Selection of reference genes for qRT-PCR normalization in Cestrum nocturnum during flowering
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摘要: 为筛选夜香树（Cestrum nocturnum L.）香气释放、生物钟等相关基因表达研究适用的内参基因，本研究采用夜香树盛花期叶片和花为实验材料，利用同源克隆和RACE技术，获得了夜香树6种经典的内参基因序列，分别为：Actb7、EF-1A、GAPDH、TUA、TUB2、UBQ；采用荧光定量PCR方法对18s rRNA和这6个内参基因的表达模式进行了分析，并通过Bestkeeper、geNorm、NormFinder 3种程序分析了内参基因的稳定性。结果表明，在花中，Actb7表达最稳定；在叶片中，EF-1A和UBQ的表达比较稳定；在2种组织中，EF-1A的表达相对稳定。3组稳定性分析中，geNorm程序确定的最佳内参基因数目均为2，最佳内参基因组合均为Actb7/EF-1A。本研究通过对稳定内参基因的筛选，以期为准确检测夜香树盛花期花瓣节律运动、香气释放、生物钟变化等相关基因的表达研究奠定基础。Abstract: To screen stable reference genes for studies on gene expression of aroma release and the circadian clock changes in Cestrum nocturnum,six traditional reference genes,Actb7,EF-1A,GAPDH,TUA,TUB2,and UBQ,were isolated from flowers and leaves in the blooming stage by homology cloning and RACE technology.The expression patterns of 18s rRNA and the six reference genes were analysed by real-time quantitative PCR (qRT-PCR) and the expression stabilities were analysed by Bestkeeper,geNorm,and NormFinder.Results showed that the expression of Actb7 was stable in flowers;EF-1A and UBQ were stable in leaves;and EF-1A was stable in both tissues.According to the analysis of the three groups by geNorm,the optimal number of reference genes was two and the best combination was Actb7 and EF-1A.The stable reference genes screened in this study provide a basis for accurate detection of the expression of genes related to petal rhythm movement,aroma release,and circadian clock changes.
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Keywords:
- Cestrum nocturnum /
- Reference gene /
- Real-time quantitative PCR
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