甘薯蚁象气味结合蛋白CforOBP8的基因表达谱及配体结合特性分析
|
摘要
【目的】明确甘薯蚁象Cylas formicarius气味结合蛋白(odorant binding protein, OBP) OBP8的基因表达谱及与配体化合物的结合特性。【方法】基于甘薯蚁象成虫转录组数据,利用RT-PCR方法从甘薯蚁象成虫触角中克隆了气味结合蛋白OBP8基因,并进行生物信息学分析;采用qPCR方法测定该基因在甘薯蚁象成虫触角、口器、翅和跗节中的表达谱;构建原核表达载体,进行诱导表达及蛋白纯化。利用荧光竞争结合实验测定纯化的重组蛋白与16种配体化合物(1种性信息素和15种植物挥发物)的结合特性。【结果】获得甘薯蚁象气味结合蛋白基因CforOBP8(GenBank登录号:MH549528)的完整编码序列,其开放阅读框长414 bp,编码138个氨基酸,预测分子量为15.56 kD,等电点为4.71,N末端具有21个氨基酸组成的信号肽序列,蛋白序列具有4个保守的半胱氨酸,CforOBP8属于Minus-C OBPs亚家族。荧光定量PCR结果表明,CforOBP8在甘薯蚁象成虫触角中特异性表达,在其他组织中微量表达。SDS-PAGE结果显示融合蛋白成功表达。CforOBP8与配体化合物的结合力测试表明,CforOBP8与性信息素(E)-2-丁烯酸顺-(Z)-3十二烯基酯结合能力最强,解离常数K_i为4.13μmol/L;其次与5种植物挥发物也有一定的结合能力,依次为乙酸顺-3-己烯酯(K_i=7.54μmol/L)、柠檬烯(K_i=15.23μmol/L)、橙花醇(K_i=16.31μmol/L)、壬醛(K_i=28.71μmol/L)和苯乙醛(K_i=29.54μmol/L)。【结论】CforOBP8可能是甘薯蚁象性信息素结合蛋白,并且在感受性信息素和寄主植物挥发物的过程中都发挥作用。
【Aim】 This study aims to clarify the gene expression patterns of the odorant binding protein(OBP) OBP8 in Cylas formicarius and its ligand binding characteristics. 【Methods】 Based on the transcriptome data of C. formicarius adults, the odorant binding protein OBP8 gene was cloned from the antenna of C. formicarius adults and subjected to bioinformatical analysis. The expression patterns of this gene in adult antennae, mouthparts, wings and tarsi of C. formicarius were assayed by qPCR. Prokaryotic expression vector was constructed, and the recombinant protein was expressed and purified. The protein binding characteristics of the purified recombinant protein with 16 ligand compounds(one sex pheromone and 15 different plant volatiles) were assayed by fluorescence competitive binding assay. 【Results】 CforOBP8(GenBank accession no.: MH549528) of C. formicarius was successfully cloned and sequenced. Its open reading frame is 414 bp in length, encoding a protein of 138 amino acids with the predicted molecular weight of 15.56 kD and the isoelectric point of 4.71. The N-terminal signal peptide of CforOBP8 consists of 21 amino acids. The mature protein possesses four conserved cysteines and can be classified into the Minus-C OBPs subfamily. The qPCR results showed that CforOBP8 was specifically expressed in the antenna of C. formicarius adults and slightly expressed in other tissues. SDS-PAGE analysis showed that the fusion protein was successfully expressed. The binding affinity assay of CforOBP8 to ligand compounds showed that CforOBP8 had the strongest binding affinity to sex pheromone(Z)-3-dodecen-1-yl(E)-2-butenoate with the dissociation constant(K_i) of 4.13 μmol/L, and a certain binding affinities to five plant volatiles including(Z)-3-hexen-1-ol acetate, limonene, nerol, nonanal and phenylacetaldehyde, with the K_i values of 7.54, 15.23, 16.31, 28.71 and 29.54 μmol/L, respectively. 【Conclusion】 CforOBP8 might be a pheromone binding protein of C. formicarius and play dual roles in the perception of sex pheromones and plant volatiles.
