本文選題：變形鏈球菌耐氟菌株 + 氟抗性��； 參考：《吉林大學》2017年碩士論文

【摘要】：目的:齲病是人類最常見的口腔疾病之一,其主要致齲菌是變形鏈球菌(Streptococcus mutans,簡稱S.mutans)。而氟化物能有效抑制變形鏈球菌生長,1g/L的氟化物濃度就能完全殺滅變形鏈球菌[1]。目前,氟化物已經(jīng)被廣泛應(yīng)用于防齲領(lǐng)域,但是長期局部高濃度氟化物的使用可能導致變形鏈球菌耐氟菌株的產(chǎn)生,為防齲領(lǐng)域帶來新的挑戰(zhàn)[2]。實驗證明,耐氟菌株具有更強的產(chǎn)酸性、耐酸性及脫礦能力,這提示耐氟菌株具有更強的致齲性[3]。對于耐氟菌株一系列性狀改變,尤其是氟抗性產(chǎn)生的原因尚無明確結(jié)論。一般認為是來源于細菌基因組的改變。本課題組前期通過對變形鏈球菌耐氟菌株進行全基因組測序[4],與親代菌株對比,發(fā)現(xiàn)有20個基因及5個基因間隔發(fā)生了突變。其中4個基因,ciaH、eno、pykF和rpl可能與細菌的氟抗性相關(guān)。ciaH、eno、pykF和rpl分別編碼組氨酸激酶、烯醇酶、丙酮酸激酶和轉(zhuǎn)錄調(diào)節(jié)因子。這四種基因都與細菌的生存密切相關(guān),并且其牽涉的耐酸性、糖酵解和糖轉(zhuǎn)運過程又與氟的抑菌機制直接相關(guān)[5]。這四種基因如果發(fā)生變化很可能引起細菌生理的變化以及氟抗性的產(chǎn)生。以往曾將變形鏈球菌親代菌株及其耐氟菌株同時置于低濃度氟環(huán)境下來探究二者性狀上的差異,本實驗直接將耐氟菌株置于其誘導時達到的最大氟濃度1g/L中,試圖在基因表達層面對這四種基因予以探討,進一步解析氟抗性產(chǎn)生的原因,為應(yīng)對耐氟菌株的產(chǎn)生及基因防齲提供理論基礎(chǔ)。方法:1.對變形鏈球菌UA159及其耐氟菌株UA159-FR復(fù)蘇、培養(yǎng),鑒定。2.實驗條件分為三組,即變形鏈球菌于無氟培養(yǎng)基培養(yǎng)、耐氟菌株分別于無氟及含氟量為1g/L的培養(yǎng)基培養(yǎng),測定三者的生長曲線。3.設(shè)計16sRNA、ciaH、eno、pykF和rpl基因的引物并合成。4.按上述實驗條件培養(yǎng),分別于對數(shù)期(11h)及穩(wěn)定期(20h)提取三組條件下菌株的總RNA并逆轉(zhuǎn)錄。5.采用16sRNA為內(nèi)參基因,用實時熒光定量PCR技術(shù)測定三組條件下菌株的ciaH、eno、pykF和rpl基因的相對表達量。結(jié)果:1.復(fù)蘇培養(yǎng)的變形鏈球菌及其耐氟菌株經(jīng)生理、生化和16SrDNA鑒定后證實均為變形鏈球菌。2.繪制三組實驗條件下菌株的生長曲線,即變形鏈球菌于無氟培養(yǎng)基培養(yǎng)、耐氟菌株分別于無氟及含氟量為1g/L的培養(yǎng)基培養(yǎng)。確定11h處于三者的生長對數(shù)期,20h處于三者的生長穩(wěn)定期。3.成功設(shè)計并合成16sRNA、ciaH、eno、pykF和rpl基因的引物。4.成功提取三組實驗條件下對數(shù)期(11h)及穩(wěn)定期(20h)菌株的總RNA并逆轉(zhuǎn)錄。5.實時熒光定量PCR結(jié)果分析顯示:(1)三組實驗條件下,菌株ciaH、eno、pykF和rpl基因在對數(shù)期的表達水平均遠遠高于穩(wěn)定期的表達水平(P0.001)。(2)與無氟培養(yǎng)的耐氟菌株比較,含氟培養(yǎng)的耐氟菌株ciaH、eno和pykF基因的表達水平在對數(shù)期及穩(wěn)定期均明顯升高(P0.001);rpl基因表達水平在對數(shù)期無明顯差異(P0.05),而在穩(wěn)定期表達升高(P0.001)。(3)無氟培養(yǎng)時,與親代菌株比較,耐氟菌株eno、pykF和rpl基因的表達水平在對數(shù)期及穩(wěn)定期均明顯下降(P0.001);ciaH基因的表達水平在對數(shù)期無明顯差異(P0.05),而在穩(wěn)定期表達升高(P0.01)。結(jié)論:氟能提高變形鏈球菌耐氟菌株ciaH、eno和pykF基因的表達,表明這些基因與耐氟菌株氟抗性的產(chǎn)生相關(guān);變形鏈球菌親代菌株及其耐氟菌株eno、pykF和rpl基因的表達水平存在內(nèi)源性差異,提示二者在糖轉(zhuǎn)運、糖酵解等方面存在差異。這些結(jié)果有助于進一步解釋耐氟菌株相較其親代菌株在生理功能上的差異以及氟抗性產(chǎn)生的原因,并為研究這些基因的具體功能提供了基礎(chǔ)。
[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.
【學位授予單位】：吉林大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R781.1

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