Abstract
Stipa grandis P. Smirn. is a dominant plant species in the typical steppe of the Xilingole Plateau of Inner Mongolia. Selection of suitable reference genes for the quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) is important for gene expression analysis and research into the molecular mechanisms underlying the stress responses of S. grandis. In the present study, 15 candidate reference genes (EF1 beta, ACT, GAPDH, SamDC, CUL4, CAP, SNF2, SKIP1, SKIP5, SKIP11, UBC2, UBC15, UBC17, UCH, and HERC2) were evaluated for their stability as potential reference genes for qRT-PCR under different stresses. Four algorithms were used: GeNorm, NormFinder, BestKeeper, and RefFinder. The results showed that the most stable reference genes were different under different stress conditions: EF1beta and UBC15 during drought and salt stresses; ACT and GAPDH under heat stress; SKIP5 and UBC17 under cold stress; UBC15 and HERC2 under high pH stress; UBC2 and UBC15 under wounding stress; EF1beta and UBC17 under jasmonic acid treatment; UBC15 and CUL4 under abscisic acid treatment; and HERC2 and UBC17 under salicylic acid treatment. EF1beta and HERC2 were the most suitable genes for the global analysis of all samples. Furthermore, six target genes, SgPOD, SgPAL, SgLEA, SgLOX, SgHSP90 and SgPR1, were selected to validate the most and least stable reference genes under different treatments. Our results provide guidelines for reference gene selection for more accurate qRT-PCR quantification and will promote studies of gene expression in S. grandis subjected to environmental stress.
Highlights
The expression levels of the candidate reference genes were determined by quantitative realtime reverse transcription polymerase chain reaction (qRT-PCR) across all samples (Fig 1), and a wide range of Ct values, from 15.25 to 30.03, were found, with the values for most of the genes lying between 20 and 26
We found that the most suitable reference genes varied under different environmental stimuli
When the target gene normalization was performed with the least stable genes, the expression patterns varied in comparison with most stable reference genes. These results suggest that appropriate selection of reference genes is necessary for the normalization of target gene expression to ensure the accuracy of qRT-PCR data
Summary
Is a C3 perennial bunchgrass [1] that is distributed widely in eastern Eurasian steppes and the middle Eurasian steppe zone [2]. This species is dominant in the typical steppe of the Xilingole Plateau of Inner Mongolia [1]. The S. grandis steppe acts as a natural. Reference Genes Selection in Stipa grandis role in study design, data collection and analysis, decision to publish, or preparation of the manuscript
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