College of Forestry, Shenyang Agricultural University, Shenyang, China.
State Key Laboratory of Tree Genetics and Breeding (Northeast Forestry University), Harbin, China.
PLoS One. 2019 Dec 3;14(12):e0225926. doi: 10.1371/journal.pone.0225926. eCollection 2019.
Selecting appropriate reference genes is vital to normalize gene expression analysis in birch (Betula platyphylla) under different abiotic stress conditions using quantitative real-time reverse transcription PCR (qRT-PCR). In this study, 11 candidate birch reference genes (ACT, TUA, TUB, TEF, 18S rRNA, EF1α, GAPDH, UBC, YLS8, SAND, and CDPK) were selected to evaluate the stability of their expression in different tissues and under different abiotic stress conditions. Three statistical algorithms (GeNorm, NormFinder, and BestKeeper) were used to analyze the stability of the 11 candidate reference genes to identify the most appropriate one. The results indicated that EF-1α was the most stable reference gene in different birch tissues, ACT was the most stable reference gene for normal conditions, ACT and TEF were the most stable reference genes for salt stress treatment, TUB was the most stable reference gene for osmotic stress treatment, and ACT was the most appropriate choice in all samples of birch. In conclusion, the most appropriate reference genes varied among different experimental conditions. However, in this study, ACT was the optimum reference gene in all experimental groups, except in the different tissues group. GAPDH was the least stable candidate reference gene in all experimental conditions. In addition, three stress-induced genes (BpGRAS1, BpGRAS16, and BpGRAS19) were chosen to verify the stability of the selected reference genes in different tissues and under salt stress. This study laid the foundation for the selection of appropriate reference gene(s) for future gene expression pattern studies in birch.
选择合适的内参基因对于使用实时荧光定量 RT-PCR（qRT-PCR）分析不同非生物胁迫条件下白桦（Betula platyphylla）基因表达的归一化至关重要。本研究中，选择了 11 个候选白桦内参基因（ACT、TUA、TUB、TEF、18S rRNA、EF1α、GAPDH、UBC、YLS8、SAND 和 CDPK），以评估其在不同组织和不同非生物胁迫条件下表达的稳定性。使用三种统计算法（GeNorm、NormFinder 和 BestKeeper）分析 11 个候选内参基因的稳定性，以确定最合适的基因。结果表明，EF-1α是不同白桦组织中最稳定的内参基因，ACT 是正常条件下最稳定的内参基因，ACT 和 TEF 是盐胁迫处理下最稳定的内参基因，TUB 是渗透胁迫处理下最稳定的内参基因，ACT 是白桦所有样本中最合适的选择。总之，最适内参基因因实验条件而异。然而，在本研究中，ACT 是所有实验组（不同组织组除外）的最佳内参基因。GAPDH 是所有实验条件下最不稳定的候选内参基因。此外，选择了三个胁迫诱导基因（BpGRAS1、BpGRAS16 和 BpGRAS19）来验证所选内参基因在不同组织和盐胁迫下的稳定性。本研究为白桦未来基因表达模式研究中选择合适的内参基因奠定了基础。
