Selection and validation of garlic reference genes for quantitative real-time PCR normalization
Selection and validation of garlic reference genes for quantitative real-time PCR normalization
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用于定量实时 PCR 标准化的大蒜内参基因的选择和验证
DOI：
10.1007/s11240-015-0780-9
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发表时间：
2015-08-01
影响因子：
3
通讯作者：
Jiang, Fangling
中科院分区：
文献类型：
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作者：
Liu, Min;Wu, Zhen;Jiang, Fangling
稳定的参考基因是获得可靠的定量实时PCR（qPCR）分析结果的先决条件。尽管大蒜的全球分布和营养，药用和经济价值，但qPCR的参考基因尚未报道。选择了8个大蒜管家基因作为候选参考基因，它们分别是18 S核糖体RNA（18 S）、肌动蛋白（ACT）、甘油醛-3-磷酸脱氢酶（GAPDH）、延伸因子（EF-1α）、α-微管蛋白（TUA）和β-微管蛋白（TUB）、多聚泛素（UBQ）和亲环蛋白（Cyclophilin）。通过GeNorm、Normalization、BestKeeper、比较ΔCt方法和Referral评价其稳定性。结果表明，TUA、GAPDH、UBQ、NAB和ACT分别是大蒜不同基因型、不同器官、不同发育阶段、不同非生物胁迫和不同基质处理的最适参考基因。为了进一步验证这些参考基因的稳定性，分析了超氧化物歧化酶基因（SOD）的表达水平。SOD的相对定量根据参考基因的稳定性而变化，因此突出了在qPCR中使用合适的参考基因的重要性。我们的研究结果也为研究大蒜的发育和抗逆机制提供了分子生物学基础。
A stable reference gene is a prerequisite to obtaining reliable quantitative real-time PCR (qPCR) analysis results. Despite the global distribution and nutritional, medicinal and economic value of garlic, reference genes for qPCR have not been reported. Eight garlic housekeeping genes, 18S ribosomal RNA (18S), actin (ACT), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), elongation factor (EF-1α), the α-tubulin (TUA) and β-tubulin (TUB), polyubiquitin (UBQ), and cyclophilin (CYP), were chosen as candidate reference genes. Their stability was evaluated by GeNorm, NormFinder, BestKeeper, the comparative ΔCt method, and RefFinder. The results demonstrated thatTUA,GAPDH,UBQ,CYP, andACTare the most suitable of the eight reference genes for different garlic genotypes, organs, developmental stages, abiotic stresses and media treatments, respectively. To further validate the stability of these reference genes, the expression levels of the superoxide dismutase gene (SOD) were analyzed. The relative quantification ofSODvaried according to the stability of the reference gene, thus highlighting the importance of using suitable reference genes in qPCR. Our results also provide a molecular biological basis for studying the mechanisms of development and stress tolerance in garlic.