中华蜜蜂气味结合蛋白基因AcerOBP14的克隆及时空表达

doi:10.3864/j.issn.0578-1752.2016.19.016

摘要/Abstract

摘要： 【目的】克隆获得中华蜜蜂（Apis cerana cerana）AcerOBP14基因序列，并对其蛋白结构进行预测，分析其在不同组织和发育阶段的时空表达差异，为该基因的功能研究提供参考。【方法】以中华蜜蜂全组织cDNA为模板利用RT-PCR技术扩增和克隆获得AcerOBP14 cDNA全长序列，并提交至GenBank数据库；利用多种生物信息学软件预测分析其编码蛋白的理化特性和结构特征；采用MEGA5.2软件中的邻位相连法（neighbor-joining，NJ）构建AcerOBP14及其同源膜翅目昆虫OBPs的系统发育树；通过实时荧光定量PCR技术分析AcerOBP14在1、5、10、15、20、25和30日龄中华蜜蜂不同组织（触角、头、胸、腹、足和翅）中mRNA的表达情况。【结果】克隆获得了中华蜜蜂气味结合蛋白基因AcerOBP14的cDNA序列（GenBank登录号为KT24648）。AcerOBP14的开放阅读框（ORF）长度为408 bp，编码135个氨基酸，预测其蛋白分子量为15.08 kD，理论等电点为5.44，有信号肽，无跨膜结构，且第18—127位氨基酸之间存在一个昆虫气味结合蛋白家族PBP-GOBP superfamily保守结构域；存在多个比较明显的疏水区域，可能是脂溶性气味分子的结合位点；含有4个保守的半胱氨酸位点，属于Minus-C OBP亚家族。亚细胞定位分析表明，AcerOBP14属于分泌型蛋白，主要集中在内质网-高尔基体-质膜分泌途径上。AcerOBP14蛋白具有7个α螺旋，α1—α6属于典型OBPs核心螺旋，α7位于C端且暴露在蛋白表面。氨基酸同源序列比对结果显示，AcerOBP14与西方蜜蜂AmelOBP14的氨基酸序列一致性最高，达到86%；其次是大蜜蜂AdorGOBP56a-like，一致性为55%。系统进化树分析表明，同为蜜蜂属的中华蜜蜂AcerOBP14、西方蜜蜂AmelOBP14、大蜜蜂AdorGOBP56a-like、中华蜜蜂AcerOBP21和小蜜蜂AfloGOBP56d-like聚为一个分支，切叶蜂属的苜蓿切叶蜂MrotGOBP56a-like、侧沟茧蜂属的毁侧沟茧蜂MdemGOBP83a-like、壁蜂属的角额壁蜂OcorOBP3和熊蜂属的BterGOBP56d-like和BimpGOBP56d-like聚为另一大分支。荧光定量PCR结果显示，AcerOBP14在成蜂不同发育期的触角、头、胸、腹、足和翅中均有表达，其表达程度有差异。1日龄工蜂的胸部表达量最高，触角次之，且两者均极显著高于头、腹、足和翅（P＜0.01）；其他日龄工蜂触角表达量均极显著高于其他组织（P＜0.01），其中20日龄工蜂的触角表达量最高。从各组织在不同发育阶段的表达量来看，触角的表达量在1日龄最低，极显著地低于其他日龄（P＜0.01）；5、10日龄逐渐增加，15日龄突然下降，20日龄达到最高，极显著高于其他日龄（P＜0.01）；之后又呈逐渐下降趋势。头、胸、腹、足和翅膀表达量总体上随日龄增加而呈下降趋势，5日龄之后大幅下降，之后趋于平稳且呈低丰度表达。【结论】AcerOBP14属于Minus-C OBP亚家族，具有PBP-GOBP家族的典型结构；组织表达谱结果暗示，AcerOBP14属普通气味结合蛋白GOBP，在中华蜜蜂中除嗅觉识别之外还参与其他生理功能。

