AUTHOR=Ma Rui , Xu Sheng , Zhao Yucheng , Xia Bing , Wang Ren 

TITLE=Selection and Validation of Appropriate Reference Genes for Quantitative Real-Time PCR Analysis of Gene Expression in Lycoris aurea

JOURNAL=Frontiers in Plant Science

VOLUME=7

YEAR=2016

URL=https://www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2016.00536

DOI=10.3389/fpls.2016.00536

ISSN=1664-462X

ABSTRACT=<p><italic>Lycoris aurea</italic> (L' Hér.) Herb, a perennial grass species, produces a unique variety of pharmacologically active Amaryllidaceae alkaloids. However, the key enzymes and their expression pattern involved in the biosynthesis of Amaryllidaceae alkaloids (especially for galanthamine) are far from being fully understood. Quantitative real-time polymerase chain reaction (qRT-PCR), a commonly used method for quantifying gene expression, requires stable reference genes to normalize its data. In this study, to choose the appropriate reference genes under different experimental conditions, 14 genes including <italic>YLS8</italic> (<italic>mitosis protein YLS8</italic>), <italic>CYP2</italic> (<italic>Cyclophilin 2</italic>), <italic>CYP 1</italic> (<italic>Cyclophilin 1</italic>), <italic>TIP41</italic> (<italic>TIP41-like protein</italic>), <italic>EXP2</italic> (<italic>Expressed protein 2</italic>), <italic>PTBP1</italic> (<italic>Polypyrimidine tract-binding protein 1</italic>), <italic>EXP1</italic> (<italic>Expressed protein 1</italic>), <italic>PP2A</italic> (<italic>Serine/threonine-protein phosphatase 2A</italic>), β<italic>-TUB</italic> (β<italic>-tubulin</italic>), α<italic>-TUB</italic> (α<italic>-tubulin</italic>), <italic>EF1-</italic>α (<italic>Elongation factor 1-</italic>α), <italic>UBC</italic> (<italic>Ubiquitin-conjugating enzyme</italic>), <italic>ACT</italic> (<italic>Actin</italic>) and <italic>GAPDH</italic> (<italic>Glyceraldehyde 3-phosphate dehydrogenase</italic>) were selected from the transcriptome datasets of <italic>L. aurea</italic>. And then, expressions of these genes were assessed by qRT-PCR in various tissues and the roots under different treatments. The expression stability of the 14 candidates was analyzed by three commonly used software programs (geNorm, NormFinder, and BestKeeper), and their results were further integrated into a comprehensive ranking based on the geometric mean. The results show the relatively stable genes for each subset as follows: (1) <italic>EXP1</italic> and <italic>TIP41</italic> for all samples; (2) <italic>UBC</italic> and <italic>EXP1</italic> for NaCl stress; (3) <italic>PTBP1</italic> and <italic>EXP1</italic> for heat stress, polyethylene glycol (PEG) stress and ABA treatment; (4) <italic>UBC</italic> and <italic>CYP2</italic> for cold stress; (5) <italic>PTBP1</italic> and <italic>PP2A</italic> for sodium nitroprusside (SNP) treatment; (6) <italic>CYP1</italic> and <italic>TIP41</italic> for methyl jasmonate (MeJA) treatment; and (7) <italic>EXP1</italic> and <italic>TIP41</italic> for various tissues. The reliability of these results was further enhanced through comparison between part qRT-PCR result and RNA sequencing (RNA-seq) data. In summary, our results identified appropriate reference genes for qRT-PCR in <italic>L. aurea</italic>, and will facilitate gene expression studies under these conditions.</p>