梨小食心虫普通气味结合蛋白2(GmolGOBP2)cDNA的克隆与原核表达  

Cloning and prokaryotic expression of general odorant binding protein 2 cDNA from oriental fruit moth,Grapholita molesta

作　　者：张国辉[1,2] 仵均祥[1] 

机构地区：[1]西北农林科技大学植物保护学院植保资源与病虫害治理教育部重点实验室,应用昆虫学重点实验室,陕西杨凌712100 [2]长江大学农学院昆虫研究所,湿地生态与农业利用教育部工程研究中心,湖北荆州434025

出　　处：《西北农林科技大学学报（自然科学版）》2016年第6期111-115,124,共6页Journal of Northwest A&F University(Natural Science Edition)

基　　金：国家自然科学基金项目(31501641,31272043);农业部公益性行业(农业)科研专项(201103024)

摘　　要：【目的】克隆梨小食心虫(Grapholita molesta)普通气味结合蛋白2(GmolGOBP2)的全长cDNA序列并进行原核表达,为研究该蛋白在梨小食心虫化学感受系统中的作用奠定基础。【方法】利用RT-PCR和RACE技术克隆GmolGOBP2的全长cDNA序列,并使用原核表达载体pET-32a在大肠杆菌BL21(DE3)中进行表达,用SDSPAGE和Western blot检测其表达情况。【结果】GmolGOBP2的cDNA全长序列为637bp(GenBank登录号:JN857940),开放性阅读框长度为483bp,编码161个氨基酸残基,成熟蛋白分子质量为15.98ku,等电点为4.85。GmolGOBP2预测蛋白的N末端具20个氨基酸残基组成的信号肽序列,并且氨基酸序列中具有6个保守半胱氨酸的典型气味结合蛋白家族标志。将GmolGOBP2编码序列重组到表达载体pET-32a中,转入大肠杆菌BL21(DE3)进行原核表达,SDS-PAGE和Western blot检测结果显示,梨小食心虫普通气味结合蛋白基因在大肠杆菌中成功地表达出分子质量约为32ku的融合蛋白,与预测的融合蛋白分子质量大小一致。【结论】克隆并原核表达了GmolGOBP2的cDNA序列,可用于研究该蛋白分子结构及其在化学感受系统中的功能。[Objective] Cloning and prokaryotic expression of a novel cDNA encoding the general odor- ant binding protein 2 （GOBP2） from the oriental fruit moth Grapholita molesta was conducted. [Method] The full-length cDNA encoding GOBP2 isolated from GraphoLita rnolesta by reverse transcription-polymer- ase chain reaction （RT-PCR） and rapid amplification of cDNA ends-PCR （RACE-PCR） was named as GmolGOBP2. It was then constructed into the expression vector pET-32a and expressed in Escherichia coli BL21 （DE3）. [Result] The full length cDNA of GmolGOBP2 was 637 bp （GenBank accession no. JN857940） ,containing a 483 bp open reading frame with 161 amino acids. The deduced molecular weight （MW） was 15. 98 ku and the PI was 4.85. Protein signature analysis indicated that the deduced Gmol- GOBP2 contained an N-terminal signal sequence of 20 amino acids. The GmolGOBP2 was then constructed into the expression vector pET-32a and expressed in Escherichia coli BL21 （DE3） after induction with IPTG. SDS-PAGE and Western blot analysis showed the molecular weight of the recombinant GmolGOBP2 was about 32 ku,in consistence with the predicted result. [Conclusion] GmolGOBP2 was cloned and ex- pressed in prokaryotic expression system,which was helpful for further studies on its molecular structure and function in the olfactory system.

关 键 词：梨小食心虫 普通气味结合蛋白2 分子克隆 原核表达 

分 类 号：S433.4[农业科学—农业昆虫与害虫防治] Q966[农业科学—植物保护]