胡子鲇Dmrt1基因全长cDNA克隆及其表达分析
cDNA CLONING AND EXPRESSION ANALYSIS OF Dmrt1 IN CLARIAS FUSCUS
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摘要: 以胡子鲇(Clarias fuscus)为研究对象, 利用RT-PCR技术和SMART RACE技术克隆获得Dmrt1基因cDNA全长, 并利用生物信息学分析其结构及功能; 利用半定量RT-PCR技术检测胡子鲇性腺(精巢/卵巢)、肌肉、肠、肝脏、心脏、头肾、鳃丝、脑和眼等10种组织以及Ⅱ-Ⅴ期精巢中Dmrt1基因表达。结果表明: 胡子鲇Dmrt1基因cDNA全长为1417 bp, 其中5非编码区(5-UTR)为35 bp, 3非编码区(3-UTR)为516 bp, 开放阅读框(ORF)包含864 bp, 编码287个氨基酸(aa), 预测所编码DMRT1为主要位于细胞核内的不稳定性亲水蛋白。氨基酸序列比对显示, 胡子鲇DMRT1与已公布的非洲胡子鲇、蟾胡子鲇、黄颡鱼等鲇形目鱼类的相似性为83.3%-96.1%。胡子鲇DMRT1中具有DMRT基因家族共有的、保守性很高的DM结构域, 此结构域具有典型的C2H2C4锌指结构, 与上述鲇形目鱼类的相似性达100%, 与斑马鱼、青鳉、虹鳟等鱼类的相似性为91.9%-97.3%, 而与鸡、鼠、猪人等的相似性达80%以上。组织表达显示, 胡子鲇Dmrt1基因仅在精巢中表达, 且Ⅱ期精巢(即精子发生期)中Dmrt1基因表达量显著高于Ⅲ、Ⅳ和Ⅴ期精巢(P0.05), 而卵巢及其他8种组织中均无表达, 表明Dmrt1是胡子鲇精巢特异性表达基因, 可能与胡子鲇的雄性性别决定、精子发生及精巢发育密切相关。Abstract: Sex of fish is determined by both genetic substance and environment. The studies on the relationship between the sex and sex-related genes expression can not only reveal the mechanisms of sex determination, but also provide the theoretical basis to control the sex differentiation in artificial propagation in the fish. Catfish (Clarias fuscus) is a common freshwater fish in China. The Dmrt1 cDNA sequences were isolated from the testes of C. fuscus by RT-PCR and SMART RACE. Structure and function of the predicted DMRT1 protein were analyzed by bioinformatics. Expressions of Dmrt1 were examined using semi-quantitative RT-PCR in ten tissues (testes, ovaries, muscles, intestine, livers, heart, head kidney, gills, brains and eyes) and the testes from stageⅡ to stageⅤ. Results showed that the Dmrt1 complete cDNA was 1417 bp in length, and the 5 un-translated region (5-UTR) and the 3 un-translated region (3-UTR) were 35 bp and 516 bp, respectively. The open reading frame (ORF) was 864 bp, and a sequence of predicted 287 amino acids residues was encoded. The C. fuscus DMRT1 shared a higher amino acid identity of 83.3% to 96.1% with three Siluriformes fishes such as C. gariepinus, C. batrachus and Pelteobagrus fulvidraco. DMRT1 protein coded by Dmrt1 gene located mainly in nucleus, and worked as an unstable hydrophobic protein. A highly conserved DM domain was found in the DMRT1 protein, which was a common characteristic in DMRT clusters. The C. fuscus shared a higher DM domain identity of 100% with the above-mentioned Siluriformes fishes, 91.9% to 97.3% with Danio rerio, Oryzias latipes and Oncorhynchus mykiss, and over 80% with chicken, rat, pig and human. In addition, the DM domain of C. fuscus had a typical C2H2C4 zinc finger structure, which bound with the downstream DNA and control the gene transcription. Expression of Dmrt1 was found only in the testes, but not in ovaries, muscles, intestine, liver, heart, head kidney, gills, brain and eyes, which suggested that Dmrt1 was a testis-specific gene in C. fuscus. The expression of Dmrt1 in testes stageⅡ(spermatogenesis) was significantly higher compared with the testes from stageⅢ to stageⅤ(P0.05), which indicated that Dmrt1 was related to sex determination, spermatogenesis and testes development in male C. fuscus.
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Keywords:
- Clarias fuscus /
- Dmrt1 /
- DM domain /
- Spermatogenesis /
- Expression analysis
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