本文關(guān)鍵詞：整體調(diào)控子PhoP直接調(diào)控鼠疫耶爾森氏菌胞內(nèi)生存能力的研究　出處：《中國人民解放軍軍事醫(yī)學(xué)科學(xué)院》2006年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 鼠疫耶爾森氏菌 二元調(diào)控系統(tǒng)PhoP-PhoQ 轉(zhuǎn)錄調(diào)控 胞內(nèi)生存能力

【摘要】：鼠疫是一種古老的自然疫源性疾病，是由鼠疫耶爾森氏菌(以下簡稱鼠疫菌)引起的。機(jī)體感染早期，鼠疫菌能在巨噬細(xì)胞內(nèi)存活并繁殖，是鼠疫菌致病的關(guān)鍵階段。PhoP-PhoQ是一種二元調(diào)控系統(tǒng)(two-component system)，它可以調(diào)控細(xì)菌的毒力，參與細(xì)菌對(duì)Mg~(2+)限制性生長環(huán)境的適應(yīng)，而且還可以調(diào)節(jié)幾種革蘭氏陰性細(xì)菌的許多細(xì)胞活性。研究發(fā)現(xiàn)△phoP突變株對(duì)小鼠J774巨噬細(xì)胞感染力下降，對(duì)巨噬細(xì)胞殺(抑)菌機(jī)制(酸脅迫、過氧化氫刺激、高滲脅迫、抗菌肽作用)敏感性大大增強(qiáng)。雖然毒力和生化表型實(shí)驗(yàn)證明了PhoP-PhoQ控制著鼠疫菌胞內(nèi)寄生能力，但是PhoP-PhoQ如何控制這個(gè)過程的機(jī)制至今尚未闡明。 本研究首先利用基于Red系統(tǒng)的一步法突變技術(shù)缺失替換鼠疫菌的phoP基因，進(jìn)而利用鼠疫菌全基因組DNA芯片進(jìn)行基因轉(zhuǎn)錄調(diào)控研究。通過比較△phoP突變株和野生株在低鎂條件下的轉(zhuǎn)錄水平差異，，界定表達(dá)上調(diào)和下調(diào)的基因，這些基因組成了PhoP調(diào)控元，這一分析可從全基因組水平篩選出所有受PhoP-PhoQ直接或間接調(diào)控的靶基因。然后，利用經(jīng)典分子生化實(shí)驗(yàn)和生物信息學(xué)分析，深入探究二元調(diào)控系統(tǒng)PhoP-PhoQ轉(zhuǎn)錄調(diào)控的分子機(jī)制。 用凝膠遷移實(shí)驗(yàn)明確46個(gè)操縱子／基因直接受PhoP的轉(zhuǎn)錄調(diào)控。這些基因有的受PhoP的上調(diào)，有的受其下調(diào)，包括PhoP調(diào)控的各方面功能的基因。PhoP正調(diào)控mgtCB，后者可以調(diào)節(jié)Mg~(2+)的轉(zhuǎn)運(yùn)；通過調(diào)節(jié)氧化應(yīng)激基因、分子伴侶和ATP-依賴性蛋白酶基因以及其他應(yīng)激基因來參與菌體的應(yīng)激防御機(jī)制；通過正調(diào)控glgPACXB操縱子來參與恢復(fù)高滲條件下細(xì)胞漿的體積、正調(diào)控pmrHFIJKLM和ugd基因來實(shí)現(xiàn)脂質(zhì)A的氨基阿拉伯糖修飾、調(diào)控代謝相關(guān)基因來參與鼠疫菌能量代謝、大分子和小分子的降解以及氨基酸、嘌呤、嘧啶、核苷酸、輔基和脂肪酸生物合成等。值得注意的是，鼠疫菌的PhoP能對(duì)包括其自身在內(nèi)的八個(gè)調(diào)節(jié)基因，phoP、crp、lacI、phoB、cyaA、ntrB、slyA和glnK，進(jìn)行直接調(diào)控從而發(fā)揮更大的級(jí)聯(lián)作用。因此，二元調(diào)控系統(tǒng)PhoP-PhoQ應(yīng)當(dāng)是鼠疫菌的一個(gè)整體調(diào)控子。 為了明確PhoP的精細(xì)調(diào)控機(jī)制，本研究對(duì)26個(gè)凝膠遷移實(shí)驗(yàn)陽性的基因的啟動(dòng)子進(jìn)行了足跡實(shí)驗(yàn)，得到了25個(gè)PhoP結(jié)合保護(hù)區(qū)域，在這個(gè)區(qū)域內(nèi)存在著PhoP box，PhoP蛋白調(diào)控靶基因主要是通過結(jié)合各靶基因啟動(dòng)子內(nèi)的PhoP box，調(diào)控各基因的轉(zhuǎn)錄。用motif sampler軟件對(duì)這25個(gè)結(jié)合保護(hù)區(qū)域進(jìn)行同源比對(duì)，并從統(tǒng)計(jì)學(xué)角度分析了18個(gè)位置上的堿基分布情況，最終將鼠疫菌的PhoP box歸納為(T／G)(A／G)TTTA(A／T)七核苷酸同向重復(fù)序列。但PhoP box存在
[Abstract]:Yersinia pestis is an ancient natural disease caused by Yersinia pestis. In the early stage of infection, Yersinia pestis can survive and reproduce in macrophages. PhoP-PhoQ is a two-component system, which can control the virulence of bacteria. To participate in the adaptation of bacteria to Mg~(2) restrictive growth environment. And it can also regulate the cell activity of several Gram-negative bacteria. It was found that the phoP mutant decreased the infectivity of J774 macrophages and killed (inhibited) the macrophages (acid stress). The sensitivity of hydrogen peroxide stimulation, hyperosmotic stress and antimicrobial peptide was greatly enhanced, although virulence and biochemical phenotype experiments proved that PhoP-PhoQ controlled the parasitic ability of Yersinia pestis. But the