【Aim】 This study aims to clarify the gene expression patterns of the odorant binding protein(OBP) OBP8 in Cylas formicarius and its ligand binding characteristics. 【Methods】 Based on the transcriptome data of C. formicarius adults, the odorant binding protein OBP8 gene was cloned from the antenna of C. formicarius adults and subjected to bioinformatical analysis. The expression patterns of this gene in adult antennae, mouthparts, wings and tarsi of C. formicarius were assayed by qPCR. Prokaryotic expression vector was constructed, and the recombinant protein was expressed and purified. The protein binding characteristics of the purified recombinant protein with 16 ligand compounds(one sex pheromone and 15 different plant volatiles) were assayed by fluorescence competitive binding assay. 【Results】 CforOBP8(GenBank accession no.: MH549528) of C. formicarius was successfully cloned and sequenced. Its open reading frame is 414 bp in length, encoding a protein of 138 amino acids with the predicted molecular weight of 15.56 kD and the isoelectric point of 4.71. The N-terminal signal peptide of CforOBP8 consists of 21 amino acids. The mature protein possesses four conserved cysteines and can be classified into the Minus-C OBPs subfamily. The qPCR results showed that CforOBP8 was specifically expressed in the antenna of C. formicarius adults and slightly expressed in other tissues. SDS-PAGE analysis showed that the fusion protein was successfully expressed. The binding affinity assay of CforOBP8 to ligand compounds showed that CforOBP8 had the strongest binding affinity to sex pheromone(Z)-3-dodecen-1-yl(E)-2-butenoate with the dissociation constant(K_i) of 4.13 μmol/L, and a certain binding affinities to five plant volatiles including(Z)-3-hexen-1-ol acetate, limonene, nerol, nonanal and phenylacetaldehyde, with the K_i values of 7.54, 15.23, 16.31, 28.71 and 29.54 μmol/L, respectively. 【Conclusion】 CforOBP8 might be a pheromone binding protein of C. formicarius and play dual roles in the perception of sex pheromones and plant volatiles.
引文
Ban L, Scalonib A, Angeli S, Zhang L, Yan Y, Pelosi P, 2003. Chemosensory proteins of Locusta migratoria. Insect Mol. Biol., 12(2): 125-134.
Cheng XJ, Cai LJ, Zheng LS, Qin JM, Huang YP, You MS, 2016. Cloning, expression profiling and binding characterization of the OBP2 gene in the diamondback moth, Plutella xylostella (Lepidoptera: Plutellidae). Acta Entomol. Sin., 59(4): 365-376. [程小娟, 蔡立君, 郑丽双, 覃江梅, 黄宇萍, 尤民生, 2016. 小菜蛾气味结合蛋白OBP2基因的克隆、表达谱及其结合特性分析. 昆虫学报, 59(4): 365-376]
Fang CQ, Zhang XX, Liu DD, Li KB, Zhang S, Cao YZ, Fan D, Yin J, 2016. Cloning and functional analysis of an odorant-binding protein HparOBP15a gene from Holotrichia parallela (Coleoptera: Melolonthidae). Acta Entomol. Sin., 59(3): 260-268. [房迟琴, 张鑫鑫, 刘丹丹, 李克斌, 张帅, 曹雅忠, 樊东, 尹姣, 2016. 暗黑鳃金龟气味结合蛋白HparOBP15a基因的克隆及功能分析. 昆虫学报, 59(3): 260-268]
Field LM, Pickett JA, Wadhams LJ, 2000. Molecular studies in insect olfaction. Insect Mol. Biol., 9(4): 545-551.
