Abstract:  【Aim】 Gene expression patterns and enzyme activities of trehalases in Antheraea pernyi pupae during diapause and diapause termination were detected so as to elucidate the relationship between carbohydrate metabolism and diapause termination during diapause of this insect. 【Methods】 The trehalase genes were cloned from A. pernyi pupa using RT-PCR technology and subjected to bioinformatics analysis. Their expression patterns in different tissues of A. pernyi pupae during diapause termination after exposure to long photoperiod (17L∶7D) and diapause (the control group) were analyzed by semi-quantitative RT-PCR. The relative expression levels of trehalase genes in the fat body of A. pernyi pupae during diapause termination under long photoperiod were measured using qPCR, the trehalase activity in the fat body was detected using 3,5-dinitrosalicylic acid method and the trehalose content in the haemolymph was measured using antrone chromametry method. 【Results】 Three trehalase genes were cloned from A. pernyi, named ApTreh1A, ApTreh1B and ApTreh2 and deposited in GenBank under accession numbers KU977455, KU977456 and KU977457, respectively. Their open reading frames (ORFs) are 1 797, 1 635 and 1 932 bp in length, encoding 598, 544 and 643 amino acids, respectively. Homologous sequence alignment and phylogenetic analysis indicated that ApTreh1A and ApTreh1B are soluble trehalases (TrehS), and ApTreh2 is a membrane-bound trehalase (TrehM). Tissue-specific mRNA expression profiling using semi-quantitative RT-PCR showed that ApTreh2 had a wider distribution and higher expression level than ApTreh1. The qPCR results indicated that the expression levels of ApTreh1A and ApTreh1B in fat body at 21 d after exposure to long photoperiod were up-regulated and significantly higher than that of the control group (12L∶12D) (2- and 4.7-fold, respectively), while down-regulated at 28 and 35 d, and then up-regulated again at 42 d. The expression levels of ApTreh2 were up-regulated gradually, and reached the maximum (2.7-fold as high as that of the control group) at 28 d. At 42 d after exposure to long photoperiod, there was another expression peak (2.3-fold as high as that of the control group), and then down-regulated gradually. Trehalase activities in fat body increased gradually, reached the peak (about 18.5 U) at 21 d after exposure to long photoperiod, while declined to the lowest level (11.2 U) at 35 d, and increased slightly at 42 d, showing the same variation trend as the gene expression. The trehalose content in the haemolymph increased under long photoperiod and reached the maximum at 21 d, and was higher than that of the control during the whole developmental stage. 【Conclusion】 The results suggest that the expression of trehalase genes shows the same variation trend as the trehalase activities in the fat body and the trehalose content in the haemolymph in A. pernyi, suggesting that the expression response of trehalase genes plays a significant role in pupal diapause and diapause termination of A. pernyi.

Key words: Antheraea pernyi, diapause, trehalase, trehalose, gene expression, enzyme activity