日本鳗鲡Dmrt2基因cDNA的克隆与表达分析

发布时间：2018-06-15 21:07

  本文选题：日本鳗鲡 + Dmrt2基因　； 参考：《上海海洋大学》2017年硕士论文

【摘要】：Dmrt(double-sex and mab3-relatated transcription factor)基因是与性别决定与性腺发育有关的一个基因家族,同时对某些器官的发育和功能维持也起着重要作用。随着分子手段的进步,Dmrt基因家族的研究也在日益增进,最新的研究表明Dmrt2基因主要参与胚胎的发育和配子的形成。本实验结果表明日本鳗鲡Dmrt2基因表达蛋白仅含有一个功能结构域,即DM结构域。其Dmrt2蛋白的分子量为46.82kD,分子式为C2037H3281N595O617S26,总原子数为6556, Dmrt2蛋白分子的氨基酸组成主要包括以下几种:Ser (S),11.1%; Leu (L),10.8%; Lys (K) 7.5%和Trp (W),0.7%。利用模型软件预测日本鳗鲡Dmrt2基因表达蛋白的二级结构包含14个α-螺旋,17个β-折叠并具有多个卷曲。通过软件比较不同物种的Dmrt2基因表达蛋白的结构域,发现其相对保守。通过Race技术对Dmrt2基因的5'和3'序列进行扩增并拼接获得Dmrt2基因的cDNA全长为1499bp。通过对氨基酸序列的分析和同源性对比显示,日本鳗鲡和斑马鱼(Danio rerio)及尼罗罗非鱼(Oreochromis niloticus)之间具有高同源性。为了研究Dmrt2在日本鳗鲡的性别分化中的功能以及在个体发育中的作用,采用RT-PCR方法对Dmrt2基因在不同组织中的表达进行了分析。采用FISH分析验证Dmrt2在性腺中的表达情况。本研究结果显示,Dmrt2基因在多种组织中表达,并且在卵巢中相对较高,在精巢,肌肉和肝组织中弱表达。对于用E2 (雌二醇-17β)处理的日本鳗鲡,Dmrt2基因在卵巢和肝脏中表达较高,而在脑和肌肉组织中弱表达。荧光原位杂交结果表明Dmrt2基因的杂交信号只在卵巢中的卵泡细胞中可见,在性成熟的精巢中没有发现杂交信号。这些数据将为日本鳗鲡性别分化的进一步提供理论基础。
[Abstract]:The double-sex and mab3-relatated transcription factor gene is a family of genes related to sex determination and gonadal development. It also plays an important role in the development and functional maintenance of some organs. With the development of molecular techniques, the study of Dmrt gene family is increasing. The latest research shows that Dmrt2 gene is mainly involved in embryo development and gamete formation. The results showed that the Dmrt2 gene expression protein of Japanese eel contained only one functional domain, DM domain. The molecular weight of Dmrt2 protein is 46.82kD. the molecular formula is C2037H3281N595O617S26.The total atomic number of Dmrt2 protein is 6556. The amino acid composition of Dmrt2 protein mainly consists of the following kinds of: Ser Schion 11. 1; Leu L10.8; Lys K1) 7.5%; The secondary structure of Dmrt2 gene expressed protein in Japanese eel Anguilla japonica contained 14 伪 -helix, 17 尾 -folds and multiple curls. The domain of Dmrt2 gene expressed in different species was found to be relatively conserved by software. The 5 'and 3' sequences of Dmrt2 gene were amplified by Race technique and the full-length cDNA of Dmrt2 gene was obtained to be 1499bp. Amino acid sequence analysis and homology comparison showed that there was a high homology between Japanese eel and Danio Rerioo) and Oreochromis niloticus of Oreochromis niloticus. In order to study the function of Dmrt2 in sex differentiation and ontogeny of Japanese eel, the expression of Dmrt2 gene in different tissues was analyzed by RT-PCR. Fish analysis was used to verify the expression of Dmrt2 in gonad. The results showed that the Dmrt2 gene was expressed in a variety of tissues and was relatively high in the ovary and weakly expressed in the testis, muscles and liver. The expression of Dmrt2 gene in Japanese eel (Anguilla japonica) treated with E2 (estradiol-17 尾) was higher in ovary and liver, but weak in brain and muscle. The results of fluorescence in situ hybridization showed that the hybridization signal of Dmrt2 gene was only found in follicular cells of ovary, but not in the testis of sexual maturity. These data will provide theoretical basis for sex differentiation of Japanese eel.
【学位授予单位】：上海海洋大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S917.4

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