【作者】 杨春；

【导师】 赵志辉；

【作者基本信息】 吉林大学 ， 动物遗传育种与繁殖， 2011， 博士

【摘要】 DNA甲基化在真核生物中是一种重要的表观遗传学修饰方法,也是基因表达调控重要影响因素之一。大量研究结果表明,胞嘧啶DNA甲基化在功能基因表达、细胞分化、X染色体失活、胚胎分化、疾病和癌症发生中都起到非常重要的作用。很多研究表明,不同的甲基化水平可能与组织特异性转录有关,并且对正常的分化和发育至关重要。本研究以猪为研究对象,利用F-MSAP的方法对莱芜猪肌肉、心脏、肝脏、脾脏、肺脏、肾脏和胃七个组织进行全基因组DNA甲基化分析,以及对具有不同肉质性状一肌间脂肪含量的我国地方品种莱芜猪和东北民猪,育成品种鲁莱猪、松辽黑猪和军牧一号以及外来品种大白猪的肌肉组织进行全基因组DNA甲基化分析。结果显示,莱芜猪肌肉、心脏、肝脏、脾脏、肺脏、肾脏和胃七个组织全基因组DNA甲基化程度分别为53.9%,51.2%,50.1%,53.3%,51.9%,51.1%和53.3%；大白猪、莱芜猪、鲁莱猪、军牧一号、松辽黑猪、东北民猪六个品种肌肉组织的甲基化程度分别为47.97%,51.75%,53.21%,50.46%,54.88%,56.98%；采用卡方检验分析不同品种肌肉组织的甲基化程度,结果表明各个品种间的甲基化程度差异显著(P<0.05)。通过数据分析,发现各品种肌肉组织DNA甲基化程度与各品种肌间脂肪含量间不具有相关性。本研究希望通过对猪不同组织和不同品种肌肉组织DNA甲基化的分析,了解猪不同组织和不同品种肌肉的全基因组DNA甲基化水平和组织间DNA甲基化模式差异。为今后猪DNA甲基化的研究积累数据,希望发现和了解甲基化在组织分化过程中的作用,以及通过对具有差异性不同肌间脂肪含量的不同品种肌肉组织全基因组DNA甲基化的测定,揭示是否不同品种肌肉组织全基因组DNA甲基化和肌肉肌间脂肪含量具有相关性,期望发现与肉质性状相关的功能基因,为更好的研究畜禽提供新的思路并奠定基础。更多还原

