小菜蛾触角结合蛋白PxylOBP31的鉴定与结合特性分析

doi:10.16380/j.kcxb.2016.08.002

昆虫学报 ›› 2016, Vol. 59 ›› Issue (8): 812-822.doi: 10.16380/j.kcxb.2016.08.002

小菜蛾触角结合蛋白PxylOBP31的鉴定与结合特性分析

覃江梅^1,2,3,4, 蔡立君^1,2,3,4,*, 郑丽双^1,2,3, 程小娟^1,2,3,5, 尤民生^1,2,3,4,*

（1. 福建农林大学应用生态研究所, 福州 350002; 2. 闽台特色作物病虫生态防控协同创新中心, 福州 350002; 3. 农业部闽台作物有害生物综合治理重点实验室, 福州 350002; 4. 福建农林大学植物保护学院, 福州 350002; 5. 福建农林大学生命科学学院, 福州 350002）

出版日期:2016-08-20 发布日期:2016-08-20

Identification and ligand binding characteristics of antennal binding protein PxylOBP31 in the diamondback moth, Plutella xylostella (Lepidoptera: Plutellidae)

QIN Jiang-Mei^1,2,3,4, CAI Li-Jun^1,2,3,4,*, ZHENG Li-Shuang^1,2,3, CHENG Xiao-Juan^1,2,3,5, YOU Min-Sheng^1,2,3,4,*

(1. Institute of Applied Ecology, Fujian Agriculture and Forestry University, Fuzhou 350002, China; 2. Fujian-Taiwan Joint Innovation Centre for Ecological Control of Crop Pests, Fujian Agriculture and Forestry University, Fuzhou 350002, China; 3. Key Laboratory of Integrated Pest Management of Fujian and Taiwan, China Ministry of Agriculture, Fuzhou 350002, China; 4. College of Plant Protection, Fujian Agriculture and Forestry University, Fuzhou 350002, China; 5. College of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou 350002, China)

Online:2016-08-20 Published:2016-08-20

摘要/Abstract

摘要： 【目的】触角结合蛋白（antennal binding proteins, ABPs）为昆虫气味结合蛋白（odorant binding proteins, OBPs）家族的一个亚类，是昆虫识别和响应外界环境中气味信号的载体之一，对昆虫的生存和繁衍有着重要的意义。明确触角结合蛋白在小菜蛾Plutella xylostella (L.)嗅觉识别中的作用，有助于揭示小菜蛾嗅觉识别分子机制。【方法】利用PCR技术克隆小菜蛾的一个触角结合蛋白基因；采用实时荧光定量PCR技术对该基因在小菜蛾不同发育阶段和成虫不同组织中的表达量进行分析；利用荧光竞争结合实验测试该触角结合蛋白与39种配基化合物的结合特性。【结果】成功克隆了一个小菜蛾触角结合蛋白基因，命名为PxylOBP31（GenBank登录号: KT156676）。序列分析结果显示，其开放阅读框全长411 bp，编码136个氨基酸，N端自起始位置开始21个氨基酸为信号肽，含有气味结合蛋白家族的6个保守半胱氨酸残基，预测分子量为14.74 kD，等电点为4.41。表达谱分析表明，PxylOBP31主要在雄蛾中表达，且交配后的雄蛾中表达量明显降低；该基因在小菜蛾触角中有较高表达，在雄蛾触角中的表达量比雌蛾触角中高近2倍。结合特性实验结果显示，PxylOBP31与醛、酮、萜品油烯以及邻苯二甲酸二异丁酯等物质的结合能力较强，与3种性信息素及其他烯烃与酯类结合能力弱。【结论】本研究明确了PxylOBP31的核苷酸序列以及发育和组织表达谱。根据qRT-PCR和荧光竞争结合实验结果，推测PxylOBP31蛋白可能与小菜蛾觅偶、定位寄主植物等行为有关。

关键词: 小菜蛾, 气味结合蛋白, 触角结合蛋白, 基因克隆, 表达谱, 荧光竞争结合试验

Abstract: 【Aim】 Antennal binding proteins (ABPs), a sub-class of odorant binding proteins (OBPs), indispensable primary carriers of chemical clues for survival and reproduction of insects. Uncovering the functions of ABPs in olfactory recognition of the diamondback moth, Plutella xylostella (L.), is fundamental to revealing the molecular mechanisms of olfaction of the moth. 【Methods】 An antennal binding protein gene was cloned by PCR, and the stage- and tissue-specific expressions of the gene were profiled using real-time quantitative PCR. Then the binding properties of the antennal binding protein to 39 ligand compounds were detected by fluorescence competitive binding assays. 【Results】 The antennal binding protein gene cloned was named as PxylOBP31 (GenBank accession no.: KT156676). Its ORF is 411 bp in length and encodes 136 amino acids, containing the six conserved cysteine residues of typical OBPs. The signal peptide consists of 21 amino acids in the N terminus. The predicted molecular weight and the isoelectric point of the encoded protein are 14.74 kD and 4.41, respectively. Expression profiling indicated that PxylOBP31 was highly expressed in male adults, and mainly expressed in antennae. While the expression level in male adult antennae was about 2-fold higher than that in female adult antennae. The expression level of PxylOBP31 decreased obviously in mated male adults. The results of binding property assays showed that PxylOBP31 had a high binding capacity to aldehydes, ketones, terpinolene, and disobutyl phthalate, but a low binding capacity to the main three sex pheromone components, olefins, and esters. 【Conclusion】 The nucleotide sequences and developmental and tissue expression profiles of PxylOBP31 were characterized. Based on the qRT-PCR and fluorescence competitive binding assay results, we inferred that PxylOBP31 is involved in such behaviors of the diamondback moth as seeking a mate and locating host plants.

Key words: Plutella xylostella, odorant binding protein, antennal binding protein, gene cloning, expression profile, fluorescence competitive binding assay

覃江梅, 蔡立君, 郑丽双, 程小娟, 尤民生. 小菜蛾触角结合蛋白PxylOBP31的鉴定与结合特性分析[J]. 昆虫学报, 2016, 59(8): 812-822.

QIN Jiang-Mei, CAI Li-Jun, ZHENG Li-Shuang, CHENG Xiao-Juan, YOU Min-Sheng. Identification and ligand binding characteristics of antennal binding protein PxylOBP31 in the diamondback moth, Plutella xylostella (Lepidoptera: Plutellidae)[J]. Acta Entomologica Sinica, 2016, 59(8): 812-822.

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小菜蛾触角结合蛋白PxylOBP31的鉴定与结合特性分析

Identification and ligand binding characteristics of antennal binding protein PxylOBP31 in the diamondback moth, Plutella xylostella (Lepidoptera: Plutellidae)

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