梨小食心虫气味结合蛋白GmolOBP3的cDNA克隆、表达谱及结合特性分析

›› 2014, Vol. 57 ›› Issue (3): 274-285.doi:

梨小食心虫气味结合蛋白GmolOBP3的cDNA克隆、表达谱及结合特性分析

宋月芹^1，2，解幸承¹，董钧锋²，仵均祥^1,*

（1. 西北农林科技大学植物保护学院，植保资源与害虫治理教育部重点实验室，陕西杨凌 712100; 2. 河南科技大学林学院，河南洛阳 471003）

出版日期:2014-03-20 发布日期:2014-03-20

cDNA cloning, expression profiling and binding properties of odorant-binding protein GmolOBP3 in the oriental fruit moth, Grapholita molesta (Lepidoptara: Tortricidae)

SONG Yue-Qin^{1, 2}, XIE Xing-Cheng¹, DONG Jun-Feng², WU Jun-Xiang^1,*

(1. Key Laboratory of Plant Protection Resources and Pest Control, Ministry of Education, College of Plant Protection, Northwest A&F University, Yangling, Shaanxi 712100, China; 2. College of Forestry, Henan University of Science and Technology, Luoyang, Henan 471003, China)

Online:2014-03-20 Published:2014-03-20

摘要/Abstract

摘要： 【目的】为了更好地了解昆虫气味结合蛋白（odorant binding proteins, OBPs）在梨小食心虫Grapholita molesta（Busck）嗅觉识别中的作用，并明确其与寄主挥发物的结合特性。【方法】利用RT-PCR和RACE技术克隆梨小食心虫OBP基因；采用RT-PCR和实时定量PCR对该基因在成虫不同组织和羽化后不同日龄成虫中的表达情况进行了测定；以N-phenyl-1-naphthylamine（1-NPN）为荧光探针，采用荧光竞争结合试验对GmolOBP3蛋白的结合特性进行了分析。【结果】得到梨小食心虫一个新的气味结合蛋白基因，命名为GmolOBP3（GenBank登录号：KF395363）。GmolOBP3开放阅读框全长492 bp，编码163个氨基酸残基，预测分子量和等电点分别为18.72 kDa和4.93，呈酸性，具有典型的6个半胱氨酸位点。GmolOBP3在雌、雄成虫触角和腹部均有表达，成虫在羽化后5 d内，雌蛾触角中GmolOBP3表达量随羽化后日龄而增加，但雄蛾在羽化后第5天触角中 GmolOBP3表达量显著降低。通过构建GmolOBP3原核表达载体，在大肠杆菌Escherichia coli中诱导表达并获得了GmolOBP3重组蛋白。荧光竞争结合实验对GmolOBP3蛋白与16种寄主挥发物及4种性信息素类似物的结合力发现，在供试的4种梨小食心虫性信息素类似物中，GmolOBP3蛋白与反-8-十二碳烯醋酸酯和十二烷-1-醇不结合，而与顺-8-十二碳烯醋酸酯和顺-8-十二碳烯醇结合，但结合力较弱，结合常数分别为83.00和103.70 μmol/L；与16种寄主挥发物结合能力也不强，其中结合最强的是β紫罗酮，结合常数为49.36 μmol/L。【结论】由此推断，GmolOBP3具有选择性识别和结合各种配基的特性。

关键词: 梨小食心虫, 气味结合蛋白, cDNA克隆, 组织表达谱, 原核表达, 荧光竞争结合

Abstract: 【Aim】 To study the function and binding characteristics with plant volatiles of the odorant-binding proteins in the oriental fruit moth, Grapholita molesta (Busck).【Methods】 The OBP cDNA from G. molesta was cloned by using reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE), its tissue and developmental expression profiles were detected by RT-PCR and Real-time PCR, and the protein binding property was analyzed using fluorescence competitive binding assay with N-phenyl-1-naphthylamine (1-NPN) as the fluorescent probe. 【Results】A novel OBP cDNA from G. molesta was obtained, which was named as GmolOBP3 (GenBank accession no.: KF395363). GmolOBP3 contains a 492 bp open reading frame encoding a 163-amino-acid residue peptide. The predicted molecular weight and isoelectric point are 18.72 kDa and 4.93, respectively. The mature protein GmolOBP3 includes six typical conservative cysteine residues, which are the hallmark of insect OBPs. GmolOBP3 was expressed in the antennae and abdomen of female and male adults. In five days after eclosion, the expression level of GmolOBP3 in antennae of female moths increased with day-old after eclosion, but on the 5th day after eclosion, the expression quantity in antennae of male moths significantly reduced. In order to obtain the recombinant protein GmolOBP3, we constructed the prokaryotic expression vector of GmolOBP3, which was successfully expressed in the optimized condition. The recombinant protein was purified by anion exchange and Superose-12. The binding affinity of GmolOBP3 with 16 plant volatiles and 4 sex pheromone analogs indicated that GmolOBP3 could not bind with (E)-8-dodecenyl acetate and 1-dodecanol, while had weaker affinity with (Z)-8-dodecenyl acetate and (Z)-8-dodecenol with the association constants of 83.00 and 103.70 μmol/L, respectively. GmolOBP3 could also weakly bind with 16 plant volatiles with the strongest affinity to β-ionone and with a association constant of 49.36 μmol/L. 【Conclusion】 We so inferred that GmolOBP3 protein have characteristics of selective binding with various ligands.

