Selection of appropriate reference genes for the detection of rhythmic gene expression via quantitative real-time PCR in Tibetan hulless barley.
Selection of appropriate reference genes for the detection of rhythmic gene expression via quantitative real-time PCR in Tibetan hulless barley.
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选择合适的内参基因用于实时定量PCR检测青稞节律性基因表达
DOI：
10.1371/journal.pone.0190559
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发表时间：
2018
期刊：
影响因子：
3.7
通讯作者：
Xu Y
中科院分区：
文献类型：
--
作者：
Cai J;Li P;Luo X;Chang T;Li J;Zhao Y;Xu Y
大麦（Hordeum vulgare L.）变种裸体。hook. f.）作为中国青藏高原的主要农作物栽培了数千年。与其他禾谷类作物相比，西藏青稞具有较强的内生抗性，能够在恶劣的生境环境中生存。为了了解这种植物独特的抗性机制，需要进行详细的遗传研究。实时定量逆转录-聚合酶链反应（qRT-PCR）是检测基因表达最常用的方法。然而，在有限的实验条件下选择稳定的参考基因被认为是在qRT-PCR中获得准确结果的必要步骤。在本研究中，10个候选参考基因-ACT（Actin）、E2（泛素结合酶2），TUBα（α-微管蛋白），TUBβ6（β-微管蛋白6），GAPDH（甘油醛3-磷酸脱氢酶），EF-1α（延伸因子1-α），SAMDC（S-腺苷甲硫氨酸脱羧酶）（蛋白激酶HvPKABA 1的基因）、PGK（磷酸甘油酸激酶）和HSP 90（热休克蛋白90）-选自大麦的NCBI基因数据库。利用qRT-PCR技术对不同胁迫条件下西藏青稞幼苗中的所有候选参考基因进行扩增，并利用geNorm、Normandum和BestKeeper软件包对这些候选参考基因的稳定性进行分析。结果表明，TUBβ6、E2、TUBα和HSP 90在所有试验条件下均为最适宜的参考基因; TUBα和HSP 90在盐胁迫下表现出峰值稳定性，TUBα和EF-1α在冷胁迫下最适宜，ACT和E2在干旱胁迫下最稳定。最后，利用已知的昼夜节律基因CCA 1验证所选参考基因的服务能力。结果证实，三种软件推荐的参考基因均适用于qRT-PCR方法在测试胁迫条件下的基因表达分析。
Hulless barley (Hordeum vulgare L. var. nudum. hook. f.) has been cultivated as a major crop in the Qinghai-Tibet plateau of China for thousands of years. Compared to other cereal crops, the Tibetan hulless barley has developed stronger endogenous resistances to survive in the severe environment of its habitat. To understand the unique resistant mechanisms of this plant, detailed genetic studies need to be performed. The quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) is the most commonly used method in detecting gene expression. However, the selection of stable reference genes under limited experimental conditions was considered to be an essential step for obtaining accurate results in qRT-PCR. In this study, 10 candidate reference genes—ACT (Actin), E2 (Ubiquitin conjugating enzyme 2), TUBα (Alpha-tubulin), TUBβ6 (Beta-tubulin 6), GAPDH (Glyceraldehyde 3-phosphate dehydrogenase), EF-1α (Elongation factor 1-alpha), SAMDC (S-adenosylmethionine decarboxylase), PKABA1 (Gene for protein kinase HvPKABA1), PGK (Phosphoglycerate kinase), and HSP90 (Heat shock protein 90)—were selected from the NCBI gene database of barley. Following qRT-PCR amplifications of all candidate reference genes in Tibetan hulless barley seedlings under various stressed conditions, the stabilities of these candidates were analyzed by three individual software packages including geNorm, NormFinder, and BestKeeper. The results demonstrated that TUBβ6, E2, TUBα, and HSP90 were generally the most suitable sets under all tested conditions; similarly, TUBα and HSP90 showed peak stability under salt stress, TUBα and EF-1α were the most suitable reference genes under cold stress, and ACT and E2 were the most stable under drought stress. Finally, a known circadian gene CCA1 was used to verify the service ability of chosen reference genes. The results confirmed that all recommended reference genes by the three software were suitable for gene expression analysis under tested stress conditions by the qRT-PCR method.
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影响因子：
5.3
作者：
Jarosová J;Kundu JK
通讯作者：
Kundu JK
影响因子：
37.3
作者：
Pagliarulo V;George B;Beil SJ;Groshen S;Laird PW;Cai J;Willey J;Cote RJ;Datar RH
通讯作者：
Datar RH
影响因子：
5.6
作者：
Choi WY;Kim JS;Park SJ;Ma CJ;Lee HY
通讯作者：
Lee HY
影响因子：
20.5
作者：
Hsu, Polly Yingshan;Harmer, Stacey L.
通讯作者：
Harmer, Stacey L.
鉴定非生物胁迫和激素处理下大麦基因表达的合适参考基因
影响因子：
2.1
作者：
Hua, Wei;Zhu, Jinghuan;Yang, Jianming
通讯作者：
Yang, Jianming