昆虫学报 ›› 2020, Vol. 63 ›› Issue (4): 390-400.doi: 10.16380/j.kcxb.2020.04.002

• 研究论文 • 上一篇    下一篇

花椒窄吉丁气味结合蛋白基因AzanOBP3的克隆、原核表达及组织表达谱分析

巩雪芳, 杨平, 王延来, 郭莉, 陈迪, 谢寿安*, 吕淑杰   

  1. (西北农林科技大学林学院, 陕西杨凌 712100)
  • 出版日期:2020-04-20 发布日期:2020-05-08

Cloning, prokaryotic expression and tissue expression profiling of odorant binding protein gene AzanOBP3 from Agrilus zanthoxylumi (Coleoptera: Buprestidae)

GONG Xue-Fang, YANG Ping, WANG Yan-Lai, GUO Li, CHEN Di, XIE Shou-An*, LÜ Shu-Jie    

  1. (Northwest A&F University, Yangling, Shaanxi 712100, China)
  • Online:2020-04-20 Published:2020-05-08

摘要:  【目的】本研究克隆花椒窄吉丁Agrilus zanthoxylumi气味结合蛋白(odorant binding protein, OBP)基因AzanOBP3,并对其进行序列和表达分析,旨在更好地了解OBP基因在花椒窄吉丁成虫触角识别气味物质过程中的作用,为农林害虫的绿色防控提供理论依据。【方法】利用RT-PCR扩增花椒窄吉丁气味结合蛋白基因AzanOBP3的cDNA序列,采用生物信息学软件分析其核苷酸和氨基酸序列;构建重组表达载体pET-28a(+)/AzanOBP3,转化到大肠杆菌Escherichia coli BL21(DE3)感受态细胞中进行融合蛋白的IPTG诱导表达,SDS-PAGE及Western blot鉴定重组表达蛋白;基于qPCR技术分析AzanOBP3基因在花椒窄吉丁雌雄成虫不同组织(头、胸、腹、足和翅)中的表达情况。【结果】克隆获得了花椒窄吉丁气味结合蛋白基因AzanOBP3的cDNA全长序列(GenBank登录号: MT318832),预测到其开放阅读框长 414 bp,共编码 137 个氨基酸,等电点为4.79,蛋白分子量为16.038 kD,N末端具有29个氨基酸组成的信号肽序列,蛋白序列中具有6个保守的半胱氨酸残基,属于典型的昆虫OBP。序列分析表明,AzanOBP3的氨基酸序列分别与苹果小吉丁Agrilus mali AmalOBP2和白蜡窄吉丁Agrilus planipennis AplaGOBP的一致性最高,分别为74.45%和76.92%,在进化关系上更加同源。构建了重组表达载体pET-28a(+)/AzanOBP3。在37℃ 180 r/min摇床培养及1 mmol/L IPTG诱导4 h条件下,AzanOBP3在大肠杆菌中成功地表达出了与预测蛋白分子质量大小一致的AzanOBP3融合蛋白。qPCR检测结果表明, AzanOBP3基因在雌性和雄性成虫的不同组织中都有表达,其中在雄成虫足中的表达量最高。【结论】明确了AzanOBP3的核苷酸和氨基酸序列组成及编码蛋白的理化特性。组织表达谱结果提示, AzanOBP3的功能可能不仅局限于嗅觉识别,在非嗅觉器官中可能也具有重要的生理功能,特别是在其寻找寄主植物与取食的过程中可能发挥着重要作用,AzanOBP3功能还需更深入的研究。本研究为今后更深入地探究气味结合蛋白的结构及其在花椒窄吉丁化学感受系统中的作用机制提供依据。

关键词: 花椒窄吉丁, 气味结合蛋白, 基因克隆, 序列分析, 原核表达, 组织表达谱

Abstract: 【Aim】 The objective of this study is to clone the coding sequence of odorant binding protein (OBP) gene Azan OBP3 in Agrilus zanthoxylumi and to analyze its structure properties and expression profile, so as to further understand the process of identifying 
odorant substances in A. zanthoxylumi adult antennae and to provide a new fundamental evidence for the pest management and control. 【Methods】 The cDNA sequence of AzanOBP3 was amplified from A. zanthoxylumi with RT-PCR, and the nucleotide and deduced amino acid sequences of the gene were analyzed using different bioinformatics software. The recombinant expression vector pET-28a(+)/AzanOBP3 was constructed and transformed into Escherichia coli BL21(DE3) competent cells for expression by IPIG iduction. The recombinant protein was identified by SDS-PAGE and Western blotting. The expression profiles of AzanOBP3 in different female and male adult tissues (head, thorax, abdomen, leg and wing) of A. zanthoxylumi were detected by qPCR. 【Results】 We cloned the full-length cDNA sequence of AzanOBP3 (GenBank accession no.: MT318832) from A. zanthoxylumi. Its ORF is 414 bp in length, encoding 137 amino acids, with a predicted molecular weight of 16.038 kD and the isoelectric point of 4.79, and a signal peptide sequence of 29 amino acids at the N terminus. The encoded protein has six conserved cysteines belonging to the typical insect OBPs. Homologous sequence alignment analyses showed that AzanOBP3 has the highest amino acid sequence identity with AmalOBP2 from Agrilus mali and AplaGOBP from Agrilus planipennis (74.45% and 76.92%, respectively). The recombinant expression vector pET-28a(+)/AzanOBP3 was successfully constructed and the target protein was stably expressed in host bacteria in the form of fusion protein after culture at 37℃ 180 r/min in a shaker incubator and induction with 1 mmol/L IPTG for 4 h. The qPCR results revealed that AzanOBPwas expressed in various tissues of male and female adults of A. zanthoxylumi, with the highest expression level in the male adult leg. 【Conclusion】 In this study, the nucleotide and amino acid sequences of AzanOBP3 and the physiochemical properties of its encoded protein were clarified. The tissue expression profile suggests that AzanOBP3 may not be limited to the recognition of olfactory odorant, also have physiological functions in non-olfactory tissues, and especially may play important roles during insect feeding and host locating. Its functions need further study. This study provides a basis for exploring the molecular structure of odorant binding proteins and their functional mechanisms in the chemical sensing system of A. zanthoxylumi.

Key words:  Agrilus zanthoxylumi, odorant binding protein, gene cloning, sequence analysis; prokaryotic expression, tissue expression profile