Abstract:

 【Aim】The aim of this study is to find out the reference genes stably expressed in different developmental stages and adult tissues of Grapolita molesta and in its adults after treatment with different concentrations of three insecticides, so as to lay a foundation for the subsequent study on target gene expression in G. molesta.【Methods】Ten candidate reference genes (β-actin, 18S rRNA, β-tubulin, EF-1α, RPL13, RPL32, RSPL40, UBC7, α-tubulin and RPS20) were selected based on G. molesta transcriptome data. The expression levels of the candidate reference genes in different developmental stages (egg, 1st-5th instar larvae, pupa and adult) and different adult tissues (head, foregut, midgut, hindgut, fat body, Malpighian tubules, testis and ovary) of G. molesta and in G. molesta adults treated with three insecticides at different concentrations (avermectin: 19.819, 72.897 and 179.663 μg/mL; imidacloprid: 17.638, 163.323 and 762.986 μg/mL; and lambda-cyhalothrin: 33.791, 96.123 and 198.282 μg/mL) with the method of residual film in glass tube were detected using real-time quantitative PCR (qRT-PCR). The expression stabilities of the 10 candidate reference genes were evaluated by geNorm, NormFinder, ΔCt, BestKeeper and RefFinder. The cytochrome P450 gene CYP354A32 of G. molesta was selected for validation. 【Results】 qRT-PCR results combined with software evaluation results revealed that the expression stabilities of the reference genes in different developmental stages of G. molesta were ranked in a descending order of β-tubulin, 18S rRNA, EF-1α, RPL13, β-actin, RPS20, UBC7, RPL32, α-tubulin and RSPL40, those in different adult tissues were ranked in a descending order of UBC7, β-tubulin, β-actin, 18S rRNA, RSPL40, EF-1α, RPS20, RPL13, RPL32 and α-tubulin, and those in adults after exposure to different concentrations of avermectin, imidacloprid and lambda-cyhalothrin were ranked in a descending order of RPS20, RPL13, β-tubulin, β-actin, RPL32, RSPL40, EF-1α, UBC7, α-tubulin and 18S rRNA. The expression characteristics of CYP354A2 analyzed by using the obtained reference gene combinations showed that CYP354A2 was highly expressed in the old larvae and adults when using the combination of β-tubulin, EF-1α and 18S rRNA as the reference genes, and was highly expressed in the testis and ovary of adults when using the combination of UBC7, β-tubulin and β-actin as the reference genes. After G. molesta adults were exposed to different concentrations of insecticides, only the expression level of CYP354A2 in the treatment with 19.819 μg/mL avermectin was higher than that in the control, while the expression levels of CYP354A2 in treatments with other concentrations of insecticides were lower than that in the control when using the combination of RPS20, RPLB and β-tubulin as the refernece genes.【Conclusion】 The combination of β-tubulin, 18S rRNA and EF-1α is recommended as the reference genes for studying the target gene expression in different developmental stages of G. molesta, that of UBC7, β-tubulin and β-actin as the reference genes for studying the target gene expression in different adult tissues of G. molesta and that of RPS20, RPL13 and β-tubulin as the reference genes for studying the target gene expression in adults of G. molesta after treatment with different concentrations of avermectin, imidacloprid and lambda-cyhalothrin.

Key words: Grapholita molesta, reference gene, gene screening, qRT-PCR, expression stability