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【目的】蜕皮激素受体(ecdysteroid receptor,EcR)是一种超家族核受体,它能与超气门蛋白USP组成异源二聚体复合物EcR/USP,调节20-羟基蜕皮酮(20E)的生物活性,进而调控昆虫的发育、变态及繁殖等生命过程。本研究旨在克隆茶尺蠖Ectropis obliqua Prout EcR基因全长,并了解该基因的编码蛋白特征和时空表达模式。【方法】通过RT-PCR方法并结合RACE技术,克隆茶尺蠖EcR的基因全长,通过生物信息学软件和在线网站分析茶尺蠖EcR的生物学特性,通过实时荧光定量PCR(real-time quantitative PCR,qRT-PCR)技术比较茶尺蠖EcR在不同发育时期和6龄幼虫不同组织中的相对表达含量。【结果】克隆并鉴定了茶尺蠖EcR基因,将其命名为Eo-EcR(基因登录号:KP869130.1),Eo-EcR全长2 268 bp,含有1 728 bp开放阅读框,编码576个氨基酸。系统进化树和氨基酸同源性比对表明,Eo-EcR具有相对保守的进化特性,特别是与鳞翅目昆虫的保守性最高。三级结构模拟和功能结构域预测表明,Eo-EcR具有3个经典的结构模型,并以α螺旋为主,功能位点单一且为C4型锌指结构。qRT-PCR结果表明,Eo-EcR在5龄和6龄幼虫期以及成虫期表达量较高,在其他龄期表达量变化不大;同时在前胸腺表达量最高,在血淋巴表达量最低。【结论】明确了Eo-EcR的核苷酸序列及编码蛋白特征,明确了Eo-EcR的时空表达特性。该研究结果为进一步研究Eo-EcR的分子功能和基于Eo-EcR为靶标杀虫剂的研制奠定分子基础。
【Objective】 Ecdysteroid receptor (EcR) is a kind of superfamily nuclear receptor that can form heterodimer complex EcR / USP with USP and regulates the expression of 20-hydroxyecdysone (20E) Biological activity, and then regulate insect development, metamorphosis and reproduction and other life processes. The aim of this study is to clone the full-length EcR gene of Ectropis obliqua Prout and to understand the characteristics of the encoded protein and the temporal-spatial expression pattern of this gene. 【Method】 The full-length cDNA of EcR was cloned by RT-PCR and RACE. The biological characteristics of EcR were analyzed by bioinformatics software and online web site. Real-time quantitative PCR , qRT-PCR) were used to compare the relative expression levels of EcR in different tissues of 6th instar larvae at different developmental stages. 【Result】 The EcR gene was cloned and identified as Eo-EcR (Gene Accession No .: KP869130.1). The full length of Eo-EcR was 2 268 bp with a 1 728 bp open reading frame encoding 576 amino acids . Phylogenetic tree and amino acid homology alignment showed that Eo-EcR has the relatively conservative evolutionary characteristics, especially the Lepidoptera. Three-level structural modeling and functional domain prediction show that Eo-EcR has three classical structural models, mainly α-helix, single functional site and C4-type zinc finger structure. The results of qRT-PCR showed that Eo-EcR was highly expressed in 5th and 6th instar larvae as well as in adult stage, while its expression did not change much at other ages. The expression of Eo-EcR was highest in the anterior thymus and lowest in the hemolymph. 【Conclusion】 The nucleotide sequence of Eo-EcR and the characteristics of the encoded protein were confirmed, and the temporal and spatial expression characteristics of Eo-EcR were confirmed. The results laid the molecular foundation for the further study of the molecular function of Eo-EcR and the development of target pesticides based on Eo-EcR.