京海黄鸡腿肌全基因组甲基化和转录组分析研究

发布时间：2018-05-05 05:01

本文选题：京海黄鸡 + 早期生长　；参考：《扬州大学》2017年博士论文

【摘要】：肉鸡早期生长发育规律尤其是生长拐点影响着肉鸡最佳上市时间和屠宰体重,进而影响着肉鸡产业的经济效益,而目前关于肉鸡早期生长发育规律遗传机理的研究却很少。DNA甲基化作为最常见的表观遗传修饰方式之一,在转录及转录后水平调控基因的表达,进而影响动物的生长发育,目前关于优质肉鸡早期生长过程中表观遗传调控机理的研究报道很少。因此本研究选取京海黄鸡生长拐点前(缓慢生长阶段)、生长拐点及拐点后(上市阶段),即对应4、12和16周龄三个特殊时间点的腿肌组织,采用转录组测序RNA-seq和全基因组甲基化测序BS-seq技术相结合,探索京海黄鸡早期生长过程中全基因组范围内基因表达水平的变化、DNA甲基化状态的变化以及DNA甲基对生长相关基因表达的调控机制,为揭示京海黄鸡生长发育规律的遗传机理以及优质肉鸡腿部肌纤维发育机制提供参考依据。主要研究结果如下:1.利用三种非线性模型对京海黄鸡0～25周龄生长发育数据进行生长曲线拟合,发现京海黄鸡生长曲线接近“S”型,且Logistic模型下计算的京海黄鸡生长拐点为11.48周龄,拐点体重为896.68g。2.对不同生长阶段京海黄鸡腿肌样本共9个(各阶段3个重复),进行转录组测序,每个样本最终获得了 8Gb(gigabases)以上的可用数据,共检测出15699个在腿肌中表达的基因,其中有14432个基因在4、12和16周龄中共同表达,而分别检测出59、724和633个基因在4周龄与12周龄(M4WvsM12W)、4周龄与16周龄(M4WvsM16W)以及12周龄与16周龄(M12WvsM16W)三组比较中差异表达(FC≥2,FDR0.05),在这些差异表达的基因中,发现了 MYOD1、FBX032、CEBPB和FOX034个转录因子,以及鸡生长轴中的一些基因,如 GH、IGF2BP1、IGF2BP2、IGF2BP3、IGFBP3、IGFBP5 和 IGFBP7等生长相关的候选基因。3.对差异表达的基因进行GO和Pathway功能富集分析,发现差异表达基因显著富集于细胞增殖和分化、细胞迁移、细胞粘着和连接、细胞生长、骨骼肌肌纤维发育、肌肉器官发育和骨骼肌细胞分化等生长发育相关的生物过程(P0.05),涉及到的基因包括MYOD1,IGFBP3,IGFBP5、1GFBP7和MYH10等已知的与生长发育相关的基因。在显著富集的信号通路中(P0.05),发现了 5个与生长发育相关的信号通路,分别为粘着斑、细胞外基质受体互作、胰岛素信号通路、紧密连接和肌动蛋白细胞骨架调节,共有42个基因包含在这5个通路中,包括FGF2、FGF16、FN1、MYH10、MAPK9等,推测这些通路及基因在京海黄鸡早期的生长和腿肌发育过程中发挥着重要的调控作用。4.本研究通过BS-seq技术构建了京海黄鸡腿肌全基因组DNA甲基化图谱,并揭示了其DNA甲基化特征:mCpG为京海黄鸡基因组DNA甲基化主要方式,极少部分DNA甲基化发生在CHG和CHH序列上;CG类型甲基化位点侧翼序列不存在碱基偏好性,而非CG类型甲基化胞嘧啶位点的下游碱基以腺嘌呤(A)出现最多;CG位点甲基化水平在TSS附近显著下降、基因本体区显著升高,而非CG位点甲基化水平在TSS附近显著升高、基因本体区内显著下降;不同功能元件的甲基化水平分析中,启动子区和CpG岛的甲基化水平最低,CDS区和外显子的甲基化水平最高;各条染色体上的DNA甲基化水平及甲基化密度存在较大变化。5.不同生长阶段京海黄鸡甲基化组比较(M4W vs M12W、M4W vs M16W和M12W vsM16W),分别检测出2141、2260和1550个差异甲基化区域(DMR),分别涉及1022、1055和797个差异甲基化基因,其中分别有263、239和229个基因的差异甲基化发生在了启动子区。6.对启动子区差异甲基化基因进行功能富集分析,发现这些差异甲基化基因显著富集于转化生长因子β受体信号通路调控、细胞迁移和细胞增殖正调控、细胞分化和细胞增殖调控、骨骼系统发育、肌动蛋白细胞骨架组织、细胞黏附等生长相关的生物过程(P0.05),以及转化生长因子β信号通路、细胞周期、溶酶体、核糖体、细胞黏附分子5个生长发育相关的信号通路(P0.05),涉及到的基因包括BMP4和SMAD4等生长发育相关基因。7.将甲基化数据与转录组数据进行整合分析,发现大部分基因启动子区DNA甲基化与表达呈负相关,并且检测出26个启动子区差异甲基化且差异表达的基因,其中有20个基因启动子区甲基化变化与基因表达变化呈负相关关系,包括IGF2BP1和IGF2BP3两个已知的生长发育相关基因。通过BSP技术对IGF2BP1和IGF2BP3启动子区甲基化水平进行验证,并结合基因表达水平进行分析,发现BSP方法与BS-seq研究结果相一致,均显示IGF2BP1和IGF2BP3启动子区甲基化水平与基因表达水平呈负相关,因此推测IGF2BP1和IGF2BP3可能通过启动子DNA甲基化抑制其自身的表达,进而发挥对京海黄鸡生长发育的调控作用。
[Abstract]:The early growth and development of broilers, especially the growth inflection point, affects the best market time and slaughter weight of broilers, and then affects the economic benefits of the broiler industry. At present, the study on the genetic mechanism of the early growth and development of broilers is rarely.DNA methylation as one of the most common epigenetic modification methods, in transcription and transcription. At present, there are few reports on the epigenetic regulation mechanism in the early growth process of high quality broilers. Therefore, this study selected the growth inflection point (slow growth stage), growth inflection point and inflection point (in the market stage), that is, three special 4,12 and 16 weeks old age. In order to reveal the change of gene expression level in the whole genome of Jinghai yellow chicken during the early growth process, the change