RgsA在铜绿假单胞菌抗氧化应激中的作用

发布时间：2018-06-03 16:27

  本文选题：铜绿假单胞菌 + RgsA　； 参考：《福建医科大学》2013年硕士论文

【摘要】：目的: 铜绿假单胞菌是临床上极为常见的条件致病菌，患代谢性疾病、血液病和恶性肿瘤的患者，以及术后或某些治疗后的患者易感染本菌，具有多重耐药的特点。寻找铜绿假单胞菌通过何种途径感知外界的变化，以何种方式改变生理学过程从而适应环境，一直是该菌的研究热点之一。 细菌小RNA(small regulatory RNA)是细菌中普遍存在的一类长度在40～500个核苷酸的，具有调控功能的小分子RNA,简称为sRNA(small RNA)，sRNA在广泛细菌种群的许多关键生命进程中发挥重要的调控作用，其调控作用在数量上和多样性上甚至超过了蛋白质。迄今已在铜绿假单胞菌中发现29种sRNA，其中RgsA是由Nicolas González在2008年进行全基因组搜索发现的，存在于铜绿假单胞菌和荧光假单胞菌中的sRNA，推测可能与铜绿假单胞菌抗氧化应激反应有关，在铜绿假单胞菌生物膜状态下高度表达。铜绿假单胞菌难治性持续性感染与生物膜形成联系密切。在人类感染性疾病中，约有65%与生物膜有关，例如牙菌斑、囊性纤维化肺病、反复发作的慢性中耳炎、慢性骨髓炎、慢性鼻窦炎及慢性伤口感染等。对本研究拟从sRNA RgsA的基本结构、功能、作用机制到调控方式进行深入研究，以探讨该sRNA在铜绿假单胞菌生命活动中的作用。 方法: （1）本研究以铜绿假单胞菌PAO1菌株作为实验菌株，根据公用数据库的序列信息和文献资料设计合成引物，采用5′RACE确定转录起始位点，结合Northern blot与计算机预测3′末端转录终止序列，以确定RgsA的基本转录本结构。 （2）在此基础上，PCR扩增rgsA同源臂与庆大霉素抗性基因插入自杀质粒pEX18-Ap，构建rgsA基因敲除载体，利用双亲杂交转化铜绿假单胞菌PAO1，构建rgsA基因缺陷株SH2-5。 （3） PCR扩增rgsA基因编码片段，插入pJN105质粒阿拉伯糖诱导型启动子的下游，构建rgsA基因表达载体，转化rgsA基因缺失缺陷株SH2-5，构建rgsA基因补偿/过表达株。 （3）以生长时间为横坐标，OD600值为纵坐标绘制生长曲线，观察野生株、rgsA基因缺陷株的生长情况。（4）利用Trizol法分别抽提不同浓度过氧化氢，有机过氧化物刺激下铜绿假单胞菌的总RNA，Real-time PCR检测RgsA表达情况。 （5）以含有不同浓度过氧化氢，有机过氧化物LB平板作为培养基进行平板实验，以不同浓度过氧化氢与不同反应时间组合，进行MTT实验，比较野生株，缺失株以及过表达菌株在浮游菌状态下抗氧化应激能力。 （6）野生株，缺失株以及过表达菌株分别进行生物膜培养，三天后利用不同浓度过氧化氢分别对形成的生物膜进行刺激， SYTO9/PI染色后荧光显微镜观察，以比较三者间在生物膜状态下抗氧化应激能力。 结果： （1）rgsA在铜绿假单胞菌中存在两个转录本，转录本长度分别约为300bp与90bp。其中短片段转录本的转录起始位点，位于NCBI注释基因片段起始下游86bp处，长片段转录本的转录起始位点，位于NCBI注释基因片段起始上游157bp处。 （2）经PCR及测序证明，铜绿假单胞菌缺陷株SH2-5中缺失rgsA基因，成功构建铜绿假单胞菌rgsA基因缺陷株。经测序证明，过表达质粒pJN105-SH和补偿/过表达菌株SH2S构建的正确性。 （3）生长曲线提示rgsA基因缺陷株生长速度明显低于野生株。 （4）经Real-time PCR检测，不同浓度过氧化氢以及有机过氧化物刺激下，RgsA表达均有不同程度的增加，尤其在有机过氧化物刺激时表达明显上升。 （5）通过计算生长于含有不同浓度过氧化氢与有机过氧化物的LB平板上的菌落数，以及MTT法检测各实验组中细菌总体活性，rgsA基因缺陷株表现出对过氧化氢及有机过氧化物的抵抗能力明显下降，而在突变菌株中导入表达载体后，在0.1%L-Arabinose的诱导下，对氧化应激的抵抗能力恢复到接近野生株的水平。 （6）野生株，缺失株，，过表达株在流动培养系统中培养3天后形成生物膜，在30mM过氧化氢刺激下，相比野生株及0.1%L-Arabinose的诱导下的过表达株细菌，缺失株出现大面积能被SYTO9染色的死菌（呈黄色）。 结论： rgsA在铜绿假单胞菌中存在两个转录本，证明RgsA在铜绿假单胞菌抗氧化应激中发挥重要作用。
[Abstract]:Objective:
Pseudomonas aeruginosa is a very common pathogenic bacteria in clinic. Patients with metabolic diseases, hematological and malignant tumors, and patients after or after some treatment are susceptible to the bacteria and have the characteristics of multiple drug resistance. To adapt to the environment, it has always been one of the research hotspots of the bacteria.
Bacterial small RNA (small regulatory RNA) is a common type of nucleotides, a small molecule with 40~500 nucleotides and regulatory functions, RNA, called sRNA (small RNA). SRNA plays an important regulatory role in many critical life processes in a wide range of bacterial populations, and its regulatory role is even in quantity and diversity. To date, 29 kinds of sRNA have been found in Pseudomonas aeruginosa, of which RgsA was found in the whole genome search by Nicolas Gonz ah lez in 2008, existed in Pseudomonas aeruginosa and