Jia XJ, Ma J, Gao B, Li XH, Zhang T, Chen SL, Wang RY, 2017. EAG and olfactory responses of Cylas formicarius (Coleoptera: Curculionidae) to volatiles from plants of different sweetpotato cultivars. Acta Entomol. Sin., 60(11): 1285-1291. [贾小俭, 马娟, 高波, 李秀花, 张涛, 陈书龙, 王容燕, 2017. 甘薯蚁象对不同甘薯品种植物挥发物的EAG和嗅觉反应. 昆虫学报, 60(11): 1285-1291]
Leal WS, 2013. Odorant reception in insects: roles of receptors, binding proteins, and degrading enzymes. Annu. Rev. Entomol., 58: 373-379.
Li L, Liu ZD, Sun JH, 2015. Olfactory cues in host and host-plant recognition of a polyphagous ectoparasitoid Scleroderma guani. BioControl, 60(3): 307-316.
Li ZQ, He P, Zhang YN, Dong SL, 2017. Molecular and functional characterization of three odorant-binding protein from Periplaneta Americana. PLoS ONE, 12(1): e0170072.
Li ZQ, Zhang S, Zhou SF, Luo JY, Cui JJ, 2017. Tissue expression profiling and ligand-binding properties of HarmOBP16 of the cotton bollworm, Helicoverpa armigera (Lepidoptera: Noctuidae). Acta Entomol. Sin., 60(8): 891-899. [李兆群, 张帅, 周淑芬, 雒珺瑜, 崔金杰, 2017. 棉铃虫气味结合蛋白HarmOBP16的组织表达谱及配及结合特性分析. 昆虫学报, 60(8): 891-899]
Li ZQ, Zhang S, Zhou SF, Luo JY, Cui JJ, Ma Y, Dong SL, 2013. Two Minus-C odorant binding proteins from Helicoverpa armigera display higher ligand binding affinity at acidic pH than neutral pH. J. Insect Physiol., 59(3): 263-272.
Ma L, Li ZQ, Bian L, Cai XM, Luo ZX, Zhang YJ, Chen ZM, 2016. Identification and comparative study of chemosensory genes related to host selection by legs transcriptome analysis in the tea geometrid Ectropis obliqua. PLoS ONE, 11(3): e0149591.
Manoharan M, Chong MNF, Va?tinadapoulé A, Frumence E, Sowdhamini R, Offmann B, 2013. Comparative genomics of odorant binding proteins in Anopheles gambiae, Aedes aegypti, and Culex quinquefasciatus. Genome Biol. Evol., 5(1): 163-180.
Schmittgen TD, Livak KJ, 2008. Analyzing real-time PCR data by the comparative CT method. Nat. Protoc., 3(6): 1101-1108.
Sun HZ, Song YQ, Du J, Wang XD, Cheng ZJ, 2016. Identification and tissue distribution of chemosensory protein and odorant binding protein genes in Athetis dissimilis (Lepidoptera: Noctuidae). Appl. Entomol. Zool., 51(3): 409-420.
Wang CQ, Zhao Y, Cao YZ, Wei HS, Li KB, Zhang S, Peng Y, Yin J, 2017. Expression and binding characterization of odorant binding protein (AcorOBP1) in Anomala corpulenta (Coleoptera: Scarabaeoidea). Acta Entomol. Sin., 60(4): 363-371. [王超群, 赵莹, 曹雅忠, 魏红爽, 李克斌, 张帅, 彭宇, 尹姣, 2017. 铜绿丽金龟气味结合蛋白AcorOBP1的表达和结合特性分析. 昆虫学报, 60(4): 363-371]
Wang GR, Guo YY, Wu KM, 2002. Progress in the studies of antenna odorant binding proteins of insects. Acta Entomol. Sin., 45(1): 131-137. [王桂荣, 郭予元, 吴孔明, 2002. 昆虫触角气味结合蛋白的研究进展. 昆虫学报, 45(1): 131-137]
Wang RY, Chen SL, Wang LP, Zhang H, Fan KJ, Zou XM, 2015. Survey on occurrence of the sweetpotato weevil Cylas formicarius and preliminary study on the causes of its outbreak in Chongqing. J. Agric., 5(1): 15-19. [王容燕, 陈书龙, 王良平, 张菡, 范开举, 邹祥明, 2015. 甘薯蚁象在重庆的发生调查及成灾原因初探. 农学学报, 5(1): 15-19]
Wang Y, Kays S, 2002. Sweetpotato volatile chemistry in relation to sweetpotato weevil (Cylas formicarius) behavior. J. Am. Soc. Hortic. Sci., 127(4): 656-662.
