本文選題：Tet1(Ten-eleven　切入點(diǎn)：translocation　出處：《西北農(nóng)林科技大學(xué)》2016年博士論文　論文類型：學(xué)位論文

【摘要】：奶山羊是我國重要的經(jīng)濟(jì)動(dòng)物,保持和優(yōu)良奶山羊品種,實(shí)現(xiàn)快速繁殖是現(xiàn)代農(nóng)業(yè)的目標(biāo),奶山羊雄性生殖干細(xì)胞(male germline stem cell,mGSCs)自我更新的維持和發(fā)育分化的相關(guān)基礎(chǔ)性研究也逐步深入,家畜mGSCs體外培養(yǎng)體系不穩(wěn)定、重編程效率低等問題都亟待解決。研究表明表觀修飾在精子發(fā)生過程中發(fā)揮著重要的調(diào)控作用。特定位點(diǎn)的DNA甲基化動(dòng)態(tài)變化是維持正常精子發(fā)生的關(guān)鍵。Tet1(Ten-eleven translocation 1)作為主要的去甲基化酶,參與精子發(fā)生的表觀修飾調(diào)控進(jìn)程。因此,開展mGSCs的表觀遺傳學(xué)研究,不僅有助于探究成體干細(xì)胞體外培養(yǎng)技術(shù)的優(yōu)化體系,推進(jìn)mGSCs自我更新與分化機(jī)理的探索;而且在畜牧業(yè)生產(chǎn)上對(duì)提高優(yōu)良種畜的精子質(zhì)量,減少疾病發(fā)生,促進(jìn)家畜大動(dòng)物優(yōu)良品種的遺傳與繁育有重要意義。本研究以奶山羊?yàn)檠芯繉?duì)象,對(duì)Tet1及產(chǎn)物5-羥甲基胞嘧啶(5hmC)進(jìn)行表達(dá)譜和定位分析,同時(shí)探究了奶山羊精子發(fā)生過程中的dimethyl histone H3 lysine 9(H3K9me2)和tri-methyl H3 lysine 9(H3K9me3)在不同年齡階段睪丸組織的甲基化模式;利用Tet1過表達(dá)的方法修飾mGSCs并篩選陽性單細(xì)胞克隆,觀察細(xì)胞形態(tài)學(xué)、基因表達(dá)差異和DNA甲基化、組蛋白甲基化的變化;通過自發(fā)分化與體內(nèi)移植對(duì)Tet1過表達(dá)的mGSCs進(jìn)行功能驗(yàn)證,并分析Tet1與蛋白互作的調(diào)控機(jī)理。結(jié)果發(fā)現(xiàn):(1)Tet1在奶山羊睪丸組織中表達(dá)水平較低,且主要分布于曲細(xì)精管基底膜的精原細(xì)胞中;對(duì)其產(chǎn)物5hmC在不同年齡階段奶山羊睪丸組織中的含量與定位進(jìn)行檢測,表明5hmC隨年齡增加而降低,且在mGSCs與支持細(xì)胞都有分布;奶山羊精子發(fā)生過程中的組蛋白H3K9me2在不同年齡階段睪丸組織呈現(xiàn)先升后降的模式,在青春期達(dá)到峰值;而H3K9me2含量則是隨年齡增長持續(xù)緩慢升高。成年睪丸組織中dimethyl histone H3 lysine 4(H3K4me2)定位于減數(shù)分裂后期的圓形精細(xì)胞,tri-methyl H3 lysine 27(H3K27me3)在mGSCs中呈現(xiàn)特殊的核周定位;在體外培養(yǎng)的mGSCs中,Tet1在維持自我更新的mGSCs中表達(dá),在分化狀態(tài)下表達(dá)降低;5hmC與其表現(xiàn)同樣的趨勢;同時(shí)對(duì)體外培養(yǎng)的mGSCs中H3K9me2和H3K9me3進(jìn)行檢測,發(fā)現(xiàn)維持自我更新的mGSCs克隆和趨于分化的克隆及未形成克隆的細(xì)胞相比含量含量較低。(2)生物信息學(xué)分析不同物種間Tet1基因的遺傳同源性和保守結(jié)構(gòu)域,通過對(duì)Tet1基因共有的發(fā)揮功能關(guān)鍵的識(shí)別結(jié)構(gòu)域和催化結(jié)構(gòu)域進(jìn)行分析,確定小鼠Tet1(mTet1)在山羊源細(xì)胞中發(fā)揮作用;篩選Dox誘導(dǎo)表達(dá)mTet1的陽性單細(xì)胞克隆,在基因、mRNA和蛋白水平進(jìn)行鑒定后觀察細(xì)胞形態(tài),發(fā)現(xiàn)mTet1陽性細(xì)胞細(xì)胞核變大,且5hmC含量增加;通過階梯濃度培養(yǎng)確定0.50μg/mL的Dox濃度為最佳的誘導(dǎo)培養(yǎng)濃度;QRT-PCR、免疫熒光染色和western blot檢測發(fā)現(xiàn)mTet1陽性細(xì)胞中Gfra1、PCNA、CCND1、Ki67、ETV5等增殖和生殖特異性標(biāo)志基因表達(dá)上調(diào),分化特異性基因REC8表達(dá)下調(diào);Tet1過表達(dá)的奶山羊mGSCs的陽性細(xì)胞,組蛋白甲基化相關(guān)的H3K9me2和H3K9me3含量有所下降,且H3K9me3由細(xì)胞核內(nèi)顆粒狀分布轉(zhuǎn)變?yōu)榫鶆蚍植?H3K27me3含量顯著降低,由細(xì)胞核內(nèi)均勻分布轉(zhuǎn)變?yōu)橄蚝酥芊植肌?3)構(gòu)建過表達(dá)Tet1慢病毒載體,對(duì)篩選出的穩(wěn)定過表達(dá)Tet1的奶山羊mGSCs陽性克隆(mGSC-pCDH-mTet1)細(xì)胞進(jìn)行QRT-PCR檢測,篩選出既能維持mGSCs自我更新又促進(jìn)細(xì)胞增殖的克隆;制備mGSC-pCDH-mTet1細(xì)胞和mGSC-pCDH細(xì)胞的類胚體(EBs),自發(fā)分化試驗(yàn)檢測體外分化能力,發(fā)現(xiàn)mGSC-pCDH-mTet1細(xì)胞自發(fā)分化后的細(xì)胞三胚層分化的速度較慢,說明mTet1具有維持自我更新、抑制分化的功能;對(duì)生殖缺陷小鼠模型進(jìn)行mGSC-pCDH-mTet1細(xì)胞和mGSC-pCDH細(xì)胞的曲細(xì)精管移植,4周采集睪丸,制成石蠟切片,并進(jìn)行PGP9.5、VASA、PCNA、Ki67等生殖與增殖相關(guān)特性標(biāo)記的檢測,證實(shí)mGSC-pCDH-mTet1細(xì)胞在生殖缺陷小鼠的曲細(xì)精管內(nèi)不僅維持mGSCs的特性,還有顯著的增殖能力。(4)對(duì)mTet1在mGSCs中發(fā)揮作用的方式進(jìn)行分析,確認(rèn)mTet1通過上調(diào)組蛋白去甲基化酶JMJD3的表達(dá),下調(diào)H3K27me3的含量并發(fā)生從核內(nèi)向核周分布的定位變化,此過程伴有MEK-ERK信號(hào)通路的抑制;mTet1通過與Hdac1形成蛋白復(fù)合體,共同調(diào)節(jié)組蛋白乙酰化狀態(tài),進(jìn)行mGSCs的表觀修飾調(diào)控;mTet1與PCNA形成蛋白復(fù)合體,通過mTet1過表達(dá)來促進(jìn)mGSCs的增殖。綜上所述,本研究從體內(nèi)和體外分析了Tet1在奶山羊曲細(xì)精管和細(xì)胞中的分布;同時(shí)研究Tet1修飾引起的形態(tài)變化和基因調(diào)控機(jī)理。這不僅為精子發(fā)生過程中Tet1表達(dá)調(diào)控進(jìn)一步的研究提供思路,為更深入探究表觀修飾調(diào)控在奶山羊mGSCs的發(fā)育與分化作用奠定了基礎(chǔ)。