of DNA methylation status and the regulation mechanism of DNA methylation on the expression of growth related genes in the early growth process of Jinghai yellow chicken, the change of the gene expression level in the whole genome of Jinghai yellow chicken, the change of DNA methylation and the regulation of the expression of DNA methyl on the growth related genes in the early growth process of Jinghai yellow chicken were studied by the combination of transcription group sequencing and whole genome methylation sequencing. The genetic mechanism of growth and development of chicken and the mechanism of high quality meat and chicken leg muscle fiber development provide reference basis. The main results are as follows: 1. using three nonlinear models to fit the growth curve of 0~25 weeks long development data of Beijing yellow chicken, it is found that the growth curve of the Beijing yellow chicken is close to the "S" type, and the Logistic model is calculated. The growth inflection point of the Jinghai yellow chicken was 11.48 weeks old, and the inflection point was 896.68g.2. to the 9 samples of the Beijing yellow chicken leg muscle at different growth stages (3 repetitions at each stage), and the transcriptional group was sequenced. Each sample finally obtained the available data above the 8Gb (gigabases). A total of 15699 genes expressed in the leg muscles were detected, of which there were 14432 genes. In 4,12 and 16 weeks of age, 59724 and 633 genes were detected at 4 weeks and 12 weeks of age (M4WvsM12W), 4 weeks and 16 weeks (M4WvsM16W), 12 weeks and 16 weeks old (M12WvsM16W) in three groups (FC > 2, FDR0.05). In these differentially expressed genes, the transcriptional causes of MYOD1, FBX032, CEBPB and FOX034 were found. Some genes in the long axis of the chicken, such as GH, IGF2BP1, IGF2BP2, IGF2BP3, IGFBP3, IGFBP5 and IGFBP7, were enriched by GO and Pathway enrichment analysis of differentially expressed genes, and found that the differentially expressed genes were significantly enriched in cell proliferation and differentiation, cell migration, cell adhesion and connection, and cell growth. The growth and development related biological processes (P0.05) of skeletal muscle fiber development, muscle organ development and skeletal muscle cell differentiation. The genes involved in the growth and development related genes, including MYOD1, IGFBP3, IGFBP5,1GFBP7 and MYH10, are known. In the significantly enriched signaling pathway (P0.05), 5 signals associated with growth and development have been found. There are 42 genes involved in the 5 pathways, including FGF2, FGF16, FN1, MYH10, MAPK9, etc., which are important in the early growth of the Beijing yellow chicken and the development of the leg muscles. .4. in this study, the whole genome DNA methylation Atlas of the Jinghai yellow chicken leg muscle was constructed by BS-seq technology, and its DNA methylation characteristics were revealed. MCpG was the main way of DNA methylation in the Jinghai yellow chicken genome, and the minimal part of DNA methylation occurred on the CHG and