本文選題：TRIM28 + DNA甲基化　； 參考：《石河子大學(xué)》2016年碩士論文

【摘要】：目的:體細(xì)胞克隆動(dòng)物因基因組重編程不完全,部分基因甲基化異常導(dǎo)致流產(chǎn)、死胎和巨嬰癥等諸多問題。TRIM28在植入前早期胚胎發(fā)育過程中對(duì)印記基因甲基化的維持起著重要作用,母源TRIM28缺失可導(dǎo)致正常受精胚胎死亡,其發(fā)育過程中具有與克隆異常胎兒相同的癥狀,而且母源合子轉(zhuǎn)換過程中TRIM28的表達(dá)量與印記基因DMR的甲基化水平密切相關(guān)。本研究通過分析過表達(dá)TRIM28細(xì)胞克隆的胚胎中與正常細(xì)胞克隆胚胎TRIM28表達(dá)和甲基化水平差異,探究TRIM28是否與綿羊早期克隆印記基因異常去甲基化有關(guān)。方法:實(shí)驗(yàn)一:通過提取中國美利奴細(xì)毛羊卵巢組織RNA,反轉(zhuǎn)錄后獲得cDNA,設(shè)計(jì)特異性引物以擴(kuò)增TRIM28基因編碼區(qū)序列,經(jīng)測(cè)序后采用生物信息學(xué)軟件對(duì)編碼區(qū)序列及氨基酸序列進(jìn)行相關(guān)生物信息學(xué)分析和結(jié)構(gòu)預(yù)測(cè),并采用RT-PCR對(duì)綿羊不同組織中TRIM28表達(dá)量進(jìn)行檢測(cè)繪制組織表達(dá)圖譜和成纖維細(xì)胞中與其互作參與甲基化調(diào)控的基因進(jìn)行了表達(dá)量的檢測(cè)。實(shí)驗(yàn)二:構(gòu)建TRIM28真核表達(dá)載體及干擾si RNA,通過電轉(zhuǎn)染綿羊成纖維細(xì)胞篩選得到能夠高效表達(dá)的重組表達(dá)載體和有效抑制TRIM28,并檢測(cè)轉(zhuǎn)染后細(xì)胞TRIM28的表達(dá)量對(duì)DNA甲基化基因和印記基因表達(dá)表達(dá)水平的影響。實(shí)驗(yàn)三:分別收集正常體細(xì)胞和過表達(dá)TRIM28體細(xì)胞核移植后不同發(fā)育時(shí)期克隆重構(gòu)胚胎,采用RT-q PCR檢測(cè)不同發(fā)育時(shí)期胚胎中TRIM28基因的m RNA表達(dá)量;收集2-cell和囊胚,提取基因組,重亞硫酸鹽處理后采用BSP法克隆印記基因H19和IGF2R的差異甲基化區(qū)域,檢測(cè)印記基因H19和IGF2R甲基化水平。結(jié)果:本研究克隆得到了綿羊TRIM28基因完整CDS序列,長度為2441 bp。獲得的該基因核苷酸序列及所編碼的氨基酸序列與人、牛、豬、小鼠等動(dòng)物核苷酸序列和氨基酸序列具有非常高的一致性。TRIM28 m RNA在綿羊的心臟、肝臟、脾臟、肺臟、腎臟、肌肉、卵巢組織中均有表達(dá),組織表達(dá)譜結(jié)果顯示,TRIM28 m RNA在中國美利奴細(xì)毛羊肺臟、脾臟和卵巢中高豐度表達(dá),且與ZFP57在所有檢測(cè)組織中表達(dá)情況一致。成功構(gòu)建了綿羊TRIM28真核過表達(dá)載體p EGFP-C1-TRIM28和篩選得到有效抑制TRIM28表達(dá)的干擾si RNA,電轉(zhuǎn)染p EGFP-C1-TRIM28和si RNA的成纖維細(xì)胞中TRIM28表達(dá)改變導(dǎo)致甲基化酶基因DNMT1、DNMT3B、ZFP57、SETDB1的表達(dá)量下調(diào),但是對(duì)DNMT3A影響不大;IGF2、IGF2R、PEG1.2、PEG3、MEG8五個(gè)印記基因的表達(dá)量隨著TRIM28表達(dá)降低均表現(xiàn)出不同水平下降,隨著TRIM28表達(dá)的升高均出現(xiàn)上調(diào)。H19和DLK1兩個(gè)印記基因的表達(dá)在TRIM28表達(dá)下降和上升中均表現(xiàn)出下調(diào)。結(jié)論:綿羊TRIM28基因完整的編碼區(qū)序列與其他物種存在較高的同源性。本研究結(jié)果表明TRIM28不僅在早期胚胎發(fā)育中印記基因甲基化維持起到重要作用,而且在高度分化的成纖維細(xì)胞增殖中DNA甲基化的維持至關(guān)重要。TRIM28過表達(dá)體細(xì)胞克隆可以有效提高早期胚胎TRIM28表達(dá)量,且TRIM28過表達(dá)核移植胚胎印記基因H19和IGF2R甲基化水平高于正常核移植組,表明TRIM28對(duì)于克隆胚胎印記基因甲基化維持起到作用。
[Abstract]:Objective: somatic cell cloned animals are incomplete because of genome reprogramming, some abnormal methylation leads to miscarriage, stillbirth and giant baby, and many problems, such as abortion, stillbirth and giant baby, play an important role in the maintenance of imprinted gene methylation during the early embryonic development process, and the loss of mother source TRIM28 can lead to the death of normal fertilized embryos and the development process of.TRIM28. In this study, the expression of TRIM28 is closely related to the level of methylation of the imprinted gene DMR during the transformation of the parent heterozygote, and the difference between the expression of TRIM28 and the methylation level of the normal cell cloned embryos in the embryo of the TRIM28 cell cloned by the overexpression of the TRIM28 cells is analyzed to explore whether or not the TRIM28 is with the sheep. Early cloning of imprinted gene abnormal demethylation. Method: Experiment 1: by extracting RNA from Chinese Merino fine wool sheep ovarian tissue, cDNA was obtained after reverse transcription, specific primers were designed to amplify the sequence of TRIM28 gene coding region, and bioinformatics software was used to carry out related