Key words: Grapholita molesta, odorant binding proteins, cDNA cloning, tissue expression pattern, prokaryotic expression, fluorescence competitive binding assay

宋月芹，解幸承，董钧锋，仵均祥. 梨小食心虫气味结合蛋白GmolOBP3的cDNA克隆、表达谱及结合特性分析[J]. , 2014, 57(3): 274-285.

SONG Yue-Qin, XIE Xing-Cheng, DONG Jun-Feng, WU Jun-Xiang. cDNA cloning, expression profiling and binding properties of odorant-binding protein GmolOBP3 in the oriental fruit moth, Grapholita molesta (Lepidoptara: Tortricidae)[J]. , 2014, 57(3): 274-285.

0
/ / 推荐

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

链接本文: http://www.insect.org.cn/CN/

http://www.insect.org.cn/CN/Y2014/V57/I3/274

[1]	陈秀琳, 苏丽, 陈丽慧, 李怡萍, 仵均祥, 李广伟. 梨小食心虫Minus-C气味结合蛋白的分子克隆、表达谱及结合特性分析[J]. , 2018, 61(7): 771-783.
[2]	孙佳斌, 刘乃勇, 李双美, 闫祺, 董双林. 斜纹夜蛾信息素结合蛋白SlitPBP4的分子克隆、组织表达谱及结合特性分析[J]. , 2018, 61(6): 657-667.
[3]	司品法, 周琼, 崔中翌 . 柑橘大实蝇气味结合蛋白基因BminOBP25的克隆、原核表达及组织表达分析[J]. , 2018, 61(5): 537-545.
[4]	李帅, 苏丽, 李伯辽, 李怡萍, 李广伟, 仵均祥. 梨小食心虫谷胱甘肽S-转移酶GmolGST6的基因克隆、原核表达和酶学特征[J]. , 2018, 61(4): 398-409.
[5]	程杏安, 林贤伟, 蒋旭红, 刘展眉, 钟国华, 胡美英. 草地贪夜蛾组织蛋白酶L的基因克隆、原核表达及多克隆抗体制备[J]. , 2018, 61(4): 410-422.
[6]	柳小龙, 张宾, 田浩楷, 宁静, 王海香, 赵莉蔺. 松墨天牛Mfe2基因的克隆、原核表达和表达谱分析[J]. , 2018, 61(3): 282-291.
[7]	杨海博, 胡镇杰, 李定旭, 朱品红, 董钧锋. 悬铃木方翅网蝽触角气味结合蛋白基因鉴定[J]. , 2018, 61(10): 1121-1131.
[8]	申利, 许川, 张蕾, 张奎, 毛景欣, 潘光照, 李重阳, 胡仁建, 杨丽群, 崔红娟. 家蚕C类清道夫受体BmSR-C的基因克隆、原核表达及亚细胞定位[J]. , 2018, 61(10): 1132-1144.
[9]	苗娅, 张甜甜, 许柏英, 陈斌. 中华按蚊谷胱甘肽-S-转移酶E6（AsGSTE6）的表达纯化与结晶[J]. , 2018, 61(1): 59-67.
[10]	史宗畔, 冉永红, 张晶晶, 张静, 闫振天, 陈斌, 何正波. 中华按蚊气味结合蛋白AsinOBP1与避蚊胺（DEET）的结合特性分析[J]. , 2018, 61(1): 139-148.
[11]	谭永安, 赵旭东, 肖留斌, 孙洋, 赵静, 柏立新, 郝德君. 绿盲蝽核受体基因AlE75D的克隆和性质分析[J]. , 2017, 60(9): 984-993.
[12]	杨静, 高越, 刘中芳, 张鹏九, 樊建斌, 牛国飞, 韩召军, 范仁俊. 梨小食心虫几丁质合成酶1基因的克隆与表达分析[J]. , 2017, 60(9): 994-1005.
[13]	赵钰, 刘忠渊. 光滑鳖甲丝氨酸蛋白酶抑制剂基因ApSerpin-FA72的克隆、表达及功能验证[J]. , 2017, 60(8): 857-864.
[14]	李兆群, 张帅, 周淑芬, 雒珺瑜, 崔金杰. 棉铃虫气味结合蛋白HarmOBP16的组织表达谱及配基结合特性分析[J]. , 2017, 60(8): 891-899.
[15]	杨叶青, 王山宁, 彭勇, 单双, 郑瑶, 李瑞军, 张永军, 郭予元. 气味结合蛋白MmedOBP19在中红侧沟茧蜂足部的表达及配体结合特征[J]. , 2017, 60(6): 613-620.