冷杉梢斑螟化学感受蛋白DabiCSP8的配体结合特性分析  

Ligand binding property of chemosensory protein 8(DabiCSP8)of Dioryctria abietella

作　　者：朱垚龙 王政全 吴春 尹宁娜 刘乃勇[1] ZHU Yaolong;WANG Zhengquan;WU Chun;YIN Ningna;LIU Naiyong(Key Laboratory of Forest Disaster Warning and Control of Yunnan Province,Southwest Forestry University,Kunming 650224,China)

机构地区：[1]西南林业大学,云南省森林灾害预警与控制重点实验室,昆明650224

出　　处：《植物保护》2023年第3期50-59,74,共11页Plant Protection

基　　金：云南省应用基础研究面上项目(202001AT070100);云南省“高层次人才培养支持计划”青年拔尖人才项目(YNWR-QNBJ-2019-057)。

摘　　要：采用分子生物学和荧光竞争性结合测定等技术,研究了冷杉梢斑螟Dioryctria abietella雌虫性腺和雌、雄成虫足中高表达的化学感受蛋白DabiCSP8基因的表达特征和功能。定量分析结果表明,DabiCSP8基因在雌虫性腺中显著高表达,表达量分别是雌、雄虫触角的1232.27倍和3130.64倍;此外,该基因在雌、雄成虫足中表达量也较高。通过原核表达系统及亲和层析技术获得了13.64 kD的目的蛋白DabiCSP8。结合测定结果表明,DabiCSP8与探针N-苯基-1-萘胺(1-NPN)具有强的结合能力,结合常数(K_(1-NPN))为(1.61±0.02)μmol/L。β-紫罗兰酮是DabiCSP8的最佳结合配基,解离常数(K_(d))为(11.43±0.62)μmol/L。DabiCSP8结合腔内的极性氨基酸精氨酸46(Arg46)能够与β-紫罗兰酮形成两个氢键,暗示Arg46在该化合物的结合中起着重要作用。此外,DabiCSP8与部分杀虫剂具有中等强度的结合能力。In this study,the expression profiles of DabiCSP8 and binding properties of DabiCSP8 enriched in female pheromone glands and female and male legs of Dioryctria abietella were characterized using molecular biological methods and fluorescence competitive binding assays.Quantitative real-time PCR results revealed that the expression of DabiCSP8 was significantly higher in female pheromone glands compare to other tissues,which was 1232.27-and 3130.64-fold higher than that in female and male antennae,respectively.In addition,the high expression of DabiCSP8 was also detected in female and male legs.By prokaryotic recombinant expression and affinity chromatography,DabiCSP8 was expressed and purified with the expected size of 13.64 kD.Binding assays showed that DabiCSP8 could strongly bind to N-phenyl-1-naphthylamine(1-NPN)with a binding constant(K_(1-NPN))of(1.61±0.02)μmol/L.Among the 100 tested compounds,β-ionone was the best ligand for DabiCSP8 with a dissociation constant(K_(d))of(11.43±0.62)μmol/L.In the binding cavity of DabiCSP8,the polar amino acid residue arginine 46(Arg46)was able to form two hydrogen bonds withβ-ionone,suggesting the importance of Arg46 in the binding of DabiCSP8 toβ-ionone.In addition,DaciCSP8 exhibited moderate binding affinities to some insecticides.

关 键 词：冷杉梢斑螟 化学感受蛋白 结合测定 Β-紫罗兰酮 分子对接 

分 类 号：S763.3[农业科学—森林保护学]