本文關鍵詞：秦川牛脂肪沉積相關基因篩選及可變剪接對基因表達和細胞定位的影響研究　出處：《西北農(nóng)林科技大學》2017年博士論文　論文類型：學位論文

  更多相關文章： 秦川牛 轉錄組測序 脂肪沉積 可變剪接 TUSC5

【摘要】：脂肪組織不僅在個體發(fā)育過程中發(fā)揮必要作用,并且在牛肉品質的研究中也扮演著重要角色。培育生產(chǎn)優(yōu)質牛肉的肉牛品種已成為目前分子育種的重要目標。牛肉中脂肪含量決定了牛肉的品質。但由于脂肪組織形成和沉積的分子作用機制復雜,在牛上對脂肪沉積相關基因的研究相對較少,有待挖掘;并且基因的可變剪接豐富了其表達產(chǎn)物的種類,加大了基因作用機制的復雜程度,同時也為分子機制的研究提供了新思路,成為當今研究的熱點。為此,本研究對不同發(fā)育階段和不同性別秦川牛的皮下脂肪組織進行轉錄組測序和基因差異表達分析,并在組織、細胞和分子水平上對差異表達基因逐步篩選得到脂肪沉積相關基因,最后利用電子克隆、基因組織表達譜構建、脂肪細胞分化基因時序表達分析、細胞共定位及亞細胞定位分析等手段進一步研究了可變剪接的組織特異性以及可變剪接對脂肪沉積相關基因功能的影響。主要研究結果如下:1.牛皮下脂肪組織轉錄組測序及基因表達分析本研究利用二代高通量測序分別對胎牛、成年公牛、成年母牛和成年閹牛皮下脂肪組織進行轉錄組測序。每個樣品獲得至少2,600萬clean reads,其中82.58%~84.41%的clean reads成功的比對到參考基因組上。我們共檢測了12,233個基因在四類皮下脂肪組織內的表達,其中109個基因在四類皮下脂肪組織中共同高表達(≥500 RPKM)。共同高表達的基因中除了含有63個編碼核糖體蛋白的基因外,還包含多個與脂肪代謝相關的基因(APOE、GAPDH、FABP4、SPARC和ADIRF等),說明共同高表達的基因可以為篩選脂肪沉積相關基因提供材料。不同性別成年牛皮下脂肪組織基因表達高度相關(r≥0.91),胎牛和各成年牛皮下脂肪組織基因表達相關程度較低(r≤0.64);并且通過對四類皮下脂肪組織兩兩比較差異基因數(shù)目統(tǒng)計分析,發(fā)現(xiàn)本研究中年齡相對性別對皮下脂肪組織基因的表達影響更大。此外,我們還在四類牛脂肪組織中檢測到大量新轉錄本,這為將來完善牛基因組注釋及非編碼RNA的研究提供資源。2.年齡和性別對脂肪組織基因表達差異的影響通過將成年公牛、成年母牛和成年閹牛皮下脂肪組織作為三個重復與胎牛皮下脂肪組織的基因表達進行比較,共檢測到2,703個差異表達基因(差異表達倍數(shù)2,P0.05)。成年牛和胎牛皮下脂肪組織差異基因GO(Gene ontology,基因功能注釋)富集分析顯示,差異基因顯著富集(P0.05)在與組織發(fā)育相關的GO條目;脂肪代謝相關的GO條目沒有顯著富集,但具有較低的P值。差異基因顯著富集在脂肪代謝關鍵信號通路PPAR pathway,其中促進脂肪沉積的基因在成年牛皮下脂肪組織中顯著上調;另外,14個脂肪細胞成脂分化標志基因中有12個基因在成年牛皮下脂肪組織內相對胎牛皮下脂肪組織高表達,說明成年牛皮下脂肪組織相對胎牛皮下脂肪組織進行著更為活躍的脂肪沉積。對成年公牛、成年母牛和成年閹牛皮下脂肪組織兩兩比較差異基因進行功能和參與的信號通路富集分析發(fā)現(xiàn),差異基因在與甾類激素刺激、脂肪代謝和細胞因子相關的go條目顯著富集,在和細胞因子相關的信號通路共同顯著富集,這初步證明性別對牛皮下脂肪組織的脂肪代謝有影響,并且可能和細胞因子的分泌相關。3.脂肪沉積相關基因的篩選基于以上對轉錄組測序數(shù)據(jù)的分析結果,選取11個在成年牛皮下脂肪組織內均為胎牛皮下脂肪組織內表達量10倍以上的基因和20個在四類牛皮下脂肪組織內共同高表達的基因,作為后續(xù)逐步篩選與脂肪沉積相關基因的初始材料。通過構建31個候選基因的組織表達譜,發(fā)現(xiàn)其中12個基因(adipor2、cidec、ghr、lipe、s100b、scd、thrsp、tusc5、anxa2、cst3、sparc和vim)在脂肪組織內相對其它組織高表達。通過羅格列酮對牛原代脂肪細胞進行脂肪沉積誘導,檢測到其中7個基因(ghr、thrsp、scd、lipe、tusc5、cidec和cst3)在脂肪沉積過程中表達呈上調趨勢。利用腺病毒在牛原代脂肪細胞中超表達pparg2,檢測到其中3個基因(cidec、tusc5和cst3)表達量顯著上調。最終,本實驗篩選出3個受pparg2調控且與脂肪沉積相關的基因,同時補充了牛pparg2基因的調控網(wǎng)絡。4.脂肪組織內表達基因可變剪接特征分析在以上轉錄組測序數(shù)據(jù)中共發(fā)現(xiàn)4,753個基因存在可變剪接現(xiàn)象,占總檢測基因數(shù)目的38.85%。但只有1,319(28%)個基因在四類牛脂肪組織中均出現(xiàn)可變剪接現(xiàn)象,并且共同出現(xiàn)可變剪接的基因只有17%的可變剪接事件在四類牛脂肪組織內同時存在,說明可變剪接在脂肪組織的不同發(fā)育時期或不同性別的個體內存在較大差異。本研究選擇牛nfix基因對可變剪接的組織特異性進行了進一步驗證。通過電子克隆及pcr方法成功預測并驗證牛nfix基因存在5種不同轉錄本。組織表達譜分析顯示不同轉錄本在牛不同發(fā)育時期和不同組織內表達出現(xiàn)多樣化的差異。以上分析說明同一基因的不同亞型可能會具有不同功能,因此有必要進一步分析可變剪接對脂肪沉積相關基因表達和分布的影響。5.脂肪沉積相關基因可變剪接模式及細胞定位分析本實驗分析了三個脂肪沉積相關基因(cebpa、cidec和tusc5)的可變剪接模式,并通過細胞定位分析或細胞共定位分析在239t或3t3-l1細胞中檢測可變剪接對以上三個基因在細胞內的表達分布影響。結果顯示:cebpa的兩種蛋白亞型均分布在細胞核內;cidec的兩種蛋白亞型均分布在細胞質內,并且在細胞質內的分布未發(fā)現(xiàn)差異;tusc5的兩種蛋白亞型均分布在細胞質內,但在細胞質內的分布出現(xiàn)明顯差異。對tusc5產(chǎn)生的兩種蛋白亞型氨基酸序列進一步分析顯示,可變剪接可能通過改變TUSC5的內質網(wǎng)滯留信號影響其在細胞質內的分布。6.可變剪接對脂肪沉積相關基因TUSC5作用的影響對TUSC5不同亞型(TUSC5a和TUSC5b)的組織表達譜和在牛原代脂肪細胞誘導分化不同階段表達變化趨勢分析,均發(fā)現(xiàn)TUSC5a和TUSC5b存在一定的差異;并且利用細胞定位載體檢測TUSC5a和TUSC5b在293T細胞內的時序表達,發(fā)現(xiàn)TUSC5a相對TUSC5b出現(xiàn)明顯的遲緩表達。但亞細胞定位分析顯示TUSC5a和TUSC5b都在內質網(wǎng)上有分布,CIDEC不在內質網(wǎng)上分布;并且TUSC5兩種亞型與CIDEC細胞共定位分析顯示都不和CIDEC在細胞質內發(fā)生互作。綜上,可變剪接會影響TUSC5在細胞質內的表達,但對TUSC5與CIDEC之間的互作關系沒有明顯影響。本研究對秦川牛皮下脂肪組織進行轉錄組測序和脂肪沉積相關基因篩選,并將脂肪沉積相關基因的分析深入到可變剪接水平,為牛脂肪組織發(fā)育分子機制的研究提供了豐富的資源和新思路。
