黄曲条跳甲短链气味结合蛋白基因PstrOBP-sc分子和功能特点分析

Molecular and functional characteristics of a short-chain odorant binding protein gene PstrOBP-sc from Phyllotreta striolata

【目的】分析黄曲条跳甲(Phyllotreta striolata)短链气味结合蛋白(Odorant binding protein,OBP)基因PstrOBP-sc分子和功能特点,为揭示昆虫短链OBP基因的功能提供参考。【方法】利用逆转录聚合酶链反应(RT-PCR)克隆黄曲条跳甲短链Classic OBP基因,并对其进行生物信息学分析;通过逆转录聚合酶链反应技术分析短链OBP基因在黄曲条跳甲雌、雄成虫不同组织中的表达情况;构建重组表达载体异源表达短链OBP基因,以聚丙烯酰胺凝胶电泳(SDS-PAGE)和蛋白质印迹法(Western blotting)验证目的蛋白;运用荧光结合试验检测短链OBP的配体结合能力。【结果】通过逆转录聚合酶链反应克隆验证,成功获得1条具有完整开放阅读框的黄曲条跳甲短链OBP基因PstrOBP-sc。PstrOBP-sc基因含有1个357 bp的开放阅读框,编码118个氨基酸残基,N末端具有1个由20个氨基酸残基组成的信号肽,成熟蛋白PSTROBP-SC仅由98个氨基酸残基组成,序列中含有6个保守的半胱氨酸,属于典型短链OBP基因。蛋白二级结构预测结果显示,PSTROBP-SC蛋白仅存在5个α-螺旋,且在第5个α-螺旋的下游仅有2个氨基酸残基。PstrOBP-sc基因组织表达模式及异源表达的PSTROBP-SC蛋白配体结合能力研究结果表明,PstrOBP-sc基因在黄曲条跳甲雌、雄成虫的所有供试组织中均有表达,而非特异性表达于嗅觉器官。同时,异源表达PSTROBP-SC不能结合荧光探针1-NPN。【结论】PstrOBP-sc基因的分子和功能特点不同于当前广泛研究的中长链OBP基因,其在生物体内很可能行使嗅觉以外的其他生理功能。

【Objective】The study aimed to analyze the molecular and functional characteristics of the short-chain odorant binding protein(OBP)gene PstrOBP-sc from Phyllotreta striolata,providing reference for exploring the function of short-chain OBP genes in insects.【Method】The short-chain Classic OBP gene from P.striolata was cloned using reverse transcription-polymerase chain reaction(RT-PCR)and subjected to bioinformatics analysis.The expression of the shortchain OBP gene in different tissues of male and female adult P.striolata was analyzed by RT-PCR.A recombinant expression vector was constructed for the heterologous expression of the short chain OBP gene,and the target protein was verified using sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE)and Western blotting techniques.The ligand-binding ability of the short-chain OBP was detected using fluorescence binding assay.【Result】A short-chain OBP gene(PstrOBP-sc)from P.striolata with a complete open reading frame was successfully obtained through RT-PCR cloning and verification.The PstrOBP-sc gene contained an open reading frame of 357 bp,encoding 118 amino acid residues,with a signal peptide of 20 amino acid residues at the N-terminal end.The mature protein PSTROBP-SC consisted of only 98 amino acid residues and contained 6 conserved cysteines in its sequence,making it a typical short-chain OBP gene.Protein secondary structure prediction showed that the PSTROBP-SC protein contained only 5α-helixes,and there were only 2 amino acid residues in the downstream of the fifthα-helix.The tissue expression pattern of the PstrOBP-sc gene and the ligand-binding ability of the heterologously expressed PSTROBP-SC protein showed that the PstrOBP-sc gene was expressed in all tested tissues of male and female adult P.striolata,and was not specifically expressed in olfactory organs.Additionally,the heterologously expressed PSTROBP-SC could not bind to the fluorescence probe 1-NPN.【Conclusion】The molecular and functional characteristics of PstrOBP-sc gene are different from those of the currently widely studied medium-long chain OBP genes,and it is likely to perform other physiological functions besides olfaction in the organism.