南亚果实蝇CSP基因鉴定及其触角高表达CSP的配体结合特性

Identification of CSP genes and ligand binding properties of antenna-enriched CSP in Bactrocera tau (Walker)

  • 摘要: 【目的】 筛选在南亚果实蝇成虫触角中高表达的化学感受蛋白(CSP)基因,并对CSP基因异源表达蛋白的配体结合特性进行研究,为揭示CSP基因在南亚果实蝇成虫嗅觉感受中的功能及研发高效引诱剂提供理论参考。【方法】 通过逆转录聚合酶链反应(RT-PCR)分析南亚果实蝇CSP基因(BtauCSP1、BtauCSP2、BtauCSP3、BtauCSP4)在雌、雄成虫触角、头(去触角)、其他组织(去头部)的表达模式;构建重组表达载体pET-28a(+)/BtauCSP3,将重组表达载体转入原核细胞BL21(DE3)中进行原核表达;通过荧光竞争结合试验分析细菌表达的BtauCSP3蛋白与16种气味配体的结合特性。【结果】 4个南亚果实蝇CSP基因BtauCSP1BtauCSP2BtauCSP3BtauCSP4均具有完整的开放阅读框(ORF),长度为336~474 bp,编码112~158个氨基酸残基。4个BtauCSPs蛋白一级结构的氨基端均含有一段由18~24个氨基酸残基组成的信号肽序列;去除信号肽的成熟BtauCSP蛋白的预测分子量为10.03~14.95 kD,理论等电点为5.04~9.32。对BtauCSPs基因在南亚果实蝇成虫不同组织中的表达模式分析结果显示,4个BtauCSPs基因在供试所有组织中均有表达,其中BtauCSP3基因在南亚果实蝇雌、雄成虫触角中高表达,暗示BtauCSP3基因可能参与南亚果实蝇的嗅觉识别过程。荧光竞争结合试验结果显示,BtauCSP3蛋白与十一醇、诱蝇酮和柠檬烯3种气味化合物表现出结合能力,抑制常数(Ki)分别为15.44、23.16和23.67 μmol/L。【结论】 南亚果实蝇成虫触角中高表达的BtauCSP3基因可能参与了南亚果实蝇对气味化合物尤其是十一醇的嗅觉识别过程。

     

    Abstract: 【Objective】 This study aimed to identify antenna-enriched chemosensory protein (CSP) genes in adult Bactrocera tau (Walker), and to investigate the ligand binding properties of heterologously expressed proteins of CSP genes, thereby providing a theoretical basis for elucidating the roles of CSP genes in the olfactory perception of adult Bactrocera tau (Walker) and developing efficient attractants. 【Method】 Reverse transcription polymerase chain reaction (RT-PCR) was used to analyze the expression patterns of CSP genes (BtauCSP1BtauCSP2BtauCSP3, BtauCSP4) in antennae, heads (without antennae) and other tissues (without heads) of male and female Bactrocera tau (Walker) adults. Recombinant expression vectors pET-28a(+)/BtauCSP3 were constructed, and transferred into the prokaryotic cell BL21 (DE3) for prokaryotic expression. The ligand binding properties of the bacterially expressed protein BtauCSP3 with 16 odorant ligands were analyzed by fluorescence competitive binding assays. 【Result】 Four CSP genes (BtauCSP1BtauCSP2BtauCSP3, and BtauCSP4) in Bactrocera tau (Walker) contained complete open reading frames (ORF) ranging from 336 to 474 bp, encoding 112-158 amino acid residues. The amino terminals of primary structure of 4 BtauCSPs proteins included signal peptide sequences (18-24 amino acid residues). The predicted molecular weight of the mature BtauCSP protein with the signal peptide removed was 10.03-14.95 kD and the theoretical isoelectric point was 5.04-9.32. Accor-ding to the expression pattern analysis of BtauCSPs genes in different tissues of adult Bactrocera tau (Walker), four BtauCSPs genes were expressed in all tested tissues, and the BtauCSP3 gene exhibited expression of high abundance in antennae of male and female Bactrocera tau (Walker) adults, implying that the BtauCSP3 gene may be involved in the olfactory recognition process of Bactrocera tau (Walker). Fluorescence competitive binding assays demonstrated that the BtauCSP3 protein exhibited binding affinities to three host volatiles: undecanol, 4-(4-acetoxyphenyl)-2-butanone, and (R)-(+)-limonene; the inhibition constant (Ki values were 15.44, 23.16 and 23.67 μmol/L, respectively. 【Conclusion】 The high-expressed BtauCSP3 gene in the antennae of adult Bactrocera tau (Walker) may be involved in the olfactory recognition of odorant compounds, particularly for odor compounds such as undecanol.

     

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