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黑腹果蝇GABA受体GRD亚基和LCCH3 亚基的克隆、表达与鉴定
引用本文:张晓敏,马彩霞,任天瑞,王丰.黑腹果蝇GABA受体GRD亚基和LCCH3 亚基的克隆、表达与鉴定[J].昆虫学报,2012,55(3):259-266.
作者姓名:张晓敏  马彩霞  任天瑞  王丰
作者单位:上海师范大学生命与环境科学学院, 资源化学教育部重点实验室, 上海 200234
基金项目:国家自然科学基金项目,上海市教委基金项目
摘    要:昆虫GABA受体(γ-aminobutyric acid receptor, GABAR)是杀虫剂的重要靶标之一。本研究以黑腹果蝇Drosophila melanogaster整体组织的cDNA作为模板, 采用RT-PCR技术扩增了黑腹果蝇GABA受体LCCH3亚基和GRD亚基的cDNA序列, 并克隆至pET-32a表达载体上, 测序结果表明获得的序列与基因库中已发表的序列一致性在99%以上, 无移码突变。在IPTG的诱导下, LCCH3基因成功在大肠杆菌Escherichia coli中表达, 而GRD基因未表达。通过包涵体洗涤、变性、Ni2+亲合层析纯化、稀释复性获得纯化的重组表达的LCCH3蛋白, 并用圆二色谱测定了目标蛋白的二级结构, 主要富含β结构。该研究结果为研究昆虫GABAR的结构和功能关系提供了重要的参考数据。

关 键 词:黑腹果蝇    GABA受体    GRD亚基    LCCH3亚基    基因克隆    表达    鉴定  

Cloning,expression and identification of GRD and LCCH3 subunits of GABA receptor in Drosophila melanogaster
ZHANG Xiao-Min,MA Cai-Xia,REN Tian-Rui,WANG Feng.Cloning,expression and identification of GRD and LCCH3 subunits of GABA receptor in Drosophila melanogaster[J].Acta Entomologica Sinica,2012,55(3):259-266.
Authors:ZHANG Xiao-Min  MA Cai-Xia  REN Tian-Rui  WANG Feng
Institution:The Key Laboratory of Resource Chemistry of Ministry of Education, College of Life and Environment Sciences, Shanghai Normal University, Shanghai 200234, China
Abstract:γ-Aminobutyric acid receptor(GABAR) is one of the most important insecticide targets.In this study,the fragments of genes of GRD and LCCH3 subunits in Drosophila melanogaster were amplified by RT-PCR and cloned into pET-32a expression vector.Homologous analysis showed that they have more than 99% identity with the published amino acid sequences of the homologous genes in GenBank,without frameshift mutation.The recombinant plasmids were transformed into Escherichia coli and expressed under IPTG induction.LCCH3 gene was expressed successfully,but GRD gene failed.The expressed LCCH3 subunit was purified by the processes of inclusion body washing,denaturation,Ni2+ affinity chromatography and renaturation.The secondary structure of LCCH3 subunit was determined by circular dichroism(CD) spectroscopy,the result showed that this subunit was rich in β-strand.The results provide important information for the study of relationship between the structure and function of GABA receptor.
Keywords:Drosophila melanogaster  GABA receptor  GRD subunit  LCCH3 subunit  gene cloning  expression  identification
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