【摘要】：群體感應(yīng)(Quorum Sensing,QS)是細(xì)菌細(xì)胞之間進(jìn)行信息交流的一種機(jī)制。乳酸菌屬于革蘭氏陽(yáng)性細(xì)菌,除了擁有寡肽介導(dǎo)的QS系統(tǒng),還同時(shí)擁有自誘導(dǎo)物2(autoinducer 2,AI-2)介導(dǎo)的種間QS系統(tǒng),研究發(fā)現(xiàn)QS系統(tǒng)與乳酸菌耐酸耐膽鹽及抑菌特性有較為密切的關(guān)系。本研究使用的植物乳桿菌(Lactobacillus plantarum)AY01與YM-4-3分別分離自石林發(fā)酵羊奶與易門豆豉,且在對(duì)AY01與YM-4-3的全基因組序列進(jìn)行分析時(shí)發(fā)現(xiàn),AY01含有一個(gè)編碼種間QS系統(tǒng)信號(hào)分子——AI-2合成酶(LuxS)的開放閱讀框(Open Reading Frame,ORF),而YM-4-3含有兩個(gè)。本論文主要針對(duì)AY01 luxS的敲除及其luxS突變株在酸、膽鹽耐受、生物膜形成能力及抑制腸出血性大腸桿菌(enterohemorrhage Escherichia coli,EHEC)生物膜形成等方面與野生型菌株的差異。此研究基礎(chǔ)上,構(gòu)建了兩種luxS基因的原核表達(dá)載體pET28a-luxS1和pET28a-luxS2,并成功在E.coli BL21(DE3)中表達(dá)。具體實(shí)驗(yàn)結(jié)果如下:(1)利用溫度敏感型敲除載體PKML05,通過同源重組方法敲除了 AY01的luxS,得到luxS突變菌株LuxSA1。(2)哈氏弧菌(Vibrio harveyi)BB170的生物發(fā)光實(shí)驗(yàn)顯示LuxSA1不能產(chǎn)生AI-2,表明luxS在AY01合成AI-2途徑中發(fā)揮關(guān)鍵性作用。(3)AY01與LuxSA1在生長(zhǎng)、產(chǎn)酸、酸與膽鹽耐受及生物膜形成能力方面沒有顯著性差異。(4)通過AY01與LuxS△1對(duì)Caco-2細(xì)胞的粘附實(shí)驗(yàn)發(fā)現(xiàn),培養(yǎng)14 h與27 h的LuxSA1菌體的粘附能力遠(yuǎn)低于AY01,證明luxS在AY01對(duì)Caco-2細(xì)胞的粘附過程中發(fā)揮重要作用。(5)AY01培養(yǎng)物對(duì)EHEC生物膜形成的抑制作用強(qiáng)于LuxSA1。(6)構(gòu)建了兩種luxS基因原核表達(dá)載體pET28a-luxS1和pET28a-luxS2,并通過優(yōu)化純化步驟成功純化出純度超過90%的LuxS 1與LuxS2蛋白。(7)純化的LuxS1與LuxS2都能催化SRH合成QS信號(hào)分子AI-2,誘導(dǎo)V.harveyi BB170 生物發(fā)光;Al3+,Ca2+和 EDTA 能增強(qiáng) LuxS1 的酶活性,Al3+,Ca2+,Fe2+,Mn2+和EDTA能增強(qiáng)LuxS2的酶活性,LuxS1與LuxS2都受到Cu2+,Zn2+,Ni2+等種金屬離子及甘油的強(qiáng)烈抑制。
[Abstract]:Group sensing (Quorum Sensing,QS) is a mechanism for the communication of information between bacterial cells. Lactic acid bacteria (lactic acid bacteria) belong to Gram-positive bacteria. In addition to the QS system mediated by oligoseptide, lactic acid bacteria also possess the interspecific QS system mediated by self-inducible agent 2 (autoinducer 2 / AI-2). It is found that the QS system is closely related to acid tolerance and bile salt tolerance and bacteriostatic characteristics of lactic acid bacteria. Lactobacillus plantarum (Lactobacillus plantarum) AY01 and YM-4-3 were isolated from fermented goat milk and Yimen Douchi, respectively. By analyzing the whole genome sequence of AY01 and YM-4-3, we found that AY01 contains an open reading frame (Open Reading Frame,ORF of AI-2 synthase (LuxS), and YM-4-3 contains two. This paper aims at the difference between AY01 luxS knockout and luxS mutant in acid, bile salt tolerance, biofilm formation and inhibition of (enterohemorrhage Escherichia coli,EHEC biofilm formation in enterohemorrhagic Escherichia coli. Based on this study, two prokaryotic expression vectors of luxS gene, pET28a-luxS1 and pET28a-luxS2, were constructed and successfully expressed in E.coli BL21 (DE3). The specific results are as follows: (1) the bioluminescence experiments of luxS mutant LuxSA1. (2) (Vibrio harveyi) BB170 showed that LuxSA1 could not produce AI-2, and luxS could not be produced in AY01 by using the thermo-sensitive knockout vector PKML05, to knock out the luxS, of AY01 by homologous recombination method. Synthesis of AI-2 pathway plays a key role. (3) AY01 and LuxSA1 in growth, There was no significant difference in acid production, acid and bile salt tolerance and biofilm formation ability. (4) Adhesion of AY01 and LuxS 1 to Caco-2 cells was observed. The adhesion ability of LuxSA1 cells cultured for 14 h and 27 h was much lower than that of AY01,. It was proved that luxS played an important role in the adhesion of AY01 to Caco-2 cells. (5) the inhibitory effect of AY01 culture on EHEC biofilm formation was stronger than that of LuxSA1. (6). Two luxS gene prokaryotes were constructed. The expression vectors pET28a-luxS1 and pET28a-luxS2, were successfully purified with purity of more than 90% of LuxS 1 and LuxS2 protein. (7) the purified LuxS1 and LuxS2 could catalyze SRH to synthesize QS signaling molecule AI-2, to induce V.harveyi BB170 bioluminescence; Al3, Ca2 and EDTA could enhance the expression of V.harveyi BB170 bioluminescence. The enzyme activity of LuxS1, Al3, Ca2, Fe2, Mn2 and EDTA enhanced the activity of LuxS2. LuxS1 and LuxS2 were strongly inhibited by Cu2, Zn2, Ni2 and other metal ions and glycerol.
【學(xué)位授予單位】：昆明理工大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：Q78

