本文選題：銅綠假單胞菌　切入點：群體感應(yīng)系統(tǒng)　出處：《廣州中醫(yī)藥大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：目的:質(zhì)粒轉(zhuǎn)移是細(xì)菌傳播耐藥基因的重要途徑。質(zhì)粒(plasmid)是的環(huán)形DNA,它不僅獨立于細(xì)菌染色體,而且能自主復(fù)制。部分質(zhì)粒具有轉(zhuǎn)移性,并能攜帶抗生素耐藥基因。廣泛宿主質(zhì)粒能穿梭于細(xì)菌間,因此質(zhì)粒被視為傳播耐藥的關(guān)鍵媒介。質(zhì)粒水平轉(zhuǎn)移的主要途徑是細(xì)菌的接合作用。接合作用(conjugation)是細(xì)菌借助性菌毛相互溝通、傳受DNA的過程。因此,細(xì)菌接合作用被視為傳播抗生素耐藥基因的重要途徑。AHL是細(xì)菌群體感應(yīng)系統(tǒng)的信號分子。群體感應(yīng)(quorum sensing,QS)是細(xì)菌感知群體數(shù)量變化,并調(diào)整群體行為的調(diào)控系統(tǒng)。其中,脂肪�；呓z氨酸內(nèi)酯(acyl homoserine lactones,AHL)是革蘭陰性菌QS系統(tǒng)的信號分子,它通過結(jié)合LuxR類受體蛋白,實現(xiàn)調(diào)控基因表達(dá)。蛋白SdiA是QS系統(tǒng)的受體蛋白,同時也是重要的轉(zhuǎn)錄因子。蛋白SdiA存在于大腸埃希菌中,能感知并結(jié)合其他細(xì)菌分泌的AHL。蛋白SdiA擁有DNA結(jié)合域,它能結(jié)合基因的啟動子,以調(diào)控基因表達(dá)。因此,QS系統(tǒng)通過AHL-SdiA調(diào)控基因表達(dá)。一般認(rèn)為,質(zhì)粒轉(zhuǎn)移的調(diào)控是由質(zhì)粒與宿主共同調(diào)控的過程,而受體菌參與接合調(diào)控則少有報道。本文推測,受體菌能通過QS系統(tǒng)調(diào)控質(zhì)粒轉(zhuǎn)移:受體菌分泌AHL,通過與宿主的蛋白SdiA結(jié)合,兩者形成復(fù)合物,以調(diào)控接合相關(guān)基因traI。其中,蛋白TraI能結(jié)合質(zhì)粒復(fù)制起始點oriT,參與質(zhì)粒的復(fù)制及轉(zhuǎn)移全過程,直接調(diào)控細(xì)菌接合作用。因此,本文探究AHL-SdiA與基因traI的調(diào)控關(guān)系。另外,本文發(fā)現(xiàn)蛋白SdiA對sRNA的調(diào)控作用,展望后續(xù)研究AHL-SdiA-sRNA對細(xì)菌接合作用的調(diào)控機(jī)制。方法:本實驗采用大腸埃希菌SM10λπ作為質(zhì)粒供體菌。該菌染色體被整合了質(zhì)粒轉(zhuǎn)移的調(diào)控元件(包含基因traI),并含有轉(zhuǎn)移性質(zhì)粒pUC24T。質(zhì)粒pUC24T具慶大霉素(Gm)抗性,作為候選實驗的篩選標(biāo)記。受體菌則選用銅綠假單胞菌PA01。PA01基因IasI、基因rhlI分別調(diào)控3-oxo-C12-HSL、C4-HSL的催化合成,后兩者為PA01最主要的AHL。實驗主要通過構(gòu)建接合模型、運用熒光定量PCR(qPCR)檢測基因表達(dá)量以及啟動子報告質(zhì)粒來驗證實驗假設(shè)。構(gòu)建接合模型。分別將供、受體菌培養(yǎng)至0.5麥?zhǔn)蠞岫葐挝?MCF)。利用尿液分析儀測細(xì)菌濃度,并調(diào)配菌液濃度至1.0×107CFU/mL。供、受體菌各取100μL,37℃混合培養(yǎng)6h。篩選合子并計數(shù),換算接合頻率。接合頻率高,則說明接合作用則強(qiáng)。qPCR檢測基因表達(dá)量。收集待測菌液,并抽提細(xì)菌全RNA組。將RNA反轉(zhuǎn)錄為cDNA。以cDNA作為模板,利用qPCR檢測目的基因相對表達(dá)量。構(gòu)建基因traI啟動子報告質(zhì)粒pQF50-PtraI。以質(zhì)粒pQF50為載體,該質(zhì)粒缺少Laz啟動子。運用分子克隆技術(shù),將基因traI啟動子及其上游317 bp片段,克隆于β-半乳糖苷酶基因上游。當(dāng)基因traI啟動子被激活時,其下游β-半乳糖苷酶基因也被激活。后續(xù)通過β-半乳糖苷酶試驗(ONPG試驗)檢測酶活性,便能反映出基因traI啟動子的表達(dá)狀況。酶活性高,則說明基因traI表達(dá)上調(diào)。通過該實驗觀察AHL-SdiA對基因traI的調(diào)控情況。結(jié)果:①SM10λ π-PA01 進(jìn)行接合反應(yīng)。敲除PA01基因lasI、基因rhlI,即AHL的缺失能提高SM10λ π接合頻率(P0.001):若在此基礎(chǔ)上,加入3-oxo-C12-HSL、C4-HSL干預(yù)接合反應(yīng),接合頻率則降低(P0.05)。敲除SM10λ π的基因sdiA,能增加SM10λ π-PA01接合頻率(P0.001)。該結(jié)果提示,AHL與蛋白SdiA共同抑制SM10λ π接合作用。②SM10λ π-EC600進(jìn)行接合反應(yīng)。其中,受體菌EC600為大腸埃希菌,自身不合成AHL。加入AHL干預(yù),接合作用受抑制,SM10λπ-EC600接合頻率降低(P0.001);而在敲除基因sdiA后,接合頻率又回復(fù)至原水平。但是,在不加AHL干預(yù)時,在敲除基因sdiA后,SM10λπ-EC600的接合頻率相近。該結(jié)果說明,AHL抑制接合作用依賴于基因sdiA的表達(dá)。至此,實驗從表型上闡明了 AHL-SdiA抑制質(zhì)粒轉(zhuǎn)移。③SM10λ π-PA01進(jìn)行接合反應(yīng)。敲除PA01基因lasI、基因rhlI,即AHL的缺失能上調(diào)SM10λ π基因traI表達(dá)(P0.05)。若在此基礎(chǔ)上,加入3-oxo-C12-HSL、C4-HSL干預(yù)接合反應(yīng),SM10λ π基因traI表達(dá)降低(P0.05)。敲除SM10λ π的基因sdiA,SM10λ π基因traI表達(dá)上調(diào)(P0.05)。該結(jié)果從分子上闡明了AHL-SdiA抑制質(zhì)粒轉(zhuǎn)移。④將含有報告質(zhì)粒pQF50-PtraI大腸埃希菌BW25113泛進(jìn)行ONPG試驗。加入AHL干預(yù)培養(yǎng)后,其β-半乳糖苷酶活性降低(P0.05)。而敲除BW25113基因sdiA或不加入AHL干預(yù)培養(yǎng)時,則均不降低酶活性。該結(jié)果說明,AHL-SdiA抑制基因traI啟動子。⑤本實驗通過生物信息學(xué)方法,預(yù)測并初步驗證4條sRNA與基因sdiA調(diào)控成正相關(guān)性。敲除SM10λ π基因sdiA,能下調(diào)RnpB、SibA、SibB、EyeA等sRNA的表達(dá)。結(jié)論:實驗通過SM10λ π-PA01、SM10λ π-EC600接合模型,從表型上闡明AHL-SdiA抑制質(zhì)粒轉(zhuǎn)移。而后,通過qPCR檢測基因traI表達(dá)變化及pQF50-PtraI啟動子報告質(zhì)粒實驗,從分子水平闡明了 AHL-SdiA抑制基因traI啟動子,從而抑制質(zhì)粒轉(zhuǎn)移。最后,實驗發(fā)現(xiàn)數(shù)條sRNA受SdiA調(diào)控,為進(jìn)一步探討AHL-SdiA-sRNA抑制質(zhì)粒轉(zhuǎn)移機(jī)制指明了方向。
