Institute of Botany, Jiangsu Province and Chinese Academy of Sciences, Nanjing, 210014, China.
College of Agro-grassland Science, Nanjing Agricultural University, Nanjing, 210095, China.
Mol Biol Rep. 2022 Feb;49(2):907-915. doi: 10.1007/s11033-021-06902-z. Epub 2022 Jan 11.
Reference genes are necessary for quantitative real-time PCR (qRT-PCR) analysis and their stability can directly influence the accuracy of gene expression result. Miscanthus sacchariflorus, a perennial C4 grass that serves as promising biofuel plant for temperate climates, has not been explored for the identification of stable reference genes yet.
Nine potential reference genes (ACT, EF1a, FBOX, GAPDH, PP2A, SAND, TIP41, TUB and UBC) of M. sacchariflorus under different abiotic (salinity, drought and cadmium) stresses, as well as in two tissues (roots and leaves) were evaluated. The expression stability of these genes were analyzed by four commonly used software programs (geNorm, NormFinder, BestKeeper, ΔCt method and RefFinder).
Our results found that FBOX and SAND are the most stable genes among all tested samples. FBOX and EF1a are suitable for gene expression normalization of cadmium-treated samples and salinity-treated leaves. FBOX and PP2A are appropriate reference genes for salt-stressed roots and PEG-treated leaves. The traditional reference gene ACT and GAPDH exhibited the most variable pattern, which would not be recommended for qRT-PCR analysis under different abiotic stresses. Furthermore, the expression levels of PIP2, NHX1 and MT2a under drought, salt and cadmium treatment were detected with above reference genes.
This work identified the appropriate reference genes for qRT-PCR in M. sacchariflorus and FBOX was recommended to be effective internal control for gene expression normalization in M. sacchariflorus in response to different abiotic stresses.
实时荧光定量 PCR（qRT-PCR）分析需要参考基因，其稳定性会直接影响基因表达结果的准确性。甜高粱是一种多年生 C4 禾本科植物，有望成为温带地区的生物燃料作物，但尚未对其稳定的参考基因进行鉴定。
本研究选用了 9 个候选参考基因（ACT、EF1a、FBOX、GAPDH、PP2A、SAND、TIP41、TUB 和 UBC），对其在不同非生物胁迫（盐胁迫、干旱胁迫和镉胁迫）及两种组织（根和叶）下的表达稳定性进行了评估。采用 4 种常用软件程序（geNorm、NormFinder、BestKeeper、ΔCt 法和 RefFinder）对这些基因的表达稳定性进行了分析。
结果表明，在所有测试样本中，FBOX 和 SAND 是最稳定的基因。FBOX 和 EF1a 适用于镉处理样品和盐处理叶片的基因表达归一化。FBOX 和 PP2A 是盐胁迫根和 PEG 处理叶片的合适参考基因。传统的参考基因 ACT 和 GAPDH 表现出最不稳定的模式，不建议在不同非生物胁迫下用于 qRT-PCR 分析。此外，还利用上述参考基因检测了 PIP2、NHX1 和 MT2a 在干旱、盐和镉处理下的表达水平。
本研究确定了适用于甜高粱 qRT-PCR 的参考基因，建议 FBOX 作为甜高粱响应不同非生物胁迫时基因表达归一化的有效内参基因。
