Selection and Validation of Appropriate Reference Genes for Quantitative Real-Time PCR Analysis of Gene Expression in Lycoris aurea
Selection and Validation of Appropriate Reference Genes for Quantitative Real-Time PCR Analysis of Gene Expression in Lycoris aurea
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选择并验证合适的内参基因，用于石蒜基因表达的定量实时 PCR 分析
DOI：
10.3359/fpls.2016.00536
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发表时间：
2016-04-25
影响因子：
5.6
通讯作者：
Wang, Ren
中科院分区：
文献类型：
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作者：
Ma, Rui;Xu, Sheng;Wang, Ren
石蒜（L' Her.）本属多年生草本植物，具有独特的药用活性。然而，Amaryllidaceae生物碱（特别是加兰他敏）生物合成的关键酶及其表达模式尚不完全清楚。定量实时聚合酶链反应（Quantitative real-time polymerase chain reaction, qRT-PCR）是一种常用的基因表达定量方法，需要稳定的内参基因来规范其数据。本研究在不同实验条件下选择合适的内参基因，包括YLS8（有丝分裂蛋白YLS8）、CYP2（亲环蛋白2）、cyp1（亲环蛋白1）、TIP41 （TIP41样蛋白）、EXP2（表达蛋白2）、PTBP1（多嘧啶束结合蛋白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（泛素偶联酶）、从金黄色葡萄球菌的转录组数据中选择ACT （Actin）和GAPDH （Glyceraldehyde 3-phosphate dehydrogenase）。然后利用qRT-PCR检测不同处理下这些基因在不同组织和根系中的表达情况。采用geNorm、NormFinder、BestKeeper三种常用软件对14个候选基因的表达稳定性进行分析，并根据几何均值对结果进行综合排序。结果表明，各亚群中相对稳定的基因为：(1)所有样本的EXP1和TIP41；(2) NaCl胁迫下的UBC和EXP1；(3) PTBP1和EXP1分别用于热应激、聚乙二醇（PEG）胁迫和ABA处理；(4) UBC和CYP2对冷应激的影响；(5) PTBP1和PP2A用于硝普钠（SNP）治疗；(6)茉莉酸甲酯（MeJA）处理的CYP1和TIP41；(7)各种组织的EXP1和TIP41。通过部分qRT-PCR结果与RNA测序（RNA-seq）数据的比较，进一步增强了这些结果的可靠性。综上所述，我们的结果确定了金黄色葡萄球菌qRT-POR的合适内参基因，将为这些条件下的基因表达研究提供便利。
Lycoris aurea (L' Her.) 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), beta-TUB (beta-tubulin), alpha-TUB (alpha-tubulin), EF1-alpha (Elongation factor 1-alpha), 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-POR in L. aurea, and will facilitate gene expression studies under these conditions.