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 崔燕麗;徐曉芬;吳正鈞;劉振民;;乳酸菌胞外多糖對(duì)人類腸道菌群的影響[J];中國(guó)微生態(tài)學(xué)雜志;2016年07期

2 鄭慧琦;龐雪輝;朱宗濤;孟祥晨;;植物乳桿菌KLDS1.0391 luxS基因敲除載體的構(gòu)建與應(yīng)用[J];中國(guó)科技論文;2016年06期

3 黃庶識(shí);盧明倩;李冰;周冰;陳麗梅;;重組大腸桿菌表達(dá)可溶性蛋白和包涵體過程的拉曼光譜實(shí)時(shí)分析[J];中國(guó)激光;2014年12期

4 王洋;由田;潘超;果馬;葛菁萍;平文祥;;不同感受態(tài)細(xì)胞制備方法對(duì)副干酪乳桿菌轉(zhuǎn)化效率的影響[J];中國(guó)食品學(xué)報(bào);2014年09期

5 徐芳;李軍;段云飛;劉曉光;;細(xì)菌群體感應(yīng)信號(hào)網(wǎng)絡(luò)及其應(yīng)用[J];生物學(xué)雜志;2014年04期

6 任曉鏷;李明楊;任少東;王群霞;;誘導(dǎo)植物乳桿菌生物膜形成的環(huán)境因素探索[J];食品與發(fā)酵工業(yè);2014年07期

7 許玉彬;張穎;王淼;王少偉;王剛;;細(xì)菌群體感應(yīng)研究進(jìn)展[J];河南農(nóng)業(yè)科學(xué);2013年12期

8 張騰;田建軍;李梓媛;賈原博;賀銀鳳;;植物乳桿菌HE-1由AI-2/LuxS介導(dǎo)的群體感應(yīng)與生物膜形成關(guān)系的研究[J];食品工業(yè)科技;2014年05期

9 陳瑞;王大力;林志芬;尹大強(qiáng);;枯草芽孢桿菌的群體感應(yīng)信號(hào)系統(tǒng)及其在環(huán)境領(lǐng)域的應(yīng)用前景[J];安全與環(huán)境學(xué)報(bào);2012年06期

10 宋協(xié)法;孫頡;;群體感應(yīng)在水產(chǎn)養(yǎng)殖中的應(yīng)用前景[J];漁業(yè)信息與戰(zhàn)略;2012年02期

相關(guān)碩士學(xué)位論文 前1條

1 陳臣;植物乳桿菌ST-Ⅲ對(duì)腸上皮細(xì)胞的粘附性質(zhì)及機(jī)理的研究[D];江南大學(xué);2008年

，

本文編號(hào)：2273472