本文選題：京海黃雞　切入點：lncRNA　出處：《揚州大學(xué)》2017年博士論文

【摘要】：肌肉中肌內(nèi)脂肪沉積與多種肉質(zhì)性狀相關(guān),如嫩度、多汁性、風(fēng)味和系水力。為了迎合消費者對高肉質(zhì)不斷增加的需求,提高肌內(nèi)脂肪含量已經(jīng)成為全世界育種工作者的目標(biāo)之一。然而,在過去幾十年中,由于人們對肉雞生長速度和飼料利用率遺傳選擇的過度追求,導(dǎo)致目前雞肉質(zhì)量和風(fēng)味降低。與此同時,對肉雞生長速度的過度選擇伴隨著腹部脂肪的過度積累,因此,增加肌內(nèi)脂肪同時減少腹部脂肪沉積已經(jīng)成為目前肉雞育種工作的目標(biāo)之一。本研究一方面旨在利用RNA-seq技術(shù)描述雞腹部和肌內(nèi)脂肪前體細(xì)胞在成脂分化過程中的轉(zhuǎn)錄組表達情況,鑒定兩種細(xì)胞中的lncRNAs,并分析lncRNAs的特點。一方面篩選雞腹部和肌內(nèi)脂肪前體細(xì)胞成脂分化過程中的差異表達基因,結(jié)合生物信息學(xué)方法,鑒定出可能影響雞腹部和肌內(nèi)脂肪沉積的重要因子和信號通路。最后,通過比較雞腹部和肌內(nèi)脂肪前體細(xì)胞成脂分化過程中基因的表達水平,研究雞腹部和肌內(nèi)脂肪前體細(xì)胞分化過程中轉(zhuǎn)錄組水平上的差異。主要研究結(jié)果如下:1.雞腹部脂肪沉積關(guān)鍵基因的篩選由雞腹部脂肪組織分離培養(yǎng)獲得雞原代腹部脂肪前體細(xì)胞,誘導(dǎo)其成脂分化,油紅O染色顯示,成功分離獲得雞腹部脂肪前體細(xì)胞。在雞腹部脂肪前體細(xì)胞成脂分化過程中共鑒定出27,023條lncRNAs,分析顯示,雞腹部脂肪前體細(xì)胞中的lncRNAs具有序列和開放閱讀框短及外顯子少的特點;功能預(yù)測發(fā)現(xiàn),雞腹部脂肪前體細(xì)胞中的lncRNAs主要參與蛋白修飾、細(xì)胞蛋白修飾和信號調(diào)控等生物學(xué)過程。在雞腹部脂肪前體細(xì)胞成脂分化過程中共篩選到4,232條差異表達lncRNAs和1,656條差異表達mRNAs;功能注釋顯示,差異表達基因主要富集在細(xì)胞周期、間葉細(xì)胞分化和細(xì)胞周期過程等生物學(xué)過程;通路分析結(jié)果顯示,差異表達基因顯著富集在細(xì)胞周期、DNA復(fù)制和PPAR等信號通路中。共表達分析共鑒定出了 6個階段特異性模塊,并利用可視化在6個模塊中篩選出了 29個高連通性的基因,這些基因可能為調(diào)控雞腹部脂肪前體細(xì)胞成脂分化的重要候選基因,包括XLOC_052948、gga-mir-30c、SCD和KLF15等基因。2.雞肌內(nèi)脂肪沉積關(guān)鍵基因的篩選由雞胸部肌肉組織分離培養(yǎng)獲得雞原代肌內(nèi)脂肪前體細(xì)胞,誘導(dǎo)其成脂分化,油紅O染色顯示,成功分離獲得雞肌內(nèi)脂肪前體細(xì)胞。在雞肌內(nèi)脂肪前體細(xì)胞中共鑒定出26,172條lncRNAs,分析顯示,雞肌內(nèi)脂肪前體細(xì)胞中的lncRNAs具有序列和開放閱讀框短及外顯子少的特點;功能預(yù)測發(fā)現(xiàn),雞肌內(nèi)脂肪前體細(xì)胞中的lncRNAs主要參與基因表達、細(xì)胞學(xué)大分子合成、RNA代謝等生物學(xué)過程。在雞肌內(nèi)脂肪前體細(xì)胞成脂分化過程中共篩選到4,694條差異表達lncRNAs和2,169條差異表達mRNAs;功能注釋顯示,差異表達基因主要富集在信號調(diào)控、細(xì)胞通信調(diào)控及信號轉(zhuǎn)導(dǎo)調(diào)控等生物學(xué)過程,通路分析結(jié)果顯示,差異表達基因顯著富集在焦點黏連、MAPK及PPAR等信號通路中。共表達分析共鑒定出了 6個階段特異性模塊,可視化分析在6個階段特異性模塊中篩選出了 28個高連通性的基因,這些基因可能為調(diào)控雞肌內(nèi)脂肪前體細(xì)胞成脂分化的重要候選基因,包括XLOC_013577、gga-mir-146b、BMP3和MYOD1等基因。3.