韭菜迟眼蕈蚊气味结合蛋白OBP11的配体结合能力及关键氨基酸分析

doi:10.3864/j.issn.0578-1752.2026.18.005

Abstract

Abstract:

【Objective】The objective of this study is to elucidate the molecular characteristics of the odorant-binding protein BodoOBP11, which is highly expressed in the antennae of adult Bradysia odoriphaga, and to investigate its olfactory function in host localization.【Method】Bioinformatics tools were used to analyze the basic characteristics and evolutionary relationships of BodoOBP11. The spatio-temporal expression patterns of BodoOBP11 across different developmental stages and tissues were then assessed using quantitative real-time PCR (qPCR). The BodoOBP11 protein was obtained through prokaryotic expression in E. coli, purification via nickel affinity chromatography, and cleavage using recombinant enterokinase. Using fluorescence competitive binding assays with the fluorescent probe 1-NPN, the binding ability of the protein to 22 volatile compounds from the host plant (Chinese chives) and one putative sex pheromone compound was subsequently evaluated. Finally, protein structure prediction, molecular docking, and site-directed mutagenesis were employed to identify the key binding sites between BodoOBP11 and ligand compounds.【Result】BodoOBP11 has an open reading frame of 426 bp, encoding 141 amino acid residues, including an N-terminal signal peptide of 20 amino acids. It exhibits the typical features of the Classic-C OBP, characterized by six conserved cysteine residues. The predicted molecular weight of the mature protein is 13.53 kDa, with an isoelectric point of 7.71. qPCR analysis revealed distinct expression patterns of BodoOBP11. Across developmental stages, its expression was highest in male adults, followed by female adults and pupae. In different tissues, BodoOBP11 showed significant high expression in the antennae of both sexes, with the highest level detected in male antennae. Fluorescence competitive binding assays demonstrated that BodoOBP11 binds to six volatiles: dipropyl trisulfide, 2-tridecanone, dipropyl disulfide, 2-undecanone, phenethyl isothiocyanate, and α-ionone. The strongest binding affinity was observed for dipropyl trisulfide. Protein structure prediction and molecular docking indicated that BodoOBP11 binds to these six ligands primarily via hydrogen bonding and hydrophobic interactions. Specifically, Thr74 forms hydrogen bonds with 2-tridecanone, 2-undecanone, and α-ionone, while residues Val76, Ile107, Leu127, Met131, Phe138, and Phe140 mainly mediate hydrophobic interactions. Site-directed mutagenesis followed by fluorescence competitive binding assays revealed that mutations of Ile107, Phe138, and Phe140 to alanine significantly reduced the binding affinity of BodoOBP11 to all six ligands, indicating their critical roles in binding.【Conclusion】BodoOBP11 exhibits strong binding affinity to six volatile compounds from Chinese chive, including dipropyl trisulfide, 2-tridecanone, and dipropyl disulfide, etc. Furthermore, residues Ile107, Phe138, and Phe140 were identified as crucial sites for binding these ligands. These findings preliminarily reveal its molecular mechanism in mediating host plant volatile recognition by adult B. odoriphaga using BodoOBP11, offering a theoretical foundation for developing green control technologies based on olfactory interference.

Key words: Bradysia odoriphaga, odorant-binding protein (OBP), fluorescence competitive binding, molecular docking, site-directed mutagenesis

WANG QinLi, TAN ShunYang, XIA Gen, LIU WenQing, YANG YuTing, TIAN LiXia. Ligand Binding Property and Key Amino Acid Analysis of Odorant- Binding Protein OBP11 in Bradysia odoriphaga[J].Scientia Agricultura Sinica, 2026, 59(18): 4007-4020.

Figures/Tables 9

Table 1

Fig. 1

Fig. 2

Fig. 3

Table 2

Binding affinity of BodoOBP11 with different volatile compounds"