【Abstract】 DNA methylation is one of the main epigenetic modification mechanisms in eukaryotic organisms, playing a crucial role in the regulation of gene expression. It entails the transfer of methyl groups from S-adenosine-L-methionine to cytosines and adenines by DNA methyltransferases following DNA duplication. In living cells this process has been reported as one of the most common covalent modifications. Many studies have shown that DNA methylation, especially methylation of cytosine, has been implicated in gene expression, genomic imprinting, X chromosome inactivation, determination of chromatin structure, disease and cancer development.Methylation of CpG dinucleotides in the 5’regulatory regions of genes often results in transcriptional inactivation, and some studies have shown that actively transcribed sequences are often methylated less than promoters and certain coding regions of silent genes. Significant differences in the levels or pattern of cytosine methylation have been observed in various tissues or under different functional states in the same tissue. Several studies suggested that various levels of DNA methylation may regulate tissue-specific transcription and be important for normal development or differentiation and trait. But how methylation regulates gene expression is still unclear. Therefore, the detection and analysis of levels and patterns of genome-wide methylation in various tissues or in same tissue of the different varieties, is essential for understanding associations between tissue-specific or variety-specific methylation or and tissue-specific or variety-specific gene expression.The fluorescence-labeled methylation-sensitive amplification polymorphism (F-MSAP) technique is a relatively new modification of the amplification fragment length polymorphism (AFLP) technique. The F-MSAP technique utilizes the restriction isoschizomer pair HpaⅡand MspⅠinstead of MseⅠin the AFLP, in which selective amplification is performed with fluorescently labeled primers instead of radiolabeled primers. The F-MSAP system consists of four major parts:digestion and ligation reactions, preamplification, selective amplification reactions and detection reactions. In F-MSAP, these enzymes recognize the same restriction site (CCGG) but have different sensitivities to certain methylation states of cytosines. HpaⅡis inactive if one or both cytosines are fully methylated (both strands are methylated) but cleaves the hemimethylated sequence (only one DNA strand is methylated), whereas MspI is inactive if the external cytosine is fully or-hemimethylated (only one DNA strand is methylated). Thus, for a given DNA sample, the full methylation of the internal cytosines and hemimethylation of the external cytosines at the assayed CCGG sites can be unequivocally distinguished using these two restriction enzymes. So, In F-MSAP the bands revealing methylation patterns of HpaⅡ-EcoRl and Mspl-EcoRI digested genomic DNA could be divided into three types of methylation status as follows:type I presents bands of the same length in both lanes, which indicates inner methylation of single-stranded DNA or no methylation; type II is present for Hpall but absent for MspI, which indicates outer methylation of single-stranded DNA and hemi-methylation at the outer cytosine nucleotide in the CCGG sequence; and typeⅢis present for MspI but absent for Hpall, which indicates inner methylation of double-stranded DNA and full methylation of the CCGG sequence.In this study, we used 16 pairs of selective primers, obtained from eight EcoRI primers in combination with two fluorescently labeled Hpall/MspI primers, to analyze DNA methylation at CCGG sites of muscle, heart, liver, spleen, lung, kidney and stomach from Laiwu pigs and the muscle from Large white, Laiwu, Lulai, Junmul, Songliaohei and Dongbei min by the F-MSAP method.In this study, we used the fluorescence-labeled methylation-sensitive amplified polymorphism (F-MSAP) method to assess the extent and pattern of cytosine methylation in muscle, heart, liver, spleen, lung, kidney and stomach from the swine strain Laiwu and the muscles from Large white, Laiwu, Lulai, Junmul, Songliaohei and Dongbei min. The main results are as follow:1 In this study, we used the fluorescence-labeled methylation-sensitive amplified polymorphism (F-MSAP) method to assess the extent and pattern of cytosine methylation in muscle, heart, liver, spleen, lung, kidney and stomach from the swine strain Laiwu, and we also examined specific methylation patterns in the seven tissues. In total,96,371 fragments, each representing a recognition site cleaved by either or both EcoRI+HpaⅡand EcoRI+MspⅠ,the HpaⅡand MspⅠare isoschizomeric enzymes, were amplified using 16 pairs of selective primers. A total of 50,094 sites were found to be methylated at cytosines in seven tissues. The incidence of DNA methylation was approximately 53.99% in muscle,51.24% in the heart, 50.18% in the liver,53.31% in the spleen,51.97% in the lung,51.15% in the kidney and 53.39% in the stomach, as revealed by the incidence of differential digestion. Additionally, differences in DNA methylation levels imply that such variations may be related to specific gene expression during tissue differentiation, growth and development. Three types of bands were generated in the F-MSAP profile, the total numbers of these three types of bands in the seven tissues were 46,277,24,801 and 25,293, respectively.2 In this study, we found that among 35 sites of TDMs, the sequence ranged from 88 to 278 bp. Sixteen are located within introns while four are located in the 5’ upstream regions, and two are located in the 3’downstream regions of genes. Moreover because the pig genome is not complete, there are 13 sites that we cannot locate in genes, but can only locate at the chromosome level. Because the number of tissues is large, we rarely found a band present only in one tissue, but we often found bands present in two or three tissues or bands present in different patterns in different tissues.3 In this study, we used the fluorescence-labeled methylation-sensitive amplified polymorphism (F-MSAP) method to assess the extent and pattern of cytosine methylation in muscle tissues from Large white, Laiwu, Lulai, Junmul, Songliaohei and Dongbei min. In total,5378 fragments, each representing a recognition site cleaved by either or both EcoRI+HpaⅡand EcoRI+Mspl, the HpaⅡand MspI are isoschizomeric enzymes, were amplified using 16 pairs of selective primers. A total of 2828 sites were found to be methylated at cytosines in muscle tissues from Large white, Laiwu.Lulai, Junmul, Songliaohei and Dongbei min. The incidence of DNA methylation was approximately 47.97% in Large white,51.75% in the Laiwu,53.21% in the Lulai,50.46% in the Junmul,54.88% in the Songliaohei, and 56.98% in the Dongbeimin. Three types of bands were generated in the F-MSAP profile, the total numbers of these three types of bands in the seven tissues were 2550,1393 and 1435 respectively. The result shown that various varieties of muscle tissues DNA methylation and the intramuscular fat content of muscle does not have correlaton.4 In this study, we found that among 22 sites of TDMs, the sequence ranged from 82 to 214bp. Thirteen are located within introns while three are located in the 5’ upstream regions, and three are located in the 3’downstream regions of genes. Moreover because the pig genome is not complete, there are three sites that we cannot locate in genes, but can only locate at the chromosome level. Because the number of tissues is large, we rarely found a band present only in one tissue, but we often found bands present in two or three tissues or bands present in different patterns in different tissues.5 In this study we use the F-MSAP method to compare the levels of DNA cytosine methylation of pigs for the first time, The results show that there are different methylation levels and patterns in various tissues and muscle of different variety. The results clearly demonstrated that F-MSAP is highly efficient for large-scale detection of cytosine methylation and can be further extended to research on genome of other animals and plants that have complex genome and are rich in methylation polymorphism.更多还原