[Abstract]:Dairy goat is an important economic animal in China, and maintain excellent dairy goat breeds, fast breeding is the modern agriculture, dairy goat male germline stem cells (male germline stem cell, mGSCs) on the self-renewal and differentiation of the relevant basic also gradually thorough, livestock mGSCs in vitro culture system is not stable reprogramming, low efficiency problems should be solved. The research showed that epigenetic modifications in spermatogenesis play an important role. Methylation sites of the DNA dynamic change is key to maintaining.Tet1 normal spermatogenesis (Ten-eleven translocation 1) as the main enzyme to methylation, participate in spermatogenesis of epigenetic modification the regulation process. Therefore, to carry out mGSCs epigenetic research not only helps to explore the training system optimization technology of adult stem cells in vitro, promote mGSCs self-renewal and differentiation mechanism Exploration; and in animal husbandry to improve sperm quality of breeding livestock, reduce disease, promote important genetic and breeding of fine varieties of livestock animal. In this study, the dairy goat as the research object, the Tet1 and 5- product hydroxymethylcytosine (5hmC) spectral analysis and positioning for expression, at the same time to explore the dairy goat during spermatogenesis of dimethyl histone H3 lysine 9 (H3K9me2) and tri-methyl H3 lysine 9 (H3K9me3) methylation patterns in the testis of different age stages; using Tet1 over expression method of modified mGSCs and screening of positive clones to observe the cell morphology, gene expression and DNA methylation changes. Histone methylation; through spontaneous differentiation and in vivo transplantation on expression of Tet1 mGSCs functional verification, and analyze the regulation mechanism of Tet1 and protein interaction. Results showed that: (1) Tet1 in dairy goats Testis tissue expression level is low, and is mainly distributed in the basal membrane of the seminiferous tubules of spermatogonial cells; to detect the content of the product and location of 5hmC in the testis of different age stages in dairy goats, showed that 5hmC reduced with the increase of age, and are distributed in mGSCs and Sertoli cells; goat in the process of spermatogenesis of histone H3K9me2 first increased and then decreased in testis of different age stages, and reached the peak in adolescence; while the content of H3K9me2 is continuously increased slowly with the growth of age. Adult testis tissue dimethyl histone H3 lysine 4 (H3K4me2) located in the postmeiotic round spermatid, tri-methyl H3 lysine 27 (H3K27me3) showed a perinuclear localization of particular in mGSCs; in mGSCs cultured in vitro. Tet1 expression in maintaining the self-renewal of mGSCs, reduce the expression of differentiation in 5hmC and its performance of the same state; The trend; at the same time. H3K9me2 and H3K9me3 mGSCs in vitro, found to maintain mGSCs self-renewal and cloning cloning tend to differentiation and did not form the cloned cells compared to content was lower. (2) analysis of Tet1 gene