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Selection and Validation of Reference Genes for Quantitative Real-Time PCR in White Clover (Trifolium repens L.) Involved in Five Abiotic Stresses.
Plants ( IF 4.658 ) Pub Date : 2020-08-05 , DOI: 10.3390/plants9080996
Qi Pu 1 , Zhou Li 1 , Gang Nie 1 , Jiqiong Zhou 1 , Lin Liu 1 , Yan Peng 1
Affiliation  

White clover (Trifolium repens L.) is a widely cultivated cool-season perennial forage legume in temperate grassland systems. Many studies have analyzed the gene expression in this grass species using quantitative real-time reverse transcription PCR (qRT-PCR). The selection of stable reference genes for qRT-PCR is crucial. However, there was no detailed study on reference genes in different tissues of white clover under various abiotic stress conditions. Herein, 14 candidate reference genes (ACT7, ACT101, TUA1109, TUB, CYP, 60SrRNA, UBQ, E3, GAPDH1, GAPDH2, PP2A, BAM3, SAMDC, and ABC) were selected and analyzed by four programs (GeNorm, NormFinder, BestKeeper, and RefFinder). Samples were taken from two tissues (leaves and roots) under five different abiotic stresses (drought, salt, heat, cold, and heavy metal stress). Our results showed that 60SrRNA and ACT101 were the two top-ranked genes for all samples. Under various experimental conditions, the most stable gene was different; however, SAMDC, UBQ, 60SrRNA, and ACT101 were always top ranked. The most suitable reference genes should be selected according to different plant tissues and growth conditions. Validation of these reference genes by expression analysis of Cyt-Cu/Zn SOD and CAT confirmed their reliability. Our study will benefit the subsequent research of gene function in this species.

中文翻译:

涉及五个非生物胁迫的白三叶草(Trifolium repens L.)中定量实时PCR的参考基因的选择和验证。

白三叶草(Trifolium repens L.)是在温带草原系统中广泛种植的冷季多年生饲草豆类。许多研究已经使用定量实时逆转录PCR(qRT-PCR)分析了该草种中的基因表达。qRT-PCR稳定参考基因的选择至关重要。然而,目前还没有关于在各种非生物胁迫条件下白三叶草不同组织中参考基因的详细研究。本文中有14个候选参考基因(ACT7,ACT101,TUA1109,TUB,CYP,60SrRNA,UBQ,E3,GAPDH1,GAPDH2,PP2A,BAM3,SAMDCABC)由四个程序(GeNorm,NormFinder,BestKeeper和RefFinder)进行选择和分析。在五个不同的非生物胁迫(干旱,盐,热,冷和重金属胁迫)下,从两个组织(叶和根)中采集样品。我们的结果表明60SrRNAACT101是所有样品中排名靠前的两个基因。在各种实验条件下,最稳定的基因是不同的。但是,SAMDC,UBQ,60SrRNAACT101始终排名最高。应根据不同的植物组织和生长条件选择最合适的参考基因。通过Cyt-Cu / Zn SODCAT的表达分析验证这些参考基因确认了其可靠性。我们的研究将有益于该物种的基因功能的后续研究。
更新日期:2020-08-05
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