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  本文關(guān)鍵詞：鼠疫耶爾森氏菌毒力相關(guān)轉(zhuǎn)錄調(diào)控子Zur和PhoP的研究　出處：《中國人民解放軍軍事醫(yī)學(xué)科學(xué)院》2008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 轉(zhuǎn)錄調(diào)控 比較轉(zhuǎn)錄譜學(xué) Zur PhoP

【摘要】： 鼠疫是一種古老的自然疫源性疾病,其病原菌鼠疫耶爾森氏菌(以下簡稱鼠疫菌)是多宿主寄生菌,體現(xiàn)在:鼠疫菌以蚤類為傳播媒介寄居在特定的宿主(主要是嚙齒動物),并在自然界宿主中造成鼠疫疫情的周期性爆發(fā),該過程中偶然與患畜接觸或被帶菌跳蚤叮咬而感染到人。面對上述的復(fù)雜因素,鼠疫菌必須能夠感應(yīng)并快速適應(yīng)每個環(huán)節(jié)的變化,才能在宿主體內(nèi)存活并在自然界中傳播最終致病,而每個適應(yīng)性反應(yīng)也必然伴隨著鼠疫菌基因轉(zhuǎn)錄改變,這體現(xiàn)在:很多特定的調(diào)控子,分別感應(yīng)特定的外界信號,介導(dǎo)特定基因(包括毒力基因)表達(dá)的上調(diào)和下調(diào),最終組成復(fù)雜的毒力調(diào)控網(wǎng)絡(luò),控制鼠疫菌在媒介和貯存宿主中生存能力和致病力。 病原菌從感應(yīng)信號刺激到表達(dá)毒力因子的過程中,毒力調(diào)控子起著不可替代的作用。廣譜或特異性的調(diào)控子,控制著毒力基因的表達(dá);另外,若干個毒力調(diào)控基因的表達(dá)產(chǎn)物可組成一個復(fù)雜系統(tǒng),執(zhí)行毒力調(diào)控的功能,常稱作毒力調(diào)控系統(tǒng)。一直作為研究熱點的細(xì)菌毒力調(diào)控系統(tǒng)主要有QS系統(tǒng)、二元調(diào)控系統(tǒng)、Fur系統(tǒng)等等。這些系統(tǒng)又分成許多子調(diào)控系統(tǒng),控制著不同類型毒力基因的表達(dá),在細(xì)菌表現(xiàn)毒力的不同階段起著調(diào)控作用。 Zur阻遏蛋白屬于Fur家族,可以調(diào)控鋅離子的轉(zhuǎn)運,鋅離子在所有生物細(xì)胞中是一種重要的蛋白質(zhì)功能組分。PhoP-PhoQ是一種二元調(diào)控系統(tǒng)(two-component system),可以調(diào)控細(xì)菌的毒力,參與細(xì)菌對Mg2+限制性生長環(huán)境的適應(yīng),從而有利于鼠疫菌在巨噬細(xì)胞內(nèi)的存活和繁殖。為了鑒定鼠疫菌毒力調(diào)控子Zur和PhoP的靶基因,尤其是直接調(diào)控的靶基因,并進一步研究Zur和PhoP對其下游直接靶基因的精細(xì)調(diào)控機制,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zur和phoP基因,進而利用鼠疫菌全基因組DNA芯片進行基因轉(zhuǎn)錄譜分析。通過比較突變株和野生株在特定條件下的轉(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Zur或PhoP調(diào)控元。這一分析可從全基因組水平篩選出所有受Zur或PhoP直接或間接調(diào)控的靶基因。轉(zhuǎn)錄譜的分析結(jié)果可以通過Real time RT-PCR進行驗證。凝膠阻滯實驗技術(shù)的應(yīng)用,可以鑒定直接調(diào)控的靶基因,選擇若干關(guān)鍵毒力相關(guān)基因,采用DNA酶I足跡實驗技術(shù)、引物延伸實驗技術(shù),深入研究Zur和PhoP是如何直接調(diào)控這些基因的轉(zhuǎn)錄和表達(dá),將毒力表型和分子實驗數(shù)據(jù)結(jié)合起來,認(rèn)識Zur和PhoP如何通過激活或抑制特定基因的表達(dá),控制著鼠疫菌的宿主適應(yīng)力和致病力。 與野生株相比,敲除了zur基因的鼠疫菌在鋅離子刺激下有154個基因發(fā)生了轉(zhuǎn)錄豐度改變,其中64個基因轉(zhuǎn)錄上調(diào),90個基因轉(zhuǎn)錄下調(diào)。匯總文獻(xiàn)預(yù)測的Zur結(jié)合位點,通過consensus-matrix和convert-matrix程序計算Zur結(jié)合序列每個位置四個堿基出現(xiàn)的權(quán)重,得到Zur結(jié)合序列的Matrix,進而用WebLogo軟件展示序列l(wèi)ogo,最終預(yù)測得到Zur結(jié)合box:GAAATGTTATAWTATAACATTTC。基于上述Zur基序分析和Zur調(diào)控子的轉(zhuǎn)錄譜分析,我們通過Matrix scan程序查找Zur保守基序,選取weight值較高的4個基因ykgM、znuC、znuA、astA進行進一步的分子生化實驗。通過凝膠阻滯實驗明確了ykgM、znuC、znuA 3個轉(zhuǎn)錄單元直接受Zur調(diào)控。為了明確Zur的精細(xì)調(diào)控機制,本研究對這三個轉(zhuǎn)錄單元的啟動子進行了足跡實驗,得到了精確的Zur結(jié)合保護區(qū)域,在這個區(qū)域內(nèi)存在著預(yù)測的Zur box,Zur蛋白直接調(diào)控的這三個轉(zhuǎn)錄單元,其Zur box均覆蓋-10序列,這樣就阻礙了RNA聚合酶的結(jié)合,可能正因為如此,使Zur表現(xiàn)出對它們的抑制特性。對鋅離子刺激下的野生株和?zur突變株同時進行引物延伸實驗,不僅可以明確受Zur轉(zhuǎn)錄調(diào)控的鼠疫菌ykgM、znuC和znuA的轉(zhuǎn)錄起始位點,也可以確定Zur對其轉(zhuǎn)錄的影響。同時結(jié)合足跡實驗得到的Zur結(jié)合位點,推斷出每個啟動子內(nèi)的RNA聚合酶結(jié)合位點(-10序列)以及-35序列,豐富了Zur調(diào)控元啟動子區(qū)的分子特性。 前期對于?phoP突變株和野生株在低鎂條件下的轉(zhuǎn)錄譜分析,已經(jīng)初步界定了PhoP調(diào)控元,同樣,我們用Real time RT-PCR對轉(zhuǎn)錄譜的分析結(jié)果進行驗證。凝膠阻滯實驗確定了30個轉(zhuǎn)錄單元直接受PhoP的轉(zhuǎn)錄調(diào)控,對其中17個基因的啟動子區(qū)做了足跡實驗,得到19個PhoP結(jié)合保護區(qū)域。通過生物信息學(xué)分析將鼠疫菌的PhoP box歸納為TGTTTAW七核苷酸同向重復(fù)序列,結(jié)合引物延伸實驗得到的基因轉(zhuǎn)錄起始位點的位置信息,根據(jù)鼠疫菌的PhoP box在靶基因啟動子內(nèi)的位置以及是否含有-35序列,我們將PhoP啟動子結(jié)構(gòu)特征歸納為三類:PhoP結(jié)合位點在-35序列上游;PhoP結(jié)合位點覆蓋-35序列;PhoP結(jié)合位點在-10序列的下游。 本研究首次較全面地鑒定了鼠疫菌Zur和PhoP直接調(diào)控的調(diào)控元,同時從分子水平上明確了鼠疫菌Zur box和PhoP box的特性,探究了Zur和PhoP轉(zhuǎn)錄調(diào)控機制,為Zur和PhoP這類毒力調(diào)控子調(diào)控網(wǎng)絡(luò)的構(gòu)建,以及這些調(diào)控元功能的進一步明確提供了實驗證據(jù),這些機制的系統(tǒng)闡述將為鼠疫菌致病機制和疫苗的研究奠定重要基礎(chǔ)。