mechanism by which PhoP-PhoQ controls this process has not yet been clarified. In this study, the phoP gene of Yersinia pestis was replaced by one step mutation technique based on Red system. Then the gene transcriptional regulation of Yersinia pestis was studied by using the whole genome DNA chip. By comparing the transcription level of phoP mutant and wild strain under low magnesium condition, the up-regulated and down-regulated genes were defined. These genes form the PhoP regulator, an analysis that selects all target genes directly or indirectly regulated by PhoP-PhoQ at the genome level. By means of classical molecular biochemistry experiment and bioinformatics analysis, the molecular mechanism of PhoP-PhoQ transcriptional regulation was deeply explored. Gel migration assay showed that 46 operons / genes were directly regulated by the transcription of PhoP. Some of these genes were up-regulated by PhoP and others were down-regulated. The gene. PhoP, which includes all aspects of PhoP regulation, regulates the transport of Mg~(2. By regulating oxidative stress genes, molecular chaperones, ATP-dependent protease genes and other stress genes, they participate in the stress defense mechanism of bacteria. Through the positive regulation of glgPACXB operon to participate in the restoration of the cytoplasm volume under hypertonic condition, and the regulation of pmrHFIJKLM and ugd genes to achieve the amino arabinose modification of lipid A. Regulation of metabolism-related genes to participate in Yersinia pestis energy metabolism, degradation of macromolecules and small molecules, amino acid, purine, pyrimidine, nucleotide, cogroups and fatty acid biosynthesis. The PhoP of Yersinia pestis can affect the eight regulatory genes, including its own, Phosphorus cryptifolia and glnK. Therefore, the dual control system PhoP-PhoQ should be an integral regulator of Yersinia pestis. In order to elucidate the fine regulation mechanism of PhoP, the promoter of 26 positive genes for gel migration test was studied by footprint test, and 25 PhoP binding protection regions were obtained. In this region, the target gene of PhoP boxphop protein is regulated mainly by PhoP box in the promoter of each target gene. The gene transcription was regulated by motif sampler software. The 25 protected regions were compared with each other, and the distribution of the bases in 18 sites was analyzed from the point of view of statistics. Finally, the PhoP box of Yersinia pestis was summed up as the homozygote sequence of T / G box. But PhoP box existed.
【學(xué)位授予單位】：中國人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2006
【分類號(hào)】：R378

【引證文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前2條

1 高永明;結(jié)核桿菌phoP突變株的構(gòu)建及鑒定[D];華北煤炭醫(yī)學(xué)院;2009年

2 付婷;豬2型鏈球菌新型雙元系統(tǒng)ResS/ResR致病機(jī)理的研究[D];華中農(nóng)業(yè)大學(xué);2008年

本文編號(hào)：1400457