本文選題：鼠疫耶爾森氏菌 + 轉錄調控��； 參考：《中國人民解放軍軍事醫(yī)學科學院》2011年碩士論文

【摘要】：鼠疫是由鼠疫耶爾森氏菌(以下簡稱鼠疫菌)引起的一種自然疫源性疾病。鼠疫菌在傳播環(huán)節(jié)中宿主動物的改變以及致宿主病變的過程中,受到諸多環(huán)境信號,比如抗菌肽、溫度、pH、滲透壓等的刺激,鼠疫菌能感應這種復雜的信號刺激并自我調節(jié),產生毒力因子,最終使鼠疫菌得以存活。鼠疫菌自我調節(jié)機制中,轉錄調控子對靶基因在轉錄水平上的調節(jié)至關重要。PhoP和RovA是鼠疫菌重要的毒力調控因子,pH6抗原是鼠疫菌重要的毒力相關因子,它們之間聯(lián)系至今并未被闡明。 PhoP是二元調節(jié)系統(tǒng)PhoP-PhoQ的調節(jié)蛋白,PhoP-PhoQ系統(tǒng)能夠感應低Mg2+、微酸性環(huán)境以及抗菌肽的刺激信號,從而激活PhoP蛋白的轉錄調節(jié)活性,上調或下調靶基因的轉錄。本實驗室前期研究發(fā)現(xiàn),鼠疫菌PhoP蛋白是整體調控子,表達譜結果表明,在低Mg2+條件下,有706個基因的轉錄受PhoP的影響,包括pH6抗原基因、PhoP-PhoQ系統(tǒng)及rovA基因。 小腸結腸炎耶爾森氏菌rovA缺失株對小鼠毒力下降,表明RovA是其重要的毒力調控因子。RovA能以二聚化的形式結合到靶基因啟動子區(qū)調控基因轉錄,在小腸結腸炎耶爾森氏菌和假結核耶爾森氏菌中,它不僅能激活侵襲因子基因inv的表達,還能激活自身的表達以及其他毒力相關基因的表達。在鼠疫菌中,也有學者進行了RovA的芯片表達譜分析,發(fā)現(xiàn)RovA是一個全局性毒力調控因子,能調控數(shù)十個基因的表達,包括pH6抗原基因,但是詳細調控機制還沒見報道。 鼠疫菌pH6抗原由基因簇psaEFABC編碼,其中psaABC編碼結構亞單位(A)及其伴侶蛋白(B)和膜引領蛋白(C);而psaEF表達產物可能對psaA的轉錄調控有關。pH6抗原最高表達是在環(huán)境pH=6、溫度在34℃或更高條件下實現(xiàn),且只有在34℃以上才能發(fā)揮毒力效應。pH6抗原是鼠疫菌的毒力相關因子,與腺鼠疫的發(fā)生密切相關。 根據(jù)表達譜和生物信息學預測,在轉錄水平上,PhoP對psaA、psaE、自身操縱子基因以及rovA可能具有調控作用,同時RovA對自身基因及psaA、psaE的轉錄也可能具有調節(jié)作用。本研究的目的在于闡明上述調控子對可能的靶基因的調控機制。 我們基于Red系統(tǒng)分別構建了鼠疫菌phoP和rovA基因的突變株,利用大腸桿菌BL21-DE3的蛋白表達系統(tǒng)分別獲得His-PhoP和His-RovA重組蛋白,再利用凝膠阻滯實驗(EMSA)、DNaseⅠ足跡實驗、β-半乳糖苷酶報告基因融合實驗(LacZ實驗)以及引物延伸實驗等來詳細研究PhoP和RovA對各自靶基因的轉錄調控機制。 實驗結果表明:在低Mg2+、對數(shù)生長中期條件下,在轉錄水平上PhoP抑制psaA、psaE、rovA的表達,而激活自身操縱子基因YPO1635、phoP的轉錄;pH5.8、對數(shù)生長中期條件下,在轉錄水平上PhoP抑制psaA的表達,而對其它所研究基因無影響;在對數(shù)生長中期,RovA能激活psaA、psaE以及rovA的轉錄;在對數(shù)生長中期,psaA的轉錄受酸的調節(jié),pH5.8較pH7.2條件下高表達,但是溫度對其無影響。 在rovA基因啟動子區(qū)發(fā)現(xiàn)了兩個轉錄起始位點,分別命名為P1和P2,其中P1靠近翻譯起始位點,它的轉錄啟動受PhoP的抑制而受RovA的激活,P2只受RovA的激活而不受PhoP的影響;DNaseⅠ足跡實驗表明RovA對自身啟動子區(qū)有兩個親和力不等的結合位點site1和site2,前者親和力高于后者,site1位于P2之前,因此我們認為site1控制著P2的轉錄,site2位于P1之后,推測RovA與site2的結合對rovA的轉錄具有負反饋調節(jié)作用,即RovA的自調控是雙重性質的。 在psaE啟動子區(qū)分別得到了PhoP和RovA的結合位點,并發(fā)現(xiàn)了三個距離很近的轉錄起始位點,PhoP的結合位點幾乎覆蓋了這三個位點,RovA的結合位點緊挨著PhoP結合位點位于其上游而遠離轉錄起始位點,體內實驗也證明,在特定條件下PhoP對psaE轉錄具有抑制作用,而對rovA具有激活作用。 PhoP和RovA對psaA啟動子的結合位點互相重疊,都位于轉錄起始位點之后,體內實驗表明,無論是高Mg~(2+)還是低Mg~(2+)以及酸性環(huán)境下,RovA對psaA的轉錄都具有促進作用,但是在低Mg~(2+)和酸性條件下,PhoP對psaA的調控具有抑制作用,由此可見,PhoP和RovA通過競爭psaA啟動子區(qū)相同的結合位點,共同調節(jié)psaA的轉錄。 低Mg~(2+)條件下,YPO1635受PhoP的正調控在其它文獻資料中已經(jīng)得到驗證,本文也證明了這種調控關系,同時也證明了PhoP確實正調控自身的轉錄,但是所發(fā)現(xiàn)的兩個轉錄起始位點P1和P2,在細菌生長對數(shù)中期時只有P2是PhoP依賴性的。 綜上,本研究利用分子生化試驗手段,首次揭示了鼠疫菌中的PhoP-RovA-psa轉錄調控環(huán)路:RovA正調控psa位點和自身的轉錄;低Mg~(2+)環(huán)境下,PhoP抑制psa位點和rovA的轉錄,而激活自身操縱子的轉錄,PhoP也能感應酸性信號,抑制psaA的轉錄;psaA的轉錄調節(jié)還受酸的調節(jié),pH5.8較pH7.2條件下高表達,但是溫度對其無影響。
