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[摘要]
目的 筛选合适的内参基因用于青天葵实时荧光定量PCR分析的校正。方法 以毛唇芋兰3个组织(叶片、叶柄和球茎)为材料,利用实时荧光定量PCR技术探讨18S rRNA、actin、ubiquitin、EF-1α和β-tubulin 5个常用内参基因在毛唇芋兰不同组织中表达差异。利用GeNorm和NormFinder软件比较各内参基因的Ct值,以分析他们在青天葵3个组织的表达稳定性。结果 5个内参基因的表达稳定性各异,GeNorm软件分析结果表明,稳定性β-tubulin=EF-1α>ubiquitin>actin>18S rRNA;NormFinder软件结果显示β-tubulin稳定性最好,EF-1α次之,18S rRNA则最差。两个不同软件分析结果一致。结论 β-tubulin可作为毛唇芋兰不同组织中基因表达差异分析的校正内参基因。
[Key word]
[Abstract]
Objective To select an appropriate reference gene for gene expression analysis using real-time fluorescence quantitative PCR (qRT-PCR). Methods Using leaf, petiole, and corm tissues of N. fordii, five common reference genes (18S rRNA, actin, ubiquitin, EF-1α, and β-tubulin) were compared. The stability of the candidate reference genes was evaluated by Ct value using GeNorm and NormFinder software. Results The stabilities of five reference genes varied from each other in three tissues of N. fordii. The analysis of GeNorm and NormFinder exhibited that the stability of β-tubulin was the most stable, followed by EF-1a, Ubiquitin, actin, and 18S rRNA in order. Conclusion The β-tubulin gene could be served as the reference gene for the normalization of gene expression in different tissues of N. fordii using qRT-PCR.
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[基金项目]
教育部博士点基金联合资助项目(200805720004);教育部留学回国人员科研项目(教外司留 [2009] 1001)