[Abstract]:The plague is a kind of ancient natural foci of the disease, the pathogen of plague bacteria Jerson S (hereinafter referred to as Yersinia pestis) is multi host parasite, reflected in the plague fleas to the media resides in the specific host (mainly rodents), and in the natural host caused by periodic outbreaks of the plague. Accidental and patient contact or by infected flea bites to infect people in the process. In the face of the complex factors mentioned above, plague bacteria must be able to sense and adapt to the quick change of each link, in order to survive in the host body and the spread of final pathogenic in nature, and each adaptive response will inevitably accompanied by Yersinia pestis gene transcription changes. This is reflected in many specific promoter, respectively sensing specific external signals, mediated by specific genes (including virulence gene) expression up-regulated and down regulated, the final composition of virulence regulatory network complex To control the viability and pathogenicity of the Yersinia pestis in the medium and in the storage host.
The pathogenic bacteria from the induction signal process to stimulate the expression of virulence factors, virulence regulator plays an irreplaceable role. The global or specific regulators that control the expression of virulence genes; in addition, a number of virulence gene expression product can form a complex system, the implementation of the regulation of virulence function, often called the virulence regulation system. As the bacterial virulence regulation system of the main QS system, two element control system, Fur system and so on. These systems are divided into many sub control systems, control different types of virulence gene expression plays a role in different phase of bacterial virulence performance.
Zur repressor protein belongs to the Fur family, can regulate zinc ion transport, zinc ion is an important component of the.PhoP-PhoQ protein function is a two element control system in all living cells (two-component system), can control the virulence of bacteria, the bacteria involved in adaptation to Mg2+ restricted growth environment, and survival and breeding for Yersinia pestis in macrophages. In order to target gene identification of Yersinia pestis virulence regulators Zur and PhoP, especially the directly regulated target genes, and further study the fine regulation mechanism of Zur and PhoP on the downstream target gene, this study firstly uses the zur and phoP genes of one-step Red mutation deletion technology system based on the replacement of Yersinia pestis, Yersinia pestis and using whole genome DNA microarrays for gene transcription through spectrum analysis. The transcriptional difference comparison of mutant and wild-type strain under specific conditions, The definition of the expression of up-regulated and down regulated genes, these genes constitute a Zur or PhoP control element. This analysis can be screened out by all Zur or PhoP directly or indirectly regulate target genes from the whole genome level. The analysis results of transcriptional profiling can be verified by Real time RT-PCR. The application of experimental techniques of gel retardation, target gene identification of direct control, select some key virulence related genes, the DNA enzyme I footprinting experiment technology, experimental technique of primer extension, in-depth study of Zur and PhoP is how to directly regulate the transcription of these genes and expression of virulence phenotype and molecular experimental data combined with the knowledge of Zur and how PhoP expression through activation or inhibition of specific genes and control of plague host adaptation and virulence.
