铜绿假单胞菌基因组中影响吩嗪合成基因phzA2表达的调节基因的筛选及研究

发布时间：2018-03-28 06:05

本文选题：铜绿假单胞菌　切入点：phzA2　出处：《西北大学》2012年硕士论文

【摘要】：铜绿假单胞菌(Pseudomonas aeruginosa, PA)是一种革兰氏阴性条件致病菌,是院内感染的主要病原菌之一,尤其易感染烧伤、手术后和免疫力低下病人。由于其对抗生素有较强的耐药性,因此临床中PA的感染很难根治。吩嗪类化合物是PA产生的一类具有氧化还原活性的小分子物质,也是PA中重要的毒性因子之一,同时还具有信号分子的作用,能够调节许多基因的表达。在铜绿假单胞菌PAO1的基因组中,负责吩嗪-1-羧酸(PCA)合成的基因簇包含两个同源的操纵子phzA1B1C1D1E1F1G1(phzA1)和phzA2B2C2D2E2F2G2(phzA2), PCA是合成吩嗪类化合物的前体物质,可通过phzM、phzS和phzH所编码的酶将其转化为其他吩嗪类物质,包括绿脓菌素、1-羟基-吩嗪(1-HP)和吩嗪-1-甲酰胺(PCN)。绿脓菌素是其中一个较为重要的吩嗪类化合物,在PA感染人体的过程中,绿脓菌素的产生会增加PA的感染率,例如囊肿性纤维化(Cystic fibrosis,CF)病人的肺部感染,同时也会引起病人死亡率的增加。因此,从分子水平上了解影响吩嗪合成的调节途径对于控制PA的致病机制具有重要的意义。本研究将phzA2的启动子连接到缺失启动子的luxCDABE报道子上,再利用噬菌体结合位点进行位点专一性重组,将phzA2-luxCDABE整合到PA的基因组上,得到phzA2启动子的发光报道菌株,其化学发光情况直接反映phzA2基因的表达情况。采取随机转座突变的方法,在PA全基因组水平上筛选影响phzA2基因表达的基因,通过发光的变化来确定筛选能够调节phzA2基因表达的调节基因。最终我们通过大量筛选转座突变体(约20000多个克隆),得到了14株phzA2基因表达发生变化的转座突变体。对这些突变体进行随机PCR、DNA测序、比对后确定了这些转座突变体在PAo1染色体上突变的具体位点。为了进一步确定相互之间的关系,我们在筛选出的14株转座突变体中,挑选了两株phzA2的表达和绿脓菌素产量都有较大变化的突变体P2G1和P2E4进行深入的研究。通过在目标基因中插入外源Gmr-lacZ片段的方法,对基因PA3043和PA1387进行了敲除突变,得到了敲除突变体PAo1(△3043)和PAO1(△1387),并对PA1387的下游基因PA1388进行了过表达。实验结果表明,在突变体PAo1(△1387)中,基因PA1387的缺失能够导致phzA2的表达和绿脓菌素的产量都发生明显的降低,说明PA1387通过某种途径调节phzA2的基因的表达,并使其最终产物的合成量也明显降低。然而,在突变体PAo1(△3043)中,基因PA3043的缺失对phzA2的表达及绿脓菌素的产生并无明显的影响,表明PA3043对phzA2的表达并没有相互的影响。这可能是由于转座突变过程中出现的两极反应影响了PA3043上下游的基因造成的,具体的关系有待进一步的研究证明。由于突变体PAO1(△1387)与转座突变体P2E4中phzA2表达水平有着一定的差异,为了排除是由于高表达PA1388基因引起绿脓菌素产量的升高,所以对基因PA1388进行了过表达,发现其对phzA2的表达并无明显影响。上述结果说明,在PA中存在未知的与phzA2有着一定关系的基因,了解这些未知基因与phzA2的具体关系有助于我们认识PA中合成吩嗪类化合物的具体调控过程。
[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.

【学位授予单位】：西北大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R378

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