花蓟马化学感受蛋白FintCSP2与聚集信息素苨肉基(S)-2-甲基丁酸酯的结合特性  

Binding characteristics of chemosensory protein FintCSP2 of Frankliniella intonsa(Thysanoptera:Thripidae)to its aggregation pheromone neryl(S)-2-methylbutanoate

作　　者：李恒 田厚军[1] 陈艺欣[1] 林硕[1] 魏辉[1] 陈勇[1] LI Heng;TIAN Hou-Jun;CHEN Yi-Xin;LIN Shuo;WEI Hui;CHEN Yong(Fujian Key Laboratory for Monitoring and Integrated Management of Crop Pests,Fuzhou Scientific Observing and Experimental Station of Crop Pests,Ministry of Agriculture,Institute of Plant Protection,Fujian Academy of Agricultural Sciences,Fuzhou 350013,China)

机构地区：[1]福建省农业科学院植物保护研究所,福建省作物有害生物监测与治理重点实验室,农业部福州作物有害生物科学观测试验站,福州350013

出　　处：《昆虫学报》2024年第7期897-908,共12页Acta Entomologica Sinica

基　　金：福建省自然科学基金项目(2023 J06040);中央引导地方科技发展专项(2023L3021);福建省农业科学院科技项目(YCZX202405,CXTD2021004-3,XTCXGC2021011,XTCXGC2021017)。

摘　　要：【目的】本研究旨在明确花蓟马Frankliniella intonsa化学感受蛋白(chemosensory protein,CSP)FintCSP2与聚集信息素苨肉基(S)-2-甲基丁酸酯[neryl(S)-2-methylbutanoate]的结合能力。【方法】利用RT-PCR法扩增花蓟马FintCSP2的开放阅读框序列,并对其进行生物信息学分析;利用RT-qPCR检测FintCSP2在花蓟马雌成虫不同组织(触角、去除触角的头、胸、腹和足)中的表达量;利用RNAi通过向花蓟马雌成虫注射dsRNA沉默FintCSP2,24 h时通过触角电位(electroantennogram,EAG)实验检测花蓟马对苨肉基(S)-2-甲基丁酸酯的反应,利用Y型嗅觉仪测定花蓟马雌成虫对苨肉基(S)-2-甲基丁酸酯的的选择性;原核表达FintCSP2重组蛋白,利用荧光竞争结合实验检测FintCSP2重组蛋白与苨肉基(S)-2-甲基丁酸酯结合力;采用分子对接模拟技术和蛋白定点突变技术分析FintCSP2与苨肉基(S)-2-甲基丁酸酯结合的关键氨基酸残基。【结果】花蓟马FintCSP2(GenBank登录号:MT211602.1)开放阅读框长390 bp,编码129个氨基酸,N端有一个包含20个氨基酸的信号肽,含有4个保守的半胱氨酸。氨基酸序列分析结果表明,FintCSP2氨基酸与花蓟马CSP1(GenBank登录号:WBW64307.1)、西花蓟马F.occidentalis CSPs(GenBank登录号:WBW64306.1,AJL33750.1)和牛角花齿蓟马Odontothrips loti CSP2(GenBank登录号:WBU77202.1)的亲缘关系最近,氨基酸序列一致性分别为99.22%,99.22%,86.05%和65.85%。RT-qPCR结果表明,FintCSP2在雌成虫各组织中均有表达,其中在触角中的表达量最高。与对照组(注射ds EGFP)比,沉默FintCSP2显著降低花蓟马对苨肉基(S)-2-甲基丁酸酯的EAG反应绝对值和选择率。分子对接预测Tyr24,Phe29,Leu38,Val71,Cys76,Cys79和Gln83这7个氨基酸残基最可能参与FintCSP2结合苨肉基(S)-2-甲基丁酸酯;定点突变和荧光竞争结合实验结果表明,与野生型蛋白相比,FintCSP2-Tyr24Ala和FintCSP2-Gln83Ala突变蛋白与苨肉基(S)-2-甲基丁酸酯结合能力【Aim】The aim of this study is to clarify the binding ability of chemosensory protein(CSP)of Frankliniella intonsa(FintCSP2)to the aggregation pheromone neryl(S)-2-methylbutanoate.【Methods】The open reading frame sequence of FintCSP2 was amplified from F.intonsa by RT-PCR and analyzed with bioinformatics methods.The expression levels of FintCSP2 in different tissues(antennae,head without antennae,thorax,abdomen and leg)of female adult of F.intonsa were analyzed by RT-qPCR.FintCSP2 was silenced using RNAi by injection of dsRNA into female adults,electroantennogram(EAG)assay was used to detect the reaction of F.intonsa to neryl(S)-2-methylbutanoate,and the selectivity of female adult of F.intonsa to neryl(S)-2-methylbutanoate was determined by Y-tube olfactometer at 24 h after RNAi.The recombinant FintCSP2 protein was obtained by prokaryotic expression,and the binding ability of the recombinant FintCSP2 protein to neryl(S)-2-methylbutanoate was determined by fluorescence competitive binding assay.The key amino acid residues of FintCSP2 protein binding to neryl(S)-2-methylbutanoate were analyzed by molecular docking simulation and site-directed mutagenesis.【Results】The open reading frame of FintCSP2(GenBank accession number:MT211602.1)of F.intonsa is 390 bp in length,encoding 129 amino acids.The FintCSP2 protein has a signal peptide containing 20 amino acids at the N-terminus and four conserved cysteines.The amino acid sequence analysis result showed that FintCSP2 was the most closely related to CSP1(GenBank accession number:WBW64307.1)of F.intonsa,CSPs(GenBank accession number:WBW64306.1,AJL33750.1)of F.occidentalis and CSP2(GenBank accession number:WBU77202.1)of Odontothrips loti,with the amino acid sequence identities of 99.22%,99.22%,86.05%and 65.85%,respectively.RT-qPCR analysis result showed that FintCSP2 was expressed in various tissues of female adult,with the highest expression level in the antennae.Silencing of FintCSP2 significantly decreased the EAG absolute value and selection rate of F.intons

关 键 词：花蓟马 聚集信息素 化学感受蛋白 RNA干扰 定点突变 荧光竞争结合实验 

分 类 号：Q964[生物学—昆虫学]