绞股蓝实时荧光定量PCR内参基因筛选和验证

Selection and Validation of Reference Genes for Quantitative Real-time PCR Analysis in Gynostemma pentaphyllum

目的:筛选适宜绞股蓝不同组织表达分析的内参基因,掌握绞股蓝皂苷生物合成关键酶基因GpFPS在绞股蓝不同组织中的表达特性。方法:基于绞股蓝转录组数据,以10个内参基因作为候选基因,采用geNorm、NormFinder、BestKeeper和RefFinder分析绞股蓝不同组织中10个候选内参基因的稳定性。利用实时荧光定量PCR技术,筛选出符合要求的内参基因,并实测基因GpFPS在绞股蓝不同组织中的表达情况。结果:候选内参基因在绞股蓝不同组织中的表达稳定性由高到低排序为PP2A、CYP、eIF4α、Actin、EF1α、TIP、18S、GAPDH、UBQ10、TUB6。GpFPS基因表达量在不同组织中由高到低分别是:嫩叶、老叶、花、卷须、成熟果、茎或根、幼果。结论:适合于绞股蓝不同组织基因表达分析的内参基因是PP2A和CYP,通过验证分析,该内参基因的可靠性强,为进一步研究绞股蓝皂苷生物合成相关基因的功能积累了必备的实验基础。

Objective:To screen suitable reference genes for expression analysis in different tissues of Gynostemma pentaphyllum,and to master the expression characteristics of GpFPS,the key enzyme gene of Gynostemma pentaphyllum saponin biosynthesis,in different tissues of Gynostemma pentaphyllum.Methods:Based on Gynostemma pentaphyllum transcriptome data,using the ten reference genes as candidate genes,geNorm,NormFinder,BestKeeper and RefFinder were used to analyze the stability of 10 candidate reference genes in Gynostemma pentaphyllum.The real-time quantitative PCR was used to screen the suitable internal reference genes.The expression of GpFPS gene in Gynostemma pentaphyllum was analyzed.Results:The sequence of expression stability of candidate reference genes in different tissues of Gynostemma pentaphyllum was PP2A,CYP,eIF4α,Actin,EF1α,TIP,18S,GAPDH,UBQ10 and TUB6.The GpFPS expression in different tissues was as follows,young leaf,old leaf,flower,tendril,mature fruit,stem or root,young fruit.Conclusion:The reference genes PP2A and CYP are suitable for gene expression analysis in different tissues of Gynostemma pentaphyllum,and the reliability of the reference genes has been proved to be strong,which accumulate the necessary experimental basis for further study on the function of Gynostemma pentaphyllum saponin biosynthesis related genes.