among species genetic homology and conserved domain of biological information analysis based on the Tet1 gene common play domain and the catalytic domain of the structure and function of the key identification, determination of mouse Tet1 (mTet1) play a role in goat derived cells; screening Dox positive single cell cloning, expression of mTet1 gene in induction, cell morphology was observed and identified by mRNA and protein levels, found that mTet1 positive nuclei become large increased, and the content of 5hmC; through the ladder concentration determine the Dox concentration of 0.50 g/mL for induction and the optimum concentration; QRT-PCR, immunofluorescence and Western blot detection found mTet1 positive cells The cell in Gfra1, PCNA, CCND1, Ki67, ETV5, proliferation and reproductive specific marker expression of differentiation specific gene REC8 expression; Tet1 positive cells expressed goat mGSCs, histone methylation of H3K9me2 and H3K9me3 content decreased, and H3K9me3 from the nucleus into a uniform distribution of particles distribution; H3K27me3 content decreased significantly, the nucleus uniform distribution into the nucleus. (3) construct Tet1 lentiviral expression vector, screened over expression of goat mGSCs positive clone Tet1 (mGSC-pCDH-mTet1) cells were detected by QRT-PCR. The selected mGSCs can maintain the self-renewal and promote cell cloning proliferation; preparation of mGSC-pCDH-mTet1 cells and mGSC-pCDH cells of the embryoid body (EBs), spontaneous differentiation differentiation assay in vitro, found that mGSC-pCDH-mTet1 cells spontaneously differentiated cells after three embryos Layer differentiation is slow, indicating that mTet1 has self-renewal and inhibits differentiation of seminiferous function; mGSC-pCDH-mTet1 cells and mGSC-pCDH cells of reproductive deficient mice transferred 4 weeks collecting testis, made of paraffin, and PGP9.5, VASA, PCNA, Ki67 detection and proliferation related markers reproductive characteristics. The characteristics showed that mGSC-pCDH-mTet1 cells in the seminiferous tube reproductive deficient mice not only maintain mGSCs, there is a significant proliferation. (4) of mTet1 play a role in mGSCs mode analysis, confirmed the expression of mTet1 through upregulation of histone demethylase JMJD3, downregulation of H3K27me3 content and changed from location within weeks of the nuclear nuclear distribution, inhibition of this process with the MEK-ERK signaling pathway by mTet1; and the formation of Hdac1 protein complex, CO regulation of histone acetylation modification, in the mGSCs table view The regulation of mTet1; and the formation of PCNA protein complex, expressed by mTet1 to promote the proliferation of mGSCs. In conclusion, this study analyzed the distribution of Tet1 in goat seminiferous tubules and cells in the in vivo and in vitro; morphological changes and gene regulation of Tet1 modified by the machine at the same time. This is not only in the process of spermatogenesis the expression of Tet1 to provide ideas for further research to further explore the regulation, epigenetic regulation in the development and differentiation of goat mGSCs laid the foundation.

