关键词:  葱蝇, 滞育, 海藻糖-6-磷酸合成酶, 克隆, 序列分析, 半定量RT-PCR

Abstract:  Trehalose-6-phosphate synthase (TPS) is a key enzyme in trehalose synthesis pathway of insects. This research aims to clarify the important significance of TPS in the process of energy synthesis and against high and low temperature environment, and further understand the molecular mechanism of diapauses in the onion maggot, Delia antiqua, through the cloning, bioinformatic analysis and diapauserelated expression profiling of TPS. Based on an EST fragment from the suppression subtractive hybridization library of D. antiqua, TPS gene specific primers were designed, and its cDNA was cloned by RACE method and designated as DaTPS1 (GenBank accession no. JX681124). The full-length cDNA of DaTPS1 is 2 904 bp in length with an opening reading frame (ORF) of 2 488 bp encoding 815 amino acids. The calculated molecular weight of the predicted protein DaTPS1 is 91.2 kDa, with an estimated pI 5.96. Homology searching showed that DaTPS1 has two conserved domains, and shares the highest amino acid sequence identity (92.1%) with TPS1 from Drosophila melanogaster. The predicted three dimensional structure of DaTPS1 consists of 15 big alpha helices and 11 anti-parallel β-strands. RT-PCR analysis showed that DaTPS1 was expressed in non-, summer- and winter-diapausing pupae. The expression level of DaTPS1 in different developmental stages of non-diapausing pupae was relatively stable. However, its expression was lifted in the prediapause stage, declined in the diapause stage, and then up-regulated in the post-diapause stage of both summer- and winter-diapausing pupae. The increased trehalose synthesis trigged by TPS1 in the prediapause stage of summer- and winter-diapausing pupae suggests an increase in resistance to environmental stresses during diapause development, and that the declined metabolism in the diapause stage results in the decrease in energy need so the expression of TPS1 is down-regulated in the stage. However, the growth and development at the postdiapause stage recover gradually and the energy need increases, so the expression of TPS1 is up-regulated again. The study has significance in understanding the role of TPS of insects in trehalose synthesis pathway and the molecular mechanism of insect diapause.

Key words: Delia antiqua, diapause, trehalose-6-phosphate synthase, cloning, bioinformatic analysis, semi-quantitative RT-PCR