[Abstract]:Yersinia pestis is a natural epidemic disease caused by the Yersinia pestis (hereinafter referred to as Yersinia pestis). In the process of the change of the host animal and the host disease, the Yersinia pestis is stimulated by a number of environmental signals, such as antimicrobial peptides, temperature, pH, osmotic pressure, and so on. Yersinia pestis can induce such complex signal stimulation and self - stimulation. I regulate, produce the virulence factor and eventually make the Yersinia pestis survive. In the self-regulation mechanism of Yersinia pestis, the regulation of the transcriptional regulator on the target gene at the transcriptional level is essential.PhoP and RovA are important virulence factors of Yersinia pestis, and the pH6 antigen is an important virulence related factor of Yersinia pestis, and the connection between them has not been elucidated.
PhoP is the regulation protein of the two element regulation system PhoP-PhoQ. The PhoP-PhoQ system can induce the low Mg2+, the micro acid environment and the stimulation signal of the antibacterial peptide, thus activating the transcriptional regulation activity of the PhoP protein and up regulating or down regulating the transcription of the target gene. Under low Mg2+ conditions, transcription of 706 genes is affected by PhoP, including pH6 antigen gene, PhoP-PhoQ system and rovA gene.
The virulence of the rovA strain rovA strain of enterocolitis shows that RovA is an important virulence regulator,.RovA, which can be combined in the form of dimerization to regulate gene transcription in the target gene promoter region. It not only activates the expression of the invasion factor gene inv in the enterocolitis of the enterocolitis, and in the Mycobacterium tuberculosis, Jerson S bacteria. It also activates its own expression and the expression of other virulence related genes. In Yersinia pestis, some scholars have carried out RovA chip expression analysis, and found that RovA is a global virulence regulator, which can regulate the expression of dozens of genes, including the pH6 antigen gene, but the detailed regulation mechanism has not yet been reported.
