Ma Rui, Xu Sheng, Zhao Yucheng, Xia Bing, Wang Ren
Institute of Botany, Jiangsu Province and Chinese Academy of Sciences Nanjing, China.
State Key Laboratory of Natural Medicines, Department of Natural Medicinal Chemistry, China Pharmaceutical University Nanjing, China.
Front Plant Sci. 2016 Apr 25;7:536. doi: 10.3389/fpls.2016.00536. eCollection 2016.
Lycoris aurea (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 YLS8 (mitosis protein YLS8), CYP2 (Cyclophilin 2), CYP 1 (Cyclophilin 1), TIP41 (TIP41-like protein), EXP2 (Expressed protein 2), PTBP1 (Polypyrimidine tract-binding protein 1), EXP1 (Expressed protein 1), PP2A (Serine/threonine-protein phosphatase 2A), β-TUB (β-tubulin), α-TUB (α-tubulin), EF1-α (Elongation factor 1-α), UBC (Ubiquitin-conjugating enzyme), ACT (Actin) and GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) were selected from the transcriptome datasets of L. aurea. 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) EXP1 and TIP41 for all samples; (2) UBC and EXP1 for NaCl stress; (3) PTBP1 and EXP1 for heat stress, polyethylene glycol (PEG) stress and ABA treatment; (4) UBC and CYP2 for cold stress; (5) PTBP1 and PP2A for sodium nitroprusside (SNP) treatment; (6) CYP1 and TIP41 for methyl jasmonate (MeJA) treatment; and (7) EXP1 and TIP41 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 L. aurea, and will facilitate gene expression studies under these conditions.
忽地笑（L' Hér.）草本植物，一种多年生草本植物，能产生多种独特的具有药理活性的石蒜科生物碱。然而，参与石蒜科生物碱（尤其是加兰他敏）生物合成的关键酶及其表达模式仍远未被完全了解。定量实时聚合酶链反应（qRT-PCR）是一种常用的定量基因表达的方法，需要稳定的内参基因来对数据进行标准化。在本研究中，为了在不同实验条件下选择合适的内参基因，从忽地笑的转录组数据集中挑选了14个基因，包括YLS8（有丝分裂蛋白YLS8）、CYP2（亲环蛋白2）、CYP 1（亲环蛋白1）、TIP41（类TIP41蛋白）、EXP2（表达蛋白2）、PTBP1（多嘧啶序列结合蛋白1）、EXP1（表达蛋白1）、PP2A（丝氨酸/苏氨酸蛋白磷酸酶2A）、β-TUB（β-微管蛋白）、α-TUB（α-微管蛋白）、EF1-α（延伸因子1-α）、UBC（泛素结合酶）、ACT（肌动蛋白）和GAPDH（甘油醛-3-磷酸脱氢酶）。然后，通过qRT-PCR评估这些基因在不同组织和不同处理下的根中的表达。利用三种常用软件程序（geNorm、NormFinder和BestKeeper）分析了这14个候选基因的表达稳定性，并根据几何平均值将其结果进一步整合为一个综合排名。结果显示了每个子集相对稳定的基因如下：（1）所有样本中的EXP1和TIP41；（2）NaCl胁迫下的UBC和EXP1；（3）热胁迫、聚乙二醇（PEG）胁迫和ABA处理下的PTBP1和EXP1；（4）冷胁迫下的UBC和CYP2；（5）硝普钠（SNP）处理下的PTBP1和PP2A；（6）茉莉酸甲酯（MeJA）处理下的CYP1和TIP41；（7）各种组织中的EXP1和TIP41。通过部分qRT-PCR结果与RNA测序（RNA-seq）数据的比较，进一步提高了这些结果的可靠性。总之，我们的结果确定了忽地笑qRT-PCR合适的内参基因，并将有助于在这些条件下进行基因表达研究。
