鸡胚给养维生素C调控肉鸡脾脏发育和免疫功能的表观遗传学机制
发布时间：2018-08-13 10:58
【摘要】：营养管理与遗传改良的不匹配导致现代肉鸡品种的遗传潜力得不到充分发挥,限制了肉鸡生产性能的提高,而营养表观遗传学为挖掘肉鸡品种的遗传潜力提供了思路。胚胎发育早期是表观基因组重编程的关键时期,容易受外界环境的影响,也因此成为表观遗传学研究的焦点时期。由于部分营养物质(如维生素C)无法在蛋中积累,加上不同于哺乳动物的胚胎发育过程,使家禽胚期营养物质干预变得困难,而鸡胚给养技术为以胚胎发育期为切入点的家禽营养表观遗传学研究提供了手段。本研究以维生素C为营养素,以鸡胚给养为手段,在获取鸡胚DNA甲基化模式的基础上,选择不同的处理时间,初步探究了胚期补充维生素C调控肉鸡生长和免疫的机理,同时借助二代测序技术研究了鸡胚给养对肉鸡脾脏发育的影响。试验一鸡胚发育过程中基因组DNA甲基化水平变化规律本试验旨在优化用于基因组DNA甲基化水平检测的高效液相色谱法(HPLC),并探索鸡胚发育过程中DNA甲基化模式。试验首先从DNA提取、酶解体系和检测条件三个方面对HPLC法进行了改进,然后以AA肉鸡胚胎为材料,检测了鸡胚发育过程中基因组DNA甲基化水平变化规律。试验结果显示,鸡胚发育过程中心脏、肝脏和肌肉的DNA甲基化水平均随胚龄逐渐升高,且可划分为三个明显的阶段:胚胎发育第2天(E2)~4,E5~13,E14~19;胚胎发育后期肝脏的DNA甲基化水平显著高于心脏和肌肉(P0.05)。本试验结果显示,优化后的HPLC法可快速、准确地测定各种组织基因组DNA甲基化水平。另外,鸡胚发育过程中基因组DNA甲基化水平阶段性逐渐升高。试验二鸡胚发育过程中特异基因甲基化模式本试验旨在研究鸡胚发育过程中特异基因的DNA甲基化模式。研究采用重亚硫酸盐修饰后测序法测定了TNF-α和IGF2启动子和基因体的DNA甲基化水平,并检测了DNA甲基转移酶(DNMTs)、TNF-α和IGF2在mRNA和蛋白水平的表达量。试验结果表明:(1)肌肉和肝脏组织中,TNF-α启动子区域的甲基化水平呈先升高后降低的变化规律,且TNF-α基因体的甲基化水平以E17显著高于E8、E11和E14(P0.05);(2)IGF2启动子区域甲基化水平随胚龄逐渐降低,而基因体甲基化水平持续升高;(3)肝脏和肌肉中,TNF-α在E17的表达量均高于其它三个胚龄(P0.05),而在E8、E11和E14的表达量无差异(P0.05);(4)肝脏和肌肉中,IGF2的表达量均随胚龄逐渐升高;(5)dnmts均随胚龄增加呈现逐渐升高的规律。本研究结果提示,不同组织基因组dna甲基化水平与其整体的基因表达活性相关,且基因表达可能同时受到启动子区域低甲基化与基因体高甲基化的调节。试验三胚期第11天注射维生素c对肉鸡生长性能及免疫功能的影响本试验旨在探索胚期第11天注射维生素c对肉鸡生长性能、抗氧化性能和免疫功能的影响。240枚aa肉鸡种蛋随机分为2个处理,于胚期第11天分别注射0和3mg维生素c,各处理挑选健康的出壳雏鸡,随机分为6个重复,每重复10只鸡。结果表明:(1)与生理盐水注射组相比,胚期第11天注射3mg维生素c提高了种蛋的孵化率(χ2=14.895,p0.001),肉鸡21-42d和1-42d的adfi(p0.05),1-21d的adg和fcr(p0.05);(2)提高了肉鸡1d血浆维生素c含量(p0.05),且有提高21d肉鸡血浆维生素c含量的趋势(p=0.060);(3)提高了35d肉鸡t淋巴细胞增殖活性(p0.05),1d血浆iga和igm含量(p0.05),21d血浆溶菌酶活性(p0.05)和igm含量(p0.05);(4)提高了42d肉鸡血浆的总抗氧化能力(p0.05);(5)提高了21d肉鸡十二指肠的隐窝深度和绒毛高度/隐窝深度(p0.05),及42d肉鸡回肠的绒毛高度/隐窝深度(p0.05);(6)提高了21d肉鸡脾脏il-4、gcn5、dnmt1和dnmt3a的mrna表达量(p0.05),同时降低了il-1β、tet2、tet3和gadd45β的mrna表达量(p0.05);(7)提高了42d肉鸡脾脏il-4、gcn5、tip60、dnmt3a和gadd45α的mrna表达量(p0.05),降低了ifn-γ、tet3、mbd4和tdg的mrna表达量(p0.05),同时有降低svct1和svct2mrna表达量的趋势(p=0.065,p=0.095)。本研究结果提示,胚期第11天注射3mg维生素c提高了种蛋的孵化率,在一定程度上改善了肉鸡的生产性能,抗氧化能力,免疫功能和肠道形态,且免疫功能的提高可能与dna甲基化和组蛋白乙酰化水平的提高有关。试验四胚期第15天注射维生素c对肉鸡生长性能及免疫功能的影响本试验旨在探索胚期第15天注射维生素c对肉鸡生长性能、抗氧化性能和免疫功能的影响。240枚aa肉鸡种蛋随机分为2个处理,于胚期第15天分别注射0和3mg维生素c,各处理挑选健康的出壳雏鸡,随机分为6个重复,每重复10只鸡。结果表明:(1)与生理盐水注射组相比,胚期第15天注射3mg维生素c提高了种蛋的孵化率(χ2=4.219,p=0.040);(2)提高了1和42d肉鸡的胸腺指数(p0.05);(3)提高1d肉鸡血浆igm含量(p0.05),21d血浆溶菌酶活性(p0.05),及igg和igm含量(p0.05);(4)提高了42d肉鸡血浆的总抗氧化能力(p0.05);(5)提高了21d肉鸡十二指肠的隐窝深度(p0.05),降低了回肠的绒毛高度/隐窝深度(p0.05),提高了42d肉鸡十二指肠的绒毛高度和绒毛高度/隐窝深度(p0.05);(6)提高了21d肉鸡脾脏dnmt1的mrna表达量(p0.05),同时降低了il-6、ifn-γ、tet1和tdg的mrna表达量(p0.05);(7)提高了42d肉鸡脾脏gcn5、dnmt1、dnmt3b和gadd45β的mrna表达量(p0.05),同时降低了il-6、tnf-α和mbd4的mrna表达量(p0.05)。本研究结果提示,胚期第15天注射3mg维生素c提高了种蛋的孵化率,在一定程度上改善了肉鸡的抗氧化能力,免疫功能和肠道形态,且免疫性能的提高可能与dna甲基化和组蛋白乙酰水平的提高有关。试验五胚期注射维生素c调控肉鸡脾脏发育的转录组分析本试验旨在借助转录组测序(rna-seq)揭示胚期注射维生素c调控鸡胚脾脏发育的差异表达基因及相关调控通路。120枚aa肉鸡种蛋随机分为2个处理,于胚期第11天分别注射0和3mg维生素c。