枣食芽象甲化学感受蛋白PyasCSP4的基因克隆、表达及配体结合特征

doi:10.16380/j.kcxb.2022.07.005

摘要/Abstract

摘要： 【目的】本研究为明确枣树Zizyphus jujuba主要害虫枣食芽象甲Scythropus yasumatsui化学感受蛋白4(chemosensory protein 4, CSP4)的表达特点及配体结合特性。【方法】基于枣食芽象甲成虫触角转录组数据，利用RT-PCR方法克隆了枣食芽象甲PyasCSP4 cDNA序列，并进行生物信息学分析。通过RT-qPCR方法测定PyasCSP4在枣食芽象甲成虫不同组织(触角、去除触角的头、胸、腹、足和翅)中的表
达水平。通过原核表达系统和镍柱纯化获得PyasCSP4重组蛋白，采用荧光竞争结合实验测定重组蛋白PyasCSP4与35种枣树挥发物的结合特性。【结果】克隆获得枣食芽象甲PyasCSP4的cDNA序列(GenBank 登录号: OK322362)，其开放阅读框全长为366 bp，编码121个氨基酸，N末端有18个氨基酸组成的信号肽序列， PyasCSP4成熟蛋白序列具有4个保守的半胱氨酸残基。RT-qPCR结果表明， PyasCSP4基因在成虫不同组织中均有表达，在触角和翅中的表达量显著高于在其他组织中的表达量。重组蛋白PyasCSP4与25种配体具有结合活性，尤其与α-蒎烯、α-水芹烯和罗勒烯等的结合能力最强，解离常数(Ki)值分别为6.29, 6.58和6.69 μmol/L。【结论】PyasCSP4能够与多种枣树挥发物结合，推测其可能在枣食芽象甲定位寄主植物的过程中发挥重要作用。本研究的结果对阐明枣食芽象甲嗅觉机制和开展绿色防控研究具有重要意义。

关键词: 枣食芽象甲, 化学感受蛋白, 配体结合特性, 原核表达, 荧光竞争结合实验

Abstract: 【Aim】 This study aims to clarify the expression features and ligand binding characteristics of chemosensory protein 4 (CSP4) in Scythropus yasumatsui, a major pest of Zizyphus jujuba. 【Methods】 Based on the antennal transcriptome data of S. yasumatsui
adults, the cDNA sequence of PyasCSP4 was cloned by using RT-PCR and then subjected to bioinformatical analysis. The expression levels of PyasCSP4 in different adult tissues (antennae, heads without antennae, thoraxes, abdomens, legs and wings) of S. yasumatsui adults were assayed by RT-qPCR. The recombinant PyasCSP4 was prokaryotically expressed and then purified by Ni-NTA resin. The binding characteristics of the recombinant PyasCSP4 with 35 jujube volatiles were assayed by fluorescence competitive binding assay. 【Results】 The cDNA sequence of PyasCSP4 (GenBank accession no. OK322362) was cloned from S. yasumatsui. It contains an open reading frame of 366 bp in length, encoding 121 amino acids with a signal peptide of 18 amino acids at the N-terminus. The mature protein of PyasCSP4 possesses four conserved cysteines. The RT-qPCR analysis result showed that PyasCSP4 was ubiquitously expressed in all tissues of S. yasumatsui adults. The expression levels of PyasCSP4 in adult antennae and wings were significantly higher than those in other tissues. The recombinant PyasCSP4 had binding activities to 25 ligands, and particularly had the strongest binding affinity to α-pinene, α-phellandrene and ocimene, with the dissociation constant (Ki) values of 6.29, 6.58 and 6.69 μmol/L, respectively. 【Conclusion】 PyasCSP4 has the capability to bind a variety of jujube volatiles, indicating that it may play an important role in locating host plants for S. yasumatsui. The results have great significance for elucidating the olfactory mechanism of S. yasumatsui and implementing green prevention and control measures.

Key words: Scythropus yasumatsui, chemosensory protein, ligand binding characteristics; prokaryotic expression, fluorescence competitive binding assay

洪波, 常青, 翟颖妍, 任博文, 谢毓芬, 张锋. 枣食芽象甲化学感受蛋白PyasCSP4的基因克隆、表达及配体结合特征[J]. 昆虫学报, 2022, 65(7): 831-842.

HONG Bo, CHANG Qing, ZHAI Ying-Yan, REN Bo-Wen, XIE Yu-Fen, ZHANG Feng. cDNA cloning, expression and ligand binding properties of the chemosensory protein PyasCSP4 in Scythropus yasumatsui (Coleoptera: Curculionidae)[J]. Acta Entomologica Sinica, 2022, 65(7): 831-842.

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枣食芽象甲化学感受蛋白PyasCSP4的基因克隆、表达及配体结合特征

cDNA cloning, expression and ligand binding properties of the chemosensory protein PyasCSP4 in Scythropus yasumatsui (Coleoptera: Curculionidae)

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