【摘要】： 銅綠假單胞菌(綠膿桿菌,Pseudomonas aeruginosa,PA)是一種條件致病菌,其很多致病性都與毒性因子的分泌有關(guān),其中吩嗪類復(fù)合物是很重要的毒性因子,包括:吩嗪-1-羧酸(phenazine-1-carboxylic acid,PCA)、綠膿菌素(pyocyanin)等。PCA等吩嗪類復(fù)合物在細(xì)胞內(nèi)可作為電子載體,增加感染細(xì)胞內(nèi)的氧化壓力,使感染的目標(biāo)細(xì)胞中毒死亡,綠膿菌素與囊腫性纖維化病人(CF)的肺部感染有關(guān)。PCA為綠膿菌素合成的中間體,而phzA1B1C1D1G1(phzA1)操縱子參與了PCA的合成過程。已有的研究報(bào)道表明次抑制濃度的抗生素可以作為信號(hào)分子調(diào)節(jié)致病基因的表達(dá)。本實(shí)驗(yàn)室前期研究表明:次抑制濃度(subinhibitory concentrations)的四環(huán)素可以調(diào)節(jié)phzA1操縱子的表達(dá);在次抑制濃度四環(huán)素條件下,以發(fā)光桿菌的熒光素酶基因操縱子luxCDABE為報(bào)道基因,對(duì)含有phzA1操縱子報(bào)道載體的轉(zhuǎn)座突變庫(kù)進(jìn)行了篩選。本實(shí)驗(yàn)對(duì)篩選出的其中三株突變體PAM0487、PAM0487R、PAM0207進(jìn)行了研究。 在三株突變體PAM0487、PAM0487R、PAM0207中,phzA1操縱子的表達(dá)降低。通過隨機(jī)PCR、基因測(cè)序及基因比對(duì)確定了突變點(diǎn)在染色體上的位置。發(fā)現(xiàn)其中的兩株突變體PAM0487、PAM0487R的突變點(diǎn)均位于PA0487基因啟動(dòng)子區(qū)內(nèi),PA0487基因?yàn)榧俣ㄣf元素的轉(zhuǎn)運(yùn)調(diào)節(jié)基因。PAM0207位于PA0207基因啟動(dòng)子區(qū)內(nèi)。 通過測(cè)定綠膿菌素在野生型PAO1、PAM0487、PAM0487R中的生成量,發(fā)現(xiàn)在突變體PAM0487和PAM0487R中綠膿菌素的生成量降低,證實(shí)PA0487基因?qū)hzA1操縱子在轉(zhuǎn)錄水平的正向調(diào)節(jié)作用表現(xiàn)在表型上。在不同濃度的四環(huán)素條件下,phzA1操縱子表達(dá)受四環(huán)素調(diào)節(jié)的現(xiàn)象在突變體PAM0487R中消失,說明PA0487是四環(huán)素對(duì)phzA1操縱子調(diào)節(jié)途徑中的一員。由于銅綠假單胞菌中吩嗪類復(fù)合物的合成受群體感應(yīng)系統(tǒng)(quorum-sensing,QS)的調(diào)控,本實(shí)驗(yàn)研究了PA0487基因與QS系統(tǒng)的關(guān)系,發(fā)現(xiàn)PA0487對(duì)群體感應(yīng)系統(tǒng)相關(guān)基因的表達(dá)有調(diào)節(jié)作用。 將Gm~r-lacZ基因插入野生型PAO1的PA0207基因中間構(gòu)建了突變體PAK0207,在突變體PAK0207中phzA1操縱子的表達(dá)無明顯變化,表明對(duì)phzA1具有調(diào)節(jié)作用的基因并不是PA0207。
[Abstract]:Pseudomonas aeruginosa (Pseudomonas aeruginosa,PA) is a conditional pathogen. Many pathogenicity of Pseudomonas aeruginosa is related to the secretion of toxic factors, among which phenazine complexes are very important toxic factors. Phenazine-1-carboxylic acid (phenazine-1-carboxylic acid,PCA), pyocyanin (pyocyanin), etc. can be used as electronic carriers in cells to increase the oxidative pressure in infected cells and cause poisoning and death of infected target cells. Pseudomonas aeruginosa is associated with pulmonary infection in patients with cystic fibrosis (CF). PhzA1B1C1D1G1 (phzA1) operon is an intermediate in the synthesis of pyogenin, and phzA1B1C1D1G1 (phzA1) operon is involved in the synthesis of PCA. Previous studies have shown that subinhibitory concentrations of antibiotics can be used as signal molecules to regulate the expression of pathogenic genes. Previous studies in our laboratory showed that tetracycline with subinhibitory concentration of (subinhibitory concentrations) could regulate the expression of phzA1 operon. Using luciferase gene operon luxCDABE as reporter gene, the transposable library containing phzA1 operon reporting vector was screened under the condition of tetracycline subinhibitory concentration. In this study, three of the selected mutants, PAM0487,PAM0487R,PAM0207, were studied. In the three mutants PAM0487,PAM0487R,PAM0207, the expression of phzA1 operon was decreased. The position of mutation point on chromosome was determined by random PCR, gene sequencing and gene comparison. Two mutants of PAM0487,PAM0487R were found to be located in the promoter region of the PA0487 gene. The PA0487 gene was supposed to regulate the transport of molybdenum. PAM0207 was located in the promoter region of the PA0207 gene. By measuring the production of pyocyanin in wild-type PAO1,PAM0487,PAM0487R, it was found that the production of pyocyanin in mutant PAM0487 and PAM0487R decreased, which confirmed that the positive regulatory effect of PA0487 gene on phzA1 operon at the transcriptional level was shown to be phenotypic. At different concentrations of tetracycline, the tetracycline-regulated expression of phzA1 operon disappeared in mutant PAM0487R, suggesting that PA0487 is one of the regulatory pathways of tetracycline on phzA1 operon. Because the synthesis of phenazine complexes in Pseudomonas aeruginosa was regulated by quorum sensing system (quorum-sensing,QS), the relationship between PA0487 gene and QS system was studied in this study. It was found that PA0487 could regulate the expression of quorum sensing system related genes. The expression of phzA1 operon in mutant PAK0207, was not changed by inserting Gm~r-lacZ gene into the PA0207 gene of wild type PAO1, indicating that the gene that regulates phzA1 is not PA0207..
【學(xué)位授予單位】：西北大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R378

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