Wu Qixuan, Deng Meixin, Zhao Xiaolan, Long Jianmei, Zhang Jianxia
Guangdong Key Laboratory for Innovative Development and Utilization of Forest Plant Germplasm, College of Forestry and Landscape Architecture, South China Agricultural University, Guangzhou, 510642, China.
Sci Rep. 2025 Apr 1;15(1):11045. doi: 10.1038/s41598-025-96110-7.
Abelmoschus Manihot is an important medicinal and edible plant known for its functional secondary metabolites. However, little is known about the key genes involved in production of secondary metabolites in A. manihot. This is largely due to the lack of effective gene expression detection systems for A. manihot, making the screening of real-time PCR reference genes a prerequisite. In this study, 11 candidate reference genes were screened and cloned from A. manihot, and their expression stability was evaluated in different tissues under different flowering stages using four algorithms: geNorm, NormFinder, BestKeeper, and RefFinder. The expression stability of eIF and PP2A1 was the highest, while that of tubulin alpha (TUA) was the lowest. The combined use of the two most stable reference genes, eIF and PP2A1, met the experimental requirements for normalizing gene expression in A. manihot. Furthermore, the gene expression of transcription factors bHLH147 and bHLH148 was further validated by data normalization. This study identified potential reference genes in different A. manihot tissues, paving the way for functional gene analysis and dissecting metabolite regulation mechanisms in A. manihot.
黄蜀葵是一种重要的药食两用植物，以其功能性次生代谢产物而闻名。然而，关于黄蜀葵次生代谢产物合成所涉及的关键基因，人们了解甚少。这在很大程度上是由于缺乏有效的黄蜀葵基因表达检测系统，使得筛选实时荧光定量PCR内参基因成为一项先决条件。在本研究中，从黄蜀葵中筛选并克隆了11个候选内参基因，并使用geNorm、NormFinder、BestKeeper和RefFinder这四种算法在不同开花阶段的不同组织中评估了它们的表达稳定性。真核起始因子（eIF）和蛋白磷酸酶2A1（PP2A1）的表达稳定性最高，而α-微管蛋白（TUA）的表达稳定性最低。联合使用两个最稳定的内参基因eIF和PP2A1，满足了黄蜀葵基因表达标准化的实验要求。此外，通过数据标准化进一步验证了转录因子bHLH147和bHLH148的基因表达。本研究鉴定了黄蜀葵不同组织中的潜在内参基因，为黄蜀葵功能基因分析和剖析代谢物调控机制铺平了道路。
