Co-IP联合质谱分析筛选中华蜜蜂气味受体OR1和OR2的互作蛋白

郭丽娜; 申红英; 王珏; 于点点; 张旭凤; 郭媛

doi:10.19303/j.issn.1008-0384.2024.01.002

Co-IP联合质谱分析筛选中华蜜蜂气味受体OR1和OR2的互作蛋白

doi: 10.19303/j.issn.1008-0384.2024.01.002

1.
山西农业大学动物科学学院，山西　太谷　030801
2.
山西农业大学园艺学院，山西　太谷　030801

基金项目: 山西省应用基础研究计划（青年）项目（20210302124360）；山西省高校科技创新项目（2021L098）；国家现代农业产业技术体系建设专项（CARS-44-KXJ2）

详细信息

作者简介:
郭丽娜（1987 —），女，博士，副教授，主要从事蜜蜂分子生物学研究，E-mail：linaguo@126.com

通讯作者:
郭媛（1975 —），女，硕士，研究员，主要从事蜜蜂授粉及蜜蜂生物学研究，E-mail：yysgy3@163.com

中图分类号: S891
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出版历程
- 收稿日期: 2023-07-14
- 修回日期: 2023-09-25
- 网络出版日期: 2023-12-21
- 刊出日期: 2024-01-28

Interacting Proteins of Olfactroy Receptor OR1and OR2 with Co-IP Approach Followed by Mass Spectrometry Analysis in Apis cerana cerana

1.
College of Animal Science, Shanxi Agricultural University, Taigu, Shanxi　030801, China
2.
College of Horticulture, Shanxi Agricultural University, Taigu, Shanxi　030801, China

摘要

摘要: 目的中华蜜蜂（Apis cerana cerana）是我国特有的蜜蜂品种，其高度灵敏的嗅觉系统能在复杂气味环境中识别群体内化学信号以及区分食物源散发的特异性气味分子，气味受体（Odorant receptors, ORs）在中蜂识别气味分子的行为过程中起到了重要而又关键的作用。通过分析筛选OR1和OR2的互作蛋白，为深入探究OR1和OR2蛋白在蜜蜂嗅觉系统中的功能提供理论依据。方法通过构建OR1和OR2基因的真核表达载体pFastBac-OR1和pFastBac-OR2载体，转染Sf9细胞，提取细胞总蛋白，利用免疫共沉淀（Co-IP）联合质谱分析技术筛选鉴定与OR1和OR2互作的细胞蛋白，并对这些互作蛋白进行GO功能注释、KEGG信号通路和蛋白互作网络分析。结果 IP组和IgG组重组蛋白在细胞内得到正确表达，利用Co-IP联合质谱分析技术共筛选到273个与OR1互作的细胞蛋白和204个与OR2互作的细胞蛋白，主要为微管蛋白、热休克蛋白、核糖体蛋白等。对这些蛋白进行GO功能富积分析，发现这些蛋白质涉及多种生物学功能，包括RNA剪接、核糖体和能量运输有关。KEGG信号通路分析结果表明互作蛋白参与调节了多条细胞内的重要通路，包括核糖体、剪接体、RNA转运等与核糖体相关的通路，丙酮酸代谢、硫胺素新陈代谢、脂肪酸生物合成、淀粉和蔗糖代谢、FoxO信号通路，Hedgehog信号通路等。结论 OR1和OR2可能通过与多种蛋白直接或间接的相互作用调控并影响其嗅觉感受。
- 中华蜜蜂 /
- 气味受体 /
- 免疫共沉淀 /
- 蛋白互作
Abstract: Objectives Roles of the proteins interacting with the odorant receptors (ORs) in the highly sensitive olfactory system of Apis cerana cerana were investigated. Methods The eukaryotic expression vectors pFastBac of OR1 and OR2 in A. cerana cerana were constructed and transfected into Sf9 cells to extract the proteins. The cellular interacting proteins were identified by co-immunoprecipitation (Co-IP) and mass spectrometry, and GO function annotation, KEGG signal pathway analysis, and protein interaction network determination. Results The recombinant proteins of the IP and IgG groups were successfully expressed in the cells. There were 273 proteins interacting with OR1, and 204 with OR2. They were mainly tubulin, heat shock proteins, and ribosomal proteins involving in a variety of biological functions, such as RNA splicing and ribosome and energy transport. The KEGG analysis also showed their association with the regulation of several important intracellular pathways related to the ribosome, splicing body, RNA transport, pyruvate metabolism, thiamine metabolism, fatty acid biosynthesis, starch and sucrose metabolism, and FoxO and Hedgehog signals. Conclusion A unique honey bee species in China, A. cerana ceranahas evolved with a highly sensitive olfactory system capable of recognizing chemical signals in the bee population and distinguishing molecules emitted from food sources. OR1 and OR2 in A. cerana cerana regulated the olfactory transduction through direct or indirect interaction with a variety of proteins.
- Apis cerana cerana /
- odorant receptors /
- co-immunoprecipitation /
- interacting proteins

