西花蓟马气味结合蛋白的cDNA克隆、序列分析及时空表达

doi:10.3864/j.issn.0578-1752.2016.06.007

摘要/Abstract

摘要： 【目的】鉴定西花蓟马(Frankliniella occidentalis)气味结合蛋白（odorant binding protein，OBP）并对其序列特征及分布情况进行研究，为阐明该虫嗅觉识别的机制、利用干扰昆虫嗅觉识别来进行害虫防治提供依据。【方法】利用RT-PCR和RACE技术克隆西花蓟马气味结合蛋白基因，用DNAMAN软件进行序列分析，用BLAST进行同源性比较，并用MEGA6.0的邻接法（neighbor-joining）构建系统进化树，应用服务器Chou & Fasman预测蛋白的二级结构，通过实时荧光定量PCR检测该基因在西花蓟马不同发育期以及成虫不同组织中的分布情况。【结果】克隆了一个西花蓟马气味结合蛋白基因，命名为FoccOBP1（GenBank登录号：KM527948）；该基因cDNA序列全长660 bp，其中开放阅读框450 bp，编码149个氨基酸，3′端非编码区长172 bp，具有真核生物polyA加尾结构，5′末端非编码区长38 bp，预测成熟蛋白分子量为16.39 kD，等电点为7.46，N端有22个氨基酸组成的信号肽序列，具有气味结合蛋白典型的6个保守半胱氨酸位点特征，其排列方式为C1-X₂₆-C2-X₃-C3-X₄₀-C4-X₉-C5-X₈-C6，符合典型OBP 6个保守的半胱氨酸位点结构模型。氨基酸序列中有3个亲脂性区域，第74—83位的氨基酸残基形成的亲脂性口袋尤为明显，可能是脂溶性气味分子的结合位点；FoccOBP1氨基酸序列与3个半翅目和10个同翅目昆虫OBP聚在同一分支，其中与绿盲蝽（Apolygus lucorum）的AlucOBP8（GenBank登录号为AFJ54049.1）、苜蓿盲蝽（Adelphocoris lineolatus）的AlinOBP5（GenBank登录号为ACZ58031.1）、牧草盲蝽（Lygus lineolaris）的LlinOBP2（GenBank登录号为AHF71029.1）同源相似性最高，其次是棉蚜（Aphis gossypii）的AgosOBP7（GenBank登录号为AGE97637.1）、大豆蚜（Aphis glycines）的AglyOBP7（GenBank登录号为AHJ80893.1）、禾谷缢管蚜（Rhopalosiphum padi）的RpadOBP7（GenBank登录号为AHL30243.1）等昆虫的OBP，表明FoccOBP1与这些基因的亲缘关系也较近；经FoccOBP1蛋白的二级结构预测，FoccOBP1以α-螺旋为主，其次是β-折叠，转角含量较少；经不同发育阶段检测发现，FoccOBP1在西花蓟马若虫期和初羽化成虫期的表达量较高，且随成虫羽化后天数的延长表达量逐渐降低，在雌雄成虫间的表达量也有差异；在初羽化成虫的不同组织检测发现，FoccOBP1几乎在初羽化成虫触角中特异性表达；构建了重组表达质粒pET-30a/FoccOBP1。【结论】明确了FoccOBP1的核苷酸、氨基酸序列特征，分析了该蛋白的二级结构特征，根据FoccOBP1在西花蓟马中的时空表达情况，推测该基因可能在西花蓟马嗅觉识别、定位寄主植物、信息素合成或性行为方面扮演重要角色；成功构建了重组表达质粒pET-30a/FoccOBP1，为下一步该蛋白表达纯化及其功能的深入研究打下基础。

