本文選題：銅綠假單胞菌　切入點：phzA2　出處：《西北大學》2012年碩士論文

【摘要】：銅綠假單胞菌(Pseudomonas aeruginosa, PA)是一種革蘭氏陰性條件致病菌,是院內感染的主要病原菌之一,尤其易感染燒傷、手術后和免疫力低下病人。由于其對抗生素有較強的耐藥性,因此臨床中PA的感染很難根治。 吩嗪類化合物是PA產生的一類具有氧化還原活性的小分子物質,也是PA中重要的毒性因子之一,同時還具有信號分子的作用,能夠調節(jié)許多基因的表達。在銅綠假單胞菌PAO1的基因組中,負責吩嗪-1-羧酸(PCA)合成的基因簇包含兩個同源的操縱子phzA1B1C1D1E1F1G1(phzA1)和phzA2B2C2D2E2F2G2(phzA2), PCA是合成吩嗪類化合物的前體物質,可通過phzM、phzS和phzH所編碼的酶將其轉化為其他吩嗪類物質,包括綠膿菌素、1-羥基-吩嗪(1-HP)和吩嗪-1-甲酰胺(PCN)。綠膿菌素是其中一個較為重要的吩嗪類化合物,在PA感染人體的過程中,綠膿菌素的產生會增加PA的感染率,例如囊腫性纖維化(Cystic fibrosis,CF)病人的肺部感染,同時也會引起病人死亡率的增加。因此,從分子水平上了解影響吩嗪合成的調節(jié)途徑對于控制PA的致病機制具有重要的意義。 本研究將phzA2的啟動子連接到缺失啟動子的luxCDABE報道子上,再利用噬菌體結合位點進行位點專一性重組,將phzA2-luxCDABE整合到PA的基因組上,得到phzA2啟動子的發(fā)光報道菌株,其化學發(fā)光情況直接反映phzA2基因的表達情況。采取隨機轉座突變的方法,在PA全基因組水平上篩選影響phzA2基因表達的基因,通過發(fā)光的變化來確定篩選能夠調節(jié)phzA2基因表達的調節(jié)基因。 最終我們通過大量篩選轉座突變體(約20000多個克隆),得到了14株phzA2基因表達發(fā)生變化的轉座突變體。對這些突變體進行隨機PCR、DNA測序、比對后確定了這些轉座突變體在PAo1染色體上突變的具體位點。為了進一步確定相互之間的關系,我們在篩選出的14株轉座突變體中,挑選了兩株phzA2的表達和綠膿菌素產量都有較大變化的突變體P2G1和P2E4進行深入的研究。通過在目標基因中插入外源Gmr-lacZ片段的方法,對基因PA3043和PA1387進行了敲除突變,得到了敲除突變體PAo1(△3043)和PAO1(△1387),并對PA1387的下游基因PA1388進行了過表達。實驗結果表明,在突變體PAo1(△1387)中,基因PA1387的缺失能夠導致phzA2的表達和綠膿菌素的產量都發(fā)生明顯的降低,說明PA1387通過某種途徑調節(jié)phzA2的基因的表達,并使其最終產物的合成量也明顯降低。然而,在突變體PAo1(△3043)中,基因PA3043的缺失對phzA2的表達及綠膿菌素的產生并無明顯的影響,表明PA3043對phzA2的表達并沒有相互的影響。這可能是由于轉座突變過程中出現的兩極反應影響了PA3043上下游的基因造成的,具體的關系有待進一步的研究證明。由于突變體PAO1(△1387)與轉座突變體P2E4中phzA2表達水平有著一定的差異,為了排除是由于高表達PA1388基因引起綠膿菌素產量的升高,所以對基因PA1388進行了過表達,發(fā)現其對phzA2的表達并無明顯影響。 上述結果說明,在PA中存在未知的與phzA2有著一定關系的基因,了解這些未知基因與phzA2的具體關系有助于我們認識PA中合成吩嗪類化合物的具體調控過程。
[Abstract]:Pseudomonas aeruginosa (Pseudomonas aeruginosa PA) is a gram negative opportunistic pathogen, is one of the primary pathogens of nosocomial infections, especially susceptible to burn, after surgery and immunocompromised patients. Due to its resistance to antibiotics in clinical PA infection is difficult to cure.
Phenazine compounds with redox active molecules such as PA, is one of the important virulence factor of PA, but also has the role of signal molecule, which can regulate the expression of many genes in Pseudomonas aeruginosa. The genome of PAO1 -1-, responsible for phenazine carboxylic acid (PCA) synthesis gene cluster contains two homologous phzA1B1C1D1E1F1G1 operon (phzA1) and phzA2B2C2D2E2F2G2 (phzA2), PCA is a precursor to synthesize phenazine compounds, through phzM, phzS and phzH encoding the enzyme converted into other phenazine compounds, including pyocyanin, 1- hydroxy - phenazine (1-HP) -1- and phenothiazine formamide (PCN). Pyocyanin is one of phenazine compounds is important, in the process of human PA infection, which will increase the rate of PA infection of Pseudomonas aeruginosa, such as cystic fibrosis (Cystic fibrosis, CF) in patients with pulmonary infection It also increases the mortality of patients. Therefore, it is very important to understand the regulation pathway of phenazine synthesis from molecular level to control the pathogenic mechanism of PA.