雞腹部和肌內(nèi)脂肪沉積機制比較分析腹部和肌內(nèi)脂肪前體細(xì)胞間lncRNAs結(jié)構(gòu)比較分析顯示,兩種細(xì)胞lncRNAs具有相同的特點,即外顯子數(shù)少、開放閱讀框短和序列短。表達量比較顯示,兩種細(xì)胞lncRNAs的表達量均遠(yuǎn)遠(yuǎn)低于mRNAs。轉(zhuǎn)錄組比較分析顯示,兩種細(xì)胞間差異基因隨著分化的延長而減少,在分化第0、2、4和6天,分別篩選到2,942(1,877個mRNAs和1,065個lncRNAs)、3,788(1,964 個 mRNAs 和 1,824 個 lncRNAs)、2,872(1,194 個 mRNAs 和 1,678 個 lncRNAs)和2,214(1,206個mRNAs和1,008個lncRNAs)個差異表達基因。異表達基因GO注釋顯示,在分化第0天,腹部和肌內(nèi)脂肪前體細(xì)胞之間差異表達基因主要顯著富集在生長因子應(yīng)答、生長因子刺激細(xì)胞應(yīng)答及細(xì)胞運動性調(diào)控等生物學(xué)過程;在分化第2天,差異表達基因主要顯著富集在肌肉結(jié)構(gòu)發(fā)育、肌肉器官發(fā)育和生長因子細(xì)胞學(xué)應(yīng)答等生物學(xué)過程中;在分化第4天,差異基因主要顯著富集在間充質(zhì)細(xì)胞分化、細(xì)胞遷移調(diào)控和細(xì)胞粘附等生物學(xué)過程中;細(xì)胞學(xué)組分分析顯;在分化第6天時,差異基因主要顯著富集在酶聯(lián)受體蛋白信號通路、運動和細(xì)胞定位等生物學(xué)過程中。Pathway分析顯示,差異表達基因顯著富集到了 4條已知與脂肪前體細(xì)胞分化相關(guān)的信號通路,如PPAR和甘油酯代謝通路等。最后,我們比較了兩種細(xì)胞成脂分化過程中的差異表達基因,結(jié)果顯示,兩種細(xì)胞間僅有253個共有的差異表達lncRNAs,而差異表達mRNAs則達到了 911個。共有基因顯著富集到了包括細(xì)胞周期、細(xì)胞周期過程、染色體分離等在內(nèi)的超過200個生物學(xué)過程中;通路分析顯示,共有差異基因顯著富集到了包括細(xì)胞周期、細(xì)胞外基質(zhì)-受體相互作用和焦點粘連等在內(nèi)的數(shù)十條信號通路,其中包括多條已知參與脂肪生成調(diào)控的信號通路,如PPAR、p53、Foxo 和 TGF-beta 信號通路。4.gga-mir-30c-2 與 XLOC_060155 和 SIX4 基因靶關(guān)系驗證高通量測序和基因共表達結(jié)果顯示,XLOC_060155、gga-mir-30c和SIX4為調(diào)控雞腹部脂肪前體細(xì)胞成脂分化的重要候選基因,XLOC_060155與gga-mir-30c及gga-mir-30c與SIX4基因的表達模式具有高度相關(guān)性。RT-qPCR結(jié)果顯示,XLOC_060155的表達模式與gga-mir-30c呈顯著負(fù)相關(guān)(r=-0.97,P0.01),gga-mir-30c的表達模式與SIX4基因呈顯著負(fù)相關(guān)(r=-0.684,P=0.0140.05)。靶基因預(yù)測顯示,SIX4和XLOC_060155基因存在gga-mir-30c-5p的結(jié)合位點。雙熒光素酶活性檢測結(jié)果顯示,gga-mir-30c-5p與XLOC_060155結(jié)合后熒光素酶的表達活性顯著降低,而將結(jié)合位點突變后,熒光素酶表達活性極顯著回升;gga-mir-30c-5P與SIX4 3'UTR區(qū)結(jié)合后熒光素酶的表達活性顯著下降,而將結(jié)合位點突變后,熒光素酶表達活性顯著回升,表明XLOC_060155和SIX4基因確實是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.

【學(xué)位授予單位】：揚州大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：S831

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