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    National Tsing Hua University Institutional Repository > 生命科學院  > 分子醫學研究所 > 博碩士論文 >  綠膿桿菌PAO1轉錄因子RpoN與RpoN活化因子Sfa2對第二套第六型分泌系統之調控


    Please use this identifier to cite or link to this item: http://nthur.lib.nthu.edu.tw/dspace/handle/987654321/85663


    Title: 綠膿桿菌PAO1轉錄因子RpoN與RpoN活化因子Sfa2對第二套第六型分泌系統之調控
    Authors: 杜韻亭
    Tu, Yun-Ting
    Description: GH02101080532
    碩士
    分子醫學研究所
    Date: 2014
    Keywords: 綠膿桿菌;轉錄因子 RpoN;轉錄活化因子 Sfa2;第六型分泌系統
    Pseudomonas aeruginosa;sigma factor RpoN;RpoN activator;Type six secretion system
    Abstract: 第六型分泌系統為革蘭氏陰性菌傳送有毒物質至目標對象的方式之一,在綠膿桿菌PAO1中,共有三套第六型分泌系統基因叢集,分別命名為H1-T6SS、H2-T6SS與H3-T6SS基因叢集。本實驗室先前的研究發現,sfa2 (PA1663) 為RpoN (σ54) 依賴性轉錄活化因子,在sfa2基因缺損突變菌株中,H2-T6SS相關基因表現量降低,因此本研究建構轉錄因子rpoN (PA4462) 基因缺損突變菌株 (ΔrpoN),探討綠膿桿菌PAO1的H2-T6SS基因叢集是否確實透過Sfa2與RpoN兩種因子調控。利用GUS報導基因系統得知Sfa2與RpoN可以正向轉錄調控H2-T6SS基因叢集,透過凝膠電泳遷移分析得知,Sfa2與RpoN分別會鍵結在H2-T6SS基因叢集之上游序列432-bp至403-bp與672-bp至503-bp的位置。此外,透過GUS報導基因系統與免疫轉漬法釐清可能由H2-T6SS基因叢集調控的三組hcp/vgrG操作子,分別為PA0263/PA0262、PA1512/PA1511與PA5267/PA5266,由結果得知除PA0263/PA0262外,其餘兩組的操作子轉錄與轉譯結果皆會受Sfa2與RpoN影響,其中以PA5267/PA5266所受到的調控最為顯著,利用凝膠電泳遷移分析發現,只有RpoN會與此三組hcp/vgrG操作子之上游序列結合,Sfa2則不會。本研究中發現ΔrpoN的抽動運動性、泳動運動性、螯鐵能力、螢光素與綠膿素產量、生物膜生成皆低於綠膿桿菌野生株PAO1,溶血能力與群體感應能力則優於綠膿桿菌PAO1。綜合本研究的結果得知Sfa2和RpoN可共同正向調控H2-T6SS基因叢集與三組hcp/vgrG操作子,且RpoN調控綠膿桿菌PAO1眾多的生理活性表現。
    Type VI secretion system (T6SS) is an apparatus for Gram-negative bacteria to deliver effector proteins directly into target cells. There are three T6SS gene clusters, named H1-, H2-, and H3-T6SS gene cluster, in Pseudomonas aeruginosa PAO1. The aim of this study is to elucidate the role of both sfa2 (PA1663), a putative RpoN transcriptional activator encoding gene located in the H2-T6SS gene cluster and rpoN (PA4462), a transcription factor in regulation of H2-T6SS. In previous studies, the expression of H2-T6SS gene cluster were down-regulated in sfa2 deletion mutant strain. This study constructed an rpoN deletion mutant strain (ΔrpoN) to elucidate the relationship between Sfa2 and RpoN in regulation of H2-T6SS gene expression. Analysis of the upstream region of H2-T6SS with β-glucuronidase (GUS) reporter gene assay confirmed that Sfa2 and RpoN co-regulate the expression of the H2-T6SS gene cluster. Using recombinant Sfa2 and RpoN in an electrophoretic motility shift assay, this study found that the Sfa2 binding site and RpoN binding site are located from nucleotide position -432 to -403 and -672 to -503, respectively in the upstream region of the H2-T6SS gene cluster. This study also showed that the expression of Hcp decreases in both the sfa2 and rpoN deletion mutant strains. By GUS reporter gene assay, PA5267 (hcpB) is the major one of three hcp genes (PA0263, PA1512 and PA5267) regulated by Sfa2 and RpoN. However, only RpoN can bind to the upstream refion of three hcp genes but not Sfa2. Compared to PAO1, twitching motility, swimming motility, iron chelation, the production of pyocyanin and pyoverdin, and biofilm formation reduced in ΔrpoN. Conversely, the hemolysis and quorum sensing ability increased in ΔrpoN. Combined results in this study, we suggest that Sfa2 interacts with RpoN to up-regulate both H2-T6SS gene cluster and hcp genes expression and RpoN plays an important role in regulating phenotypic traits in P. aeruginosa PAO1.
    URI: http://nthur.lib.nthu.edu.tw/dspace/handle/987654321/85663
    Source: http://thesis.nthu.edu.tw/cgi-bin/gs/hugsweb.cgi?o=dnthucdr&i=sGH02101080532.id
    Appears in Collections:[分子醫學研究所] 博碩士論文

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