[Abstract]:Adipose tissue not only play a necessary role in the individual growth process, and in the study of beef quality also plays an important role for the cultivation of beef cattle production of high quality beef has become the important goal of molecular breeding. The beef fat content determines the quality of beef. But the molecular mechanism of the formation and deposition of adipose tissue in the complex the research, in cattle on fat deposition related genes is relatively small, needs mining; alternative splicing and gene expression products enrich its species, increase the complexity of gene mechanism, but also provides a new way for the study of molecular mechanism, has become a hot research topic. Therefore, the study of different transcription group and sequencing of genes in different developmental stages and different gender of Qinchuan cattle subcutaneous adipose tissue expression analysis, and in the tissue, cellular and molecular level of differential expression Gene screening genes related to fat deposition gradually, finally using electronic cloning, expression profiles of genes, expression analysis of adipocyte differentiation genes, cellular co localization and subcellular localization analysis further studied the effects of tissue specific alternative splicing and alternative splicing of genes related to the function of fat deposition. The main results are as follows: transcriptome sequencing and gene expression in adipose tissue by using the two generation high-throughput sequencing of fetal bovine, 1. adult bull leather, adipose tissue of adult cow and adult STEERHIDE by transcriptome sequencing. Each sample obtained at least 26 million clean reads 82.58%~84.41% clean reads, the ratio of success to the reference genome. We to detect the expression of 12233 genes in four types of subcutaneous fatty tissue, including 109 genes in four types of subcutaneous fat group The fabric in common high expression (over 500 RPKM). The common high expression genes contain 63 genes encoding ribosomal proteins, but also contains a number of genes related to lipid metabolism (APOE, GAPDH, FABP4, SPARC and ADIRF), indicating that both high expression genes can provide materials for screening of fat the deposition of different sex related genes. Adult cow adipose tissue gene expression were highly correlated (r = 0.91), fetal bovine and adult bovine adipose tissue gene expression level is low (r < 0.64); and through the analysis of more than the number of differentially expressed genes in subcutaneous adipose tissue of 22 class four statistics, this study found that age the relative expression of sex genes in subcutaneous adipose tissue more influence. In addition, we are still four kinds of bovine adipose tissue was detected in a large number of new transcripts, which provide resources for future research to improve the.2. age of bovine genome annotation and non encoding RNA And the influence of gender differences on the expression of adipose tissue gene by adult bulls, adipose tissue of adult