京海黄鸡脂肪沉积关键基因的筛选

发布时间：2018-04-07 19:01

本文选题：京海黄鸡　切入点：lncRNA　出处：《扬州大学》2017年博士论文

【摘要】：肌肉中肌内脂肪沉积与多种肉质性状相关,如嫩度、多汁性、风味和系水力。为了迎合消费者对高肉质不断增加的需求,提高肌内脂肪含量已经成为全世界育种工作者的目标之一。然而,在过去几十年中,由于人们对肉鸡生长速度和饲料利用率遗传选择的过度追求,导致目前鸡肉质量和风味降低。与此同时,对肉鸡生长速度的过度选择伴随着腹部脂肪的过度积累,因此,增加肌内脂肪同时减少腹部脂肪沉积已经成为目前肉鸡育种工作的目标之一。本研究一方面旨在利用RNA-seq技术描述鸡腹部和肌内脂肪前体细胞在成脂分化过程中的转录组表达情况,鉴定两种细胞中的lncRNAs,并分析lncRNAs的特点。一方面筛选鸡腹部和肌内脂肪前体细胞成脂分化过程中的差异表达基因,结合生物信息学方法,鉴定出可能影响鸡腹部和肌内脂肪沉积的重要因子和信号通路。最后,通过比较鸡腹部和肌内脂肪前体细胞成脂分化过程中基因的表达水平,研究鸡腹部和肌内脂肪前体细胞分化过程中转录组水平上的差异。主要研究结果如下:1.鸡腹部脂肪沉积关键基因的筛选由鸡腹部脂肪组织分离培养获得鸡原代腹部脂肪前体细胞,诱导其成脂分化,油红O染色显示,成功分离获得鸡腹部脂肪前体细胞。在鸡腹部脂肪前体细胞成脂分化过程中共鉴定出27,023条lncRNAs,分析显示,鸡腹部脂肪前体细胞中的lncRNAs具有序列和开放阅读框短及外显子少的特点;功能预测发现,鸡腹部脂肪前体细胞中的lncRNAs主要参与蛋白修饰、细胞蛋白修饰和信号调控等生物学过程。在鸡腹部脂肪前体细胞成脂分化过程中共筛选到4,232条差异表达lncRNAs和1,656条差异表达mRNAs;功能注释显示,差异表达基因主要富集在细胞周期、间叶细胞分化和细胞周期过程等生物学过程;通路分析结果显示,差异表达基因显著富集在细胞周期、DNA复制和PPAR等信号通路中。共表达分析共鉴定出了 6个阶段特异性模块,并利用可视化在6个模块中筛选出了 29个高连通性的基因,这些基因可能为调控鸡腹部脂肪前体细胞成脂分化的重要候选基因,包括XLOC_052948、gga-mir-30c、SCD和KLF15等基因。2.鸡肌内脂肪沉积关键基因的筛选由鸡胸部肌肉组织分离培养获得鸡原代肌内脂肪前体细胞,诱导其成脂分化,油红O染色显示,成功分离获得鸡肌内脂肪前体细胞。在鸡肌内脂肪前体细胞中共鉴定出26,172条lncRNAs,分析显示,鸡肌内脂肪前体细胞中的lncRNAs具有序列和开放阅读框短及外显子少的特点;功能预测发现,鸡肌内脂肪前体细胞中的lncRNAs主要参与基因表达、细胞学大分子合成、RNA代谢等生物学过程。在鸡肌内脂肪前体细胞成脂分化过程中共筛选到4,694条差异表达lncRNAs和2,169条差异表达mRNAs;功能注释显示,差异表达基因主要富集在信号调控、细胞通信调控及信号转导调控等生物学过程,通路分析结果显示,差异表达基因显著富集在焦点黏连、MAPK及PPAR等信号通路中。共表达分析共鉴定出了 6个阶段特异性模块,可视化分析在6个阶段特异性模块中筛选出了 28个高连通性的基因,这些基因可能为调控鸡肌内脂肪前体细胞成脂分化的重要候选基因,包括XLOC_013577、gga-mir-146b、BMP3和MYOD1等基因。3.鸡腹部和肌内脂肪沉积机制比较分析腹部和肌内脂肪前体细胞间lncRNAs结构比较分析显示,两种细胞lncRNAs具有相同的特点,即外显子数少、开放阅读框短和序列短。表达量比较显示,两种细胞lncRNAs的表达量均远远低于mRNAs。转录组比较分析显示,两种细胞间差异基因随着分化的延长而减少,在分化第0、2、4和6天,分别筛选到2,942(1,877个mRNAs和1,065个lncRNAs)、3,788(1,964 个 mRNAs 和 1,824 个 lncRNAs)、2,872(1,194 个 mRNAs 和 1,678 个 lncRNAs)和2,214(1,206个mRNAs和1,008个lncRNAs)个差异表达基因。异表达基因GO注释显示,在分化第0天,腹部和肌内脂肪前体细胞之间差异表达基因主要显著富集在生长因子应答、生长因子刺激细胞应答及细胞运动性调控等生物学过程;在分化第2天,差异表达基因主要显著富集在肌肉结构发育、肌肉器官发育和生长因子细胞学应答等生物学过程中;在分化第4天,差异基因主要显著富集在间充质细胞分化、细胞迁移调控和细胞粘附等生物学过程中;细胞学组分分析显;在分化第6天时,差异基因主要显著富集在酶联受体蛋白信号通路、运动和细胞定位等生物学过程中。Pathway分析显示,差异表达基因显著富集到了 4条已知与脂肪前体细胞分化相关的信号通路,如PPAR和甘油酯代谢通路等。最后,我们比较了两种细胞成脂分化过程中的差异表达基因,结果显示,两种细胞间仅有253个共有的差异表达lncRNAs,而差异表达mRNAs则达到了 911个。共有基因显著富集到了包括细胞周期、细胞周期过程、染色体分离等在内的超过200个生物学过程中;通路分析显示,共有差异基因显著富集到了包括细胞周期、细胞外基质-受体相互作用和焦点粘连等在内的数十条信号通路,其中包括多条已知参与脂肪生成调控的信号通路,如PPAR、p53、Foxo 和 TGF-beta 信号通路。4.gga-mir-30c-2 与 XLOC_060155 和 SIX4 基因靶关系验证高通量测序和基因共表达结果显示,XLOC_060155、gga-mir-30c和SIX4为调控鸡腹部脂肪前体细胞成脂分化的重要候选基因,XLOC_060155与gga-mir-30c及gga-mir-30c与SIX4基因的表达模式具有高度相关性。RT-qPCR结果显示,XLOC_060155的表达模式与gga-mir-30c呈显著负相关(r=-0.97,P0.01),gga-mir-30c的表达模式与SIX4基因呈显著负相关(r=-0.684,P=0.0140.05)。靶基因预测显示,SIX4和XLOC_060155基因存在gga-mir-30c-5p的结合位点。双荧光素酶活性检测结果显示,gga-mir-30c-5p与XLOC_060155结合后荧光素酶的表达活性显著降低,而将结合位点突变后,荧光素酶表达活性极显著回升;gga-mir-30c-5P与SIX4 3'UTR区结合后荧光素酶的表达活性显著下降,而将结合位点突变后,荧光素酶表达活性显著回升,表明XLOC_060155和SIX4基因确实是gga-mir-30c的靶基因。
[Abstract]:The intramuscular fat deposition and meat quality traits related to muscle, such as tenderness, juiciness, flavor and water. In order to cater to consumers for high quality increasing demand, improve the content of intramuscular fat has become one of the objectives of breeders all over the world. However, in the past few years in ten, due to the excessive pursuit of people use the rate of genetic selection on growth and feed of broiler chicken, leading to the current quality and flavor reduced. At the same time, the excessive choice of the growth rate of broilers with excessive accumulation of abdominal fat, therefore, increased intramuscular fat and reduce abdominal fat deposition has become one of the goals of broiler breeding work at present. On the one hand to describe chicken abdomen and intramuscular preadipocytes in adipogenic differentiation process of transcriptome expression using RNA-seq technology, identification of two kinds of cells in the lncRNAs and lncRNAs analysis The characteristics of a screening. Chicken