Lin Ying, Liu Guofeng, Rao Ying, Wang Bo, Tian Ruifeng, Tan Yuanyuan, Peng Ting
College of Agriculture/Key Laboratory Plant Resources Conservation and Germplasm Innovation in Mountainous Region (Ministry of Education), Guizhou University, Guiyang, 550025, China.
Department of Botany, Guangzhou Institute of Forestry and Landscape Architecture, Guangzhou, 510405, China.
J Plant Physiol. 2023 Feb;281:153925. doi: 10.1016/j.jplph.2023.153925. Epub 2023 Jan 14.
Himalayan onion (Allium wallichii) is a perennial bulbous herb with high ornamental value and has long been used as traditional medicines in Nepal and China because of the anti-cancer and anti-microbial activities. Wild Allium wallichii features different flower colors, including purple, pink, deep purple and white. However, little is known about the molecular mechanisms of color formation during A. wallichii flower development stages due to the lack of optimal reference genes. Quantitative real-time polymerase chain reaction (qRT-PCR) is a powerful tool for quantifying expression levels of target genes. The accuracy of qRT-PCR analyses is largely dependent on the identification of stable reference genes for data normalization. The stability of reference gene expression may vary with plant species and environmental conditions. The aim of this study was to select stable reference genes for qRT-PCR analyses of target genes at flower development stages, in different flower colors and organs for Allium wallichii. The CDSs of eight potential reference genes (TUB2, ACT1, GAPC, EF1α, UBQ, UBC, SAND and CYP1) were cloned and their stability was evaluated by four programs (Delta Ct, geNorm, NormFinder and BestKeeper), and the results were further integrated into a comprehensive rank by RefFinder. The results showed that TUB2 and GAPC were the most stable two reference genes at different developmental stages of purple- and white-flower genotypes and across all samples. UBC and TUB2 expression was stable at different developmental stages of purple flowers. CYP1 and TUB2 were stably expressed at different developmental stages of white flowers. GAPC and SAND showed the highest rankings in different flower colors. TUB2 and EF1α performed the best in different tissues. ACT1 was the least stable gene in all tested samples. Moreover, DIHYDROFLAVONOL-4-REDUCTASE (DFR) gene that involved in anthocyanin synthesis was used to evaluate the effectiveness of the selected candidates. This study identified the first set of suitable reference genes for qRT-PCR analyses, which will lay the foundation for gene function study in A. wallichii.
喜马拉雅洋葱（Allium wallichii）是一种具有高观赏价值的多年生球根草本植物，因其抗癌和抗菌活性，长期以来在尼泊尔和中国被用作传统药物。野生葱莲具有不同的花色，包括紫色、粉色、深紫色和白色。然而，由于缺乏合适的内参基因，关于葱莲花朵发育阶段颜色形成的分子机制知之甚少。实时定量聚合酶链反应（qRT-PCR）是一种用于定量目标基因表达水平的强大工具。qRT-PCR分析的准确性在很大程度上取决于用于数据标准化的稳定内参基因的鉴定。内参基因表达的稳定性可能因植物物种和环境条件而异。本研究的目的是为葱莲花朵发育阶段、不同花色和器官中目标基因的qRT-PCR分析选择稳定的内参基因。克隆了八个潜在内参基因（TUB2、ACT1、GAPC、EF1α、UBQ、UBC、SAND和CYP1）的编码序列（CDS），并通过四个程序（Delta Ct、geNorm、NormFinder和BestKeeper）评估它们的稳定性，结果通过RefFinder进一步整合为一个综合排名。结果表明，TUB2和GAPC是紫色和白色花基因型不同发育阶段以及所有样本中最稳定的两个内参基因。UBC和TUB2在紫色花的不同发育阶段表达稳定。CYP1和TUB2在白色花的不同发育阶段稳定表达。GAPC和SAND在不同花色中排名最高。TUB2和EF1α在不同组织中表现最佳。ACT1是所有测试样本中最不稳定的基因。此外，使用参与花青素合成的二氢黄酮醇-4-还原酶（DFR）基因来评估所选候选基因的有效性。本研究确定了第一组适用于qRT-PCR分析的内参基因，这将为葱莲的基因功能研究奠定基础。
