AbstractAgrilus 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 full-length 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 with 14 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 ç-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 insect feeding and host locating and would lay a necessary foundation for revealing the molecular mechanism of olfaction in A. zanthoxylumi.
GUO Li,CHEN Di,GAO Xiao-Jin, et al. Cloning of Odorant Binding Protein Gene AzanOBP4 in Agrilus zanthoxylumi and Its Molecular Docking with Host Volatiles[J]. 农业生物技术学报, 2021, 29(7): 1364-1377.
[1] 党心德, 陈孝达, 王明春, 等.1988.花椒主要害虫的初步研究[J].陕西林业科技, (02): 57-62. (Dang X D, Chen X D, Wang M C, et al.1988.Preliminary study on the main pests of Z.bungeanum[J].Shaanxi Forest Science and Technology, (02): 57-62.) [2] 付宁宁, 刘佳, 渠成, 等.2017.悬铃木方翅网蝽化学感受蛋白CcilCSP1的结构及其结合寄主挥发物的预测分析[J].林业科学, 53(10): 109-117. (Fu N N, Liu J, Qu C, et al.2017.Analysis of Corythucha ciliata CcilCSP1 structure and prediction of its binding to host-plant volatiles[J].Scientia Silvae Sinicae.53(10): 109-117.) [3] 巩雪芳, 谢寿安, 杨平, 等.2020a.花椒窄吉丁触角转录组及嗅觉相关基因分析[J].昆虫学报, 63(10): 1159-1170. (Gong X F, Xie S A, Yang P, et al.2020.Analysis of the antennal transcriptome and olfaction-related genes of A.zanthoxylumi (Coleoptera: Buprestidae)[J].Acta Entomologica Sinica, 63(10): 1159-1170.) [4] 巩雪芳, 杨平, 王延来, 等.2020b.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[J].昆虫学报, 63(04): 390-400. (Gong X F, Yang P, Wang Y L, et al.2020.Cloning,prokaryotic expression and tissue expression profiling of odorant binding protein gene AzanOBP3 from A.zanthoxylumi (Coleoptera: Buprestidae)[J].Acta Entomologica Sinica, 63(04): 390-400.) [5] 郭冰, 郝恩华, 王菁桢, 等.2019.入侵害虫松树蜂气味结合蛋白与其相关信息化学物质的分子对接[J].植物保护学报, 46(05): 1004-1017. (Guo B, Hao E H, Wang Q Z, et al.2019.Molecular docking of odorant binding proteins and its related semiochemicals of sirex woodwasp Sirex noctilio, an invasive insect pest[J].Journal of Plant Protection, 46(05):1004-1017.) [6] 吉波, 张耀华, 银航, 等.2020.花椒病虫害生物防治的研究综述[J].陕西农业科学, 2020, 66(10): 72-75+104. (Ji B, Zhang Y H, Yin H, et al.2020.A review of researches on biological control of Z.bungeanum[J].Shaanxi Journal of Agricultural Sciences, 66(10): 72-75+104.) [7] 李玲, 谭瑶, 周晓榕, 等.2019.沙葱萤叶甲气味结合蛋白GdauOBP20的基因克隆、原核表达及其结合特性[J].中国农业科学, 52(20): 3705-3712. (Li L, Tan Y, Zhou X R, et al.2019.Molecular cloning, prokaryotic expression and binding characterization of odorant binding protein GdauOBP20 in Galeruca daurica[J].Scientia Agricultura Sinica, 52(20): 3705-3712.) [8] 李孟楼, 李宗明, 焦爱叶.1990.花椒窄吉丁的危害规律及其分布型[J].西北林学院学报, (01): 34-38. (Li M L, Li Z M, Jiao A Y.1990.The harm law and distribution pattern of A.zanthoxylumi[J].Journal of Northwest Forestry University, (01):34-38.) [9] 刘绥鹏, 谢寿安, 袁丽芳, 等.2016.花椒窄吉丁的入侵对花椒挥发物成分的影响[J].西北林学院学报, 31(05): 246-254. (Liu S P, Xie S A, Yuan L F, et al.2016.Effect of A.zanthoxylumi infection on the volatile constituents of Z.bungeanum[J].Journal of Northwest Forestry University, 31(05): 246-254.) [10] 司品法, 周琼, 崔中翌.2018.柑橘大实蝇气味结合蛋白基因BminOBP25的克隆、原核表达及组织表达分析[J].昆虫学报, 61(05): 537-545. (Si P F, Zhou Q, Cui Z Y, et al.2018.Cloning,prokaryotic expression and tissue expression profiling of an odorant binding protein gene BminOBP25 from Bactrocera minax (Diptera: Tephritidae)[J].Acta Entomologica Sinica, 61(05): 537-545.) [11] 孙小洁, 李仔博, 张强, 等.2020.绿盲蝽气味结合蛋白AlucOBP33配体结合特性[J].中国生物防治学报, 36(02): 203-210. (Sun X J, Li Z B, Zhang Q, et al.2020.Ligands binding characteristics of odorant binding proteins AlucOBP33 of Apolygus lucorum (Hemiptera: Miridae)[J].Chinese Journal of Biological Control, 36(02): 203-210.) [12] 孙亚兰, 吕琪卉, 杨海博, 等.2020.疆夜蛾Plus-C气味结合蛋白PsauOBP7的组织表达谱及配体结合特性分析[J].昆虫学报, 63(07): 807-816. (Sun Y L, Lv Q H, Yang H B, et al.2020.Tissue expression profiling and ligand binding characterization of the Plus-C odorant binding protein PsauOBP7 of Peridroma saucia (Lepidoptera: Noctuidae)[J].Acta Entomologica Sinica, 63(07):807-816.) [13] 王延来, 谢寿安, 赖青, 等.2020.花椒窄吉丁对寄主果实挥发物的触角电位及行为反应[J].西北林学院学报, 35(01): 150-157. (Wang Y L, Xie S A, Lai Q, et al.2020.Elecrtoantennogram and behavioral responses of A.zanthoxylumi to volatiles from Z.bungeanum fruit[J].Journal of Northwest Forestry University, 35(01): 150-157.) [14] 尹传林, 李美珍, 贺康, 等.2017.昆虫基因组及数据库研究进展[J].