Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/21235
標題: 十字花科黑腐病菌 sigma E 因子 (rpoE) 下游基因的特性分析
作者: 許朝欽
Hsu, Chao-Chin
關鍵字: rpoE
十字花科病菌
RNA polymerase
sigma E
RseA
unfolding protein
extracytoplasmic function
熱休克
未摺疊蛋白
出版社: 分子生物學研究所
摘要: 中 文 摘 要 Extracytoplasmic function (ECF) sigma 因子,在分類上是屬於 70 類sigma 因子的次家族,屬於這次家族的 sigma 因子, E , 在已知的菌種間具有調控不同基因表現的功能,其中包括在 E. coli 中調控著熱休克基因的表現。目前發現緊鄰在 ECF sigma 因子基因下游,一般都存在著一個 anti-sigma 因子的基因rseA,通常這個下游基因都具有負向調控 sigma 因子的功能。本研究室之前在 Xanthomonas campestris pv. campestris strain 11 (簡稱 Xc11) 中已經選殖到了 rpoE 基因,經定序與比對分析後,推測此基因是屬於ECF 類sigma因子的成員之一。在本研究中,首先經由 DNA 定序及比對分析確認了Xc11 rpoE 基因下游亦存在有rseA 與 mucD 兩基因。再經由北方墨點分析顯示,Xc11的 rpoE與rseA基因是位於一個由 E 所自我調控的操縱組當中 ,這表示Xc11 mucD 基因並不屬於rpoE-rseA操縱組而具有自己的啟動子,同時mucD基因上游的轉錄起始點也在引子延伸實驗中被確認,並且具有啟動子的功能,顯示 Xc11 與其它菌株在基因組成上不相同。在本研究中,進一步藉由yeast two hybrid 與co-purification 實驗,證實了Xc11 E 與 anti-sigma 因子RseA 兩蛋白在生體內與生體外具有交互作用。為了分析 rpoE 基因表現的調控機制,將含有rpoE基因啟動子區域的DNA片段構築在以lacZ為報導基因的promoter probing vector 上,藉由分析 -galactosidase 的活性,來觀察rpoE啟動子在熱休克的刺激下與在Xc11 mucD突變株中的表現情形,結果顯示,rpoE基因啟動子在上述的情形表現量皆會增加。然而,西方墨點法分析卻顯示細胞內 E蛋白量並不會隨著熱休克的刺激而增加,而RseA蛋白卻因熱休克的刺激而快速被分解。綜合以上的實驗結果顯示,熱休克會導致胞內游離態 E蛋白含量的增加,以及 E 活性的高低主要是決定於 RseA 與 E 蛋白量的比率。最後也發現Xc11 mucD 基因突變株的致病性有減弱的情形。
Abstract Extracytoplasmic function (ECF) sigma factors belong to a subfamily of the 70 class sigma subunit . One such class of sigma factors, E, appears to control a variety of functions, including expression of heat shock genes in E. coli. Many ECF sigma factors are negatively regulated by an anti-sigma factor that is encoded by the gene immediately downstream of the ECF sigma factor gene. We have previously cloned and sequenced the rpoE gene of Xanthomonas campestris pv. campestris strain 11 (Xcll) that is predicted to be member of ECF sigma factor family. In this study, two genes, rseA and mucD, were identified immediately downstream of the Xcll rpoE gene. Northern blot analysis revealed that the genes encoding E and RseA lie in a single operon and are transcribed primarily from a E—dependent promoter. This indicated that the Xc11 mucD gene is transcribed from its own promoter and not belong to the rpoE-rseA operon. In addition, the transcriptional start site of mucD was determined by primer-extension. The in vivo and in vitro interactions of Xc11 RseA and E proteins were demonstrated by yeast two hybrid and co-purification experiments in this study. In order to analyze the promoter activity of Xc11 rpoE gene, the promoter region of rpoE was cloned into pFY13-9 promoter-probing vector. By measuring the activity of -galactosidase, the effects of heat shock on prpoE-lacZ gene fusion in Xc11 and Xc11 mucD null mutant strain were determined. The results revealed that the expression of rpoE promoter were increased under heat shock and in mucD mutant. However, Western blot analysis revealed that the level of E in the cell was not increased under heat shock condition and RseA was rapidly degraded in response to heat shock stress. This indicated that E activity is primarily determined by the ratio of RseA to E and heat shock stress leads to an increase in the free pool of E. In addition, mucD-null mutant of Xc11 showed attenuated virulence.
URI: http://hdl.handle.net/11455/21235
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