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

摘要/Abstract

摘要：

目的中华蜜蜂（Apis cerana cerana,简称中蜂）是我国本土蜂种,也是重要的传粉昆虫和经济昆虫。气味结合蛋白（odorant binding protein,OBP）是蜜蜂嗅觉感知中的关键蛋白。在前人对中蜂AcerOBP7基因序列特征分析的基础上,本研究进一步对其表达特性及与气味物质的结合能力进行探究,为揭示气味结合蛋白在中蜂嗅觉系统中的作用提供基础数据。方法采用qRT-PCR检测AcerOBP7在工蜂不同组织和发育阶段的表达特性;通过原核表达系统获得AcerOBP7融合蛋白,使用Ni-NTA柱进行蛋白纯化;采用荧光竞争结合法,以1-NPN（N-phenyl-1-naphthylamine）为荧光探针,测定AcerOBP7与信息素及植物挥发物的结合能力;设计并合成dsAcerOBP7,采用饲喂法对该基因进行沉默,通过qRT-PCR测定沉默效率;结合RNAi,利用EAG检测并比较对照组与干扰组中蜜蜂触角对待测物质反应值的差异。结果 qRT-PCR结果显示,AcerOBP7 mRNA在工蜂触角中的表达量极显著高于其他组织（P<0.01）,且在20日龄时表达量最高,在1日龄时表达量最低。成功构建了pET28a/AcerOBP7表达载体,通过大肠杆菌原核表达系统获得了高纯度的重组蛋白。与37种配体分子的荧光竞争结合试验表明,AcerOBP7与9-ODA、1-壬醇结合能力最强,Ki值均为1.85 µmol·L^-1,其次与(+)-柠檬烯、1-辛烯-3-醇、芳樟醇、反式肉桂酸乙酯、桉树脑、(+)-3-蒈烯及壬醛有较强的结合活性,Ki 值分别为1.87、2.66、2.72、3.05、3.88、4.14和4.40 µmol·L^-1,与所选择的幼虫信息素均无结合能力。使用饲喂dsRNA的方法成功干扰了AcerOBP7的表达,干扰效率最高可达70.63%。干扰后的EAG试验表明,中蜂触角对所测气味物质的反应强度均有所下降,其中1-辛烯-3-醇、壬醛、桉树脑和9-ODA的EAG相对值显著降低（P<0.05）。结论 AcerOBP7在中蜂采集蜂触角中高表达,重组蛋白能与多种气味分子结合,表明AcerOBP7是一种广谱型结合蛋白,推测其可能在工蜂采集和哺育蜂王的行为中发挥着重要作用。另外,1-辛烯-3-醇、壬醛、桉树脑和9-ODA可能是AcerOBP7结合特异性较高的配体物质。

关键词: 中华蜜蜂, 气味结合蛋白, 表达模式, 蛋白纯化, 结合特性

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

赵慧婷,彭竹,姜玉锁,赵淑果,黄丽,杜亚丽,郭丽娜. 中华蜜蜂气味结合蛋白AcerOBP7的表达及结合特性[J]. 中国农业科学, 2022, 55(3): 613-624.

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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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	-	-