Abstract:

【Aim】 This study aims to better understand the sex pheromone perception mechanisms by identifying and characterizing a sex pheromone binding protein (PBP) in the yellow peach moth, Conogethes punctiferalis (CpunPBP3). 【Methods】 The cDNA sequence of CpunPBP3 of C. punctiferalis was amplified and analyzed, and the amino acid sequence was compared to those of the homologous proteins in other Crambidae species. The day-age-dependent changes and circadian fluctuations in the expression levels of CpunPBP3 in the male adult antenna of C. punctiferalis, and the changes in the expression level of CpunPBP3 in the antenna over 24 h-period following exposure of adult males to the sex pheromones E10-16∶Ald (150 ng) and Z10-16∶Ald (6 ng) were examined by qRT-PCR. The recombinant expression vector pET-30a(+)/CpunPBP3 was constructed, and the recombinant CpunPBP3 was expressed in Escherichia coli. The binding capacity of the purified recombinant protein CpunPBP3 with the above two sex pheromones was evaluated by fluorescence competitive binding assay. 【Results】 The phylogenetic analysis result revealed that CpunPBP3 and the previously identified C. punctiferalis PBP genes CpunPBP2 and CpunPBP5 clustered in different branches, but CpunPBP3 is similar to PBP genes in other insect species. The qRT-PCR results showed that the expression level of CpunPBP3 in the male adult antenna increased first and then decreased from day 0 to 8 after adult eclosion, with significantly higher expression level at 17∶00 than at 1∶00, but with no significant difference at other time points within 24-h photoperiod. However, the expression level of CpunPBP3 in the male adult antenna significantly decreased after induction by 150 ng E10-16∶Ald for 3 and 6 h, and significantly increased after induction by 6 ng Z10-16∶Ald for 6 and 24 h. Fluorescence competitive binding assay result showed that the recombinant CpunPBP3 had strong binding capacity with E10-16∶Ald and Z10-16∶Ald, with the K_i values of 9.267 and 8.656 μmol/L, respectively. 【Conclusion】 The study determined the nucleotide and amino acid sequences and the expression pattern of CpunPBP3, and CpunPBP3 was expressed in response to the sex pheromone induction. The recombinant CpunPBP3 has strong binding capacity with sex pheromone, indicating that CpunPBP3 is a sex pheromone binding protein in C. punctiferalis.