关键词: 沙葱萤叶甲, 海藻糖酶, 基因克隆, 夏滞育, RT-qPCR

Abstract:  【Aim】 Trehalase (Tre), a key enzyme in trehalose metabolism of insects, plays important roles in the energy regulation and development of insects. This study aims to clone a trehalase gene from Galeruca daurica, and to analyze its expression patterns in order to investigate the functions of trehalase in the development of G. daleruca and summer diapause of its adults. 【Methods】 Based on the transcriptome data of G. daleruca, the full-length cDNA of the Tre gene was cloned using the rapid amplification of cDNA ends (RACE)-PCR and analyzed by bioinformatics. Real-time quantitative PCR (RT-qPCR) was performed to detect the expression levels of the Tre gene in different developmental stages (egg, 1st-3rd instar larva, prepupa, pupa, and 3, 7, 10, 15, 25, 40, 60, 80 and 100 day-old adult) and various adult tissues (head, thorax and abdomen) of G. daleruca, and the 3 day-old adults under different stress temperature (15, 20, 25, 30, 35 and 40℃). The Tre activity in different day-old adults was assayed by the 3, 5-dinitrosalicylic acid colorimetry. 【Results】 A soluble trehalase gene was cloned from G. daleruca, and named GdTre1 (GenBank accession no.: KY697913). Its fulllength cDNA is 1 933 bp containing a 1 704 bp open reading frame (ORF) that encodes 567 amino acids with the predicted molecular weight of 66.56 kD and pI of 6.62. The coded protein contains typical functional domains in the trehalase superfamily with a signal peptide but without transmembrane domain. Homology and phylogenetic analyses showed that GdTre1 has the highest amino acid sequence identity (70.25%) with Leptinotarsa decemlineata Tre1b. RT-qPCR results revealed that GdTre1 was expressed throughout various developmental stages of G. daleruca, with higher expression levels in the egg and diapause adult, and lower expression levels in the larva, pre-pupa, pupa and pre-diapause adult. Tissue expression profiles revealed that the expression level of GdTre1 was the highest in the abdomen, second high in the thorax and the lowest in the head of adults. The expression level of GdTre1 in 3 day-old adults increased as the stress temperature increased, with the highest level at 30℃, and afterwards decreased a little with the temperature increasing. The expression levels of GdTre1 and the Tre activity were significantly different among different day-old adults and showed the same change trend. 【Conclusion】 Trehalase has a close relationship with the development and summer diapause in G. daleruca. The results provide a necessary foundation for further investigation on the molecular mechanisms of summer diapause in this insect.

Key words: Galeruca daurica, trehalase, gene cloning, summer diapause, RT-qPCR