胚期第19天,每处理选取发育良好的种蛋15枚,完整取脾脏,混合后提取rna进行转录组测序。测序结果显示,处理组与对照组共得到12gb有效且高质量的数据,与参考基因组的比对效率分别达到90.76%和91.41%。差异表达分析共发现184个差异表达基因,其中117个表达上调,67个表达下调。go和kegg等生物信息学分析表明,差异表达基因涉及免疫调节、胚胎发育、组蛋白修饰和繁殖等生物过程。本研究结果提示,以鸡胚给养为手段施加胚期维生素c干预可影响胚胎发育、免疫调节和表观遗传学修饰等生物过程。试验六胚期注射维生素c调控肉鸡脾脏发育的全基因组甲基化分析本试验旨在借助全基因组甲基化测序(wgbs)揭示胚期注射维生素c调控鸡胚脾脏发育的差异甲基化基因及相关调控通路。120枚aa肉鸡种蛋随机分为2个处理,于胚期第11天分别注射0和3mg维生素c。胚期第19天,每处理选取发育良好的种蛋15枚,完整取脾脏,混合后提取dna进行全基因组甲基化测序。测序结果显示,对照组与处理组共得到98gb有效且高质量的数据,平均测序深度32x。甲基化分析发现,胚期注射维生素c改变了脾脏组织的甲基化模式,且其去甲基化作用主要体现在chg和chh位点及基因的蛋白质编码区。生物信息学分析共得到5514个差异甲基化基因,涉及免疫调节、胚胎发育、表观遗传学修饰、繁殖和氧化还原等生物过程。本研究结果提示,胚期补充维生素c改变了参与胚胎发育、免疫调节和表观遗传学修饰等生物过程相关基因的甲基化状态。另外,研究揭示了鸡胚脾脏全基因组甲基化图谱,为后续相关研究奠定了基础。试验七胚期注射维生素c调控肉鸡脾脏发育的rna-seq与wgbs组合分析本试验旨在将rna-seq数据与wgbs数据进行组合分析,找出共有差异基因,并进行相关功能分析。组合分析共发现59个差异甲基化且差异表达基因,其中处理组相比对照组的去甲基化率为98.3%,涉及胚胎发育、免疫功能和表观遗传学修饰,表明胚期注射维生素c通过广泛的去甲基化作用参与调控胚胎发育、免疫功能和表观遗传学修饰等生物过程。本研究结果揭示了胚期维生素c干预下dna甲基化与基因表达的关系,为维生素C参与调控的DNA甲基化模式研究提供了参考。另外,研究发现维生素C在动物(肉鸡)体内同样可发挥广泛的去甲基化作用,并调控基因表达,且基因体的去甲基化既可调控基因表达上调,也可调控基因表达下调。本研究表明,鸡胚给养维生素C可通过广泛的去甲基化作用调控脾脏发育,且可提高饲养阶段肉鸡的生产性能和免疫功能。
[Abstract]:The mismatch between nutritional management and genetic improvement results in the inadequate utilization of genetic potential of modern broiler breeds, which limits the improvement of broiler performance. Nutritional epigenetics provides a way to tap the genetic potential of broiler breeds. Because some nutrients (such as vitamin C) can not be accumulated in eggs and the embryonic development process is different from that of mammals, it is difficult to interfere with nutrients in the embryonic stage of poultry. Vitamin C was used as nutrient and chicken embryo as feeding medium. On the basis of acquiring DNA methylation pattern of chicken embryo and choosing different treatment time, the mechanism of vitamin C supplementation in embryo regulating growth and immunity of broiler chickens was preliminarily explored. At the same time, the effect of chicken embryo feeding on spleen hair of broiler chickens was studied by second-generation sequencing technique. The purpose of this experiment is to optimize the high performance liquid chromatography (HPLC) for the detection of genomic DNA methylation level and to explore the DNA methylation pattern during chicken embryo development. The results showed that the DNA methylation levels in heart, liver and muscle of AA broiler embryos increased gradually with the embryonic age and could be divided into three distinct stages: the second day (E2) ~ 4, E5 ~ 13, E14 ~ 19 of embryonic development. The results showed that the optimized HPLC method could rapidly and accurately determine the DNA methylation level of various tissues. In addition, the DNA methylation level of chicken embryos increased gradually