基于转录组和全基因组甲基化联合分析的牛肉质性状候选基因的鉴定与功能验证

发布时间：2018-06-13 00:17

  本文选题：牛 + 肉质性状　； 参考：《吉林大学》2017年博士论文

【摘要】：牛肉富含蛋白质,铁,锌,维生素B和必需的多不饱和脂肪酸,是人类高品质营养的来源。随着现代生活水平的提高和健康消费意识的增强,我国的消费者对牛肉的数量和品质的需求也呈逐年递增趋势。肉质性状是一个复杂经济性状,大量基因参与调控肉质性状的形成过程,但目前牛肉品质遗传标记和相关功能基因的信息非常有限,严重阻碍我国肉牛优良品种选育和新品种培育进程,因此遗传标记筛查、功能基因挖掘及基因网络调控和作用机理的研究是我国优质、高效肉牛品种培育过程中必须突破的分子基础理论和技术的关键。本研究针对目前我国肉牛研究领域中功能基因较少等突出问题,以肉质性状和肌间脂肪沉积能力存在显著差异的日本和牛与草原红牛为研究对象,采用全基因组DNA甲基化测序(Whole Genome Bisulfite Sequencing,WGBS)和RNA-seq对两个品种牛背最长肌DNA甲基化和转录组进行分析,筛选牛肉质性状相关候选基因和候选区域,并对部分候选基因和候选区域进行了功能验证和作用机制分析。甲基化分析结果显示,日本和牛与草原红牛背最长肌之间,8596个基因中存在差异甲基化区域(Differentially methylated regions,DMRs),差异甲基化基因(Differentially methylated genes,DMGs)显著富集在1046个GO terms(p0.05),DMGs富集在276个KEGG信号通路中,但无信号通路显著富集(p0.05)。亚硫酸氢钠处理测序法(BSP)的方法验证了部分DMRs的甲基化水平,结果与测序结果趋势一致。RNA-seq分析结果显示,日本和牛与草原红牛背最长肌之间存在388个表达量差异基因(Differentially expressed genes,DEGs)(Log2FC0.585或-0.585,FDR0.05),其中,205个基因在日本和牛的表达水平高于草原红牛(p0.05),183个基因在日本和牛的表达水平低于草原红牛(p0.05)。差异基因显著富集在475个GO terms(p0.05);KEGG分析显示,下调基因显著富集在20个通路中(p0.05),上调基因显著富集在7个通路中(p0.05),荧光定量PCR和Western Blot的方法分别验证了部分DEGs的m RNA和蛋白的表达量,结果与测序结果一致。全基因组甲基化与转录组联合分析结果显示,共有331个DMRs与DEGs呈现负相关,其中21个DMRs位于DEGs的启动子区,在牛和猪的4个群体中,对6个负相关基因启动子区序列的CG岛分布情况和遗传多态性进行了分析和转录因子结合位点预测,为进一步研究启动子DNA甲基化对肉质性状调控的机制奠定了基础。为进一步验证候选基因对脂肪生成和脂肪酸代谢的作用及调控机制,本研究利用基因过表达技术,在牛胎儿成纤维细胞中验证了HSL,CD44,KLF5,CRYAB,ANKRD2,ALDH9A1,EHHADH对细胞内甘油三酯和脂肪酸含量的影响,并利用q PCR array分析和STRING预测了以上7个基因的表达水平对脂肪和脂肪酸代谢通路相关基因的调控作用,结果表明HSL,CD44,KLF5过表达导致牛胎儿成纤维细胞内甘油三酯含量显著增加(p0.05),而CRYAB,ANKRD2,ALDH9A1,EHHADH过表达导致细胞内甘油三酯含量显著减少(p0.05)。脂肪酸含量检测分析发现,HSL过表达导致细胞内己酸、辛酸、棕榈酸、硬脂酸以及总脂肪酸含量增加,亚油酸和顺式-4,7,10,13,16,19-二十二碳六烯酸含量降低(p0.05);过表达CRYAB导致己酸含量升高,但辛酸、棕榈酸、硬脂酸、亚油酸和顺式-4,7,10,13,16,19-二十二碳六烯酸的含量以及总的脂肪酸含量低于对照组(p0.05);过表达ANKRD2的细胞内检测的各脂肪酸成分以及总的脂肪酸降低(p0.05);ALDH9A1,CD44和KLF5过表达导致细胞内各类脂肪酸含量和总脂肪酸含量均增加,但以上基因的过表达组与对照组相比,细胞内各类脂肪酸含量和总脂肪酸含量差异均不显著(p0.05)。q PCR array检测脂肪和脂肪酸相关基因表达水平显示,HSL过表达导致CPT1C,FABP2,BDH2,PECR,PRKAB2,EHHADH表达量上调(FC1.5,p0.05);CD44过表达导致HMGCS2和CPT1C基因表达量上调(FC1.5,p0.05),EHHADH,FABP4,GK2,HSL基因表达量下调(FC1/1.5,p0.05);KLF5过表达导致HMGCS2基因表达量上调(FC1.5,p0.05),ACOT12,ACOT6,ACSBG2等12个基因表达水平下调(FC1/1.5,p0.05);CRYAB过表达导致GPD1基因表达量上调(FC1.5,p0.05),ACSL6,MCEE,PRKAG1基因表达水平下调(FC1/1.5,p0.05);ANKRD2过表达导致HMGCS2,MUT基因表达量上调(FC1.5,p0.05),GK2基因表达水平下调(FC1/1.5,p0.05);ALDH9A1过表达,导致HMGCS2,FABP5,SLC27A1,ACADSB,ACAD11,MUT,FABP4,CRAT,PECR,ACSL5基因表达水平上调(FC1.5,p0.05),HSL基因表达水平下调(FC1/1.5,p0.05);EHHADH过表达导致ACAA1,ACAD10,ACAD11,ACADM,ACADS,ACADSB等33个基因表达水平下调(FC1/1.5,p0.05)。此外,蛋白相互作用预测结果显示,CD44,KLF5,CRYAB和ANKRD2与脂肪和脂肪酸代谢通路中表达水平上调和下调的基因不存在蛋白间互作关系,而HSL,ALDH9A1,EHHADH基因与部分基因存在相互作用。本研究获得了日本和牛与草原红牛之间存在的大量DMRs和DEGs,系统筛选和挖掘决定肉牛肌肉生长、脂肪沉积等肉质性状的关键候选基因,通过不同物种间基因序列比对以及CG岛预测和遗传多态性检测,分析和预测了SNPs位点对基因启动子序列甲基化水平,转录因子结合位点和候选基因表达可能存在的调控作用,在细胞水平上,采用基因过表达,验证了候选基因对脂肪和脂肪酸代谢的功能,全面解析候选基因对细胞内脂肪含量和脂肪酸含量的影响,以及对脂肪和脂肪酸代谢的调控机制。为进一步挖掘与肉质性状相关的功能基因和完善脂肪代谢调控网络奠定理论基础。此外,获得具有重要育种价值的功能基因和调控元件,将为转基因肉牛新品种培育及其产业化提供基因资源和技术支撑。
[Abstract]:Beef is rich in protein, iron, zinc, vitamin B and essential polyunsaturated fatty acids. It is the source of high quality nutrition in human. With the improvement of the modern living standard and the increasing awareness of health consumption, the demand for the quantity and quality of beef is increasing year by year in our country. The meat quality is a complex economic character, a large number of people. Gene participates in the process of regulating the formation of meat quality traits, but