关键词: 中华蜜蜂, AcerOBP14, 基因克隆, 生物信息学, 时空表达

Abstract: 【Objective】The objective of this study is to clone the cDNA sequence of AcerOBP14 from Apis cerana cerana, predict the protein structure, explore its expression profiles in different tissues and at different developmental stages of A. c. cerana, and to provide a fundamental evidence for the future study of the physiological function of this gene.【Method】 The full-length cDNA sequence of AcerOBP14 was cloned from the entire tissues of A. c. cerana by RT-PCR and submitted to the GenBank database. Physiochemical properties and structure characteristics of the deduced amino acid sequence were analyzed by multiple bioinformatics methods. Phylogenetic tree between AcerOBP14 and its homologous OBPs from Hymenoptera insects was constructed using neighbor-joining of MEGA5.2. The expression levels of AcerOBP14 mRNA in different tissues (antenna, head, thorax, abdomen, legs and wings) at 1, 5, 10, 15, 20, 25 and 30 days were detected by real-time PCR.【Result】The full-length cDNA sequence of AcerOBP14 was obtained (GenBank accession number is KT24648). The open reading frame (ORF) of AcerOBP14 is 408 bp, encoding a protein of 135 aa with an estimated molecular weight of 15.08 kD and pI of 5.44. The encoding protein has a signal peptide and no transmembrance structure, possesses a PBP-GOBP superfamily domain between 18-127 residues, exists multiple obviously hydrophobic regions binding liposoluble odorant and contains four conserved cysteins, suggesting that it belongs to the Minus-C OBP subfamily. Subcellular localization results showed that AcerOBP14 was located on the secretory pathway about endoplasmic reticulum, golgi and plasmalemma which belongs to the secretory protein. AcerOBP14 possesses 7 α-helixs, among which the α1-α6 belong to the core helixs of classical OBPs and α7 is exposed to the surface of protein at the C-terminus. The results of amino acid sequence alignment showed that AcerOBP14 was homologous to AmelOBP14 with 86% amino acid sequence identity, and then had 55% sequence identity with AdorGOBP56a-like. Phylogenetic analysis revealed that AcerOBP14, AmelOBP14, AdorGOBP56a-like, AcerOBP21 and AfloGOBP56d-like belonging to the Apis were clustered into one group, MrotGOBP56a-like (Meqachile), MdemGOBP83a-like (Microplitis), OcorOBP3 (Osmia), BterGOBP56d-like (Bombus) and BimpGOBP56d-like (Bombus) were clustered into the other group. Besides, the results of real-time PCR showed that the AcerOBP14 transcripts were differentially expressed in the antenna, head, thorax, abdomen, legs and wings of the adult workers. The thorax of 1-day-old workers had the highest expression level of AcerOBP14, the antenna had the second highest expression level, and both of them were significantly higher than the head, abdomen, legs and wings (P＜0.01). Moreover, the expression profiles in worker bees at other days were significantly higher in antenna than that in other tissues (P＜0.01), of which the antenna of 20-day-old workers had the highest expression level. According to the expression profiles of AcerOBP14 at different developmental stages, the antenna of workers had the lowest expression level at 1-day-old which was significantly lower than other days (P＜0.01), increased gradually at 5-day-old and 10-day-old, then decreased suddenly at 15-day-old. The expression level at 20-day-old was significantly higher than other days (P＜0.01), afterwards, the expression level gradually reduced. Generally, the expression profiles of the head, thorax, abdomen, legs and wings appeared a downward trend with the days of age increasing, and maintained a lower level after decreasing remarkably at 5-day-old.【Conclusion】AcerOBP14 belongs to the Minus-C OBP subfamily with a PBP-GOBP superfamily domain. The results of expression profiling suggested that AcerOBP14 belongs to general odorant binding protein (GOBP) and is involved in not only the olfactory recognition, but also other physiological functions.

Key words: Apis cerana cerana, AcerOBP14, gene cloning, bioinformatics analysis, temporal-spacial expression

杜亚丽，张中印，潘建芳，王树杰，杨爽，赵慧婷，姜玉锁. 中华蜜蜂气味结合蛋白基因AcerOBP14的克隆及时空表达[J]. 中国农业科学, 2016, 49(19): 3852-3862.

