林业科学2016，Vol.52Issue(8)：29-37,9.DOI:10.11707/j.1001-7488.20160804
光皮桦实时荧光定量PCR内参基因的筛选
The Selection of Reference Genes for Quantitative PCR in Betula luminifera
摘要
Abstract
[Objective]Real-time quantitative PCR ( RT-qPCR) has become the preferred approach to the quantification of gene expression. Besides its simplicity and efficiency,this method requires suitable reference genes to guarantee the accuracy of the quantification. Betula luminifera is not only a high-quality timber species with excellent wood properties, but also an ideal species for genetic study. Stability of candidate reference genes in different tissues was investigated using RT-qPCR and expression analysis software to select suitable reference genes,the reliability of the reference genes was further verified through expression analysis of two functional genes in different tissues. [Method]16 housekeeping genes were chosen as candidate reference genes,and specificity of the primers was investigated by agarose gel electrophoresis and melting curve of target amplified fragments. The software of geNorm,NormFinder and BestKeeper were used to analyze expression stability of reference genes in 16 different tissues. Furthermore,two functional genes were selected to verify the reliability of the suitable reference genes. [Result]Electrophoresis results showed distinct PCR products with the expected size,and the single-peak melting curves further indicated the specificity of the selected primers. Except 18S,the Ct values of the candidate genes were all between 25 to 30 ,indicating the expressions of these candidate genes were quite stable in different tissues. EF1α was characterized as the most stable gene based on NormFinder and BestKeeper analysis, while TATA was ranked in the first place according to geNorm. UBi-lp showed the most unstable expression. Furthermore, three top-stable genes ( EF1α,TATA and UBi4 ) and the unstable gene UBi-lp were chosen to verify the reliability of the reference genes. As results,the two target genes showed consistent expression profiles when normalized by the three top-ranked reference genes,and UBi-lp failed to standardize the expression data. [Conclusion]EF1α,TATA and UBi4 were most stable reference genes in different tissues,the reliability of these reference genes were further confirmed by expression analysis of the target genes. Therefore,EF1α,TATA and UBi4 could serve as reference genes for gene expression among different tissues in B. luminifera.关键词
光皮桦/内参基因/实时荧光定量 PCRKey words
Betula luminifera/reference gene/real-time quantitative PCR分类
农业科技引用本文复制引用
刘文哲,牛明月,李秀云,林二培,黄华宏,童再康..光皮桦实时荧光定量PCR内参基因的筛选[J].林业科学,2016,52(8):29-37,9.基金项目
国家自然科学基金项目(31100494) （31100494）
浙江省竹木农业新品种选育重大科技专项(2012C12908-8)。 （2012C12908-8）
