Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/21679
標題: Cloning and Analysis of the dnaK Operon Promoer in Xanthomonas campestris pv. campestris 17
十字花科黑腐病菌dnaK操縱組啟動子之選殖與分析
作者: 楊政華
關鍵字: dnaK;熱休克;grpE;hrcA;heat shock;stress;promoter;sigma 32;逆境;操縱組;啟動子
出版社: 分子生物學研究所
摘要: 
Abstract
The dnaK and its flanking regions were cloned and sequenced from the plant pathogenic bacterium Xanthomonas campestris pv. campestris 17. Analysis of the nucleotide sequences of the dnaK and its flanking sequences indicated a gene organization of 5'-hrcA-grpE-dnaK-dnaJ-pdxK-3'. Analysis of upstream of grpE and dnaK revealed sequences very similar to consensus sequence for Eσ32 binding. Putative Rho-independent
terminators were found in the grpE/dnaK and dnaK/dnaJ intergenic regions, as well as downstream of dnaJ. Northern blotting analysis detected the heat-induced transcripts of 2.6 kb and 0.6 kb using probe E which carrying part of grpE gene, transcripts of 3.3 kb, 2.6 kb, 2.0kb and 1.1 kb using probe K containing part of dnaK gene, and transcripts of 3.3 kb, 2.6 kb and 0.8 kb using probe J containing part of dnaJ gene. Primer extension experiment showed that the grpE transcriptional start site located at 34 nt upstream of the translational initiation site and the dnaK transcriptional start site locats at 46 nt upstream of the translational start site. The DNA fragment, containing the putative grpE and dnaK promoter regions were amplified by PCR, and cloned into the promoter proving vector pFY13-9, forming pEZ+ and pKZ+, respectively. Results showed that these two promoters are induced under heat stress, ethanol stress and hydrogen peroxide stress .

中文摘要
植物致病菌Xanthomonas campestris pv. campestris 17 (Xc17) 之熱休克基因dnaK及其上下游部份以被選殖及定序完成。 經核酸分析比對後,發現其基因排列順序為5’-hrcA-grpE-dnaK-dnaJ-pdxK-3’。 在grpE與dnaK基因上游帶有類似E. coliσ32所認辨之啟動子區域。 在grpE與dnaK之間,dnaK與dnaJ之間及dnaJ基因下游,可能具有轉錄終止子功能之二級結構。 以包含grpE及dnaK部分基因之片段作為探針,進行北方轉漬分析,在生長於正常環境下的Xc17,無法偵測到該等基因之mRNA。 但是,經熱休克處理後,所抽取之Xc 17總RNA,則可偵測到2.6 kb及0.6 kb的mRNA。 以包含 dnaK部分基因之片段作為探針,於經熱休克處理後,所抽取之Xc 17總RNA,亦可偵測到3.3 kb、2.6 kb及2.0 kb以及1.1 kb 的mRNA。 以包含dnaJ部分基因之片段作為探針,則可偵測到3.3 kb、1.1 kb 及0.8 kb 的mRNA。 引子延伸分析結果顯示,grpE轉錄起始點,位於grpE基因轉譯起始點上游34 nt G的位置,其上游10 nt為所推測之σ32所認辨啟動子-10區域。 dnaK轉錄起始點,位於dnaK基因轉譯起始點上游46 nt G的位置,其上游9 nt為所推測之σ32所認辨啟動子-10區域。於包含grpE及dnaK啟動子區域上下游分別設計引子,以PCR擴增包含grpE及dnaK啟動子區域,分別黏接於啟動子選殖載體pFY13-9的報導基因lacZ上游之選殖區後,分析發現啟動子受到35℃熱休克逆境、4%乙醇逆境和2mM雙氧水逆境之誘導而表現。 最後觀察Xc11之σ32與dnaK啟動子於E. coli中之表現,當Xc11σ32受到誘導表現時,dnaK啟動子之表現也增加,證明該啟動子確實受到σ32之調控。 於E. coli中Xc17 grpE及dnaK上游之啟動子也能表現,但相較於Xc17菌體中之表現,E. coli之表現較弱。
URI: http://hdl.handle.net/11455/21679
Appears in Collections:分子生物學研究所

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