中国农业科学 ›› 2019, Vol. 52 ›› Issue (15): 2616-2623.doi: 10.3864/j.issn.0578-1752.2019.15.006

• 植物保护 • 上一篇    下一篇



  1. 1 华中农业大学植物科学技术学院,武汉 430070
    2 北京市农林科学院植物保护环境保护研究所北方果树病虫害绿色防控北京市重点实验室, 北京100097
  • 收稿日期:2019-04-02 接受日期:2019-04-29 出版日期:2019-08-01 发布日期:2019-08-06
  • 通讯作者: 牛长缨,李峰奇
  • 作者简介:李都,
  • 基金资助:

Binding Characterization of Odorant Binding Protein OBP56h in Drosophila suzukii with Small Molecular Compounds

LI Du1,2,NIU ChangYing1(),LI FengQi2(),LUO Chen2   

  1. 1 College of Plant Science & Technology, Huazhong Agricultural University, Wuhan 430070;
    2 Beijing Key Laboratory of Environment Friendly Management on Fruit Diseases and Pests in North China, Institute of Plant and Environment Protection, Beijing Academy of Agriculture and Forestry Sciences, Beijing 100097
  • Received:2019-04-02 Accepted:2019-04-29 Online:2019-08-01 Published:2019-08-06
  • Contact: ChangYing NIU,FengQi LI


【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2 μmol·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′-disulfonic acid 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 -1IPTG、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0.9568 μmol·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17.93 μmol·L -1,柚皮素与DsuzOBP56h的解离常数为25.32 μmol·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,其解离常数为31.37 μmol·L -1。【结论】斑翅果蝇气味结合蛋白OBP56h能与测试的多种植物苦味物质和挥发性气味物质结合,表明DsuzOBP56h很有可能参与斑翅果蝇对食物味觉和嗅觉的识别行为,研究结果可为理解斑翅果蝇的取食行为提供理论依据,并为开展斑翅果蝇的生态防控提供新思路。

关键词: 斑翅果蝇, 气味结合蛋白, 原核表达, 竞争结合


【Objective】The objective of this study is to clone the odorant binding protein 56h (OBP56h) gene from Drosophila suzukii, get the recombinant DsuzOBP56h protein, and characterize the binding profiles of DsuzOBP56h 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 DsuzOBP56h. The sequences of insect OBPs with high similarity were downloaded from NCBI database for sequence alignment and analysis. Using NdeⅠand XhoⅠas restriction sites, OBP56h was ligated into pET-30a (+) prokaryotic expression vector, and transformed into Escherichia coli BL21 (DE3). IPTG was applied to induce the expression of recombinant DsuzOBP56h 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 2 μmol·L -1, and the ligand was diluted with chromatography-grade methanol to a final concentration of 1 mmol·L -1. The binding characterization of DsuzOBP56h with 18 small molecular compounds was investigated using bis-ANS as fluorescence probe. 【Result】 The full-length ORF of OBP56h 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 OBP56h. OBP56h was successfully inserted into pET-30a (+) 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 0.9568 μmol·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 berberine chloride and coumarin with DsuzOBP56h was strong, and the dissociation constant is 12.16 and 17.93 μmol·L -1, respectively. The dissociation constant of naringenin with DsuzOBP56h is 25.32 μmol·L -1. A volatile odorant β-cyclocitral, which is attractive to D. suzukii produced by strawberry leaves, can also bind to DsuzOBP56h, with the dissociation constant of 31.37 μmol·L -1.【Conclusion】The OBP56h in D. suzukii can bind with a variety of bitter tastants and volatile odors from plants, indicating that OBP56h may be involved in the gustatory and olfactory recognition of food in D. suzukii. The results can provide a theoretical basis for understanding the feeding behavior of D. suzukii, and provide a new idea for the ecological prevention and control of D. suzukii.

Key words: Drosophila suzukii, odorant-binding protein, prokaryotic expression, competitive binding