Selection and Validation of Reference Genes for Quantitative Real-Time PCR During the Developmental Stages of Seeds in Sophora davidii The

Jingjing Li¹Shanrong Han¹Zongren Xu²Bin Deng³Na Zheng¹Yaqiong Su¹Ziyao Qiao¹Yun Yang¹Hong Zhang²Zongsuo Liang³Jing Liu^2*Shuai Liu^2*

• ¹Northwest University, Xi'an, China
• ²Shaanxi Academy of Traditional Chinese Medicine, Xi'an, China
• ³Guizhou University of Traditional Chinese Medicine, Guiyang, Guizhou Province, China

The seeds of Sophora davidii are abundant in quinolizidine alkaloids, including matrine and sophocarpine, which exhibit significant anticancer potential. However, the biosynthetic pathway of these alkaloids is not fully understood yet, and the expressions of key enzyme genes involved in this pathway still needs further exploration.Quantitative real-time polymerase chain reaction (qRT-PCR), a highly sensitive method for gene expression analysis, holds great application value in relevant research.However, selecting a suitable reference gene is a critical step in guaranteeing the accuracy and reliability of qRT-PCR results. In this study, 10 candidate genes commonly used as reference genes (18S, ACT13, RL15B, RL74, RLA2, RL182, RL291, EF1-α, EF1G, and YLS8) were assessed. Five commonly used software programs, namely GeNorm, NormFinder, BestKeeper, ΔCt, and RefFinder, were used to comprehensively analyze the expression stability of these candidate reference genes in seeds harvested at five different developmental stages after flowering. During these stages, the seeds underwent significant morphological changes. The results revealed that EF1G and RL291 were identified as the most stable reference genes, whereas RL182 was deemed unsuitable as a reference gene. Validation experiments further confirmed that using stable reference genes can accurately normalize the expressions of target genes. These findings not only facilitate a deeper understanding of the biosynthetic pathway of quinolizidine alkaloids but also emphasize the significance of selecting reliable reference genes in gene expression studies.