二点委夜蛾非典型嗅觉受体AlepOrco的基因克隆、原核表达及多克隆抗体制备

doi:10.16380/j.kcxb.2020.06.002

昆虫学报 ›› 2020, Vol. 63 ›› Issue (6): 667-678.doi: 10.16380/j.kcxb.2020.06.002

二点委夜蛾非典型嗅觉受体AlepOrco的基因克隆、原核表达及多克隆抗体制备

田彩红¹, 刘晓光², 黄建荣¹, 王瑛¹, 封洪强^1，*

(1. 河南省农业科学院植物保护研究所, 河南省农作物病虫害防治重点实验室, 农业部华北南部作物有害生物综合治理重点实验室, 河南省作物保护国际联合实验室, 河南省生物农药工程研究中心, 郑州 450002; 2. 河南农业大学植物保护学院, 省部共建小麦玉米作物学重点实验室, 郑州 450002)

出版日期:2020-06-20 发布日期:2020-07-02

cDNA cloning, prokaryotic expression and polyclonal antibody preparation of the olfactory receptor co-receptor AlepOrco from Athetis lepigone (Lepidoptera: Noctuidae)

TIAN Cai-Hong¹, LIU Xiao-Guang², HUANG Jian-Rong¹, WANG Ying¹, FENG Hong-Qiang^1,*

(1. Henan Key Laboratory of Crop Pest Control, Key Laboratory of Integrated Pest Management on Crops in Southern Region of Northern China, Ministry of Agriculture, International Joint Research Laboratory for Crop Protection of Henan, Biological Pesticides Engineering Research Center of Henan Province, Institute of Plant Protection, Henan Academy of Agricultural Sciences, Zhengzhou 450002, China; 2. State Key Laboratory of Wheat and Maize Crop Science, College of Plant Protection, Henan Agricultural University, Zhengzhou 450002, China)

Online:2020-06-20 Published:2020-07-02

摘要/Abstract

摘要： 【目的】非典型嗅觉受体(olfactory receptor co-receptor, Orco)与典型嗅觉受体共同形成离子通道，在昆虫嗅觉识别中具有至关重要的作用。本研究旨在克隆和表达二点委夜蛾Athetis lepigone Orco基因，明确其分子特性，为进一步研究该基因在二点委夜蛾中的功能奠定基础。【方法】将二点委夜蛾雌雄成虫触角转录组数据建立本地数据库，通过生物信息学分析获得二点委夜蛾Orco同源基因AlepOrco；利用RT-PCR方法克隆二点委夜蛾AlepOrco基因全长，并在pGEX-6P-1/BL21(DE3)系统中进行了该基因开放阅读框(ORF)的原核表达, 制备多克隆抗体，用Western blot检测抗体特异性；利用qPCR技术检测该基因在二点委夜蛾雌雄成虫不同组织(喙、触角、去除触角和喙的头、胸、腹、足和翅)中的表达谱。【结果】获得了二点委夜蛾AlepOrco的cDNA(GenBank登录号: MN583125)全长序列，开放阅读框长1 422 bp，编码473个氨基酸，序列中有7个跨膜结构区，预测等电点为8.59，分子量为53.40 kD。SDS-PAGE和Western blot分析结果表明AlepOrco能够在大肠杆菌Escherichia coli中高效表达。利用制备的多克隆抗体对二点委夜蛾AlepOrco进行Western blot检测，其能够特异识别成虫触角中AlepOrco蛋白。qPCR结果表明，AlepOrco在二点委夜蛾雌雄成虫不同组织间具有相似的表达模式，都是在触角中的相对表达量最大，在翅中的表达量最小。【结论】克隆并原核表达了二点委夜蛾非典型嗅觉受体基因AlepOrco，制备的多克隆抗体能够特异识别二点委夜蛾成虫触角中的AlepOrco。结果为深入了解二点委夜蛾AlepOrco基因的结构和功能奠定了基础。

关键词: 二点委夜蛾, 非典型嗅觉受体, 基因克隆, 原核表达, 多克隆抗体, 组织表达

Abstract: 【Aim】 The olfactory receptor co-receptor (Orco) and the typical olfactory receptor constitute the ion channel together, playing critical roles in the olfactory activity in insects. This study aims to clone, express and characterize the Orco gene from Athetis lepigone so as to provide a basis for further study on the function of this gene in A. lepigone. 【Methods】 One local transcriptome database was established based on the antenna transcriptome data of female and male adults of A. lepigone, and a homologous gene of Orco in A. lepigone, AlepOrco, was obtained from bioinformatic analysis of the local transcriptome database. The full-length cDNA sequence of AlepOrco was cloned from A. lepigone using RT-PCR. The open reading frame of AlepOrco was further subcloned into prokaryotic expression vector pGEX-6P-1/BL21(DE3) system. Anti-AlepOrco polyclonal antibody was prepared, and its specificity was examined by Western blot. The expression patterns of AlepOrco in different tissues (proboscis, antennae, head without antennae and proboscis, thorax, abdomen, legs and wings) of female and male adults of A. lepigone were also analyzed by qPCR. 【Results】 The full-length cDNA of AlepOrco (GenBank accession no.: MN583125) of A. lepigone is 1 422 bp in length, encoding 473 amino acids, with seven transmembrane regions, the predicted isoelectric point of 8.59 and the molecular weight of 53.40 kD. The SDS-PAGE and Western blot results showed that AlepOrco was successfully expressed in Escherichia coli. The Western blot results indicated that the prepared antibody could specifically recognize AlepOrco from the antenna of A. lepigone adults. The qPCR results revealed that AlepOrco exhibited a similar expression pattern in different tissues of female and male adults, with the highest expression level in the antenna and the lowest expression level in the wing. 【Conclusion】 AlepOrco was cloned and successfully expressed in E. coli. The prepared polyclonal antibody can specifically identify the AlepOrco in the antenna of A. lepigone adults. The results provide a basis for further study on the structure and function of AlepOrco in A. lepigone.

Key words: Athetis lepigone; olfactory receptor co-receptor, gene cloning, prokaryotic expression, polyclonal antibody, tissue expression

田彩红, 刘晓光, 黄建荣, 王瑛, 封洪强. 二点委夜蛾非典型嗅觉受体AlepOrco的基因克隆、原核表达及多克隆抗体制备[J]. 昆虫学报, 2020, 63(6): 667-678.

TIAN Cai-Hong, LIU Xiao-Guang, HUANG Jian-Rong, WANG Ying, FENG Hong-Qiang. cDNA cloning, prokaryotic expression and polyclonal antibody preparation of the olfactory receptor co-receptor AlepOrco from Athetis lepigone (Lepidoptera: Noctuidae)[J]. Acta Entomologica Sinica, 2020, 63(6): 667-678.

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二点委夜蛾非典型嗅觉受体AlepOrco的基因克隆、原核表达及多克隆抗体制备

cDNA cloning, prokaryotic expression and polyclonal antibody preparation of the olfactory receptor co-receptor AlepOrco from Athetis lepigone (Lepidoptera: Noctuidae)

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