[Abstract]:Objective: plasmid transfer is an important way to the spread of bacteria resistant gene. The plasmid (plasmid) DNA is a ring, it is not only independent of the bacterial chromosome, and can self replicate. Some plasmids with metastasis, and can carry antibiotic resistance genes. Extensive host plasmid shuttling between bacteria, the plasmid is seen as key media the main way of resistance. Horizontal plasmid transfer is conjugation. Bacterial conjugation (conjugation) is a bacterial pili by means of mutual communication, transmission by the DNA process. Therefore, bacterial conjugation is regarded as an important way to spread the antibiotic resistant gene in.AHL is a signal molecule of quorum sensing systems. Quorum sensing (quorum sensing, QS) is the number of perceived changes in bacterial groups, control system and adjust the group behavior. Among them, fatty acyl homoserine lactone (acyl homoserine, lactones, AHL) is a gram negative bacteria The signal molecules of the QS system, using a combination of LuxR receptor like protein, can regulate gene expression. SdiA protein is a receptor protein of the QS system, but also an important transcription factor. SdiA protein in Escherichia coli, and combined with other bacteria can sense the secretion of AHL. protein with SdiA DNA binding domain, it can be combined with gene the promoter to regulate gene expression. Therefore, QS AHL-SdiA system through the regulation of gene expression. It is generally considered that the regulation of plasmid transfer is by plasmid and host common regulatory process, and participate in the regulation of receptor engagement is rarely reported. It is suggested that receptor bacteria can utilize QS system to control plasmid transfer: the receptor bacteria secrete AHL and through the combination with the host protein SdiA, both the complex formation, in order to control the joint related gene traI., TraI protein can bind to plasmid replication starting point oriT in plasmid replication and transfer process, Direct regulation of bacterial conjugation. Therefore, this paper explores the relationship of AHL-SdiA and regulation of traI gene. In addition, this paper found that the regulation effect of SdiA protein on sRNA and prospects for future research AHL-SdiA-sRNA on the regulation mechanism of bacterial conjugation. Methods: this experiment used Escherichia coli SM10 k'rr53 as donor bacteria. The bacteria chromosome is integrated regulatory elements (including the transfer of plasmid traI gene), and contains a transfer plasmid pUC24T. pUC24T with gentamicin (Gm) resistance as a selective marker candidate experiment. The recipient bacterium Pseudomonas aeruginosa PA01.PA01 gene IasI and gene rhlI were regulated by 3-oxo-C12-HSL, catalytic synthesis of C4-HSL, both for AHL. PA01 the main experiment mainly through the construction of joint model, using fluorescence quantitative PCR (qPCR) to detect gene expression and promoter reporter plasmids to verify the hypotheses. The construction joint model. The supply of bacteria to 0.5 receptor Maxwell turbidity unit (MCF). The measured concentration of bacteria by urine analyzer, and the deployment of the concentration of bacteria to 1 x 107CFU/mL. for each receptor strain 100 L, 37 c mixed culture 6h. screening and zygote counting, conversion joint frequency. The high frequency of engagement, engagement effect of strong detection of.QPCR gene expression. The collection to be tested bacteria, and bacterial RNA extraction