秦川牛脂肪沉积相关基因筛选及可变剪接对基因表达和细胞定位的影响研究

发布时间：2018-01-12 06:13

本文关键词：秦川牛脂肪沉积相关基因筛选及可变剪接对基因表达和细胞定位的影响研究　出处：《西北农林科技大学》2017年博士论文　论文类型：学位论文

【摘要】：脂肪组织不仅在个体发育过程中发挥必要作用,并且在牛肉品质的研究中也扮演着重要角色。培育生产优质牛肉的肉牛品种已成为目前分子育种的重要目标。牛肉中脂肪含量决定了牛肉的品质。但由于脂肪组织形成和沉积的分子作用机制复杂,在牛上对脂肪沉积相关基因的研究相对较少,有待挖掘;并且基因的可变剪接丰富了其表达产物的种类,加大了基因作用机制的复杂程度,同时也为分子机制的研究提供了新思路,成为当今研究的热点。为此,本研究对不同发育阶段和不同性别秦川牛的皮下脂肪组织进行转录组测序和基因差异表达分析,并在组织、细胞和分子水平上对差异表达基因逐步筛选得到脂肪沉积相关基因,最后利用电子克隆、基因组织表达谱构建、脂肪细胞分化基因时序表达分析、细胞共定位及亚细胞定位分析等手段进一步研究了可变剪接的组织特异性以及可变剪接对脂肪沉积相关基因功能的影响。主要研究结果如下:1.牛皮下脂肪组织转录组测序及基因表达分析本研究利用二代高通量测序分别对胎牛、成年公牛、成年母牛和成年阉牛皮下脂肪组织进行转录组测序。每个样品获得至少2,600万clean reads,其中82.58%~84.41%的clean reads成功的比对到参考基因组上。我们共检测了12,233个基因在四类皮下脂肪组织内的表达,其中109个基因在四类皮下脂肪组织中共同高表达(≥500 RPKM)。共同高表达的基因中除了含有63个编码核糖体蛋白的基因外,还包含多个与脂肪代谢相关的基因(APOE、GAPDH、FABP4、SPARC和ADIRF等),说明共同高表达的基因可以为筛选脂肪沉积相关基因提供材料。不同性别成年牛皮下脂肪组织基因表达高度相关(r≥0.91),胎牛和各成年牛皮下脂肪组织基因表达相关程度较低(r≤0.64);并且通过对四类皮下脂肪组织两两比较差异基因数目统计分析,发现本研究中年龄相对性别对皮下脂肪组织基因的表达影响更大。此外,我们还在四类牛脂肪组织中检测到大量新转录本,这为将来完善牛基因组注释及非编码RNA的研究提供资源。2.年龄和性别对脂肪组织基因表达差异的影响通过将成年公牛、成年母牛和成年阉牛皮下脂肪组织作为三个重复与胎牛皮下脂肪组织的基因表达进行比较,共检测到2,703个差异表达基因(差异表达倍数2,P0.05)。成年牛和胎牛皮下脂肪组织差异基因GO(Gene ontology,基因功能注释)富集分析显示,差异基因显著富集(P0.05)在与组织发育相关的GO条目;脂肪代谢相关的GO条目没有显著富集,但具有较低的P值。差异基因显著富集在脂肪代谢关键信号通路PPAR pathway,其中促进脂肪沉积的基因在成年牛皮下脂肪组织中显著上调;另外,14个脂肪细胞成脂分化标志基因中有12个基因在成年牛皮下脂肪组织内相对胎牛皮下脂肪组织高表达,说明成年牛皮下脂肪组织相对胎牛皮下脂肪组织进行着更为活跃的脂肪沉积。对成年公牛、成年母牛和成年阉牛皮下脂肪组织两两比较差异基因进行功能和参与的信号通路富集分析发现,差异基因在与甾类激素刺激、脂肪代谢和细胞因子相关的go条目显著富集,在和细胞因子相关的信号通路共同显著富集,这初步证明性别对牛皮下脂肪组织的脂肪代谢有影响,并且可能和细胞因子的分泌相关。3.脂肪沉积相关基因的筛选基于以上对转录组测序数据的分析结果,选取11个在成年牛皮下脂肪组织内均为胎牛皮下脂肪组织内表达量10倍以上的基因和20个在四类牛皮下脂肪组织内共同高表达的基因,作为后续逐步筛选与脂肪沉积相关基因的初始材料。