TRIM28对绵羊早期克隆胚胎印记基因甲基化作用的研究

发布时间：2018-06-04 22:56

  本文选题：TRIM28 + DNA甲基化　； 参考：《石河子大学》2016年硕士论文

【摘要】：目的:体细胞克隆动物因基因组重编程不完全,部分基因甲基化异常导致流产、死胎和巨婴症等诸多问题。TRIM28在植入前早期胚胎发育过程中对印记基因甲基化的维持起着重要作用,母源TRIM28缺失可导致正常受精胚胎死亡,其发育过程中具有与克隆异常胎儿相同的症状,而且母源合子转换过程中TRIM28的表达量与印记基因DMR的甲基化水平密切相关。本研究通过分析过表达TRIM28细胞克隆的胚胎中与正常细胞克隆胚胎TRIM28表达和甲基化水平差异,探究TRIM28是否与绵羊早期克隆印记基因异常去甲基化有关。方法:实验一:通过提取中国美利奴细毛羊卵巢组织RNA,反转录后获得cDNA,设计特异性引物以扩增TRIM28基因编码区序列,经测序后采用生物信息学软件对编码区序列及氨基酸序列进行相关生物信息学分析和结构预测,并采用RT-PCR对绵羊不同组织中TRIM28表达量进行检测绘制组织表达图谱和成纤维细胞中与其互作参与甲基化调控的基因进行了表达量的检测。实验二:构建TRIM28真核表达载体及干扰si RNA,通过电转染绵羊成纤维细胞筛选得到能够高效表达的重组表达载体和有效抑制TRIM28,并检测转染后细胞TRIM28的表达量对DNA甲基化基因和印记基因表达表达水平的影响。实验三:分别收集正常体细胞和过表达TRIM28体细胞核移植后不同发育时期克隆重构胚胎,采用RT-q PCR检测不同发育时期胚胎中TRIM28基因的m RNA表达量;收集2-cell和囊胚,提取基因组,重亚硫酸盐处理后采用BSP法克隆印记基因H19和IGF2R的差异甲基化区域,检测印记基因H19和IGF2R甲基化水平。结果:本研究克隆得到了绵羊TRIM28基因完整CDS序列,长度为2441 bp。获得的该基因核苷酸序列及所编码的氨基酸序列与人、牛、猪、小鼠等动物核苷酸序列和氨基酸序列具有非常高的一致性。TRIM28 m RNA在绵羊的心脏、肝脏、脾脏、肺脏、肾脏、肌肉、卵巢组织中均有表达,组织表达谱结果显示,TRIM28 m RNA在中国美利奴细毛羊肺脏、脾脏和卵巢中高丰度表达,且与ZFP57在所有检测组织中表达情况一致。成功构建了绵羊TRIM28真核过表达载体p EGFP-C1-TRIM28和筛选得到有效抑制TRIM28表达的干扰si RNA,电转染p EGFP-C1-TRIM28和si RNA的成纤维细胞中TRIM28表达改变导致甲基化酶基因DNMT1、DNMT3B、ZFP57、SETDB1的表达量下调,但是对DNMT3A影响不大;IGF2、IGF2R、PEG1.2、PEG3、MEG8五个印记基因的表达量随着TRIM28表达降低均表现出不同水平下降,随着TRIM28表达的升高均出现上调。H19和DLK1两个印记基因的表达在TRIM28表达下降和上升中均表现出下调。结论:绵羊TRIM28基因完整的编码区序列与其他物种存在较高的同源性。本研究结果表明TRIM28不仅在早期胚胎发育中印记基因甲基化维持起到重要作用,而且在高度分化的成纤维细胞增殖中DNA甲基化的维持至关重要。TRIM28过表达体细胞克隆可以有效提高早期胚胎TRIM28表达量,且TRIM28过表达核移植胚胎印记基因H19和IGF2R甲基化水平高于正常核移植组,表明TRIM28对于克隆胚胎印记基因甲基化维持起到作用。
[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.
【学位授予单位】：石河子大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S826
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本文编号：1979201