中华绒螯蟹蜕皮激素受体基因(Ers-EcR)的克隆和组织表达分析

Cloning and expression analysis of ecdysteriod receptor gene ( Ers-EcR) in Eriocheir sinensis

蜕皮激素受体(ecdysteroid receptor,EcR)介导调控甲壳动物蜕皮生长、附肢再生等重要生命活动。为了解EcR在人工控制甲壳动物的繁殖和生长中的作用,采用RACE方法结合同源克隆技术,首次从中华绒螯蟹Y-器官中克隆得到蜕皮激素受体基因全长cDNA序列(Ers-EcR,登录号:KF736985),并进行了结构解析和组织表达分析。结果发现,Ers-EcR编码基因全长2 176 bp,开放阅读框为1 638 bp,编码545个氨基酸,具有DNA结合域(DBD)和配体结合域(LBD)等典型的核受体超家族结构域,但不具有信号肽结构。其中,DBD含有8个保守的Cys残基,可以形成2个锌指结构(C156-C159-C173-C176、C192-C198-C208-C211),是典型的DBD特征。多重序列比对分析表明,Ers-EcR氨基酸序列与拳手招潮蟹同源性最高,达到91%。荧光定量PCR结果显示成体中华绒螯蟹ErsEcR基因在Y-器官和肌肉组织中表达量最高,在血液、肠道、卵巢、眼柄、心脏和肝胰腺中有一定表达,在鳃、胸神经节和精巢表达量较低。这表明Ers-EcR基因在中华绒螯蟹各组织器官中的表达不具有典型的特异性,提示Ers-EcR基因可能参与体内多种生命活动的调控。

Ecdysteroid receptor（ EcR） could mediate the regulation effect of ecdysteroid hormone（ EH）,and plays important roles in molting and growth of Arthropod. To study the structure and function of ecdysteroid receptor（ EcR） gene in Eriocheir sinensis,a complete cDNA sequence encoding EcR（ Ers-EcR,GenBank Accession No： KF736985） was cloned from Y-organ of E. sinensis by 5’ and 3’ RACEs. The full-length cDNA consists of 2 176 bp with a 1 638 bp open reading frame,encoding 545 amino acids without signal peptide. Typical domains of nuclear receptor super family： DNA binding domain（ DBD） and ligand binding domain（ LBD） were identified by SMART（ simple modular architecture research tool） in Ers-EcR. DNA binding domain contains 8 conserved cysteines forming two zinc finger motifs（ C156-C159-C173-C176,C192-C198-C208-C211）,which is the typical character of DNA binding domain. Multi-sequence alignments analysis showed Ers-EcR shared the highest identity with Uca pugilator（ 91%）,also shared high identities with Callinectes sapidus（ 88%）,Scylla paramamosain（ 87%） and Portunus trituberculatus（ 83%）,however,it showed lower identity with EcRs from shrimps and insects. Phylogenetic analysis revealed that Ers-EcR was closely related to U. pugilator and Gecarcinus lateralis. Tissue distribution of Ers-EcR was detected by quantitative real-time PCR,although strong expression of Ers-EcR was detected in muscles and Y-organ,and its expression could also be found in eyestalk,intestine,hepatopancreas,heart,haemocyte,ovary,testis,thoracic nerve cord and gill. This study would facilitate the study of the roles of Ers-EcR in molt and growth regulation in crustacean.