葱蝇海藻糖-6-磷酸合成酶基因的克隆、序列分析及滞育相关表达

Cloning, bioinformatic analysis and diapause-related expression of trehalose-6-phosphate synthase gene from the onion maggot, Delia antiqua (Diptera: Anthomyiidae)

海藻糖-6-磷酸合成酶(trehalose-6-phosphate synthase,TPS)是昆虫海藻糖合成途径中的关键酶之一。本研究通过对葱蝇Delia antiqua海藻糖-6-磷酸合成酶基因的克隆、序列分析及滞育相关表达的分析,旨在证明该基因在能源合成以及抵御高温和低温环境方面6发挥重要作用,为进一步弄清葱蝇滞育分子机制提供理论依据。根据葱蝇抑制消减杂交文库中的EST序列信息,设计特异性引物,并通过RACE技术克隆了葱蝇海藻糖-6-磷酸合成酶基因全长cDNA,命名为DaTPS1(GenBank登录号:JX681124),其全长为2904bp,开放阅读框2448bp,编码815个氨基酸,推测其相对分子质量为91.2kD,等电点为5.96。生物信息学分析表明,该基因编码的氨基酸序列具有两个保守结构域,与其他物种TPS具有较高的同源性,其中和黑腹果蝇Drosophila melanogaster亲缘关系最近,氨基酸序列一致性为92.1%;其蛋白质三维结构有15条大的α螺旋和11股反向平行的β链折叠。RT-PCR分析表明,DaTPS1在葱蝇非滞育、夏滞育和冬滞育期蛹中都有表达,但是非滞育期各时期表达量基本没有变化,而在夏滞育和冬滞育蛹的滞育前期表达量较高,滞育保持期表达量较低,滞育期后期表达量又有所升高。推断在葱蝇蛹夏滞育和冬滞育期前期,TPS1开始催化合成较多的海藻糖以提高滞育期抵御不良环境的能力,滞育保持期蛹的新陈代谢降低,所需能量较少,所以TPS1处于低水平表达状态,而滞育期结束后,蛹生长发育逐渐恢复,所需能量有所增加,TPS1的表达量再次升高。本研究对揭示昆虫TPS在能量代谢通路中的作用及昆虫滞育的分子机理具有一定的科学意义。

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.