the information of genetic markers and related functional genes of beef quality is very limited, which seriously hinders the breeding and breeding process of beef cattle. Therefore, genetic markers screening, functional gene mining and gene network regulation and mechanism research are high quality and high efficiency in China. Molecular basis theory and technology must be broken through in the breeding process of beef cattle. This study aims at the prominent problems in the field of beef cattle research in our country, such as less functional genes and other prominent problems, which have significant differences in meat quality and intermuscular fat deposition ability in Japan and cattle and Grassland Red cattle, using full genome DNA methylation. Sequencing (Whole Genome Bisulfite Sequencing, WGBS) and RNA-seq were used to analyze the DNA methylation and transcriptome of the longest muscle of two varieties of cattle, screening candidate genes and candidate regions for beef quality traits, and functional verification and mechanism analysis of some candidate genes and candidate regions. Between the cattle and the longest muscles of the prairie Red Bull, the 8596 genes differ in the methylation region (Differentially methylated regions, DMRs), and the differential methylation gene (Differentially methylated genes, DMGs) is enriched in 1046 GO terms (P0.05), and DMGs is enriched in 276 signaling pathways, but there is no significant enrichment of the signal pathway. The methylation level of partial DMRs was verified by the method of sodium bisulfate sequencing (BSP), and the results showed that there were 388 differentially expressed genes (Differentially expressed genes, DEGs) (Log2FC0.585 or -0.585, FDR0.05) between Japanese and cattle and prairie red cattle, and 205 of them (Log2FC0.585 or -0.585, FDR0.05). The expression level of genes in Japan and cattle was higher than that of grassland red cattle (P0.05). The expression level of 183 genes in Japan and cattle was lower than that of grassland red cattle (P0.05). The difference genes were significantly enriched in 475 GO terms (P0.05); KEGG analysis showed that the down regulated genes were enriched in 20 pathways (P0.05), and the up-regulated genes were significantly enriched in 7 pathways (P0.05), fluorescence (P0.05). Quantitative PCR and Western Blot methods were used to verify the m RNA and protein expression of part of DEGs, and the results were in accordance with the sequencing results. The combined analysis of whole genome methylation and transcriptional group showed that 331 DMRs and DEGs were negatively correlated, of which 21 DMRs were in the promoter region of DEGs, and 6 negatively correlated in 4 cattle and pigs. The CG island distribution and genetic polymorphisms of the gene promoter sequence were analyzed and the transcription factor binding sites were predicted, which laid the foundation for further research on the mechanism of the promoter DNA methylation in the regulation of meat quality traits. Gene overexpression techniques have verified the effects of HSL, CD44, KLF5, CRYAB, ANKRD2, ALDH9A1, EHHADH on intracellular triglycerides and fatty acids in bovine fetal fibroblasts, and used Q PCR array analysis and STRING to predict the regulation of the expression levels of the above 7 genes on the genes related to fat and