为筛选出沙地云杉(Picea mongolica)种子成熟过程中均可稳定表达的内参基因,本研究以沙地云杉种子不同发育时期的样本为研究对象,采用RT-qPCR技术,检测沙地云杉种子转录组数据库中GAPDH、EF1α、CYC、Actin等8个候选内参基因在种子发育过程中呈现的综合表达水平,并通过内参基因分析软件(geNorm,NormFinder,BestKeeper)对其稳定性统一评估.结果表明,候选内参基因中稳定性最好的是CYC、EF1α和GAPDH.分别以CYC、EF1α和GAPDH作为内参基因对沙地云杉胚胎发育关键调控基因LEA1(Late embryogenesis abundant protein)和 WOX9(WUSCHEL related homebox)进行了 定量分析,其表达趋势基本一致.本研究结果将对沙地云杉及近缘物种的分子育种研究提供参考.
Screening and Verification of Internal Reference Genes in Seed of Picea mongolica
In order to select the internal reference genes that can be stably expressed during the maturation of the Picea mongolica seeds,in this study,the samples of the Picea mongolica seeds at different development stages were used as the research object,and the RT-qPCR technology was used to detect the sandy spruce.The comprehensive expression levels of 8 candidate internal reference genes,including GAPDH,EF1α,CYC,Actin,in the seed tran-scriptome database during seed development,are uniformly evaluated for their stability through internal reference gene analysis software(geNorm,NormFinder,BestKeeper).The results showed that the most stable candidate internal reference genes were CYC,EF1α and GAPDH.CYC,EF1α and GAPDH were used as internal reference genes to quantitatively analyze the key regulatory genes LEA1(Late embryogenesis abundant protein)and WOX9(WUSCHEL related homebox),and their expression trends were basically the same.The results of this study will provide reference for the molecular breeding research of Picea mongolica and related species.
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