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標題: 十字花科黑腐病菌 rpoH 基因表現的調控研究
Studies on the Regulation of Xanthomonas campestris rpoH Gene Expression
作者: Chen, Yung-Da
關鍵字: 十字花科黑腐病菌
Xanthomonas campestris
sigma H
出版社: 分子生物學研究所
摘要: Abstract The aim of this work was to study the regulation of rpoH gene expression at transcriptional and translational level. The rpoH gene of X. campestris, which encodes an alternative sigma factor (sigma H), had been cloned and sequenced in this laboratory. Northern blot analysis indicated that the amount of rpoH mRNA could be greatly enhanced during 42C heat shock and the size of the induced mRNA was about 900 nt. In agreement with this, an inverted repeat sequence at downstream of the rpoH gene was proved to function as a transcription terminator. Results of Northern blot and terminator assays suggested that Xc11 rpoH gene has its own promoter and terminator and this gene was thus predicted to be monocistronic. The transcriptional start site of Xc11 rpoH gene was determined to locate at 20 nt (G) and 21 nt (C) preceding the ATG start codon. Sequence similar to consensus sigma E and sigma 70 type promoters were observed at the upstream region of transcriptional start sites. However, no transcript initiated from the putative sigma 70 type promoter was identified by primer-extension. In order to analyze the promoter activity of Xc11 rpoH gene, the putative promoter region of rpoH was amplified by PCR and cloned into pFY13-9 promoter-probing vector. By measuring the activity of beta-galactosidase, the effects of heat shock and ethanol shock on PrpoH-lacZ gene fusion in Xc11 and E. coli were determined. The results revealed that the putative promoter region has promoter activity at normal growth temperature and could be induced under heat shock and ethanol stress. In addition, the construct with Xc11 sigma E-type promoter was shown to have a higher b-galactosidase activity when transformed into E. coli ER2566 harboring a plasmid with an inducible E. coli sigma E gene than the control strain without the plasmid. In vitro transcription studies demonstrated that Xc11 rpoH promoter could be recognized by E. coli RNA polymerase holoenzyme containing sigma E or sigma 70. However, recognition of Xc11 rpoH promoter by Xc11 RNA polymerase holoenzyme reconstituted with Xc11 sigma 70 or E. coli could only be verified by gel retardation analysis. The effect of heat shock on the level of rpoH gene expression was further analyzed by Northern blot analysis. A 7-fold increase in the amount of rpoH mRNA was detectable 5 min after heat shock at 42C and up to 14-fold increase was observed 30 and 60 min after heat shock. However, Western blot analysis indicated that the level of sigma H increase rapidly 5 min after heat shock, reached to its maximal level between 10 and 20 min, and then declined rapidly after 30 min.
中文摘要 本研究是針對實驗室已選殖之 X. campestris 的次要 sigma H 因子基因 (rpoH) 在轉錄及轉譯上表現的調控進行探討。首先由北方墨點分析得知,rpoH mRNA 的轉錄在熱休克時會大量表現,其長度約為 900 nt。由於 rpoH 基因的全長為 876 bp,故推測 rpoH 基因為單一基因形成的操縱組。進一步藉由 lacZ 報導基因的實驗分析,證實其轉譯終止碼下游的 inverted repeat DNA 序列具有轉錄終止子的活性。接著利用引子延伸實驗來偵測 rpoH 基因在正常生長溫度及 42 oC 熱休克時的轉錄起始位置,結果在 ATG 上游第 20 (G) 及 21 (C) 鹼基有訊號產生,而且熱休克時訊號更加的顯著。此轉錄起始點上游的 -10 區域 (TCCAA) 及 -35區域 (GAACCT) 有與 E. coli sigma E 因子辨識的啟動子相似的序列。 將含有 rpoH 基因 sigma 70 及 sigma E 啟動子區域的 DNA 片段構築在以 lacZ 為報導基因的 promoter probing vector 上,並分別在 Xc11 及 E. coli 菌體中偵測其啟動子的活性。結果顯示,在正常生長溫度下,當 Xc11 及 E. coli 菌體中含有 sigma 70 與 sigma E 啟動子的 DNA 片段之 pFYRP3 質體,及只含有 sigma E 啟動子的 DNA 片段 之 pFYRP1質體時,均具有啟動子的活性;而只有含 pFYRP3 質體者才有受熱休克誘導的現象。在 5% 的酒精的環境下,則 Xc11(pFYRP3) 及 Xc11(pFYRP1) 菌株之 rpoH 啟動子的表現亦會受到誘導。故 rpoH 啟動子的表現可受酒精及熱休克的誘導,此結果與北方墨點分析相符。在 E. coli 中若含有大量表現的 E. coli sigma E 因子則 rpoH sigma E-type 的啟動子的活性較一般表現量為高。此外,由 gel retardation 及體外轉錄的結果顯示,Xc11 rpoH 基因含有可被 Xc11 及 E. coli sigma 70 之全、Xc11 及 E. coli 之核心與 E. coli sigma E 重組之全所辨識的啟動子。由北方墨點法分析顯示,rpoH 受 42 oC 熱休克時其 mRNA,在5 分鐘之內的表現量即高達 7 倍,而在 30-60 分鐘內均維持在高達 14倍的量,顯示 rpoH mRNA 在高溫時相當穩定。而以西方墨點法偵測42 oC熱休克時 sigma H 的濃度變化,顯示在5分鐘之內 RpoH 蛋白即大量的表現,在10-20 分鐘之間達到最高,但在 30 分鐘即下降持續至 60 分鐘仍可被偵測到。故推測 Xc11 rpoH 基因受熱誘導時,其轉錄的表現主要可能由 sigma E-like 因子所調控。
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