家蚕羧酸酯酶基因Bmae35的克隆、序列分析及表达  

Cloning,sequence analysis and expression of carboxylesterase gene Bmae35 in the silkworm,Bombyx mori

作　　者：杨微[1] 齐登伟[2] 余泉友[1] 张泽[1,2] 

机构地区：[1]重庆大学农学及生命科学研究院,重庆400044 [2]西南大学农业部蚕桑学重点开放实验室,重庆400715

出　　处：《昆虫学报》2011年第6期634-641,共8页Acta Entomologica Sinica

基　　金：中央高校基本科研业务费项目(CDJZR10290002);国家高技术研究发展计划项目(2006AA10A117)

摘　　要：昆虫羧酸酯酶是一类能对外源化合物解毒和气味分子降解的重要酶系。本研究选取在家蚕Bombyx mori幼虫嗅觉感器中有表达,并与蛀茎夜蛾Sesamia nonagrioides触角酯酶基因Snon-EST可能为直系同源基因的Bmae35进行克隆和外源表达研究。结果表明:该基因编码区长1581bp,共编码526个氨基酸。与其他昆虫触角酯酶的多序列比对分析发现,Bmae35编码的蛋白具有酯酶活性必须的催化残基Ser191,Glu313和His429,也保持着α-酯酶家族特征基序Gly-x-Ser-x-Gly。RT-PCR分析显示,Bmae35在家蚕5龄第3天各组织中均有表达,其中在头、脂肪体、马氏管、体壁和丝腺中的表达量较高。Bmae35在雌蛾性信息腺中表达,并与性信息素合成呈正相关,暗示其在信息素合成中起重要作用。构建Bmae35与pET28(a)重组载体,经异丙基-β-D-硫代半乳糖苷诱导,电泳检测发现该基因以包涵体形式表达,以镍亲和层析柱纯化,Western blotting鉴定证实Bmae35在大肠杆菌Escherichiacoli中正确表达并得以纯化。本实验通过对家蚕Bmae35基因的克隆、原核表达与纯化,为进一步深入研究其表达定位和气味降解等功能奠定基础。Insect carboxylesterases are important enzymes involved in xenobiotic metabolism and degradation of odorants.In the present study,Bmae35 gene,which is expressed in larval olfactory tissues of the silkworm and the putative ortholog of antennal esterase Snon-EST in Sesamia nonagrioides,was cloned and exogenously expressed.Bmae35 comprises a complete coding sequence of 1 581 bp and codes 526 amino acids.The multiple sequence alignment of Bmae35 with other esterases revealed that Bmae35 has the structure characteristics of insect esterases,including the catalytic triad （Ser191,Glu313 and His429） and the conserved pentapeptide Gly-x-Ser-x-Gly in the α-esterase family.Based on the semi-quantitative RT-PCR analysis,the expression pattern of Bmae35 gene showed that it was highly expressed in head,fat body,Malpighian tubules,integument,and silk glands in day-3 5th instar larvae.In addition,the expression of Bmae35 showed a positive correlation with sex pheromone content.The results suggest that Bmae35 might play important roles in pheromone synthesis.The Bmae35 gene was sub-cloned into the expression vector pET28（a） and the recombinant protein was obtained by isopropyl β-D-1-thiogalactopyranoside （IPTG） induction.Electrophoresis analysis showed that Bmae35 was expressed in Escherichia coli as inclusion body.The recombinant protein Bmae35 was purified by immobilized Ni2＋ absorption chromatograph column.Western blotting analysis indicated that Bmae35 was correctly expressed in E.coli and purified.The results of this study provide useful data for further understanding the odorant detoxification and expression orientation.

关 键 词：家蚕 羧酸酯酶 基因克隆 表达模式 原核表达 蛋白免疫印迹 

分 类 号：Q966[生物学—昆虫学]