亚洲玉米螟气味受体识别产卵化合物的分子机制

Odorant receptors (ORs) plays an important role in insect olfactory function, is the center of insect olfaction response. In addition, the ORs, which is highly expressed in female moths, is generally involved in controlling female specific behaviors such as spawning selection. Previous researches found that, a variety of compounds selected from maize leaves, which having a regulatory role of the Asian corn borer (Ostrinia furnacalis, ACB) oviposition behavior (referred to as oviposition compounds). However, the mechanism of recognizing these oviposition compounds by ACB is still unclear. In addition, the three ORs which were highly expressed in female moths were selected from the transcriptome data of the female and male moth of ACB. In this study, these three ORs genes will be cloned, and the identification of ORs candidate genes of oviposition compounds will be detected by vitro expression method for the establishment of identification system. The expression pattern of ORs in antennal sensilla will be studied by in situ hybridization, and the antennal nerve mechanism of female oviposition will be identified by Electroantennogram (EAG), single-sensillum recording (SSR). At last, these candidate genes were knocked out by CRISPR/Cas9 technology, and their functions in the ACB were analyzed by comparing their physiological and behavioral differences. Based on these tests, the molecular mechanisms of ACB female identification oviposition compounds will be elucidated. The study will provide a theoretical basis for the use of oviposition compounds for prevention of ACB.

气味受体是昆虫嗅觉识别反应的中心，在嗅觉功能中发挥着重要作用，其中雌蛾高表达气味受体一般参与调控产卵选择等雌性特异行为。申请人前期从玉米叶片中筛选出多种调控亚洲玉米螟雌蛾产卵行为的化合物（简称产卵化合物），但雌蛾识别这些产卵化合物的机制尚不清楚。为此，申请人从亚洲玉米螟雌、雄蛾触角转录组数据中筛选了雌蛾高表达的3个气味受体基因。本项目拟克隆这3个气味受体基因，利用双电极电压钳技术检测识别产卵化合物的气味受体候选基因；并采用原位杂交技术研究气味受体在触角感受器中的表达模式，通过触角电位（EAG）及单细胞记录（SSR）技术明确雌蛾识别产卵化合物的触角神经机制；最后利用CRISPR/Cas9技术敲除候选基因，比对基因突变型与野生型的电生理和行为学差异，确定其在亚洲玉米螟体内的功能。该研究将阐明亚洲玉米螟气味受体识别产卵化合物的分子机制，为利用产卵行为发展引诱剂和驱避剂防治亚洲玉米螟提供理论依据。

寄主植物气味对昆虫的寄主识别和产卵等行为具有重要作用；而气味受体是昆虫嗅觉识别反应的中心，在嗅觉功能中发挥着重要作用。在本项目中，申请人鉴定了35种玉米果穗挥发物，明确了亚洲玉米螟触角上嗅觉器官类型，确定了亚洲玉米螟对玉米挥发物的触角电位反应和单细胞记录反应，明确了其对法尼醇等61种化合物具有电生理反应；进一步分析了亚洲玉米螟对玉米挥发物的行为反应，结果表明其对法尼醇等18种化合物具有显著的行为反应。在此基础上，申请者在爪蟾卵母细胞中异源表达了OfurOR26 受体蛋白，利用双电极电压钳系统记录了该受体对活性气味的反应，OfurOR26/Orco能特异性结合法尼醇，对10-4 M 法尼醇电压钳计量反应值最大（280 nA），EC50 = 3.606 × 10-6 M。通过触角电位（EAG）技术检测发现，随着法尼醇浓度的增加，亚洲玉米螟EAG反应值逐渐增大，具有明显的EAG反应；行为实验结果表明，法尼醇在20和200 μg均能够驱避雌蛾产卵，且随着剂量增加，对其产卵驱避效果更好。本研究明确了亚洲玉米螟气味受体识别产卵化合物的分子机制，为利用产卵行为发展引诱剂和驱避剂防治亚洲玉米螟提供了理论依据。