Zhang RZ, 2015. Cylas formicarlus (F.). Chin. J. Appl. Entomol., 52(2): 418. [张润志, 2015. 甘薯蚁象Cylas formicarlus (F.). 应用昆虫学报, 52(2): 418]
Zhou JJ, Robertson G, He XL, Dufour S, Hooper AM, Pickett JA, Keep NH, Field LM, 2009. Characterisation of Bombyx mori odorant-binding proteins reveals that a general odorant-binding protein discriminates between sex pheromone components. J. Mol. Biol., 389(3): 529-545.
Cheng XJ, Cai LJ, Zheng LS, Qin JM, Huang YP, You MS, 2016. Cloning, expression profiling and binding characterization of the OBP2 gene in the diamondback moth, Plutella xylostella (Lepidoptera: Plutellidae). Acta Entomol. Sin., 59(4): 365-376. [程小娟, 蔡立君, 郑丽双, 覃江梅, 黄宇萍, 尤民生, 2016. 小菜蛾气味结合蛋白OBP2基因的克隆、表达谱及其结合特性分析. 昆虫学报, 59(4): 365-376]
Fang CQ, Zhang XX, Liu DD, Li KB, Zhang S, Cao YZ, Fan D, Yin J, 2016. Cloning and functional analysis of an odorant-binding protein HparOBP15a gene from Holotrichia parallela (Coleoptera: Melolonthidae). Acta Entomol. Sin., 59(3): 260-268. [房迟琴, 张鑫鑫, 刘丹丹, 李克斌, 张帅, 曹雅忠, 樊东, 尹姣, 2016. 暗黑鳃金龟气味结合蛋白HparOBP15a基因的克隆及功能分析. 昆虫学报, 59(3): 260-268]
Field LM, Pickett JA, Wadhams LJ, 2000. Molecular studies in insect olfaction. Insect Mol. Biol., 9(4): 545-551.
Jia XJ, Ma J, Gao B, Li XH, Zhang T, Chen SL, Wang RY, 2017. EAG and olfactory responses of Cylas formicarius (Coleoptera: Curculionidae) to volatiles from plants of different sweetpotato cultivars. Acta Entomol. Sin., 60(11): 1285-1291. [贾小俭, 马娟, 高波, 李秀花, 张涛, 陈书龙, 王容燕, 2017. 甘薯蚁象对不同甘薯品种植物挥发物的EAG和嗅觉反应. 昆虫学报, 60(11): 1285-1291]
Leal WS, 2013. Odorant reception in insects: roles of receptors, binding proteins, and degrading enzymes. Annu. Rev. Entomol., 58: 373-379.
Li L, Liu ZD, Sun JH, 2015. Olfactory cues in host and host-plant recognition of a polyphagous ectoparasitoid Scleroderma guani. BioControl, 60(3): 307-316.
Li ZQ, He P, Zhang YN, Dong SL, 2017. Molecular and functional characterization of three odorant-binding protein from Periplaneta Americana. PLoS ONE, 12(1): e0170072.
