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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DOI：
10.3389/fpls.2016.00536
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发表时间：
2016
影响因子：
5.6
通讯作者：
Wang R
中科院分区：
文献类型：
--
作者：
Ma R;Xu S;Zhao Y;Xia B;Wang R
黄花石蒜Lycoris aurea（L' Hér.）草本植物是一种多年生禾本科植物，能产生多种独特的石蒜科生物碱。然而，石蒜科生物碱（尤其是加兰他敏）生物合成中的关键酶及其表达模式还远未完全了解。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，需要稳定的参考基因来标准化其数据。为了在不同的实验条件下选择合适的参考基因，本研究选择了YLS 8等14个基因，（有丝分裂蛋白YLS 8），CYP 2（亲环素2），（亲环素1），TIP 41（TIP 41样蛋白），EXP 2（表达蛋白2），PTBP 1（多聚嘧啶片段结合蛋白1），EXP 1（表达蛋白1），PP 2A（丝氨酸/苏氨酸-蛋白磷酸酶2A），β-TUB从L. tubulin（β-微管蛋白）、α-TUB（α-微管蛋白）、EF 1-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）的转录组数据集中选择了5个转录因子。金黄色。然后通过qRT-PCR检测这些基因在不同组织和不同处理下的根中的表达。通过三种常用的软件程序（geNorm、Normalization和BestKeeper）分析14个候选物的表达稳定性，并且将它们的结果进一步整合到基于几何平均值的综合排名中。结果表明：（1）所有样品中相对稳定的基因为EXP 1和TIP 41，（2）NaCl胁迫中相对稳定的基因为UBC和EXP 1，（3）热胁迫、聚乙二醇（PEG）胁迫和阿坝处理中相对稳定的基因为PTBP 1和EXP 1，（4）冷胁迫中相对稳定的基因为UBC和CYP 2，（5）硝普钠（SNP）处理中相对稳定的基因为PTBP 1和PP 2A，（6）盐胁迫中相对稳定的基因为UBC和EXP 1，（7）盐胁迫中相对稳定的基因为PTBP 1和EXP 1，（8）盐胁迫中相对稳定的基因为UBC和CYP 2，（9）盐胁迫中相对稳定的基因为PTBP 1和PP 2A，（10）盐胁迫中相对稳定的基因为UBC和EXP 1。（6）茉莉酸甲酯（MeJA）处理的CYP 1和TIP 41;以及（7）各种组织的EXP 1和TIP 41。通过比较部分qRT-PCR结果和RNA测序（RNA-seq）数据，进一步增强了这些结果的可靠性。总之，我们的结果确定了合适的参考基因的qRT-PCR在L。Aurea，并将促进在这些条件下的基因表达研究。
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.