中华蜜蜂气味结合蛋白AcerOBP7的表达及结合特性

doi:10.3864/j.issn.0578-1752.2022.03.015

Abstract

Abstract:

【Objective】 Apis cerana cerana is the indigenous bee of China, which is also one of the important pollinator and economic insect. Odorant binding proteins (OBPs) are the key proteins in the olfactory perception of A. c. cerana. Based on the previous analysis about the sequence characteristics of AcerOBP7, the present study intends to further research its expression profiles and binding properties, so as to provide basic data for revealing the role of OBPs in the olfactory system of A. c. cerana.【Method】 In this paper, qRT-PCR was used to detect the spatio-temporal expression characteristics of AcerOBP7 in worker bees. AcerOBP7 protein was obtained by prokaryotic expression system, and the recombinant protein was purified using Ni-NTA column. Competitive fluorescence binding method was adopted, with 1-NPN (N-phenyl-1-naphthylamine) was used as a fluorescent probe, to determine the binding affinity of AcerOBP7 with pheromone and plant volatiles. dsAcerOBP7 was designed and synthesized, and the gene was silenced by feeding method, the silencing efficiency was detected by qRT-PCR. Combined with RNAi method, EAG was conducted to test and compare the difference of response values of AcerOBP7 to the candidate volatiles between the control and RNAi group.【Result】 qRT-PCR results showed that AcerOBP7 expressed significantly higher in the antennae of worker bees than that in other tissues (P<0.01), and the expression reached to the highest level at 20-day-old and the lowest at 1-day-old. The pET28a/AcerOBP7 expression vector was successfully constructed, and the high-purity recombinant protein was obtained using the Escherichia coli prokaryotic expression system. Fluorescence competition binding assay showed that AcerOBP7 had the strongest binding affinities with 9-ODA and 1-nonanol among 37 ligand molecules, with the Ki values was both 1.85 µmol·L^-1, followed by (+)-limonene, 1-octene-3-ol, linalool, trans-ethyl-cinnamate, eucalyptol, (+)-3-carene and nonanal, and the Ki values were 1.87, 2.66, 2.72, 3.05, 3.88, 4.14 and 4.40 µmol·L^-1, respectively, while AcerOBP7 had no binding ability with all the tested larval pheromone components. AcerOBP7 was successfully silenced by feeding dsRNA, and the highest interference efficiency could reach to 70.63%. The results of EAG assay after RNAi showed that the EAG values response to the tested odorant chemicals were all decreased, and the relative EAG values of 1-octen-3-ol, nonanal, eucalyptol and 9-ODA decreased significantly (P<0.05).【Conclusion】 AcerOBP7 is highly expressed in antennae of the forager bees and the recombinant protein can bind to a variety of odor molecules, suggesting that AcerOBP7 is a binding protein with broad spectrum, which may play an important role in the foraging behavior and feeding the queen of the worker bees. In addition, 1-octen-3-ol, nonanal, eucalyptol and 9-ODA are the ligands with high binding specificity to AcerOBP7.

Key words: Apis cerana cerana, odorant binding protein (OBP), expression profile, protein purification, binding property

ZHAO HuiTing,PENG Zhu,JIANG YuSuo,ZHAO ShuGuo,HUANG Li,DU YaLi,GUO LiNa. Expression and Binding Properties of Odorant Binding Protein AcerOBP7 in Apis cerana cerana[J].Scientia Agricultura Sinica, 2022, 55(3): 613-624.

