College of Life Sciences, University of Chinese Academy of Sciences, Beijing, 100049, China.
Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, 100101, China.
BMC Plant Biol. 2019 Nov 14;19(1):498. doi: 10.1186/s12870-019-2108-0.
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. 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.
Twelve 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 [GA], 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 GA 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.
We 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.
蓼科虎杖Polygonum cuspidatum 是一种传统药用植物，含有多种生物活性化合物，对人类健康和应激反应具有重要作用。研究人员试图鉴定该物种中的生物合成基因和代谢途径，而定量实时 PCR（RT-qPCR）因其速度、灵敏度和特异性而常用于检测基因表达。然而，尚未鉴定出虎杖的参考基因，这阻碍了基因表达研究。本研究旨在鉴定合适的参考基因，以准确可靠地对虎杖 RT-qPCR 数据进行归一化。
分析了 12 个候选参考基因，包括 9 个常用基因（ACT、TUA、TUB、GAPDH、EF-1γ、UBQ、UBC、60SrRNA 和 eIF6A）和 3 个新基因（SKD1、YLS8 和 NDUFA13），分别在未经处理的根、茎和叶以及叶片在非生物胁迫（盐、紫外线[UV]、冷、热和干旱）和激素刺激（脱落酸[ABA]、乙烯[ETH]、赤霉素[GA]、茉莉酸甲酯[MeJA]和水杨酸[SA]）下的表达情况。在 65 个样本中使用△CT 法、geNorm、NormFinder、BestKeeper 和 RefFinder 计算了表达稳定性。在所有样本组中，两个参考基因（NDUFA13 和 EF-1γ）足以归一化基因表达。它们也是在非生物胁迫和不同组织中最稳定的基因，而 NDUFA13 和 SKD1 是激素刺激的首选。考虑到个别实验集，在 ABA、ETH 和 GA 处理下，GAPDH 是排名最高的基因，而在 MeJA 和冷处理下，60SrRNA 表现出良好的稳定性。在干旱、UV 和 ABA 处理下，ACT、UBC 和 TUB 是合适的基因，而 TUA 由于在不同条件下表达变化较大，因此不适合使用。在 UV 和 SA 处理以及不同组织中，用稳定和不稳定的参考基因归一化的 PcPAL、PcSTS 和 PcMYB4 的表达模式表明了最佳参考基因的适用性。
我们建议使用 NDUFA13 和 EF-1γ 作为参考基因来归一化虎杖的表达数据。据我们所知，这是对虎杖参考基因进行的首次系统研究，这有助于推进虎杖和相关物种的分子生物学研究。