DU Ya-li, ZHANG Zhong-yin, PAN Jian-fang, WANG Shu-jie, YANG Shuang, ZHAO Hui-ting, JIANG Yu-suo. Cloning and Expression Analysis of Odorant Binding Protein Gene AcerOBP14 from Apis cerana cerana[J]. Scientia Agricultura Sinica, 2016, 49(19): 3852-3862.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2016.19.016

https://www.chinaagrisci.com/CN/Y2016/V49/I19/3852

参考文献

[1] Carey A F, Carlson J R. Insect olfaction from model systems to disease control. Proceedings of the National Academy of Sciences of the United States of America, 2011, 108(32): 12987-12995.

[2] Forstner M, Breer H, Krieger J. A receptor and binding protein interplay in the detection of a distinct pheromone component in the silkmoth Antheraea polyphemus. International Journal of Biological Sciences, 2009, 5(7): 745-757.

[3] Laughlin J D, Ha T S, Jones D N, Smith D P. Activation of pheromone-sensitive neurons is mediated by conformational activation of pheromone-binding protein. Cell, 2008, 133(7): 1255-1265.

[4] Zhao H T, Zhao W M, Gao P F, Zhang G X, Jiang Y S. Sequence and expression characterization of an OBP1 gene in the Asian honeybee, Apis cerana cerana (Hymenoptera: Apidae). Applied Entomology and Zoology, 2014, 49(1): 189-196.

[5] Field L M, Pickett J A, Wadhams L J. Molecular studies in insect olfaction. Insect Molecular Biology, 2000, 9(6): 545-551.

[6] 赵慧婷, 高鹏飞, 张桂贤, 姜玉锁. 蜜蜂嗅觉相关蛋白的研究进展. 应用昆虫学报, 2012, 49(5): 1366-1371.

Zhao H T, Gao P F, Zhang G X, Jiang Y S. Advances in research on honeybee olfactory proteins. Chinese Journal of Applied Entomology, 2012, 49(5): 1366-1371. (in Chinese)

[7] 胡颖颖, 徐书法, WUBIE A J, 李薇, 国占宝, 周婷. 昆虫嗅觉相关蛋白及嗅觉识别机理研究概述. 基因组学与应用生物学, 2013, 32(5): 667-676.

Hu Y Y, Xu S F, WUBIE A J, Li W, Guo Z B, Zhou T. Research advance of olfactory proteins and olfactory mechanism in insects. Genomics and Applied Biology, 2013, 32(5): 667-676. (in Chinese)

[8] Hekmat-Scafe D S, Scafe C R, McKinney A J, Tanouye M A. Genome-wide analysis of the odorant-binding protein gene family in Drosophila melanogaster. Genome Research, 2002, 12(9): 1357-1369.

[9] Forêt S, Maleszka R. Function and evolution of a gene family encoding odorant binding-like proteins in a social insect, the honey bee (Apis mellifera). Genome Research,2006,16(11): 1404-1413.

[10] Richards S, Gibbs R A, Weinstock G M, Brown S J, Denell R, Beeman R W, Gibbs R, Beeman R W, Brown S J,Bucher G, Friedrich M, Grimmelikhuijzen C J, Klingler M. The genome of the model beetle and pest Tribolium castaneum. Nature, 2008, 452(7190): 949-955.

[11] Gong D P, Zhang H J, Zhao P, Xia Q Y, Xiang Z H. The odorant binding protein gene family from the genome of silkworm, Bombyx mori. BMC Genomics, 2009, 10: 332.

[12] Li K M, Yang X B, Xu G Y, Cao Y, Lu B Q, Peng Z Q. Identification of putative odorant binding protein genes in Asecodes hispinarum, a parasitoid of coconut leaf beetle (Brontispa longissima) by antennal RNA-Seq analysis. Biochemical and Biophysical Research Communication, 2015, 467(3): 514-520.

[13] Zhou J J, Huang W, Zhang G A, Pickett J A, Field L M. “Plus-C” odorant-binding protein genes in two Drosophila species and the malaria mosquito Anopheles gambiae. Gene, 2004, 327(1): 117-129.

[14] Xu P X, Zwiebel L J, Smith D P. Identification of a distinct family of genes encoding atypical odorant-binding proteins in the malaria vector mosquito, Anopheles gambiae. Insect Molecular Biology, 2003, 12(6): 549-560.

