Identification of CSP genes and ligand binding properties of antenna-enriched CSP in Bactrocera tau （Walker）

【Objective】 This study aimed to identify antenna-enriched chemosensory protein （CSP） genes in adult Bactrocera tau （Walker）， and to investigate the ligand binding properties of heterologously expressed proteins of CSP genes， thereby providing a theoretical basis for elucidating the roles of CSP genes in the olfactory perception of adult Bactrocera tau （Walker） and developing efficient attractants. 【Method】 Reverse transcription polymerase chain reaction （RT-PCR） was used to analyze the expression patterns of CSP genes （BtauCSP1， BtauCSP2， BtauCSP3， BtauCSP4） in antennae， heads （without antennae） and other tissues （without heads） of male and female Bactrocera tau （Walker） adults. Recombinant expression vectors pET-28a（+）/BtauCSP3 were constructed， and transferred into the prokaryotic cell BL21 （DE3） for prokaryotic expression. The ligand binding properties of the bacterially expressed protein BtauCSP3 with 16 odorant ligands were analyzed by fluorescence competitive binding assays. 【Result】 Four CSP genes （BtauCSP1， BtauCSP2， BtauCSP3， and BtauCSP4） in Bactrocera tau （Walker） contained complete open reading frames （ORF） ranging from 336 to 474 bp， encoding 112-158 amino acid residues. The amino terminals of primary structure of 4 BtauCSPs proteins included signal peptide sequences （18-24 amino acid residues）. The predicted molecular weight of the mature BtauCSP protein with the signal peptide removed was 10.03-14.95 kD and the theoretical isoelectric point was 5.04-9.32. Accor-ding to the expression pattern analysis of BtauCSPs genes in different tissues of adult Bactrocera tau （Walker）， four BtauCSPs genes were expressed in all tested tissues， and the BtauCSP3 gene exhibited expression of high abundance in antennae of male and female Bactrocera tau （Walker） adults， implying that the BtauCSP3 gene may be involved in the olfactory recognition process of Bactrocera tau （Walker）. Fluorescence competitive binding assays demonstrated that the BtauCSP3 protein exhibited binding affinities to three host volatiles： undecanol， 4-（4-acetoxyphenyl）-2-butanone， and （R）-（+）-limonene； the inhibition constant （K_i） values were 15.44， 23.16 and 23.67 μmol/L， respectively. 【Conclusion】 The high-expressed BtauCSP3 gene in the antennae of adult Bactrocera tau （Walker） may be involved in the olfactory recognition of odorant compounds， particularly for odor compounds such as undecanol.