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

【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。

Cloning,prokaryotic expression and tissue expression profiling of odorant binding proteingeneAzanOBP3 fromAgrilus zanthoxylumi(Coleoptera: Buprestidae)

【Aim】 The objective of this study is to clone the coding sequence of odorantbinding protein (OBP) gene Azan OBP3 in Agrilus zanthoxylumi and to analyze its structureproperties and expression profile, so as to further understand the process of identifying odorant substances in A. zanthoxylumi adult antennae and to provide a new fundamentalevidence for the pest management and control. 【Methods】 The cDNA sequence of AzanOBP3 wasamplified from A. zanthoxylumi with RT-PCR, and the nucleotide and deduced amino acidsequences of the gene were analyzed using different bioinformatics software. Therecombinant expression vector pET-28a(+)/AzanOBP3 was constructed and transformed intoEscherichia coliBL21(DE3) competent cells for expression by IPIG iduction. The recombinantprotein was identified by SDS-PAGE and Western blotting. The expression profiles ofAzanOBP3 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 cDNAsequence of AzanOBP3 (GenBank accession no.： MT318832) from A. zanthoxylumi. Its ORF is414 bp in length, encoding 137 amino acids, with a predicted molecular weight of 16.038 kDand the isoelectric point of 4.79, and a signal peptide sequence of 29 amino acids at theN terminus. The encoded protein has six conserved cysteines belonging to the typical insectOBPs. Homologous sequence alignment analyses showed that AzanOBP3 has the highest aminoacid sequence identity with AmalOBP2 from Agrilus mali and AplaGOBP from Agrilusplanipennis (74.45% and 76.92%, respectively). The recombinant expression vector pET-28a(+)/AzanOBP3 was successfully constructed and the target protein was stably expressed inhost bacteria in the form of fusion protein after culture at 37℃ 180 r/min in a shakerincubator and induction with 1 mmol/L IPTG for 4 h. The qPCR results revealed that AzanOBP3was expressed in various tissues of male and female adults of A. zanthoxylumi, with thehighest expression level in the male adult leg. 【Conclusion】 In this study, thenucleotide and amino acid sequences of AzanOBP3 and the physiochemical properties of itsencoded protein were clarified. The tissue expression profile suggests that AzanOBP3 maynot be limited to the recognition of olfactory odorant, also have physiological functionsin non-olfactory tissues, and especially may play important roles during insect feedingand host locating. Its functions need further study. This study provides a basis forexploring the molecular structure of odorant binding proteins and their functionalmechanisms in the chemical sensing system of A. zanthoxylumi.