摘要
【目的】克隆斑翅果蝇(Drosophila suzukii)气味结合蛋白56h(odorant binding protein 56h,OBP56h)基因,诱导表达斑翅果蝇OBP56h重组蛋白(DsuzOBP56h),研究其与小分子化合物的结合特性。【方法】通过RT-PCR并设计特异性引物克隆斑翅果蝇OBP56h ORF全长,从NCBI数据库中下载相似度高的昆虫气味结合蛋白序列,进行序列比对和分析。以NdeⅠ和XhoⅠ为酶切位点,将OBP56h连入pET-30a(+)原核表达载体,将重组质粒转入BL21(DE3)大肠杆菌感受态细胞。扩大培养阳性菌株,并用IPTG诱导表达DsuzOBP56h重组蛋白。收集菌液,通过超声波破碎细胞得到蛋白,利用Ni-NTA柱纯化蛋白,进行Tris-HCl透析,用BCA法测定蛋白浓度。蛋白用50 mmol·L-1 Tris-HCl(pH 7.4)稀释至终浓度2μmol·L-1,配基用色谱级甲醇稀释至终浓度1 mmol·L-1,以4,4′-二苯胺基-1,1′-联萘-5,5′-二磺酸二钾盐(4,4′-dianilino-1,1′-binaphthyl-5,5′-disulfonicacid dipotassium salt,bis-ANS)荧光探针为报告子,利用荧光竞争结合试验检测DsuzOBP56h蛋白与18种候选小分子化合物配基的结合特性。【结果】克隆获得了斑翅果蝇OBP56h的ORF全长,共405 bp,N-端含有19个氨基酸组成的信号肽,具有6个保守半胱氨酸位点,符合OBP的典型特征,与其同属的黑腹果蝇OBP56h进化关系最近。成功连入pET-30a(+)表达载体,在1 mmol·L-1 IPTG、28℃条件下诱导DsuzOBP56h蛋白表达,并过柱纯化得到目的蛋白。荧光光谱试验显示,荧光探针bis-ANS与DsuzOBP56h的解离常数为0.9568μmol·L-1,适合作为本试验中竞争性荧光结合试验的报告子;进一步的荧光竞争结合试验表明,在18种候选配基中,苦味物质盐酸小蘖碱和香豆素与DsuzOBP56h的结合亲和性较强,解离常数分别为12.16和17.93μmol·L-1,柚皮素与DsuzOBP56h的解离常数为25.32μmol·L-1,草莓叶片产生的一种对斑翅果蝇具有吸引作用的挥发性气味物质β-环柠檬醛也能与DsuzOBP56h结合,其解离常数�
【Objective】The objective of this study is to clone the odorant binding protein 56 h(OBP56 h)gene from Drosophila suzukii,get the recombinant DsuzOBP56 h protein,and characterize the binding profiles of DsuzOBP56 h with some small molecular compounds.【Method】By means of specific primer,reverse transcription PCR(RT-PCR)was used to clone the full-length ORF of Dsuz OBP56 h.The sequences of insect OBPs with high similarity were downloaded from NCBI database for sequence alignment and analysis.Using NdeⅠand XhoⅠas restriction sites,OBP56 h was ligated into pET-30 a(+)prokaryotic expression vector,and transformed into Escherichia coli BL21(DE3).IPTG was applied to induce the expression of recombinant Dsuz OBP56 h protein.The bacterial solution was collected and the protein was obtained through breaking cells by ultrasound,and then the protein was purified by the method of Ni-NTA resin.The purified protein was dialyzed by Tris-HCl and the concentration was determined by the method of BCA.The protein was diluted with 50 mmol·L-1 Tris-HCl(pH 7.4)to a final concentration of 2μmol·L-1,and the ligand was diluted with chromatography-grade methanol to a final concentration of 1 mmol·L-1.The binding characterization of DsuzOBP56 h with 18 small molecular compounds was investigated using bis-ANS as fluorescence probe.【Result】The full-length ORF of OBP56 h in D.suzukii was amplified,which is 405 bp in total,including 19 amino acids of signal peptide in the N-terminal.It has 6 conserved cysteine sites,which is consistent with the typical characteristics of OBPs,and has the closest evolutionary relationship with D.melanogaster OBP56 h.OBP56 h was successfully inserted into pET-30 a(+)and expressed at 1 mmol·L-1 IPTG and 28℃,then purified by the method of Ni-NTA resin.In the competitive fluorescence assay,the dissociation constant Kbis-ANS was 0.9568μmol·L-1,indicating that bis-ANS is suitable to be a reporter of competitive fluorescence binding assay.Among 18 ligands,the binding affinity of bitter tastants
作者
李都
牛长缨
李峰奇
罗晨
LI Du;NIU ChangYing;LI FengQi;LUO Chen(College of Plant Science&Technology,Huazhong Agricultural University,Wuhan430070;Beijing Key Laboratory ofEnvironment Friendly Management on Fruit Diseases and Pests in North China,Institute of Plant and EnvironmentProtection,Beijing Academy of Agriculture and Forestry Sciences,Beijing100097)
出处
《中国农业科学》
CAS
CSCD
北大核心
2019年第15期2616-2623,共8页
Scientia Agricultura Sinica
基金
国家重点研发计划(2017YFD0200900)
国家自然科学基金(31661143045)
国家公益性行业(农业)科研专项(201503137)
关键词
斑翅果蝇
气味结合蛋白
原核表达
竞争结合
Drosophila suzukii
odorant-binding protein
prokaryotic expression
competitive binding
