Abstract
BackgroundPolygonum cuspidatum of the Polygonaceae family is a traditional medicinal plant with many bioactive compounds that play important roles in human health and stress responses. Research has attempted to identify biosynthesis genes and metabolic pathways in this species, and quantitative real-time PCR (RT-qPCR) has commonly been used to detect gene expression because of its speed, sensitivity, and specificity. However, no P. cuspidatum reference genes have been identified, which hinders gene expression studies. Here, we aimed to identify suitable reference genes for accurate and reliable normalization of P. cuspidatum RT-qPCR data.ResultsTwelve candidate reference genes, including nine common (ACT, TUA, TUB, GAPDH, EF-1γ, UBQ, UBC, 60SrRNA, and eIF6A) and three novel (SKD1, YLS8, and NDUFA13), were analyzed in different tissues (root, stem, and leaf) without treatment and in leaves under abiotic stresses (salt, ultraviolet [UV], cold, heat, and drought) and hormone stimuli (abscisic acid [ABA], ethylene [ETH], gibberellin [GA3], methyl jasmonate [MeJA], and salicylic acid [SA]). Expression stability in 65 samples was calculated using the △CT method, geNorm, NormFinder, BestKeeper, and RefFinder. Two reference genes (NDUFA13 and EF-1γ) were sufficient to normalize gene expression across all sample sets. They were also the two most stable genes for abiotic stresses and different tissues, whereas NDUFA13 and SKD1 were the top two choices for hormone stimuli. Considering individual experimental sets, GAPDH was the top-ranked gene under ABA, ETH, and GA3 treatments, while 60SrRNA showed good stability under MeJA and cold treatments. ACT, UBC, and TUB were suitable genes for drought, UV, and ABA treatments, respectively. TUA was not suitable because of its considerable variation in expression under different conditions. The expression patterns of PcPAL, PcSTS, and PcMYB4 under UV and SA treatments and in different tissues normalized by stable and unstable reference genes demonstrated the suitability of the optimal reference genes.ConclusionsWe propose NDUFA13 and EF-1γ as reference genes to normalize P. cuspidatum expression data. To our knowledge, this is the first systematic study of reference genes in P. cuspidatum which could help advance molecular biology research in P. cuspidatum and allied species.
Highlights
Polygonum cuspidatum of the Polygonaceae family is a traditional medicinal plant with many bioactive compounds that play important roles in human health and stress responses
Our results provide some useful resources for the future quantification of gene expression in P. cuspidatum and allied species
We found that the rankings by △cycle threshold (CT), geNorm, and NormFinder, were similar, especially for individual sets, but different from those by BestKeeper; similar findings were reported in other studies [19, 34]
Summary
Polygonum cuspidatum of the Polygonaceae family is a traditional medicinal plant with many bioactive compounds that play important roles in human health and stress responses. No P. cuspidatum reference genes have been identified, which hinders gene expression studies. Its first medicinal application records date back over 1800 years in Mingyi Bielu [1]. It has long been used in Chinese folk medicine for the treatment of abdominal masses, postpartum blood stasis, urethritis, suppuration, ulcers, sore throats, toothache, chronic bronchitis, hemorrhoids, and other ailments [1, 2]. Numerous active compounds have been isolated and identified from P. cuspidatum to date, such as anthraquinones, flavonoids, stilbenes, organic acids, coumarins, catechins, and lignans [3]. The mechanism of P. cuspidatum responses to abiotic stresses and hormone stimuli as well as in various tissues needs to be thoroughly explored to better understand the production of these active ingredients
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