氟环境下变形链球菌耐氟菌株ciaH、eno、pykF和rpl基因的差异表达及其意义

发布时间：2018-06-19 11:31

本文选题：变形链球菌耐氟菌株 + 氟抗性　；参考：《吉林大学》2017年硕士论文

【摘要】：目的:龋病是人类最常见的口腔疾病之一,其主要致龋菌是变形链球菌(Streptococcus mutans,简称S.mutans)。而氟化物能有效抑制变形链球菌生长,1g/L的氟化物浓度就能完全杀灭变形链球菌[1]。目前,氟化物已经被广泛应用于防龋领域,但是长期局部高浓度氟化物的使用可能导致变形链球菌耐氟菌株的产生,为防龋领域带来新的挑战[2]。实验证明,耐氟菌株具有更强的产酸性、耐酸性及脱矿能力,这提示耐氟菌株具有更强的致龋性[3]。对于耐氟菌株一系列性状改变,尤其是氟抗性产生的原因尚无明确结论。一般认为是来源于细菌基因组的改变。本课题组前期通过对变形链球菌耐氟菌株进行全基因组测序[4],与亲代菌株对比,发现有20个基因及5个基因间隔发生了突变。其中4个基因,ciaH、eno、pykF和rpl可能与细菌的氟抗性相关。ciaH、eno、pykF和rpl分别编码组氨酸激酶、烯醇酶、丙酮酸激酶和转录调节因子。这四种基因都与细菌的生存密切相关,并且其牵涉的耐酸性、糖酵解和糖转运过程又与氟的抑菌机制直接相关[5]。这四种基因如果发生变化很可能引起细菌生理的变化以及氟抗性的产生。以往曾将变形链球菌亲代菌株及其耐氟菌株同时置于低浓度氟环境下来探究二者性状上的差异,本实验直接将耐氟菌株置于其诱导时达到的最大氟浓度1g/L中,试图在基因表达层面对这四种基因予以探讨,进一步解析氟抗性产生的原因,为应对耐氟菌株的产生及基因防龋提供理论基础。方法:1.对变形链球菌UA159及其耐氟菌株UA159-FR复苏、培养,鉴定。2.实验条件分为三组,即变形链球菌于无氟培养基培养、耐氟菌株分别于无氟及含氟量为1g/L的培养基培养,测定三者的生长曲线。3.设计16sRNA、ciaH、eno、pykF和rpl基因的引物并合成。4.按上述实验条件培养,分别于对数期(11h)及稳定期(20h)提取三组条件下菌株的总RNA并逆转录。5.采用16sRNA为内参基因,用实时荧光定量PCR技术测定三组条件下菌株的ciaH、eno、pykF和rpl基因的相对表达量。结果:1.复苏培养的变形链球菌及其耐氟菌株经生理、生化和16SrDNA鉴定后证实均为变形链球菌。2.绘制三组实验条件下菌株的生长曲线,即变形链球菌于无氟培养基培养、耐氟菌株分别于无氟及含氟量为1g/L的培养基培养。确定11h处于三者的生长对数期,20h处于三者的生长稳定期。3.成功设计并合成16sRNA、ciaH、eno、pykF和rpl基因的引物。4.成功提取三组实验条件下对数期(11h)及稳定期(20h)菌株的总RNA并逆转录。5.实时荧光定量PCR结果分析显示:(1)三组实验条件下,菌株ciaH、eno、pykF和rpl基因在对数期的表达水平均远远高于稳定期的表达水平(P0.001)。(2)与无氟培养的耐氟菌株比较,含氟培养的耐氟菌株ciaH、eno和pykF基因的表达水平在对数期及稳定期均明显升高(P0.001);rpl基因表达水平在对数期无明显差异(P0.05),而在稳定期表达升高(P0.001)。(3)无氟培养时,与亲代菌株比较,耐氟菌株eno、pykF和rpl基因的表达水平在对数期及稳定期均明显下降(P0.001);ciaH基因的表达水平在对数期无明显差异(P0.05),而在稳定期表达升高(P0.01)。结论:氟能提高变形链球菌耐氟菌株ciaH、eno和pykF基因的表达,表明这些基因与耐氟菌株氟抗性的产生相关;变形链球菌亲代菌株及其耐氟菌株eno、pykF和rpl基因的表达水平存在内源性差异,提示二者在糖转运、糖酵解等方面存在差异。这些结果有助于进一步解释耐氟菌株相较其亲代菌株在生理功能上的差异以及氟抗性产生的原因,并为研究这些基因的具体功能提供了基础。
[Abstract]:Objective: caries are one of the most common oral diseases in humans. The main cariogenic bacteria are Streptococcus mutans (Streptococcus mutans, S.mutans). Fluorides can effectively inhibit the growth of Streptococcus mutans. The fluoride concentration of 1g/L can completely kill Streptococcus mutans [1].. Fluorides have been widely used in the field of prevention and treatment, but the fluoride has been widely used in the field of prevention and treatment, but the fluoride has been widely used in the field of prevention and treatment. The use of local high concentration fluorides may lead to the production of fluorine resistant strains of Streptococcus mutans and bring new challenges to the field of caries prevention. [2]. experiments have proved that fluorine resistant strains have stronger acidity, acid resistance and demineralization. This suggests that fluorine resistant strains have stronger cariogenic [3]. for a series of fluorine resistant strains, especially fluorine. There is no clear conclusion for the cause of resistance. It is generally believed to be derived from the change of bacterial genome. The group of Streptococcus mutans was sequenced by genome sequencing [4], and compared with parental strains, 20 genes and 5 gene intervals had been mutated. 4 of them, ciaH, Eno, pykF and RPL, may be with bacteria. Fluorine resistance related.CiaH, Eno, pykF and RPL respectively encode histidine kinase, enolase, pyruvate kinase and transcriptional regulator. These four genes are closely related to the survival of bacteria, and their acid resistance, glycolysis, and sugar transport processes are directly related to the bacteriostasis mechanism of fluorine. These four genes are likely to change if changes are likely to occur. In the past, the strain of Streptococcus mutans and their fluorine resistant strains were placed at the Low Concentration Fluorine environment to explore