鲫鱼Dnmt1基因cDNA的克隆及表达分析
MOLECULAR CLONING AND EXPRESSION ANALYSIS OF Dnmt1 IN GOLDFISH, CARASSIUS AURATUS
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摘要: 研究克隆了编码鲫鱼Dnmt1蛋白的cDNA序列,并对Dnmt1基因的时空表达模式和表达丰度差异进行了分析。该cDNA全长4912bp,编码1503个氨基酸。鲫鱼Dnmt1蛋白与其他物种Dnmt1蛋白的同源性分析表明,N端调节区的保守性较低,但C端的催化区是高度保守的。整胚原位杂交显示,Dnmt1 mRNA在胚胎发育早期是广泛分布的,随着胚胎的发育,Dnmt1 mRNA在眼睛、脑部和已成熟体节的信号较强烈,显示了明显的区域性差异。实时定量PCR结果显示在鲫鱼胚胎发育早期有丰富的母源Dnmt1 mRNA存在,在早期发育过程中其丰度逐渐降低,在胚胎发育至2d后,Dnmt1 mRNA丰度又升到较高水平。RT-PCR检测表明在肝、心、脾、肾、脑、肌肉、视网膜等成体组织中都有Dnmt1的表达,但实时定量PCR结果显示在细胞分裂增殖旺盛的组织中表达水平明显较高。上述结果提示鲫鱼Dnmt1可能参与了胚胎基因组DNA甲基化的重编程,甲基化表观遗传学式样的确立和合子核基因正确时空表达模式形成的调节控制。Abstract: Fish is the lowest vertebrate. Its external fertilization and development, large reproductive potential, and optically clear embryos give fish a number of distinct advantages over other vertebrates as an exploitable experimental system for the study of developmental processes as well as the mechanism and function of epigenetic modification in vertebrates. Studying the mechanism of epigenetic modification by DNA methylation and the function of epigenetic modification in regulating gene expression in fish will provide insights into the biological function of DNA methylation, the evolution of epigenetic modification mechanism in vertebrates and the role of epigenetic modification in the evolution of vertebrates. In vertebrates, DNA methyltransferase (Dnmt1) Ⅰ plays a pivotal role in maintaining the correct genome DNA methylation pattern. In low vertebrate including fish, however, Dnmt1 has still been poorly investigated. Here, the full length cDNA of goldfish Dnmt1, Carassius auratus, was cloned by reverse transcription polymerase chain reaction(RT-PCR)method. The temporal and spatial expression patterns of goldfish Dnmt1 were examined by real-time PCR and whole mount in situ hybridization. The entire Dnmt1 cDNA sequence was 4912bp long and the open read frame was 4512bp long, which encoded a protein with 1503 amino acids. Amino acid sequence alignment of Dnmt1s (Carcasses auratus, Danio rerio, Xenopus laevis, Gallus gallus and Homo sapiens) revealed that the structure of Dnmt1 protein also consisted of an N-terminal regulatory domain and a C-terminal catalytic domain. The conservation of N-terminal domain among Carcasses auratus, Danio rerio, Xenopus laevis, Gallus gallus and Homo sapiens was very low, but C-terminal domain was highly conserved. Phylogenetic tree analysis showed that the homology of Dnmt1 protein between Carassius auratus and Danio rerio was the highest, Xenopus laevis was the second and Gallus gallus and Homo sapiens took the last. The high conservation of C-terminal domain implied that the catalytic function might be crucial for the survival of the species. The variation of N-terminal domain may reflect adaptive synergistic evolutions between the Dnmt1 structure and chromatin in different species.Whole mount in situ hybridization and real-time PCR results reveled that Dnmt1 mRNA was of abundantly maternal origin in Carassius auratus. During cleavage and blastula stages, the level of Dnmt1 mRNA was gradually reduced. As a result, it hit the lowest during gastrula stage. But in 2-day old embryos, the transcription level of Dnmt1 was gradually increased. No regional difference was detected in the pre-neurula stage embryos by in situ hybridization, but it was observed in the embryos after period of organogenesis. Dnmt1 mRNA expression was significantly higher in eyes, brain and matured somites than in other tissues. RT-PCR and real-time PCR results demonstrated that Dnmt1 expressed in all the examined adult tissues such as liver, heart, spleen, kidney, brain, muscle and retina. However, the transcription level of Dnmt1 mRNA was higher in those tissues with vigorous cell proliferation. The difference is apparently in association with the copying and maintenance of cell lineage specific methylation patterns in the genome of the newly forming cells.
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Keywords:
- Dnmt1 /
- Carassius auratus /
- Whole mount in situ hybridization /
- Real-time PCR
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