本文選題：京海黃雞 + 早期生長(zhǎng)��； 參考：《揚(yáng)州大學(xué)》2017年博士論文

【摘要】：肉雞早期生長(zhǎng)發(fā)育規(guī)律尤其是生長(zhǎng)拐點(diǎn)影響著肉雞最佳上市時(shí)間和屠宰體重,進(jìn)而影響著肉雞產(chǎn)業(yè)的經(jīng)濟(jì)效益,而目前關(guān)于肉雞早期生長(zhǎng)發(fā)育規(guī)律遺傳機(jī)理的研究卻很少。DNA甲基化作為最常見(jiàn)的表觀遺傳修飾方式之一,在轉(zhuǎn)錄及轉(zhuǎn)錄后水平調(diào)控基因的表達(dá),進(jìn)而影響動(dòng)物的生長(zhǎng)發(fā)育,目前關(guān)于優(yōu)質(zhì)肉雞早期生長(zhǎng)過(guò)程中表觀遺傳調(diào)控機(jī)理的研究報(bào)道很少。因此本研究選取京海黃雞生長(zhǎng)拐點(diǎn)前(緩慢生長(zhǎng)階段)、生長(zhǎng)拐點(diǎn)及拐點(diǎn)后(上市階段),即對(duì)應(yīng)4、12和16周齡三個(gè)特殊時(shí)間點(diǎn)的腿肌組織,采用轉(zhuǎn)錄組測(cè)序RNA-seq和全基因組甲基化測(cè)序BS-seq技術(shù)相結(jié)合,探索京海黃雞早期生長(zhǎng)過(guò)程中全基因組范圍內(nèi)基因表達(dá)水平的變化、DNA甲基化狀態(tài)的變化以及DNA甲基對(duì)生長(zhǎng)相關(guān)基因表達(dá)的調(diào)控機(jī)制,為揭示京海黃雞生長(zhǎng)發(fā)育規(guī)律的遺傳機(jī)理以及優(yōu)質(zhì)肉雞腿部肌纖維發(fā)育機(jī)制提供參考依據(jù)。主要研究結(jié)果如下:1.利用三種非線性模型對(duì)京海黃雞0～25周齡生長(zhǎng)發(fā)育數(shù)據(jù)進(jìn)行生長(zhǎng)曲線擬合,發(fā)現(xiàn)京海黃雞生長(zhǎng)曲線接近“S”型,且Logistic模型下計(jì)算的京海黃雞生長(zhǎng)拐點(diǎn)為11.48周齡,拐點(diǎn)體重為896.68g。2.對(duì)不同生長(zhǎng)階段京海黃雞腿肌樣本共9個(gè)(各階段3個(gè)重復(fù)),進(jìn)行轉(zhuǎn)錄組測(cè)序,每個(gè)樣本最終獲得了 8Gb(gigabases)以上的可用數(shù)據(jù),共檢測(cè)出15699個(gè)在腿肌中表達(dá)的基因,其中有14432個(gè)基因在4、12和16周齡中共同表達(dá),而分別檢測(cè)出59、724和633個(gè)基因在4周齡與12周齡(M4WvsM12W)、4周齡與16周齡(M4WvsM16W)以及12周齡與16周齡(M12WvsM16W)三組比較中差異表達(dá)(FC≥2,FDR0.05),在這些差異表達(dá)的基因中,發(fā)現(xiàn)了 MYOD1、FBX032、CEBPB和FOX034個(gè)轉(zhuǎn)錄因子,以及雞生長(zhǎng)軸中的一些基因,如 GH、IGF2BP1、IGF2BP2、IGF2BP3、IGFBP3、IGFBP5 和 IGFBP7等生長(zhǎng)相關(guān)的候選基因。3.對(duì)差異表達(dá)的基因進(jìn)行GO和Pathway功能富集分析,發(fā)現(xiàn)差異表達(dá)基因顯著富集于細(xì)胞增殖和分化、細(xì)胞遷移、細(xì)胞粘著和連接、細(xì)胞生長(zhǎng)、骨骼肌肌纖維發(fā)育、肌肉器官發(fā)育和骨骼肌細(xì)胞分化等生長(zhǎng)發(fā)育相關(guān)的生物過(guò)程(P0.05),涉及到的基因包括MYOD1,IGFBP3,IGFBP5、1GFBP7和MYH10等已知的與生長(zhǎng)發(fā)育相關(guān)的基因。在顯著富集的信號(hào)通路中(P0.05),發(fā)現(xiàn)了 5個(gè)與生長(zhǎng)發(fā)育相關(guān)的信號(hào)通路,分別為粘著斑、細(xì)胞外基質(zhì)受體互作、胰島素信號(hào)通路、緊密連接和肌動(dòng)蛋白細(xì)胞骨架調(diào)節(jié),共有42個(gè)基因包含在這5個(gè)通路中,包括FGF2、FGF16、FN1、MYH10、MAPK9等,推測(cè)這些通路及基因在京海黃雞早期的生長(zhǎng)和腿肌發(fā)育過(guò)程中發(fā)揮著重要的調(diào)控作用。4.本研究通過(guò)BS-seq技術(shù)構(gòu)建了京海黃雞腿肌全基因組DNA甲基化圖譜,并揭示了其DNA甲基化特征:mCpG為京海黃雞基因組DNA甲基化主要方式,極少部分DNA甲基化發(fā)生在CHG和CHH序列上;CG類型甲基化位點(diǎn)側(cè)翼序列不存在堿基偏好性,而非CG類型甲基化胞嘧啶位點(diǎn)的下游堿基以腺嘌呤(A)出現(xiàn)最多;CG位點(diǎn)甲基化水平在TSS附近顯著下降、基因本體區(qū)顯著升高,而非CG位點(diǎn)甲基化水平在TSS附近顯著升高、基因本體區(qū)內(nèi)顯著下降;不同功能元件的甲基化水平分析中,啟動(dòng)子區(qū)和CpG島的甲基化水平最低,CDS區(qū)和外顯子的甲基化水平最高;各條染色體上的DNA甲基化水平及甲基化密度存在較大變化。5.不同生長(zhǎng)階段京海黃雞甲基化組比較(M4W vs M12W、M4W vs M16W和M12W vsM16W),分別檢測(cè)出2141、2260和1550個(gè)差異甲基化區(qū)域(DMR),分別涉及1022、1055和797個(gè)差異甲基化基因,其中分別有263、239和229個(gè)基因的差異甲基化發(fā)生在了啟動(dòng)子區(qū)。6.對(duì)啟動(dòng)子區(qū)差異甲基化基因進(jìn)行功能富集分析,發(fā)現(xiàn)這些差異甲基化基因顯著富集于轉(zhuǎn)化生長(zhǎng)因子β受體信號(hào)通路調(diào)控、細(xì)胞遷移和細(xì)胞增殖正調(diào)控、細(xì)胞分化和細(xì)胞增殖調(diào)控、骨骼系統(tǒng)發(fā)育、肌動(dòng)蛋白細(xì)胞骨架組織、細(xì)胞黏附等生長(zhǎng)相關(guān)的生物過(guò)程(P0.05),以及轉(zhuǎn)化生長(zhǎng)因子β信號(hào)通路、細(xì)胞周期、溶酶體、核糖體、細(xì)胞黏附分子5個(gè)生長(zhǎng)發(fā)育相關(guān)的信號(hào)通路(P0.05),涉及到的基因包括BMP4和SMAD4等生長(zhǎng)發(fā)育相關(guān)基因。7.將甲基化數(shù)據(jù)與轉(zhuǎn)錄組數(shù)據(jù)進(jìn)行整合分析,發(fā)現(xiàn)大部分基因啟動(dòng)子區(qū)DNA甲基化與表達(dá)呈負(fù)相關(guān),并且檢測(cè)出26個(gè)啟動(dòng)子區(qū)差異甲基化且差異表達(dá)的基因,其中有20個(gè)基因啟動(dòng)子區(qū)甲基化變化與基因表達(dá)變化呈負(fù)相關(guān)關(guān)系,包括IGF2BP1和IGF2BP3兩個(gè)已知的生長(zhǎng)發(fā)育相關(guān)基因。通過(guò)BSP技術(shù)對(duì)IGF2BP1和IGF2BP3啟動(dòng)子區(qū)甲基化水平進(jìn)行驗(yàn)證,并結(jié)合基因表達(dá)水平進(jìn)行分析,發(fā)現(xiàn)BSP方法與BS-seq研究結(jié)果相一致,均顯示IGF2BP1和IGF2BP3啟動(dòng)子區(qū)甲基化水平與基因表達(dá)水平呈負(fù)相關(guān),因此推測(cè)IGF2BP1和IGF2BP3可能通過(guò)啟動(dòng)子DNA甲基化抑制其自身的表達(dá),進(jìn)而發(fā)揮對(duì)京海黃雞生長(zhǎng)發(fā)育的調(diào)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.

