多硫化物对大肠杆菌OxyR regulon活性状态的影响

发布时间：2018-01-06 08:30

  本文关键词：多硫化物对大肠杆菌OxyR regulon活性状态的影响　出处：《山东大学》2017年硕士论文　论文类型：学位论文

  更多相关文章： OxyR polysulfides 信号功能

【摘要】：H_2S作为第三种气体信号分子的假说由来已久,H_2S的生理作用表现为两重性,一方面,H_2S能抑制细胞色素氧化酶的活性[1],影响电子传递链上的电子传递,抑制生物呼吸;另一方面,适量的H_2S又对生物体的心血管系统、中枢神经系统及炎症反应的调节等方面有重要功能[2]。现有研究表明,H_2S的信号作用与靶向蛋白的硫巯基化(-SSH)修饰有关。但是这一修饰仍然存在争议,也有研究者认为H_2S是通过硫烷(S0,sulfane sulfur)对蛋白进行了-SSH的修饰。H_2S作用的两重性还表现在其与活性氧自由基(ROS)的关系上。有研究表明,H_2S的积累可以使生物体内ROS提高;但也有文章证明,敲除大肠杆菌中主要的负责产生H_2S的基因3-巯基丙酮酸转移酶(sseA),会导致大肠杆菌对抗生素(通过氧化压力升高造成细菌死亡的抗生素类型)的抗性降低。可见,H_2S与生物体内的ROS息息相关,本论文以大肠杆菌为载体,构建了一系列不同H_2S产量的菌株,在此基础上对H_2S与氧化应激之间的关系作了相应的研究。通过survival test、抑菌圈的测定等实验,证实H_2S在改变菌体内的氧化还原状态及应对外界氧化压力方面都有相应功能。选取了大肠杆菌主要应激系统—ArcAB系统、SoxRS调控系统及OxyR调节子进行研究,结果表明,多硫化物(polysulfides)可以使OxyR的活性状态发生改变,进而影响菌体内若干基因的表达。本论文首次提出OxyR除了受H_2O_2诱导外,还受到polysulfides的调控。论文选取已有文献报道的受OxyR调控的四个基因katG、dps、grxA、trxC进行实验,用PCR的方法.扩增这些基因的启动子区,构建含有egfp/mkate荧光基因的报告质粒,利用诱导物诱导大肠杆菌本底水平或经质粒过量表达的OxyR,通过荧光值的改变来判定OxyR的状态变化。氧化态和还原态的OxyR虽然都能与DNA结合但只有氧化态OxyR才能激活下游基因的表达。利用H_2O_2和polysulfides作为诱导物进行比较分析,我们得出,在对OxyR的诱导方面,polysulfides所起作用与H_2O_2相似,不同在于,较低浓度的polysulfides就可以使OxyR产生构象的改变,并且敲除oxyR基因,polysulfides对菌体生长的抑制作用比H_2O_2更强烈。这说明,OxyR对polysulfides更为敏感。通过对其反应机理的分析,我们发现通过polysulfides处理的OxyR更易以四聚体的形式与DNA结合,说明polysulfides对OxyR活性的调节可能是通过改变蛋白的聚合状态来实现的。本论文还对OxyR蛋白受诱导后的结构改变做了分析,通过DTT、H_2O_2、polysulfides分别对纯蛋白进行了处理,并进行了MALDI-TOF分析。结果表明,H_2O_2和polysulfides对蛋白的修饰是两种不同的形式。并且无论何种处理,OXyR活性状态时两个活性位点的半胱氨酸之间都没有二硫键的生成。除此之外,我们对OxyR氧化过后还原型状态的恢复做了相应研究,据已有文献报道grxA、trxC基因对OxyR的还原起到重要作用。但是我们对这两个基因进行敲除发现OxyR的活性没有发生明显改变。本论文首次提出polysulfides对OxyR具有诱导作用,为OxyR的研究开辟了新的方向,同时,我们也证明不同的信号分子对OxyR的修饰形式不同,不同的修饰形式对OxyR功能的影响不同,打破了传统意义上OxyR只通过二硫键的形成进行活性转变的说法。
[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.

【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q78

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1 阎珍珍;多硫化物对大肠杆菌OxyR regulon活性状态的影响[D];山东大学;2017年

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