Cloning And Characteristics Of Trehalose Metabolism-relative Genes, And Their Expression During Diapause In The Onion Maggot, Delia Antiqua

Diapause is a physiological phenomenon of insects into a state of arrested development under adverse environments such as low temperature, high temperature, drought and food shortage, to ensure the survival of species and individual. Delia antiqua is a worldwide liliaceae pest belonging to Delia, Anthomyiidae of Diptera, with winter diapause and summer diapause happening at same pupal developmental stage separately in subject of different induce conditions, so it is an ideal model species for studying the diapause mechanism of insect.Trehalose, as the blood sugar of insects, plays a very important role in diapause and development of insects. In the research, through cloning and characteristics of trehalose-6-phosphate synthase (TPS), trehalose-6-phosphate phosphatase (TPP) and trehalase (TRE) genes, and their expression during diapause in the onion maggot were conducted. This research aims to prove that these three genes play very important roles in the synthesis and degradation of trehalose as well as resistance to high temperature and low temperature environments. The purpose of the research provided with a theoretical basis for further clarifying the molecular mechanism of onion fly diapause. The results are as follows:①Cloning and analysis of TPS1in Delia antiquaBased on an EST fragment from the suppression subtractive hybridization library of Delia antiqua. TPS gene specific primers were designed, and its cDNA (designated as DaTPSl) was cloned by RACE method. The full-length cDNA is2904bp in length with an opening reading frame (ORF) of2488bp encoding815amino acids. The calculated molecular weight is91.2kDa and the estimated pI is5.96. Homology searching showed that DaTPS has four conserved amino acid sequences of TPS family, the catalytic and substrate-binding residues, and two conserved domains, and shares the highest sequence similarity (92.1%) with the TPS1in Drosophila melanogaster. Semi-quantitative PCR and Real-time quantitative PCR analysis showed that it was expressed in non-. summer-and winter-diapausing pupae. The expression through stages in non-diapausing pupae was relatively stable. However, its expression was lifted in pre-diapause stage, declined in maintenance period, and then up-regulated in post-diapause stage of both summer-and winter-diapause pupae. The increased trehalose synthesis trigged by TPS1in the pre-diapause stage of summer-and winter-diapause pupae would suggest an increase resistance to environment stresses during diapause development, and then declined metabolism in diapause maintenance stage would result in decreasing in energy need and so downregulating in the stage. However, development activities through the post-diapause stage recovered gradually and the energy need was increasing, so TPS1was up-regulated again.②Cloning and analysis of TPP in Delia antiquaBased on transcriptome datas of Delia antiqua obtained in our laboratory, TPP gene specific primers were designed, and its cDNA (designated as DaTPP) was cloned by RACE method. The full-length cDNA is1414bp in length with an opening reading frame (ORF) of822bp encoding273amino acids. The calculated molecular weight is30.7kDa and the estimated pI is5.77. Homology searching showed that DaTPP had four conserved amino acid sequences of TPP family, the catalytic and substrate-binding residues, and one conserved domain, and shares the highest sequence similarity (85.0%) with the TPP from Drosophila simulans. Semi-quantitative PCR and Real-time quantitative PCR analysis showed that it was expressed in non-. summer-and winter-diapausing pupae. The expression through stages in non-diapausing pupae was relatively stable. However, its expression was lifted in pre-diapause stage, declined in maintenance period, and then up-regulated in post-diapause stage of both summer-and winter-diapause pupae. TPP with TPS1together conducts the catalytic synthesis of trehalose. so the expression patterns are the very similar.③Cloning and analysis of TRE in Delia antiquaBased on transcriptome datas of Delia antiqua from our laboratory. TRE gene specific primers were designed, and its cDNA (designated as DaTRE) was cloned by RACE method. The full-length cDNA is2082bp in length with an opening reading frame (ORF) of1788bp encoding595amino acids. The calculated molecular weight is68.6kDa and the estimated pI is5.19. Homology searching showed that DaTRE had two tag sequence of membrane-penetrating trehalase. one Gly enrichment region, and two conserved domains, and shares the highest sequence similarity (78.7%) with the TRE from Bactrocera dorsalis. Semi-quantitative PCR and Real-time quantitative PCR analysis showed that it was expressed in non-. summer-and winter-diapausing pupae. The expression through stages in non-diapausing pupae was relatively stable. However, its expression was lifted in pre-diapause stage, declined in maintenance period, and then up-regulated in post-diapause stage of both summer-and winter-diapause pupae.The increased high expression of TRE in the pre-diapause stage of summer-and winter-diapause pupae would suggest producing an excess quantity of trehalose into storage carbohydrates for stress resistance during diapause development, and then declined metabolism in diapause maintenance stage would result in the high levels of trehalose, increasing resistance of the pupa to high and low temperature, so TRE downregulated in the stage. However, development activities through the post-diapause stage recovered gradually and trehalose is degraded into glucose, which is conducive to breaking diapause, restoring growth, so TRE was up-regulated again.