海蜇Frizzled1基因的克隆及在无性繁殖中的表达  

Identification of Frizzled1 from Rhopilema esculentum and its role in asexual reproduction

作　　者：潘滢[1,2] 朱玲[2,3] 周春娅[2,4] 陈四清 庄志猛[2,3] PAN Ying;ZHU Ling;ZHOU Chunya;CHEN Siqing;ZHUANG Zhimeng(College of Ocean and Earth Sciences,Xiamen University,Xiamen 361005,China;Key Laboratory of Sustainable Development of Marine Fisheries,Ministry of Agriculture,Yellow Sea Fisheries Research Institute,Chinese Academy of Fishery Sciences,Qingdao 266071,China;Marine Biology and Biotechnology Laboratory,Qingdao National Laboratory for Marine Science and Technology,Qingdao 266237,China 4.College of Fisheries and Life Science,Shanghai Ocean University,Shanghai 201306,China)

机构地区：[1]厦门大学海洋与地球学院,福建厦门361005 [2]农业部海洋渔业可持续发展重点实验室中国水产科学研究院黄海水产研究所,山东青岛266071 [3]青岛海洋科学与技术国家实验室,山东青岛266237 [4]上海海洋大学水产与生命学院,上海201306

出　　处：《中国水产科学》2018年第6期1335-1346,共12页Journal of Fishery Sciences of China

基　　金：国家自然科学基金项目(31372507);鳌山科技创新计划项目(2016ASKJ02);中央级公益性科研院所基本科研业务费专项资金项目(20603022016001)

摘　　要：采用RACE技术解析了海蜇(Rhopilema esculentum)Frizzled1基因的cDNA和基因组结构:Re-Fzd1基因的全长cDNA为2387 bp,其中编码区为1761bp,编码586个氨基酸的多肽。SMART分析表明,Re-Fzd1基因具备Fzd家族共同的结构特征,包括:一个由23个氨基酸组成的信号肽,一个位于N-末端富含10个保守半胱氨酸残基的半胱氨酸富集域(CRD),一个含有7个跨膜片段的跨膜结构域,以及一个含有5个重要的磷酸化位点的C端尾巴。多序列比对表明,Re-Fzd1基因与刺胞动物贝螅(Hydra echinata)、水螅(Hydra vulgaris)、半球美螅水母(Clytia hemisphaerica)和海葵(Nematostella vectensis)Fzd1具有高度相似性,与来自脊椎动物人(Homo sapiens)、鼠(Mus musculus)、爪蟾(Xenopus laevis)和斑马鱼(Danio rerio)的Fzd1、Fzd2和Fzd7家族基因也具有较高的同源性。基于N-J法,将人、鼠、爪蟾、斑马鱼和果蝇(Drosophila melanogaster)所有Fzd家族基因系统进化分析显示,除果蝇外,所有Fzd家族成员聚类成4个类群,11个亚家族,海蜇Re-Fzd1基因首先与刺胞动物门的Fzd1聚类在一起,然后与脊椎动物Fzd1、Fzd2和Fzd7三个家族聚成一个类群,表明脊椎动物Fzd1、Fzd2和Fzd7家族可能与刺胞动物门的Fzd1起源于同一个共同祖先。Re-Fzd1基因组序列中不含有内含子。实时荧光定量PCR结果显示,Re-Fzd1基因在海蜇无性繁殖的4个发育阶段均有表达,其中,表达量最高的横裂体阶段是表达量最低的稚水母阶段的3.67倍。整体原位杂交显示,在海蜇横裂体时期,Re-Fzd1原位表达在触手、基座及发生横裂的部位。这些结果都表明,Re-Fzd1不但参与了海蜇的早期发育过程,还调控了海蜇无性繁殖的发生。The cDNA and genome of Frizzled1 from Rhopilema esculentum was cloned by the RACE technique. The full-length cDNA of Re-Fzd1 was 2387 bp, containing an open reading frame （ORF） of 1761 bp encoding a poly-peptide with 586 amino acid residues. SMART analysis showed that Re-Fzd1 shared common features of the Frizzled family, including a putative signal peptide with 23 amino acid residues, the N-terminal extracellular cysteine-rich domain （CRD） with 10 conserved cysteine residues, a transmembrane region composed of seven transmembrane segments, and a C-terminal “tail” with five key phosphorylation sites. The deduced amino acid sequence of Re-Fzd1 had high homology and may share a common ancestor with that of Frizzled1 in Cnidaria from Hydra echinata, H. vulgaris, Clytia hemisphaerica, and Nematostella vectensis, and that of Frizzled1, Frizzled2, and Frizzled7 in vertebrates, including Homo sapiens, Mus musculus, Xenopus laevis, and Danio rerio, by the multiple sequence alignment and phylogenetic analysis. None of the introns were included in the genome of Re-Fzd1. Quantitative real-time PCR analysis revealed that the expression of Re-Fzd1 transcript was detected in all four developmental stages. The mRNA expression level of Re-Fzd1 was the highest in strobila, which was 3.67-fold （P〈0.01） of that in the medusa. Furthermore, the Re-Fzd1 transcript was revealed in the tentacle and occurrence sites of strobilation in strobila by whole mount in situ hybridization. These results suggested that Re-Fzd1 should be involved in the regulation of asexual reproduction and occurrence of strobilation in R. esculentum.

关 键 词：海蜇 FRIZZLED CDNA 基因组结构 整体原位杂交 无性繁殖 

分 类 号：S917[农业科学—水产科学]