豌豆蚜实时荧光定量PCR内参基因的评估

doi:10.16380/j.kcxb.2023.05.007

摘要/Abstract

摘要： 【目的】本研究旨在通过对豌豆蚜Acyrthosiphon pisum内参基因在不同实验条件下的表达稳定性进行评估，为豌豆蚜基因表达分析奠定基础。【方法】利用qPCR测定昆虫常用14种候选内参基因(EF1α, Tubulin, NADH, RPL12, SDHB, 18S rRNA, 28S rRNA, 16S rRNA, ATPase, Actin, TATA, RPL32, GAPDH和RPL7)在豌豆蚜不同发育阶段(1-4龄若蚜和成蚜)、有翅和无翅孤雌成蚜、孤雌无翅成蚜不同组织(头、胸和腹)、不同地理种群(美国种群、甘肃种群、云南种群和德令哈种群)的孤雌无翅成蚜、不同寄主植物(苜蓿、三叶草和蚕豆)饲养的孤雌无翅成蚜、不同光周期(24L∶0D, 0L∶24D和16L∶8D)下饲养的孤雌无翅成蚜、不同温度(4, 18和35 ℃)下饲养的孤雌无翅成蚜和吡虫啉(200 g/L)处理下孤雌无翅成蚜中的表达量；利用RefFinder, ΔCt法, GeNorm, NormFinder和BestKeeper对上述14种内参基因表达稳定性进行分析；以CYP6CY3为靶标基因，探究不同内参基因对其在吡虫啉(200 g/L)处理下孤雌无翅成蚜中表达量分析的影响。【结果】依据qPCR检测结果，通过RefFinder对ΔCt法, GeNorm, NormFinder和BestKeeper结果的综合分析显示，在不同生物条件下(发育阶段、翅型、组织、地理种群和寄主植物)，18S rRNA和GAPDH是表达最稳定的内参基因，而16S rRNA和Actin的表达稳定性最差。在非生物条件下(光周期、温度和杀虫剂)，18S rRNA和EF1α的表达最稳定，而Tubulin和TATA的表达稳定性最差。基于GeNorm最佳内参基因数分析和不同内参基因对靶标基因CYP6CY3表达影响的分析，推荐使用2个表达最稳定内参基因18S rRNA和EF1α用于豌豆蚜的进一步研究。【结论】在豌豆蚜qPCR分析中，推荐同时使用18S rRNA和EF1α作为内参基因。

关键词: 豌豆蚜, 内参基因, 稳定性, 实时荧光定量PCR, 表达分析

Abstract: 【Aim】The aim of this study is to lay a foundation for the gene expression analysis of Acyrthosiphon pisum by identifying the expression stablility of reference genes in A. pisum under different experimental conditions. 【Methods】The expression levels of 14 candidate reference genes commonly used in insects (EF1α, Tubulin, NADH, RPL12, SDHB, 18S rRNA, 28S rRNA, 16S rRNA, ATPase, Actin, TATA, RPL32, GAPDH and RPL7)in different developmental stages (1st-4th instar nymphs and adult), winged and wingless parthenogenetic adults, different tissues (head, thorax and abdomen) of parthenogenetic wingless adults, parthenogenetic wingless adults of different geographical populations (American population, Gansu population, Yunan population and Delingha population), parthenogenetic wingless adults fed on different host plants (alfalfa, clover and broad bean), parthenogenetic wingless adults reared under different photoperiods (24L∶0D, 0L∶24D and 16L∶8D), parthenogenetic wingless adults reared under different temperatures (4, 18 and 35 ℃) and parthenogenetic wingless adults treated with 200 g/L imidacloprid were detected by qPCR. The expression stability of the above 14 reference genes was evaluated using RefFinder, ΔCt method, GeNorm, NormFinder, and BestKeeper. Using CYP6CY3 as the target gene to explore the influence of different reference genes on its expression level analysis in parthenogenetic wingless adults treated with 200 g/L imidacloprid. 【Results】 18S rRNA and GAPDH were recommended as the most stably expressed reference genes, whereas 16S rRNA and Actin showed the least expression stability under biotic conditions (developmental stage, wing morph, tissue, geographical population and host plant) through comprehensive analysis for the results of ΔCt method, GeNorm, NormFinder and BestKeeper by RefFinder, based on the qPCR results. Meanwhile, 18S rRNA and EF1α were recommended as the most stably expressed reference genes, while Tubulin and TATA showed the least expression stablility under abiotic conditions (photoperiod, temperature and insecticide). According to the GeNorm analysis data of the optimal number of reference genes and analysis of the influence of different reference genes on the expression of target gene CYP6CY3, 18S rRNA and EF1α were recommended as the most stably expressed reference genes for further studies in A. pisum. 【Conclusion】It is recommended to use the combination of 18S rRNA and EF1α in qPCR analyses in A. pisum.

Key words: Acyrthosiphon pisum, reference gene, stability, real-time quantative PCR, expression analysis

高雨晴, 马子淇, 李真祥, 陈珍珍, 刘芳华, 康志伟, 许永玉. 豌豆蚜实时荧光定量PCR内参基因的评估[J]. 昆虫学报, 2023, 66(5): 663-675.

GAO Yu-Qing, MA Zi-Qi, LI Zhen-Xiang, CHEN Zhen-Zhen, LIU Fang-Hua, KANG Zhi-Wei, XU Yong-Yu. Evaluation of reference genes for real-time quantitative PCR in the pea aphid, Acyrthosiphon pisum (Hemiptera: Aphididae)[J]. Acta Entomologica Sinica, 2023, 66(5): 663-675.

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豌豆蚜实时荧光定量PCR内参基因的评估

Evaluation of reference genes for real-time quantitative PCR in the pea aphid, Acyrthosiphon pisum (Hemiptera: Aphididae)

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