【摘要】 羧酸酯酶(Carboxylesterase,CarE)是一类在生物中广泛分布的多功能家族酶系,在昆虫抗药性形成机制中发挥重要作用。为探讨飞蝗Locusta migratoria(Meyen)羧酸酯酶基因LmCarE25的生物学功能,本文采用荧光实时定量PCR技术进行研究,发现其在飞蝗成虫各组织部位均有表达,其中胃盲囊、翅和肌肉中表达量较低,中肠、马氏管和脂肪体表达量较高。本文尝试在大肠杆菌和昆虫细胞体外重组表达该酶,构建重组表达载体pET32a-LmCarE25,SDS-PAGE结果显示,LmCarE25可在大肠杆菌BL21(DE3)和JM109中表达,蛋白分子量约为60 ku,但为包涵体;将LmCarE25基因插入真核表达载体pFastBacHTA中,借助Bac-to-Bac体系获得重组Bacmid,以sf9细胞系作为宿主细胞表达目的蛋白,Western-Blot检测结果显示,LmCarE25获得可溶性表达。上述结果为进一步深入研究飞蝗羧酸酯酶家族功能提供了基础资料。更多还原

【Abstract】 Carboxylesterases( CarE) are a family of widely distributed,multifunctional enzymes that are distinctly implicated in insecticide resistance. The physiological functions of the carboxylesterase member LmCarE25 was investigated in Locusta migratoria( Meyen). Real-time PCR results show that LmCarE25 was expressed in all adult tissues,with lower levels in the gastric caecum,wings and muscles,and higher expression in the midgut,Malpighian tubules and fat body. In addition,colibacillus and insect cell expression were used to express this enzyme in vitro. Recombinant LmCarE25 was successfully expressed by the recombinant vector pET32a-LmCarE25 in Escherichia coli BL21( DE3) and JM109 with a molecular weight of approximately 60 ku,although it was in the form of inclusion bodies. LmCarE25 was then subcloned into the eukaryotic expression vector pFastBacHTA. The recombinant bacmid DNA was obtained using the Bac-to-Bac system,and was transfected into sf9 cells by lipid mediated transfection. Western-blot results suggested that LmCarE25 was expressed in soluble form at low levels. These results could provide the basis for further study of the function of the carboxylesterase family in L. migratoria.更多还原