Selection of Reference Genes for Gene Expression Normalization in Peucedanum praeruptorum Dunn under Abiotic Stresses, Hormone Treatments and Different Tissues

• Published in: PloS one 2016-03, Vol.11 (3), p.e0152356-e0152356
• Main Authors: Zhao, Yucheng, Luo, Jun, Xu, Sheng, Wang, Wei, Liu, Tingting, Han, Chao, Chen, Yijun, Kong, Lingyi
• Format: Article
• Language: English
• Subjects: 1-Aminocyclopropane-1-carboxylate oxidase; Abiotic stress; Actin; Apiaceae - drug effects; Apiaceae - genetics; Apiaceae - physiology; Biology and Life Sciences; Biosynthesis; Chemistry; Coumarins; Design; Flowers & plants; Gene expression; Gene Expression Profiling; Gene Expression Regulation, Plant - drug effects; Genes; Genes, Plant; Genetic aspects; Genomics; Glyceraldehyde; Glyceraldehyde 3-phosphate; Glyceraldehyde-3-phosphate dehydrogenase; Herbal medicine; Identification; Laboratories; Lolium perenne; Metabolites; Normalizing; Pharmaceutical sciences; Phosphates; Phosphoprotein phosphatase; Physical Sciences; Physiological aspects; Plant Growth Regulators - pharmacology; Plant species; Polyethylene glycol; Polymerase chain reaction; Polypyrimidine tract-binding protein; Protein phosphatase; Real-Time Polymerase Chain Reaction; Reference Standards; Reproducibility of Results; Research and Analysis Methods; Reverse transcription; RNA, Messenger - genetics; RNA, Messenger - metabolism; Sand; Software; Stress, Physiological - drug effects; Stress, Physiological - genetics; Studies; Tissues; Traditional Chinese medicine; Tubulin; Ubiquitin; Ubiquitin-conjugating enzyme
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0152356

Peucedanum praeruptorum Dunn is one of the main traditional Chinese medicines producing coumarins and plenty of literatures are focused on the biosynthesis of coumarins. Quantitative real-time reverse transcription PCR (qRT-PCR) is a widely used method in studying the biosynthesis pathway and the selection of reference genes plays a crucial role in accurate normalization. To facilitate biosynthesis study of coumarins, twelve candidate reference genes were selected from the transcriptome database of P. praeruptorum according to previous studies. Then, BestKeeper, geNoFrm and NormFinder were used for selecting stably expressed reference genes in different tissues and under various stress treatments. The results indicated that, among the twelve candidate reference genes, the SAND family protein (SAND), actin 2 (ACT2), ubiquitin-conjugating enzyme 9 (UBC9), protein phosphatase 2A gene (PP2A) and polypyrimidine tract-binding protein (PTBP1) were the most stable reference genes under different experimental treatments, while glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and tubulin beta-6 (TUB6) were the least stable genes. In addition, the suitability of SAND, TIP41-like protein (TIP41), UBC9, ACT2, TUB6 and their combination as reference genes were confirmed by normalizing the expression of 1-aminocyclopropane-1-carboxylate oxidase (ACO) in different treatments. This work is the first survey of the stability of reference genes in P. praeruptorum and provides guidelines to obtain more accurate qRT-PCR results in P. praeruptorum and other plant species.