Sahni Azita, Hajjari Mohammadreza, Raheb Jamshid, Foroughmand Ali Mohammad, Asgari Morteza
Nour Danesh Institute of Higher Education, Department of Biology, Isfahan, Iran.
Department of Genetics, Faculty of Science, Shahid Chamran University of Ahvaz, Ahvaz, Iran.
Bioinformation. 2017 Jan 31;13(1):21-24. doi: 10.6026/97320630013021. eCollection 2017.
Recent reports have indicated that small RNAs have key roles in the response of the E.coli to stress and also in the regulating of virulence factors. It seems that some small non-coding RNAs are involved in multidrug resistance. Previous studies have indicated that rprA can increase the tolerance to Kanamycin in RcsB-deficient Escherichia coli K-12 following osmotic shock. The current study aims to clone and over-express the non-coding RNA rprA in E.coli and investigate its effect on the bacterial resistance to Kanamycin without any osmotic shock. For this purpose, rprA gene was amplified by the PCR and then cloned into the PET-28a (+) vector. The recombinant plasmid was transformed into wild type E.coli BL21 (DE3). The over expression was induced by IPTG and confirmed by qRT-PCR. The resistance to the kanamycin was then measured in different times by spectrophotometry. The statistical analysis showed that the rprA can increase the resistance to Kanamycin in Ecoli K12. The interaction between rprA and rpoS was reviewed and analyzed by in silico methods. The results showed that the bacteria with over-expressed rprA were more resistant to Kanamycin. The present study is an important step to prove the role of non-coding RNA rprA in bacterial resistance. The data can be the basis for future works and can also help to develop and deliver next-generation antibiotics.
最近的报告表明，小RNA在大肠杆菌应对压力以及调节毒力因子方面发挥着关键作用。似乎一些小的非编码RNA参与了多药耐药性。先前的研究表明，在渗透休克后，rprA可以提高RcsB缺陷型大肠杆菌K-12对卡那霉素的耐受性。本研究旨在克隆非编码RNA rprA并在大肠杆菌中过表达，研究其在无任何渗透休克情况下对细菌抗卡那霉素能力的影响。为此，通过PCR扩增rprA基因，然后将其克隆到PET-28a(+)载体中。将重组质粒转化到野生型大肠杆菌BL21(DE3)中。通过IPTG诱导过表达，并通过qRT-PCR进行确认。然后在不同时间通过分光光度法测量对卡那霉素的抗性。统计分析表明，rprA可以提高大肠杆菌K12对卡那霉素的抗性。通过计算机模拟方法对rprA与rpoS之间的相互作用进行了综述和分析。结果表明，rprA过表达的细菌对卡那霉素更具抗性。本研究是证明非编码RNA rprA在细菌抗性中作用的重要一步。这些数据可为未来的研究奠定基础，也有助于开发和提供下一代抗生素。