biological information on the sequence of coding region and the sequence of amino acids after sequencing. Study and structure prediction, and use RT-PCR to detect the expression of TRIM28 expression in different sheep tissues and detect the expression of tissue expression map and the genes involved in methylation in the fibroblasts. Experiment two: construct TRIM28 eukaryotic expression vector and interference Si RNA, and transfect sheep fibroblasts by electrotransfection We screened the recombinant expression vector and effective inhibition of TRIM28, and detected the effect of TRIM28 expression on the expression level of DNA methylation gene and imprinted gene in the transfected cells. Experiment three: to collect the normal somatic cells and the overexpressed TRIM28 body nuclei after the cell nucleus migration and to clone the reconstructed embryos at different developmental stages, and use R T-q PCR was used to detect the m RNA expression of TRIM28 gene in different developmental stages, collect 2-cell and blastocyst, extract genomes, and then clone the differential methylation region of the imprinted gene H19 and IGF2R by BSP method, and detect the level of H19 and IGF2R methylation of the imprinted genes. DS sequence, the nucleotide sequence of the gene and the encoded amino acid sequence obtained by the length of 2441 bp., and the nucleotide sequence and amino acid sequence of human, bovine, pig and mouse have very high consistency of.TRIM28 m RNA in sheep's heart, liver, spleen, lung, kidney, muscle, and ovarian tissue, and the tissue expression profile results TRIM28 m RNA is highly expressed in the lungs, spleen and ovary of the Chinese Merino sheep, and is in accordance with the expression of ZFP57 in all the detected tissues. The TRIM28 eukaryotic overexpression vector of the sheep P EGFP-C1-TRIM28 and the dry disturbance Si RNA which effectively inhibit the expression of TRIM28 are successfully constructed. The expression of TRIM28 in fibrous cells led to the downregulation of the expression of methylase gene DNMT1, DNMT3B, ZFP57, SETDB1, but little effect on DNMT3A. The expression of five imprinted genes, IGF2, IGF2R, PEG1.2, PEG3, MEG8, decreased with the decrease of TRIM28 expression. The expression of the imprinting gene was downregulated in the decline and rise of TRIM28 expression. Conclusion: the complete coding region sequence of the sheep TRIM28 gene has high homology with other species. This study shows that TRIM28 not only plays an important role in the maintenance of imprinted gene methylation in early embryonic development, but also in highly differentiated fibroblast. The maintenance of DNA methylation in vascular proliferation is crucial to.TRIM28 overexpressed somatic cell cloning, which can effectively improve the expression of TRIM28 in early embryo, and the level of H19 and IGF2R methylation of TRIM28 overexpressed nuclear transfer embryos is higher than that of normal nuclear transplantation group, which indicates that TRIM28 plays a role in maintaining the methylation of the cloned embryo imprinting gene.
【學(xué)位授予單位】：石河子大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：S826
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本文編號(hào)：1979201