昆虫学报 ›› 2015, Vol. 58 ›› Issue (10): 1063-1071.

• 研究论文 • 上一篇    下一篇

尺蠖蜕皮激素受体基因Eo-EcR 的克隆、生物信息学分析和表达检测

李良德, 王定锋, 李慧玲, 张辉, 吴光远*   

  1. (福建省农业科学院茶叶研究所, 福建福安 355015)
  • 出版日期:2015-10-20 发布日期:2015-10-20
  • 作者简介:李良德, 男, 1988年01月生, 福建泉州人, 硕士, 研究实习员, 研究方向为昆虫生理学和毒理学, E-mail: liliangde_scau@163.com

Molecular cloning, bioinformatics analysis and expression profiling of ecdysteroid receptor gene Eo-EcR in Ectropis obliqua (Lepidoptera: Geometridae)

LI Liang-De, WANG Ding-Feng, LI Hui-Ling, ZHANG Hui, WU Guang-Yuan*   

  1. (Tea Research Institute, Fujian Academy of Agricultural Sciences, Fu’an, Fujian 355015, China)
  • Online:2015-10-20 Published:2015-10-20

摘要: 【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。

关键词: 茶尺蠖, 蜕皮激素受体, 克隆, 生物信息学, 时空表达, 杀虫剂

Abstract: 【Aim】 Ecdysteroid receptor (EcR) was a superfamily nuclear receptor, and it can form a heterodimeric complex EcR/USP with ultraspiracle (USP) protein to adjust the biological activity of 20-hydroxy molting ketone (20E) and regulate the developmental, abnormal and reproductive processes in insect lifecycle. This study aims to clone the full-length cDNA of EcR gene from Ectropis obliqua Prout, and to understand the characteristics of its encoding protein and its expression patterns. 【Methods】 The complete cDNA was cloned from E. obliqua by RT-PCR and RACE technologies. The bioinformatics analysis was carried out by bioinformatics software and editing on online website. The mRNA levels of EcR in different developmental stages and different tissues of the 6th instar larvae were investigated by real-time quantitative PCR (qRT-PCR). 【Results】 The EcR cDNA was cloned successfully from E. obliqua and named Eo-EcR (GenBank accession no. KP869130.1). Its complete length cDNA is 2 268 bp, including a 1 728 bp open reading frame (ORF), which encodes 576 amino acids. Phylogenetic treeanalysis and alignment of amino acid sequences showed that Eo-EcR is highly conserved among insects, especially in Lepidoptera. The results from tertiary structure and function domain prediction indicated that Eo-EcR has three classic structure models and the alpha helix is the dominant structure. It also contains a single functional site with a uni-C4 type zinc finger structure. qRT-PCR results showed that the expression level of Eo-EcR was higher in the 5th and 6th instar larval stage and adult stage compared to other stages. Tissue expression profiling showed that the expression level of Eo-EcR was the highest in prothoracic gland while lowest in hemolymph. 【Conclusion】 The sequence of nucleotide and characteristics of the encoded amino acid sequence of Eo-EcR were clarified. These results provide a foundation for further researches on the molecular function of Eo-EcR, and bring a new insight for the design of target insecticide to better control E. obliqua.

Key words: Ectropis obliqua, ecdysteroid receptor, clone, bioinformatics, temporal expression, insecticide