山羊Tet1基因克隆及其在胎盘的表达谱

发布时间：2018-05-07 23:01

本文选题：山羊 + Tet1　；参考：《西南大学》2016年硕士论文

【摘要】：生产优质羔羊是养羊业中取得高经济效益的重要条件。山羊繁殖性能的充分发挥,不仅体现在产羔率高,还体现在羔羊品质和成活率等性状上。母羊的妊娠维持、胎儿的发育乃至出生后生长情况都依赖于早期胎盘的正常发育。DNA甲基羟化酶TET1蛋白是Fe2+和2-氧化戊二酸依赖性型的双加氧酶家族成员之一,参与调节DNA主动、被动去甲基化机制,将DNA 5-甲基胞嘧啶(5m C)催化羟化为5-羟甲基胞嘧啶(5hm C),并可继续将5hm C氧化为5-醛基胞嘧啶(5f C)及5-羧基胞嘧啶(5ca C),被TDG特异结合进行碱基切除修复达到DNA主动脱甲基化的目的,在维持胚胎干细胞的自我更新和多能性,保证干细胞分化成不同的组织器官有重要意义。但该分子调控妊娠期胎盘发育的分子机制研究尚属空白,鉴于此,本研究首先克隆了山羊Tet1基因,然后探索了其在山羊胎盘不同发育阶段的表达模式,对明确其调控山羊胎盘发育的机理,为山羊的分子育种及相关应用基础研究奠定基础。主要研究结果如下:利用RT-PCR和RACE末端扩增技术,首次克隆得到山羊Tet1基因c DNA,全长序列7056 bp(Gen Bank:KU870424),开放阅读框(ORF)长6507 bp,除终止密码子外翻译2168个氨基酸,预测山羊TET1蛋白分子量为239677.2 Da,是一种亲水性非分泌性、非膜结合的球状蛋白。以DNA为模板扩增得到山羊Tet1基因5’非翻译区启动子序列856 bp,对其进行Cp G岛预测显示所扩增的该序列无明显的Cp G岛,预测得到两个核心启动子区序列分别是GATCAGACCTG TGTCCCCTGCACTGGCAGCCAGATTCTTATCTACTGTAC和TTCTTGAAGTGC ACAGGCTTCTCATTGTGGTGGCTTCTTTTATTTTGGAT。山羊TET1蛋白与TET家族其它成员间的同源性不高,与TET2为24.9%,与TET3的同源性为23.9%,与牛、马、人、猴、小鼠、猩猩、大鼠和猪的同源性分别是95.4%、86.1%、78.3%、78.3%、56.7%、76.5%、57.9%和87.3%,与牛的同源性最高,与小鼠的同源性最低。山羊Tet1组织表达谱分析结果表明,Tet1基因在肌肉中表达最高,在睾丸、子叶、卵巢等与繁殖相关的组织中也有较高的表达,在肝和肾中表达最低,这一结果说明Tet1基因可能参与调控了山羊相关的繁殖生理过程。通过实时荧光定量对Tet1基因在山羊胎盘不同发育阶段中的表达模式分析表明,Tet1在妊娠25 d山羊胎儿胎盘中的相对表达水平极显著高于60 d、90~120 d和150 d的表达(P0.01),显著高于妊娠20 d和30 d的表达水平(P0.05),Tet1基因表达量在妊娠25 d、30 d、60 d、90~120 d和150 d期间呈下降趋势。这一结果初步表明Tet1基因在妊娠早期(胚胎植入和胎儿形成)中可能发挥了重要作用。另对Dnmts的定量PCR结果表明,在妊娠早期20、25和30 d山羊胎盘中,甲基化标记物Dnmt3a的相对表达量呈增加趋势,在妊娠30 d的胎盘中Dnmt1的表达极显著高于其他任何阶段的Dnmt1;免疫荧光组织化学实验发现,DNA甲基化标记物5m C在山羊妊娠早期(20 d、25 d、30 d)中表达较弱。本研究首次成功克隆了山羊Tet1基因,c DNA全长为7056 bp(Gen Bank:KU870424),初步发现Tet1基因在妊娠早期山羊胎盘发育和去甲基化机制中发挥了重要作用,为进一步研究该基因生物学功能及其在胎儿胎盘上的相关调控机制奠定了基础。
[Abstract]:The production of high quality lambs is an important condition for obtaining high economic benefits in the sheep industry. The full play of the reproductive performance of goats is not only reflected in the high lambing rate, but also in the traits of the lamb quality and survival rate. The pregnancy maintenance of the ewes, the development of the fetus and the growth of the post birth are all dependent on the normal development of the early placenta,.DNA methyl hydroxyl. The enzyme TET1 protein is one of the members of the Fe2+ and 2- diacid dependent dioxygenase family, which participates in the regulation of DNA active and passive demethylation mechanism, and catalyzes the hydroxylation of DNA 5- methyl cytosine (5m C) to 5- hydroxymethyl cytosine (5hm C), and can continue to oxidize 5hm to oxidize to aldehyde based cytosine and carboxylic cytosine. It is important to maintain the self renewal and pluriability of the embryonic stem cells in order to maintain the self renewal and pluriability of the embryonic stem cells and to ensure the differentiation of the stem cells into different tissues and organs. However, the molecular mechanism of the molecular regulation of placental development in pregnancy is still blank. In view of this, this study first cloned the goat Tet in this study. The 1 gene and then explore its expression pattern in the different developmental stages of the goat placenta, to clarify the mechanism of its regulation of goat placenta