Compared with wild strains, strains with zur gene deletion of 154 genes transcript abundance changes under stimulation of zinc ions, including 64 up-regulated and 90 genes down regulated. Summary to predict Zur binding sites, through the consensus-matrix and convert-matrix program to calculate Zur weight with four nucleotide sequence for each position the Zur binding sequence of Matrix, and then use WebLogo software to show the sequence of logo, to predict the Zur combined with box:GAAATGTTATAWTATAACATTTC. spectrum analysis of the Zur transcription motif analysis and Zur regulator based on Matrix scan Zur by our program to find conserved motifs, we selected 4 genes ykgM, weight and higher znuC znuA. AstA molecular and biochemical experiments further. The gel retardation experiments confirmed that ykgM, znuC, znuA 3 transcription units directly regulated by Zur. In order to clear the fine tuning of Zur The control mechanism, the research on the promoter of the three transcription units of footprinting experiments, we obtain precise Zur combination of protected areas, in the area of memory in the prediction of Zur box, the three Zur protein directly regulate the transcription unit, the Zur box are covered with -10 sequence, thus impeding with RNA the polymerase, probably because of this, the Zur showed inhibitory properties of zinc ions on them. Under the stimulation of wild-type and mutant zur? And primer extension experiments can not only clear by Yersinia pestis ykgM Zur transcription, transcription initiation site of znuC and znuA, can also determine the effect of Zur on at the same time with the footprint of transcription. The experimentally obtained Zur binding sites inferred from each promoter within the RNA polymerase binding site (-10 sequence) and -35 sequence, enrich the molecular properties of Zur promoter regulatory elements.
For the pre? PhoP mutant and wild-type transcription in low magnesium under the condition of spectral analysis, has been defined in PhoP regulatory element, similarly, our results with Real time RT-PCR on the transcriptome verified. EMSA identified 30 transcription units by PhoP transcription regulation of the promoter. 17 genes do footprinting experiments, 19 PhoP with protection area. Through bioinformatics analysis of Yersinia pestis PhoP box will be summarized as TGTTTAW with seven nucleotide repeat sequences to the location information, gene transcription initiation site by primer extension obtained from the experiment, according to PhoP box of Yersinia pestis in target gene promoter in the position and the presence of -35 sequences, we will PhoP promoter structure is divided into three categories: PhoP binding sites in the upstream sequence of -35; PhoP binding sites covering -35 sequence; PhoP binding sites in -10 sequences Downstream.
This is the first study to comprehensively identify regulatory elements of Yersinia pestis Zur and PhoP directly regulated at the same time, from the molecular level to clear the characteristics of Yersinia pestis Zur box and PhoP box, Zur and PhoP on the mechanism of transcriptional regulation of Zur and PhoP, for the construction of this kind of virulence regulators, regulatory networks, and these regulatory elements to further clarify the function and provide experimental evidence, the system mechanism will lay an important foundation for the study of Yersinia pestis pathogenesis and vaccine.