sRNA in Pseudomonas fluorescens, presumably related to the antioxidant stress response of Pseudomonas aeruginosa and high in the state of Pseudomonas aeruginosa biofilm. In human infectious diseases, about 65% are associated with biofilms, such as plaque, cystic fibrosis, chronic otitis media, chronic osteomyelitis, chronic sinusitis, and chronic wound infection. This study is intended to be based on the base of sRNA RgsA. This structure, function, action mechanism and regulation mode were studied in depth to explore the role of sRNA in the life activities of P. aeruginosa.
Method:
(1) in this study, Pseudomonas aeruginosa PAO1 strain was used as an experimental strain. The primers were designed according to the sequence information and literature of the public database. The transcriptional starting site was determined by 5 'RACE, and the 3' terminal transcriptional terminating sequence was predicted with Northern blot and computer to determine the basic transcriptional structure of RgsA.
(2) on this basis, the rgsA homologous and gentamicin resistant genes were inserted into the suicide plasmid pEX18-Ap, and the rgsA gene knockout vector was constructed by PCR, and the rgsA gene defective strain SH2-5. was constructed by using parental hybridization to transform the PAO1 of Pseudomonas aeruginosa.
(3) PCR amplified rgsA gene encoding fragment, inserted the downstream of the pJN105 plasmid Arabia sugar inducible promoter, constructed the rgsA gene expression vector, transformed the rgsA gene deletion defect strain SH2-5, and constructed the rgsA gene compensation / overexpression strain.
(3) taking the growth time as the horizontal coordinate and the OD600 value as the longitudinal coordinate to draw the growth curve to observe the growth of the wild plant and the rgsA gene defective strain. (4) the Trizol method was used to extract the different concentration of hydrogen peroxide, and the total RNA of Pseudomonas aeruginosa was stimulated by organic peroxide, and the Real-time PCR was used to detect the expression of RgsA.
(5) the experiment was carried out with different concentrations of hydrogen peroxide and organic peroxide LB plate as medium. The MTT experiment was carried out at different concentrations of hydrogen peroxide and different reaction time. The anti oxidative stress ability of wild plant, missing strain and overexpressed strain in the state of planktonic bacteria was compared.
(6) wild plants, missing strains and overexpressed strains were cultured for biofilm respectively. After three days, the biofilms were stimulated by different concentrations of hydrogen peroxide. After SYTO9/PI staining, the fluorescence microscope was observed to compare the antioxidant activity of the three in the biofilm state.
Result锛

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