昆虫学报 ›› 2020, Vol. 63 ›› Issue (9): 1048-1058.doi: 10.16380/j.kcxb.2020.09.002

• 研究论文 • 上一篇    下一篇

绿盲蝽八个普通气味受体基因的克隆及功能鉴定

李彬, 张赛, 王晨蕊, 王桂荣*, 刘杨*   

  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)
  • 出版日期:2020-09-20 发布日期:2020-09-30

Cloning and functional characterization of eight odorant receptor genes in Apolygus lucorum (Hemiptera: Miridae)

LI Bin, ZHANG Sai, WANG Chen-Rui, WANG Gui-Rong*, LIU Yang*   

  1.  (State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China)
  • Online:2020-09-20 Published:2020-09-30

摘要: 【目的】本研究旨在克隆绿盲蝽Apolygus lucorum的气味受体基因,明确这些气味受体基因在绿盲蝽成虫不同组织中的表达水平及对主要寄主植物挥发物的电生理反应,为揭示绿盲蝽对寄主植物的识别机制提供理论基础。【方法】在绿盲蝽成虫触角转录组测序与分析的基础上,通过PCR技术克隆得到8个具有完整ORF的气味受体基因序列。用qPCR研究这8个基因在雌雄成虫不同组织(触角、无触角的头、胸、腹、足和翅)中的表达水平。然后通过爪蟾卵母细胞体外表达结合双电极电压钳技术测试这些气味受体对56种气味化合物的电生理反应。【结果】克隆得到了绿盲蝽8个气味受体基因AlucOR9, AlucOR16, AlucOR38, AlucOR53, AlucOR55, AlucOR56, AlucOR57和AlucOR58的cDNA全长序列(GenBank登录号: MN905538-MN905545)。这些气味受体含有7个跨膜结构域,且N末端位于胞内,C末端位于胞外,符合昆虫气味受体的典型特征。qPCR结果表明,这8个气味受体基因均在绿盲蝽成虫触角中高表达,而在其他组织中低表达,其中除AlucOR38外的其他7个气味受体基因在雌雄成虫触角间存在显著的表达差异。双电极电压钳记录结果显示,AlucOR57对其中15种气味化合物(苯甲醛、氧化石竹烯、庚醛、反-2-己烯醛、乙酸苯甲酯、桃金娘烯醛、4-乙基苯甲醛、乙酸壬酯、四氢芳樟醇、十三烷、反-3-己烯醇、2-丙烯酸丁酯、丙酸丁酯、乙酸辛酯和乙酸戊酯)有反应,其余7个气味受体对测试的全部气味化合物均无反应。【结论】AlucOR57对测试的一些气味化合物有反应,推测其在绿盲蝽的寄主识别过程中发挥重要作用;其他7个气味受体对测试气味化合物均无反应,其功能有待进一步研究。

关键词: 绿盲蝽, 触角, 气味受体, 电生理反应, qPCR, 双电极电压钳

Abstract: 【Aim】 This study aims to clone odorant receptor (OR) genes in Apolygus lucorum and to assay their expression levels in different adult tissues and their electrophysiological responses to host plant volatiles, so as to provide the theoretical basis for understanding the mechanism of host plant recognition in A. lucorum. 【Methods】 Based on the sequencing and analysis of the antennal transcriptome of A. lucorum, the full-length sequences of eight OR genes with intact ORF were cloned by PCR. The expression levels of these genes in different adult tissues (antenna, head without antenna, thorax, abdomen, leg and wing) were detected by using qPCR. The electrophysiological responses of these ORs to 56 odor compounds were tested in Xenopus oocytes as a heterologous expression system using two-electrode voltage clamp recording. 【Results】 The full-length cDNA sequences of 8 OR genes AlucOR9, AlucOR16, AlucOR38, AlucOR53, AlucOR55, AlucOR56, AlucOR57 and AlucOR58 (GenBank accession no.: MN905538-MN905545)of A. lucorum were cloned. These ORs contain seven transmembrane domains, with the intracellular N-terminus and extracellular C-terminus, consistent with the typical characteristics of insect ORs. The qPCR results showed that all the eight OR genes were highly expressed in adult antennae and lowly expressed in all the other tissues of A. lucorum. Moreover, the expression of the other seven OR genes except AlucOR38 in the antennae showed significant differences between male and female. The results of two-electrode voltage clamp recording showed that only AlucOR57 responded to 15 odor compounds [benzaldehyde, (-)-caryophyllene oxide, heptanal, (E)-2-hexenal, benzyl acetate, (1R)-(-)-myrtenal, 4-ethylbenzaldehyde, nonyl acetate, 3,7-dimethyl-3-octanol, tridecane, (E)-3-hexenol, butyl acrylate, butyl propionate, octyl acetate and amyl acetate], while the other seven ORs did not respond to all the odor compounds tested. 【Conclusion】 AlucOR57 responds to partial odor compounds tested, suggesting that it might be involved in the recognition of host plant volatiles in A. lucorum. The other seven ORs fail to respond to all the tested odor compounds, and their
functions need further study.

Key words:  Apolygus lucorum, antennae, odorant receptor, electrophysiological response; qPCR, two-electrode voltage clamp