【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：S827

【相似文獻(xiàn)】

相關(guān)期刊論文 前10條

1 張伯鈞,張洪芝,孫宏武,曲喜峰;綏棱縣奶山羊改良情況調(diào)查[J];黑龍江動(dòng)物繁殖;2000年03期

2 付吉青;如何養(yǎng)好奶山羊[J];吉林農(nóng)業(yè);2000年06期

3 巴特,白素文;怎樣選擇奶山羊[J];當(dāng)代畜禽養(yǎng)殖業(yè);2000年05期

4 ;奶山羊高產(chǎn)十招[J];當(dāng)代畜禽養(yǎng)殖業(yè);2000年06期

5 高文波;淺談提高奶山羊產(chǎn)奶量的措施[J];遼寧畜牧獸醫(yī);2001年06期

6 李健民,方惠文,趙伶;我靠養(yǎng)奶山羊發(fā)了家[J];農(nóng)村新技術(shù);2001年06期

7 金江;提高奶山羊產(chǎn)奶量四要點(diǎn)[J];飼料博覽;2001年04期

8 高文波;如何提高奶山羊的產(chǎn)奶量[J];新農(nóng)業(yè);2001年11期

9 王功勝;提高奶山羊產(chǎn)奶量的關(guān)鍵措施[J];農(nóng)村百事通;2002年23期

10 張兆順;奶山羊的管理技術(shù)[J];農(nóng)村養(yǎng)殖技術(shù);2002年03期

相關(guān)會(huì)議論文 前10條

1 白躍宇;譚旭信;;河南奶山羊培育技術(shù)[A];中國畜牧獸醫(yī)學(xué)會(huì)養(yǎng)羊?qū)W分會(huì)全國養(yǎng)羊生產(chǎn)與學(xué)術(shù)研討會(huì)議論文集[C];2010年

