沙葱萤叶甲气味结合蛋白GdauOBP20的基因克隆、 原核表达及其结合特性

doi:10.3864/j.issn.0578-1752.2019.20.020

Abstract

Abstract:

【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

Ling LI,Yao TAN,XiaoRong ZHOU,BaoPing PANG. Molecular Cloning, Prokaryotic Expression and Binding Characterization of Odorant Binding Protein GdauOBP20 in Galeruca daurica[J].Scientia Agricultura Sinica, 2019, 52(20): 3705-3712.

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名称 Name	引物序列Primer sequence (5′-3′)	引物用途 Use of primers
GdauOBP20-5′ GSP	CACGGTCGAAAATTCCTTTTTTGCC	RACE末端扩增 RACE end amplification
GdauOBP20-3′ GSP	TTGGCCCCATCTAAAGTTGCCGACG	RACE末端扩增 RACE end amplification
GdauOBP20-5′ NGSP	TGGATCCGTAATCTCCATTAAGGGA	巢氏PCR Nested PCR
GdauOBP20-3′ NGSP	TGGAGCTTGTCACAACAAAGTCGCA	巢氏PCR Nested PCR
GdauOBP20-F1	ATGTTTCGGGAGCTTCTA	中间片段扩增 Middle fragment amplification
GdauOBP20-R1	TTAGAATATTATAAAGAGTTC	中间片段扩增 Middle fragment amplification
GdauOBP20-F2	AGTCACACACCATGTTTCG	cDNA全长验证 Verification of full-length cDNA
GdauOBP20-R2	GATTTTTATTTATTTCTACAAAGAG	cDNA全长验证 Verification of full-length cDNA
GdauOBP20-F3	GAATTCAATCCCTTAATGGAGAT	原核表达 Prokaryotic expression
GdauOBP20-R3	CTCGAGTTAGAATATTATAAAGAGTT	原核表达 Prokaryotic expression

化学配基 Chemical ligand	IC₅₀ (μmol·L^-1)	解离常数Ki 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

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

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