CHH sequences; the CG type methylation site flanking sequences did not exist base preference. The downstream base of the CG methylation cytosine site was most likely to occur with adenine (A), and the level of methylation at the CG site decreased significantly near TSS, and the gene body region increased significantly, while the methylation level of the non CG site increased significantly near TSS and decreased significantly in the gene body region; in the analysis of the methylation level of different functional components, the promoter was started. The methylation level of the subregion and CpG island is the lowest, the methylation level of the CDS and exons is the highest, and the DNA methylation level and the methylation density on the chromosomes have great changes in the methylation group of the Beijing yellow chicken in different stages of.5. (M4W vs M12W, M4W vs M16W and M12W vsM16W), respectively, to detect 21412260 and 1550 differential methylation respectively. Region (DMR), involving 10221055 and 797 differentially methylated genes, respectively, of which 263239 and 229 differentially methylation occurred in the promoter region.6. for the functional enrichment analysis of the differentially methylated genes in the promoter region. It was found that these differentially methylated genes were significantly enriched in the regulation of transforming growth factor beta receptor signaling pathway. Cell migration and cell proliferation regulation, cell differentiation and cell proliferation regulation, skeletal system development, actin cytoskeleton, cell adhesion and other growth related biological processes (P0.05), as well as transforming growth factor beta signal pathway, cell cycle, lysosome, ribosome, cell adhesion molecule 5 growth related signaling pathways (P0.05), the genes involved in the gene including BMP4 and SMAD4 and other growth related genes.7. integrated the methylation data with the transcriptional data. It was found that most of the gene promoter regions were negatively correlated with DNA methylation and expression, and 26 genes were detected for differentially methylation and differential expression in the promoter region, including 20 gene promoters. The change of methylation was negatively correlated with the change of gene expression, including two known growth and development related genes of IGF2BP1 and IGF2BP3. The methylation level of the promoter region of IGF2BP1 and IGF2BP3 was verified by BSP technology and the gene expression level was analyzed. It was found that BSP formula was consistent with the results of BS-seq research, and all showed IGF2BP1. There is a negative correlation between the level of methylation in the promoter region of IGF2BP3 and the level of gene expression, so it is presumed that IGF2BP1 and IGF2BP3 may inhibit the expression of its own by the promoter DNA methylation, and then play a role in regulating the growth and development of Jinghai yellow chicken.

【学位授予单位】：扬州大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S831

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