关键词: 沙葱萤叶甲, 海藻糖合成酶, 基因克隆, 温度胁迫, 实时荧光定量PCR

Abstract: 【Aim】 Trehalose-6-phosphate synthase (TPS) is one of key enzymes in the trehalose synthesis pathway. This study aims to clone the TPS gene from Galeruca daurica and to analyze its expression levels in response to temperature stress so as to investigate the molecular regulation mechanisms of TPS in temperature tolerance in G. daurica. 【Methods】 The full-length cDNA of the TPS gene was cloned from G. daurica by rapid amplification of cDNA ends (RACE), and its sequence was subjected to bioinformatics analysis. The expression profiles of the TPS gene in the 2nd instar larvae of G. daurica at different temperatures were detected by real-time quantitative PCR. 【Results】 A TPS gene was cloned from G. daurica, and named GdTPS (GenBank accession no.: KY460114), which is 2 706 bp in length with an open reading frame (ORF) of 2 496 bp, encoding a protein of 831 amino acids with the predicted molecular weight of 94.05 kD and pI of 6.82. The encoded protein contains three potential N-glycosylation sites, without signal peptide and transmembrane domain. GdTPS has two conserved domains, shares higher amino acid sequence identity with TPSs of other insect species and has the highest amino acid sequence identity (88%) with TPS from Leptinotarsa decemlineata. Real-time quantitative PCR results showed that when the stress temperature was lower than 25℃ (the control), the expression level of GdTPS increased as the temperature decreased, and reached the peak at -10℃. However, when the stress temperature was higher than 25℃, its expression level increased as the temperature increased, and reached the peak at 40℃.【Conclusion】 The expression of GdTPS in G. daurica larvae is up-regulated significantly in response to low and high temperaturestresses. The results provide a foundation for the functional research of TPS in the regulation of temperature stress in insects.

Key words: Galeruca daurica, trehalose-6-phosphate synthase, gene cloning, temperature stress, real-time quantitative PCR