Selection of Reference Genes for qRT-PCR Analysis of Gene Expression in Stipa grandis during Environmental Stresses

PLoS One. 2017 Jan 5;12(1):e0169465. doi: 10.1371/journal.pone.0169465. eCollection 2017.

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.

MeSH terms

  • Droughts
  • Gene Expression Regulation, Plant / drug effects
  • Gene Expression Regulation, Plant / genetics
  • Hot Temperature
  • Plant Proteins / genetics*
  • Poaceae / drug effects
  • Poaceae / genetics*
  • Reverse Transcriptase Polymerase Chain Reaction
  • Sodium Chloride / toxicity

Substances

  • Plant Proteins
  • Sodium Chloride

Grants and funding

This work was supported by the National Basic Research Program of China (973 program, 2014CB138804), the Natural Science Foundation of Inner Mongolia Autonomous Region of China (2014BS0330), and the Central Non-profit Research Institutes Fundamental Research Funds of China (1610332015004). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.