[15] Zhou J J, Vieira F G, He X L, Smadja C, Liu R, Rozas J, Field L M.Genome annotation and comparative analyses of the odorant-binding proteins and chemosensory proteins in the pea aphid Acyrthosiphon pisum. Insect Molecular Biology, 2010, 19(Suppl. 2): 113-122.

[16] Vieira F G, Rozas J. Comparative genomics of the odorant-binding and chemosensory protein gene families across the Arthropoda: origin and evolutionary history of the chemosensory system. Genome Biology and Evolution, 2011, 3: 476-490.

[17] Spinelli S, Lagarde A, Iovinella I, Legrand P, Tegoni M, Pelosi P, Cambillau C. Crystal structure of Apis mellifera OBP14, a C-minus odorant-binding protein, and its complexes with odorant molecules. Insect Biochemistry and Molecular Biology, 2012, 42(1): 41-50.

[18] Gong Z J, Zhou W W, Yu H Z, Mao C G, Zhang C X, Cheng J A, Zhu Z R. Cloning, expression and functional analysis of a general odorant-binding protein 2 gene of the rice striped stem borer, Chilo suppressalis (Walker) (Lepidoptera: Pyralidae). Insect Molecular Biology, 2009, 18(3): 405-417.

[19] Wang G R, Wu K M, Guo Y Y. Molecular cloning and bacterial expressional of pheromone binding protein in the antennae of Helicoverpa armigera (Hübner). Archives of Insect Biochemistry and Physiology, 2004, 57(1): 15-27.

[20] Xu P X, Atkinson R, Jones D N, Smith D P. Drosophila OBP LUSH is required for activity of pheromone sensitive neurons. Neuron, 2005, 45(2): 193-200.

[21] Gu S H, Wang S P, Zhang X Y, Wu K M, Guo Y Y, Zhou J J, Zhang Y J. Identification and tissue distribution of odorant binding protein genes in the lucerne plant bug Adelphocoris lineolatus (Goeze). Insect Biochemistry and Molecular Biology, 2011, 41(4): 254-263.

[22] Li S, Picimbon J F, Ji S D, Kan Y C, Qiao C L, Zhou J J, Pelosi P. Multiple functions of an odorant-binding protein in the mosquito Aedes aegypti. Biochemical and Biophysical Research Communications, 2008, 372(3): 464-468.

[23] Li Z Q, Zhang S, Luo J Y, Cui J J, Ma Y, Dong S L. Two minus-C odorant binding proteins from Helicoverpa armigera display higher ligand binding affinity at acidic pH than neutral pH. Journal of Insect Physiology, 2013, 59(3): 263-272.

[24] 钱凯, 冯波, 巫诩亮, 劳冲, 沈幼莲, 杜永钧. 松墨天牛OBP基因MaltOBP2和MaltOBP6的克隆、序列分析及组织表达谱和原核表达研究. 昆虫学报, 2015, 58(5): 496-506.

Qian K, Feng B, Wu X L, Lao C, Shen Y L, Du Y J. Cloning, sequence analysis, tissue expression profiling and prokaryotic expression of odorant binding protein genes MaltOBP2 and MaltOBP6 from Monochamus alternatus (Coleoptera: Cerambycidae). Acta Entomologica Sinica, 2015, 58(5): 496-506. (in Chinese)

[25] 李红亮, 高其康, 程家安. 中华蜜蜂信息素结合蛋白ASP1 cDNA的克隆及时空表达. 昆虫学报, 2008, 51(7): 689-693.

Li H L, Gao Q K, Cheng J A. Cloning and spatio-temporal expression of cDNA encoding pheromone binding protein ASP1 in Apis cerana cerana (Hymenoptera: Apidae). Acta Entomologica Sinica, 2008, 51(7): 689-693. (in Chinese)

[26] 吉挺, 沈芳, 梁勤, 吴黎明, 刘振国, 罗岳雄. 中华蜜蜂OBP3基因的克隆、原核表达及组织表达谱. 昆虫学报, 2014, 57(8): 897-904.

