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