Acta Entomologica Sinica ›› 2020, Vol. 63 ›› Issue (6): 667-678.doi: 10.16380/j.kcxb.2020.06.002

• RESEARCH PAPERS • Previous Articles     Next Articles

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-Qiang1,*    

  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

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