鲤鱼(Cyprinus carpio)外异蛋白A受体Edar基因的克隆及表达定位

生物技术通报 ›› 2013, Vol. 0 ›› Issue (12): 99-107.

鲤鱼(Cyprinus carpio)外异蛋白A受体Edar基因的克隆及表达定位

蒋丽¹ 王阳阳^{1, 2} 程安达^1，3, 张保勇^{1, 2}, 马龙^1，4, 王书¹ 刘永新¹, 孙效文^1，5

1.中国水产科学研究院水产生物应用基因组研究中心，北京 100141
2.大连海洋大学生命科学与技术学院，大连 116023
3.上海海洋大学水产与生命学院，上海 201306
4.河南师范大学水产学院，新乡 453200
5.中国水产科学研究院黑龙江水产研究所农业部淡水水产生物技术与遗传育种重点实验室，哈尔滨 150070

收稿日期:2013-07-22 出版日期:2013-12-20 发布日期:2013-12-20
作者简介:蒋丽，女，博士，助理研究员，研究方向：水产生物发育生物学；E-mail：jiangl@cafs.ac.cn
基金资助:
中央公益级基本科研业务费项目（2011C015）

Preliminary Study of Edar Gene Cloning and Localization of Expression in Cyprinus carpio

Jiang Li¹, Wang Yangyang^{1, 2}, Cheng Anda^1，3, Zhang Baoyong ^{1, 2}, Ma Long^1，4, Wang Shu¹, Liu Yongxin¹， Sun Xiaowen^1，5

1. The Center for Applied Aquatic Genomics, Chinese Academy of Fishery Sciences, Beijing 100141
2. Dalian Ocean University College of Life Science and Technology, Dalian Institute of Fisheries and Life, Dalian 116023
3. Shanghai Ocean University, Shanghai 201306
4. College of Henan Normal University, Xinxiang 453200
5. Heilongjiang Fisheries Research Institute Chinese Academy of Fishery Sciences Department of Agriculture Freshwater Aquaculture Biotechnology Key Laboratory of Genetics and Breeding, Harbin 150070

Received:2013-07-22 Published:2013-12-20 Online:2013-12-20

摘要/Abstract

摘要：

外异蛋白A受体(Ectodysplasin-A receptor，Edar)基因最早是在哺乳动物中发现的，其在皮肤附属物的发育中具有重要的生物学功能。利用Genfishing差异筛选技术对建鲤和镜鲤的皮肤转录表达产物进行筛选，得到Edar 基因的部分片段。通过克隆镜鲤mRNA全长发现，镜鲤该基因全长CDS为1 389 bp，编码一个含有462个氨基酸的蛋白质，包含一个信号肽位点、肿瘤坏死因子受体结合位点序列、跨膜区和死亡结构域。序列比对结果表明，镜鲤Edar 蛋白与斑马鱼相似度最高，达88.31%，而与其亲缘关系较远的爪蟾，鸡，人类，小鼠相似度较低，分别是64.88%，63.79%，64.36%，63.50%。对5'-UTR和3'-UTR区进行分析表明，镜鲤与斑马鱼、青鳉 5'-UTR的相似度只有23.78%，24.73%，而与非洲爪蟾、鸡、人和老鼠的相似度更低，仅为11.73%、18%、6.50%、10.04%；3'-UTR与青鳉的相似度为35.49%，与非洲爪蟾、人和小鼠的相似度分别为9.42%、8.05%、9.37%。整体原位杂交结果表明，该基因在建鲤和镜鲤鳞片发生时在鳞片着生的皮肤基质中特异表达，而在非鳞片发生区域表达较弱或者不表达，到所有鳞片发育形成后，该基因的表达消失，表明该基因可能参与鲤鱼鳞片的起始发育而不是后期的鳞片模式维持过程。

关键词: 外异蛋白A受体, RACE, 整体原位杂交

Abstract:

Ectodysplasin-A receptor(Edar)was firstly found in mammal, and plays vital roles in developmental events of the skin appendages. The screening for transcripts of skin from jian carp and mirror carp by Genefishing differential found Edar is one of the targeted different expressed in both materials, and the partial fragment of Edar gene was recovered from differential expressed products by sequencing. Then by cloning, the mirror carp Edar CDS consisted of 1 389 base pair nucleotides which encodes a 462 amino acid protein, has a signal peptide, a tumor necrosis factor receptor region, a transmembrane region and death domain. The alignment result showed that the carp shares the highest similarity(88.31%)with the zebrafish. However, it has the relative lower similarity with Xenopus laevis (64.88%)chicken(63.79%), human(64.36%), mouse(63.50%)due to evolutionarily distant relationships. The 5'-UTR shares the high variation, even with the medaka(24.73%)and zebrafish(23.78%). Also, in the 3'-UTR, it varies significantly with medaka(35.49%), let alone with Xenopus laevis (11.73% and 9.42%), human(6.50% and 8.05%), mouse(10.04% and 10.04%). The in situ results showed that the Edar unique expression signal was detected in skin matrix which the scale formed, but weak or no expression signal was detected in other positions on skin, later disappeared in full developed scaled body. All of these results showed that this gene may involve the initiation of the scales not the maintenance of the scale pattern formation.

Key words: Ectodysplasin-A receptor, RACE, Whole mount in situ hybridization

蒋丽, 王阳阳, 程安达, 张保勇, 马龙，王书, 刘永新, 孙效文. 鲤鱼(Cyprinus carpio)外异蛋白A受体Edar基因的克隆及表达定位[J]. 生物技术通报, 2013, 0(12): 99-107.

Jiang Li, Wang Yangyang, Cheng Anda, Zhang Baoyong,Ma Long, Wang Shu, Liu Yongxin， Sun Xiaowen. Preliminary Study of Edar Gene Cloning and Localization of Expression in Cyprinus carpio[J]. Biotechnology Bulletin, 2013, 0(12): 99-107.

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