荻草谷网蚜唾液蛋白SmHMp1的基因克隆及基于HIGS技术的功能分析  

Gene cloning of salivary protein SmHMp1 of Sitobion miscanthi(Hemiptera:Aphididae)and functional analysis based on the HIGS technology

作　　者：王晗 尚哲明 刘晓明 刘德广[1] WANG Han;SHANG Zhe-Ming;LIU Xiao-Ming;LIU De-Guang(College of Plant Protection,Northwest A&F University,Yangling 712100,China)

机构地区：[1]西北农林科技大学植物保护学院,杨凌712100

出　　处：《昆虫学报》2023年第2期158-170,共13页Acta Entomologica Sinica

基　　金：国家自然科学基金项目(31971431)。

摘　　要：【目的】荻草谷网蚜Sitobion miscanthi是我国农业生产中的重大害虫之一,其唾液中存在许多功能各异的效应蛋白,在取食过程中参与蚜虫-植物互作。本研究旨在探索荻草谷网蚜唾液蛋白SmHMp1在该蚜虫取食和繁殖过程中的功能,探索其作为沉默靶标用于防治荻草谷网蚜的可行性。【方法】基于荻草谷网蚜唾液腺转录组数据,克隆荻草谷网蚜SmHMp1的cDNA全长序列,并进行生物信息学分析;以RT-qPCR技术测定荻草谷网蚜不同发育阶段(1-4龄若虫以及无翅成蚜)、无翅成蚜不同组织(唾液腺、头、胸、腹、胚胎以及整虫)、不同翅型成蚜(有翅成蚜和无翅成蚜)以及取食不同饲料(人工饲料和小麦苗)的无翅成蚜中SmHMp1基因表达量;以农杆菌Agrobacterium tumefaciens介导的烟草瞬时表达确定SmHMp1蛋白的亚细胞定位;构建大麦条纹花叶病毒(barley stripe mosaic virus,BSMV)重组病毒载体,利用寄主诱导的基因沉默技术(host-induced gene silencing,HIGS)沉默荻草谷网蚜SmHMp1基因后,通过解剖、生命表和刺探电位(electrical penetration graph,EPG)技术分析SmHMp1基因沉默对荻草谷网蚜生长发育、繁殖和取食的影响。【结果】克隆获得荻草谷网蚜SmHMp1(GenBank登录号:OP021692)cDNA全长序列,其开放阅读框长426 bp,编码141个氨基酸残基;预测其蛋白质分子质量为16.2 kD,理论等电点8.74,N端具有长度为19个氨基酸残基的信号肽。系统发育分析显示,SmHMp1与豌豆蚜Acyrthosiphon pisum未注释蛋白LOC100165393 precursor(GenBank登录号:NP_001155659.1)亲缘关系最近。RT-qPCR结果表明,荻草谷网蚜SmHMp1在各发育阶段均有表达,在1龄若虫期表达量最高,随发育时间总体呈先下降后升高趋势;SmHMp1在无翅成蚜唾液腺中表达量显著高于在其他组织中的;不同翅型成蚜中,SmHMp1表达量无显著性差异;取食不同饲料(人工饲料和小麦苗)的荻草谷网蚜无翅成蚜中SmHMp1表达量无�【Aim】The grain aphid,Sitobion miscanthi,is one of the most important pest insects in agricultural production in China.Its saliva contains many effector proteins with various functions,which are involved in aphid-plant interactions during the feeding process.The aim of this study is to clarify the functions of the salivary protein SmHMp1 in the processes of feeding and reproduction of S.miscanthi,and to explore the feasibility of using this gene as a silencing target to control S.miscanthi.【Methods】Based on salivary gland transcriptome data of S.miscanthi,the full-length cDNA sequence of SmHMp1 of S.miscanthi was cloned,and bioinformatically analyzed.The RT-qPCR was used to determine the expression levels of SmHMp1 in different development stages(1st-4th instar nymphs and wingless adult),tissues(salivary gland,head,thorax,abdomen,embryo and whole body)of wingless adults,adults of different wing morphs(winged and wingless adults)and wingless adults of S.miscanthi fed with different diets(artificial diet and wheat seedlings).Subcellular localization of the SmHMp1 protein was analyzed by Agrobacterium tumefaciens-mediated transient expression in tobacco.The recombinant virus vector of barley stripe mosaic virus(BSMV)was constructed and the host-induced gene silencing(HIGS)technology was used to silence SmHMp1 of S.miscanthi,and the silencing effects on the growth and development,reproduction and feeding of S.miscanthi were analyzed by anatomy,life table and electrical penetration graph(EPG)techniques.【Results】The full-length cDNA sequence of SmHMp1(GenBank accession no.:OP021692)of S.miscanthi was cloned.SmHMp1 has an open reading frame(ORF)of 426 bp in length,encoding 141 amino acid residues with the predicted molecular weight of 16.2 kD,theoretical isoelectric point of 8.74,and an N-terminal signal peptide with 19 amino acid residues.The phylogenetic analysis showed that SmHMp1 is most closely to the uncharacterized protein LOC100165393 precursor(GenBank accession no.:NP_001155659.1)of the pea aphid,Acy

关 键 词：荻草谷网蚜 唾液蛋白 效应子 亚细胞定位 基因沉默 

分 类 号：Q966[生物学—昆虫学]