during the development of chicken embryos. The aim of this study was to investigate the DNA methylation patterns of specific genes during chicken embryo development. The DNA methylation levels of TNF-a and IGF2 promoters and gene bodies were determined by bisulfite Modified Sequencing method, and the expression levels of DNA methyltransferase (DNMTs), TNF-a and IGF2 at mRNA and protein levels were detected. The results showed that: (1) the methylation level of TNF-a promoter region increased first and then decreased in muscle and liver tissues, and the methylation level of TNF-a gene was significantly higher in E17 than in E8, E1 1 and E14 (P 0.05); (2) the methylation level of IGF2 promoter region decreased gradually with embryonic age, while the methylation level of gene body continued to increase; (3) liver In the viscera and muscle, the expression of TNF-alpha in E17 was higher than that in the other three embryonic ages (P 0.05), but there was no difference in the expression of E8, E11 and E1 4 (P 0.05); (4) The expression of IGF2 in liver and muscle increased gradually with the embryonic age; (5) The expression of DNMTs increased gradually with the embryonic age. The effect of vitamin C injection on growth performance and immune function of broilers at the 11th day of triembryonic stage was studied in order to explore the effect of vitamin C injection on growth performance and anti-hypermethylation of broilers at the 11th day of embryonic stage. 240 AA broiler eggs were randomly divided into two treatments. On the 11th day of embryo, 0 and 3 mg vitamin C were injected. Healthy chickens were randomly divided into 6 replicates, 10 chickens per replicate. Eggs hatching rate (_2 = 14.895, p0.001), broiler 21-42 days and 1-42 days ADFI (p0.05), 1-21 days ADG and FCR (p0.05); (2) increased the 1-day plasma vitamin C content (p0.05), and increased the 21-day plasma vitamin C content of broiler (p = 0.060); (3) increased the 35-day broiler T lymphocyte proliferation activity (p0.05), 1-day plasma IgA and IgM content (p0.05), 21-day blood plasma vitamin C content (p0.05), 21-day blood vitamin C content (p0.05). Plasma lysozyme activity (p0.05) and IgM content (p0.05); (4) increased plasma total antioxidant capacity (p0.05); (5) increased the duodenal recess depth and villus height / recess depth (p0.05), and villus height / recess depth of ileum (p0.05); (6) increased spleen il-4, gcn5, DNMT1 and Dnmt3a mRNA in 21 d broilers (p0.05). The expression of IL-1 beta, tet-2, tet-3 and gadd-45 beta was decreased (p 0.05); (7) the expression of il-4, gcn-5, tip-60, dnmt-3a and gadd-45a in spleen was increased (p 0.05), and the expression of ifn-gamma, tet-3, mbd-4 and TDG was decreased (p 0.05), and the expression