Acta Entomologica Sinica ›› 2022, Vol. 65 ›› Issue (7): 831-842.doi: 10.16380/j.kcxb.2022.07.005

• RESEARCH PAPERS • Previous Articles     Next Articles

cDNA cloning, expression and ligand binding properties of the chemosensory protein PyasCSP4 in Scythropus yasumatsui (Coleoptera: Curculionidae)

HONG Bo1, CHANG Qing1, ZHAI Ying-Yan1, REN Bo-Wen2, XIE Yu-Fen2, ZHANG Feng1,*    

  1. (1. Bio-Agriculture Institute of Shaanxi, Xi′an 710043, China; 2. Shaanxi Academy of Forestry, Xi′an 710082, China)
  • Online:2022-07-20 Published:2022-08-10

Abstract:  【Aim】 This study aims to clarify the expression features and ligand binding characteristics of chemosensory protein 4 (CSP4) in Scythropus yasumatsui, a major pest of Zizyphus jujuba. 【Methods】 Based on the antennal transcriptome data of S. yasumatsui 
adults, the cDNA sequence of PyasCSP4 was cloned by using RT-PCR and then subjected to bioinformatical analysis. The expression levels of PyasCSP4 in different adult tissues (antennae, heads without antennae, thoraxes, abdomens, legs and wings) of S. yasumatsui adults were assayed by RT-qPCR. The recombinant PyasCSP4 was prokaryotically expressed and then purified by Ni-NTA resin. The binding characteristics of the recombinant PyasCSP4 with 35 jujube volatiles were assayed by fluorescence competitive binding assay. 【Results】 The cDNA sequence of PyasCSP4 (GenBank accession no. OK322362) was cloned from S. yasumatsui. It contains an open reading frame of 366 bp in length, encoding 121 amino acids with a signal peptide of 18 amino acids at the N-terminus. The mature protein of PyasCSP4 possesses four conserved cysteines. The RT-qPCR analysis result showed that PyasCSP4 was ubiquitously expressed in all tissues of S. yasumatsui adults. The expression levels of PyasCSP4 in adult antennae and wings were significantly higher than those in other tissues. The recombinant PyasCSP4 had binding activities to 25 ligands, and particularly had the strongest binding affinity to α-pinene, α-phellandrene and ocimene, with the dissociation constant (Ki) values of 6.29, 6.58 and 6.69 μmol/L, respectively. 【Conclusion】 PyasCSP4 has the capability to bind a variety of jujube volatiles, indicating that it may play an important role in locating host plants for S. yasumatsui. The results have great significance for elucidating the olfactory mechanism of S. yasumatsui and implementing green prevention and control measures.

Key words: Scythropus yasumatsui, chemosensory protein, ligand binding characteristics; prokaryotic expression, fluorescence competitive binding assay