Identification of candidate reference genes for quantitative RT-PCR in Miscanthus sinensis subjected to various abiotic stresses
Identification of candidate reference genes for quantitative RT-PCR in Miscanthus sinensis subjected to various abiotic stresses
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DOI：
10.1007/s11033-020-05392-9
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发表时间：
2020-03
影响因子：
2.8
通讯作者：
Minyi Zhong;Xinying Yang;Yiyue Hu;Linkai Huang;Yan Peng;Zhou Li;Qiuxu Liu;Xia Wang;
中科院分区：
文献类型：
--
作者：
Minyi Zhong;Xinying Yang;Yiyue Hu;Linkai Huang;Yan Peng;Zhou Li;Qiuxu Liu;Xia Wang;
实时定量 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标准化的参考基因。芒草是一种多年生C4草，生物量高，抗逆性强，常被用作高价值能源作物。然而，尚未研究可靠的参考基因来标准化该物种的基因表达。本研究选取12个候选内参基因，利用geNorm、NormFinder、BestKeeper和RefFinder软件，鉴定其在5种不同非生物胁迫处理（干旱、盐、镉、铬和砷）下的稳定性。结果表明，18S rRNA和Unigene33312是干旱处理下最佳的内参基因。Unigene33312和Unigene33024是盐胁迫和Cd胁迫下表达最稳定的基因。此外，Unigene33024和PP2A是Cr胁迫下最合适的内参基因，Unigene33024和Sb09g019750被认为是As胁迫下更合适的内参基因。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是内参最不稳定的内参基因。两个目标基因（Cu/Zn SOD和CAT）的表达模式用于进一步验证所选参考基因在不同条件下的表达模式。结果表明，最稳定和最不稳定的内参基因具有明显不同的表达模式。本工作全面评估了M中内参基因的稳定性。 sinensis 可能有助于了解其他组织以及其他相关品种中参考基因的选择。这些建议的参考基因将有助于进一步验证 M 中假定的基因表达。中华。
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 that18S rRNAandUnigene33312were the best reference genes under drought treatments.Unigene33312andUnigene33024were found to be the most stably expressed genes under salt stress and Cd stress. Moreover,Unigene33024andPP2Awere the most suitable reference genes under Cr stress andUnigene33024andSb09g019750were deemed more suitable reference genes under As stress. In total, considering all the samples,Unigene33024andPP2Awere the most stable genes whileACTINandUnigene26576were the least stable reference genes for internal control. The expression patterns of two target genes (Cu/Zn SODandCAT) 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 inM. sinensiswhich 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 inM. sinensis.