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標題: 十字花科黑腐病菌 rpoE 基因之選殖及其啟動子之探討
Cloning and Promoter Analysis of the rpoE Gene from Xanthomonas campestris pv. campestris
作者: shieh, Sheau-yann
關鍵字: RNA 聚合;rpoE;熱休克蛋白;次要 sigma 因子;ECF;σE;Xanthomonas campestris;rseA;algU;rpoH
出版社: 分子生物學研究所
The level of gene expression is tightly coupled to the metabolic and environmental status of the cell. This is achieved primarily by regulation at the transcription level. RNA polymerases of eubacteria are essential component of the transcriptional apparatus. They are multiple subunits enzymes composed of a, b, b¢, and s subunit. The s subunit of RNA polymerase confers to core enzyme the specificity to initiate transcription. We have previously cloned and sequenced the rpoH gene encoding the s32 of Xanthomonas campestris pv. campestris strain 11 (Xc11). Interestingly, a putative sE-type promoter was recognized at the regulatory region of the X. campestris rpoH gene. The presence of this heat shock sigma factor (sE) in X. campestris and the expression of rpoE gene at normal growth temperature and under heat shock conditions were investigated in this study. Two degenerated oligonucleotides, SigE-N and SigE-C, which correspond to the conserved regions of RpoE were designed and used in polymerase chain reaction. The 300-bp PCR amplification product was then used as probe to select rpoE-containing l phages from the constructed Xc11 genomic library. After conducting in vivo excision, three pBK-CMV derivated phagemids were generated and designated as pBK-CMV1A3, pBK-CMV5B, and pBK-CMV6A3, respectively. Restriction map and Southern blot analysis indicated that the 1.5-kb EcoRI-EcoRI fragment of pBK-CMV1A3 containing the intact rpoE gene. The nucleotide sequences on both strands of the 1.5-kb insert were determined. Results of DNA sequence analysis revealed one intact and one incomplete open reading frames (ORF). The first ORF (ORF206) starts at nt 474 (ATG) and ends at nt 1094 (TGA), from which a 206 amino acids with molecular weight of 24 kDa could be translated. The deduced amino acids of ORF206 showed 56% identity with E. coli RpoE. The second ORF begins at nt 1109 and an incomplete 111 amino acids polypeptide could be encoded. The predicated amino acids have 35% identity with the N-terminal region of E. coli RseA protein. The transcriptional start site of ORF206 was determined to locate at 33 nt (G) preceding the ATG start codon by primer-extension. Sequence similar to consensus sE type promoter was observed at the upstream region of the transcriptional start site. Recognition of this promoter by Xc11 RNA polymerase core enzyme reconstituted with Xc11 or E. coli sE could be verified by gel retardation analysis. The effect of heat shock on the level of rpoE expression was analyzed by Northern blot analysis. A small increase in the amount of rpoE mRNA was detectable 40 min after heat shock at 37℃. However, Western blot analysis showed no detectable increase in the level of RpoE during heat shock at 42℃.

細胞內基因的表現和其本身的代謝情況與所處的環境狀態有密切的相關性, 其主要的調控是在轉錄作用的階段。 原核生物的 RNA 聚合是轉錄作用時的主要執行者。 它是由多種的次單元酵素,包括 α,β,β''及 σ 所組成,其中 σ 次單元提供 RNA 聚合的核心可專一性地去進行轉錄的起始作用。 本實驗室過去曾對負責轉錄作用的 RNA 聚合各次單元及 sigma 因子的基因進行選殖的工作。 其中,在與熱休克表現有關的 σH 因子基因上游發現有類似 E. coli σE 辨識的起動子序列,且含此啟動子序列的 DNA 模板,可被含有 E. coli σE 因子的 RNA 聚合辨識與結合。 因此推測 X. campestris pv. campestris strain 11 (簡稱Xc11) 菌株內可能含有次要 sigma 因子 σE,並參與調控 rpoH 基因的表現。 本研究即在尋找 Xc11 菌株裡的 rpoE 基因,同時對 rpoE 基因上游的啟動子進行研究分析。 首先,以 RpoE的蛋白保留性區域,設計二條退化性引子,分別命名為 SigE-N 及 SigE-C,進行聚合鏈鎖反應,增幅出 300-bp 的 PCR 產物,接著以此 DNA 片段為探針 (probe),對所構築好的 Xc11 的基因庫進行菌斑雜交法,篩選到包含有rpoE基因的 l 噬菌體,經菌體內質體切割法得到三個衍生的質體,分別命名為 pBK-CMV1A3、pBK-CMV5B 及 pBK-CMV6A3。 進一步以鑑識圖譜及南方墨點法分析,顯示在質體 pBK-CMV1A3 的內嵌約 1.5 kb EcoRI-EcoRI DNA 片段中內包含有完整的 rpoE 基因。 同時完成此 1. 5 kb 片段雙股 DNA 的核酸定序工作。由 DNA 序列分析結果顯示,存在一個完整的及一個不完整的 ORF,第一個 ORF (ORF206) 起始於第 474 個核酸 (ATG), 終止於第 1094 個核酸 (TGA),可轉譯出 206 個胺基酸,分 子量為 24 kDa 的蛋白,經比對 ORF206 與 E. coli 的 rpoE 蛋白有 56 % 的相同性。 第二個不完整的 ORF 起始於第 1109 個核酸, 可轉譯出 111 個胺基酸, 它與 E. coli RseA 蛋白 N 端區域有 35 % 的相同性。此外,亦藉由引子延伸反應偵測 ORF206 轉錄作用的起始點,為距離轉譯起始點 ATG 上游第 33 個核酸 2G2 處,並在轉錄起始點上游找到保留性高的 sE-type 啟動子序列。 在啟動子的鑑定方面, 乃是藉由所得之 Xc11 核心與 Xc11 或 E. coli 的sE 因子重組後的 RNA 聚合, 進行 gel-retardation 實驗分析而證實。 並由北方墨點法偵測 rpoE 基因在受到熱休克刺激後的表現情形,結果顯示 rpoE 的 mRNA 在 37 ℃ 熱休克 40 分鐘時, 可偵測到最高的表現量。 然而利用抗RpoE的抗體進行西方墨點分析時發現,Xc11 RpoE 蛋白並不隨著熱休克時間延長而有明顯的變化。
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