化合物名称 Compound name	分子式 Molecular formula	分子量 Molecular weight	CAS号 CAS number	BodoOBP11 (pH 7.4)
化合物名称 Compound name	分子式 Molecular formula	分子量 Molecular weight	CAS号 CAS number	IC₅₀(μmol·L^-1)	K_i (μmol·L^-1)
假定的性信息素Putative sex pheromone
正十七烷Heptadecane	C₁₇H₃₆	240.47	629-78-7	—	—
韭菜挥发物Chinese chives’ volatile
酮类Ketones
2-十一烷酮2-Undecanone	C₁₁H₂₂O	170.29	112-12-9	21.12±7.38	19.15±6.69
2-十三烷酮2-Tridecanone	C₁₃H₂₆O	198.34	593-08-8	15.58±2.95	14.13±2.67
α-紫罗酮α-Ionone	C₁₃H₂₀O	192.30	127-41-3	26.38±0.86	23.91±0.79
醇类Alcohols
3-甲基-2-丁烯-1-醇3-Methyl-2-buten-1-ol	C₅H₁₀O	86.13	556-82-1	—	—
芳樟醇Linalool	C₁₀H₁₈O	154.25	78-70-6	—	—
香茅醇Citronellol	C₁₀H₂₀O	156.27	106-22-9	—	—
醛类Aldehydes
苯甲醛Benzaldehyde	C₇H₆O	106.12	100-52-7	—	—
壬醛1-Nonanal	C₉H₁₈O	142.24	124-19-6	—	—
庚醛Heptaldehyde	C₇H₁₄O	114.19	111-71-7	—	—
挥发性硫类化合物Volatile sulfur compounds
二丙基二硫Propyl disulfide	C₆H₁₄S₂	150.31	629-19-6	18.33±2.80	16.62±2.54
二丙基三硫醚Dipropyl trisulfide	C₆H₁₄S₃	182.37	6028-61-1	5.25±0.47	4.76±0.43
二甲基三硫Dimethyl trisulfide	C₂H₆S₃	126.26	3658-80-8	—	—
二烯丙基二硫Diallyl disulfide	C₆H₁₀S₂	146.27	2179-57-9	—	—
甲基丙基二硫Methyl propyl disulfide	C₄H₁₀S₂	122.25	2179-60-4	—	—
不饱和烃类Unsaturated hydrocarbons
1-庚烯1-Heptene	C₇H₁₄	98.18	1161-13-3	—	—
石竹烯Caryophyllene	C₅₅H₂₄	204.36	87-44-5	—	—
β-蒎烯β-Pinene	C₁₀H₁₆	136.23	127-91-3	—	—
酯类Esters
异硫氰酸苯乙酯Phenylethyl isothiocyanate	C₉H₉NS	163.24	2257-09-2	21.67±1.66	19.64±1.51
3-（甲硫基）丙基异硫酸酯 3-(methyl thioyl) propyl isosulfate	C₅H₉NS₂	147.26	505-79-3	—	—
水杨酸甲酯Methyl salicylate	C₈H₈O₃	152.15	119-36-8	—	—
烯丙基硫氢酸酯Allyl thiocyanate	C₄H₅NS	99.15	764-49-8	—	—
月桂酸乙酯Ethyl dodecanoate	C₁₄H₂₈O₂	228.37	106-33-2	—	—

Table 2

Fig. 4

Table 3

Fig. 5

Fig. 6

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引物名称Primer name	引物序列Primer sequence
用于克隆 For cloning	F: CGCGGATCCGGTGTAACGATGGAGCAAAT
用于克隆 For cloning	R: GGGAAGCTTTTAAGCAAACGTGAACTTTTTAT
用于实时荧光定量分析 For qPCR analysis	F: AACTATGAGGCATCTCTGAA
用于实时荧光定量分析 For qPCR analysis	R: TTACTCCATTAGCAGCATCT
内参基因 Reference gene	RPS15-F: ATCGTGGCGTCGATTTGGAT
	RPS15-R: CTCATTTGGTGGGGCTTCCT
	RPL18-F: TTCCCTTATCCATCAAACATT
	RPL18-R: CTTAGAATAACTTCAGGTAATGC
用于原核表达 For heterologous expression	F: CGCGGATCCGGTGTAACGATGGAGCAAAT
用于原核表达 For heterologous expression	R: GGGAAGCTTTTAAGCAAACGTGAACTTTTTAT
基因定点突变 Site-directed mutant	T74A-F: GCCGGAGTCATGAAGAAAAACAAACT
	T74A-R: CATTTCCAACACACATTGCATGT
	V76A-F: GCCATGAAGAAAAACAAACT
	V76A-R: TCCTGTCATTTCCAACACACAT
	I107A-F: GCCACGCAGTGCAAAGATGC
	I107A-R: ACCATTTTTATTTTCTTCCACAATGTCGT
	L127A-F: GCCCTTCCTAATAAGAT
	L127A-R: TATGTATGCTGCTTCGCAGGCAT
	M131A-F: GCCTACAAAAACAATAAAAAGTTCACGT
	M131A-R: GCATTGAACTAATATGTATGCTGCT
	P138A-F: CGCGGATCCGGTGTAACGATGGAGCAAAT
	P138A-R: GGGAAGCTTTTAAGCAAACGTGGCCTTTT
	P140A-F: CGCGGATCCGGTGTAACGATGGAGCAAAT
	P140A-R: GGGAAGCTTTTAAGCGGCCGTGAACT

配体名称 Ligand name	结合能 Binding energy (kJ·mol^-1)	氢键 Conventional hydrogen bond	疏水相互作用 Hydrophobic interaction
二丙基二硫Propyl disulfide	-15.23	—	Lys81, Leu82, Ala114, Leu127
二丙基三硫醚Dipropyl trisulfide	-16.61	—	Val76, Leu82, Ile107, Phe138, Phe140
2-十三烷酮2-Tridecanone	-21.67	Thr74	Leu71, Val76, Leu82, Gln90, Ile107, Leu127, Met131, Phe138, Phe140
2-十一烷酮2-Undecanone	-20.71	Thr74	Leu71, Val76, Gln90, Ile107, Leu127, Val128, Met131, Phe138, Phe140
α-紫罗酮α-Ionone	-26.02	Thr74	Val76, Gln90, Leu94, Ile107, Met131, Phe138, Phe140
异硫氰酸苯乙酯Phenylethyl isothiocyanate	-21.46	—	Leu71, Ile107, Leu127, Val128, Met131, Phe138, Phe140

Ligand Binding Property and Key Amino Acid Analysis of Odorant- Binding Protein OBP11 in Bradysia odoriphaga

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