development, and lay the foundation for the molecular breeding and related basic research of goat. The main results are as follows: RT-PCR and RACE terminal amplification technology were used to clone the goat Tet1 gene C DNA for the first time. Column 7056 BP (Gen Bank:KU870424), open reading frame (ORF) long 6507 BP, except for terminating codon 2168 amino acids, predicting the molecular weight of TET1 protein in goat is 239677.2 Da, it is a kind of non secretory, non membrane binding globular protein. The 5 'non translation region promoter sequence 856 BP of goat Tet1 gene is obtained by DNA as the template. The Cp G Island prediction showed that the amplified Cp G island had no obvious Cp G island. It was predicted that the sequence of two core promoter regions was GATCAGACCTG TGTCCCCTGCACTGGCAGCCAGATTCTTATCTACTGTAC and TTCTTGAAGTGC ACAGGCTTCTCATTGTGGTGGCTTCTTTTATTTTGGAT. goats, and the homology between the other members of the TET family was not high, and the TET2 was 24.9. Homology with TET3 is 23.9%. Homology with cattle, horses, people, monkeys, mice, orangutans, rats, rats and pigs are 95.4%, 86.1%, 78.3%, 78.3%, 56.7%, 76.5%, 57.9% and 87.3%, and the highest homology with the cow. The Tet1 expression analysis of goats shows that the Tet1 gene is the highest in the muscles, in the testicles, cotyledon, and eggs. The nests are also highly expressed in the reproduction related tissues and the lowest expression in the liver and kidney. This result indicates that the Tet1 gene may participate in the regulation of the related reproductive physiological processes of the goat. The expression of the Tet1 gene in the different developmental stages of the goat placenta by real-time fluorescence quantitative analysis shows that Tet1 is in the pregnancy of 25 D goats in pregnancy. The relative expression level in the disc was significantly higher than that of 60 d, 90~120 D and 150 D (P0.01), significantly higher than the expression level of 20 D and 30 d in pregnancy (P0.05). The expression of Tet1 gene was decreased in 25 D, 30 d, 60 d, 60, and 150. This result showed that the gene was initially in the early pregnancy (embryo implantation and fetal formation). The quantitative PCR results of Dnmts showed that the relative expression of Dnmt3a was increased in the 20,25 and 30 d goat placenta in the early pregnancy, and the expression of Dnmt1 in the placenta of 30 d pregnancy was significantly higher than that of any other Dnmt1; the immunofluorescence histochemical experiment found that the DNA methylation marker was marked. The expression of 5m C was weak in the early pregnancy (20 D, 25 D, 30 d). The first successful cloning of the goat Tet1 gene and the full length of C DNA were 7056 BP (Gen Bank:KU870424). It was preliminarily found that the Tet1 gene played a important role in the mechanism of placenta development and demethylation in early gestation. It has laid a foundation for the regulation of fetal placenta.

【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S827

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