关键词: 西花蓟马, 气味结合蛋白, 分子克隆, 序列分析, 时空表达

Abstract: 【Objective】The objective of this study is to clarify the sequence properties and distribution of odorant binding protein (OBP) in the western flower thrips (Frankliniella occidentalis), which would be favorable for throwing light on the mechanism of insect olfaction perception and lay a basis for controlling the target pests by interfering with insect chemoreception.【Method】The OBP gene from F. occidentalis (FoccOBP1) was cloned using RT-PCR and RACE-PCR strategies. Nucleotide sequence was analyzed using DNAMAN software. Homology was analyzed using BLAST. A phylogenetic tree was constructed using neighbor-joining of MEGA 6.0. The secondary structure and three-dimensional model of FoccOBP1 were predicted using Chou & Fasman and SWISS MODEL, respectively. Expression profiles of FoccOBP1 at different developmental stages and in different tissues of the adults were assayed using real-time quantitative PCR. 【Result】 The OBP gene in F. occidentalis was cloned and named as FoccOBP1. The number in GenBank is KM527948. The full length of FoccOBP1 cDNA is 660 bp, contains a 450 bp open reading frame (ORF) encoding a putative protein of 149 amino acids with a molecular mass of 16.39 kD and an isoelectric point of 7.46. The non-coding district of 3′ and 5′ end is 172 bp and 38 bp, respectively, there is a polyA structure of eukaryotes. The deduced amino acid sequence possesses a putative signal peptide of 22 amino acid residues at the N terminus and contains the typical six-cysteine signature of insect OBPs. The arrangement of C1-X₂₆-C2-X₃-C3-X₄₀-C4-X₉-C5-X₈-C6 conform to six conservative cysteine site of the typical OBPs structural model. There are three lipotropism areas in amino acid sequence. A lipotropy pocket significantly was formed by 74-83 amino acid residues, which could be a fat-soluble binding site of odor molecules. Three OBPs of Hemiptera insects, 10 OBPs of Homoptera insects and FoccOBP1 amino acid sequence were clustered into one group by distance tree, and among them FoccOBP1 protein had high homolog with Apolygus lucorum AlucOBP8 (GenBank number is AFJ54049.1), Adelphocoris lineolatus AlinOBP5 (GenBank number is ACZ58031.1) and Lygus lineolaris LlinOBP2 (GenBank number is AHF71029.1) of Hemiptera insects, and Aphis gossypii AgosOBP7（GenBank number is AGE97637.1), Aphis glycines AglyOBP7 (GenBank number is AHJ80893.1), Rhopalosiphum padi RpadOBP7 (GenBank number is AHL30243.1) and so on of Homoptera insects, suggesting that these genes likely developed from a common ancestral gene. Secondary structure of FoccOBP1 predicted showed that FoccOBP1 had more α-helices, followed by beta-fold and less angle. The results of FoccOBP1 expression at different development stages revealed that this gene was highly expressed in nymph and 1-day-old adult. Expression levels reduced gradually with ages of adult and had differences with adult sex. The results of FoccOBP1 expression in different tissues revealed this gene was specifically expressed in the antennae. The reconstruction of expression plasmid pET-30a (+)/FoccOBP1 was constructed successfully. 【Conclusion】The sequence characteristics of nucleotides and amino acids of FoccOBP1 were clarified. The secondary structure and three-dimensional model of FoccOBP1 were analyzed. Temporal and spatial expression pattern of FoccOBP1 was clarified, indicating that FoccOBP1 may play an important role in olfactory reception, locating food sources, synthesis of pheromone and mating of F. occidentalis. The reconstruction of expression plasmid pET-30a (+)/ FoccOBP1 was constructed successfully, which is a basis of expression, purity and function of FoccOBP1 protein.

Key words: Frankliniella occidentalis, odorant binding protein (OBP), molecular cloning, sequence analysis, expression profile

张治科，吴圣勇，雷仲仁. 西花蓟马气味结合蛋白的cDNA克隆、序列分析及时空表达[J]. 中国农业科学, 2016, 49(6): 1106-1116.

ZHANG Zhi-ke, WU Sheng-yong, LEI Zhong-ren. Cloning, Sequence Analysis and Expression Profile of an Odorant Binding Protein Gene in Western Flower Thrips (Frankliniella occidentalis)[J]. Scientia Agricultura Sinica, 2016, 49(6): 1106-1116.

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.0578-1752.2016.06.007

https://www.chinaagrisci.com/CN/Y2016/V49/I6/1106

参考文献

[1] Yudin L S, Cho J J, Mitchell W C. Host range of western flower thrips, Frankliniella occidentalis (Thysanoptera: Thripidae), with special references to Leucaena glauca. Environmental Entomology, 1986, 15(6): 1292-1295.

