Selection of suitable reference genes for quantitative real-time PCR gene expression analysis in Salix matsudana under different abiotic stresses.
Selection of suitable reference genes for quantitative real-time PCR gene expression analysis in Salix matsudana under different abiotic stresses.
复制标题
在不同的非生物应力下，选择合适的参考基因用于Salix Matsudana中定量实时PCR基因表达分析。
DOI：
10.1038/srep40290
复制
发表时间：
2017-01-25
影响因子：
4.6
通讯作者：
Zhuo R
中科院分区：
文献类型：
--
作者：
Zhang Y;Han X;Chen S;Zheng L;He X;Liu M;Qiao G;Wang Y;Zhuo R
旱柳是中国常见的落叶速生柳树品种，能耐旱、耐盐、耐重金属胁迫条件。选择合适的参考基因进行实时定量聚合酶链式反应，对于使与各种胁迫相关的关键基因的表达正常化具有重要意义。为了验证合适的参考基因，我们选择了11个候选参考基因(5个传统看家基因和6个新基因)，并分析了它们在不同组织和不同非生物胁迫处理下的表达稳定性。使用geNorm、Normfinder、BestKeeper、ΔCt和Reffinder五个程序确定这些基因的表达。结果表明，α-TUB2(α-微管蛋白2)和DNAJ(伴侣蛋白DNAJ49)是所有供试样本中最稳定的参考基因。我们使用两个最稳定的和一个最不稳定的参考基因来测量防御反应基因SmCAT(过氧化氢酶)在所有样品中的表达谱。SmCAT的相对定量因参考基因的不同而有较大差异。我们认为α-TUB2和DNAJ应该成为未来各种非生物胁迫条件下柳树基因表达研究中转录水平标准化和量化的首选参考基因。
Salix matsudana is a deciduous, rapidly growing willow species commonly cultivated in China, which can tolerate drought, salt, and heavy metal stress conditions. Selection of suitable reference genes for quantitative real-time PCR is important for normalizing the expression of the key genes associated with various stresses. To validate suitable reference genes, we selected 11 candidate reference genes (five traditional housekeeping genes and six novel genes) and analyzed their expression stability in various samples, including different tissues and under different abiotic stress treatments. The expression of these genes was determined using five programs—geNorm, NormFinder, BestKeeper, ΔCt, and RefFinder. The results showed that α-TUB2 (alpha-tubulin 2) and DnaJ (chaperone protein DnaJ 49) were the most stable reference genes across all the tested samples. We measured the expression profiles of the defense response gene SmCAT (catalase) using the two most stable and one least stable reference genes in all samples of S. matsudana. The relative quantification of SmCAT varied greatly according to the different reference genes. We propose that α-TUB2 and DnaJ should be the preferred reference genes for normalization and quantification of transcript levels in future gene expression studies in willow species under various abiotic stress conditions.