猪DNMT1基因的免疫生物学初步研究

发布时间：2018-03-10 22:09

本文选题：猪　切入点：表观遗传学　出处：《华中农业大学》2015年硕士论文　论文类型：学位论文

【摘要】：表观遗传学又称“拟遗传学”、“表遗传学”、“外遗传学”、“后遗传学”,是软遗传的重要组成部分。虽然表观遗传涉及多个内容,但DNA甲基化却是表观遗传研究的最核心、最主要的内容之一。DNMT1是一种甲基化转移酶,在细胞中主要承担DNA甲基化维持的生物学功能。目前,在人、小鼠、拟南芥等动植物中,对DNMT1基因开展了大量的研究,得到了一系列丰硕的结果。不过,这研究主要集中在模式动植物,在家畜中的研究则不够深入,而系统地从免疫角度探讨DNMT1基因功能的研究则更加不足。DNMT1基因参与了机体内绝大多数生物学过程的调控,与免疫调控也有密切的关系。鉴于此,本研究主要以猪为研究对象,以免疫为重点视角,拟对猪的DNMT1基因开展初步的研究,分别从系统发育树、可变剪切体、偶联转录组等角度,对猪DNMT1基因的系统发生关系,该基因在鼻粘膜、脑淋巴结、骨髓、胸腺、脾等免疫组织中的可变剪切情况,以及基于外周血细胞模型的DNMT1基因的偶联转录组开展了较为系统的研究,初步得到研究结果如下:1.用mega3.1构建DNMT1基因在多个物种间的系统发生树,从进化角度揭示了猪DNMT1基因与其他物种DNMT1基因的分化关系,发现DNMT1基因在各物种间的系统遗传距离各不相同,但在家养农业动物之间具有更高的保守性。2.利用RT-PCR技术,鉴定了猪DNMT1基因的可变剪切体DNMTo1和DNMTo2,并系统地检测了鼻粘膜、脑淋巴结、骨髓、胸腺、脾等免疫组织及对照组织中的DNMT1基因m RNA及其可变剪切体的表达情况,结果发现Dnmt1s、Dnmt1o1、Dnmt1o2在各个免疫组织中均有表达,但其丰度和相互间比例在各免疫组织间呈现出差异。3.以猪外周血细胞为模型,利用猪Affymetrix表达谱芯片建立了DNMT1基因的偶联转录组(coupling transcriptome)的分析框架,包括基于DNMT1基因对应探针组(probesets)的分布检测、质量控制分析、差异表达基因鉴定、聚类分析、GO注释等。4.以探针组Ssc.24344.1.S1_a_at表达值的两尾为基础,鉴定出差异表达转录本共161个;以探针组Ssc.24344.1.S1_at表达值为协变量,鉴定出差异表达转录本135个。5.利用降维策略初步探讨了DNMT1基因表达变异对基因组表达模式的影响。我们采用非负矩阵因子化方法对经过非特异性过滤后的表达矩阵进行降维处理,构建出10个新的因子化“性状”,并估计了DNMT1基因与各因子之间的相关系数与回归系数,从宏观上揭示了DNMT1基因的表达变异与基因组的整体表达模式相偶联。本研究以免疫生物学为视角,从多个角度对猪DNMT1基因开展了研究,得到了多个有意义的研究结果,积累了猪DNMT1基因的基础数据,为进一步深入研究猪DNMT1基因的免疫调控功能奠定了数据基础。
[Abstract]:Epigenetics, also known as "pseudogenetics", "epigenetics", "extrinsic genetics" and "post-genetics", is an important part of soft genetics. Although epigenetics involves many aspects, DNA methylation is the core of epigenetic research. DNMT1 is a kind of methylation transferase, which is mainly responsible for the biological function of DNA methylation in cells. At present, DNMT1 gene has been widely studied in animals and plants, such as human, mouse, Arabidopsis thaliana and so on. A series of fruitful results have been obtained. However, this study is mainly focused on model animals and plants, but not enough on domestic animals. However, the systematic study of the function of DNMT1 gene from the immune angle is even less. DNMT1 gene is involved in the regulation of most biological processes in the organism and has close relationship with immune regulation. In view of this, the study mainly focuses on pigs. From the point of view of immunity, this paper intends to carry out a preliminary study on the DNMT1 gene of pigs. From the perspectives of phylogenetic tree, variable shearing body and coupling transcriptome, the phylogenetic relationship of porcine DNMT1 gene was studied. The gene was found in nasal mucosa, brain lymph node, bone marrow, and so on. The variable shearing of thymus and spleen and the coupling transcriptome of DNMT1 gene based on peripheral blood cell model have been studied systematically. The results are as follows: 1. The phylogenetic tree of DNMT1 gene was constructed with mega3.1, and the relationship between porcine DNMT1 gene and DNMT1 gene of other species was revealed from the perspective of evolution. It was found that the phylogenetic distance of DNMT1 gene varied from species to species, but there was a higher conservation among farm animals raised at home. Using RT-PCR technique, the variable shearing bodies DNMTo1 and DNMTo2 of porcine DNMT1 gene were identified, and the nasal mucosa was systematically detected. The expression of DNMT1 gene m RNA and its variable shearing bodies in brain lymph nodes, bone marrow, thymus, spleen and control tissues were observed. The results showed that Dnmt1snmt1o1, Dnmt1o1 and Dnmt1o2 were expressed in all immune tissues. However, the abundance and proportion of DNMT1 gene were different among different immune tissues. Using porcine peripheral blood cells as a model, the analysis framework of coupling transcriptome of DNMT1 gene was established by using porcine Affymetrix expression microarray. Including distribution detection, quality control analysis, differential expression gene identification, cluster analysis and so on. Based on the two ends of Ssc.24344.1.S1_a_at expression value of probe group, 161 differentially expressed transcripts were identified. The Ssc.24344.1.S1_at expression value of probe group was used as covariable. 135 differentially expressed transcripts were identified. The effects of DNMT1 gene expression variation on genomic expression patterns were preliminarily discussed by dimensionality reduction strategy. We used non-negative matrix factorization method to analyze the expression moments after non-specific filtering. The array is processed by dimensionality reduction, Ten new factorization "traits" were constructed, and the correlation coefficient and regression coefficient between DNMT1 gene and each factor were estimated. It is revealed that the expression variation of DNMT1 gene is coupled with the overall expression pattern of the genome. From the perspective of immunobiology, the porcine DNMT1 gene was studied from several angles, and many meaningful results were obtained. The basic data of porcine DNMT1 gene were accumulated, which laid a data foundation for further study on the immunological regulation function of porcine DNMT1 gene.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S828

【参考文献】