悬铃木方翅网蝽化学感受蛋白CcilCSP1的结构及其结合寄主挥发物的预测分析

doi:10.11707/j.1001-7488.20171012

摘要/Abstract

摘要： [目的]分析悬铃木方翅网蝽化学感受蛋白编码基因CcilCSP1的序列和表达特征，重点研究CcilCSP1结合二球悬铃木寄主植物挥发物的能力，旨在为CcilCSP1的功能研究提供理论参考，为通过化学感受蛋白反向模拟筛选悬铃木方翅网蝽的信息素物质提供依据。[方法]用分子克隆技术获得悬铃木方翅网蝽化学感受蛋白CcilCSP1基因ORF的全长序列，用RT-qPCR研究该基因的表达模式，并通过生物信息学方法分析其序列特征。用Swiss-model构建CcilCSP1蛋白的三维结构，并通过AutoDock 4.2开展CcilCSP1蛋白与寄主植物9种挥发物成份的分子对接，用Amber 14对其中结合最为稳定的反式-β-石竹烯与CcilCSP1蛋白的复合物开展了10 000 ps的分子动力学模拟，并进一步通过"Y"型嗅觉仪测定反式-β-石竹烯对悬铃木方翅网蝽成虫的行为学影响。[结果]成功克隆到该虫的化学感受蛋白基因CcilCSP1，且该基因在成虫中显著高表达。序列分析表明，CcilCSP1具有CSPs家族的典型特征，该基因与绿盲蝽的AlucCSP1，AlucCSP3，AlucCSP8以及苜蓿盲蝽的AlinCSP1亲缘关系较近。同源建模和分子对接发现，在二球悬铃木9种植物挥发物中反式-β-石竹烯与CcilCSP1的结合能力最强（k_l=2.63 μm，Binding Energy=-7.61），通过分析二者的结合特性，推测TYR-29、GLN-83、LEU-64、ASP-30可能是CcilCSP1蛋白结合反式-β-石竹烯所需的关键氨基酸位点。分子动力学模拟结果显示，CcilCSP1蛋白能够与反式-β-石竹烯稳定结合。进一步的行为学试验表明，0.1 μg的反式-β-石竹烯对悬铃木方翅网蝽成虫有显著的驱避作用。[结论]本研究克隆到在成虫中显著高表达的悬铃木方翅网蝽化学感受蛋白基因CcilCSP1。用分子对接和分子动力学模拟方法发现CcilCSP1能与二球悬铃木寄主挥发物成份反式-β-石竹烯稳定结合，并通过进一步的行为学试验进行证实。该研究可为悬铃木方翅网蝽化学感受蛋白CcilCSP1的功能研究奠定基础，也为基于计算机反向筛选获得悬铃木方翅网蝽信息素物质研发提供依据。

关键词: 悬铃木方翅网蝽, 化学感受蛋白, 寄主植物挥发物, 分子对接, 分子动力学模拟

Abstract: [Objective] Corythucha ciliata (Say) is an important invasive forest pest, and seriously damages Platanus spp. This paper analyzes the sequence and expression characteristics of chemosensory proteins 1 (CcilCSP1), focus on the ability of CcilCSP1 binding to host volatiles of P. acerifolia. This study gims to provide a foundation for studying function of the chemosensory protein and a useful reference for searching the pheromone substance.[Method] The full-length ORF sequence of CcilCSP1 was obtained by the molecular cloning technique. The expression pattern of CciCSP1 was analyzed by RT-qPCR. The three-dimensional structure of chemosensory protein CcilCSP1 was mimicked, and nine plant volatiles were docked to the CcilCSP1 by AutoDock 4.2. The molecular dynamics simulation analysis was carried out to the docking complex of trans-β-caryophyllene and CcilCSP1. The effects of trans-β-caryophyllene on the C. ciliata were assayed by Y-tube olfactometer.[Result] The CcilCSP1 gene of C. ciliata was cloned and sequenced, and it was highly expressed in adults. The sequence analyses showed that CcilCSP1 had the typical characteristics of CSPs, and closely related with AlucCSP1, AlucCSP3, AlucCSP8 and AlinCSP1. The binding capacity of trans-β-caryophyllene to CcilCSP1 was strongest (k_l=2.63 μm, Binding Energy=-7.61) in nine volatiles. It is suggested that TYR-29, GLN-83, LEU-64 and ASP-30 may be ligand-binding active sites. Molecular dynamics simulations showed that CcilCSP1 protein could bind to trans-β-caryophyllene stably. Furthermore, behavioral experiments showed that 0.1 μg trans-β-caryophyllene had a significant evading effect on the adults of C. ciliata.[Conclusion] CcilCSP1, a chemosensory protein gene highly expressed in C. ciliata adults, was cloned and sequenced in this study. Molecular docking and molecular dynamics simulations were used to investigate the interaction between CcilCSP1 and trans-β-caryophyllene, which was confirmed by the behavioral experiment. This study lays a foundation for the functional study of the protein CcilCSP1 of C. ciliata, and provid a useful reference for searching for the pheromone substance.

Key words: Corythucha ciliata, chemosensory proteins(CSPs), homology modeling, host-plant volatiles, molecular docking, dynamics simulation

中图分类号:

S718.7

付宁宁, 刘佳, 渠成, 王然, 许奕华, 罗晨, 李峰奇. 悬铃木方翅网蝽化学感受蛋白CcilCSP1的结构及其结合寄主挥发物的预测分析[J]. 林业科学, 2017, 53(10): 109-117.

Fu Ningning, Liu Jia, Qu Cheng, Wang Ran, Xu Yihua, Luo Chen, Li Fengqi. Analysis of Corythucha ciliata CcilCSP1 Structure and Prediction of Its Binding to Host-Plant Volatiles[J]. Scientia Silvae Sinicae, 2017, 53(10): 109-117.

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