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图 1 重组表达载体pFastBac1-OR1（A）和pFastBac1-OR2（B）酶切验证

1：质粒DNA；2：Bam HI/Hind III酶切片段；M：DNA Marker。

Figure 1. Plasmids pFastBac1-OR1 (A) and pFastBac1-OR2 (B) verified by restriction enzyme digestion

1: plasmid DNA; 2: digested by Bam HI/Hind III1; M: DNA marker.

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图 2 OR1（A）和OR2（B）蛋白的免疫共沉淀验证

M：DNA Marker；Input：全细胞裂解液。

Figure 2. Co-IP validation of OR1(A) and OR2(B

M：DNA Marker；Input：Whole cell lysate.

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图 3 OR1-IP(A)/OR2-IP(B)与IgG组免疫共沉淀蛋白质的银染胶图比较

M：DNA Marker；Input：全细胞裂解液。

Figure 3. Silver staining gel graphs on Co-IP protein of OR1-IP(A)/OR2-IP(B) and IgG groups

M：DNA Marker；Input：Whole cell lysate.

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图 4 OR1-IP(A)/OR2-IP(B)与IgG组鉴定的蛋白质数量的韦恩图

Figure 4. Venn diagram on number of proteins identified in OR1-IP(A)/OR2-IP(B) and IgG groups

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图 5 互作蛋白的GO注释（A、B)和KEGG富积分析（C、D)

Figure 5. Go annotations (A and B) and KEGG analysis (C and D) on interacting proteins

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表 1 蛋白质鉴定信息统计

Table 1. Statistical information on protein identification

样本 Sample	总谱图数 Total spectrograms	鉴定图数 Matched spectrum	鉴定肽段数 Peptide	鉴定蛋白数 Identified protein
OR1_Flag	79 137	2 734	1 487	565
OR1_IgG	76 029	1 153	699	317
OR2_Flag	75 160	2 055	1 280	481
OR2_IgG	76 020	1 212	767	318

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表 2 OR1蛋白相互作用的细胞蛋白（部分蛋白）

Table 2. Cellular proteins interacting with OR1 (partial protein)

