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

【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显著下降,FintCSP2-Phe29Ala突变体蛋白失去与苨肉基(S)-2-甲基丁酸酯的结合能力。【结论】花蓟马FintCSP2蛋白在识别苨肉基(S)-2-甲基丁酸酯过程中起关键作用,Tyr24,Phe29和Gln83是FintCSP2结合苨肉基(S)-2-甲基丁酸酯的3个关键氨基酸残基。

【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.intonsa to neryl(S)-2-methylbutanoate compared with the control group(ds EGFP injection).Molecular docking predicted that seven residues,Tyr24,Phe29,Leu38,Val71,Cys76,Cys79 and Gln83 were most likely involved in the process of FintCSP2 binding to neryl(S)-2-methylbutanoate.Site-directed mutagenesis and fluorescence competitive binding assay result showed that compared to the wild-type protein,the two mutants,FintCSP2-Tyr24Ala and FintCSP2-Gln83Ala showed significantly decreased binding ability to neryl(S)-2-methylbutanoate,and FintCSP2-Phe29Ala lost its binding ability to neryl(S)-2-methylbutanoate.【Conclusion】FintCSP2 of F.intonsa plays a key role in recognition of neryl(S)-2-methylbutanoate,and Tyr24,Phe29 and Gln83 are the three key amino acid residues in FintCSP2 that affect its binding to neryl(S)-2-methylbutanoate.