桃蛀螟性信息素结合蛋白Cpun-PBP1的cDNA克隆、表达谱及其与配体化合物的结合特性分析

›› 2015, Vol. 58 ›› Issue (11): 1167-1176.doi:

桃蛀螟性信息素结合蛋白Cpun-PBP1的cDNA克隆、表达谱及其与配体化合物的结合特性分析

贾小俭^1,2, 郝少东², 杜艳丽^2,* , 张民照², 覃晓春², 王进忠², 王海香¹, 冀卫荣^1,*

（1. 山西农业大学林学院, 山西太谷 030801; 2. 北京农学院植物科学技术学院, 农业应用新技术重点实验室, 北京 102206）

出版日期:2015-11-20 发布日期:2015-11-20
作者简介:贾小俭, 女, 1989年8月生, 河北邯郸人, 研究方向为昆虫与害虫防治, E-mail: jiaxiaojian0@163.com

cDNA cloning, expression profiling and binding affinity assay of the pheromone binding protein Cpun-PBP1 in the yellow peach moth, Conogethes punctiferalis (Lepidoptera: Crambidae)

JIA Xiao-Jian ^1,2, HAO Shao-Dong ², DU Yan-Li ^2,* , ZHANG Min-Zhao ² , QIN Xiao-Chun ², WANG Jin-Zhong ² , WANG Hai-Xiang ¹ , JI Wei-Rong ^1,*

(1. College of Forestry, Agricultural University of Shanxi, Taigu, Shanxi 030801, China; 2. Beijing Key Laboratory for Agricultural Application and New Technique, College of Plant Science and Technology, Beijing University of Agriculture, Beijing 102206, China)

Online:2015-11-20 Published:2015-11-20

摘要/Abstract

摘要： 【目的】为了更好地了解性信息素结合蛋白（pheromone binding proteins, PBPs）在桃蛀螟Conogethes punctiferalis （Guenée）嗅觉识别过程中的作用，明确其与配体化合物的结合特性。【方法】本研究利用RT-PCR结合RACE方法克隆了桃蛀螟一个性信息素结合蛋白基因；采用Real-time PCR方法分析了该蛋白在桃蛀螟不同发育阶段及雌雄蛾间的表达差异；利用荧光竞争结合实验对Cpun-PBP1蛋白与16种配基化合物的结合特性进行了分析。【结果】克隆了一个桃蛀螟性信息素结合蛋白基因，命名为Cpun-PBP 1（GenBank登录号：KP027486）。Cpun-PBP 1开放阅读框全长510 bp，编码 169个氨基酸，预测分子量为19.12 kDa，等电点为5.09，N-末端包括由起始位置开始的30个氨基酸组成的信号肽。蛋白特征分析显示，该氨基酸序列具有昆虫气味结合蛋白的典型特征，即含有6个保守的半胱氨酸残基。Cpun-PBP 1在桃蛀螟成虫阶段表达量最高，且几乎全部在触角中表达，卵期微量表达，幼虫期和蛹期均不表达。通过构建Cpun-PBP 1原核表达载体，诱导并获得Cpun-PBP 1重组蛋白。荧光竞争结合实验对2种性信息素组分和14种寄主植物挥发物的结合力发现，Cpun-PBP1不但能有效地与桃蛀螟性信息素组分（顺-10-十六碳烯醛和十六醛）结合，结合常数分别为7.32和9.39 μmol/L；还能与8种寄主植物挥发物有效结合；其中，与莰烯的结合能力最强，结合常数为3.76 μmol/L。【结论】根据这些结果，我们推测Cpun-PBP1在桃蛀螟感受性信息素和寄主植物挥发物的过程中发挥着双重作用。

关键词: 桃蛀螟, 性信息素结合蛋白, 基因克隆, 表达谱分析, 原核表达, 荧光竞争结合

Abstract: 【Aim】 This study aims to study the function and ligand binding characteristics of pheromone binding proteins (PBPs) in the olfactory system of the yellow peach moth, Conogethes punctiferalis (Guenée). 【Methods】 A cDNA encoding pheromone binding protein of C. punctiferalis was cloned by RT-PCR and RACE. The expression pattern of the PBP gene in different sexes and developmental stages of C. punctiferalis was analyzed by real-time PCR, and the protein binding affinity was analyzed using fluorescence competitive binding assay. 【Results】 A novel PBP cDNA was obtained from C. punctiferalis and named as CpunPBP 1 (GenBank accession no. KP027486). Its open reading frame is 510 bp in length, encoding a protein of 169 amino acids with the predicted molecular weight of 19.12 kDa and theisoelectric point of 5.09. The 30 amino residues at the N-terminal hydrophobic region display the characteristics of a signal peptide. Protein signature analysis revealed that Cpun-PBP 1 shares the typical structural features of odorant binding proteins with other insects, including six conservative cysteine residues. Real-time quantitative PCR results indicated that CpunPBP1 was dominantly expressed in adult antennae, whereas scarcely expressed in egg stage and not expressed in larval and pupal stages. In addition, the recombinant protein of CpunPBP1 was prokaryotically expressed in Escherichia coli and purified. The binding affinity of CpunPBP1 to two sex pheromones and 14 plant volatiles showed that CpunPBP1 had a strong capability of binding two pheromone odorants (cis-10-hexadecenal and hexadecanoyl), with the binding constants of 7.32 and 9.39 μmol/L, respectively. Especially, CpunPBP1 could also bind with 8 plant volatiles, with the highest binding specificity to camphene (the binding constant: 3.76 μmol/L). 【Conclusion】 Based on these results, we inferred that CpunPBP1 play important roles in the process of discriminating odorants of pheromones and host plant volatiles of C. punctiferalis.

Key words: Conogethes punctiferalis, pheromone binding protein (PBP), gene cloning, expression pattern analysis, prokaryotic expression, fluorescence competitive binding assay

贾小俭, 郝少东, 杜艳丽, 张民照, 覃晓春, 王进忠, 王海香, 冀卫荣. 桃蛀螟性信息素结合蛋白Cpun-PBP1的cDNA克隆、表达谱及其与配体化合物的结合特性分析[J]. , 2015, 58(11): 1167-1176.

JIA Xiao-Jian, HAO Shao-Dong, DU Yan-Li, ZHANG Min-Zhao, QIN Xiao-Chun, WANG Jin-Zhong, WANG Hai-Xiang, JI Wei-Rong. cDNA cloning, expression profiling and binding affinity assay of the pheromone binding protein Cpun-PBP1 in the yellow peach moth, Conogethes punctiferalis (Lepidoptera: Crambidae)[J]. , 2015, 58(11): 1167-1176.

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