Main Article Content
Carposina sasakii is one of the most important pests on the quality of stone and pome fruits. Investigation of a gene expression level in the species is hampered because of the gap of validated reference genes. The expression variation in the transcription levels of eight candidate reference genes, Actin (ACT), Tubulinbeta-1 (TUB), Ribosomal protein 49 (RP49), Elongation factor1-alpha (EF-1a), Elongation factor1-b (EF-1b), Elongation factor1-d (EF-1d), Ribosomal proteinL13 (RPL13) and Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), were analyzed by quantitative real-time PCR (qPCR). The stability and ranking of these gene expression profiles in three organ types (head, thorax and abdomen), three developmental stages (larva, pupa and moth), and five diapause states (non-diapause, pre-diapause, diapause 0 d, diapause 20 d and diapause 60 d) were assessed using two algorithm-based methods, geNorm and NormFinder. EF-1a, ACT and GAPDH were evaluated to be the three stable reference genes based on the important observations and comprehensive analysis, whereas TUB and EF-1b showed low expression stability. Best gene combinations for different qPCR analysis in C. sasakii could be chosen from the three stable reference genes, the using of two reference genes is sufficient to effectively normalize qPCR data in C. sasakii. The study laid the foundation for gene expression analysis in C. sasakii and provided new information for the selection of reference genes.
Quan LF, Qiu GS, Zhang HJ, Sun LN, Li YY, Yan WT. Sublethal concentration of beta-cypermethrin influences fecundity and mating behavior of Carposina sasakii (Lepidoptera: Carposinidae) adults. J. Eco. Entomolo. 2016;109(5):2196–2204.
Tian ZQ, Qiu G, Li YY, Zhang HJ, Yan WT, Yue Q. Molecular characterization and functional analysis of pheromone binding proteins and general odorant binding proteins from Carposina sasakii matsumura (Lepidoptera: Carposinidae). Pest Manag. Sci. 2019;75(1):234-245.
Zhang B, Peng Y, Zhao XJ, Hoffmann A, Li R, Ma CS. Emergence of the overwintering generation of peach fruit moth (Carposina sasakii) depends on diapause and spring soil temperatures. J.Insect Physio. 2016;86(3):32–39.
Toshima A, Honma K, Masaki S. Factors influencing the seasonal incidence and breaking of diapause in Carposina niponensis Walshingham. Jan. J. Appl. Entomol. Z. 1961;5(4):260–269.
Tian ZQ, Sun LN, Li Y, Quan LF, Zhang HJ, Yan WT. Antennal transcriptome analysis of the chemosensory gene families in Carposina sasakii (lepidoptera: carposinidae). BMC Genomics. 2018;19(1):544.
Li J, Wang X, Zhang L. Identification of putative odorant binding proteins in the peach fruit borer Carposina sasakii Matsumura (Lepidoptera: Carposinidae) by transcriptome analysis and their expression profile. Biochem. Bioph. Res. Co. 2019;508(4):1024-1030.
Shen GM, Jiang HB, Wang XN, Wang JJ. Evaluation of endogenous references for gene expression profiling in different organs of the oriental fruit fly Bactrocera dorsalis (Diptera: Tephritidae). BMC Mol. Biol. 2010;11(1):76.
Li RM, Xie W, Wang SL, Wu QJ, Yang N, Yang NN. Reference gene selection for qRT-PCR analysis in the sweet potato whitefly, Bemisia tabaci (Hemiptera: Aleyrodidae). PLoS One. 2013;8(1):53006.
Lu YH, Yuan M, Gao XW, Kang TH, Zhan S, Wan H. Identification and validation of reference genes for gene expression analysis using quantitative PCR in Spodoptera litura (Lepidoptera: Noctuidae). PLoS One. 2013;8(7):68059.
Rubio RO, Suzuki A, Mitsumasu K, Homma T, Niimi T, Yamashita O. Cloning of cDNAs encoding sorbitol dehydrogenase-2a and b, enzymatic characterization, and up-regulated expression of the genes in Bombyx mori diapause eggs exposed to 5 °C, Insect Biochem. Mol. Biol. 2011;41(6):378-387.
Tan QQ, Zhu L, Li Y, Liu W, Ma WH, Lei CL. A de novo transcriptome and valid reference genes for quantitative real-time PCR in Colaphellus bowringi. PLoS One. 2015;10(2):0118693.
Gutierrez L, Mauriat M, GuNin S, Pelloux J, Lefebvre JF, Louvet R. The lack of a systematic validation of reference genes: A serious pitfall undervalued in reverse transcription‐polymerase chain reaction (RT‐PCR) analysis in plants. Plant Biotechnol. J. 2008;6(6):609–618.
Arun A, Bauml ÉV, Amelot G, Nieberding CM. Selection and validation of reference genes for qRT-PCR expression analysis of candidate genes involved in olfactory communication in the butterfly Bicyclus anynan. PLoS One. 2015;10(3):0120401.
Teste MA, Duquenne M, François JM, Parrou JL. Validation of reference genes for quantitative expression analysis by real-time RT-PCR in Saccharomyces cerevisiae. BMC Mol. Bio. 2009;10(10):99.
Shi XQ, Guo WC, Wan PJ, Zhou LT, Ren XL, Ahmat T. Validation of reference genes for expression analysis by quantitative real-time PCR in Leptinotarsa decemlineata (Say). BMC Res. N. 2011;6(1):93.
Pfaffl MW. A new mathematical model for relative quantification in real-time RT–PCR. Nucleic Acids Res. 2001;29(9):45.
Vandesompele J, Preter K, De Pattyn F, Poppe B, Van Roy N, De Paepe A. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 2002;3(7):003411.
Zhu X, Li X, Chen W, Chen J, Lu W, Chen L. Evaluation of new reference genes in papaya for accurate transcript normalization under different experimental conditions. PLoS One, 2012;7(8):44405.
Teng XL, Zhang Z, He GL, Yang LW, Li F. Validation of reference genes for quantitative expression analysis by real-time RT-PCR in four lepidopteran insects. J. Insect Sci. 2012;12(60):1-17.
Fu W, Xie W, Zhang Z, Wang S, Wu Q, Liu Y. Exploring valid reference genes for quantitative real-time PCR analysis in Plutella xylostella (Lepidoptera: Plutellidae). Int. J. Biol. Sci. 2013;9(8):792–802.