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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

郭丽娜,申红英,王珏,等. 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 [J]. 福建农业学报,2024,39(1):7−16 doi: 10.19303/j.issn.1008-0384.2024.01.002
引用本文: 郭丽娜,申红英,王珏,等. 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 [J]. 福建农业学报,2024,39(1):7−16 doi: 10.19303/j.issn.1008-0384.2024.01.002
GUO L N, SHEN H Y, WANG J, et al. Interacting Proteins of Olfactroy Receptor OR1and OR2 with Co-IP Approach Followed by Mass Spectrometry Analysis in Apis cerana cerana [J]. Fujian Journal of Agricultural Sciences,2024,39(1):7−16 doi: 10.19303/j.issn.1008-0384.2024.01.002
Citation: GUO L N, SHEN H Y, WANG J, et al. Interacting Proteins of Olfactroy Receptor OR1and OR2 with Co-IP Approach Followed by Mass Spectrometry Analysis in Apis cerana cerana [J]. Fujian Journal of Agricultural Sciences,2024,39(1):7−16 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
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

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

  • 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OR1OR2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。
  • 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.

    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.

    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.

    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

    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

    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_Flag79 1372 7341 487565
    OR1_IgG76 0291 153699317
    OR2_Flag75 1602 0551 280481
    OR2_IgG76 0201 212767318
    下载: 导出CSV

    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
    A0A8B6Z0H7ATP合酶β亚单位 ATP synthase subunit beta 51655.15.41150.99
    A0A8B8H9N3微管蛋白β链 Tubulin beta chain44749.94.89137.54
    A0A8B9B4M6微管蛋白β链 Tubulin beta chain455514.86135.05
    A0A8B9B2J4微管蛋白α链 Tubulin alpha chain45050.25.2133.21
    A0A8B6WZM5肌动蛋白相关蛋白1 Actin related protein 137641.85.48132.96
    A0A8B6Z7B8微管蛋白α链 Tubulin alpha chain45049.95.14127.76
    A0A8B8GXQ7Actin肌动蛋白37641.75.48105.7
    A0A8B6Z7F0延伸因子1-α异构体X1 Elongation factor 1-alpha isoform X146150.39.03105.16
    A0A7M6UVC2未知蛋白 Uncharacterized protein64070.35.8282.67
    A0A8B6WZH6热休克蛋白同源3前体 Heat shock protein cognate 3 precursor65872.85.4379.69
    A0A8B6WZD4热休克蛋白83 Heat shock protein 8372483.35.0679.38
    A0A8B6YS79微管蛋白α链 Tubulin alpha chain44850.45.4374.64
    A0A8B9AWD0ATP合酶亚单位 ATP synthase subunit alpha 54759.58.974.6
    A0A7M7GZK8热休克蛋白83 Heat shock protein 8371882.75.0573.46
    A0A7M7GKN6未知蛋白 Uncharacterized protein35736.99.3873.41
    A0A8B6WZI3热休克同源蛋白4 Heat shock protein cognate 4650715.5870.79
    A0A8B6Z8U0组蛋白H4 Histone H410311.411.3669.62
    A0A7M7MSJ5ATP依赖性解旋酶brm ATP-dependent helicase brm 2019217.67.1457.85
    A0A8B9B3I360S核糖体蛋白L23 60S ribosomal protein L23 14014.810.5855.71
    A0A8B7KRE5SWI/SNF复合亚基SMARCC2 SWI/SNF complex subunit SMARCC2 1026114.46.0550.7
    A0A7M7LPS540S核糖体蛋白S5 40S ribosomal protein S5 21624.19.4547.67
    A0A8B7KKF5前信使RNA加工剪接因子8 Pre-mRNA-processing-splicing factor 8 23742778.8844.2
    A0A8B6Z1L060S核糖体蛋白L10a 60S ribosomal protein L10a 21724.69.7641.69
    A0A7M7R4Y7未知蛋白 Uncharacterized protein35838.57.0241.56
    A0A7M7IR0740S核糖体蛋白S9 40S ribosomal protein S9 19322.510.7440.63
    A0A8B6Z285囊泡融合ATP酶 Vesicle-fusing ATPase80088.85.3340.6
    A0A8B6Z28014-3-3蛋白zeta异构体X1 14-3-3 protein zeta isoform X1 24728.14.8939.95
    A0A7M7GMY7含有14_3_3结构域的蛋白质 14_3_3 domain-containing protein25629.14.8939.62
    A0A8B6Z1F626S蛋白酶体调节亚基8 26S proteasome regulatory subunit 8 40545.78.538.87
    A0A8B7KJ60平移伸长因子2亚型X1 Translation elongation factor 2 isoform X184494.56.5236.68
    A0A8B8H899钙转运ATP酶肌浆/内质网型X1亚型
    Calcium-transporting ATPase sarcoplasmic/
    endoplasmic reticulum type isoform X1
    1020112.15.5435.55
    A0A7M7TG4540S核糖体蛋白S18 40S ribosomal protein S18 18121.19.9635.13
    A0A8B6ZA3026S蛋白酶体调节亚基7 26S proteasome regulatory subunit 7 43448.46.0734.05
    A0A8B6YVR3RNA解旋酶 RNA helicase 72882.29.4233.23
    A0A7M7RB98网格蛋白重链 Clathrin heavy chain16781925.9432.91
    A0A7M7R7J9含有AAA结构域的蛋白质 AAA domain-containing protein44049.26.7632.26
    A0A7M7MT55微管蛋白α链 Tubulin alpha chain43248.25.0631.9
    A0A8B9B3X8RNA解旋酶 RNA helicase 40346.26.2431.23
    A0A8B6YTH0α链异构体X2 Spectrin alpha chain isoform X2 2418278.25.2631.03
    A0A8B6YUN0钙调素依赖性蛋白激酶 Calcium/calmodulin-dependent protein kinase47453.97.8830.31
    A0A7M7R795RNA解旋酶 RNA helicase 42348.15.4930.17
    A0A7M7L992ras相关蛋白Rab-5C Ras-related protein Rab-5C 21423.48.1330.16
    A0A8B9B6N2v型质子ATP酶催化亚基A V-type proton ATPase catalytic subunit A 61668.35.4329.78
    下载: 导出CSV