UniProt登录号 Accession No.	蛋白名称 Description	长度 Length/aa	蛋白质分子量 Molecular weight/kDa	理论等电点 pI	分值 Score
A0A8B9AYG1	微管蛋白β链 Tubulin beta chain	447	50.1	4.86	230.92
A0A8B6Z0H7	ATP合酶β亚单位 ATP synthase subunit beta	516	55.1	5.41	150.99
A0A8B8H9N3	微管蛋白β链 Tubulin beta chain	447	49.9	4.89	137.54
A0A8B9B4M6	微管蛋白β链 Tubulin beta chain	455	51	4.86	135.05
A0A8B9B2J4	微管蛋白α链 Tubulin alpha chain	450	50.2	5.2	133.21
A0A8B6WZM5	肌动蛋白相关蛋白1 Actin related protein 1	376	41.8	5.48	132.96
A0A8B6Z7B8	微管蛋白α链 Tubulin alpha chain	450	49.9	5.14	127.76
A0A8B8GXQ7	Actin肌动蛋白	376	41.7	5.48	105.7
A0A8B6Z7F0	延伸因子1-α异构体X1 Elongation factor 1-alpha isoform X1	461	50.3	9.03	105.16
A0A7M6UVC2	未知蛋白 Uncharacterized protein	640	70.3	5.82	82.67
A0A8B6WZH6	热休克蛋白同源3前体 Heat shock protein cognate 3 precursor	658	72.8	5.43	79.69
A0A8B6WZD4	热休克蛋白83 Heat shock protein 83	724	83.3	5.06	79.38
A0A8B6YS79	微管蛋白α链 Tubulin alpha chain	448	50.4	5.43	74.64
A0A8B9AWD0	ATP合酶亚单位 ATP synthase subunit alpha	547	59.5	8.9	74.6
A0A7M7GZK8	热休克蛋白83 Heat shock protein 83	718	82.7	5.05	73.46
A0A7M7GKN6	未知蛋白 Uncharacterized protein	357	36.9	9.38	73.41
A0A8B6WZI3	热休克同源蛋白4 Heat shock protein cognate 4	650	71	5.58	70.79
A0A8B6Z8U0	组蛋白H4 Histone H4	103	11.4	11.36	69.62
A0A7M7MSJ5	ATP依赖性解旋酶brm ATP-dependent helicase brm	2019	217.6	7.14	57.85
A0A8B9B3I3	60S核糖体蛋白L23 60S ribosomal protein L23	140	14.8	10.58	55.71
A0A8B7KRE5	SWI/SNF复合亚基SMARCC2 SWI/SNF complex subunit SMARCC2	1026	114.4	6.05	50.7
A0A7M7LPS5	40S核糖体蛋白S5 40S ribosomal protein S5	216	24.1	9.45	47.67
A0A8B7KKF5	前信使RNA加工剪接因子8 Pre-mRNA-processing-splicing factor 8	2374	277	8.88	44.2
A0A8B6Z1L0	60S核糖体蛋白L10a 60S ribosomal protein L10a	217	24.6	9.76	41.69
A0A7M7R4Y7	未知蛋白 Uncharacterized protein	358	38.5	7.02	41.56
A0A7M7IR07	40S核糖体蛋白S9 40S ribosomal protein S9	193	22.5	10.74	40.63
A0A8B6Z285	囊泡融合ATP酶 Vesicle-fusing ATPase	800	88.8	5.33	40.6
A0A8B6Z280	14-3-3蛋白zeta异构体X1 14-3-3 protein zeta isoform X1	247	28.1	4.89	39.95
A0A7M7GMY7	含有14_3_3结构域的蛋白质 14_3_3 domain-containing protein	256	29.1	4.89	39.62
A0A8B6Z1F6	26S蛋白酶体调节亚基8 26S proteasome regulatory subunit 8	405	45.7	8.5	38.87
A0A8B7KJ60	平移伸长因子2亚型X1 Translation elongation factor 2 isoform X1	844	94.5	6.52	36.68
A0A8B8H899	钙转运ATP酶肌浆/内质网型X1亚型 Calcium-transporting ATPase sarcoplasmic/ endoplasmic reticulum type isoform X1	1020	112.1	5.54	35.55
A0A7M7TG45	40S核糖体蛋白S18 40S ribosomal protein S18	181	21.1	9.96	35.13
A0A8B6ZA30	26S蛋白酶体调节亚基7 26S proteasome regulatory subunit 7	434	48.4	6.07	34.05
A0A8B6YVR3	RNA解旋酶 RNA helicase	728	82.2	9.42	33.23
A0A7M7RB98	网格蛋白重链 Clathrin heavy chain	1678	192	5.94	32.91
A0A7M7R7J9	含有AAA结构域的蛋白质 AAA domain-containing protein	440	49.2	6.76	32.26
A0A7M7MT55	微管蛋白α链 Tubulin alpha chain	432	48.2	5.06	31.9
A0A8B9B3X8	RNA解旋酶 RNA helicase	403	46.2	6.24	31.23
A0A8B6YTH0	α链异构体X2 Spectrin alpha chain isoform X2	2418	278.2	5.26	31.03
A0A8B6YUN0	钙调素依赖性蛋白激酶 Calcium/calmodulin-dependent protein kinase	474	53.9	7.88	30.31
A0A7M7R795	RNA解旋酶 RNA helicase	423	48.1	5.49	30.17
A0A7M7L992	ras相关蛋白Rab-5C Ras-related protein Rab-5C	214	23.4	8.13	30.16
A0A8B9B6N2	v型质子ATP酶催化亚基A V-type proton ATPase catalytic subunit A	616	68.3	5.43	29.78

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表 3 与OR2蛋白相互作用的细胞蛋白（部分蛋白）

Table 3. Cellular proteins interacting with OR2 (partial protein)

