Li Jingjing, Han Shanrong, Xu Zongren, Deng Bin, Zheng Na, Su Yaqiong, Qiao Ziyao, Yang Yun, Zhang Hong, Liang Zongsuo, Liu Jing, Liu Shuai
Key Laboratory of Resource Biology and Biotechnology in Western China, Ministry of Education/College of Life Science, Northwest University, Xi'an, Shaanxi, China.
Institute of Chinese Materia Medica, Shaanxi Academy of Traditional Chinese Medicine, Xi'an, Shaanxi, China.
Front Plant Sci. 2025 May 16;16:1485586. doi: 10.3389/fpls.2025.1485586. eCollection 2025.
Quinolizidine alkaloids, such as matrine and sophocarpine, enriched in seeds, demonstrate notable anticancer properties. However, the biosynthetic pathway of these alkaloids remains incompletely elucidated, and the expression patterns of key enzyme genes involved in this pathway require further investigation. Quantitative real-time PCR (qRT-PCR) serves as a highly sensitive method for gene expression analysis, yet selecting appropriate reference genes is crucial to ensure the accuracy and reliability of results.
Ten candidate reference genes (, and ) were evaluated for their expression stability in seeds collected at five distinct developmental stages post-flowering, characterized by significant morphological changes. Five computational tools-GeNorm, NormFinder, BestKeeper, ΔCt, and RefFinder-were employed to comprehensively analyze the stability of these genes.
Among the candidate genes, and exhibited the highest expression stability, whereas proved unsuitable as a reference gene. Validation experiments confirmed that normalization using stable reference genes (e.g., and ) yielded accurate quantification of target gene expression.
This study identifies and as optimal reference genes for qRT-PCR analysis during Sophora davidii seed development, addressing a critical methodological gap in alkaloid biosynthesis research. These findings underscore the necessity of rigorous reference gene validation to ensure reliable gene expression data. The results advance our understanding of quinolizidine alkaloid biosynthesis and highlight the broader importance of reference gene selection in plant molecular studies.
喹诺里西啶生物碱，如苦参碱和槐果碱，在种子中含量丰富，具有显著的抗癌特性。然而，这些生物碱的生物合成途径仍未完全阐明，参与该途径的关键酶基因的表达模式需要进一步研究。定量实时聚合酶链反应（qRT-PCR）是一种用于基因表达分析的高度灵敏方法，但选择合适的内参基因对于确保结果的准确性和可靠性至关重要。
对10个候选内参基因（……）在开花后五个不同发育阶段采集的苦参种子中的表达稳定性进行评估，这些阶段具有显著的形态变化。使用五种计算工具——GeNorm、NormFinder、BestKeeper、ΔCt和RefFinder——全面分析这些基因的稳定性。
在候选基因中，……表现出最高的表达稳定性，而……被证明不适合作内参基因。验证实验证实，使用稳定的内参基因（如……）进行标准化可准确量化靶基因的表达。
本研究确定了……作为苦参种子发育过程中qRT-PCR分析的最佳内参基因，填补了生物碱生物合成研究中一个关键的方法学空白。这些发现强调了严格验证内参基因以确保可靠基因表达数据的必要性。研究结果增进了我们对喹诺里西啶生物碱生物合成的理解，并突出了内参基因选择在植物分子研究中的更广泛重要性。
