昆虫学报 ›› 2022, Vol. 65 ›› Issue (3): 364-385.doi: 10.16380/j.kcxb.2022.03.012

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昆虫气味受体的研究方法与进展

白鹏华1,2,3, 王冰1,*, 张仙红3, 王桂荣1,*   

  1.  (1. 中国农业科学院植物保护研究所, 植物病虫害生物学国家重点实验室, 北京100193; 2. 天津市农业科学院植物保护研究所, 天津300384; 3. 山西农业大学植物保护学院, 山西太谷030801)
  • 出版日期:2022-03-20 发布日期:2022-03-24

Research methods and advances of odorant receptors in insects

BAI Peng-Hua1, 2, 3, WANG Bing1,*, ZHANG Xian-Hong3, WANG Gui-Rong1,*   

  1.  (1. State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China; 2. Institute of Plant Protection, Tianjin Academy of Agricultural Sciences, Tianjin 300384, China; 3. College of Plant Protection, Shanxi Agricultural University, Taigu, Shanxi 030801, China)
  • Online:2022-03-20 Published:2022-03-24

摘要:

昆虫气味受体(odorant receptors, ORs)在外周嗅觉系统识别气味信号过程中起着关键作用,参与昆虫取食、交配和产卵等重要生命活动过程。随着测序技术飞速发展以及生物信息学研究的深入,昆虫气味受体基因实现了大规模鉴定。本文详述了昆虫气味受体基因的研究方法及其原理和优缺点,归纳总结了自2015年以来已发表文章中采用不同分析方法进行ORs鉴定和功能研究的数量和比例,列举了重要害虫气味受体的研究进展。2015-2019年利用基因组和转录组测序技术分别鉴定了2 543和5 111个OR基因。基因表达和蛋白定位研究有助于分析OR基因在不同组织以及不同发育阶段的表达特异性,荧光原位杂交和免疫组织化学方法常用于分析细胞和组织定位;半定量反转录PCR(semi-quantitative reverse transcription PCR, RT-PCR)技术和实时荧光定量PCR(quantitative real-time PCR, qRT-PCR)技术常用于研究OR基因的时空表达模式。利用冷冻电镜技术解析昆虫OR蛋白的超微晶体结构,以明确OR关键氨基酸残基与配体化合物互作规律。ORs功能研究手段不断丰富,体外异源表达系统在2015-2019年间的占比为75.76%,以爪蟾卵母细胞Xenopus oocytes系统结合双电极电压钳技术(two-electrode voltage clamp, TEVC)为主(占比43.94%),辅以转基因果蝇异源表达系统结合单感器记录技术以及细胞系异源表达ORs结合钙离子成像技术;体内功能研究方法包括RNA干扰(RNAi)技术和CRISPR/Cas9基因编辑技术,后者在ORs功能研究中取得了突破性进展并具有广泛的应用前景。最后本文对今后的研究方向提出了以下建议:(1)开展更多重大害虫及天敌昆虫嗅觉识别机制研究;(2)阐明昆虫性信息素和寄主植物挥发物协同作用的内在机理;(3)解析气味受体蛋白超微晶体结构并揭示ORs与气味分子特异性识别机制;(4)基于RNAi技术开发新型昆虫行为调节剂。

关键词: 气味受体, 基因鉴定, 表达定位, 双电极电压钳, 单感器记录, RNA干扰, 基因编辑技术

Abstract: Insect odorant receptors (ORs) play critical roles in the peripheral olfactory system and are involved in such vital life events in insects as feeding, mating and oviposition. With the development of sequencing technologies and bioinformation tools, insect OR genes have been widely identified in recent years. In this article, we comprehensively reviewed the research methods of insect OR genes, and their principles, advantages and disadvantages. Furthermore, we summerized the numbers and proportions of research methods published for identification and functional studies of ORs since 2015. Besides, we listed the research advances of ORs in serious insect pests. A total of 2 543 and 5 111 OR genes had been respectively identified through genome and transcriptome sequencing analyses during 2015 to 2019. Gene expression and protein localization analysis are used to analyze the expression specificities of OR genes in different tissues and developmental stages. The in situ hybridization and immunohistochemistry are used for analyzing cellular and tissue localization, while the semi-quantitative reverse transcription PCR (RT-PCR) and quantitative real-time PCR (qRT-PCR) are often used to study the spatiotemporal expression profiles of OR genes. Otherwise, cryo-electron microscopy (Cryo-EM) is employed to elucidate the OR micro-crystal structure and to demonstrate the interactions between critical amino acid residues and ligands. Multiple approaches are developed for functional characterization of ORs. In vitro heterologous expression systems are commonly used to study the function of insect ORs, representing 75.76% of the research methods published from 2015 to 2019. The most abundantly used in vitro systems are through heterologous expression in Xenopus oocytes with two-electrode voltage clamp system (43.94%), followed by transgenic Drosophila with single sensillum recording (SSR) technique and cell line expression systems with calcium imaging. In vivo research methods include RNA interference (RNAi) and clustered regularly interspaced short palindromic repeats (CRISPR)-associated nuclease Cas9 (CRISPR/Cas9) gene edting technique. The latter has made great breakthroughs in OR deorphanization, and has a great prospective application for further studies. Finally, we suggest the future research directions for insect ORs including, (1) studying the molecular mechanism of olfaction in serious insect pests and natural enemies; (2) elucidating the molecular mechanism of synergism to insect sex pheromones and host plant volatiles; (3) analyzing the micro-crystal structure of ORs and explore the specific recognition mechanisms of ORs and odorants; and (4) developing behavioral regulation products in insects through RNAi technique.

Key words: Odorant receptor, gene identification, expression and localization, two-electrode voltage clamp, single sensillum recording, RNA interference, gene editing technology