Ji T, Shen F, Liang Q, Wu L M, Liu Z G, Luo Y X. Cloning, prokaryotic expression and tissue expression profiling of an OBP3 gene in the Chinese honeybee, Apis cerana cerana (Hymenoptera: Apidae). Acta Entomologica Sinica, 2014, 57(8): 897-904. (in Chinese)

[27] 张林雅. 中华蜜蜂两种嗅觉相关蛋白的基因克隆与功能研究[D]. 杭州: 中国计量学院, 2013.

Zhang L Y. Molecular cloning and functional analysis of two kinds of olfactory related genes in the Chinese honeybee, Apis cerana cerana[D]. Hangzhou: China Jiliang University, 2013. (in Chinese)

[28] Goldberg T, Hecht M, Hamp T, Karl T, Yachdav G, Ahmed N, Altermann U, Angerer P, Ansorge S, Balasz K. LocTree3 prediction of localization. Nucleic Acids Research, 2014, 42: W350-W355.

[29] Park D, Jung J W, Choi B S, Jayakodi M, Lee J, Lim J, Yu Y, Choi Y S, Lee M Y, Park Y, Choi I Y, Yang T J, Edwards O R, Nah G, Kwon H W. Uncovering the novel characteristics of Asian honey bee, Apis cerana, by whole genome sequencing. BMC Genomics, 2015, 16(1): 1.

[30] Robertson H M, Gadau J, Wanner K W. The insect chemoreceptor superfamily of the parasitoid jewel wasp Nasonia vitripennis. Insect Molecular Biology, 2010, 19(Suppl. 1): 121-136.

[31] Croset V, Rytz R, Cummins S F, Budd A, Brawand D, Kaessmann H, Gibson T J, Benton R. Ancient protostome origin of chemosensory ionotropic glutamate receptors and the evolution of insect taste and olfaction. PLoS Genetics, 2010, 6(8): e1001064.

[32] 赵亚华. 基础分子生物学教程. 2版. 北京: 科学出版社, 2006: 351-355.

Zhao Y H. Fundamental Molecular Biology Course. 2nd ed. Beijing: Science Press, 2006: 351-355. (in Chinese)

[33] 彭佳师, 龚继明. 信号肽与蛋白质的分选转运. 植物生理学报, 2011, 47(1): 9-17.

Peng J S, Gong J M. The mechanisms of protein sorting and translocation regulated by signal peptides. Plant Physiology Journal, 2011, 47(1): 9-17. (in Chinese)

[34] 谷少华, 张雪莹, 张永军, 吴孔明, 郭予元. 苜蓿盲蝽气味结合蛋白基因Alin-OBP1的克隆及表达谱分析. 昆虫学报, 2010, 53(5): 487-496.

Gu S H, Zhang X Y, Zhang Y J, Wu K M, Guo Y Y. Cloning and expression pattern analysis of an odorant binding protein gene Alin-OBP1 in the lucerne plant bug, Adelphocoris lineolatus (Goeze) (Hemiptera: Miridae). Acta Entomologica Sinica, 2010, 53(5): 487-496. (in Chinese)

[35] Tsuruda J M, Harris J W, Bourgeois L, Danka R G, Hunt G J. High-resolution linkage analyses to identify genes that influence Varroa sensitive hygiene behavior in honey bees. PLoS One, 2012, 7(11): e48276.

[36] 赵红霞, 曾鑫年, 梁勤, 张学锋, 黄文忠, 陈华生, 罗岳雄. 蜜蜂气味结合蛋白的生化特性、系统进化、表达部位和生理功能研究. 环境昆虫学报, 2015, 37(2): 420-425.

Zhao H X, Zeng X N, Liang Q, Zhang X F, Huang W Z, Chen H S, Luo Y X. Research on honeybee odorant-binding proteins biochemical characteristics, phyletic evolution, expression location and physiological function. Journal of Environmental Entomology, 2015, 37(2): 420-425. (in Chinese)

[37] 匡邦郁, 匡海鸥. 蜜蜂生物学. 昆明: 云南科技出版社, 2002: 101-105.

Kuang B Y, Kuang H O. Biology of the Honey Bee. Kunming: Yunnan Science and Technology Press, 2002: 101-105. (in Chinese)