大鲵Dmrt2基因cDNA的克隆与表达分析  

Molecular cloning and expression analysis of Dmrt2 gene in Andrias davidianus

作　　者：许宝红[1] 肖真明[1] 肖调义[1] 陈开健[1] 刘巧林[1] 周伟[1] 刘敏[1] 姚一彬[1] 

机构地区：[1]湖南农业大学动物科学技术学院,湖南长沙410128

出　　处：《水产学报》2012年第8期1179-1184,共6页Journal of Fisheries of China

基　　金：国家自然科学基金项目(31040083);湖南省科技厅攻关项目(06NK3049);湖南省教育厅重点项目(07A012)

摘　　要：Dmrt2是参与性腺发育最古老的发育基因家族Dmrt家族成员之一,在维持胚胎组织的正常发育、精子的发生、神经系统和感觉器官发育等方面起重要作用。目前在哺乳动物人类、鸟类红原鸡以及水生动物青鳉中都克隆到Dmrt家族成员并证明其与性腺和生长发育的密切相关性。研究采用RACE方法克隆了大鲵Dmrt2基因全长cDNA序列共2 026 bp,开放阅读框长1 581 bp,5′非编码区长58 bp,3′非编码区长387 bp,基因序列提交GenBank的登录号为FJ859987。该基因编码蛋白含526个氨基酸,依生物信息学方法预测该氨基酸为非跨膜蛋白,无信号肽,定位在细胞质内,无分泌蛋白。CDD数据库分析该ORF框翻译的氨基酸,在93~146位含DM保守结构域（c102557）的两个成员pfam00751和smart003010,与哺乳类人mab-3、鸟类红原鸡Dmrt2、两栖类非洲爪蟾Dmrt2和水生类鲐mab-3 DM结构域存在I（异亮氨酸）、R（精氨酸）、M（甲硫氨酸）、T（苏氨酸）、C（半胱氨酸）5个氨基酸的变异;系统进化树分析表明：大鲵Dmrt2与以上四物种首先聚类,据此推测Dmrt基因在进化上高度保守,DM结构域上5个氨基酸的变异对蛋白功能的发挥可能无重要影响。实时荧光定量组织差异表达显示,Dmrt2基因在大鲵的精巢和肌肉组织中高表达,预示该基因可能在性腺发育和生长发育方面起重要作用。Dmrt family is one of the oldest developmental gene family, and is important to the embryo normal growth, spermatogenesis, and the nervous system and sense organ development. Dmrt2 is one of the members of Dmrt family, which was cloned in mammals such as human, birds such as Gallus gallus, aquatic animals such as Oryzias latipes, is closely related to gonad development and growth. RACE -PCR technique was used to clone Dmrt2 gene in Andrias davidianus, 2 026 bp full-length cDNA was obtained, containing 1 581 bp open reading frame（ORF）, 58 bp 5＇UTR, 387 bp 3＇UTR, GenBank number is FJ859987. This sequence encoded 526 amino acids, and the amino acid sequence was across the membrane protein, no signal peptide, and the protein positioning in the cytoplasm. The amino acid sequence was ana- lyzed by the Conserved Domain Database（CDD）, surveyed that single amino-terminal DM domain is from 93 bp to 146 bp, contains a pattern of conserved zinc chelating residues C2H2C4, super-family pfam00751 and smart003010. Blast DM domain with Homo sapiens mab-3, G. gallus Dmrt2, Xenopus （Silurana） tropicalis Dmrt2 and Danio rerio mab-3, five amino acid had variation, include lie, Arg, Met, Thr, Cys. Phylogenetic analysis indicated that the five species of Homo sapiens, G. gallus, X. （Silurana） tropicalis and D. rerio were clustered first, that means Dmrt family is highly conserved during evolution, and the five amino acids variation had little effect on protein functions. RT-PCR was used to detect the ex- pression of Dmrt2 in skin, intestines, gills, muscle, stomach, spermary and kidney of A. davidianus, and results showed it is high level expression only in spermary and muscle, which indicated that the Dmrt2 gene may play an important role in gonad development and growth development.

关 键 词：大鲵 Dmrt2 实时荧光定量PCR 

分 类 号：Q785[生物学—分子生物学] S917.4[农业科学—水产科学]