Stable reference gene selection for quantitative real-time PCR normalization in passion fruit (Passiflora edulis Sims.)
Stable reference gene selection for quantitative real-time PCR normalization in passion fruit (Passiflora edulis Sims.)
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DOI：
10.1007/s11033-022-07382-5
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发表时间：
2022-03-31
影响因子：
2.8
通讯作者：
Xu, Meng
中科院分区：
文献类型：
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作者：
Zhao, Meiqi;Fan, Hang;Xu, Meng
背景：西番莲是我国中国南部地区广泛种植的热带水果，具有很高的营养价值和药用价值。然而，对美味拟青霉的分子生物学研究尚不多见。关于西番莲果基因表达研究中参考基因的选择的报道很少。方法与结果通过在geNorm、NormFinder和BestKeeper中实现的三种算法，我们选择了10个候选参考基因，以探讨它们在不同组织和冷应激条件下转录表达的稳定性。EF1和His在5种组织中稳定表达。TS和OTU在营养器官中稳定存在。50只S和Liom在生殖器官中表现稳定。EF1和UBQ在冷处理和恢复处理的叶片样品中的转录丰度是稳定的。在所有样本中，EF1和TS表现出最高的表达稳定性。使用简单的统计方法(单因素方差分析和后处理分析)评估选定的基因。总体而言，EF1基因是青霉qRT-PCR正常化的最佳参考基因。此外，qRT-PCR分析表明，ICE1的表达随冷处理时间的延长而增加。结论在本研究中，我们成功地从10个菜青霉候选基因中筛选出稳定的参考基因，并通过分析ICE1的表达水平对结果进行了验证。本研究结果为进一步开展食用菌基因表达分析提供了可靠有效的参考基因，为后续食用菌功能基因的研究奠定了基础。
Background Passiflora edulis is a tropical fruit with high nutrient and medicinal values that is widely planted in southern China. However, the molecular biology of P. edulis has not been well studied. There are few reports regarding the choice of reference genes for gene expression studies of passion fruit. Methods and results By using three algorithms, implemented in geNorm, NormFinder and BestKeeper, we have selected ten candidate reference genes to explore their transcriptional expression stability in various tissues and under cold stress conditions. EF1 and HIS were stably expressed in five tissues. Ts and OTU were stably in vegetative organs. 50 S and Liom were stably in reproductive organs. The transcriptional abundance of EF1 and UBQ was stable in cold-treated and recovery treated leaf samples of P. edulis. In all samples, EF1 and Ts exhibited the highest expression stability. Evaluation of selected genes using simple statistical methods (ANOVA and post hoc analysis). Overall, EF1 emerged as the optimum reference gene for qRT-PCR normalize in P. edulis. In addition, the qRT-PCR analysis revealed that expression of ICE1 increases with the duration of cold treatment. Conclusions In this study, we successfully screened stable reference genes from 10 candidates in P. edulis and verified the results by analyzing the expression level of ICE1. The results provide reliable and effective reference genes for future research on gene expression analysis in P. edulis, and lay a foundation for follow-up research on functional genes in P. edulis.