花椒窄吉丁气味结合蛋白AzanOBP4基因克隆及其与寄主挥发物分子对接  

Cloning of Odorant Binding Protein Gene AzanOBP4 in Agrilus zanthoxylumi and Its Molecular Docking with Host Volatiles

作　　者：郭莉 陈迪 高晓进 贾任航 张泽腾 谢寿安[1] 吕淑杰[1] 陈正斌 GUO Li;CHEN Di;GAO Xiao-Jin;JIA Ren-Hang;ZHANG Ze-Teng;XIE Shou-An;LV Shu-Jie;CHEN Zheng-Bin(College of Forestry,Northwest A&F University,Yangling 712100,China;Forestry and Grassland Bureau of Dingxi City,Dingxi 743000,China)

机构地区：[1]西北农林科技大学林学院,杨陵712100 [2]定西市安定区林业和草原局,定西743000

出　　处：《农业生物技术学报》2021年第7期1364-1377,共14页Journal of Agricultural Biotechnology

基　　金：国家公益性行业(林业)科研项目(201504324);云南省林业科技推广示范项目(云[2019]TG01号);陕西省第二批特支计划领军人才项目(陕组通字2020-44号)。

摘　　要：花椒窄吉丁(Agrilus zanthoxylumi)是花椒(Zanthoxylum bungeanum)树的专一性蛀干害虫,为了分析其与寄主花椒挥发物的结合模式及能力,本研究基于花椒窄吉丁转录组数据,利用cDNA末端快速扩增技术(rapid amplification of cDNA ends,RACE)对花椒窄吉丁气味结合蛋白(odorant binding protein,OBP)基因AzanOBP4进行全长克隆;采用生物信息学软件预测分析其核酸序列及氨基酸序列;通过q RTPCR检测AzanOBP4基因在成虫不同组织中(头,胸,腹,足和翅)的表达情况;I-TASSER在线软件对AzanOBP4氨基酸序列三维建模,通过SAVES v6.0以及PROSA软件评价模型,应用AutoDock软件对AzanOBP4蛋白模型和14种寄主挥发物进行分子对接分析。AzanOBP4的cDNA全长序列为691 bp(GenBank No.MT318833),5’端非编码区182 bp,3’端非编码区89 bp,开放阅读框(ORF)为423 bp,编码140个氨基酸,含有6个保守半胱氨酸,属于典型的OBP。序列分析表明,AzanOBP4氨基酸序列与苹果小吉丁(Agrilus mali)的AmalOBP3氨基酸序列一致性最高为83.57%,两者在系统发育树中以99%的置信度聚为一支。AzanOBP4在雌雄成虫的不同组织中均有表达,其中在雌性成虫的足部表达量最高。分子对接显示,AzanOBP4与14种寄主挥发物主要以氢键、疏水作用和范德华力相结合,发现4种环状烯类寄主挥发物(葎草烯,β-石竹烯,荜澄茄油烯和?-依兰油烯)与AzanOBP4的结合能力较好。研究表明花椒窄吉丁基因AzanOBP4参与识别寄主植物的嗅觉机制,推测AzanOBP4可能在花椒窄吉丁寻找寄主与取食行为中发挥重要作用。本研究为揭示花椒窄吉丁的嗅觉识别机制提供参考依据。Agrilus zanthoxylumi is a significant trunk-boring pest of the Zanthoxylum bungeanum tree. In order to analyze its binding mode and ability with the host volatiles, the objective of this study is to clone the full-length cDNA sequence of the odor-binding protein gene(AzanOBP4), and analyze its binding mode and ability to host volatiles. Rapid amplification of cDNA ends(RACE) technique was used to clone the fulllength cDNA of AzanOBP4 based on the transcriptome database of A. zanthoxylumi, and the nucleotide and deduced amino acid sequences of the gene were analyzed using different bioinformatics software. Moreover,the expression levels of AzanOBP4 in different tissues of adult were determined by qRT-PCR. I-TASSER software was used to develop the homologous model of AzanOBP4 and the model equality was evaluated using SAVES v6.0 and PROSA software. Using AutoDock to perform molecular docking analysis on the AzanOBP4 model and 14 host volatiles. The full-length cDNA of AzanOBP4 was 691 bp(GenBank No. MT318833), with the non-coding regions of 5’ and 3’ ends of 182 and 89 bp, respectively. ORF was 423 bp, encoding 140 amino acids. The encoded protein had 6 conserved cysteines belonging to the typical insect OBPs. Homologous sequence alignment analyses showed that AzanOBP4 had the highest amino acid sequence identity(83.57%)with AmalOBP3 from Agrilus mali, and the two sequence were clustered into one branch with 99% confidence in the phylogenetic tree. The AzanOBP4 gene was expressed in various tissues of both male and female adults,and the highest expression level was in female leg. Molecular docking showed that AzanOBP4 combined with14 host volatiles mainly through hydrogen bonding, hydrophobic interaction and van der Waals force, and 4 cyclic host volatiles(α-caryophyllene, β-caryophyllene, α-cubebene and ?-muurolene) had a strong binding ability with AzanOBP4. Studies showed that AzanOBP4 was involved in the olfactory mechanism of identifying host plants, suggesting that it may play an important role during

关 键 词：花椒窄吉丁 气味结合蛋白 基因克隆 寄主挥发物 分子对接 

分 类 号：S763.38[农业科学—森林保护学]