of svct 1 and SVCT2 mRNA was decreased (p = 0.065, P = 0.095). The results suggested that 3 mg vitamin C Injection on the 11th day of embryo increased hatchability of broiler eggs, improved production performance, antioxidant capacity, immune function and intestinal morphology to a certain extent, and the improvement of immune function might be related to the increase of DNA methylation and histone acetylation level. Effects of vitamin C Injection on growth performance, antioxidant capacity and immune function of broilers were studied. 240 AA broiler eggs were randomly divided into two treatments. On the 15th day of embryo, 0 and 3 mg vitamin C were injected into broilers. Healthy shelled chickens were selected and randomly divided into 6 treatments. The results showed that: (1) compared with saline injection group, 3 mg vitamin C Injection on the 15th day of embryonic stage increased hatching rate of eggs (_2 = 4.219, P = 0.040); (2) increased thymus index (p0.05); (3) increased plasma IgM content (p0.05), plasma lysozyme activity (p0.05) and IgG and IgM content (p0.05); (2) increased thymus index (p0.05) increased 1 and 42 days of Broilers (p0.05). 4) increased the total antioxidant capacity of plasma (p0.05); (5) increased the depth of duodenal recess (p0.05), decreased the villus height / recess depth of ileum (p0.05), increased the villus height and villus height / recess depth of duodenum (p0.05); (6) increased the expression of DNMT1 mRNA in spleen (p0.05) of 21-day broilers. At the same time, the expression of il-6, ifn-gamma, tet-1 and TDG mRNA was decreased (p0.05); (7) the expression of gcn-5, dnmt-1, dnmt-3b and gadd-45 beta in spleen of 42-day broilers was increased (p0.05), and the expression of il-6, TNF-a and mbd-4 mRNA was decreased (p0.05). The results showed that the antioxidant capacity, immune function and intestinal morphology of broilers were improved, and the improvement of immune function might be related to the increase of DNA methylation and histone acetyl level. 120 AA broiler eggs were randomly divided into two treatments. On the 11th day of embryonic stage, 0 and 3 mg vitamin C were injected into the eggs. On the 19th day of embryonic stage, 15 well-developed eggs were selected for each treatment. The whole spleen was harvested and the RNA was extracted after mixing and sequenced. A total of 184 differentially expressed genes were found, of which 117 were up-regulated and 67 were down-regulated. Bioinformatics analysis such as go and KEGG showed that the differentially expressed genes involved in immune regulation, embryonic development, and egg formation. The results of this study suggest that embryonic vitamin C intervention may affect embryonic development, immune regulation and epigenetic modification by feeding chicken embryos. The whole genome methylation analysis of spleen development regulated by vitamin C injection at six embryonic stages was carried out. Group methylation sequencing (wgbs) revealed differential methylation genes and related regulatory pathways in spleen development of embryonic chicken embryos regulated by vitamin C injection. 