Dataset Information

Selection and Validation of Reference Genes for Quantitative Real-Time PCR Analysis of Development and Tissue-Dependent Flower Color Formation in Cymbidium lowianum.

ABSTRACT: 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 Cymbidium lowianum (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 C. lowianum. Our results show that the two most stable reference genes in different tissues of C. lowianum bud and flower are EF1δ and 60S, the most unstable reference gene is 26S. The expression profiles of the CHS and BCH genes were similar to FPKM value profiles after normalization to the two most stable reference genes, EF1δ and 60S, with the upregulated CHS and BCH 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, 26S, was used to normalize the qRT-PCR data, the expression profiles of CHS and BCH differed from FPKM value profiles, indicating the necessity of selecting stable reference genes. Moreover, CHS and BCH 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 C. lowianum, and also provide useful information for studies that aim to understand the molecular mechanisms of flower color formation in C. lowianum.

SUBMITTER: Dong XM

PROVIDER: S-EPMC8776083 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Similar Datasets

Project description:Lycoris aurea (L' Hér.) Herb, a perennial grass species, produces a unique variety of pharmacologically active Amaryllidaceae alkaloids. However, the key enzymes and their expression pattern involved in the biosynthesis of Amaryllidaceae alkaloids (especially for galanthamine) are far from being fully understood. Quantitative real-time polymerase chain reaction (qRT-PCR), a commonly used method for quantifying gene expression, requires stable reference genes to normalize its data. In this study, to choose the appropriate reference genes under different experimental conditions, 14 genes including YLS8 (mitosis protein YLS8), CYP2 (Cyclophilin 2), CYP 1 (Cyclophilin 1), TIP41 (TIP41-like protein), EXP2 (Expressed protein 2), PTBP1 (Polypyrimidine tract-binding protein 1), EXP1 (Expressed protein 1), PP2A (Serine/threonine-protein phosphatase 2A), ?-TUB (?-tubulin), ?-TUB (?-tubulin), EF1-? (Elongation factor 1-?), UBC (Ubiquitin-conjugating enzyme), ACT (Actin) and GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) were selected from the transcriptome datasets of L. aurea. And then, expressions of these genes were assessed by qRT-PCR in various tissues and the roots under different treatments. The expression stability of the 14 candidates was analyzed by three commonly used software programs (geNorm, NormFinder, and BestKeeper), and their results were further integrated into a comprehensive ranking based on the geometric mean. The results show the relatively stable genes for each subset as follows: (1) EXP1 and TIP41 for all samples; (2) UBC and EXP1 for NaCl stress; (3) PTBP1 and EXP1 for heat stress, polyethylene glycol (PEG) stress and ABA treatment; (4) UBC and CYP2 for cold stress; (5) PTBP1 and PP2A for sodium nitroprusside (SNP) treatment; (6) CYP1 and TIP41 for methyl jasmonate (MeJA) treatment; and (7) EXP1 and TIP41 for various tissues. The reliability of these results was further enhanced through comparison between part qRT-PCR result and RNA sequencing (RNA-seq) data. In summary, our results identified appropriate reference genes for qRT-PCR in L. aurea, and will facilitate gene expression studies under these conditions.

Dataset Information

Selection and Validation of Reference Genes for Quantitative Real-Time PCR Analysis of Development and Tissue-Dependent Flower Color Formation in Cymbidium lowianum.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets