Department of Grassland Science, Animal Science and Technology College, Sichuan Agricultural University, Chengdu, 611130, Sichuan, China.
Mol Biol Rep. 2020 Apr;47(4):2913-2927. doi: 10.1007/s11033-020-05392-9. Epub 2020 Mar 28.
Quantitative real-time PCR (qRT-PCR) has been widely used for studying gene expression at the transcript level. Its accuracy usually relies on the reference genes that are utilized for data normalization. Miscanthus sinensis, a perennial C4 grass with high biomass and strong resistance to adversities, is often utilized as a high value energy crop. However, no reliable reference genes have been investigated for normalizing gene expression for this species. In this study, 12 candidate reference genes were selected to identify their stability under five different abiotic stress treatments (drought, salt, cadmium, chromium and arsenic) by using geNorm, NormFinder, BestKeeper and RefFinder softwares. The results showed that 18S rRNA and Unigene33312 were the best reference genes under drought treatments. Unigene33312 and Unigene33024 were found to be the most stably expressed genes under salt stress and Cd stress. Moreover, Unigene33024 and PP2A were the most suitable reference genes under Cr stress and Unigene33024 and Sb09g019750 were deemed more suitable reference genes under As stress. In total, considering all the samples, Unigene33024 and PP2A were the most stable genes while ACTIN and Unigene26576 were the least stable reference genes for internal control. The expression patterns of two target genes (Cu/Zn SOD and CAT) were used to further verify those selected reference genes under different conditions. The results showed that the most and the least stable reference genes had clearly different expression patterns. This work comprehensively estimated the stability of reference genes in M. sinensis which may give insight to the reference genes selection in other tissues as well as other related varieties. These suggested reference genes would assist in further putative gene expression validation in M. sinensis.
实时荧光定量 PCR（qRT-PCR）已广泛用于研究转录水平的基因表达。其准确性通常依赖于用于数据归一化的参考基因。芒，一种具有高生物量和强逆境抗性的多年生 C4 草，常被用作高价值能源作物。然而，尚未针对该物种进行可靠的参考基因研究以进行基因表达的正常化。在这项研究中，选择了 12 个候选参考基因，通过使用 geNorm、NormFinder、BestKeeper 和 RefFinder 软件，在五种不同的非生物胁迫处理（干旱、盐、镉、铬和砷）下鉴定其稳定性。结果表明，18S rRNA 和 Unigene33312 是干旱处理下最佳的参考基因。在盐胁迫和 Cd 胁迫下，Unigene33312 和 Unigene33024 被发现是表达最稳定的基因。此外，Unigene33024 和 PP2A 是 Cr 胁迫下最适合的参考基因，而 Unigene33024 和 Sb09g019750 是 As 胁迫下更适合的参考基因。总的来说，考虑到所有样本，Unigene33024 和 PP2A 是最稳定的基因，而 ACTIN 和 Unigene26576 是最不稳定的内参基因。两种靶基因（Cu/Zn SOD 和 CAT）的表达模式进一步验证了不同条件下所选参考基因的表达模式。结果表明，最稳定和最不稳定的参考基因的表达模式明显不同。这项工作综合评估了芒参考基因的稳定性，这可能为其他组织以及其他相关品种的参考基因选择提供参考。这些建议的参考基因将有助于进一步验证芒中的推定基因表达。
