蜂巢小甲虫气味结合蛋白基因AtOBP1的分子特性及功能研究  

Molecular characterization and functional analysis of odorant binding protein gene AtOBP1 in Aethina tumida(Coleoptera:Nitidulidae)

作　　者：李良斌 武丽仙 许雅静 刘芳 陈大福 刘家莉 赵红霞 LI Liang-Bin;WU Li-Xian;XU Ya-Jing;LIU Fang;CHEN Da-Fu;LIU Jia-Li;ZHAO Hong-Xia(Guangdong Public Laboratory of Wild Animal Conservation and Utilization,Guangdong Key Laboratory of Animal Conservation and Resource Utilization,Institute of Zoology,Guangdong Academy of Sciences,Guangzhou 510260,China;College of Plant Protection,South China Agricultural University,Guangzhou 510642,China;College of Animal Sciences(College of Bee Science),Fujian Agriculture and Forestry University,Fuzhou 350002,China)

机构地区：[1]广东省科学院动物研究所,广东省野生动物保护与利用公共实验室,广东省动物保护与资源利用重点实验室,广州510260 [2]华南农业大学植物保护学院,广州510642 [3]福建农林大学动物科学学院(蜂学学院),福州350002

出　　处：《昆虫学报》2023年第1期19-26,共8页Acta Entomologica Sinica

基　　金：广东省自然科学基金面上项目(2021A1515012102,2021A1515010630);国家蜂产业技术体系专项资金(CARS-44-SYZ11);广州市科技计划项目(202003000009)。

摘　　要：【目的】本研究旨在明确蜂巢小甲虫Aethina tumida气味结合蛋白1(odorant binding protein 1,OBP1)的表达模式,分析AtOBP1在蜂巢小甲虫嗅觉识别中的作用。【方法】基于蜂巢小甲虫转录组和基因组数据库扩增AtOBP1的cDNA全长序列,并进行生物信息学分析;利用RT-qPCR检测该基因在蜂巢小甲虫不同发育阶段(卵、幼虫、蛹和雌雄成虫)、羽化后第7天成虫不同组织(头、表皮、翅、足、脂肪体、肠道、马氏管、精巢和卵巢)以及羽化后不同日龄成虫头部的表达量;采用RNA干扰技术和Y型管行为选择实验,解析AtOBP1在蜂巢小甲虫嗅觉识别中的生物学功能。【结果】AtOBP1基因(GenBank登录号:MT211982.1)的cDNA全长序列包含6个外显子,其开放阅读框(ORF)长447 bp,编码148个氨基酸残基,预测分子量和等电点分别为15.9 kD和4.73,具有PBP_GOBP亚家族的保守结构域;AtOBP1蛋白是由6个α螺旋组成的二聚体,且有6个保守的半胱氨酸形成3个二硫键;系统发育进化树显示该蛋白与鞘翅目(Coleoptera)黄粉虫Tenebrio molitor的TmOBP8聚在同一分支。RT-qPCR结果显示AtOBP1在蜂巢小甲虫蛹期及雄成虫期表达量较高,且在成虫头部和精巢中特异性高表达;AtOBP1在成虫头部的表达量随着日龄而逐渐升高,分别在羽化后第5和7天达到2个高峰,羽化后第8天降低。RNAi技术结合Y型管行为选择行为实验结果表明,沉默AtOBP1后,蜂巢小甲虫成虫对花粉挥发性化合物棕榈酸乙酯和亚麻酸乙酯的选择偏好性显著降低。【结论】蜂巢小甲虫AtOBP1属于Classical OBPs家族,AtOBP1主要在蜂巢小甲虫成虫头部和精巢中高表达而且可能参与蜂巢小甲虫对花粉挥发性化合物棕榈酸乙酯和亚麻酸乙酯的识别过程。【Aim】This study aims to clarify the expression pattern of odorant binding protein 1(OBP1)in Aethina tumida and analyze the role of AtOBP1 in olfactory recognition of A.tumida.【Methods】The cDNA full-length sequence of AtOBP1 was cloned based on the transcriptome and genome database of A.tumida and analyzed by bioinformatics.RT-qPCR was used to detect the expression levels of AtOBP1 in different developmental stages(egg,larva,pupa,female adult and male adult),different tissues(head,cuticle,wing,leg,fat body,gut,Malpighian tubules,testis and ovary)of the 7-day-old adult and in the head of different day-old adults of A.tumida after eclosion.The biological function of AtOBP1 in olfactory recognition of A.tumida was studied by RNA interference(RNAi)and Y-tube behavior choice experiment.【Results】The cDNA full-length sequence of AtOBP1 gene(GenBank accession no.:MT211982.1)has six exons and an open reading frame(ORF)of 447 bp in length.AtOBP1 encodes 148 amino acid residues with PBP_GOBP superfamily conserved domain,and the predicted molecular mass and isoelectric point are 15.9 kD and 4.73,respectively.AtOBP1 protein is a dimer composed of sixα-helics with six conserved cysteines that form three disulfide bonds.The phylogenetic tree also showed that AtOBP1 was closely clustered into one branch with TmOBP8 from Tenebrio molitor of Coleoptera.The RT-qPCR results showed that AtOBP1 was highly expressed in the pupal stage and the male adult stage,and was highly specifically expressed in the head and testis of adults.In addition,the expression level of AtOBP1 in adult head increased gradually with the day-old age,reached two peaks in the 5-and 7-day-old adult stages,respectively,and decreased in the 8-day-old adult stage.RNAi in combination with Y-tube behavior choice experiment results revealed that silencing of AtOBP1 resulted in significantly reduced preference of A.tumida adults to the pollen volatile compounds ethyl palmitate and ethyl linolenate.【Conclusion】AtOBP1 belongs to Classical OBPs family.AtOBP

关 键 词：蜂巢小甲虫 气味结合蛋白 AtOBP1 RNAI 嗅觉行为 

分 类 号：Q965[生物学—昆虫学]