• 中文核心期刊
  • CSCD来源期刊
  • 中国科技核心期刊
  • CA、CABI、ZR收录期刊

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

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

    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。

     

    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.

     

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