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

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中文摘要：[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.

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

外文摘要：[Objective]The study aimed to analyze the molecular and functional characteristics of the short-chain odo-rant 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 short-chain OBP gene in different tissues of male and female adult P.striolata was analyzed by RT-PCR.A recombinant expres-sion vector was constructed for the heterologous expression of the short chain OBP gene,and the target protein was veri-fied 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 clo-ning and verification.The PstrOBP-sc gene contained an open reading frame of 357 bp,encoding 118 amino acid resi-dues,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 5 α-helixes,and there were only 2 amino acid residues in the downstream of the fifth α-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 olfac-tory 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.

外文关键词：

Phyllotreta striolatashort-chain odorant binding proteinmolecular characteristicstissue expression patternfluorescence binding assay

作者：

田晓丽、宋旭荣、毛永娜、李传仁、张国辉

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作者单位：

长江大学生命科学学院,湖北荆州 434025

长江大学农学院/长江大学昆虫研究所,湖北荆州 434025

湖北省林业科学研究院荆州分院,湖北荆州 434020

关键词：

黄曲条跳甲短链气味结合蛋白分子特征组织表达模式荧光结合试验

基金：

国家自然科学基金项目湖北省高等学校优秀中青年科技创新团队项目湖北省高等学校实验室研究项目

项目编号：

31972274T2022009HBSY2023-016

出版年：

2024

DOI：

10.3969/j.issn.2095-1191.2024.08.024

南方农业学报

广西壮族自治区农业科学院

南方农业学报

CSTPCD北大核心

影响因子：0.83

ISSN：2095-1191

年,卷(期)：2024.55(8)

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