本文選題：山羊 + Tet1��； 參考：《西南大學》2016年碩士論文

【摘要】：生產(chǎn)優(yōu)質(zhì)羔羊是養(yǎng)羊業(yè)中取得高經(jīng)濟效益的重要條件。山羊繁殖性能的充分發(fā)揮,不僅體現(xiàn)在產(chǎn)羔率高,還體現(xiàn)在羔羊品質(zhì)和成活率等性狀上。母羊的妊娠維持、胎兒的發(fā)育乃至出生后生長情況都依賴于早期胎盤的正常發(fā)育。DNA甲基羥化酶TET1蛋白是Fe2+和2-氧化戊二酸依賴性型的雙加氧酶家族成員之一,參與調(diào)節(jié)DNA主動、被動去甲基化機制,將DNA 5-甲基胞嘧啶(5m C)催化羥化為5-羥甲基胞嘧啶(5hm C),并可繼續(xù)將5hm C氧化為5-醛基胞嘧啶(5f C)及5-羧基胞嘧啶(5ca C),被TDG特異結(jié)合進行堿基切除修復達到DNA主動脫甲基化的目的,在維持胚胎干細胞的自我更新和多能性,保證干細胞分化成不同的組織器官有重要意義。但該分子調(diào)控妊娠期胎盤發(fā)育的分子機制研究尚屬空白,鑒于此,本研究首先克隆了山羊Tet1基因,然后探索了其在山羊胎盤不同發(fā)育階段的表達模式,對明確其調(diào)控山羊胎盤發(fā)育的機理,為山羊的分子育種及相關應用基礎研究奠定基礎。主要研究結(jié)果如下:利用RT-PCR和RACE末端擴增技術,首次克隆得到山羊Tet1基因c DNA,全長序列7056 bp(Gen Bank:KU870424),開放閱讀框(ORF)長6507 bp,除終止密碼子外翻譯2168個氨基酸,預測山羊TET1蛋白分子量為239677.2 Da,是一種親水性非分泌性、非膜結(jié)合的球狀蛋白。以DNA為模板擴增得到山羊Tet1基因5’非翻譯區(qū)啟動子序列856 bp,對其進行Cp G島預測顯示所擴增的該序列無明顯的Cp G島,預測得到兩個核心啟動子區(qū)序列分別是GATCAGACCTG TGTCCCCTGCACTGGCAGCCAGATTCTTATCTACTGTAC和TTCTTGAAGTGC ACAGGCTTCTCATTGTGGTGGCTTCTTTTATTTTGGAT。山羊TET1蛋白與TET家族其它成員間的同源性不高,與TET2為24.9%,與TET3的同源性為23.9%,與牛、馬、人、猴、小鼠、猩猩、大鼠和豬的同源性分別是95.4%、86.1%、78.3%、78.3%、56.7%、76.5%、57.9%和87.3%,與牛的同源性最高,與小鼠的同源性最低。山羊Tet1組織表達譜分析結(jié)果表明,Tet1基因在肌肉中表達最高,在睪丸、子葉、卵巢等與繁殖相關的組織中也有較高的表達,在肝和腎中表達最低,這一結(jié)果說明Tet1基因可能參與調(diào)控了山羊相關的繁殖生理過程。通過實時熒光定量對Tet1基因在山羊胎盤不同發(fā)育階段中的表達模式分析表明,Tet1在妊娠25 d山羊胎兒胎盤中的相對表達水平極顯著高于60 d、90~120 d和150 d的表達(P0.01),顯著高于妊娠20 d和30 d的表達水平(P0.05),Tet1基因表達量在妊娠25 d、30 d、60 d、90~120 d和150 d期間呈下降趨勢。這一結(jié)果初步表明Tet1基因在妊娠早期(胚胎植入和胎兒形成)中可能發(fā)揮了重要作用。另對Dnmts的定量PCR結(jié)果表明,在妊娠早期20、25和30 d山羊胎盤中,甲基化標記物Dnmt3a的相對表達量呈增加趨勢,在妊娠30 d的胎盤中Dnmt1的表達極顯著高于其他任何階段的Dnmt1;免疫熒光組織化學實驗發(fā)現(xiàn),DNA甲基化標記物5m C在山羊妊娠早期(20 d、25 d、30 d)中表達較弱。本研究首次成功克隆了山羊Tet1基因,c DNA全長為7056 bp(Gen Bank:KU870424),初步發(fā)現(xiàn)Tet1基因在妊娠早期山羊胎盤發(fā)育和去甲基化機制中發(fā)揮了重要作用,為進一步研究該基因生物學功能及其在胎兒胎盤上的相關調(diào)控機制奠定了基礎。
[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.

【學位授予單位】：西南大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：S827

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