Abstract: 【Aim】 This study aims to screen and verify stably expressed genes under given conditions as reference genes for quantitative real-time PCR (qRT-PCR) in Conogethes punctiferalis, so as to provide the basis for quantitative studies of genes of this moth. 【Methods】 Based on transcriptomics sequencing results in C. punctiferalis and reported reference genes in other insect species, six candidate genes including β-actin gene (ACT), glyceraldehyde 3-phosphate dehydrogenase gene (GAPDH), ribosomal protein 49 gene (RP49), alpha tubulin gene (α-tub), ribosomal protein L13 gene (RPL13) and vacuolar-type ATPase gene (V-ATPase) were cloned, and their expression levels in different developmental stages and different adult tissues were measured by qRT-PCR. Then the stabilities of these candidate genes were evaluated by a series of programs including ΔCt method, GeNorm, NormFinder, BestKeeper and an online program, RefFinder. Finally, the stabilities of selected reference genes were validated with an olfactory receptor co-receptor gene (Orco) and pheromone binding protein 1 gene (PBP1). 【Results】 Analyzed by four programs including ΔCt method, GeNorm, NormFinder and BestKeeper, similar rankings of six candidate genes were obtained, among which RP49, RPL13 and GAPDH were the most stable genes in different developmental stages and different adult tissues, and ACT was ranked as the least stable gene despite of experimental conditions by all programs. Comprehensive analysis with RefFinder further showed that in different adult tissues, RPL13 was the most stable gene, followed by RP49, and in different developmental stages, RP49 was the most stable gene, followed by GAPDH. Additionally, the optimal number of reference genes was calculated by GeNorm as 2. Finally, the stabilities of selected reference genes were validated with Orco and PBP1 as target genes. The results indicated that when two pairs of genes, RPL13 and RP49, and RP49 and GAPDH, were respectively used as reference genes, Orco and PBP1 showed reliable expression patterns, which were consistent with life habits of C. punctiferalis and the results of the previous research. The expression patterns of Orco and PBP1, however, were irregular when ACT was used as the reference gene. 【Conclusion】 In C. punctiferalis, two pairs of genes, RPL13 and RP49, and RP49 and GAPDH, are recommended as reference genes in different adult tissues and different developmental stages, respectively.

Key words: Conogethes punctiferails, reference genes, gene screening, expression stability, quantitative real-time PCR