The pH6 antigen of Yersinia pestis is encoded by the gene cluster psaEFABC, in which psaABC encodes the structural subunit (A) and its chaperone protein (B) and membrane leading protein (C); while the psaEF expression product may express the highest expression of the.PH6 antigen on psaA's transcriptional regulation in environmental pH=6, the temperature is at 34 or more, and the virulence can be exerting only at 34 degrees centigrade. The effect.PH6 antigen is a virulence related factor of Yersinia pestis, and is closely related to the occurrence of bubonic plague.
According to the expression spectrum and bioinformatics, PhoP may play a regulatory role on psaA, psaE, the operon gene and rovA at the transcriptional level, and RovA may also regulate the transcription of its own genes and psaA and psaE. The purpose of this study is to elucidate the regulatory mechanism of the above regulator on the possible target genes.
The mutant strains of Yersinia pestis phoP and rovA gene were constructed based on the Red system. The recombinant protein of His-PhoP and His-RovA was obtained by the protein expression system of Escherichia coli BL21-DE3, and then the gel block experiment (EMSA), DNase I footprint experiment, beta galactosidase report gene fusion experiment (LacZ experiment) and primer extension experiment were used. To study the transcriptional regulation mechanism of PhoP and RovA on their respective target genes.
The experimental results showed that PhoP inhibited psaA, psaE, rovA expression at the transcriptional level at low Mg2+, at the transcriptional level, and activated the autoperon gene YPO1635, phoP transcription; pH5.8, at the medium-term condition of logarithmic growth, the PhoP inhibited psaA, but had no influence on other studied genes; in the middle period of logarithmic growth, Ro VA activates the transcription of psaA, psaE and rovA; in the middle of logarithmic growth, the transcription of psaA is regulated by acid, and pH5.8 is highly expressed in the pH7.2 condition, but the temperature has no effect on it.
Two transcriptional starting sites were found in the promoter region of the rovA gene, named P1 and P2, in which P1 was close to the translation initiation site, and its transcriptional initiation was activated by RovA, and P2 was only activated by RovA and was not affected by PhoP; DNase I footprint experiments showed that RovA had two unequal affinity to the self promoter region. Loci site1 and site2, the former is higher than the latter, and site1 is before P2. Therefore, we think site1 controls the transcription of P2. After site2 is located in P1, it is assumed that the combination of RovA and site2 has a negative feedback regulation on rovA transcription, that is, the self regulation of RovA is dual properties.
The binding sites of PhoP and RovA were obtained at the psaE promoter region, and three close transcriptional starting sites were found. The binding site of PhoP was almost covered by these three loci. The binding site of RovA was close to the PhoP binding site at its upstream and far away from the transcriptional starting site. In vivo experiments also proved that PhoP against PS under specific conditions. AE transcription has inhibitory effect on rovA.
The binding sites of PhoP and RovA overlapped with the psaA promoter, both at the starting site of the transcriptional starting point. In vivo experiments showed that RovA had a promoting effect on the transcription of psaA in both high Mg~ (2+) or low Mg~ (2+) and acidic environments, but under the low Mg~ (2+) and acid conditions, PhoP had an inhibitory effect on the regulation. OP and RovA co regulate psaA transcription by competing for the same binding sites in psaA promoter region.
Under the condition of low Mg~ (2+), the positive regulation of YPO1635 by PhoP has been verified in other literature. This paper also proves this regulation relationship, and also proves that PhoP does regulate its own transcription, but the two transcriptional starting sites, P1 and P2, are PhoP dependent in the mid-term logarithm of bacterial growth.
To sum up, this study revealed the PhoP-RovA-psa transcriptional regulation loop in Yersinia pestis for the first time: RovA is regulating the PSA locus and its own transcription. In low Mg~ (2+) environment, PhoP inhibits the transcription of PSA loci and rovA, and activates the transcription of the operon, PhoP can also induce acid signals, inhibit the transcription of psaA; psaA turn. The regulation of pH5.8 was also regulated by acid. The expression of pH5.8 was higher than that of pH7.2, but temperature had no effect on it.
【學位授予單位】：中國人民解放軍軍事醫(yī)學科學院
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R378

【共引文獻】

相關期刊論文 前10條

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本文編號：1971997