abdomen and intramuscular preadipocytes into lipid differences during the differentiation of gene expression, combined with bioinformatics methods, identified important factors and signaling pathways may affect fat deposition in chicken abdominal and muscle. Finally, through the comparison of the chicken abdomen and intramuscular preadipocytes into expression the level of lipid gene in the differentiation process of different transcriptional level of chicken abdominal and intramuscular preadipocyte differentiation. The main results are as follows: 1. screening key genes in chicken abdominal fat deposition from chicken adipose tissue separation culture obtained primary chicken abdominal fat precursor cells, adipogenic differentiation induction, oil red O staining showed that the success of the chicken abdominal fat precursor cells isolated. Display analysis in chicken abdominal fat precursor cells adipogenic differentiation process identified 27023 lncRNAs, abdominal fat precursor fine chicken Cellular lncRNAs has sequence and open reading frame and short exon less features; function prediction showed that the chicken abdominal fat precursor cells in lncRNAs cells mainly involved in protein modification, protein modification and signal regulation and other biological processes. In chicken abdominal fat precursor cells adipogenic differentiation process of the screening to 4232 the differential expression of mRNAs lncRNAs and 1656 differentially expressed; functional annotation showed that differentially expressed genes mainly enriched in cell cycle, mesenchymal cell differentiation and cell cycle process and biological process; pathway analysis showed that the differentially expressed genes were significantly enriched in cell cycle, DNA replication and PPAR signaling pathway. Co expression analysis identified the 6 stage specific modules, and in the 6 module selected 29 high connectivity gene using visualization, these genes may be regulated in chicken abdominal fat precursor cells into adipocytes An important candidate gene for differentiation, including XLOC_052948, gga-mir-30c, SCD and KLF15 gene screening of.2. chicken intramuscular fat deposition in key gene from chicken breast muscle tissue isolated from chicken primary intramuscular preadipocytes, the adipogenic induction, oil red O staining showed that the chicken intramuscular preadipocytes the successful separation in chicken. Intramuscular preadipocytes were identified in 26172 lncRNAs analysis showed that the chicken fat in muscle precursor cells in the lncRNAs sequence and open reading frame and short exon less features; function prediction showed that chicken intramuscular preadipocytes in lncRNAs mainly involved in gene expression cytology, molecular synthesis, metabolism and other biological processes. RNA in chicken fat in muscle precursor cells adipogenic differentiation process of the screened 4694 differentially expressed mRNAs lncRNAs and 2169 differentially expressed functional annotation showed differential expression; The genes mainly enriched in signal regulation, cell communication and signal transduction regulation of biological process, pathway analysis showed that the differentially expressed genes were significantly enriched in the focal adhesions, MAPK and PPAR signalingpathways. Co expression analysis identified a total of 6 of the die stage specific block, visual analysis in the 6 stage specific the module selected 28 high connectivity genes, these genes may be an important candidate gene controlling chicken intramuscular preadipocytes adipogenic differentiation including XLOC_013577, gga-mir-146b, BMP3 and MYOD1 genes of.3. chicken abdomen and intramuscular fat deposition mechanism analysis of abdominal and intramuscular preadipocytes between lncRNAs structure comparison analysis showed