    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 chain44750.14.86186.38
    A0A8B6Z0H7ATP合酶β亚单位 ATP synthase subunit beta 51655.15.41118.08
    A0A8B6WZM5肌动蛋白相关蛋白1 Actin related protein 1 37641.85.48112.87
    A0A8B6Z7B8微管蛋白α链 Tubulin alpha chain 45049.95.14110.42
    A0A8B8H9N3微管蛋白β链 Tubulin beta chain 44749.94.89106.87
    A0A8B9B2J4微管蛋白α链 Tubulin alpha chain 45050.25.2100.02
    A0A8B8GXQ7肌动蛋白 Actin 37641.75.4892.02
    A0A8B6Z7F0延伸因子1- α异构体X1 Elongation factor 1-alpha isoform X1 46150.39.0375.26
    A0A8B6WZD4热休克蛋白83 Heat shock protein 83 72483.35.0664.73
    A0A7M7GZK8热休克蛋白83 Heat shock protein 83 71882.75.0563.46
    A0A7M6UVC2未知蛋白 Uncharacterized protein 64070.35.8260.39
    A0A7M7GKN6未知蛋白 Uncharacterized protein 35736.99.3857.52
    A0A8B6WZH6热休克蛋白同源3前体 Heat shock protein cognate 3 precursor 65872.85.4351.8
    A0A8B6YS79微管蛋白α链 Tubulin alpha chain 44850.45.4351.66
    A0A8B6WZI3热休克同源蛋白4 Heat shock protein cognate 4 650715.5846.41
    A0A8B6Z8U0组蛋白H4 Histone H4 10311.411.3643.62
    A0A7M7MSJ5ATP依赖性解旋酶brm ATP-dependent helicase brm 2019217.67.1443.24
    A0A8B9AWD0ATP合酶亚单位 ATP synthase subunit alpha 54759.58.942.65
    A0A8B9B3I360S核糖体蛋白L23 60S ribosomal protein L23 14014.810.5834.65
    A0A7M7IR0740S核糖体蛋白S9 40S ribosomal protein S9 19322.510.7432.99
    A0A7M7R4Y7未知蛋白 Uncharacterized protein 35838.57.0230.64
    A0A8B6Z1F626S蛋白酶体调节亚基8 26S proteasome regulatory subunit 8 40545.78.529.63
    A0A7M7LPS540S核糖体蛋白S5 40S ribosomal protein S5 21624.19.4529.41
    A0A8B9B6N2V型质子ATP酶催化亚基A V-type proton ATPase catalytic subunit A 61668.35.4328.38
    A0A7M6UPZ640S核糖体蛋白S14亚型X1 40S ribosomal protein S14 isoform X1 15116.210.4527.66
    A0A8B7KRE5SWI/SNF复合亚基SMARCC2 SWI/SNF complex subunit SMARCC2 1026114.46.0527.6
    A0A8B6YVR3RNA解旋酶 RNA helicase 72882.29.4227.19
    A0A7M7RB98网格蛋白重链 Clathrin heavy chain 16781925.9425.73
    A0A8B8H899钙转运ATP酶肌浆/内质网型X1亚型
    Calcium-transporting ATPase sarcoplasmic/
    endoplasmic reticulum type isoform X1
    1020112.15.5424.84
    A0A7M7IK02RNA解旋酶 RNA helicase 56364.310.1424.52
    A0A7M7L992Rab-5C ras相关蛋白Rab-5C Ras-related protein 21423.48.1324.48
    A0A8B6Z1L060S核糖体蛋白L10a 60S ribosomal protein L10a 21724.69.7624.42
    A0A8B7KKF5前信使RNA加工剪接因子8 Pre-mRNA-processing-splicing factor 8 23742778.8823.78
    A0A8B6YTH0α链异构体X2 Spectrin alpha chain isoform X2 2418278.25.2623.34
    A0A7M7H2S3未知蛋白 Uncharacterized protein 1970227.15.522.78
    A0A8B6YUN0钙调素依赖性蛋白激酶 Calcium/calmodulin-dependent protein kinase 47453.97.8822.64
    A0A7M7TG0840S核糖体蛋白S23 40S ribosomal protein S23 1431610.5622.61
    A0A8B9B2J1依赖ATP的RNA解旋酶WM6 ATP-dependent RNA helicase WM6 42448.85.9522.47
    A0A7M7TG4540S核糖体蛋白S18 40S ribosomal protein S18 18121.19.9622.04
    A0A7M7R7J9含有AAA结构域的蛋白质 AAA domain-containing protein 44049.26.7621.88
    A0A8B8HD04Putative ATP-dependent RNA helicase me31b44450.67.7421.18
    A0A7M7LKA7组蛋白H2B Histone H2B 12313.710.3921.12
    A0A7M7R836类驱动蛋白 Kinesin-like protein 988112.46.1120.87
    A0A7M7LK91未知蛋白 Uncharacterized protein 13014.810.0520.81
    A0A8B6Z285囊泡融合ATP酶 Vesicle-fusing ATPase 80088.85.3320.56
    下载: 导出CSV
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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

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