This study will be connected to the phzA2 promoter deletion promoter luxCDABE reported on the phage binding sites for site-specific recombination, phzA2-luxCDABE will be integrated into the genome of PA, has been reported phzA2 promoter luminescent strains, the chemiluminescence situation directly reflect the expression of phzA2 gene. By random transposon the mutation screening effect of phzA2 gene expression in PA gene on the whole genome level, through the light changes to determine screening can regulate the gene regulation of phzA2 gene expression.
Finally through a large number of screening mutants (approximately more than 20000 clones), the expression of transposase mutants changed 14 strains of phzA2 gene. The random PCR of these mutants, DNA sequencing, alignment identified these mutants in the PAo1 chromosome mutation with loci. In order to further determine the relationship between each other in the US 14 strains of mutants screened, selected two strains of phzA2 expression and pyocyanin production have larger changes in mutants P2G1 and P2E4 were studied in detail. Through the method of insertion of exogenous Gmr-lacZ fragments in the target gene in the gene of PA3043 and PA1387 were knockout mutations, the knockout mutant PAo1 (delta 3043) and PAO1 (delta 1387), and the downstream gene PA1388 of PA1387 overexpression. The experimental results show that the mutant PAo1 (delta 1387), missing the PA1387 gene could cause p The expression of hzA2 and production of pyocyanin are significantly decreased, the expression of PA1387 phzA2 gene regulated by some way, and the amount of synthesis of the final product was also decreased. However, the mutant PAo1 (delta 3043), which has no significant influence on the loss of gene PA3043 on phzA2 surface and pyocyanin, indicates the expression of PA3043 on phzA2 did not influence each other. This may be due to two reactions in the process of transposon mutation affected PA3043 downstream genes, the specific relationship needs to be studied further proof. Because of the mutant PAO1 (delta 1387) there are certain differences between and transposon mutant P2E4 expression level of phzA2, in order to exclude is due to the high expression of PA1388 gene increased pyocyanin production, so the PA1388 gene was overexpressed, we found that the expression of phzA2 had no significant effect.
The above results indicate that there is an unknown gene related to phzA2 in PA. Understanding the specific relationship between these unknown genes and phzA2 will help us to understand the specific regulation process of phenazine compounds in PA.

【學位授予單位】：西北大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R378

【參考文獻】

相關碩士學位論文 前1條

1 段佳麗;銅綠假單胞菌中影響RsmA調節(jié)基因的篩選及調節(jié)途徑研究[D];西北大學;2011年

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本文編號：1675059