本文選題：牛 + 肉質(zhì)性狀��； 參考：《吉林大學(xué)》2017年博士論文

【摘要】：牛肉富含蛋白質(zhì),鐵,鋅,維生素B和必需的多不飽和脂肪酸,是人類高品質(zhì)營養(yǎng)的來源。隨著現(xiàn)代生活水平的提高和健康消費意識的增強(qiáng),我國的消費者對牛肉的數(shù)量和品質(zhì)的需求也呈逐年遞增趨勢。肉質(zhì)性狀是一個復(fù)雜經(jīng)濟(jì)性狀,大量基因參與調(diào)控肉質(zhì)性狀的形成過程,但目前牛肉品質(zhì)遺傳標(biāo)記和相關(guān)功能基因的信息非常有限,嚴(yán)重阻礙我國肉牛優(yōu)良品種選育和新品種培育進(jìn)程,因此遺傳標(biāo)記篩查、功能基因挖掘及基因網(wǎng)絡(luò)調(diào)控和作用機(jī)理的研究是我國優(yōu)質(zhì)、高效肉牛品種培育過程中必須突破的分子基礎(chǔ)理論和技術(shù)的關(guān)鍵。本研究針對目前我國肉牛研究領(lǐng)域中功能基因較少等突出問題,以肉質(zhì)性狀和肌間脂肪沉積能力存在顯著差異的日本和牛與草原紅牛為研究對象,采用全基因組DNA甲基化測序(Whole Genome Bisulfite Sequencing,WGBS)和RNA-seq對兩個品種牛背最長肌DNA甲基化和轉(zhuǎn)錄組進(jìn)行分析,篩選牛肉質(zhì)性狀相關(guān)候選基因和候選區(qū)域,并對部分候選基因和候選區(qū)域進(jìn)行了功能驗證和作用機(jī)制分析。甲基化分析結(jié)果顯示,日本和牛與草原紅牛背最長肌之間,8596個基因中存在差異甲基化區(qū)域(Differentially methylated regions,DMRs),差異甲基化基因(Differentially methylated genes,DMGs)顯著富集在1046個GO terms(p0.05),DMGs富集在276個KEGG信號通路中,但無信號通路顯著富集(p0.05)。亞硫酸氫鈉處理測序法(BSP)的方法驗證了部分DMRs的甲基化水平,結(jié)果與測序結(jié)果趨勢一致。RNA-seq分析結(jié)果顯示,日本和牛與草原紅牛背最長肌之間存在388個表達(dá)量差異基因(Differentially expressed genes,DEGs)(Log2FC0.585或-0.585,FDR0.05),其中,205個基因在日本和牛的表達(dá)水平高于草原紅牛(p0.05),183個基因在日本和牛的表達(dá)水平低于草原紅牛(p0.05)。差異基因顯著富集在475個GO terms(p0.05);KEGG分析顯示,下調(diào)基因顯著富集在20個通路中(p0.05),上調(diào)基因顯著富集在7個通路中(p0.05),熒光定量PCR和Western Blot的方法分別驗證了部分DEGs的m RNA和蛋白的表達(dá)量,結(jié)果與測序結(jié)果一致。全基因組甲基化與轉(zhuǎn)錄組聯(lián)合分析結(jié)果顯示,共有331個DMRs與DEGs呈現(xiàn)負(fù)相關(guān),其中21個DMRs位于DEGs的啟動子區(qū),在牛和豬的4個群體中,對6個負(fù)相關(guān)基因啟動子區(qū)序列的CG島分布情況和遺傳多態(tài)性進(jìn)行了分析和轉(zhuǎn)錄因子結(jié)合位點預(yù)測,為進(jìn)一步研究啟動子DNA甲基化對肉質(zhì)性狀調(diào)控的機(jī)制奠定了基礎(chǔ)。為進(jìn)一步驗證候選基因?qū)χ旧珊椭舅岽x的作用及調(diào)控機(jī)制,本研究利用基因過表達(dá)技術(shù),在牛胎兒成纖維細(xì)胞中驗證了HSL,CD44,KLF5,CRYAB,ANKRD2,ALDH9A1,EHHADH對細(xì)胞內(nèi)甘油三酯和脂肪酸含量的影響,并利用q PCR array分析和STRING預(yù)測了以上7個基因的表達(dá)水平對脂肪和脂肪酸代謝通路相關(guān)基因的調(diào)控作用,結(jié)果表明HSL,CD44,KLF5過表達(dá)導(dǎo)致牛胎兒成纖維細(xì)胞內(nèi)甘油三酯含量顯著增加(p0.05),而CRYAB,ANKRD2,ALDH9A1,EHHADH過表達(dá)導(dǎo)致細(xì)胞內(nèi)甘油三酯含量顯著減少(p0.05)。