Abstract: 【Objective】The objective of this study is to clone and clarity odorant binding protein gene in the leafminer (Liriomyza sativae), analyze the sequence properties, gene expression, phylogeny and protein function, which would provide a basis for further study of the olfactory mechanism.【Method】The whole coding region of OBP gene from L. sativae was cloned by PCR strategy. Nucleotide sequence was analyzed using DNAMAN and homology comparison was analyzed using BLAST. An evolutionary tree was constructed by MEGA 6.0 to analyze phylogeny. Expression situation of LsatOBP13 in different tissues was assayed by the real-time quantitative PCR. Prokaryotic expression vector was constructed and then the recombinant protein was expressed and purified. The excitation wavelength of the fluorescence spectrophotometer was 337 nm and the 1-NPN was used as fluorescent probe to study the binding properties of OBP13 with 25 different odor ligands. The distribution of the LsatOBP13 was located by the indirect immuno-fluorescent staining and specific polyclonal antibody which was already prepared. The antenna of L.sativae was embedded, sliced up and observed under laser scanning confocal microscope to study the subcellular distribution of OBP13.【Result】An OBP gene in L.sativae was obtained and named as LsatOBP13. The accession number in GenBank ID is KT250751. The whole length of LsatOBP13 coding region is 462 bp that encodes a putative protein of 153 amino acids with a molecular mass of 17.80 kD and an isoelectric point of 5.75, and deduced amino acid sequence possesses a putative signal peptide of 17 amino acid residues at the N terminus. There are four conservative cysteine sites in the protein sequence, so that the LsatOBP13 is a Minus-C OBP. Phylogenetic analysis showed that the OBP13 and CcapOBP99a-like were clustered into one branch and had a high homology. According to the expression level in different tissues, the expression level in the antenna was much higher than that in other tissues. The recombinant expression vector was successfully constructed and the recombinant protein of high purity was obtained. After testing combining capacity of 25 odor ligands, it was found that LsatOBP13 had a good combining capacity with trans-2-hexenal, linalool, 1-octen-3-ol, α-ionone, benzothiazole and β-ionone, and the dissociation constants were 12.592, 10.995, 11.165, 11.224, 10.336, 9.218 μmol·L^-1, respectively. In these 6 kinds of odors, the affinity of β-ionone was the strongest. By immunofluorescence localization, it was found that it was mainly located in trichoid sensilla and basiconic sensilla on the crosscut flagellum section, and also in the olfactory pit and the joint of arista and funicular section.【Conclusion】The LsatOBP13 is a Minus-C OBP which belongs to the atypical OBPs. Expression situation, odorant binding characteristic and immunofluorescence localization showed that OBP13 existed mainly in the antenna of L. sativae and involved in the recognition process of odors in green leafed plants. It is presumed that it plays a role in olfactory recognition and host plant location of L.sativae.

Key words: Liriomyza sativae, odorant binding protein, sequence analysis, expression situation, odorant binding characteristic, immunofluorescence localization