Fan Wenna, Shi Yaqi, Shi Pengfei, Yang Yixin, Zhang Mengyao
Animal Science and Technology College, Henan University of Science and Technology, Luoyang, 471003, Henan, China.
Sci Rep. 2025 Feb 21;15(1):6324. doi: 10.1038/s41598-025-90664-2.
Compared with the traditional gene expression techniques of quantitative analysis, RT-qPCR is most widely used because of its low cost, time-saving, and accuracy. It is essential to introduce suitable internal reference genes as reference corrections in RT-qPCR experiments to reduce the RNA quality and reverse transcription efficiency of different samples. In our study, we chose the candidate internal reference genes of alfalfa from transcriptome sequence datasets (162 RNA-seq sequencing data) through comparative analysis. Finally, 10 candidate reference genes were selected. These candidate reference gene expressions were determined by RT-qPCR under five abiotic stresses of drought, alkali, high temperature, low temperature, and acid treatments. The stability index of these candidate genes was calculated and evaluated correspondently using specific software and different lgorithms, such as GeNorm, Normfinder, Bestkeeper, ΔCt method, and an online analysis tool RefFinder. Then the appropriate candidate genes were screened strictly; and validated by the phyA gene. GAPDH and Actin are taken as traditional reference genes on gene expression of quantitative analysis commonly used in alfalfa, Our results showed GAPDH and Actin aren't the most appropriate reference genes of alfalfa under different abiotic stresses, under alkaline stress, the optimal reference gene is UBL-2a, and the optimal combination of reference genes is MS.65,463 (some candidate reference genes haven't been annotated yet, using gene ID abbreviation number of Medicago sativa L. instead )and UBL-2a; Under drought stress, the optimal reference gene is Ms.33,066, and the optimal combination of reference genes is MS.65,463 and UBL-2a; Under high-temperature stress, the optimal reference gene is Actin, and the optimal combination of reference genes is Rer1, Actin, MS.00617, MS.74,923, UBL-2a, MS.33,066, MS.99,505, and MS.65,463; Under low-temperature stress, the optimal reference gene is Actin, and the optimal combination of reference genes is Rer1, Actin, MS.99,505, MS.073307, and UBL-2a. The optimal reference genes and their combinations need further validation under acid stress. This paper provides scientific evidence for quantitative analysis of the genes of alfalfa.
与传统的基因表达定量分析技术相比，RT-qPCR因其成本低、省时且准确性高而被广泛应用。在RT-qPCR实验中引入合适的内参基因作为参照校正，对于降低不同样本的RNA质量和逆转录效率至关重要。在我们的研究中，通过比较分析从转录组序列数据集（162个RNA-seq测序数据）中选择了苜蓿的候选内参基因。最终，筛选出10个候选内参基因。通过RT-qPCR测定这些候选内参基因在干旱、盐碱、高温、低温和酸处理这五种非生物胁迫下的表达情况。使用GeNorm、Normfinder、Bestkeeper、ΔCt法等特定软件和不同算法以及在线分析工具RefFinder相应地计算并评估这些候选基因的稳定性指数。然后严格筛选出合适的候选基因；并通过phyA基因进行验证。GAPDH和肌动蛋白通常被用作苜蓿基因表达定量分析中的传统内参基因，我们的结果表明，在不同非生物胁迫下，GAPDH和肌动蛋白并非苜蓿最合适的内参基因，在盐碱胁迫下，最佳内参基因是UBL-2a，最佳内参基因组合是MS.65,463（部分候选内参基因尚未注释，用紫花苜蓿的基因ID缩写编号代替）和UBL-2a；在干旱胁迫下，最佳内参基因是Ms.33,066，最佳内参基因组合是MS.65,463和UBL-2a；在高温胁迫下，最佳内参基因是肌动蛋白，最佳内参基因组合是Rer1、肌动蛋白、MS.00617、MS.74,923、UBL-2a、MS.33,066、MS.99,505和MS.65,463；在低温胁迫下，最佳内参基因是肌动蛋白，最佳内参基因组合是Rer1、肌动蛋白、MS.99,505、MS.073307和UBL-2a。最佳内参基因及其组合在酸胁迫下还需进一步验证。本文为苜蓿基因的定量分析提供了科学依据。