UniProt登录号 Accession No.	蛋白名称 Description	长度 Length/aa	蛋白质分子量 Molecular weight/kDa	理论等电点 pI	分值 Score
A0A8B9AYG1	微管蛋白β链 Tubulin beta chain	447	50.1	4.86	186.38
A0A8B6Z0H7	ATP合酶β亚单位 ATP synthase subunit beta	516	55.1	5.41	118.08
A0A8B6WZM5	肌动蛋白相关蛋白1 Actin related protein 1	376	41.8	5.48	112.87
A0A8B6Z7B8	微管蛋白α链 Tubulin alpha chain	450	49.9	5.14	110.42
A0A8B8H9N3	微管蛋白β链 Tubulin beta chain	447	49.9	4.89	106.87
A0A8B9B2J4	微管蛋白α链 Tubulin alpha chain	450	50.2	5.2	100.02
A0A8B8GXQ7	肌动蛋白 Actin	376	41.7	5.48	92.02
A0A8B6Z7F0	延伸因子1- α异构体X1 Elongation factor 1-alpha isoform X1	461	50.3	9.03	75.26
A0A8B6WZD4	热休克蛋白83 Heat shock protein 83	724	83.3	5.06	64.73
A0A7M7GZK8	热休克蛋白83 Heat shock protein 83	718	82.7	5.05	63.46
A0A7M6UVC2	未知蛋白 Uncharacterized protein	640	70.3	5.82	60.39
A0A7M7GKN6	未知蛋白 Uncharacterized protein	357	36.9	9.38	57.52
A0A8B6WZH6	热休克蛋白同源3前体 Heat shock protein cognate 3 precursor	658	72.8	5.43	51.8
A0A8B6YS79	微管蛋白α链 Tubulin alpha chain	448	50.4	5.43	51.66
A0A8B6WZI3	热休克同源蛋白4 Heat shock protein cognate 4	650	71	5.58	46.41
A0A8B6Z8U0	组蛋白H4 Histone H4	103	11.4	11.36	43.62
A0A7M7MSJ5	ATP依赖性解旋酶brm ATP-dependent helicase brm	2019	217.6	7.14	43.24
A0A8B9AWD0	ATP合酶亚单位 ATP synthase subunit alpha	547	59.5	8.9	42.65
A0A8B9B3I3	60S核糖体蛋白L23 60S ribosomal protein L23	140	14.8	10.58	34.65
A0A7M7IR07	40S核糖体蛋白S9 40S ribosomal protein S9	193	22.5	10.74	32.99
A0A7M7R4Y7	未知蛋白 Uncharacterized protein	358	38.5	7.02	30.64
A0A8B6Z1F6	26S蛋白酶体调节亚基8 26S proteasome regulatory subunit 8	405	45.7	8.5	29.63
A0A7M7LPS5	40S核糖体蛋白S5 40S ribosomal protein S5	216	24.1	9.45	29.41
A0A8B9B6N2	V型质子ATP酶催化亚基A V-type proton ATPase catalytic subunit A	616	68.3	5.43	28.38
A0A7M6UPZ6	40S核糖体蛋白S14亚型X1 40S ribosomal protein S14 isoform X1	151	16.2	10.45	27.66
A0A8B7KRE5	SWI/SNF复合亚基SMARCC2 SWI/SNF complex subunit SMARCC2	1026	114.4	6.05	27.6
A0A8B6YVR3	RNA解旋酶 RNA helicase	728	82.2	9.42	27.19
A0A7M7RB98	网格蛋白重链 Clathrin heavy chain	1678	192	5.94	25.73
A0A8B8H899	钙转运ATP酶肌浆/内质网型X1亚型 Calcium-transporting ATPase sarcoplasmic/ endoplasmic reticulum type isoform X1	1020	112.1	5.54	24.84
A0A7M7IK02	RNA解旋酶 RNA helicase	563	64.3	10.14	24.52
A0A7M7L992	Rab-5C ras相关蛋白Rab-5C Ras-related protein	214	23.4	8.13	24.48
A0A8B6Z1L0	60S核糖体蛋白L10a 60S ribosomal protein L10a	217	24.6	9.76	24.42
A0A8B7KKF5	前信使RNA加工剪接因子8 Pre-mRNA-processing-splicing factor 8	2374	277	8.88	23.78
A0A8B6YTH0	α链异构体X2 Spectrin alpha chain isoform X2	2418	278.2	5.26	23.34
A0A7M7H2S3	未知蛋白 Uncharacterized protein	1970	227.1	5.5	22.78
A0A8B6YUN0	钙调素依赖性蛋白激酶 Calcium/calmodulin-dependent protein kinase	474	53.9	7.88	22.64
A0A7M7TG08	40S核糖体蛋白S23 40S ribosomal protein S23	143	16	10.56	22.61
A0A8B9B2J1	依赖ATP的RNA解旋酶WM6 ATP-dependent RNA helicase WM6	424	48.8	5.95	22.47
A0A7M7TG45	40S核糖体蛋白S18 40S ribosomal protein S18	181	21.1	9.96	22.04
A0A7M7R7J9	含有AAA结构域的蛋白质 AAA domain-containing protein	440	49.2	6.76	21.88
A0A8B8HD04	Putative ATP-dependent RNA helicase me31b	444	50.6	7.74	21.18
A0A7M7LKA7	组蛋白H2B Histone H2B	123	13.7	10.39	21.12
A0A7M7R836	类驱动蛋白 Kinesin-like protein	988	112.4	6.11	20.87
A0A7M7LK91	未知蛋白 Uncharacterized protein	130	14.8	10.05	20.81
A0A8B6Z285	囊泡融合ATP酶 Vesicle-fusing ATPase	800	88.8	5.33	20.56

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