Abstract: [Objective] In this study, the stably expressed genes under given conditions, including different temperature stresses, different developmental stages and different tissues of larva, were screened and verified as internal reference genes for quantitative real-time PCR (qRT-PCR) in Hyphantria cunea, to provide a basis for quantitative studies of genes of the moth.[Method] Based on transcriptomics sequencing results in H. cunea (the data was constructed by our laboratory and not published), eight candidate internal reference genes of GAPDH, ACT, RPL12, RPL18a, RPS16, EF1α, EF1β and 18S rRNA were selected, and their base sequences were obtained. The above sequences were cloned, sequenced and compared, and finally proved to be the gene of H. cunea and the base sequence was correct. Then the sequence was uploaded to NCBI to obtain GenBank accession No. Real-time fluorescent quantitative PCR primers (qRT-PCR) of the eight candidate reference genes were designed. The qRT-PCR technique was applied to measure the eight candidate reference genes at different developmental stages (1-6 age of larvae), under the different temperature stress (-10, -5, 0, 25, 35, 40, 45℃ with 2 h), and in different tissues of larva (head, the gut, fat body, malpighian tube, skin), and the C_t values were recorded. The C_t values of the eight candidate reference genes under different conditions were analyzed by four methods of ΔC_t, GeNorm, NormFinder and BestKeeper, finally. The most stable reference genes were chosen by RelFinder. The value of V_n/(n+1) was calculated by GeNorm to determine the most suitable number of reference genes.[Result] The ΔC_t showed that the most stable reference gene was RPL12 at different development stages, the most stable reference gene was RPL12 under different temperature stresses, and the most stable reference gene was RPS16 in different tissues of larva. The results obtained by GeNorm analysis were similar to those obtained by the C_t analysis. NormFinder showed that RPL18a and EF1α were the most stable reference genes at different developmental stages and under different temperatures, respectively. ACT was the most table reference gene in different tissues of larva. According to BestKeeper analysis, ACT and GAPDH were not suitable as reference genes at different development stages, ACT, RPL18a, RPL12, RPS16 and EF1β were not suitable as reference genes in different temperature treatments, and RPL18a, EF1α, EF1β, and 18S rRNA were not suitable as reference genes in different tissues of larva. At last, RelFinder showed that the most stable reference gene combination was RPL12 and EF1β, while the most unstable reference gene was ACT at different development stages; the most stable reference gene combination was EF1, GAPDH, while ACT was the most unstable reference gene in different temperature conditions; the most stable reference gene combination was ACT and RPS16, while the most unstable candidate reference gene was RPL18a. The most suitable number of reference genes should be 2 by GeNorm.[Conclusion] In H. cunea, three pairs of genes, RPL12 and EF1β, EF1α and GAPDH, and ACT and RPS16, are recommended as reference genes in different developmental stages, different temperature stresses and different tissues of larvae.

Key words: reference genes, gene screening, qRT-PCR