Quantitative real-time PCR(qPCR)is an effective and widely used method to analyze expression patterns of target genes.Selection of stable reference genes is a prerequisite for accurate normalization of target gene exp...Quantitative real-time PCR(qPCR)is an effective and widely used method to analyze expression patterns of target genes.Selection of stable reference genes is a prerequisite for accurate normalization of target gene expression by qRT-PCR.In Iris germanica L.,no studies have yet been published regarding the evaluation of potential reference genes.In this study,nine candidate reference genes were assessed at different flower developmental stages and in different tissues by four different algorithms(GeNorm,NormFinder,BestKeeper,and RefFinder).The results revealed that ACT11(Actin 11)and EF1α(Elongation factor 1 alpha)were the most stable reference genes in different tissues,whereas TUA(Tubulin alpha)and UBC9(Ubiquitin-protein ligase 9)were the most stable ones in different flower developmental stages.UBC9 and ACT11 were the most stable reference genes in all of the tested samples,while the SAMDC(S-Adenosylmethionine decarboxylase)showed the least stability.Finally,to validate the suitability of the selected reference genes,the relative expression level of IgTPS(beta-caryophyllene synthase)was assessed and highlighted the importance of suitable reference gene selection.This work constitutes the first systematic evaluation of potential reference genes in I.germanica and provides guidelines for future research on gene function and molecular mechanisms on I.germanica and related species.展开更多
基金The work was supported by the National Natural Science Foundation of China(31801901)the Natural Science Foundation of Jiangsu(BK20180314)+1 种基金the Foundation of Key Laboratory of Landscaping(KF201901),Ministry of Agriculture and Rural Affairs,Chinathe Jiangsu Key Laboratory for the Research and Utilization of Plant Resources(JSPKLB201814).
文摘Quantitative real-time PCR(qPCR)is an effective and widely used method to analyze expression patterns of target genes.Selection of stable reference genes is a prerequisite for accurate normalization of target gene expression by qRT-PCR.In Iris germanica L.,no studies have yet been published regarding the evaluation of potential reference genes.In this study,nine candidate reference genes were assessed at different flower developmental stages and in different tissues by four different algorithms(GeNorm,NormFinder,BestKeeper,and RefFinder).The results revealed that ACT11(Actin 11)and EF1α(Elongation factor 1 alpha)were the most stable reference genes in different tissues,whereas TUA(Tubulin alpha)and UBC9(Ubiquitin-protein ligase 9)were the most stable ones in different flower developmental stages.UBC9 and ACT11 were the most stable reference genes in all of the tested samples,while the SAMDC(S-Adenosylmethionine decarboxylase)showed the least stability.Finally,to validate the suitability of the selected reference genes,the relative expression level of IgTPS(beta-caryophyllene synthase)was assessed and highlighted the importance of suitable reference gene selection.This work constitutes the first systematic evaluation of potential reference genes in I.germanica and provides guidelines for future research on gene function and molecular mechanisms on I.germanica and related species.