cow and adult STEERHIDE as three replicates with fetal bovine adipose tissue gene expression under comparison, detected a total of 2703 differentially expressed genes (differentially expressed in multiples of 2, P0.05). The adult and fetal bovine leather fat the difference of GO gene (Gene tissue ontology, gene annotation) enrichment analysis showed that the differences were significantly enriched (P0.05) in the organization and development related GO entry; entry GO related to fat metabolism has no significant enrichment, but with lower P values. The differences were significantly enriched in fat metabolism key signaling pathways PPAR pathway, which promote fat deposition in adipose tissue were significantly up-regulated in the adult cattle; in addition, there are 12 genes in adult cattle under the fat tissues in 14 fat differentiation into fat cells in gene The high expression of the fetal bovine adipose tissue, adipose tissue that relative fetal bovine adipose tissue of adult cow under a more active. Fat deposition of adult bulls, functions and participate in signal pathway enrichment analysis found that the difference between the 22 genes in adipose tissue of adult cow and adult STEERHIDE, genetic differences in stimulating steroid hormone, lipid metabolism and related cytokines were significantly enriched in go entry, signaling pathways and cytokines related to common significant enrichment, which proved that the fat metabolism sex on adipose tissue under the influence of leather, screening of secretion related.3. fat deposition related genes and cytokines and may be more than the transcriptome sequencing based on the results of data analysis, we selected 11 in adult adipose tissue in leather are expressed in the fetal bovine adipose tissue of more than 10 genes and 20 in four Adipose tissue of cattle leather under high co expression of genes, the initial material as subsequent stepwise selection of fat deposition related genes. By constructing 31 candidate gene expression profile, found that 12 genes (AdipoR2, cidec, GHR, LIPE, S100B, SCD, THRSP, tusc5, anxa2, CST3, and SPARC VIM) is highly expressed in adipose tissue relative to other tissues. Fat deposition induced by rosiglitazone in bovine adipocytes, which detected 7 genes (GHR, THRSP, SCD, LIPE, tusc5, cidec and CST3) were up-regulated expression in fat deposition process. The expression of PPARG2 in bovine adipocytes in the use of adenovirus, which detected 3 genes (cidec, tusc5 and CST3) expression was up-regulated. Finally, the experiment selected 3 PPARG2 regulated and fat deposition related genes, while complementing the bovine PPARG2 gene regulatory network of.4. in adipose tissue Analysis of gene splicing characteristics over transcriptome sequencing data identified 4753 genes expression of alternative splicing occurs, the total number of accounts for gene detection of 38.85%. but only 1319 (28%) splicing phenomenon appeared in the four types of bovine genes in adipose tissue, and a common alternative splicing event is only 17% of the gene splicing at the same time there