【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)位級別】：博士
【學(xué)位授予年份】：2008
【分類號】：R378

【共引文獻(xiàn)】

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1 王琪;朱延明;王冬冬;;酵母單雜交系統(tǒng)在植物基因工程研究中的應(yīng)用[J];北京林業(yè)大學(xué)學(xué)報;2008年01期

2 丁寧;肖慧;許立新;佘守章;;利用生物素-鏈親和素系統(tǒng)篩選呼吸機所致肺損傷HMGB1啟動子結(jié)合蛋白[J];第四軍醫(yī)大學(xué)學(xué)報;2009年21期

3 黃敏;;基因敲除技術(shù)及其應(yīng)用[J];廣東輕工職業(yè)技術(shù)學(xué)院學(xué)報;2009年04期

4 Taiho Kambe;Tomoyuki Suzuki;Masaya Nagao;Yuko Yamaguchi-Iwai;;Sequence Similarity and Functional Relationship Among Eukaryotic ZIP and CDF Transporters[J];Genomics Proteomics & Bioinformatics;2006年01期

5 趙鐵;方幸幸;劉曉露;彭亮;龍敏;張文炳;羅軍;曹虹;;尿路致病性大腸埃希菌外膜蛋白T敲除株的構(gòu)建及功能評價[J];南方醫(yī)科大學(xué)學(xué)報;2012年07期

6 徐龍鑫;楊勝林;楊海兵;李愛萍;王忠霞;;西南矮馬肌肉生長抑制素基因啟動子區(qū)PCR-RFLP多態(tài)性檢測[J];中國畜牧獸醫(yī);2012年09期

7 吳順華;鐘彥偉;成軍;鄭玉建;;篩選肝細(xì)胞p53啟動子結(jié)合蛋白的噬菌體表面展示技術(shù)[J];環(huán)境與健康雜志;2011年07期

8 李俊峰;姚淑敏;李紅芳;田智剛;郭慶杰;;一株海洋細(xì)菌鐵吸收調(diào)節(jié)蛋白(Fur)基因的克隆和表達(dá)[J];海洋湖沼通報;2009年04期

9 王妍妍;李貴陽;李杰;肖鵬;莫照蘭;;遲緩愛德華氏菌OppA蛋白的免疫原性及免疫保護分析[J];海洋科學(xué);2011年05期

10 金銳;;同源重組的好工具,Red重組系統(tǒng)[J];科教文匯(下旬刊);2007年10期

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