Department of Ecological Science and Engineering, College of Resources and Environmental Sciences, China Agricultural University, Beijing, China.
Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing, China.
J Appl Microbiol. 2019 Apr;126(4):1096-1107. doi: 10.1111/jam.14215. Epub 2019 Feb 27.
Rahnella aquatilis is a Gram-negative bacterial species with potential for agricultural and industrial applications, as well as a human pathogen. This study aims to identify an optimal reference gene to explore antibiotic resistance and stress tolerance in R. aquatilis using reverse-transcription quantitative polymerase chain reaction (RT-qPCR).
Expression levels of 14 housekeeping genes in R. aquatilis were estimated by RT-qPCR under six different conditions: exponential phase, stationary phase, acid, salinity, antibiotic and oxidative stresses. BestKeeper and the ΔCt method were used to evaluate the stability of each gene. The atpD gene was stably expressed in all conditions, thus was selected and validated as an optimal reference gene. Transcript levels of 17 putative ampicillin-resistance genes in R. aquatilis strain HX2 were evaluated using the proposed RT-qPCR. Six genes encoding efflux transporters and β-lactamase were overexpressed after ampicillin treatment. Additionally, the expression of seven putative stress response genes in strain HX2 was assessed, and five genes were up-regulated by respective stress treatments.
The atpD gene has been identified as an optimal reference gene for expression analysis of R. aquatilis responses to abiotic stresses by RT-qPCR.
The proposed RT-qPCR is suitable for gene expression analysis in R. aquatilis, thus useful for studying antimicrobial resistance and stress tolerance in this bacterium and others closely related.
海栖拉恩菌是一种革兰氏阴性细菌，具有农业和工业应用以及人类病原体的潜力。本研究旨在通过逆转录定量聚合酶链反应 (RT-qPCR) 鉴定一种最佳的参考基因，以探讨海栖拉恩菌的抗生素耐药性和应激耐受性。
通过 RT-qPCR 在六种不同条件下（指数期、静止期、酸性、盐度、抗生素和氧化应激）估计了海栖拉恩菌中 14 种管家基因的表达水平。使用 BestKeeper 和 ΔCt 方法评估每个基因的稳定性。在所有条件下，atpD 基因表达稳定，因此被选为最佳参考基因并进行验证。使用建议的 RT-qPCR 评估了海栖拉恩菌菌株 HX2 中 17 种推定氨苄青霉素抗性基因的转录水平。氨苄青霉素处理后，编码外排转运蛋白和β-内酰胺酶的 6 个基因表达上调。此外，还评估了菌株 HX2 中 7 种推定应激反应基因的表达，5 种基因分别受到各自的应激处理而上调。
atpD 基因已被确定为通过 RT-qPCR 分析海栖拉恩菌对非生物应激反应的最佳参考基因。
所提出的 RT-qPCR 适用于海栖拉恩菌基因表达分析，因此可用于研究该细菌及其他密切相关细菌的抗菌耐药性和应激耐受性。
