Screening Reliable Reference Genes for RT-qPCR Analysis of Gene Expression in Moringa oleifera

PLoS One. 2016 Aug 19;11(8):e0159458. doi: 10.1371/journal.pone.0159458. eCollection 2016.

Abstract

Moringa oleifera is a promising plant species for oil and forage, but its genetic improvement is limited. Our current breeding program in this species focuses on exploiting the functional genes associated with important agronomical traits. Here, we screened reliable reference genes for accurately quantifying the expression of target genes using the technique of real-time quantitative polymerase chain reaction (RT-qPCR) in M. oleifera. Eighteen candidate reference genes were selected from a transcriptome database, and their expression stabilities were examined in 90 samples collected from the pods in different developmental stages, various tissues, and the roots and leaves under different conditions (low or high temperature, sodium chloride (NaCl)- or polyethyleneglycol (PEG)- simulated water stress). Analyses with geNorm, NormFinder and BestKeeper algorithms revealed that the reliable reference genes differed across sample designs and that ribosomal protein L1 (RPL1) and acyl carrier protein 2 (ACP2) were the most suitable reference genes in all tested samples. The experiment results demonstrated the significance of using the properly validated reference genes and suggested the use of more than one reference gene to achieve reliable expression profiles. In addition, we applied three isotypes of the superoxide dismutase (SOD) gene that are associated with plant adaptation to abiotic stress to confirm the efficacy of the validated reference genes under NaCl and PEG water stresses. Our results provide a valuable reference for future studies on identifying important functional genes from their transcriptional expressions via RT-qPCR technique in M. oleifera.

MeSH terms

  • Adaptation, Physiological / genetics*
  • Gene Expression Profiling*
  • Moringa oleifera / genetics*
  • Moringa oleifera / growth & development
  • Plant Leaves / genetics*
  • Plant Leaves / growth & development
  • Plant Proteins / genetics*
  • Plant Roots / genetics*
  • Plant Roots / growth & development
  • Real-Time Polymerase Chain Reaction / methods*
  • Reference Standards
  • Stress, Physiological / genetics*
  • Transcriptome

Substances

  • Plant Proteins

Grants and funding

This work was supported by the Forestry Sciences and Technology Innovation Project in Guangdong Province (2015KJCX009) and the Department of Tropical crops, State Bureau for Land-Reclamation (15RZNJ-62), P.R. China. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.