the difference between the two characters. This experiment directly placed the fluorine resistant strain in the maximum fluorine concentration 1g/L of its induction, trying to face these four groups in the gene expression layer. To further analyze the causes of fluorine resistance, and to provide a theoretical basis for the production of fluorine resistant strains and genetic prevention of caries. Methods: 1. groups of Streptococcus mutans UA159 and their fluorine resistant strains UA159-FR resuscitation, culture and identification of.2. were divided into three groups, namely, Streptococcus mutans in fluorine free medium culture, fluorine resistant strains in fluorine free and The growth curve.3. containing the fluorine content was 1g/L, and the growth curve of the three was determined by.3.. The primers of 16sRNA, ciaH, Eno, pykF and RPL were primed and cultured in accordance with the experimental conditions. The total RNA of the strains under the logarithmic phase (11h) and the stable period (20h) were extracted respectively. The relative expression of ciaH, Eno, pykF and RPL genes in the three groups were measured. Results: 1. resuscitation Streptococcus and its fluorine resistant strains were confirmed by physiological, biochemical and 16SrDNA identification for the growth curve of the strain of Streptococcus mutans under the three experimental conditions, namely, Streptococcus mutans in fluorine free medium and fluorine resistance. The strains were cultured in medium with no fluorine and fluorine content of 1g/L. The logarithmic growth period of 11h was determined in three, and 20h was in the stable period of three..3. was successfully designed and synthesized by 16sRNA, ciaH, Eno, pykF and RPL gene primers. The logarithmic period (11h) and the stable phase (20h) strain were successfully extracted from three groups of experimental conditions. The results of fluorescence quantitative PCR analysis showed that: (1) the expression level of ciaH, Eno, pykF and RPL genes in the logarithmic phase was much higher than that in the stable period (P0.001). (2) the expression level of fluorine resistant strains of fluorine resistant strains ciaH, eno and pykF were both in logarithmic and stable periods compared with fluorine resistant strains without fluorine culture. There was no significant difference in the expression level of RPL gene in the logarithmic phase (P0.05), but the expression level in the stable period increased (P0.001). (3) the expression level of Eno, pykF and RPL genes in fluorine resistant strains decreased significantly at logarithmic and stable periods (P0.001), and the expression level of ciaH gene was not significantly worse in logarithmic phase than that of the parent strain (3). The expression of fluorine resistant strains of Streptococcus mutans ciaH, eno and pykF genes were enhanced by the expression of ciaH, eno and pykF in the fluorine resistant strain of Streptococcus mutans. The results showed that these genes were related to fluorine resistance of fluorine resistant strains, and the expression levels of Eno, pykF and RPL genes of Streptococcus mutans and their fluorine resistant strains of Streptococcus mutans had endogenous differences, suggesting that the two were in two There are differences in sugar transport and glycolysis. These results help to further explain the differences in physiological functions of fluorine resistant strains and the causes of fluorine resistance, and provide a basis for the study of the specific functions of these genes.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R781.1

【参考文献】