环境昆虫学报, 39(01): 1-18. (Yin C L, Li M Z, He K, et al.The progress of insec genomic research and the gene database[J].Journal of Environmental Entomology, 39(01): 1-18.) [15] 袁丽芳.2016.花椒窄吉丁对寄主挥发物化学感受机制的初步研究[D].硕士论文, 西北农林科技大学, 导师:谢寿安,pp.20-25. (Yuan L F.2016.The preliminary study on mechanism of odor perception of A.zanthoxylumi to volatiles from Z.bungeanum[D].Thesis for M.S., Northwest A&F University, Supervisor: Xie S A, pp.20-25.) [16] 张玉, 杨斌,王桂荣.2019.昆虫嗅觉相关可溶性蛋白的研究进展[J].环境昆虫学报, 41(02): 229-240. (Zhang Y, Yang B, Wang G R.2019.Research progress of soluble proteins on chemosensation in insects[J].Journal of Environmental Entomology, 41(02): 229-240.) [17] Guo H, Wang C Z.2019.The ethological significance and olfactory detection of herbivore-induced plant volatiles in interactions of plants, herbivorous insects, and parasitoids[J].Arthropod-Plant Interactions, 13(2): 161-179. [18] Grosse W E, Svatos A, Krieger J.2006.A pheromone-binding protein mediates the bombykol-induced activation of a pheromone receptor in vitro[J].Chemical Senses, 31(6): 547-55. [19] Kaissling K E.2009.Olfactory perireceptor and receptor events in moths: A kinetic model revised[J].Journal of comparative physiology, 195(10): 895-922. [20] Laughlin J D, Ha T S, Jones D N M,et al.2008.Activation of pheromone-sensitive neurons is mediated by conformational activation of pheromone-binding protein[J].Cell, 133(7): 1255-1265. [21] Leal W S.2013.Odorant reception in insects: Roles of receptors, binding proteins, and degrading enzymes[J].Annual Review of Entomology, 58: 373-91. [22] Li J, Zhang L, Wang X.2016 An odorant-bining-protein involved in perception of host plant odorants in Locusta migratoria[J].Archives of Insect Biochemistry and Physiology, 91(4): 221-9. [23] Li L, Zhou Y T, Tan Y, et al.2017.Identification of odorant-binding protein genes in Galeruca daurica (Coleoptera: Chrysomelidae) and analysis of their expression profiles[J].Bulletin of Entomological Research, 107(4): 550-561. [24] Mao Y, Xu X Z, Xu W, et al.2010.Crystal and solution structures of an odorant-binding protein from the southern house mosquito complexed with an oviposition pheromone[J].Proceedings of the National Academy of Sciences of the USA, 107(44): 19102-19107. [25] Paolini S, Scaloni A, Amoresano A, et al.1998.Amino acid sequence, post-translational modifications, binding and labelling of porcine odorant-binding protein[J].Chemical Senses, 23(6): 689-98. [26] Picimbon J F.2020.Interpopulational variations of odorant-binding protein expression in the black cutworm Moth, Agrotis ipsilon[J].Insects, 11(11): 798. [27] Swarup S, Williams T I, Anholt R R.2011.Functional dissection of odorant binding protein genes in Drosophila melanogaster[J].Genes, Brain and Behavior, 10(6): 648-57. [28] Vogt R G, Riddiford L M.1981.Pheromone binding and inactivation by moth antennae[J].Nature, 293(5828): 161-163. [29] War A R, Paulraj M G, Ahmad T, et al.2012.Mechanisms of plant defense against insect herbivores[J].Plant Signaling & Behavior, 7(10): 1306-20. [30] Wang P, Lyman R F, Mackay T F, et al.2010.Natural variation in odorant recognition among odorant-binding proteins in Drosophila melanogaster[J].Genetics, 184(3): 759-67. [31] Wiederstein M, Sippl M J.2007.ProSA-web: Interactive web service for the recognition of errors in three-dimensional structures of proteins[J].Nucleic Acids Research, 35(Web Server issue): W407-10. [32] Wogulis M, Morgan T, Ishida Y, et al.2006.The crystal structure of an odorant binding protein from Anopheles gambiae: Evidence for a common ligand release mechanism[J].Biochemical and Biophysical Research Communications, 339(1): 157-164. [33] Xu P, Atkinson R, Jones D N, et al.2005.Drosophila OBP LUSH is required for activity of pheromone-sensitive neurons[J].Neuron, 45(2): 193-200. [34] Zhao Y, Ding J, Zhang Z, et al.2018.Sex and tissue specific expression profiles of odorant binding protein and chemosensory protein genes in Bradysia odoriphaga (Diptera: Sciaridae)[J].Frontiers in Physiology, 9: 107. [35] Zhou Y N, Xie S, Chen J N, et al.2020.Expression and functional characterization of odorant-binding protein genes in the endoparasitic wasp Cotesia vestalis[J].Insect Science, https://doi.org/10.1111/1744-7917.12861.
