Scientia Agricultura Sinica ›› 2019, Vol. 52 ›› Issue (20): 3705-3712.doi: 10.3864/j.issn.0578-1752.2019.20.020

• RESEARCH NOTES • Previous Articles    

Molecular Cloning, Prokaryotic Expression and Binding Characterization of Odorant Binding Protein GdauOBP20 in Galeruca daurica

Ling LI,Yao TAN,XiaoRong ZHOU,BaoPing PANG()   

  1. Research Center for Grassland Entomology, Inner Mongolia Agricultural University, Hohhot 010020
  • Received:2019-03-11 Accepted:2019-04-18 Online:2019-10-16 Published:2019-10-28
  • Contact: BaoPing PANG


【Objective】 Galeruca daurica is a new pest with outbreak status in the Inner Mongolia grasslands in recent years. The objective of this study is to clone the full-length cDNA sequence of GdauOBP20, and clarify the binding property of the recombinant protein to main host plant volatiles, which will lay a necessary foundation for revealing the molecular mechanism of olfaction in G. daurica.【Method】 RACE technique was used to clone the full-length cDNA of GdauOBP20 based on the transcriptome database of G. daurica. The physicochemical properties and structural characteristics of the encoded protein were predicted and analyzed by bioinformatics software. The recombinant protein GdauOBP20 was induced to express by constructing prokaryotic expression system, and purified by using the Ni-NTA Agarose affinity column. Finally, the fluorescence competitive assay was applied, and N-phenyl-1-naphthylamine (1-NPN) was selected as the fluorescence probe to measure the binding profiles of GdauOBP20 recombinant protein with 13 main host plant volatiles.【Result】 The full-length cDNA of GdauOBP20 is 567 bp (GenBank accession number: MK250532), with the non-coding regions of 5′ and 3′ ends of 24 bp and 123 bp, respectively, and a ployA tail structure. The open reading frame (ORF) is 420 bp, encoding 139 amino acids. The amino acid sequence of GdauOBP20 contains 4 conserved cysteine residues, indicating that it belongs to Minus-C OBP subfamily. The three-dimensional structure prediction of GdauOBP20 contains six alpha helix and two pairs of disulfide bonds formed by cysteine. The recombinant expression vector was successfully constructed, and the recombinant protein with high purity was obtained. The binding capacity of the recombinant protein GdauOBP20 to the fluorescence probe 1-NPN was strong with a binding constant of 12.8 μmol·L -1, indicating that it could be used as the fluorescence reporter in this experiment. Affinities of recombinant protein GdauOBP20 with 13 main host plant volatiles were tested. Among them, except diallyl trisulfide, other 12 volatiles showed certain binding capacities with the recombinant protein, and p-xylene and 1,3,5-cycloheptatriene displayed the strongest affinity with the dissociation constants of 22.91 and 26.55 μmol·L -1, respectively, whereas myrcene exhibited the weakest binding affinity with the dissociation constant of 116.29 μmol·L -1.【Conclusion】 GdauOBP20 has a certain binding capacity with main host plant volatiles, suggesting that it may play an important role in the localization of host plants.

Key words: Galeruca daurica, odorant binding protein, RACE cloning, prokaryotic expression, fluorescence competitive binding assay

Table 1

Primers used in this study"

名称 Name 引物序列Primer sequence (5′-3′) 引物用途 Use of primers
RACE end amplification
Nested PCR
Middle fragment amplification
Verification of full-length cDNA
Prokaryotic expression

Fig. 1

Nucleotide and amino acid sequences of GdauOBP20 The start and stop codons are boxed, signal peptide is underlined, and conversed cysteine residues are circled"

Fig. 2

Three-dimensional prediction structure of GdauOBP20 protein"

Fig. 3

Expression induced by IPTG (A) and purification (B) of recombinant protein GdauOBP20"

Fig. 4

Binding curves of GdauOBP20 to 1-NPN (A, B) and fluorescence competition with ligands (C, D)"

Table 2

Binding affinities of selected ligands with recombinant GdauOBP20"

Chemical ligand
Dissociation constant (μmol·L-1)
二烯丙基硫醚 Diallyl sulfide 42.30 42.10
二烯丙基二硫 Diallyl disulphide 50.97 50.73
二烯丙基三硫醚 Diallyl trisulfide
二甲基二硫醚 Dimethyl disulfide 31.79 31.64
二甲基三硫醚 Dimethyl trisulfide 31.16 31.01
1,3-二噻烷 1,3-Dithiane 49.86 49.63
己醛Hexanal 49.05 48.83
2-己烯醛2-Hexenal 37.97 37.80
苯甲酸甲酯 Methyl benzoate 37.37 37.19
对二甲苯p-Xylene 23.07 22.91
顺-2-己烯-1-醇 (Z)-2-Hexen-1-ol 47.01 46.80
月桂烯Myrcene 116.83 116.29
环庚三烯 1,3,5-Cycloheptatriene 26.68 26.55
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