Validation of reference genes for real-time quantitative PCR normalisation in non-heading Chinese cabbage.
Validation of reference genes for real-time quantitative PCR normalisation in non-heading Chinese cabbage.
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DOI：
10.1071/fp11246
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发表时间：
2012-05
期刊：
影响因子：
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通讯作者：
D. Xiao;Ningwen W. Zhang;Jian-Jun Zhao;G. Bonnema;X. Hou
中科院分区：
文献类型：
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作者：
D. Xiao;Ningwen W. Zhang;Jian-Jun Zhao;G. Bonnema;X. Hou
不结球白菜是一种重要的蔬菜作物，包括小白菜，菜心和几种日本蔬菜，如水菜，水布纳和小松菜。基因表达研究经常被用来揭示复杂性状的遗传学，在这些研究中，正确选择标准化的参考基因是至关重要的。我们检测了13个候选参考基因的表达，包括ACTIN、ACTIN-1、ACTIN-2、GAPDH、Tub_α、CyP、EF1-α、18S rRNA、UBQ、UBC 30、PPR、PP2A和MDH。他们的表达稳定性进行了分析，使用两个程序，geNorm和Normalware，在20个不同的样品，代表四个战略组。结果表明，没有单一基因在所有测试样品中均匀表达。ACTIN和CyP被认为是研究发育阶段的良好参考基因。CyP、Tub_α和UBC_（30）是研究不同组织（从开花到结实）的良好参考基因。CyP和Tub_α是在霜霉和芸苔链格孢菌生物胁迫下最稳定的参考基因。UBC 30、EF1-α和ACTIN被推荐用于非生物胁迫研究中的标准化，包括激素、盐、干旱、寒冷和健康处理。此外，在研究所有供试样本的基因表达时，需要至少5个参考基因（ACTIN、CyP、UBC 30、EF1-α和UBQ）进行准确的qRT-PCR数据标准化。
Non-heading Chinese cabbage is an important vegetable crop that includes pak choi, caixin and several Japanese vegetables like mizuna, mibuna and komatsuna. Gene expression studies are frequently used to unravel the genetics of complex traits and in such studies the proper selection of reference genes for normalisation is crucial. We assessed the expression of 13 candidate reference genes including ACTIN, ACTIN-1, ACTIN-2, GAPDH, Tub_α, CyP, EF1-α, 18S rRNA, UBQ, UBC30, PPR, PP2A and MDH. Their expression stabilities were analysed using two programs, geNorm and NormFinder, in 20 different samples that represent four strategic groups. Results showed that no single gene was uniformly expressed in all tested samples. ACTIN and CyP are proposed as good reference genes when studying developmental stages. CyP, Tub_α and UBC30 are good reference genes when studying different tissues (from flowering to seed set). CyP and Tub_α are the most stable reference genes under biotic stress treatments using the fungi Peronospora parasitica and Alternaria brassicicola. UBC30, EF1-α and ACTIN are recommended for normalisation in abiotic stress studies, including hormone, salt, drought, cold and heath treatments. Moreover, at least five reference genes (ACTIN, CyP, UBC30, EF1-α and UBQ) are required for accurate qRT-PCR data normalisation when studying gene expression across all tested samples.