group. RNA is transcribed into cDNA. using cDNA as template, the relative expression detected by qPCR gene. The gene traI promoter reporter plasmid pQF50-PtraI. with plasmid pQF50, the plasmid Laz promoter. The lack of molecular cloning, gene traI promoter and its upstream 317 bp fragment cloned into upstream beta galactosidase gene. When traI gene promoter is activated, downstream of the beta galactosidase gene has also been activated. Follow through beta galactose glucoside enzyme test Test (ONPG test) to detect enzyme activity, can reflect the expression status of traI gene promoter. High enzyme activity, the expression of traI gene. Through the experimental observation on regulation of AHL-SdiA gene of traI. Results: 1. SM10 k'rr53 -PA01 conjugation reaction. PA01 gene knockout lasI gene rhlI. The loss of AHL can improve SM10 k'rr53 joint frequency (P0.001): 3-oxo-C12-HSL based on C4-HSL, intervention of conjugation reaction decreased, joint frequency (P0.05). SdiA gene knock in SM10 lambda PI, lambda PI -PA01 SM10 can increase the joint frequency (P0.001). The results suggest that the effect of AHL and protein SdiA inhibits SM10 k'rr53 bonding. The SM10 k'rr53 -EC600 conjugation reaction. Among them, EC600 receptor bacteria were Escherichia coli, their synthesis of AHL. into AHL intervention, joint action by inhibiting SM10, lambda PI -EC600 joint (P0.001); reduce the frequency in sdiA gene knockout, Joint frequency and return to the original level. However, in the absence of AHL intervention, in the knockout of sdiA gene, SM10 k'rr53 -EC600 joint. The results show that the similar frequency, AHL suppressed the expression of conjugation depends on the gene of sdiA. Thus, experiment illustrates the inhibition of AHL-SdiA plasmid transfer from the phenotype of the SM10. K'rr53 -PA01 conjugation reaction. PA01 gene knockout lasI gene, rhlI, that is the absence of AHL could up regulate the expression of SM10 lambda PI gene traI (P0.05) 3-oxo-C12-HSL. Based on C4-HSL, joint intervention reaction, reduce the expression of SM10 k'rr53 gene traI (P0.05). SdiA gene knock in SM10 lambda PI expression of SM10 gene, lambda PI traI (P0.05). The results demonstrated that AHL-SdiA inhibited plasmid transfer from the molecule. The report will contain plasmid pQF50-PtraI of Escherichia coli BW25113 pan ONPG test with AHL after intervention training, reduce the glycosidase activity of - galactosidase (P0.05). Knockdown of BW25113 sdiA or without AHL intervention training, which does not reduce the enzyme activity. The results showed that AHL-SdiA inhibited traI gene promoter. In this experiment by using bioinformatics methods, prediction and preliminary validation of 4 sRNA and sdiA gene regulation into a positive correlation. The knockout of SM10 gene can k'rr53 sdiA the down-regulation of RnpB, SibA, SibB, EyeA and sRNA expression. Conclusion: through the experiment of SM10 lambda PI -PA01, PI lambda SM10 -EC600 joint model, clarify the inhibition of AHL-SdiA plasmid transfer from the phenotype. Then, changes and pQF50-PtraI promoter report to plasmid traI gene qPCR expression was detected by experiments, from the molecular level to clarify the inhibition of AHL-SdiA gene the traI promoter, thereby inhibiting plasmid transfer. Finally, the experiment found that the number of sRNA regulated by SdiA, pointed out the direction for further study of AHL-SdiA-sRNA inhibiting plasmid transfer mechanism.

