阴沟肠杆菌Enterobacter cloacae FRM抗氟机制的解析

发布时间：2018-03-23 05:02

本文选题：阴沟肠杆菌FRM　切入点：氟抗性　出处：《中国农业科学院》2017年博士后论文　论文类型：学位论文

【摘要】：氟元素是一种广泛分布在自然界中的活跃的非金属元素,且高浓度的氟离子对生物体具有一定的抑制或杀死作用。氟离子具有防龋功能,因而被广泛应用于口腔清洁产品中。氟离子的普遍存在和应用以及其生物毒性导致了自然界中出现了许多具有氟抗性的生物,然而到目前为止,人们对生物氟抗性机制并没有完全了解。我们实验室前期分离得到一株能在高达4,000 mg/L的氟离子浓度下生存的高氟抗性菌株Enterobacter cloacae FRM,并且通过比较基因组学,转录组学等分析发现,该菌株基因组中的基因岛3对其高氟抗性具有至关重要的作用。本研究旨在寻找基因岛3上参与氟抗性的基因,解析菌株E.cloacae FR]M中高氟抗性的具体机制。本研究通过RT-PCR及qRT-PCR确认了基因岛3上的六个基因(ppaC、uspA、eno、gpmA、crcB和orf5249,分别编码氟离子通道蛋白、抗压力蛋白及氟离子抑制的酶等)组成一个操纵子,并且通过启动子区域的氟离子核糖体开关在氟离子诱导下转录成两组mRNA。为进一步探究此六个基因的作用,我们利用CRISPR-Cas9技术构建了菌株E.cloacae FRM的基因敲除系统。六个基因的敲除与回补实验数据表明:此六个基因相互作用,共同赋予了E.cloacae FRM极高的抗氟能力。本研究阐明了E.cloacac FRM的抗氟机制,加深了我们对微生物抵抗高浓度氟离子的生理机制的认识,同时也为抗氟微生物的进一步应用,如开发更有效的防龋口腔清洁用品,氟污染的微生物治理等提供了理论基础。
[Abstract]:Fluorine is an active nonmetallic element widely distributed in nature, and high concentration of fluoride can inhibit or kill organisms. Therefore, it is widely used in oral cleaning products. The widespread presence and application of fluorine ions and their biological toxicity have led to the emergence of many fluorine-resistant organisms in nature, but so far, The mechanism of biological fluorine resistance was not fully understood. We isolated a high fluorine resistant strain, Enterobacter cloacae FRM, which could survive at concentrations of up to 4000 mg/L in our laboratory, and found it by comparative genomics, transcriptome and other analyses. The gene island 3 in the genome of this strain plays an important role in its high fluorine resistance. The specific mechanism of high fluorine resistance in strain E.cloacae FR] M was identified by RT-PCR and qRT-PCR. Six genes on gene island 3, PPA CuspAgno gpmAfcrcB and orf5249, which encode fluorine channel protein, anti-stress protein and fluorine ion-inhibiting enzyme, respectively, constitute an operon. And two groups of mRNAs were transcribed into two sets of mRNAs by the fluorine ribosome switch in the promoter region to further explore the role of these six genes. The gene knockout system of strain E.cloacae FRM was constructed by using CRISPR-Cas9 technique. In this study, the anti-fluorine mechanism of E.cloacac FRM has been elucidated, which has deepened our understanding of the physiological mechanism of microbial resistance to high concentration of fluorine ions, and is also the further application of fluorine-resistant microbes. For example, the development of more effective dental cleaning products, fluoride pollution of microbial treatment provides a theoretical basis.
【学位授予单位】：中国农业科学院
【学位级别】：博士后
【学位授予年份】：2017
【分类号】：R78

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