120 AA broiler eggs were randomly divided into two treatments, 0 and 3 mg vitamin C were injected on the 11th day of embryonic development and 15 eggs were selected from each treatment on the 19th day of embryonic development. DNA was taken for whole genome methylation and sequencing. The results showed that 98 GB effective and high quality data were obtained in the control group and the treatment group. The average sequencing depth was 32 X. Methylation analysis showed that vitamin C injection during embryonic period changed the methylation pattern of spleen tissue, and its demethylation effect was mainly reflected in the CHG and CHH sites and gene protein. 5 514 differentially methylated genes were identified by bioinformatics analysis, involving biological processes such as immune regulation, embryonic development, epigenetic modification, reproduction and redox. In addition, the whole genome methylation map of chicken embryo spleen was revealed, which laid the foundation for the follow-up study. In the experiment, the combination analysis of RNA-seq and wgbs by vitamin C injection during the seven embryonic stages was carried out. The purpose of this experiment was to combine the RNA-seq data with wgbs data, find out the common differentially expressed genes and carry out the analysis. A total of 59 differentially methylated and differentially expressed genes were identified by combinatorial analysis. The demethylation rate of the treated group was 98.3% compared with the control group, which involved embryonic development, immune function and epigenetic modification. The results showed that vitamin C injection during embryonic stage participated in embryonic development, immune function and table regulation through extensive demethylation. This study revealed the relationship between DNA methylation and gene expression under the intervention of vitamin C during embryonic stage, and provided a reference for the study of DNA methylation patterns regulated by vitamin C. In addition, it was found that vitamin C could also play an extensive demethylation role in animals (broilers) and regulate genes. The results showed that vitamin C from chicken embryo could regulate the spleen development through extensive demethylation and improve the production performance and immune function of broilers in feeding stage.