[2] Jones T, Scott-Dupree C, Harris R, Shipp L, Harris B. The efficacy of spinosad against the western flower thrips, Frankliniella occidentalis, and its impact on associated biological control agents on greenhouse cucumbers in southern Ontario. Pest Management Science, 2005, 61: 179-185.

[3] 孟祥钦, 闵亮, 万方浩, 周忠实, 王文凯, 张桂芬. 西花蓟马的SCAR分子检测技术. 昆虫学报, 2010, 53(3): 323-330.

Meng X Q, Min L, Wan F H, Zhou Z S, Wang W K, Zhang G F. SCAR marker for rapid identification of the western flower thrips, Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae). Acta Entomologica Sinica, 2010, 53(3): 323-330. (in Chinese)

[4] Cockfield S D, Beers E H, Zack R S. Phenology of western flower thrips Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae) on plant species in and near apple orchards in Washington State. Journal of the Entomological Society of British Columbia, 2007, 104: 35-44.

[5] 程俊峰, 万方浩, 郭建英. 入侵昆虫西花蓟马的潜在适生区分析. 昆虫学报, 2006, 49(3): 438-446.

Cheng J F, Wan F H, Guo J Y. Analysis of potential distribution of the invaded insect Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae) in China. Acta Entomologica Sinica, 2006, 49(3): 438-446. (in Chinese)

[6] Zhang Z K, Lei Z R. Identification, expression profiling and fluorescence-based binding assays of a chemosensory protein gene from the western flower thrips, Frankliniella occidentalis. PLoS ONE, 2015, 10(1): e0117726.

[7] 张治科, 吴圣勇, 雷仲仁. 西花蓟马化学感受蛋白的cDNA克隆、时空表达分析及组织定位. 昆虫学报, 2015, 58(1): 1-14.

Zhang Z K, Wu S Y, Lei Z R. cDNA cloning, expression profiling and immunolocalization of a chemosensory protein in the western flower thrips, Frankliniella occidentalis (Thysanoptera: Thtipidae). Acta Entomologica Sinica, 2015, 58(1): 1-14. (in Chinese)

[8] Zeng F F, Zhou S S, Ding Y H, Wang M Q. Analysis of an antennal cDNA library and the expression patterns of two olfactory genes in Frankliniella occidentalis (Thysanoptera: Thripidae). Applied Entomology and Zoology, 2015, 50: 109-116.

[9] Vogt R G, Riddiford L M, Prestwich G D. Kinetic properties of a sex pheromone-degrading enzyme: the sensillar esterase of Antheraea polyphemus. Proceedings of the National Academy of Sciences of the United States of America, 1985, 82: 8827-8831.

[10] Pelosi P, Zhou J J, Ban L, Calvello M. Soluble proteins in insect chemical communication. Cellular and Molecular Life Sciences, 2006, 63: 1658-1676.

[11] Vogt R G, Riddiford L M. Pheromone binding and inactivation by moth antennae. Nature, 1981, 293(5828): 161-163.

[12] 纪萍, 刘靖涛, 谷少华, 朱晓强, 张永军, 郭予元. 绿盲蝽气味结合蛋白AlucOBP7的表达及气味结合特性. 昆虫学报, 2013, 56(6): 575-583.

Ji P, Liu J T, Gu S H, Zhu X Q, Zhang Y J, Guo Y Y. Expression and binding specificity analysis of odorant binding protein AlucOBP7 from Apolygus lucorum (Hemiptera: Miridae). Acta Entomologica Sinica, 2013, 56(6): 575-583. (in Chinese)

[13] Krieger J, Breer H. Olfactory reception in invertebrates. Science, 1999, 286(5440): 720-728.

[14] Allen J E, Wanner K W. Asian corn borer pheromone binding protein 3, a candidate for evolving specificity to the 12-tetradecenyl acetate sex pheromone. Insect Biochemistry and Molecular Biology, 2011, 41(3): 141-149.

[15] Calvello M, Guerra N, Brandazza A, D’Ambrosio C, Scaloni A, Dani F R, Turillazzi S, Pelosi P. Soluble proteins of chemical communication in the social wasp Polistes dominulus. Cellular and Molecular Life Science, 2003, 60(9): 1933-1943.

