苹果蠹蛾气味结合蛋白基因CpomOBP20的克隆及表达分析

Cloning and expression analysis of the odorant-binding protein gene CpomO BP20 in the codling moth,Cydia pomonella (Lepidoptera: Tortricidae)

【目的】通过克隆苹果蠹蛾气味结合蛋白CpomO BP20基因cD NA序列,分析其序列特征和表达谱,旨在更好地了解OBP基因在苹果蠹蛾生命活动过程中的作用,为该害虫的绿色防控提供理论支撑。【方法】采用RT-PCR法扩增苹果蠹蛾气味结合蛋白CpomO BP20基因cD NA序列,并使用生物信息学软件对其核苷酸和氨基酸序列进行分析;基于qP CR技术分析CpomO BP20基因在苹果蠹蛾4龄幼虫不同组织(头、血淋巴、表皮、脂肪体、中肠、马氏管和唾液腺)以及雌雄成虫不同末端组织(头、触角、下唇须、喙、足和翅)中的表达情况,利用分子对接研究了CpomO BP20与3种保幼激素的结合能力。【结果】苹果蠹蛾气味结合蛋白CpomO BP20基因的开放阅读框长459 bp,共编码152个氨基酸,等电点为6.30,蛋白分子质量为16.264 ku,N末端具有20个氨基酸组成的信号肽序列,蛋白质序列中具有6个保守的半胱氨酸残基,属于Classical OBP。序列分析表明,CpomO BP20的氨基酸序列与小菜蛾OBP (XP;11557123.1)的一致性最高,在亲缘关系上更加接近。qP CR结果表明,CpomO BP20基因在苹果蠹蛾4龄幼虫以及雌雄成虫不同组织中均有表达,其中在4龄幼虫的血淋巴中表达量最高,在雌雄成虫表达量最高的分别是翅和足,其次是头部。分子对接结果表明,CpomO BP20与3种保幼激素均具有较好的结合能力,可能参与保幼激素的结合与转运。【结论】本研究明确了CpomO BP20的核苷酸和氨基酸序列的组成及编码蛋白的理化性质,并推测CpomO BP20的作用可能不仅局限于嗅觉识别,在非嗅觉器官中也可能起着重要的生理作用,为今后更深入地探究气味结合蛋白在苹果蠹蛾生命活动中的作用机理提供数据支撑。

【Aim】 To better understand the function of odorant-binding proteins(OBPs) in Cydia pomonella and provide a theoretical foundation for managing and controlling fruit pests, we analyzed the sequence features and expression profiles of CpomOBP20. 【Method】 We first amplified the cDNA sequence of CpomOBP20 using reverse transcription-PCR, and then analyzed the features of its nucleotide and amino acid sequences using bioinformatics software. We also studied the expression profile of CpomOBP20 in the tissues of 4 th instar larvae(including head, hemolymph, integument, fat body, midgut, Malpighian tubules, and salivary glands) and terminal tissues of female and male adults(including head, antenna, labial palps, beak, leg, and wing). We evaluated the binding capacity of CpomOBP20 to the three juvenile hormones. 【Result】 The open reading frame encoded by CpomOBP20 was 459 base pairs and encoded 152 amino acids. The predicted molecular weight of CpomOBP20 was 16.264 ku and its isoelectric point 6.30. The first 20 amino acids at the N-terminus are signal peptide sequences. The encoded protein had six conserved cysteines, indicating that it is a classical OBP. Multiple sequence alignments showed that CpomOBP20 had the highest amino acid sequence identity with the orthologous OBP(XP;11557123.1) in Plutella xylostella. Quantitative PCR analysis showed that CpomOBP20 was expressed in almost all tissues of 4 th instar larvae and in adult C. pomonella females and males. In 4 th instar larvae, CpomOBP20 showed the highest expression level in the hemolymph. In adults, the gene was highly expressed in the wings and legs, followed by that in the head, and molecular docking showed that CpomOBP20 can bind to three juvenile hormones, suggesting that it plays an important role in the recognition and translocation of juvenile hormones. 【Conclusion】 We determined the physiochemical properties of the nucleotide and amino acid sequences of CpomOBP20 and further predicted that the function of CpomOBP20 was not limited to odor recognition but may also have physiological effects in non-olfactory tissues. This study provides supporting data for exploring the physiological functions of OBPs in C. pomonella.