Scientia Agricultura Sinica ›› 2019, Vol. 52 ›› Issue (15): 2616-2623.doi: 10.3864/j.issn.0578-1752.2019.15.006

• PLANT PROTECTION • Previous Articles     Next Articles

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 E-mail:niuchangying88@163.com;pandit@163.com

Abstract:

【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

Fig. 1

Sequences alignment of DsuzOBP56h with other insects OBP56h The 6 conserved cysteines are labeled in black"

Fig. 2

Phylogenetic tree of DsuzOBP56h and other insects OBP"

Fig. 3

Expression and purification of the recombinant DsuzOBP56h protein M:蛋白Marker Protein molecular weight;1:未诱导的pET30a-OBP56h的BL21菌体Bacterial products containing pET30a-OBP56h without induction;2:BL21菌体中DsuzOBP56h蛋白诱导表达Expression products after induction;3:纯化后的OBP56h蛋白Purified recombinant protein OBP56h"

Fig. 4

Binding of DsuzOBP56h and bis-ANS"

Fig. 5

Competitive combining of DsuzOBP56h protein between candidate ligands and bis-ANS"

Table 1

Binding affinities of DsuzOBP56h protein with candidate ligands"

化学配基 Chemical ligand CAS number IC50 (μmol·L-1) 解离常数Ki Dissociation constant (μmol·L-1)
味觉物质 Tastant
苯甲地那铵Denatonium benzoate 3734-33-6
N-苯基硫脲N-Phenylthiourea 103-85-5
盐酸小檗碱Berberine chloride 633-65-8 21.06 12.16
香豆素Coumarin 91-64-5 29.22 17.93
蔗糖Sucrose 57-50-1
海藻糖Trehalose 6138-23-4
黄酮类化合物Flavonoids
柚皮素Naringenin 67604-48-2 36.20 25.32
植物挥发物Plant volatiles
叶绿醇Phytol 7541-49-3
乙偶姻Acetoin 513-86-0
乙酸乙酯Ethyl acetate 141-78-6
β-环柠檬醛β-Cyclocitral 432-25-7 44.54 31.37
乙酸芳樟酯Linalyl acetate 115-95-7
果蝇信息素 Drosophila pheromones
肉豆蔻酸甲酯Methyl myristate 124-10-7
月桂酸甲酯Methyl laurate 111-82-0
棕榈酸甲酯Methyl palmitate 112-39-0
顺-9-二十三烯9Z-Tricosene 27519-02-4
顺-7-二十三烯7Z-Tricosene 52078-42-9
顺-11-十八碳烯酸乙酯11-cis-Vaccenyl acetate 6186-98-7
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