【學(xué)位授予單位】：廣州中醫(yī)藥大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R446.5

【參考文獻(xiàn)】

相關(guān)期刊論文 前5條

1 陳向東;王素霞;汪輝;魏洪璇;謝少朵;潘云;;多尼培南對銅綠假單胞菌生物被膜形成和群體感應(yīng)系統(tǒng)的影響[J];藥學(xué)與臨床研究;2016年03期

2 張yN博;孫景勇;倪語星;俞云松;林潔;楊青;徐英春;張小江;孫自鏞;陳中舉;汪復(fù);朱德妹;胡付品;蔣曉飛;王傳清;王愛敏;卓超;蘇丹虹;胡云建;艾效曼;黃文祥;賈蓓;張朝霞;季萍;張泓;孔菁;魏蓮花;吳玲;徐元宏;沈繼錄;單斌;杜艷;胡志東;李金;謝軼;康梅;韓艷秋;郭素芳;褚云卓;田素飛;;2005—2014年CHINET銅綠假單胞菌耐藥性監(jiān)測[J];中國感染與化療雜志;2016年02期

3 吳彬彬;王文昭;顏曉慶;陳宏運;劉春花;潘永龍;梁巖;;植物發(fā)酵液提取物對銅綠假單胞菌生物膜的作用[J];微生物學(xué)報;2016年07期

4 張安;吳潔;儲衛(wèi)華;;中藥群體感應(yīng)抑制劑的篩選及對銅綠假單胞菌的作用[J];藥物生物技術(shù);2016年01期

5 陳樹林;陳茶;黃彬;張妮;鄂順梅;蔡壬辛;袁慧;曲平華;林冬玲;;接合轉(zhuǎn)移質(zhì)粒pUCP24T的構(gòu)建與性能研究[J];中國微生態(tài)學(xué)雜志;2013年07期

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