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

Abstract:  【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 (dsEGFP 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.

Key words: Frankliniella intonsa, aggregation pheromone, chemosensory protein, RNA interference, site-directed mutagenesis, fluorescence binding assay