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  本文關(guān)鍵詞：PmrA對傷寒沙門菌高滲應激后期基因表達調(diào)節(jié)作用　出處：《江蘇大學》2009年碩士論文　論文類型：學位論文

  更多相關(guān)文章： 傷寒沙門菌 PmrA 基因芯片 高滲應激 侵襲

【摘要】： 目的 PmrA為傷寒沙門菌雙組份調(diào)節(jié)系統(tǒng)的效應調(diào)節(jié)蛋白,在高滲應激的后期其基因表達明顯下調(diào)。本研究的目的是進一步探討傷寒沙門菌調(diào)節(jié)因子PmrA在高滲應激后期對基因表達調(diào)節(jié)的影響。 方法 (1)傷寒沙門菌pmrA基因缺陷變異株制備:采用自殺質(zhì)粒pGMB151介導的同源重組方法制備傷寒沙門菌調(diào)節(jié)因子PmrA的基因缺陷變異株,根據(jù)傷寒沙門菌pmrA基因序列設(shè)計引物,擴增pmrA基因上、下游同源性片段,定向連接成pmrA基因缺損型同源核苷酸片段,并與自殺質(zhì)粒pGMB151相連后經(jīng)電擊法導入傷寒沙門菌野生株,在5%蔗糖LB平板上進行同源重組,通過篩選獲得pmrA基因缺陷變異株; (2)傷寒沙門菌基因轉(zhuǎn)錄表達譜分析:利用傷寒沙門菌全基因組芯片分析技術(shù),體外模擬高滲環(huán)境應激,在低滲(50 mmol/L NaCl)LB培養(yǎng)液中分別培養(yǎng)傷寒沙門菌野生株和pmrA基因缺陷變異株4h(37℃,250r/min)至對數(shù)生長期,后轉(zhuǎn)入高滲(300 mmol/L NaCl)LB培養(yǎng)液中在同樣條件下繼續(xù)培養(yǎng),在高滲應激后期(120min),分別提取傷寒沙門菌野生株和pmrA基因缺陷變異株的總RNA,反轉(zhuǎn)錄成cDNA并標記熒光(cy3或cy5),與基因組芯片雜交,掃描后據(jù)熒光信號分析各基因轉(zhuǎn)錄表達水平,比較傷寒沙門菌野生株和pmrA基因缺陷變異株在高滲應激后期的基因表達譜差異; (3)實時熒光定量PCR(qRT-PCR)分析:選擇部分表達差異顯著的基因,設(shè)計并合成特異性引物,進行實時熒光定量PCR,驗證DNA芯片分析結(jié)果。, 結(jié)果 (1)PCR及序列分析證實,pmrA基因缺陷變異株中pmrA基因351bp被6bp取代,表明成功構(gòu)建pmrA基因缺陷變異株; (2)基因表達譜比較分析結(jié)果表明傷寒沙門菌pmrA基因缺陷變異株在高滲應激后期有81個基因表達上調(diào),有22個基因表達下調(diào)。其主要涉及ABC轉(zhuǎn)運系統(tǒng)相關(guān)蛋白、鞭毛蛋白、侵襲蛋白、外膜蛋白及一些酶類等; (3)對pmrA基因缺陷變異株中表達差異基因(yliC,putA),利用實時熒光定量PCR進行分析其mRNA水平,結(jié)果顯示其在pmrA基因缺陷變異株和野生株中差異與基因芯片分析結(jié)果基本一致。 結(jié)論 成功構(gòu)建傷寒沙門菌pmrA基因缺陷變異株;PmrA在傷寒沙門菌高滲應激后期,對基因表達調(diào)節(jié)與物質(zhì)轉(zhuǎn)運及侵襲力的提高等發(fā)揮重要作用。
[Abstract]:objective
PmrA is the regulation protein of two components of Salmonella typhi, and its gene expression is down regulated at the late stage of hyperosmotic stress. The purpose of this study is to further explore the effect of Salmonella typhimurium regulatory factor PmrA on gene expression regulation in the late stage of hyperosmotic stress.
Method
(1) pmrA gene deleted mutant of Salmonella typhi was prepared by Dutch act plasmid mediated pGMB151 of Salmonella typhi regulator PmrA homologous recombination method for the mutant, the primers were designed according to the Salmonella pmrA gene sequence, pmrA gene amplification, downstream homologous fragment, connection oriented pmrA gene defect type homologous nucleotide fragment and plasmid pGMB151, and Dutch act after electroporation is connected into the wild strains of Salmonella typhi, homologous recombination in 5% sucrose LB tablet, obtained by pmrA mutant screening;
(2) the expression of Salmonella typhi gene transcription analysis: spectrum analysis technique using Salmonella typhi genome gene chip. The in vitro hypertonic environment stress in low permeability (50 mmol/L NaCl) were cultured in LB culture of Salmonella typhimurium wild strain and the pmrA mutant 4H solution (at 37 250r/min) to the logarithmic growth phase, then transferred to hypertonic (300 mmol/L NaCl) in LB medium under the same conditions continue to develop, in the later stage of hyperosmotic stress (120min), total RNA was extracted from Salmonella typhimurium wild-type and pmrA mutant, reverse transcription cDNA and labeled (Cy3 or Cy5), and hybridized with the gene chip, according to fluorescence scanning signal analysis of each gene expression, comparison of Salmonella typhimurium wild strain and the pmrA mutant at later stage of hyperosmotic stress the difference of gene expression profile;
(3) real-time fluorescence quantitative PCR (qRT-PCR) analysis: select partial genes that express significant differences, design and synthesize specific primers, real-time fluorescence quantitative PCR, verify the results of DNA chip analysis.
Result
(1) PCR and sequence analysis confirmed that the pmrA gene 351bp in the pmrA gene defective mutant was replaced by 6BP, indicating that the pmrA gene defective mutant was successfully constructed.
(2) expression profiles analysis revealed that pmrA gene deleted mutant of Salmonella typhi at later stage of hyperosmotic stress expression of 81 genes, 22 genes were down regulated. It mainly involves the related protein, ABC transporter flagellin, invasion protein, membrane protein and enzymes;
(3) differentially expressed genes (yliC, putA) in pmrA gene variant mutant were analyzed by real-time fluorescence quantitative PCR. The results showed that the difference between pmrA gene deficient mutant and wild strain was basically the same with that of gene chip analysis.
conclusion
We successfully constructed pmrA gene deficient mutant of Salmonella typhi. PmrA played an important role in the regulation of gene expression, transport and invasion of Salmonella typhi in the late stage of hyperosmotic stress.

【學位授予單位】：江蘇大學
【學位級別】：碩士
【學位授予年份】：2009
【分類號】：R378.23

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本文編號：1391558