翘嘴鲌生长激素(GH)基因与侧翼区的克隆及分析

doi:10.3969/j.issn.1004-1524.2017.08.08

浙江农业学报 ›› 2017, Vol. 29 ›› Issue (8): 1281-1289.DOI: 10.3969/j.issn.1004-1524.2017.08.08

翘嘴鲌生长激素(GH)基因与侧翼区的克隆及分析

刘士力^{1, 2}, 贾永义¹, 蒋文枰¹, 迟美丽¹, 程顺¹, 赵金良², 顾志敏^{1, *}, 傅建军³

1.浙江省淡水水产研究所农业部淡水渔业健康养殖重点实验室/浙江省淡水水产遗传育种重点实验室,浙江湖州 313001；
2.上海海洋大学水产与生命学院,上海 201306；
3.中国水产科学研究院淡水渔业研究中心,农业部淡水渔业与种质资源利用重点实验室,江苏无锡 214081

收稿日期:2017-03-17 出版日期:2017-08-20 发布日期:2017-09-06
通讯作者: 顾志敏,E-mail: guzhimin2006@163.com
作者简介:刘士力(1985-),男,湖北洪湖人,博士研究生,主要从事水生动物遗传育种研究。E-mail: liushili1212@126.com
基金资助:
浙江省农业新品种选育重大科技专项(2012C12907-7, 2016C02055-1)

Cloning and sequence analysis of growth hormone gene and its flanking region in topmouth culter (Culter alburnus Basilewsky)

LIU Shili^{1, 2}, JIA Yongyi¹, JIANG Wenping¹, CHI Meili¹, CHENG Shun¹, ZHAO Jinliang², GU Zhimin^{1, *}, FU Jianjun³

1. Agriculture Ministry Key Laboratory of Healthy Freshwater Aquaculture / Key Laboratory of Freshwater Aquatic Animal Genetic and Breeding of Zhejiang Province, Zhejiang Institute of Freshwater Fisheries, Huzhou 313001, China;
2. College of Fisheries and Life Science, Shanghai Ocean University, Shanghai 201306, China;
3. Key Laboratory of Freshwater Fisheries and Germplasm Resources Utilization, Ministry of Agriculture, Freshwater Fisheries Research Center, Chinese Academy of Fishery Sciences, Wuxi 214081, China

Received:2017-03-17 Online:2017-08-20 Published:2017-09-06

摘要/Abstract

摘要： 采用保守引物进行PCR成功扩增了编码翘嘴鲌生长激素的基因,该基因全长5 966 bp,其中转录单元长1 648 bp,由5个外显子和4个内含子组成。5'端和3'端侧翼序列长度分别为2 282 bp和2 036 bp,分别包含 (AAT)₈ 和(TTC)₅T(TAA)₈的微卫星序列。上游区域包含TATA框,还有一些重要的转录因子结合位点,如Pit-1、Pit-1a、CREB、AP1、GR、HNF-3、HNF-3B等转录因子。翘嘴鲌的5个外显子长度分别为64、140、117、162 和255 bp。推测的阅读框为603 bp,编码由22个氨基酸的信号肽和178个氨基酸成熟肽组成的多肽。在这个多肽中发现了5个保守的半胱氨酸残基(Cys⁷¹、Cys¹³⁵、 Cys¹⁷³、Cys¹⁹⁰和Cys¹⁹⁸)和2个可能的N-糖基化位点(145^th和197^th)。翘嘴鲌GH氨基酸序列与团头鲂完全相同,与草鱼只有一个氨基酸残基的差异,构建的鱼类进化树符合基本分类地位。翘嘴鲌生长激素基因4个内含子长度分别为229、103、565 和103 bp。相对外显子来说,种间内含子变异较大,其中第三内含子变异最大。该结果为进一步研究翘嘴鲌GH基因的表达、功能及其转录调控特征奠定了分子基础。

关键词: 翘嘴鲌, 生长激素基因, 克隆, 序列分析

Abstract: Using conserved primers and PCR, a gene encoding topmouth culter (Culter alburnus Basilewsky) growth hormone (CaGH) was amplified and sequenced. The gene spans 5 966 bp, including 2 282 bp of 5'- and 2 036 bp of 3'-flanking sequences and a 1.7-kb transcription unit comprised of five exons and five introns. (AAT)₈ and (TTC)₅T(TAA)₈ microsatellite sequences were found in the 5'- and 3'-flanking regions, respectively. The upstream region contains TATA boxes, and binding sites of important transcription factors such as Pit-1, Pit-1a, CREB, AP1, GR, HNF-3, and HNF-3B. The five exons in Culter alburnus were 64 bp, 140 bp, 117 bp, 162 bp and 255 bp in length, respectively. The complete coding sequence was 603 bp and encodes a protein with a 22 amino acid signal peptide and a 178 amino acid mature peptide. Five conserved Cys residues (Cys⁷¹, Cys¹³⁵, Cys¹⁷³, Cys¹⁹⁰, and Cys¹⁹⁸) and two possible sites of N-glycosylation (residues 145 and 197) were detected in the GH polypeptide. The amino acid sequence of GH in Culter alburnus was identical to that in Megalobrama amblycephala, and only one amino acid residue differs from Ctenpharyngodon idellus. The phylogenetic relationships among the GH amino acid sequences in fish were in accord with traditional classification. The lengths of the four introns in Culter alburnus GH gene were 229 bp, 103 bp, 565 bp and 103 bp, respectively. The variation of the introns among species was greater than that of the exons, and the variation of the third intron is the highest. These results provided the molecular basis for study of function and transcriptional regulation of the GH gene in C.alburnus, as well as the temporal expression in different developmental stages and at various nutritional levels.

Key words: Culter alburnus, growth hormone gene, clone, sequence analysis

中图分类号:

刘士力, 贾永义, 蒋文枰, 迟美丽, 程顺, 赵金良, 顾志敏, 傅建军. 翘嘴鲌生长激素(GH)基因与侧翼区的克隆及分析[J]. 浙江农业学报, 2017, 29(8): 1281-1289.

LIU Shili, JIA Yongyi, JIANG Wenping, CHI Meili, CHENG Shun, ZHAO Jinliang, GU Zhimin, FU Jianjun. Cloning and sequence analysis of growth hormone gene and its flanking region in topmouth culter (Culter alburnus Basilewsky)[J]. , 2017, 29(8): 1281-1289.

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翘嘴鲌生长激素(GH)基因与侧翼区的克隆及分析

Cloning and sequence analysis of growth hormone gene and its flanking region in topmouth culter (Culter alburnus Basilewsky)

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