朱砂叶螨硒代谢通路基因筛选及硒磷酸合成酶 TcSPS1的原核表达和特性分析

【目的】本研究旨在筛选朱砂叶螨Tetranychus cinnabarinus硒代谢通路的关键基因，探究其在朱砂叶螨硒代谢中的功能。【方法】使用基因克隆技术对8条朱砂叶螨硒代谢通路基因进行克隆；利用qPCR技术检测这些基因在不同浓度(0, 5, 20和50 μmol/L)亚硒酸钠(Na₂SeO₃)处理的豇豆苗上饲养3 d的朱砂叶螨品系(短期硒饲养品系)雌成虫间以及朱砂叶螨普通品系(豇豆苗饲养)和长期硒饲养品系(20 μmol/L Na₂SeO₃处理的豇豆苗饲养超1年)雌成虫间的表达量差异，并分析它们对硒诱导的表达响应；原核表达差异表达基因TcSPS1，并测定获得的重组蛋白生化特性。【结果】成功克隆了朱砂叶螨硒代谢通路的8条基因TcTxnrd1, TcTxnrd2, TcSPS1, TcSPS2, TcSPS2-1, TcSPS2-2, TcSG和TcPSTK。qPCR结果表明TcSPS1和TcTxnrd2在短期硒饲养和长期硒饲养朱砂叶螨品系中均上调表达，且TcSPS1的表达量变化与硒浓度变化密切相关。原核表达系统成功获得TcSPS1可溶性重组蛋白，测得其重组蛋白比活力为2.366±0.046 nmol/mg pro·min，K_m和V_max值分别为10.054±0.062 μmol/L和29.633±1.777 nmol/mg pro·min。【结论】通过硒代谢通路基因分析，初步解释了朱砂叶螨对硒的适应的分子机制，并证明了上调表达的TcSPS1基因在朱砂叶螨产生对硒的适应中起着重要的功能。

Screening of selenium metabolism pathway genes in Tetranychus cinnabarinus (Acari: Tetranychidae) and the prokaryotic expression and characterization of selenophosphate synthetase TcSPS1

【Aim】 This study aims to screen the key genes in the selenium metabolism pathway of Tetranychus cinnabarinus and to explore their functions in selenium metabolism in T. cinnabarinus. 【Methods】 The gene cloning technology was used to clone eight genes in the selenium metabolism pathway of T. cinnabarinus. The differences in the expression levels of these genes in female adults of the short-term selenium-reared strain of T. cinnabarinus with different concentrations of Na₂SeO₃ (fed with cowpea seedlings treated with 0, 5, 20 and 50 μmol/L Na₂SeO₃ for 3 d), and in female adults of the normal strain (fed with cowpea seedlings) and the long-term selenium-reared strain (fed with cowpea seedlings treated with 20 μmol/L Na₂SeO₃ for over one year) were detected by qPCR, and the expression responses of these genes to selenium induction were analyzed. Finally, the prokaryotic expression system was used to express one differentially expressed gene TcSPS1, and the biochemical characteristics of the obtained recombinant protein were assayed. 【Results】 Eight selenium metabolism pathway genes of T. cinnabarinus were successfully cloned, including TcTxnrd1, TcTxnrd2, TcSPS1, TcSPS2, TcSPS2-1, TcSPS2-2, TcSG, and TcPSTK. qPCR results showed that only TcSPS1 and TcTxnrd2 were up-regulated in both the short-term selenium-reared strain and long-term selenium-reared strain of T. cinnabarinus. Among them, the expression level of TcSPS1 was closely dependent on the selenium concentration. The soluble recombinant protein of TcSPS1 was successfully obtained using the prokaryotic expression system. The specific activity and the Km and Vmaxvalues of the recombinant TcSPS1 were measured to be 2.366±0.046 nmol/mg pro·min, 10.054±0.062 μmol/L and 29.633±1.777 nmol/mg pro·min, respectively. 【Conclusion】 Through the analysis of selenium metabolism pathway genes, the molecular mechanism of the adaptation of T. cinnabarinus to selenium was preliminarily explained, and it was proved that the up-regulated expression of the TcSPS1 gene plays an important function in the adaptation of T. cinnabarinus to selenium.