College of Agriculture, Shanxi Agricultural University, Jinzhong, Shanxi, China.
The Department of Biological Science and Technology, Changzhi University, Changzhi, Shanxi, China.
PLoS One. 2020 Jan 27;15(1):e0227927. doi: 10.1371/journal.pone.0227927. eCollection 2020.
Pseudomonas brassicacearum GS20 is an antagonistic strain of bacteria recently isolated from the rhizosphere of Codonopsis pilosula. No validated reference gene has been indentified from P. brassicacearum to use in the normalization of real-time quantitative reverse transcription-PCR data. Therefore, in this study, nine candidate reference genes (recA, gyrA, rpoD, proC, gmk, rho, 16S, ftsz, and secA) were assessed at different growth phases and under various growth conditions. The expression stability of these candidate genes was evaluated using BestKeeper, NormFinder and GeNorm. In general, the results showed rho, rpoD and gyrA were the most suitable reference genes for P. brassicacearum GS20. The relative expression of iron-regulated gene (fhu) was normalized to verify the reliability of the proposed reference genes under iron-replete and iron-limited conditions. The trend in relative expression was consistent with the change in siderophore production under different iron conditions. This study presents reliable reference genes for transcriptional studies in P. brassicacearum GS20 under the chosen experimental conditions.
铜绿假单胞菌 GS20 是一种从党参根际中分离出来的拮抗细菌。目前尚未从铜绿假单胞菌中鉴定出经过验证的参考基因，用于实时定量逆转录 PCR 数据的标准化。因此，在这项研究中，评估了 9 个候选参考基因（recA、gyrA、rpoD、proC、gmk、rho、16S、ftsZ 和 secA）在不同生长阶段和不同生长条件下的表达稳定性。使用 BestKeeper、NormFinder 和 GeNorm 评估这些候选基因的表达稳定性。一般来说，结果表明 rho、rpoD 和 gyrA 是铜绿假单胞菌 GS20 最适合的参考基因。用铁调节基因（fhu）的相对表达来验证在铁充足和铁限制条件下建议的参考基因的可靠性。在不同铁条件下，相对表达的趋势与铁载体产生的变化一致。这项研究为在所选实验条件下进行铜绿假单胞菌 GS20 的转录研究提供了可靠的参考基因。
