本文關(guān)鍵詞：多硫化物對大腸桿菌OxyR regulon活性狀態(tài)的影響　出處：《山東大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： OxyR polysulfides 信號功能

【摘要】：H_2S作為第三種氣體信號分子的假說由來已久,H_2S的生理作用表現(xiàn)為兩重性,一方面,H_2S能抑制細(xì)胞色素氧化酶的活性[1],影響電子傳遞鏈上的電子傳遞,抑制生物呼吸;另一方面,適量的H_2S又對生物體的心血管系統(tǒng)、中樞神經(jīng)系統(tǒng)及炎癥反應(yīng)的調(diào)節(jié)等方面有重要功能[2]�，F(xiàn)有研究表明,H_2S的信號作用與靶向蛋白的硫巰基化(-SSH)修飾有關(guān)。但是這一修飾仍然存在爭議,也有研究者認(rèn)為H_2S是通過硫烷(S0,sulfane sulfur)對蛋白進(jìn)行了-SSH的修飾。H_2S作用的兩重性還表現(xiàn)在其與活性氧自由基(ROS)的關(guān)系上。有研究表明,H_2S的積累可以使生物體內(nèi)ROS提高;但也有文章證明,敲除大腸桿菌中主要的負(fù)責(zé)產(chǎn)生H_2S的基因3-巰基丙酮酸轉(zhuǎn)移酶(sseA),會(huì)導(dǎo)致大腸桿菌對抗生素(通過氧化壓力升高造成細(xì)菌死亡的抗生素類型)的抗性降低�？梢�,H_2S與生物體內(nèi)的ROS息息相關(guān),本論文以大腸桿菌為載體,構(gòu)建了一系列不同H_2S產(chǎn)量的菌株,在此基礎(chǔ)上對H_2S與氧化應(yīng)激之間的關(guān)系作了相應(yīng)的研究。通過survival test、抑菌圈的測定等實(shí)驗(yàn),證實(shí)H_2S在改變菌體內(nèi)的氧化還原狀態(tài)及應(yīng)對外界氧化壓力方面都有相應(yīng)功能。選取了大腸桿菌主要應(yīng)激系統(tǒng)—ArcAB系統(tǒng)、SoxRS調(diào)控系統(tǒng)及OxyR調(diào)節(jié)子進(jìn)行研究,結(jié)果表明,多硫化物(polysulfides)可以使OxyR的活性狀態(tài)發(fā)生改變,進(jìn)而影響菌體內(nèi)若干基因的表達(dá)。本論文首次提出OxyR除了受H_2O_2誘導(dǎo)外,還受到polysulfides的調(diào)控。論文選取已有文獻(xiàn)報(bào)道的受OxyR調(diào)控的四個(gè)基因katG、dps、grxA、trxC進(jìn)行實(shí)驗(yàn),用PCR的方法.擴(kuò)增這些基因的啟動(dòng)子區(qū),構(gòu)建含有egfp/mkate熒光基因的報(bào)告質(zhì)粒,利用誘導(dǎo)物誘導(dǎo)大腸桿菌本底水平或經(jīng)質(zhì)粒過量表達(dá)的OxyR,通過熒光值的改變來判定OxyR的狀態(tài)變化。氧化態(tài)和還原態(tài)的OxyR雖然都能與DNA結(jié)合但只有氧化態(tài)OxyR才能激活下游基因的表達(dá)。利用H_2O_2和polysulfides作為誘導(dǎo)物進(jìn)行比較分析,我們得出,在對OxyR的誘導(dǎo)方面,polysulfides所起作用與H_2O_2相似,不同在于,較低濃度的polysulfides就可以使OxyR產(chǎn)生構(gòu)象的改變,并且敲除oxyR基因,polysulfides對菌體生長的抑制作用比H_2O_2更強(qiáng)烈。這說明,OxyR對polysulfides更為敏感。通過對其反應(yīng)機(jī)理的分析,我們發(fā)現(xiàn)通過polysulfides處理的OxyR更易以四聚體的形式與DNA結(jié)合,說明polysulfides對OxyR活性的調(diào)節(jié)可能是通過改變蛋白的聚合狀態(tài)來實(shí)現(xiàn)的。本論文還對OxyR蛋白受誘導(dǎo)后的結(jié)構(gòu)改變做了分析,通過DTT、H_2O_2、polysulfides分別對純蛋白進(jìn)行了處理,并進(jìn)行了MALDI-TOF分析。結(jié)果表明,H_2O_2和polysulfides對蛋白的修飾是兩種不同的形式。并且無論何種處理,OXyR活性狀態(tài)時(shí)兩個(gè)活性位點(diǎn)的半胱氨酸之間都沒有二硫鍵的生成。除此之外,我們對OxyR氧化過后還原型狀態(tài)的恢復(fù)做了相應(yīng)研究,據(jù)已有文獻(xiàn)報(bào)道grxA、trxC基因?qū)xyR的還原起到重要作用。但是我們對這兩個(gè)基因進(jìn)行敲除發(fā)現(xiàn)OxyR的活性沒有發(fā)生明顯改變。本論文首次提出polysulfides對OxyR具有誘導(dǎo)作用,為OxyR的研究開辟了新的方向,同時(shí),我們也證明不同的信號分子對OxyR的修飾形式不同,不同的修飾形式對OxyR功能的影響不同,打破了傳統(tǒng)意義上OxyR只通過二硫鍵的形成進(jìn)行活性轉(zhuǎn)變的說法。
[Abstract]:H_2S is the third gaseous signal molecule of the long-standing hypothesis, the physiological role of H_2S is duality, on the one hand, [1] H_2S can inhibit the activity of cytochrome oxidase, effect of electron transfer chain electronic transfer, inhibition of biological respiration; on the other hand, the amount of H_2S and the biological regulation of the cardiovascular system, central nervous system the nervous system and the inflammatory response has an important function to the existing [2]. research shows that the H_2S signal and targeting protein thiol sulfur (-SSH) modification. But this modification is still controversial, some researchers believe that the H_2S is by sulforaphane (S0, sulfane, sulfur) on the duality of the modified.H_2S protein effect of -SSH is also reflected in the active oxygen free radical (ROS) relationship. Studies have shown that H_2S can make the accumulation of ROS in organism increased; but also the paper demonstrates that the main knockout Escherichia coli negative Enzyme gene 3- mercaptopyruvate transfer of responsibilities to produce H_2S (sseA), will lead to Escherichia coli to antibiotics (by elevated oxidative stress