Stability evaluation of reference genes for real-time quantitative PCR normalization in Spodoptera frugiperda (Lepidoptera: Noctuidae)

doi:10.1016/S2095-3119(20)63298-1

摘要/Abstract

摘要：

本文选取Actin，EF1α，EF2，GAPDH，RPL3，RPL13，α-TUB和β-1-TUB基因作为候选内参基因；采用实时荧光定量PCR分析了8个候选内参基因在草地贪夜蛾不同龄期，6龄幼虫不同组织，幼虫在不同温度和饥饿处理等实验条件下的转录水平表达量；采用ΔCt，BestKeeper，geNorm，NormFinder和RefFinder对各个候选内参基因进行表达稳定性评估。GeNorm分析结果表明在本研究中不同实验条件下用于分析靶标基因转录水平表达量的最适内参基因个数均为2个。综合分析结果表明草地贪夜蛾不同发育阶段最稳定的内参基因为EF2和RPL13, 6龄幼虫不同组织中最稳定的内参基因为RPL13和β-1-TUB，不同温度处理下三龄幼虫中最稳定的内参基因为EF2和EF1α，饥饿处理条件下三龄幼虫中最稳定的内参基因为RPL3和EF1α，所有样本中较为稳定的内参基因为RPL13和EF1α。本研究为草地贪夜蛾不同实验条件下内参基因选择提供了参考，同时也有助于保证后续靶标基因转录水平表达研究的准确性。

Abstract:

Real-time quantitative PCR (qPCR) is a reliable and widely used technique for analyzing the expression profiles of target genes in different species, and reference genes with stable expressions have been introduced for the normalization of the data. Therefore, stability evaluation should be considered as the initial step for qPCR experiments. The fall armyworm Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae) is a polyphagous pest that consumes many plant species and seriously threatens corn production around the world. However, no studies thus far have examined the stability of reference genes in this pest. In this study, the expression profiles of the eight candidate reference genes of Actin, elongation factor 1 alpha (EF1α), elongation factor 2 (EF2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribosomal protein L3 (RPL3), ribosomal protein L13 (RPL13), alpha-tubulin (α-TUB), and beta-1-tubulin (β-1-TUB) were obtained from S. frugiperda in different samples and the stability was evaluated by ΔCt, BestKeeper, geNorm, NormFinder, and RefFinder methods. The results of pairwise variation (V) calculated by GeNorm indicated two reference genes could be selected for normalization. Therefore, the combinations of the most stable reference genes for different experimental conditions of S. frugiperda were shown as follows: EF2 and RPL13 for developmental stages, RPL3 and β-1-TUB for larval tissue samples, EF2 and EF1α for the larval samples treated with different temperatures, RPL3 and EF1α for the larval samples under starvation stress, and RPL13 and EF1α for all the samples. Our results lay the foundation for the normalization of qPCR analyses in S. frugiperda and could help guarantee the accuracy of subsequent research.

Key words: Spodoptera frugiperda , reference genes , qPCR , stability evaluation , different experimental conditions

. [J]. Journal of Integrative Agriculture, 2021, 20(9): 2471-2482.

SHU Ben-shui, YU Hai-kuo, DAI Jing-hua, XIE Zi-ge, QIAN Wan-qiang, LIN Jin-tian. Stability evaluation of reference genes for real-time quantitative PCR normalization in Spodoptera frugiperda (Lepidoptera: Noctuidae)[J]. Journal of Integrative Agriculture, 2021, 20(9): 2471-2482.

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