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References 53

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引物 Primer		序列 Sequence	产物长度 Product length (bp)	退火温度 Annealing temperature (℃)
用于荧光定量 For qRT-PCR
OBP7	F	CTTTCCGTTGCCGTAATCAT	165	60
OBP7	R	TTCCTCCGATATGTCTTCCTCT	165
Arp1	F	ACTACGGCCGAACGTGAAAT	144
Arp1	R	GGAAAAGAGCCTCGGGACAA	144
用于原核表达 For prokaryotic expression
OBP7	F	cgcggatccAATGGAATAAACGAAATCTTG	438	58
OBP7	R	cccaagcttTTACATATCGCTTAAGAATTTC	438	58
用于dsRNA的合成 For dsRNA synthesis
OBP7	T7 F1	taatacgactcactatagggAGAGGAAGACATATCGGAGGA	219	58
	R1	CCATTCTCGCATTTGTCCGT
	F1	AGAGGAAGACATATCGGAGGA
	T7 R1	taatacgactcactatagggCCATTCTCGCATTTGTCCGT
	T7 F2	taatacgactcactatagggTGCATGATACACATGGGCTTG	265
	R2	TGTCCGTACCTTTGTTCGCT
	F2	TGCATGATACACATGGGCTTG
	T7 R2	taatacgactcactatagggTGTCCGTACCTTTGTTCGCT
GFP	T7 F	taatacgactcactatagggCACAAGTTCAGCGTGTCC	539	56
	R	CTGGGTGCTCAGGTAGTG
	F	CACAAGTTCAGCGTGTCC
	T7 R	taatacgactcactatagggCTGGGTGCTCAGGTAGTG

种类 Category	CAS登记号 CAS number	IC₅₀(μmol·L^-1)	Ki(μmol·L^-1)
蜂王信息素Queen pheromone
反式-9-氧代-2-癸烯酸 9-ODA	14436-32-9	1.98	1.85
告警信息素 Alarm pheromone
2-庚酮2-Heptanone	110-43-0	17.12	14.60
乙酸异戊酯Isoamyl acetate	123-92-2	-	-
那氏信息素Nasonov pheromone
香叶醇Geraniol	106-24-1	8.21	7.00
橙花醇Nerol	106-25-2	12.95	11.04
法呢醇Farnesol	4602-84-0	10.76	9.18
柠檬醛Citral	5392-40-5	8.75	8.20
幼虫信息素 Brood pheromone
油酸甲酯Methyl oleate	112-62-9	-	-
硬脂酸甲酯Methyl stearate	112-61-8	-	-
棕榈酸甲酯Methyl palmitate	112-39-0	-	-
油酸乙酯Ethyl oleate	111-62-6	-	-
亚油酸乙酯Ethyl linoleate	544-35-4	-	-
亚油酸甲酯 Methyl linoleate	112-63-0	-	-
亚麻酸甲酯Methyl linolenate	310-00-8	-	-
植物挥发物 Plant volatiles
1-壬醇 1-Nonanol	143-08-8	1.97	1.85
1-辛烯-3-醇1-Octen-3-ol	3391-86-4	3.12	2.66
月桂烯Myrcene	123-35-3	6.93	5.91
β-石竹烯β-Caryophllene	87-44-5	6.27	5.35
香茅醇Citronellol	106-22-9	11.08	10.38
亚麻酸Linolenic acid	463-40-1	8.98	8.42
胡椒酮Piperitone	89-81-6	6.50	6.09
桉树脑Eucalyptol	470-82-6	4.55	3.88
丁香酚Eugenol	97-53-0	8.11	6.92
反式肉桂酸乙酯Ethyl-trans-cinnamate	4192-77-2	3.58	3.05
(+)-3-蒈烯 (+)-3-Carene	13466-78-9	4.85	4.14
β-罗勒烯 β-Ocimene	13877-91-3	6.67	5.69
壬醛Nonanal	124-19-6	5.16	4.40
肉桂酸乙酯Ethyl cinnamate	103-36-6	7.13	6.08
香草醇Vanillyl alcohol	498-00-0	7.62	7.14
α-亚麻酸乙酯α-Ethyl linolenic	1191-41-9	9.31	7.94
芳樟醇Linalool	78-70-6	3.19	2.72
(+)-柠檬烯 (+)-Cinene	138-86-3	1.99	1.87
β-蒎烯 β-Pinene	127-91-3	9.44	8.85
水杨酸甲酯Methyl salicylate	119-36-8	6.83	6.40
1-辛醇1-Octanol	111-87-5	8.83	8.27
β-紫罗酮β-Ionone	14901-07-6	-	-
乙酸乙酯Ethyl acetate	141-78-6	-	-

Expression and Binding Properties of Odorant Binding Protein AcerOBP7 in Apis cerana cerana

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