【学位授予单位】：西北农林科技大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S831
本文编号：2180769
[Abstract]:The mismatch between nutritional management and genetic improvement results in the inadequate utilization of genetic potential of modern broiler breeds, which limits the improvement of broiler performance. Nutritional epigenetics provides a way to tap the genetic potential of broiler breeds. Because some nutrients (such as vitamin C) can not be accumulated in eggs and the embryonic development process is different from that of mammals, it is difficult to interfere with nutrients in the embryonic stage of poultry. Vitamin C was used as nutrient and chicken embryo as feeding medium. On the basis of acquiring DNA methylation pattern of chicken embryo and choosing different treatment time, the mechanism of vitamin C supplementation in embryo regulating growth and immunity of broiler chickens was preliminarily explored. At the same time, the effect of chicken embryo feeding on spleen hair of broiler chickens was studied by second-generation sequencing technique. The purpose of this experiment is to optimize the high performance liquid chromatography (HPLC) for the detection of genomic DNA methylation level and to explore the DNA methylation pattern during chicken embryo development. The results showed that the DNA methylation levels in heart, liver and muscle of AA broiler embryos increased gradually with the embryonic age and could be divided into three distinct stages: the second day (E2) ~ 4, E5 ~ 13, E14 ~ 19 of embryonic development. The results showed that the optimized HPLC method could rapidly and accurately determine the DNA methylation level of various tissues. In addition, the DNA methylation level of chicken embryos increased gradually during the development of chicken embryos. The aim of this study was to investigate the DNA methylation patterns of specific genes during chicken embryo development. The DNA methylation levels of TNF-a and IGF2 promoters and gene bodies were determined by bisulfite Modified Sequencing method, and the expression levels of DNA methyltransferase (DNMTs), TNF-a and IGF2 at mRNA and protein levels were detected. The results showed that: (1) the methylation level of TNF-a promoter region increased first and then decreased in muscle and liver tissues, and the methylation level of TNF-a gene was significantly higher in E17 than in E8, E1 1 and E14 (P 0.05); (2) the methylation level of IGF2 promoter region decreased gradually with embryonic age, while the methylation level of gene body continued to increase; (3) liver In the viscera and muscle, the expression of TNF-alpha in E17 was higher than that in the other three embryonic ages (P 0.05), but there was no difference in the expression of E8, E11 and E1 4 (P 0.05); (4) The expression of IGF2 in liver and muscle increased gradually with the embryonic age; (5) The expression of DNMTs increased gradually with the embryonic age. The effect of vitamin C injection on growth performance and immune function of broilers at the 11th day of triembryonic stage was studied in order to explore the effect of vitamin C injection on growth performance and anti-hypermethylation of broilers at the 11th day of embryonic stage. 240 AA broiler eggs were randomly divided into two treatments. On the 11th day of embryo, 0 and 3 mg vitamin C were injected. Healthy chickens were randomly divided into 6 replicates, 10 chickens per replicate. Eggs hatching rate (_2 = 14.895, p0.001), broiler 21-42 days and 1-42 days ADFI (p0.05), 1-21 days ADG and FCR (p0.05); (2) increased the 1-day plasma vitamin C content (p0.05), and increased the 21-day plasma vitamin C content of broiler (p = 0.060); (3) increased the 35-day broiler T lymphocyte proliferation activity (p0.05), 1-day plasma IgA and IgM content (p0.05), 21-day blood plasma vitamin C content (p0.05), 21-day blood vitamin C content (p0.05). Plasma lysozyme activity (p0.05) and IgM content (p0.05); (4) increased plasma total antioxidant capacity (p0.05); (5) increased the duodenal recess depth and villus height / recess depth (p0.05), and villus height / recess depth of ileum (p0.05); (6) increased spleen il-4, gcn5, DNMT1 and Dnmt3a mRNA in 21 d broilers (p0.05). The expression of IL-1 beta, tet-2, tet-3 and gadd-45 beta was decreased (p 0.05); (7) the expression of il-4, gcn-5, tip-60, dnmt-3a and gadd-45a in spleen was increased (p 0.05), and the expression of ifn-gamma, tet-3, mbd-4 and TDG was decreased (p 0.05), and the expression of svct 1 and SVCT2 mRNA was decreased (p = 0.065, P = 0.095). The results suggested that 3 mg vitamin C Injection on the 11th day of embryo increased hatchability of broiler eggs, improved production performance, antioxidant capacity, immune function and intestinal morphology to a certain extent, and the improvement of immune function might be related to the increase of DNA methylation and histone acetylation level. Effects of vitamin C Injection on growth performance, antioxidant capacity and immune function of broilers were studied. 