斜纹夜蛾信息素结合蛋白SlitPBP4的分子克隆、组织表达谱及结合特性分析  

Molecular cloning, tissue expression profiling and binding characterization of the pheromone binding protein SlitPBP4 from Spodoptera litura( Lepidoptera: Noctuidae)

作　　者：孙佳斌 刘乃勇[2] 李双美 闫祺 董双林[1] SUN Jia-Bin;LIU Nai-Yong;LI Shuang-Mei;YAN Qi;DONG Shuang-Lin(Key Laboratory of Monitoring and Management of Crop Diseases and Pest Insects,Ministry of Agriculture,College of Plant Protection,Nanjing Agricultural University,Nanjing 210095,China;Key Laboratory of Forest Disaster Warning and Control of Yunnan Province,Southwest Forestry University,Kunming 650224,China)

机构地区：[1]南京农业大学植物保护学院农业部作物病虫害监测与防控重点开放实验室,南京210095 [2]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：《昆虫学报》2018年第6期657-667,共11页Acta Entomologica Sinica

基　　金：国家重点研发计划项目(2017YFD0200900);国家自然科学基金项目(31672350)

摘　　要：【目的】鉴定斜纹夜蛾Spodoptera litura新的信息素结合蛋白(pheromone binding protein,PBP)基因,明确其在斜纹夜蛾不同组织中的表达水平并探讨其功能。【方法】基于已报道的烟草天蛾Manduca sexta、家蚕Bombyx mori、君主斑蝶Danaus plexippus和庆网蛱蝶Melitaea cinxia 4种非夜蛾科昆虫PBP4同源基因的序列特点及与其他PBP基因在染色体上的相邻关系,通过分析实验室先前克隆到的斜纹夜蛾PBP/GOBP基因序列,克隆斜纹夜蛾PBP4同源基因;通过RT-PCR和q PCR技术测定该基因在斜纹夜蛾雌雄成虫不同组织中的表达水平;利用体外表达和荧光竞争性结合实验测定斜纹夜蛾PBP4蛋白对性信息素组分和植物气味物质的结合能力。【结果】在夜蛾科昆虫斜纹夜蛾中鉴定到首个PBP4基因,命名为Slit PBP4(Gen Bank登录号:MG356847),c DNA编码210个氨基酸,具有N末端信号肽、疏水性气味分子结合域及6个保守半胱氨酸等PBP的典型序列特征,其基因组DNA在保守位置也含有2个内含子;但和已报道的斜纹夜蛾3个PBP相比,PBP4的C末端明显较长。Slit PBP4在雄成虫腹部(生殖系统)极高表达,在雌雄成虫触角及其他组织中仅微弱表达或不表达。荧光竞争性结合实验结果表明,Slit PBP4蛋白与被测定的信息素组分及植物气味物质均没有明显结合能力。【结论】报道了夜蛾科昆虫的首个PBP4基因,该基因可能主要参与雄虫生殖相关的生理过程而非嗅觉功能。【Aim 】 This study aims to identify the new pheromone binding protein（ PBP） gene of Spodoptera litura,to clarify its tissue expression pattern and to explore its functions. 【Methods】Based on the sequence characteristics of PBP4 homologues and their vicinity with other PBP genes on the chromosome in four non-noctuid species（ Manduca sexta,Bombyx mori,Danaus plexippus and Melitaeacinxia） reported previously,a PBP4 gene was obtained from S. litura by analyzing S. litura PBP/GOBP genes previously cloned by our laboratory. The tissue expression patterns of this gene in male and female adults were determined by RT-PCR and q PCR. The binding properties of the recombinant PBP4 protein to sex pheromone components and plant odorants were measured by fluorescence competitive binding assay. 【Results】The first PBP4 gene in Noctuidae was identified from S. litura and named Slit PBP4,which was deposited in Gen Bank under accession number MG356847. The c DNA sequence of Slit PBP4 encodes 210 amino acids,with the typical sequence features of PBPs,including an N terminal signal peptide,several hydrophobic regions,six conserved cysteines,and two introns inserted at the conserved positions in the genomic DNA. However,Slit PBP4 has a C terminus much longer than that of the other three reported Slit PBPs. Slit PBP4 showed predominantly high expression level in the male abdomen（ the reproductive systems） but nearly undetectable in the antennae and other adult tissues. Fluorescence competitive binding assay showed that Slit PBP4 protein had no obvious binding ability to the tested pheromone components and plant odors. 【Conclusion】The first PBP4 gene of noctuids was reported.Slit PBP4 is likely to be involved primarily in the reproduction-related physiological processes in male adults,rather than the olfactory function of other reported PBP genes in noctuids.

关 键 词：斜纹夜蛾 SlitPBP4 组织表达分析 原核表达 荧光竞争结合实验 

分 类 号：Q966[生物学—昆虫学]