in the four types of bovine adipose tissue, that alternative splicing in adipose tissue at different developmental stages or different gender differences within individuals. This research chooses tissue specific alternative splicing of bovine Nfix gene was further verified. The success of electronic clone and PCR method to predict and verify the existence of bovine Nfix gene 5 different transcripts. Expression profile analysis showed that different transcripts in the various developmental stages and tissues expression of diverse differences. The above analysis said The different isoforms of the same gene may have different functions, so it is necessary to further analysis of alternative splicing on expression of genes related to fat deposition and distribution of.5. gene related to fat deposition in splicing patterns and cellular localization analysis of this experiment analyzed three fat deposition related genes (CEBPA, cidec and tusc5) of the splicing pattern and, through cellular localization analysis or cellular co localization analysis to detect the influence of alternative splicing on the expression of more than three genes in cells distributed in 239t or 3T3-L1 cells. The results showed that CEBPA two protein isoforms were found in the nucleus; cidec two protein isoforms were distributed in the cytoplasm, and distribution no differences were found in the cytoplasm; tusc5 two protein isoforms were distributed in the cytoplasm, but distributed in the cytoplasm. The obvious difference of tusc5 produced two kinds of egg white sub Type of amino acid sequence analysis further indicates that the alternative splicing may alter TUSC5 endoplasmic reticulum retention signal affects the distribution of.6. in the cytoplasm of the splicing effect on fat deposition related gene TUSC5 of different subtypes of TUSC5 (TUSC5a and TUSC5b) the variation trend of the spectrum and differentiation in different stages of bovine primary adipocyte expression the organization, there are certain differences were found in TUSC5a and TUSC5b; and the use of temporal expression of cellular localization of TUSC5a and TUSC5b carrier detection in 293T cells, TUSC5a TUSC5b was relatively slow. But the expression of subcellular localization showed that TUSC5a and TUSC5b are distributed in the endoplasmic reticulum, CIDEC in endoplasmic reticulum and distribution; two types of TUSC5 and CIDEC cell co localization analysis showed that CIDEC did not occur in the cytoplasm interaction. Therefore, alternative splicing in the fine effect of TUSC5 The expression in the cytoplasm, but has no obvious effect on the interaction between TUSC5 and CIDEC. The study of adipose tissue in Qinchuan screened leather transcriptome sequencing and fat deposition related genes and analysis of fat deposition related genes into the splicing level, providing abundant resources and new ideas on the development of molecular the mechanism for tallow fat tissue.

【學位授予單位】：西北農(nóng)林科技大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：S823

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