Li ZQ, Zhang S, Zhou SF, Luo JY, Cui JJ, 2017. Tissue expression profiling and ligand-binding properties of HarmOBP16 of the cotton bollworm, Helicoverpa armigera (Lepidoptera: Noctuidae). Acta Entomol. Sin., 60(8): 891-899. [李兆群, 张帅, 周淑芬, 雒珺瑜, 崔金杰, 2017. 棉铃虫气味结合蛋白HarmOBP16的组织表达谱及配及结合特性分析. 昆虫学报, 60(8): 891-899]
Li ZQ, Zhang S, Zhou SF, Luo JY, Cui JJ, Ma Y, Dong SL, 2013. Two Minus-C odorant binding proteins from Helicoverpa armigera display higher ligand binding affinity at acidic pH than neutral pH. J. Insect Physiol., 59(3): 263-272.
Ma L, Li ZQ, Bian L, Cai XM, Luo ZX, Zhang YJ, Chen ZM, 2016. Identification and comparative study of chemosensory genes related to host selection by legs transcriptome analysis in the tea geometrid Ectropis obliqua. PLoS ONE, 11(3): e0149591.
Manoharan M, Chong MNF, Va?tinadapoulé A, Frumence E, Sowdhamini R, Offmann B, 2013. Comparative genomics of odorant binding proteins in Anopheles gambiae, Aedes aegypti, and Culex quinquefasciatus. Genome Biol. Evol., 5(1): 163-180.
Schmittgen TD, Livak KJ, 2008. Analyzing real-time PCR data by the comparative CT method. Nat. Protoc., 3(6): 1101-1108.
Sun HZ, Song YQ, Du J, Wang XD, Cheng ZJ, 2016. Identification and tissue distribution of chemosensory protein and odorant binding protein genes in Athetis dissimilis (Lepidoptera: Noctuidae). Appl. Entomol. Zool., 51(3): 409-420.
Wang CQ, Zhao Y, Cao YZ, Wei HS, Li KB, Zhang S, Peng Y, Yin J, 2017. Expression and binding characterization of odorant binding protein (AcorOBP1) in Anomala corpulenta (Coleoptera: Scarabaeoidea). Acta Entomol. Sin., 60(4): 363-371. [王超群, 赵莹, 曹雅忠, 魏红爽, 李克斌, 张帅, 彭宇, 尹姣, 2017. 铜绿丽金龟气味结合蛋白AcorOBP1的表达和结合特性分析. 昆虫学报, 60(4): 363-371]
Wang GR, Guo YY, Wu KM, 2002. Progress in the studies of antenna odorant binding proteins of insects. Acta Entomol. Sin., 45(1): 131-137. [王桂荣, 郭予元, 吴孔明, 2002. 昆虫触角气味结合蛋白的研究进展. 昆虫学报, 45(1): 131-137]
Wang RY, Chen SL, Wang LP, Zhang H, Fan KJ, Zou XM, 2015. Survey on occurrence of the sweetpotato weevil Cylas formicarius and preliminary study on the causes of its outbreak in Chongqing. J. Agric., 5(1): 15-19. [王容燕, 陈书龙, 王良平, 张菡, 范开举, 邹祥明, 2015. 甘薯蚁象在重庆的发生调查及成灾原因初探. 农学学报, 5(1): 15-19]
Wang Y, Kays S, 2002. Sweetpotato volatile chemistry in relation to sweetpotato weevil (Cylas formicarius) behavior. J. Am. Soc. Hortic. Sci., 127(4): 656-662.
Zhang RZ, 2015. Cylas formicarlus (F.). Chin. J. Appl. Entomol., 52(2): 418. [张润志, 2015. 甘薯蚁象Cylas formicarlus (F.). 应用昆虫学报, 52(2): 418]
Zhou JJ, Robertson G, He XL, Dufour S, Hooper AM, Pickett JA, Keep NH, Field LM, 2009. Characterisation of Bombyx mori odorant-binding proteins reveals that a general odorant-binding protein discriminates between sex pheromone components. J. Mol. Biol., 389(3): 529-545.