Li Yongmei, Yao Yuan, Xu Tiancheng, Yang Xue, He Yuting, Zhang Shengmin, Xie Qingbiao, Cao Jie, Tang Chaorong, Liu Hui
School of Breeding and Multiplication (Sanya Institute of Breeding and Multiplication), School of Tropical Agriculture and Forestry, Hainan University, Sanya, 572025, China; Department of Biological Science and Technology, Jinzhong University, Jinzhong, 030619, China.
School of Breeding and Multiplication (Sanya Institute of Breeding and Multiplication), School of Tropical Agriculture and Forestry, Hainan University, Sanya, 572025, China.
J Plant Physiol. 2025 Jun;309:154501. doi: 10.1016/j.jplph.2025.154501. Epub 2025 Apr 24.
Quantitative real-time polymerase chain reaction (qRT-PCR) is a highly sensitive and widely used method for analyzing gene expression profiles. Accurate qRT-PCR normalization requires the identification of stable reference genes under specific experimental conditions. Although seven reference genes have been used in Taraxacum kok-saghyz (TKS), an alternative natural rubber-producing crop, a systematic identification of reliable internal references for gene expression analysis across tissues at distinct developmental stages of TKS has not been conducted. In this study, we screened 12 candidate reference genes (CRGs) based on RNA-seq data from 26 TKS samples, representing five tissue types and nine developmental stages. The expression levels of the 12 CRGs, along with 7 previously reported reference genes (RRGs), were quantified by qRT-PCR across various tissues and developmental stages. The expression stability of the 19 genes was further evaluated by four commonly used algorithms (geNorm, NormFinder, comparative delta Ct, and BestKeeper), and their results were integrated by RefFinder to generate a comprehensive stability ranking. The final results revealed that TkADF1 and TkRPT6A were the most suitable internal control genes for the all-tissue group and leaf samples. TkUPL and TkSIZ1 were found to be optimal for root samples, while TkADF1 and TkSRPRA were preferred choices for latex samples. Moreover, validation using two rubber biosynthesis-related genes (TkFPS1 and TkSRPP2) confirmed the reliability of these recommended genes, showing a strong positive correlation with the RNA-seq data. This study provides reliable reference genes for qRT-PCR normalization in TKS, facilitating future research on developmental regulation and natural rubber biosynthesis.
定量实时聚合酶链反应（qRT-PCR）是一种高度灵敏且广泛应用于分析基因表达谱的方法。准确的qRT-PCR标准化需要在特定实验条件下鉴定稳定的内参基因。尽管已经在橡胶草（TKS）——一种替代性天然橡胶生产作物中使用了7个内参基因，但尚未对橡胶草不同发育阶段各组织中用于基因表达分析的可靠内参进行系统鉴定。在本研究中，我们基于来自26个橡胶草样本的RNA-seq数据筛选了12个候选内参基因（CRGs），这些样本代表了5种组织类型和9个发育阶段。通过qRT-PCR对12个CRGs以及7个先前报道的内参基因（RRGs）在各种组织和发育阶段的表达水平进行了定量。通过四种常用算法（geNorm、NormFinder、比较ΔCt和BestKeeper）进一步评估了这19个基因的表达稳定性，并通过RefFinder整合它们的结果以生成综合稳定性排名。最终结果表明，TkADF1和TkRPT6A是全组织组和叶片样本最适合的内参基因。发现TkUPL和TkSIZ1对根样本是最优的，而TkADF1和TkSRPRA是乳胶样本的首选。此外，使用两个橡胶生物合成相关基因（TkFPS1和TkSRPP2）进行的验证证实了这些推荐基因的可靠性，显示出与RNA-seq数据有很强的正相关性。本研究为橡胶草qRT-PCR标准化提供了可靠的内参基因，有助于未来对发育调控和天然橡胶生物合成的研究。
