Neolamarckia cadamba, a member of the Rubiaceae family, is widely distributed throughout South Asia and South China. In order to acquire reliable and repeatable results, the use of a suitable internal reference gene to normalize the RT-qPCR data is essential. In this study, we reported the validation of housekeeping genes to identify the most suitable internal reference gene(s) for normalization of qPCR data obtained among different tissues (bud, leaf, cambium region) under different hormone stresses. Here, ΔCt, geNorm, NormFinder, and BestKeeper analyses were carried out to analyze the normalization of qPCR data of twenty-one reference gene families (ACT, CAC, CYP, EF1α, eIF, FPS1, FBK, GAPDH, RAN, PEPKR1, PP2A, RPL, RPS, RuBP, SAMDC, TEF, Tub-α, Tub-β, UBCE, UBQ, UPL) including 43 genes. The results showed that FPS1, RPL, and FBK were the most stable reference genes across all of the tested samples. In addition, the expression of NcEXPA8, one gene of interest that plays an important role in regulating cell wall extension, under different phytohormone stresses was used to further confirm the validated reference genes. Taken together, our results provide guidelines for reference gene selection under different phytohormone stresses and a foundation for more accurate and widespread use of RT-qPCR in N. cadamba.

中文翻译：

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多用途木材产量树Neolamarckia cadamba不同激素胁迫下内部参考基因的选择

茜草科的Neolamarckia cadamba，在南亚和华南地区广泛分布。为了获得可靠和可重复的结果，必须使用合适的内部参考基因对RT-qPCR数据进行标准化。在这项研究中，我们报告了看家基因的验证，以鉴定最合适的内部参考基因，以标准化在不同激素胁迫下不同组织（芽，叶，形成层区域）之间获得的qPCR数据。在这里，进行了ΔCt，geNorm，NormFinder和BestKeeper分析，以分析二十一个参考基因家族（ACT，CAC，CYP，EF1α，eIF，FPS1，FBK，GAPDH，RAN，PEPKR1，PP2A）的qPCR数据的标准化。，RPL，RPS，RuBP，SAMDC，TEF，Tub-α，Tub-β，UBCE，UBQ，UPL），包括43个基因。结果表明，FPS1，RPL和FBK是所有测试样品中最稳定的参考基因。此外，在不同的植物激素胁迫下，NcEXPA8的表达（一种在调节细胞壁延伸中起重要作用的重要基因）的表达被用于进一步确认经过验证的参考基因。综上所述，我们的结果为在不同植物激素胁迫下选择参考基因提供了指导，并为在卡迪巴猪笼草中更准确和广泛地使用RT-qPCR提供了基础。