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【目的】筛选稳定表达的内参基因,用于阳春砂不同发育时期的果皮和种子团中基因表达量实时荧光定量PCR(qRTPCR)检测的校正。【方法】以阳春砂3个不同发育时期的果实(分为果皮和种子团)为材料,根据高通量测序得到的转录组和表达谱数据,选择5个表达稳定的常用内参基因β-actin、EF-1α、GAPDH、PGK和TUA作为候选基因,并利用qPCR技术检测它们在不同样品中表达水平的变化,使用GeNorm和NormFinder软件对基因的稳定性进行分析。【结果】5个内参基因在不同发育时期的果皮和种子团中的表达稳定性有明显的差异,其中GeNorm分析得到的内参基因的稳定顺序为:EF-1α=TUA>PGK>GAPDH>β-actin,NormFinder的分析结果中稳定性最好的是EF-1α,其次为TUA,其他3个内参基因稳定性的排列顺序与GeNorm软件的结果一致。【结论】可选用EF-1α和TUA作为阳春砂果实发育过程中基因表达量差异分析的双内参基因。
【Objective】 The aim of this study was to screen the stable expression of internal control gene for the correction of qRTPCR in peel and seed mass at different developmental stages. 【Method】 The results showed that β-actin, a common internal control gene with stable expression, was selected from the transcriptome and expression profile data of three different developmental stages of Yangchun sands (divided into peel and seed mass) , EF-1α, GAPDH, PGK and TUA were selected as candidate genes and their expression levels in different samples were detected by qPCR. The gene stability was analyzed by using GeNorm and NormFinder software. 【Result】 The results showed that there were significant differences in the expression stability of five reference genes in the pericarp and seed mass at different developmental stages. The stability order of the reference genes obtained by GeNorm was EF-1α = TUA> PGK> GAPDH> β- The best stability of actin and NormFinder was EF-1α, followed by TUA. The order of the stability of the other three internal reference genes was consistent with that of GeNorm software. 【Conclusion】 Both EF-1α and TUA can be used as double-internal reference genes for differential analysis of gene expression during the development of P. eriocheir.