[16] Sun M J, Liu Y, Wang G R. Expression patterns and binding properties of three pheromone binding proteins in the diamondback moth, Plutella xyllotella. Journal of Insect Physiology, 2013, 59: 46-55.

[17] Kyte J, Doolittle R F. A simple method for displaying the hydropathic character of a protein. Journal of Molecular Biology, 1982, 157: 105-132.

[18] Livak K J, Schmittgen T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2^-ΔΔCtmethod. Methods, 2001, 25: 402-408.

[19] Campanacci V, Krieger J, Bette S, Sturgis J N, Lartigue A, Cambillau C, Breer H, Tegoni M. Revisiting the specificity of Mamestra brassicae and Antheraea polyphemus pheromone-binding proteins with a fluorescence binding assay. The Journal of Biological Chemistry, 2001, 276(23): 20078-20084.

[20] Zhang T T, Gu S H, Wu K M, Zhang Y J, Guo Y Y. Construction and analysis of cDNA libraries from the antennae of male and female cotton bollworms Helicoverpa armigera (Hübner) and expression analysis of putative odorant-binding protein genes. Biochemical and Biophysical Research Communications, 2011, 407(2): 393-399.

[21] Gu S H, Sun Y, Ren L Y, Zhang X Y, Zhang Y J, Wu K M, Guo Y Y. Cloning, expression and binding specificity analysis of odorant binding protein 3 of the lucerne plant bug, Adelphocoris lineolatus (Goeze). Chinese Science Bulletin, 2010, 55: 3911-3921.

[22] Vandermoten S, Francis F, Haubruge E, Leal W S. Conserved odorant-binding proteins from aphids and eavesdropping predators. PloS ONE, 2011, 6(8): e23608.

[23] 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.

[24] Yasukawa J, Tomioka S, Aigaki T, Matsuo T. Evolution of expression patterns of two odorant-binding protein genes, Obp57d and Obp57e, in Drosophila. Gene, 2010, 467(1/2): 25-34.

[25] Krieger M J, Ross K G. Identification of a major gene regulating complex social behavior. Science, 2002, 295(5553): 328-332.

[26] Ban L, Scaloni A, D’ambrosio C, Zhang L, Yan Y, Pelosi P. Biochemical characterization and bacterial expression of an odorant- binding protein from Locusta migratoria. Cellular and Molecular Life Science, 2003, 60(2): 390-400.

[27] Deng S S, Yin J, Zhong T, Cao Y Z, Li K B. Function and immunocytochemical localization of two novel odorant-binding proteins in olfactory sensilla of the scarab beetle Holotrichia oblita Faldermann (Coleoptera: Scarabaeidae). Chemical Senses, 2012, 37: 141-150.

[28] 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.

[29] Zhou J J, Vieira F G, He X L, Smadja C, Liu R H, 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.

[30] Krieger J, Von Nickisch E, Mameli M, Pelosi P, Breer H. Binding proteins from the antennae of Bombyx mori. Insect Biochemistry and Molecular Biology, 1996, 26(3): 297-307.

[31] Yin J, Feng H, Sun H, Xi J, Cao Y, Li K B. Functional analysis of general odorant binding protein 2 from the meadow moth, Loxostege sticticalis L. (Lepidoptera: Pyralidae). PLoS ONE, 2012, 7(3): e33589.

[32] Widmayer P, Heifetz Y, Breer H. Expression of a pheromone receptor in ovipositor sensilla of the female moth (Heliothis virescens). Insect Molecular Biology, 2009, 18(4): 541-547.

[33] Hua J F, Zhang S, Cui J J, Wang D J, Wang C Y, Luo J Y, Lv L M. Identification and binding characterization of three odorant binding proteins and one chemosensory protein from Apolygus lucorum (Meyer-Dur). Journal of Chemical Ecology, 2012, 38: 1163-1170.

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

[35] Zhu J Y, Zhang L F, Ze S Z, Wang D W, Yang B. Identification and tissue distribution of odorant binding protein genes in the beet armyworm, Spodoptera exigua. Journal of Insect Physiology, 2013, 59(7): 722-728.