2 李芳娥;;奶山羊場、小區(qū)生產(chǎn)技術(shù)規(guī)范[A];《2011中國羊業(yè)進(jìn)展》論文集[C];2011年

3 楊春珂;;奶山羊飼養(yǎng)配套技術(shù)[A];云南省首屆無公害豬肉生產(chǎn)研討會(huì)、云南省奶業(yè)發(fā)展對(duì)策研討會(huì)論文集[C];2004年

4 羅軍;;奶山羊良種繁育體系建設(shè)現(xiàn)狀與思路[A];《2008中國羊業(yè)進(jìn)展》論文集[C];2008年

5 張花菊;;莎能奶山羊改良本地山羊效果試驗(yàn)[A];河南省畜牧獸醫(yī)學(xué)會(huì)第七屆理事會(huì)第二次會(huì)議暨2008年學(xué)術(shù)研討會(huì)論文集[C];2008年

6 曹斌云;;養(yǎng)好奶山羊十要訣[A];《2009中國羊業(yè)進(jìn)展》論文集[C];2009年

7 褚建剛;劉自建;鄶明玉;;文登奶山羊品種選育報(bào)告[A];《2009中國羊業(yè)進(jìn)展》論文集[C];2009年

8 魏志杰;;奶山羊規(guī)模養(yǎng)殖場生產(chǎn)經(jīng)營管理體會(huì)[A];中國畜牧獸醫(yī)學(xué)會(huì)養(yǎng)羊?qū)W分會(huì)全國養(yǎng)羊生產(chǎn)與學(xué)術(shù)研討會(huì)議論文集[C];2010年

9 喬文麗;褚建剛;鄶明玉;崔軍明;;文登奶山羊良種繁育產(chǎn)業(yè)化技術(shù)體系研究報(bào)告[A];2010中國羊業(yè)進(jìn)展[C];2010年

10 彭燕;;奶山羊精液冷凍保存試驗(yàn)[A];《2011中國羊業(yè)進(jìn)展》論文集[C];2011年

相關(guān)重要報(bào)紙文章 前10條

1 謝紅江;千陽莎能奶山羊全國走俏[N];寶雞日?qǐng)?bào);2006年

2 陜西省千陽縣委宣傳部 謝紅江;把奶山羊做成大產(chǎn)業(yè)[N];中國畜牧獸醫(yī)報(bào);2007年

3 鐘石;陜西做大做強(qiáng)奶山羊產(chǎn)業(yè)[N];中國畜牧獸醫(yī)報(bào);2007年

4 鄭綜文 元莉華;做好規(guī)劃 加快關(guān)中奶山羊產(chǎn)業(yè)發(fā)展[N];中國畜牧獸醫(yī)報(bào);2007年

5 省農(nóng)業(yè)廳廳長 梁鳳民;重新認(rèn)識(shí)奶山羊產(chǎn)業(yè) 把朝陽產(chǎn)業(yè)做大做強(qiáng)[N];陜西日?qǐng)?bào);2007年

6 鄭綜文邋記者 元莉華;把奶山羊發(fā)展成關(guān)中畜牧業(yè)的主導(dǎo)產(chǎn)業(yè)[N];陜西日?qǐng)?bào);2007年

7 高東風(fēng)邋通訊員 周煥;3年建設(shè)120個(gè)奶山羊服務(wù)站[N];渭南日?qǐng)?bào);2007年

8 李爾平 張釗 記者 程娟;三原奶山羊產(chǎn)業(yè)發(fā)展勢頭強(qiáng)勁[N];咸陽日?qǐng)?bào);2007年

9 葛偉剛;淳化莎能奶山羊助農(nóng)發(fā)『羊財(cái)』[N];咸陽日?qǐng)?bào);2007年

10 殷際輝 陳沛強(qiáng);富平奶山羊欲與奶牛試比“高”[N];中國特產(chǎn)報(bào);2008年

相關(guān)博士學(xué)位論文 前10條

1 李香子;丙酸前體與亞麻酸對(duì)奶山羊瘤胃發(fā)酵與乳脂共軛亞油酸形成機(jī)理研究[D];中國農(nóng)業(yè)科學(xué)院;2014年

