梨小食心虫GmolNPC2的基因克隆、表达谱及配体结合特征  

Gene cloning, expression profiling and ligand binding characteristics of GmolNPC2 from Grapholita molesta (Lepidoptera: Tortricidae)

作　　者：李春沁 张钰析 徐世才[1,2] 李伯辽 陈秀琳 罗坤 李广伟 LI Chun-Qin;ZHANG Yu-Xi;XU Shi-Cai;LI Bo-Liao;CHEN Xiu-Lin;LUO Kun;LI Guang-Wei(Shaanxi Key Laboratory of Chinese Jujube(Yan′an University),Yan′an 716000,China;College of Life Science,Yan′an University,Yan′an 716000,China)

机构地区：[1]陕西省红枣重点实验室(延安大学),延安716000 [2]延安大学生命科学学院,延安716000

出　　处：《昆虫学报》2023年第7期870-884,共15页Acta Entomologica Sinica

基　　金：国家自然科学基金项目(31860506,32260669);陕西省重点研发计划项目(一般项目——农业领域)(2022 NY-129);延安大学产学研合作培育项目(CXY202118);2021年延安大学校级大学生创新创业训练计划项目(D2021099)。

摘　　要：【目的】本研究旨在为深入研究梨小食心虫Grapholita molesta尼曼-匹克C2型蛋白(Niemann-Pick type C2 protein,NPC2)GmolNPC2的嗅觉感受功能提供理论参考。【方法】基于梨小食心虫触角转录组数据,利用RT-PCR扩增GmolNPC 2的cDNA全长序列,进行系统进化分析和3D结构模型预测;利用RT-qPCR检测GmolNPC 2在梨小食心虫不同发育阶段(卵、1-5龄幼虫、蛹和雌雄成虫)、3日龄雌雄成虫不同组织(触角、去除触角的头、胸、腹、足和翅)中的相对表达量;利用荧光竞争结合实验测定重组GmolNPC2蛋白对包括6种性信息素和38种寄主植物挥发化合物在内的44种气味配体的抑制常数K i值,分析结合能力;通过分子对接模拟实验计算GmolNPC2与不同气味配体的结合能,预测蛋白和配体相互作用的关键氨基酸残基。【结果】获得梨小食心虫GmolNPC 2(GenBanK登录号:OQ054801)的cDNA全长序列,开放阅读框(ORF)长438 bp,编码145个氨基酸;多序列比对发现GmolNPC2具有昆虫NPC2典型的结构特征;系统进化分析表明已知鳞翅目(Lepidoptera)昆虫的NPC2s聚类至2个分枝,GmolNPC2与大豆食心虫Leguminivora glycinivorella、红铃虫Pectinophora gossypiella和烟草天蛾Manduca sexta等的NPC2氨基酸序列一致性较高并聚类至同一分枝。RT-qPCR检测结果表明,GmolNPC 2在梨小食心虫雄成虫和卵中的表达量显著高于其他发育阶段的;GmolNPC 2在3日龄雌雄成虫翅中的表达量最高,在触角中的次之,在胸、腹和足中的最低。重组蛋白GmolNPC2的结合谱较窄,仅能够结合44种气味配体中的17种,对其中的乙酸-顺-8-十二碳烯酯、苯甲醇和乙酸-顺-3-己烯酯表现出强烈的结合能力,K i值分别为(4.117±0.046),(4.845±0.079)和(3.979±0.167)μmol/L。分子对接模拟结果显示,GmolNPC2与不同气味配体之间的结合能存在差异,与上述荧光结合实验的结果较一致。疏水性氨基酸残基和氢键在GmolNPC2结合不同气味配体中�【Aim】The purpose of this study is to provide a theoretical reference for the further study on the function of the Niemann-Pick type C2 protein(NPC2)of Grapholita molesta GmolNPC2 in olfactory sensing.【Methods】Based on the antennal transcriptome data of G.molesta,the full-length cDNA sequence of GmolNPC 2 was amplified by RT-PCR,and the phylogenetic analysis and 3D structure model prediction of GmoNPC2 were performed.The relative expression levels of GmolNPC 2 in different developmental stages(egg,1st-5th instar larvae,pupa,and female and male adults)and different tissues(antenna,head without antenna,thorax,abdomen,leg,and wing)of the 3-day-old male and female adults were detected by RT-qPCR.The inhibitory constant(K i)values of the recombinant GmolNPC2 to 44 odorant ligands including six sex pheromones and 38 host plant volatile compounds were determined by fluorescence competitive binding assays to analyze the binding ability of GmolNPC2 to odorant ligands.By using molecular docking simulations,the binding energy of GmolNPC2 with different odorant ligands was calculated and the key amino acid residues of protein-ligand interactions were predicted.【Results】The full-length cDNA sequence of GmolNPC2(GenBank accession no.:OQ054801)of G.molesta was obtained and the open reading frame(ORF)is 438 bp in length,encoding 145 amino acids.Multiple sequence alignment showed that GmolNPC2 has the typical structural characteristics of NPC2 of insects.Phylogenetic analysis showed that the reported NPC2s of lepidopteran insects were clustered into two branches,and the GmolNPC2 sequence was highly identic to the NPC2 sequences of Leguminivora glycinivorella,Pectinophora gossypiella,and Manduca sexta,which were clustered into one branch.RT-qPCR detection result showed that the expression levels of GmolNPC 2 in the male adult and egg were significantly higher than those in other developmental stages.The expression level of GmolNPC 2 was the highest in the wing of the 3-day-old male and female adults followed by that in

关 键 词：梨小食心虫 尼曼-匹克C2型蛋白 嗅觉 化学感受 寄主植物挥发物 

分 类 号：Q966[生物学—昆虫学]