Junqin Zong1, Jingbo Chen1, Ling Li1, Jianjian Li1, Dandan Li1, Jingjing Wang1, Jun Liu2, Jianxiu Liu3. 1. Institute of Botany, Jiangsu Province and Chinese Academy of Sciences, Nanjing, 210014, China. 2. College of Agro-grassland Science, Nanjing Agricultural University, Nanjing, 210095, China. liujun825@njau.edu.cn. 3. Institute of Botany, Jiangsu Province and Chinese Academy of Sciences, Nanjing, 210014, China. turfunit@aliyun.com.
Abstract
BACKGROUND: 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. MATERIALS AND METHODS: 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). RESULTS: 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. CONCLUSIONS: 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.
BACKGROUND: 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. MATERIALS AND METHODS: 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). RESULTS: 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. CONCLUSIONS: 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.