240 AA broiler eggs were randomly divided into two treatments. On the 15th day of embryo, 0 and 3 mg vitamin C were injected into broilers. Healthy shelled chickens were selected and randomly divided into 6 treatments. The results showed that: (1) compared with saline injection group, 3 mg vitamin C Injection on the 15th day of embryonic stage increased hatching rate of eggs (_2 = 4.219, P = 0.040); (2) increased thymus index (p0.05); (3) increased plasma IgM content (p0.05), plasma lysozyme activity (p0.05) and IgG and IgM content (p0.05); (2) increased thymus index (p0.05) increased 1 and 42 days of Broilers (p0.05). 4) increased the total antioxidant capacity of plasma (p0.05); (5) increased the depth of duodenal recess (p0.05), decreased the villus height / recess depth of ileum (p0.05), increased the villus height and villus height / recess depth of duodenum (p0.05); (6) increased the expression of DNMT1 mRNA in spleen (p0.05) of 21-day broilers. At the same time, the expression of il-6, ifn-gamma, tet-1 and TDG mRNA was decreased (p0.05); (7) the expression of gcn-5, dnmt-1, dnmt-3b and gadd-45 beta in spleen of 42-day broilers was increased (p0.05), and the expression of il-6, TNF-a and mbd-4 mRNA was decreased (p0.05). The results showed that the antioxidant capacity, immune function and intestinal morphology of broilers were improved, and the improvement of immune function might be related to the increase of DNA methylation and histone acetyl level. 120 AA broiler eggs were randomly divided into two treatments. On the 11th day of embryonic stage, 0 and 3 mg vitamin C were injected into the eggs. On the 19th day of embryonic stage, 15 well-developed eggs were selected for each treatment. The whole spleen was harvested and the RNA was extracted after mixing and sequenced. A total of 184 differentially expressed genes were found, of which 117 were up-regulated and 67 were down-regulated. Bioinformatics analysis such as go and KEGG showed that the differentially expressed genes involved in immune regulation, embryonic development, and egg formation. The results of this study suggest that embryonic vitamin C intervention may affect embryonic development, immune regulation and epigenetic modification by feeding chicken embryos. The whole genome methylation analysis of spleen development regulated by vitamin C injection at six embryonic stages was carried out. Group methylation sequencing (wgbs) revealed differential methylation genes and related regulatory pathways in spleen development of embryonic chicken embryos regulated by vitamin C injection. 120 AA broiler eggs were randomly divided into two treatments, 0 and 3 mg vitamin C were injected on the 11th day of embryonic development and 15 eggs were selected from each treatment on the 19th day of embryonic development. DNA was taken for whole genome methylation and sequencing. The results showed that 98 GB effective and high quality data were obtained in the control group and the treatment group. The average sequencing depth was 32 X. Methylation analysis showed that vitamin C injection during embryonic period changed the methylation pattern of spleen tissue, and its demethylation effect was mainly reflected in the CHG and CHH sites and gene protein. 5 514 differentially methylated genes were identified by bioinformatics analysis, involving biological processes such as immune regulation, embryonic development, epigenetic modification, reproduction and redox. In addition, the whole genome methylation map of chicken embryo spleen was revealed, which laid the foundation for the follow-up study. In the experiment, the combination analysis of RNA-seq and wgbs by vitamin C injection during the seven embryonic stages was carried out. The purpose of this experiment was to combine the RNA-seq data with wgbs data, find out the common differentially expressed genes and carry out the analysis. A total of 59 differentially methylated and differentially expressed genes were identified by combinatorial analysis. The demethylation rate of the treated group was 98.3% compared with the control group, which involved embryonic development, immune function and epigenetic modification. The results showed that vitamin C injection during embryonic stage participated in embryonic development, immune function and table regulation through extensive demethylation. This study revealed the relationship between DNA methylation and gene expression under the intervention of vitamin C during embryonic stage, and provided a reference for the study of DNA methylation patterns regulated by vitamin C. In addition, it was found that vitamin C could also play an extensive demethylation role in animals (broilers) and regulate genes. The results showed that vitamin C from chicken embryo could regulate the spleen development through extensive demethylation and improve the production performance and immune function of broilers in feeding stage.
【学位授予单位】：西北农林科技大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S831
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