Cloning, sequencing and prokaryotic expression of an odorant-binding protein gene PstrOBP1 from Phyllotreta striolata(Fabricius)

【Objective】 The purpose of the study was to clone and sequence the odorant-binding protein gene PstrOBP1 from Phyllotreta striolata(Fabricius) and conduct prokaryotic expression,in order to provide an important basis for in-depth investigation of the molecular characterization,functional mechanism,and heterologous expression conditions of PstrOBP1 gene.【Method】 Based on the head transcriptome data of P. striolata,RT-PCR was used to clone PstrOBP1 gene which encoded the odorant-binding protein of P. striolata. Bioinformatics analysis was performed and semi-quantitative PCR was used to detect the tissue expression pattern. PstrOBP1 fusion protein was purified by using a prokaryotic expression system and Ni-NTA His·bind resin column. Western blotting was used to validate it,and homology comparison and construction of phylogenetic evolutionary tree were performed for PstrOBP1.【Result】 The full-length open reading frame(ORF) of PstrOBP1 was 459 bp encoding 152 amino acid residues. It contained a signal peptide sequence of 19 amino acid residues at the N-terminal end,including 6 conserved cysteine residues,and belonged to Classic OBPs subfamily. The amino acid sequence of PstrOBP1 had high homology with Pyrrhalta aenescens PaenOBP13 (52.3%) and Pyrrhalta maculicollis PmacOBP13(51.5%), suggesting PstrOBP1 had a closer relationship with PaenOBP13 and PmacOBP13,and might share similar functions. Tissue expression analysis showed that PstrOBP1 gene was specifically expressed in the head of adult P. striolata,implying that the gene was involved in the process of chemical communication of adult P. striolata. After several optimizations of prokaryotic expression conditions,a large amount of soluble PstrOBP1 protein was expressed. The result of Western blotting confirmed the PstrOBP1 gene was successfully expressed about 32 kD of recombinant protein in Escherichia coli. 【Conclusion】 The PstrOBP1 gene is successfully cloned. Its encoded protein may be a significant olfactory protein involves in the process of chemical communication of P. striolata. After several optimizations of prokaryotic expression conditions,a large amount of soluble protein has been expressed,which provides a reference for the preparation of protein samples required for subsequent functional studies of this protein.