、pilC 及 pilD 基因啟動子活性在 Xc17 及 AU56E 菌體中亦是約略相同,推測 pilB、pilC 及 pilD 基因可能不受 Clp 調控。 另一方面,將 pil 系列基因可能帶有啟動子的 DNA 片段選殖於以 lacZ 為報導基因的起動子選殖載體,將此系列重組質體以電孔法 (electroporation) 分別送入 Xc17 (野生株) 及 AU56E (突變株) 中,並加入 fLf 進行感染,偵測其啟動子的活性。 結果顯示,此系列轉形株的生長速度及啟動子活性都並不會受 fLf 之感染影響。 當 Xc17 菌體中含有 pilA1 及 pilC 基因 DNA 片段之質體時,均具有啟動子的活性而且高於在 AU56E 菌體中,推測 pilA1 及 pilC 基因則可能受 Clp 調控。 而 pilA2、pilB 及 pilD 基因的啟動子活性在 Xc17及 AU56E 菌體中都具有活性但並無差異,推測 Clp 不會對 pilA2、pilB 及 pilD 基因進行調控。 此外,藉由引子延伸 (primer extension) 偵測諸 pil 基因轉錄起始點之位置, 顯示 pilB 基因轉錄起始位置,為轉譯起始點 ATG 上游第 15 個核酸 ”T” 處,pilC 基因轉錄起始位置,為轉譯起始點 ATG 上游第 21 個核酸 ”G” 處。 由北方墨點法之結果顯示,pilA1 及 pilC基因皆是以單一基因的方式存在。
URI: http://hdl.handle.net/11455/21599
Appears in Collections:分子生物學研究所

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