通过构建31个候选基因的组织表达谱,发现其中12个基因(adipor2、cidec、ghr、lipe、s100b、scd、thrsp、tusc5、anxa2、cst3、sparc和vim)在脂肪组织内相对其它组织高表达。通过罗格列酮对牛原代脂肪细胞进行脂肪沉积诱导,检测到其中7个基因(ghr、thrsp、scd、lipe、tusc5、cidec和cst3)在脂肪沉积过程中表达呈上调趋势。利用腺病毒在牛原代脂肪细胞中超表达pparg2,检测到其中3个基因(cidec、tusc5和cst3)表达量显著上调。最终,本实验筛选出3个受pparg2调控且与脂肪沉积相关的基因,同时补充了牛pparg2基因的调控网络。4.脂肪组织内表达基因可变剪接特征分析在以上转录组测序数据中共发现4,753个基因存在可变剪接现象,占总检测基因数目的38.85%。但只有1,319(28%)个基因在四类牛脂肪组织中均出现可变剪接现象,并且共同出现可变剪接的基因只有17%的可变剪接事件在四类牛脂肪组织内同时存在,说明可变剪接在脂肪组织的不同发育时期或不同性别的个体内存在较大差异。本研究选择牛nfix基因对可变剪接的组织特异性进行了进一步验证。通过电子克隆及pcr方法成功预测并验证牛nfix基因存在5种不同转录本。组织表达谱分析显示不同转录本在牛不同发育时期和不同组织内表达出现多样化的差异。以上分析说明同一基因的不同亚型可能会具有不同功能,因此有必要进一步分析可变剪接对脂肪沉积相关基因表达和分布的影响。5.脂肪沉积相关基因可变剪接模式及细胞定位分析本实验分析了三个脂肪沉积相关基因(cebpa、cidec和tusc5)的可变剪接模式,并通过细胞定位分析或细胞共定位分析在239t或3t3-l1细胞中检测可变剪接对以上三个基因在细胞内的表达分布影响。结果显示:cebpa的两种蛋白亚型均分布在细胞核内;cidec的两种蛋白亚型均分布在细胞质内,并且在细胞质内的分布未发现差异;tusc5的两种蛋白亚型均分布在细胞质内,但在细胞质内的分布出现明显差异。对tusc5产生的两种蛋白亚型氨基酸序列进一步分析显示,可变剪接可能通过改变TUSC5的内质网滞留信号影响其在细胞质内的分布。6.可变剪接对脂肪沉积相关基因TUSC5作用的影响对TUSC5不同亚型(TUSC5a和TUSC5b)的组织表达谱和在牛原代脂肪细胞诱导分化不同阶段表达变化趋势分析,均发现TUSC5a和TUSC5b存在一定的差异;并且利用细胞定位载体检测TUSC5a和TUSC5b在293T细胞内的时序表达,发现TUSC5a相对TUSC5b出现明显的迟缓表达。但亚细胞定位分析显示TUSC5a和TUSC5b都在内质网上有分布,CIDEC不在内质网上分布;并且TUSC5两种亚型与CIDEC细胞共定位分析显示都不和CIDEC在细胞质内发生互作。综上,可变剪接会影响TUSC5在细胞质内的表达,但对TUSC5与CIDEC之间的互作关系没有明显影响。本研究对秦川牛皮下脂肪组织进行转录组测序和脂肪沉积相关基因筛选,并将脂肪沉积相关基因的分析深入到可变剪接水平,为牛脂肪组织发育分子机制的研究提供了丰富的资源和新思路。
[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.

【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】：S823

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