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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。
Carboxylesterase (CarE) is a type of multifunctional family of enzymes widely distributed in organisms and plays an important role in the mechanism of insect resistance. In order to investigate the biological function of the carboxylesterase gene LmCarE25 of locusta migratoria (Meyen), this study was carried out by real-time fluorescence quantitative PCR and found that it was expressed in all tissues of Locusta migratoria, Low expression in muscle, midgut, Markov tube and fat body expression is higher. The recombinant plasmid pET32a-LmCarE25 was constructed in E. coli and insect cells in vitro. SDS-PAGE analysis showed that LmCarE25 was expressed in E. coli BL21 (DE3) and JM109. The molecular weight of LmCarE25 was about 60 ku As the inclusion body. The LmCarE25 gene was inserted into the eukaryotic expression vector pFastBacHTA. The recombinant Bacmid was obtained by Bac-to-Bac system and the sf9 cell line as the host cell. Western-Blot showed that LmCarE25 was soluble. The above results provide the basic information for further study on the function of carboxylesterase family in Locusta.