摘要
白刺粗角叶甲是我国西北荒漠草原危害防风固沙先锋植物白刺的重要害虫之一,开发和利用基于性信息素或寄主挥发物介导的昆虫化学通讯手段是防治该害虫的新途径。本研究以白刺粗角叶甲为对象,通过分子克隆技术获得白刺粗角叶甲成虫触角的非典型气味受体(atypical odorant receptor co-receptor,Orco)基因序列;利用同源建模方法预测蛋白三级结构,并构建系统发育树;借助荧光定量PCR技术检测DrybOrco基因在叶甲成虫各组织中的表达量差异。结果显示:克隆获得的白刺粗角叶甲非典型气味受体基因DrybOrco的cDNA全长为1918 bp,其中开放阅读框为1440 bp,编码479个氨基酸,蛋白分子量为53.94 kD,含有7个跨膜结构域,为疏水性膜蛋白。系统发育表明,Orco基因在6目68种昆虫间保守性较高(相似度大于68%),聚类分为3个分支,相同目的昆虫聚集到同一支。白刺粗角叶甲与其他鞘翅目昆虫聚为一支,其中与玉米根萤叶甲的Orco基因亲缘关系最近,核酸相似度高达92.28%。荧光定量表明,白刺粗角叶甲DrybOrco基因在成虫触角中特异性表达,且雄虫显著高于雌虫,表达量比值为2.27;此外,该基因在雄虫足和雌虫翅中少量表达,其他组织中几乎不表达。本研究明确了白刺粗角叶甲非典型气味受体基因DrybOrco的序列特征、蛋白结构和组织表达情况,这为阐明DrybOrco在该害虫化学通讯过程中的生理功能,以及探究白刺粗角叶甲寄主专化的嗅觉分子机制研究提供重要依据。
The leaf beetle Diorhabda rybakowi is one of the most important insect pests causing serious damage to the pioneer plant Nitraria spp.,which has windbreak and sand-fixation functions in the desert grasslands of Northwest China.The development and utilization of a chemical method based on insect perception of sex pheromones or host volatiles is a promising pest control strategy.In this study,we obtained an atypical odorant receptor co-receptor(Orco)gene sequence from the antennae of D.rybakowi adults using a molecular cloning method.We predicted the tertiary structure of the DrybOrco protein using homology modeling,constructed a phylogenetic tree,and detected the transcript levels of the DrybOrco gene in different tissues of male and female adults by real-time fluorescence quantitative PCR.It was found that the DrybOrco full-length gene sequence was 1918 bp long,with an open reading frame of 1440 bp encoding 479 amino acids.The putative protein had a predicted molecular weight of 53.94 kD,contained seven transmembrane domains,and was a hydrophobic membrane protein.The phylogenetic analysis revealed high conservation of Orco genes among 68 species of insects in six different orders(similarity>68%).In the phylogenetic tree,these genes grouped into three branches,with genes from the same insect order clustered on the same branch.The DrybOrco gene was clustered on the branch with sequences from other coleoptera species,and showed the highest nucleotide sequence similarity(92.28%)to the gene from Diabrotica virgifera.The qRT-PCR results showed that there were high transcript levels of the DrybOrco gene in the adult antennae,and with significantly higher levels(2.27 times higher)in the antennae of males than in the antennae of females.Low levels of DrybOrco transcripts were also detected in the legs of male adults and the wings of female adults,but its transcripts were barely detectable or absent from the other sampled tissues.The results of this study reveal the sequence characteristics and transcript profiles of an Orco gene in D.rybakowi adults,and details of the structure and characteristics of the putative protein.These findings provide a theoretical basis for elucidating the physiological function of the DrybOrco protein in chemosensory processes,and for further detailed studies on the olfactory molecular mechanism of host specialization in D.rybakowi.
作者
席驳鑫
崔晓宁
尚素琴
胡桂馨
王彦
李昌宁
彭斌
史薛强
XI Bo-xin;CUI Xiao-ning;SHANG Su-qin;HU Gui-xin;WANG Yan;LI Chang-ning;PENG Bin;SHI Xue-qiang(College of Plant Protection,Gansu Agricultural University,Lanzhou 730070,China;College of Pratacultural Science,Gansu Agricultural University,Key Laboratory for Grassland Ecosystem of Education Ministry,Lanzhou 730070,China)
出处
《草业学报》
CSCD
北大核心
2024年第5期204-215,共12页
Acta Prataculturae Sinica
基金
国家自然科学基金(32060397)
甘肃农业大学博士科研启动基金(GAU-KYQD-2018-24)
国家现代农业产业技术体系(CARS-34)资助。
关键词
白刺粗角叶甲
嗅觉
非典型气味受体
克隆
组织表达
Diorhabda rybakowi
olfactory
atypical odorant receptor co-receptor
cloning
tissue expression
作者简介
席驳鑫(1995-),男,甘肃镇原人,在读硕士。E-mail:sxqxbx@outlook.com;通信作者:崔晓宁.E-mail:cxn213@qq.com。