fatty acid metabolism pathway. The overexpression of HSL, CD44 and KLF5 led to a significant increase in triglyceride content in bovine fetal fibroblasts (P0.05), while CRYAB, ANKRD2, ALDH9A1, and EHHADH overexpressed significantly reduced triglyceride content in the cells (P0.05). The analysis of fatty acid content found that the HSL over table reached caproic acid, octanoic acid, palmitic acid, stearic acid, and total fat. The content of oleic acid and CIS -4,7,10,13,16,19- twenty-two carbon six enoic acid decreased (P0.05), and overexpression of CRYAB resulted in the increase of caproic acid content, but the content of octanoic acid, palmitic acid, stearic acid, linoleic acid and CIS -4,7,10,13,16,19- twenty-two carbon six enoic acid and total fatty acid content were lower than that of the control group (P0.05); the overexpression of ANKRD2 was fine. The contents of fatty acids and total fatty acids decreased (P0.05), and the overexpression of ALDH9A1, CD44 and KLF5 increased all kinds of fatty acid content and total fatty acid content in the cells, but the over expression group of the above gene had no significant difference in all kinds of fatty acid content and total fatty acid content (P0.05).Q PCR arra compared with the control group. Y detected the expression level of fat and fatty acid related genes, which showed that HSL overexpression led to CPT1C, FABP2, BDH2, PECR, PRKAB2, EHHADH expression up regulation (FC1.5, P0.05). Up regulation (FC1.5, P0.05), ACOT12, ACOT6, ACSBG2, and other 12 gene expression levels down (FC1/1.5, P0.05); CRYAB overexpression leads to the up-regulation of GPD1 gene expression (FC1.5, P0.05). FC1/1.5 (P0.05); ALDH9A1 overexpression, resulting in HMGCS2, FABP5, SLC27A1, ACADSB, ACAD11, MUT, FABP4, CRAT, PECR, down regulation. The protein interaction prediction results show that CD44, KLF5, CRYAB and ANKRD2 have no interactivity relationship with the protein expression levels up and down in the fatty and fatty acid metabolic pathways, while the HSL, ALDH9A1, and EHHADH genes interact with some genes. This study obtained a large number of DMRs and D existing between Japan and cattle and grassland red cattle. EGs, system screening and mining key candidate genes that determine meat quality of beef cattle, fat deposition and other meat quality traits. Through the alignment of gene sequences among different species, CG Island prediction and genetic polymorphism detection, the methylation level, transcription factor binding site and candidate gene expression of SNPs loci in the gene promoter sequence are analyzed and predicted. The existing regulatory role, at the cell level, using gene overexpression, validates the function of the candidate genes on fat and fatty acid metabolism, comprehensively analyzes the effect of the candidate genes on the intracellular fat content and fatty acid content, as well as the regulation mechanism on the metabolism of fat and fatty acids, in order to further explore functional groups related to meat quality traits. In addition, the acquisition of functional genes and regulatory elements with important breeding value will provide genetic resources and technical support for the breeding and industrialization of new varieties of genetically modified beef cattle.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S823
，

本文编号：2011717