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 AzanOBP3 was 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