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。
Carboxylesterases （CarE） are a family of multifunctional enzymes widely distributed among organism, and distinctly related to insecticide resistance. In this present study the physiological functions of the carboxylesterase member LmCarE25 was investigated from Locusta migratoria（Meyen）. Real-time PCR rresults showed that the LmCarE25 was expressed in all of the adult tissues, while had lower expression levels in gastric cecum, wings and muscles, and higher expression levels in midgut, Malpighian tubules and fat body. In addition, colibacillus and insect cell expression system were used to express this enzyme in vitro. The recombinant LmCarE25 was successfully expressed by recombinant vector pET32a-LmCarE25 in E. coli BL21（DE3） and JM109, and its molecular weight was approximately 60 kDa, although it was inclusion bodies. Then LmCarE25 was subcloned into eukaryotic expression vector pFastBacHTA, the recombinant bacmid DNA was obtained by Bac-to-Bac？ system, and was transfected into sf9 cells by lipid mediated transfection. Western-blot results suggested LmCarE25 expressed in soluble form at low level. These results might provide information for further study the function of carboxylesterase family of Locusta migratoria.
出处
《应用昆虫学报》
CAS
CSCD
北大核心
2013年第3期659-665,共7页
Chinese Journal of Applied Entomology
基金
国家自然科学基金项目(31101463)
高等学校博士学科点专项科研基金(20111401110006
20111401120009)
公益性行业(农业)科研专项(200903021)
山西省自然科学基金(2011011033-1)
山西省青年科技基金(2012021027-1)
关键词
飞蝗
羧酸酯酶
real—time
PCR
原核表达
真核表达
Locusta migratoria
carboxylesterase
real-time PCR
prokaryotic expression
eukaryotic expression
