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

• Published in: PloS one Vol. 11; no. 8; p. e0159458
• Main Authors: Deng, Li-Ting, Wu, Yu-Ling, Li, Jun-Cheng, OuYang, Kun-Xi, Ding, Mei-Mei, Zhang, Jun-Jie, Li, Shu-Qi, Lin, Meng-Fei, Chen, Han-Bin, Hu, Xin-Sheng, Chen, Xiao-Yang
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 19.08.2016; Public Library of Science (PLoS)
• Subjects: Acyl carrier protein; Adaptation, Physiological - genetics; Agronomy; Algorithms; Architectural engineering; Biology and Life Sciences; Brassicaceae; Breeding; Chlorine compounds; Computer simulation; Developmental stages; Engineering; Forestry; Gene expression; Gene Expression Profiling; Genes; Genetic aspects; Genetic improvement; Genomes; Germplasm; High temperature; Isotypes; Laboratories; Landscape architecture; Leaves; Medical research; Medical screening; Medicine and Health Sciences; Moringa oleifera; Moringa oleifera - genetics; Moringa oleifera - growth & development; Physical Sciences; Plant breeding; Plant Leaves - genetics; Plant Leaves - growth & development; Plant Proteins - genetics; Plant Roots - genetics; Plant Roots - growth & development; Plant species; Polyethylene glycol; Polymerase chain reaction; Real-Time Polymerase Chain Reaction - methods; Reference Standards; Research and Analysis Methods; Ribosomal protein L1; Sodium; Sodium chloride; Stress, Physiological - genetics; Studies; Superoxide dismutase; Tissues; Transcription; Transcriptome; Water stress
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0159458

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.