脂肪酸含量檢測分析發(fā)現(xiàn),HSL過表達(dá)導(dǎo)致細(xì)胞內(nèi)己酸、辛酸、棕櫚酸、硬脂酸以及總脂肪酸含量增加,亞油酸和順式-4,7,10,13,16,19-二十二碳六烯酸含量降低(p0.05);過表達(dá)CRYAB導(dǎo)致己酸含量升高,但辛酸、棕櫚酸、硬脂酸、亞油酸和順式-4,7,10,13,16,19-二十二碳六烯酸的含量以及總的脂肪酸含量低于對照組(p0.05);過表達(dá)ANKRD2的細(xì)胞內(nèi)檢測的各脂肪酸成分以及總的脂肪酸降低(p0.05);ALDH9A1,CD44和KLF5過表達(dá)導(dǎo)致細(xì)胞內(nèi)各類脂肪酸含量和總脂肪酸含量均增加,但以上基因的過表達(dá)組與對照組相比,細(xì)胞內(nèi)各類脂肪酸含量和總脂肪酸含量差異均不顯著(p0.05)。q PCR array檢測脂肪和脂肪酸相關(guān)基因表達(dá)水平顯示,HSL過表達(dá)導(dǎo)致CPT1C,FABP2,BDH2,PECR,PRKAB2,EHHADH表達(dá)量上調(diào)(FC1.5,p0.05);CD44過表達(dá)導(dǎo)致HMGCS2和CPT1C基因表達(dá)量上調(diào)(FC1.5,p0.05),EHHADH,FABP4,GK2,HSL基因表達(dá)量下調(diào)(FC1/1.5,p0.05);KLF5過表達(dá)導(dǎo)致HMGCS2基因表達(dá)量上調(diào)(FC1.5,p0.05),ACOT12,ACOT6,ACSBG2等12個基因表達(dá)水平下調(diào)(FC1/1.5,p0.05);CRYAB過表達(dá)導(dǎo)致GPD1基因表達(dá)量上調(diào)(FC1.5,p0.05),ACSL6,MCEE,PRKAG1基因表達(dá)水平下調(diào)(FC1/1.5,p0.05);ANKRD2過表達(dá)導(dǎo)致HMGCS2,MUT基因表達(dá)量上調(diào)(FC1.5,p0.05),GK2基因表達(dá)水平下調(diào)(FC1/1.5,p0.05);ALDH9A1過表達(dá),導(dǎo)致HMGCS2,FABP5,SLC27A1,ACADSB,ACAD11,MUT,FABP4,CRAT,PECR,ACSL5基因表達(dá)水平上調(diào)(FC1.5,p0.05),HSL基因表達(dá)水平下調(diào)(FC1/1.5,p0.05);EHHADH過表達(dá)導(dǎo)致ACAA1,ACAD10,ACAD11,ACADM,ACADS,ACADSB等33個基因表達(dá)水平下調(diào)(FC1/1.5,p0.05)。此外,蛋白相互作用預(yù)測結(jié)果顯示,CD44,KLF5,CRYAB和ANKRD2與脂肪和脂肪酸代謝通路中表達(dá)水平上調(diào)和下調(diào)的基因不存在蛋白間互作關(guān)系,而HSL,ALDH9A1,EHHADH基因與部分基因存在相互作用。本研究獲得了日本和牛與草原紅牛之間存在的大量DMRs和DEGs,系統(tǒng)篩選和挖掘決定肉牛肌肉生長、脂肪沉積等肉質(zhì)性狀的關(guān)鍵候選基因,通過不同物種間基因序列比對以及CG島預(yù)測和遺傳多態(tài)性檢測,分析和預(yù)測了SNPs位點對基因啟動子序列甲基化水平,轉(zhuǎn)錄因子結(jié)合位點和候選基因表達(dá)可能存在的調(diào)控作用,在細(xì)胞水平上,采用基因過表達(dá),驗證了候選基因?qū)χ竞椭舅岽x的功能,全面解析候選基因?qū)?xì)胞內(nèi)脂肪含量和脂肪酸含量的影響,以及對脂肪和脂肪酸代謝的調(diào)控機(jī)制。為進(jìn)一步挖掘與肉質(zhì)性狀相關(guān)的功能基因和完善脂肪代謝調(diào)控網(wǎng)絡(luò)奠定理論基礎(chǔ)。此外,獲得具有重要育種價值的功能基因和調(diào)控元件,將為轉(zhuǎn)基因肉牛新品種培育及其產(chǎn)業(yè)化提供基因資源和技術(shù)支撐。
[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.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：S823
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本文編號：2011717