Research & Development Center of Biorational Pesticides, Northwest A & F University, Yangling, 712100, Shaanxi Province, China.
Research Center of Biopesticide Technology & Engineering Center, Yangling, 712100, Shaanxi Province, China.
Mol Biol Rep. 2019 Aug;46(4):4161-4174. doi: 10.1007/s11033-019-04867-8. Epub 2019 May 20.
Validation of suitable reference genes is critical in quantitative real-time polymerase chain reaction (qRT-PCR) analysis. Suitable and reliable reference genes for the normalization of gene expression data are characterized by high gene expression stability across tissues and different experimental conditions. This study evaluated the gene expression stability of ten reference genes commonly used in Arabidopsis thaliana for their suitability in qRT-PCR analysis in Tripterygium wilfordii Hook.f. The orthologous sequences of these ten candidate genes were identified from T. wilfordii transcriptomic data (Project No. SRX472292). Five algorithms including GeNorm, NormFinder, BestKeeper, ΔCt, and RefFinder were used to assess the gene expression stability of these putative reference genes in different plant tissues and different stress conditions. The results identified ACTINT7 and TBP as the most suitable reference genes across all samples. The gene expressions of TwHMGR (3-hydroxy-3-methylglutaryl coenzyme A reductase, KU246037.1) and of TwDXR (1-deoxy-D-xylulose-5-phosphate reductoisomerase, KJ174341.1) were investigated to validate the suitability of the reference genes. The validation analysis confirmed the suitability of ACTINT7 and TBP as the best reference genes for elucidating secondary metabolite biosynthesis pathway in T. wilfordii. In summary, this study identified the most suitable and reliable reference genes for future qRT-PCR- based studies in T. wilfordii.
在定量实时聚合酶链反应 (qRT-PCR) 分析中，验证合适的参考基因至关重要。适合且可靠的参考基因用于标准化基因表达数据的特征是在组织和不同实验条件下具有高基因表达稳定性。本研究评估了拟南芥中十种常用参考基因在雷公藤 qRT-PCR 分析中的适用性，这些基因在雷公藤转录组数据（项目编号：SRX472292）中鉴定出这些候选基因的直系同源序列。使用 GeNorm、NormFinder、BestKeeper、ΔCt 和 RefFinder 五种算法评估这些假定参考基因在不同植物组织和不同胁迫条件下的基因表达稳定性。结果表明，ACTINT7 和 TBP 是所有样品中最适合的参考基因。TwHMGR（3-羟基-3-甲基戊二酰辅酶 A 还原酶，KU246037.1）和 TwDXR（1-脱氧-D-木酮糖-5-磷酸还原异构酶，KJ174341.1）的基因表达被用来验证参考基因的适用性。验证分析证实了 ACTINT7 和 TBP 作为阐明雷公藤次生代谢物生物合成途径的最佳参考基因的适用性。总之，本研究确定了最适合和可靠的参考基因，用于未来雷公藤的 qRT-PCR 研究。