caused by the death of bacteria antibiotic resistance type) decreased. Obviously, H_2S and ROS are closely related to the organism, the Escherichia coli as a carrier to build a series of different H_2S yield strains on this basis, the research on the relationship between H_2S and oxidative stress. By survival test, the experimental determination of bacteriostatic ring, confirmed H_2S redox state and respond to oxidative stress in the oxidative changes in bacteria are selected. The corresponding function of Escherichia coli main stress system - ArcAB system, SoxRS control system and OxyR regulator was studied. The results show that the polysulfide (polysulfides) can make the OxyR active state change, thereby affecting the expression of some genes of bacteria in the first. A OxyR addition induced by H_2O_2, but also by the regulation of polysulfides. The paper selects four katG genes regulated by OxyR have been reported in the literature DPS, grxA, trxC experiments were conducted by using the method of PCR. Amplification of the promoter regions of these genes, construction of a reporter plasmid egfp/mkate fluorescent gene, using inducer the level of Escherichia coli induced by plasmid or overexpression of OxyR, to determine the change of OxyR status by fluorescence value. The change of oxidation state and reduction state of OxyR although can express only oxidized OxyR to activate downstream gene combined with DNA as inducer. Compared with H_2O_2 and polysulfides we obtained in the aspect of OxyR, induced by polysulfides, the effect is similar to H_2O_2, is different, low concentrations of polysulfides can make the OxyR conformational change and oxyR gene knockout, polysulfi The inhibitory effect of Des on cell growth than H_2O_2 more strongly. This shows that OxyR is more sensitive to polysulfides. Through the analysis of the reaction mechanism, we found that the combination of form and DNA by polysulfides OxyR easier to dimerization of four polysulfides on the activity of OxyR may be adjusted by changing the polymerization state of protein to achieve. This paper also studies the structure of OxyR protein by induced changes were analyzed by DTT, H_2O_2, polysulfides respectively were treated with purified protein, and MALDI-TOF analysis. The results showed that the H_2O_2 and polysulfides of the protein is modified in two different forms. And no matter what kind of treatment, OXyR the activity state between cysteine two active sites are not generated two disulfide bonds. In addition, some researches have been done on OxyR oxidation after restoration of our prototype state, according to the literature report GrxA, trxC gene plays an important role in the reduction of up to OxyR. But we found that knockout OxyR activity had no significant changes of the two genes for the first time in this paper. Polysulfides showed induction effect on OxyR, OxyR research has opened up a new direction, at the same time, we also demonstrated that the modified form of signal molecules different to the different OxyR, the modification of different forms of OxyR different functions, breaking the traditional sense OxyR only by forming two disulfide bonds in the activity change statement.

【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：Q78

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1 閻珍珍;多硫化物對大腸桿菌OxyR regulon活性狀態(tài)的影響[D];山東大學(xué);2017年

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本文編號：1387095