Key Laboratory of Economic Plants and Biotechnology, Kunming Institute of Botany, Chinese Academy of Sciences, 132# Lanhei Road, Kunming 650201, China.
Yunnan Key Laboratory for Wild Plant Resources, 132# Lanhei Road, Kunming 650201, China.
Int J Mol Sci. 2022 Jan 10;23(2):738. doi: 10.3390/ijms23020738.
The development and tissue-dependent color formation of the horticultural plant results in various color pattern flowers. Anthocyanins and carotenoids contribute to the red and yellow colors, respectively. In this study, quantitative real-time polymerase chain reaction (qRT-PCR) is used to analyze the expression profiles of anthocyanin and carotenoids biosynthesis genes in (Rchb.f.) Rchb.f. Appropriate reference gene selection and validation are required before normalization of gene expression in qRT-PCR analysis. Thus, we firstly selected 12 candidate reference genes from transcriptome data, and used geNorm and Normfinder to evaluate their expression stability in lip (divided into abaxial and adaxial), petal, and sepal of the bud and flower of . Our results show that the two most stable reference genes in different tissues of bud and flower are and , the most unstable reference gene is . The expression profiles of the and genes were similar to FPKM value profiles after normalization to the two most stable reference genes, and , with the upregulated and expression in flower stage, indicating that the ABP and CBP were activated across the stages of flower development. However, when the most unstable reference gene, , was used to normalize the qRT-PCR data, the expression profiles of and differed from FPKM value profiles, indicating the necessity of selecting stable reference genes. Moreover, and expression was highest in the abaxial lip and adaxial lip, respectively, indicating that the ABP and CBP were activated in abaxial and adaxial lip, respectively, resulting in a presence of red or yellow segments in abaxial and adaxial lip. This study is the first to provide reference genes in , and also provide useful information for studies that aim to understand the molecular mechanisms of flower color formation in .
园艺植物的发育和组织依赖性颜色形成导致了各种具有不同颜色图案的花朵。花青苷和类胡萝卜素分别为红色和黄色的形成做出贡献。在这项研究中，我们使用定量实时聚合酶链反应（qRT-PCR）来分析 （Rchb.f.） Rchb.f. 中花色苷和类胡萝卜素生物合成基因的表达谱。在 qRT-PCR 分析中对基因表达进行归一化之前，需要选择合适的参考基因并对其进行验证。因此，我们首先从转录组数据中选择了 12 个候选参考基因，并使用 geNorm 和 Normfinder 评估它们在芽和花的背腹（分为背腹）、花瓣和萼片中的表达稳定性。我们的结果表明，在芽和花的不同组织中，最稳定的两个参考基因是 和 ，最不稳定的参考基因是 。归一化到两个最稳定的参考基因 和 后， 和 基因的表达谱与 FPKM 值谱相似，表明 ABP 和 CBP 在花发育的各个阶段都被激活。然而，当使用最不稳定的参考基因 对 qRT-PCR 数据进行归一化时， 和 基因的表达谱与 FPKM 值谱不同，表明选择稳定的参考基因的必要性。此外， 和 基因在背唇和腹唇中的表达量最高，分别表明 ABP 和 CBP 在背唇和腹唇中被激活，导致背唇和腹唇上出现红色或黄色片段。这项研究首次为 提供了参考基因，也为研究花色形成的分子机制提供了有用的信息。