2 王維;奶山羊擴(kuò)繁技術(shù)、XY精子分選及Y染色體ZNF280BY基因拷貝數(shù)變異研究[D];西北農(nóng)林科技大學(xué);2015年

3 張強(qiáng);體細(xì)胞核移植技術(shù)制備轉(zhuǎn)GH基因奶山羊的研究[D];南京農(nóng)業(yè)大學(xué);2014年

4 宋玉潔;人乳鐵蛋白基因定點(diǎn)敲入β乳球蛋白位點(diǎn)基因打靶奶山羊的研制[D];西北農(nóng)林科技大學(xué);2015年

5 鄭麗明;Tet1對(duì)奶山羊雄性生殖干細(xì)胞自我更新與增殖的表觀修飾調(diào)控[D];西北農(nóng)林科技大學(xué);2016年

6 侯金星;奶山羊產(chǎn)奶性狀候選基因的篩選及其多基因聚合效應(yīng)的研究[D];西北農(nóng)林科技大學(xué);2013年

7 朱廣琴;多羔和單羔奶山羊發(fā)情期卵巢組織差異表達(dá)基因的篩選、鑒定及分析[D];西北農(nóng)林科技大學(xué);2011年

8 尹多;核移植介導(dǎo)生產(chǎn)轉(zhuǎn)基因延邊奶山羊供核細(xì)胞體系的研究[D];延邊大學(xué);2013年

9 李術(shù);奶山羊氟病毒理學(xué)的研究[D];東北農(nóng)業(yè)大學(xué);2001年

10 丁武;波爾山羊與關(guān)中奶山羊雜交后代產(chǎn)肉性能及羊肉品質(zhì)研究[D];西北農(nóng)林科技大學(xué);2005年

相關(guān)碩士學(xué)位論文 前10條

1 唐琳;妊娠期奶山羊黃體中褪黑素膜受體MT1和MT2的分布和表達(dá)[D];西北農(nóng)林科技大學(xué);2015年

2 宋愛愛;奶山羊均衡產(chǎn)奶配套技術(shù)研究與應(yīng)用（Ⅰ）[D];西北農(nóng)林科技大學(xué);2015年

3 高特仰;miRNA-1296 和miRNA-370對(duì)奶山羊MTHFR基因表達(dá)的調(diào)控作用研究[D];西北農(nóng)林科技大學(xué);2015年

4 郭曉盼;富平縣奶山羊產(chǎn)業(yè)發(fā)展?fàn)顩r及技術(shù)研究分析[D];東北農(nóng)業(yè)大學(xué);2015年

5 張桂芳;不同精粗比日糧對(duì)泌乳期奶山羊肝臟氨基酸和葡萄糖代謝的影響[D];南京農(nóng)業(yè)大學(xué);2014年

6 白成斌;1～90d關(guān)中奶山羊能量營養(yǎng)需要研究[D];西北農(nóng)林科技大學(xué);2010年

7 高建偉;不同鋅源和水平在奶山羊腸道的消化吸收及對(duì)相關(guān)內(nèi)分泌的影響[D];河南農(nóng)業(yè)大學(xué);2010年

8 陳珊珊;中國奶山羊遺傳多樣性的微衛(wèi)星分析[D];山東農(nóng)業(yè)大學(xué);2011年

9 黃漢軍;農(nóng)戶飼養(yǎng)條件下關(guān)中奶山羊典型日糧與生產(chǎn)性能關(guān)系的研究[D];西北農(nóng)林科技大學(xué);2005年

10 郭本玲;西農(nóng)薩能奶山羊和關(guān)中奶山羊乳鐵蛋白基因遺傳多態(tài)性及其與經(jīng)濟(jì)性狀的關(guān)聯(lián)分析[D];西北農(nóng)林科技大學(xué);2011年

，

本文編號(hào)：1558354