Selection and Validation of qRT-PCR Reference Genes in Different Tissues of Nervilia plicata
Nervilia plicata is a rare southern Chinese herb.The study on the plant at molecular level is hardly seen.Real-time quantitative PCR(qRT-PCR)has been widely used in plant gene expression analysis,but there is a lack of research on it in N.plicata,which limits the progress of related work.Based on the previous researches,nine candidate reference genes,including Actin,GAPDH,TUA,UBC,UBQ,EF-1α,EF-1β,CYP and RPL,were screened out from the transcriptomic sequencing data.In order to select suitable reference gene in N.plicata,qRT-PCR technique was em-ployed to detect the expression levels of the candidate reference genes in leaf,petiole and corm tissues of the plant.Af-ter analyzing the expression levels,the stability and comprehensive analysis of nine genes in different tissues,the most suitable reference gene was obtained.Finally,the most suitable reference gene was validated using NpDFR,Np3GT that related to anthocyanin synthesis.Results showed that the expression stability of EF-1 α and CYP was similar,with the best overall stability.When using either EF-1α or CYP,or the combination of them,as reference,the expression patterns of NpDFR and Np3GT in different tissues were consistent with the transcriptome sequencing results.This indicates that both EF-1 α and CYP can be used as reference genes in N.plicata for qRT-PCR.This study would provide a basis for fur-ther research on the molecular mechanism of genes related to the pharmacological effects of N.plicata.
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