that two cell lncRNAs has the same characteristics, namely the exon number is less, the open reading frame of short and short sequences. A comparison showed that the expression level of expression, two lncRNAs cells were much Below the mRNAs. comparative transcriptome analysis showed that differences between the two kinds of cells decreased with the prolongation of gene differentiation, differentiation in the 0,2,4 and 6 days, respectively screened 2942 (1877 mRNAs and 1065 lncRNAs), 3788 (1964 mRNAs and 1824 lncRNAs), 2872 (1194 mRNAs and 1678 lncRNAs) and 2214 (1206 mRNAs and 1008 lncRNAs) differentially expressed genes. Different expression of GO gene annotation revealed that differentiation in zeroth days, abdomen and intramuscular fat before the difference between cells expressed genes were significantly enriched in response to growth factor, cell growth factor stimulation response and cell motility regulation in biological process; second days of differentiation, differentially expressed genes were significantly enriched in the development of muscle structure, muscle organ development and growth factor cytogenetic response in many biological processes; in fourth days of differentiation, these genes mainly significantly enriched in mesenchymal Differentiation of mesenchymal cells, regulation of cell migration and cell adhesion in many biological processes; cytological component analysis; differentiation in sixth days, these genes mainly significantly enriched in enzyme linked receptor protein signaling pathway,.Pathway and cellular localization in many biological processes analysis showed that differentially expressed genes were significantly enriched to 4 known and fat precursor cell differentiation related signaling pathways, such as PPAR and glycerol metabolism pathway. Finally, we compared the two difference of lipid in the differentiation of cell gene expression, results showed that two kinds of cells only 253 common differentially expressed lncRNAs, while the expression of mRNAs reached 911 total. Genes were significantly enriched to include cell cycle, cell cycle, chromosome segregation, more than 200 biological processes; pathway analysis showed that there were differences between genes were significantly enriched to include The cell cycle, dozens of signal pathway of ECM receptor interaction and focal adhesion, including multiple signaling pathways, known to be involved in adipogenesis regulation such as PPAR, p53, co expression of Foxo and TGF-beta signal pathways of.4.gga-mir-30c-2 and XLOC_060155 and SIX4 genes to verify high-throughput sequencing and gene showed that XLOC_060155 gga-mir-30c, and SIX4 as an important candidate gene of chicken abdominal fat precursor cells adipogenic differentiation, the expression pattern of XLOC_060155 and gga-mir-30c and gga-mir-30c and SIX4 gene is highly related to the results of.RT-qPCR showed that the expression pattern was negatively correlated with gga-mir-30c XLOC_060155 (r=-0.97, P0.01), was negatively related to the expression pattern of SIX4 gene and gga-mir-30c the (r=-0.684, P=0.0140.05). The target gene prediction show that the combination of SIX4 and XLOC_060155 genes in gga-mir-30c-5p Site. Dual luciferase activity assay showed that the expression of gga-mir-30c-5p and XLOC_060155 binding activity of luciferase was significantly decreased, while the binding site mutation, expression of luciferase activity was significantly reduced; gga-mir-30c-5P and SIX4 3'UTR binding activity of luciferase expression was significantly decreased, while the binding site mutation, expression of luciferase activity rebounded significantly, showed that XLOC_060155 and the SIX4 gene is the target gene of gga-mir-30c.

【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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