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题名: 硝化细菌群体感应信号分子及其产生效应的研究
作者: 申秋璇1
学位类别: 博士
答辩日期: 2017-05
授予单位: 中国科学院大学
授予地点: 北京
导师: 庄国强
关键词: 群体感应,酰基高丝氨酸内酯,硝化细菌,维氏硝化杆菌,遗传操作 ; Quorum sensing, Acyl-homoserine lactone, Nitrite-oxidizing bacteria, Nitrobacter winogradskyi, Genetic operation
其他题名: Study on the Quorum Sensing Molecule and Its Effect in Nitrite-oxidizing Bacteria
学位专业: 环境科学
中文摘要: 在地球的氮化学循环过程中,微生物是主要的驱动力,包括固氮作用、氨氧化作用、硝化作用、反硝化作用和氨化作用。氮循环过程中微生物之间的相互作用机制是目前环境微生物学家最为关注的领域,理解物质循环及细菌间的协调控制也是其中最为重要的科学问题之一。微生物的群体感应(Quorum Sensing,QS)是通过向环境中释放信号分子并感应其浓度变化,当信号分子在环境中积累到一定阈值时可以调控微生物生理代谢的行为。然而目前对氮循环过程中细菌群体感应调控过程的认识仍不完整。本论文以典型的硝化模式细菌维氏硝化杆菌(Nitrobacter winogradskyi)为研究对象,利用生物信息学及分子生物学等研究方法对预估的酰基高丝氨酸内酯(acyl-homoserine lactones,AHL)信号分子合成酶基因进行了异源表达,完成了硝化细菌群体感应系统的验证工作。同时在硝化细菌中发现一种新的信号分子,这种信号分子对细菌的硝化过程有一定的影响。本研究获得了以下创新性结果: 1)首次在N. winogradskyi中发现了功能性的AHLs合成酶基因。本研究选取了三种硝化过程模式细菌(Nitrosomonas europaea,Nitrobacter winogradskyi,Nitrobacter hamburgensis)进行AHLs信号分子合成酶基因的验证。利用生物信息学手段比对出三种细菌中可能的AHLs信号分子合成酶基因,导入到大肠杆菌中进行异源表达,发现位于N. winogradskyi中的AHLs信号分子合成酶序列(nwiI)能够成功表达,并催化合成C7-HSL、C8-HSL、C9-HSL、C10-HSL、C8:1-HSL、C9:1-HSL、C10:1-HSL和C11:1-HSL八种不同链长及不同饱和度的信号分子。 2)在N. winogradskyi中发现了一种新的信号分子:7,8-trans-N-(decanoyl) homoserine lactone(C10:1-HSL)。通过LC-MS技术对nwiI异源表达所产生的信号分子种类进行鉴定的过程中,发现有一种高表达量的信号分子与已知的标准品无法对应。通过核磁共振氢谱(1H NMR)和碳谱(13C NMR)的测定,发现是一种具有C=C双键的信号分子,最终通过二维化学位移相关谱(COSY)确定了酰基侧链中的双键位于C7和C8分子中间。结合LC-MS中离子碎片的结果,确定该信号分子所对应的结构为7,8-trans-N-(decanoyl) homoserine lactone (C10:1-HSL)。通过外源添加该信号分子,发现其对硝化过程有一定影响。
英文摘要: In the process of nitrogen chemical cycling, microbes are the main driving forces, including nitrogen fixation, ammoxidation, nitrification, denitrification and ammoniation. The mechanism of interaction between microorganisms in the process of nitrogen cycling is one of the most important areas of environmental microbiologists, and it is one of the most important scientific problems to understand the material circulation and the coordination control between bacteria. In quorum sensing system,bacteria can regulate bacteria behaviors at population level by releasing and sensing autoinducers that accumulate to a threshold. The understanding of the process of quorum sensing during the nitrogen cycle is still incomplete. In this paper, the typical nitrification model bacteria was used as the research object, and the predicted signal molecular acyl-homoserine lactones synthase gene was expressed successfully in Escherichia coli. At the same time in the nitrifying bacteria found a new signal molecules, this signal molecules on the nitrification process of bacteria have a certain impact. The major contents and findings of the dissertation are as follows: 1) For the first time, the functional AHLs synthase gene was found in the nitrifying bacteria Nitrobacter winogradskyi. In this study, three nitrification process bacteria (N. europaea, N. winogradskyi, N. hamburgensis) were selected to validate the AHLs signal molecular synthase gene. By bioinformatics in comparison with the possible synthesis of AHLs signal molecular synthase gene from three kinds of bacteria and introduced into E. coli, the results showed that the AHLs signal molecule synthesis enzyme sequence (nwiI) in N. winogradskyi could be successfully expressed, and it can synthesize eight different chain lengths and different saturated signal molecules (C7-HSL, C8-HSL, C9-HSL, C10-HSL, C8:1-HSL, C9:1-HSL, C10:1-HSL and C11:1-HSL). 2) A new signaling molecule, 7,8-trans-N- (decanoyl) homoserine lactone (C10:1-HSL), was found in N. winogradskyi. In the process of identifying the signal molecular species produced by the nwiI heterologous expression by LC-MS, it was found that a high level of signal molecules could not correspond to the known standard. A molecule with a C=C double bond was found according to the NMR spectroscopy (1H NMR) and carbon spectrum (13C NMR). Finally, the acyl side chain was determined by two-dimensional chemical shift correlation spectroscopy (COSY). The structure of this new signal molecule was 7,8-trans-N- (decanoyl) homoserine lactone (C10:1-HSL). The affinity of the bacterium to nitrite was slightly increased by adding 2μM of C10:1-HSL signal molecule to N. winogradskyi, but there was no significant difference in the Km value of nitrification.
内容类型: 学位论文
URI标识: http://ir.rcees.ac.cn/handle/311016/38687
Appears in Collections:中科院环境生物技术重点实验室_学位论文

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申秋璇. 硝化细菌群体感应信号分子及其产生效应的研究[D]. 北京. 中国科学院大学. 2017.
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