Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/21792
標題: 十字花科蔬菜黑腐病菌受 sigma E 辨識啟動子序列之探討
Characterization of the Xanthomonas campestris pv. campestris sigma E-regulated promoters
作者: Chuo, Dai-Fang
周黛芳
關鍵字: sigma E
聚合酶
出版社: 分子生物學研究所
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摘要: The sigma E factor (σE) of Xanthomonas campestris pv. campestris (Xc) is a member of the extracytoplasmic function sigma factor (ECF) family. The members of this family have been shown to recognize conserved promoter sequences and regulate gene expression in response to changes in the extracytoplasmic environment. To date, seven σE regulons (rseA, XCC1535, XCC3074, mopB, hrpB1, hrcU, and avrBs2) have been identified in Xc11. However, the exact mechanism that induces the expression of the σE regulons remains to be elucidated. In this study, sixteen potential σE-regulated promoter sequences in the Xc17 genome were identified by computer search and ten of these putative σE-regulated promoters were chosen for further studies. The DNA fragments contain the potential promoter sequences were amplified by PCR and fused to a promoterless lacZ reporter gene carried on the pFY13-9 plasmid. Only the β-galactosidase activities of XCC1588 and rpoH gene promoters were found to be induced in Xc11 rseA mutant strain and repressed in Xc11 rpoE mutant stain, indicating that LacZ expression is in an σE-dependent manner. Gel retardation assay showed that Xc11 EσE can specifically bind to XCC1588 and rpoH gene promoters. The transcriptional start sites just downstream of the two promoters were verified by primer-extension and S1 nuclease protection assays. The putative -35 (5'-GAACTT-3') and -10 (5'-GTCYMA-3') promoter consensus sequences were identified in the upstream of the transcriptional start site. The ability of the purified Xc11 EσE to specifically recognize the promoter fragments of the seven σE regulons was further investigated by in vitro transcription assays. Results showed that no specific transcripts could be identified from XCC3074, mopB, hrpB1, hrcU, and avrBs2 DNA template, suggesting that these genes might be indirectly regulated by σE. Consequently, the consensus -35 and -10 promoter sequences recognized by Xc11 ΕσE are proposed to be 5'-GAACWW -3' (W = A or T) and 5'-GTCHHA-3' (H = A or C or T), respectively.
十字花科黑腐病菌(Xc)中的σE為 ECF sigma factor 家族的一員,此類的sigma 因子能辨識具保留性的啟動子序列的特殊基因,而這些特殊基因的表現能使菌體適應細胞外環境的改變。目前為止,已經知道Xc11中有七個基因會受到σE調控而表現,分別為rseA、XCC1535、XCC3074、mopB、hrpB1、hrcU及avrBs2,但是σE如何誘導這些基因表現的機制仍然是未知。因此,在本實驗中,利用電腦搜尋挑選出十六個在Xc17中具有可能受σE所辨識的啟動子區域的基因,針對其中fkpA、mdoG、rpoH、XCC1588、XCC2152、XCC3436、XCC1437、cheA、XCC2627與colR等十個基因的啟動子片段進行分析探討。利用PCR增幅出含有預期啟動子區域的DNA片段,並承接於含有lacZ報導基因的pFY13-9載體上,偵測其啟動子β-galactosidase活性,發現僅有XCC1588及rpoH基因的啟動子片段,在rseA突變株時,啟動子活性會受σE誘導而上升,進而帶動LacZ的表現;在rpoE突變株時,活性會明顯的下降。以膠體遲滯移動分析實驗證實Xc核酶和RpoE重組的RNA聚合酶全酶(EσE holoenzyme)會與XCC1588及rpoH基因的啟動子片段具有專一性的結合。利用引子延伸實驗及S1 nuclease protection assay確證XCC1588及rpoH基因啟動子片段受σE辨識的轉錄起始點,並推測上游可能的-35 區域的保留性序列為5’-GAACTT-3’;-10區域的保留性區域為5’-GTCYMA-3’。論文中亦延續前人的研究,以體外轉錄實驗探討上述七個已知會受σE調控基因的啟動子片段,是否會受Xc11 EσE RNA聚合酶辨識並進行轉錄,結果發現XCC3074、mopB、hrpB1、hrcU及avrBs2基因,雖然會受到σE所調控,但並不會直接受Xc EσE RNA聚合酶辨識並進行轉錄反應。因此推測這些基因可能受σE間接的調控。針對rpoE、rseA、XCC1535、XCC1588及rpoH等五個直接受σE所調控基因啟動子區域,可以歸納出具有高度保留性序列在 -35區域為5’-GAACWW-3’,而W為A或T;而 -10保留性區域為5’-GTCHHA-3’,而H為A或C或T。
URI: http://hdl.handle.net/11455/21792
其他識別: U0005-2908200612414800
文章連結: http://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-2908200612414800
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