邓培渊 王林青 袁洪哲 李长看

摘要 为阐明甜菜夜蛾Spodoptera exigua化学感受蛋白识别外源化合物的特性和作用机制，运用Western blotting法分析SexiCSP2蛋白在成虫各组织的表达谱，荧光竞争结合试验法测定SexiCSP2蛋白与25种外源配基的结合特性，运用分子对接、分子动力学、结合自由能计算等方法研究SexiCSP2与配基的结合模式。结果显示SexiCSP2在成虫接触和非接触性器官中均有表达，与己醛结合能力最强，解离常数为1.3 μmol/L;分子模拟结果显示SexiCSP2与己醛结合形成1个氢键，两者结合自由能为-32.94 kJ/mol，主要驱动力为范德华力，主要抑制力为非极性溶剂化能。这些结果说明SexiCSP2在甜菜夜蛾识别外源化合物中起重要作用，可以作为防控甜菜夜蛾的有效靶标。

关键词 甜菜夜蛾; 化学感受蛋白; Western blotting; 荧光竞争结合试验; 分子动力学

中图分类号： Q 966

文献标识码： A

DOI： 10.16688/j.zwbh.2020372

The binding specificity and mechanism of chemosensory protein SexiCSP2 in the beet armyworm， Spodoptera exigua

DENG Peiyuan， WANG Linqing， YUAN Hongzhe， LI Changkan*

（Zhengzhou Normal University， Research Centre of Biological Species Resources， Zhengzhou 450044， China）

Abstract

To determine the binding characteristics and mechanism of Spodoptera exigua chemosensory proteins （CSPs） in discriminating exogenous compounds， the expression profiles of SexiCSP2 in adult tissues were analyzed by Western blotting， the binding specificities of SexiCSP2 and 25 foreign ligands were tested through fluorescence competitive binding assay. Moreover， the binding pattern was studied by using molecular docking， molecular dynamics simulation and binding free energy calculation methods. The results showed that SexiCSP2 was expressed in both contact and non-contact organs. It had a strongest binding ability to hexanal with a dissociation constant of 13 μmol/L. Molecular simulation results showed that hexanal formed a hydrogen bond with SexiCSP2， and the binding free energy was -32.94 kJ/mol; the van der Wals was the main driving force， and the nonpolar solvation energy was the main resistance force. These data indicated that SexiCSP2 might play an important role in the process of discriminating exogenous compounds and could be used as an effective target for the prevention and control of S.exigua.

Key words

Spodoptera exigua; chemosensory protein; Western blotting; fluorescence competitive binding assay; molecular dynamics

昆蟲之间以及昆虫与外界环境之间联系主要靠化学通讯来完成，研究发现昆虫触角感受器中存在几类在感受外界化学信息过程中起关键作用的蛋白，如气味结合蛋白（odorant binding protein， OBP）[1-2]和化学感受蛋白（chemosensory protein， CSP）[3]等。它们与昆虫寻找寄主、交配产卵、躲避天敌等行为密切相关[4]。OBP主要结合和转运挥发性的化合物，CSP主要结合和转运非挥发性的化合物，与OBP相比，CSP的分布和功能更加多样化[5]，如烟芽夜蛾Heliothis virescens的CSP在足部有较高的表达量[6];东亚飞蝗Locusta migratoria的部分CSP在生殖器中特异表达[7];美洲大蠊Periplaneta americana的PameP10在新生足中的表达量是正常足的30倍，可能参与了足的再生[8];甜菜夜蛾SexiCSP3在雌虫卵巢中的表达量远高于其在雄虫精巢中的表达量，参与调控产卵行为[9];意大利蜜蜂Apis mellifera ligustica的CSP5在卵中特异表达，影响了幼虫体壁的发育[10]等。……