【學(xué)位授予單位】：揚(yáng)州大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：S831

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2 蘇改秀;兒童系統(tǒng)性紅斑狼瘡基因組甲基化水平及LINE-1序列甲基化水平的研究[D];北京協(xié)和醫(yī)學(xué)院;2016年

3 李旭;基于組學(xué)數(shù)據(jù)的模擬空間環(huán)境健康監(jiān)測(cè)及其應(yīng)用研究[D];哈爾濱工業(yè)大學(xué);2016年

4 張媛媛;全基因組DNA甲基化模式及其在復(fù)雜疾病分析中的應(yīng)用研究[D];西安電子科技大學(xué);2016年

5 陸曉筱;ACTL6A/c-Myc交互介導(dǎo)的ACTL6B甲基化促進(jìn)肝細(xì)胞肝癌EMT的機(jī)制[D];浙江大學(xué);2017年

6 薛倩;京海黃雞腿肌全基因組甲基化和轉(zhuǎn)錄組分析研究[D];揚(yáng)州大學(xué);2017年

7 胡元晶;應(yīng)用焦磷酸測(cè)序技術(shù)HPV分型檢測(cè)及甲基化研究[D];天津醫(yī)科大學(xué);2010年

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9 周慶兵;含砷中藥對(duì)骨髓增生異常綜合征異�；蚣谆{(diào)控作用研究[D];中國(guó)中醫(yī)科學(xué)院;2014年

10 王俊杰;卵巢癌中EZH2和DNA甲基化調(diào)控hMLH1基因表達(dá)的實(shí)驗(yàn)研究[D];華中科技大學(xué);2013年

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5 郭蓉;甲基化作用調(diào)節(jié)miR-200b表達(dá)及對(duì)胃腺癌細(xì)胞增殖、侵襲能力的影響[D];天津醫(yī)科大學(xué);2015年

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10 艾雪;不同細(xì)胞系和組織基因的甲基化差異分析及其序列與CpG甲基化程度的關(guān)聯(lián)[D];內(nèi)蒙古大學(xué);2016年

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