云岭牛和文山牛背最长肌肉质特性及DNA甲基化分析
发布时间：2019-06-03 03:25
【摘要】：本研究以南方黄牛云岭牛和文山牛的背最长肌组织为实验材料,以西门塔尔牛的背最长肌组织为对照,比较不同牛品种的肉品质的差异,利用甲基化修饰依赖性内切酶测序法(MethylRAD-seq)和亚硫酸氢盐测序PCR(BSP)技术分析比较不同品种牛背最长肌的甲基化图谱,分析甲基化对不同品种牛肌肉肉质的调控影响。结合MethylRAD-seq和实验室已有的RNA-seq的数据,分析甲基化对不同牛种肉质基因的表达调控,进一步研究甲基化对牛背最长肌的调控机制。背最长肌肉品质测定结果表明,牛肉中pH值和失水率在云岭牛、文山牛和西门塔尔牛之间差异不显著(P0.05),云岭牛和文山牛的脂肪百分含量显著高于西门塔尔牛(P0.05),西门塔尔牛的蛋白质含量显著高于文山牛(P0.05)。本次实验测定的4种常见脂肪酸(肉豆蔻酸、棕榈酸、棕榈油酸和硬脂酸)在云岭牛,文山牛和西门塔尔牛之间的含量差异不显著(P0.05)。MethylRAD-seq分析结果表明,云岭牛、文山牛和西门塔尔牛分别有7027017(57.74%),6405515(56.37%),6734119(58.12%),6092960(54.82%),5510367(56.94%),7204540(62.06%),6829702(61.38%),6759345(54.58%),6901684(57.14%)个reads可以比对上牛参考基因组的唯一位置,这三种牛基因组中主要的甲基化模式是CpG型。唯一比对上的reads主要分布在gene和mRNA上。不同牛品种之间比较,以西门塔尔牛为对照组,在云岭牛肌肉组织中共检测到39个甲基化差异基因,在文山牛肌肉组织中共检测到123个甲基化差异基因,以文山牛为对照组,云岭牛肌肉组织中共检测到104个甲基化差异基因,只有一个差异基因(ALDH3A1)在西门塔尔牛、文山牛和云岭牛之间甲基化水平均显著差异(P0.05)。KEGG pathway分析表明,这些甲基化差异基因显著富集的信号通路主要是氨基酸代谢,Ras信号通路、α-亚麻酸代谢、血管平滑肌收缩、脂肪消化吸收、醚类脂质代谢等(P0.05)。MethylRAD-seq和实验室已有的RNA-seq结果的联合分析表明,西门塔尔牛和文山牛之间有21个基因甲基化程度和表达量均差异,西门塔尔牛和云岭牛之间有24个差异甲基化和表达基因,文山牛和云岭牛之间有80个差异甲基化和表达基因。本研究利用亚硫酸氢盐测序技术(BSP)检测3个候选基因(ACAD11,FADS6,FASN)启动子区甲基化程度,并分析基因表达量与启动子区甲基化程度之间的关系。结果表明,FADS6和FASN整体的DNA甲基化程度与表达量呈现负相关,西门塔尔牛FASN的DNA甲基化程度显著高于云岭牛和文山牛(P0.05),FADS6的DNA甲基化程度在三个品种的牛之间差异不显著,FADS6的CpG4位点的甲基化程度西门塔尔牛品种显著高于云岭牛品种,CpG7位点的甲基化程度西门塔尔牛和云岭牛品种显著高于文山牛品种,除了 CpG7、CpG9、CpG19、CpG14、CpG15位点以外,其余位点的甲基化水平和表达量之间的关系为负相关。FASN的CpG1、CpG2、CpG3、CpG5、CpG7、CpG10位点甲基化水平和表达量之间的关系为负相关。
[Abstract]:In this study, the longissimus dorsi muscle tissue of Yunling cattle and Wenshan cattle in Southern Yellow cattle and Wenshan cattle were used as experimental materials, and the longest muscle tissues of Simmental cattle were used as control to compare the differences of meat quality among different breeds. The methylated patterns of longissimus dorsi muscle of different breeds were analyzed and compared by MethylRAD-seq and bisulfite sequencing PCR (BSP), and the effects of methylation on muscle quality of different breeds were analyzed. Based on the data of MethylRAD-seq and RNA-seq in laboratory, the regulation of methylated meat quality gene expression in different beef breeds was analyzed, and the mechanism of methylated regulation on bovine longissimus dorsi muscle was further studied. The results of the longest muscle quality of the back showed that there was no significant difference in pH value and water loss rate among Yunling cattle, Wenshan cattle and Simmental cattle (P 0.05). The percentage of fat in Yunling cattle and Wenshan cattle was significantly higher than that in Simmental cattle (P 0.05), and the protein content in Simmental cattle was significantly higher than that in Wenshan cattle (P 0.05). There was no significant difference in the contents of four common fatty acids (myristic acid, palmitic acid and stearic acid) among Yunling cattle, Wenshan cattle and Simmental cattle (P 0.05). The results of MethylRAD-seq analysis showed that Yunling cattle, Wenshan cattle and Simmental cattle were 7027017 (57.74%), 6405515 (56.37%), 6734119 (58.12%), 6092960 (54.82%), 5510367 (56.94%), 7204540 (62.06%), 6829702 (61.38%), 6759345 (54.58%), respectively. 6901684 (57.14%) of the reads could be compared to the unique position of the bovine reference genome. the main methylation pattern of the three bovine genomes was CPG type. The reads on the only comparison is mainly distributed on gene and mRNA. Compared with different cattle breeds, 39 different genes were detected in muscle tissue of Yunling cattle and 123 genes in muscle tissue of Wenshan cattle, with Simmental cattle as control group and Wenshan cattle as control group. A total of 104 differentially methylated genes were detected in muscle tissue of Yunling cattle, and only one differential gene (ALDH3A1) was significantly different among Simmental cattle, Wenshan cattle and Yunling cattle (P 0.05). KEGG pathway analysis showed that there was a significant difference in the level of methylation between Simmental cattle, Wenshan cattle and Yunling cattle. The main signal pathways of these methylated differentially enriched genes are amino acid metabolism, Ras signal pathway, 伪-linolenic acid metabolism, vascular smooth muscle contraction, fat digestion and absorption. The combined analysis of MethylRAD-seq and RNA-seq results showed that there were differences in the degree of methylation and expression of 21 genes between Simmental cattle and Wenshan cattle. There were 24 differentially methylated and expressed genes between Simmental cattle and Yunling cattle, and 80 differentially methylated and expressed genes between Wenshan cattle and Yunling cattle. In this study, bisulfite sequencing technique (BSP) was used to detect the degree of methylation in the promoter region of three candidate genes (ACAD11,FADS6,FASN), and the relationship between the expression of the gene and the degree of promoter region was analyzed. The results showed that the degree of DNA methylation of FADS6 and FASN was negatively correlated with the expression of DNA. The degree of DNA methylation of Simmental cattle FASN was significantly higher than that of Yunling cattle and Wenshan cattle (P 0.05). There was no significant difference in the degree of DNA methylation of FADS6 among the three breeds, but the degree of CpG4 methylation of FADS6 in Simmental cattle breeds was significantly higher than that in Yunling cattle breeds. The degree of methylation of CpG7 sites in Simmental cattle and Yunling cattle breeds was significantly higher than that in Wenshan cattle breeds. Except for CpG7,CpG9,CpG19,CpG14,CpG15 loci, there was a negative correlation between the methylated level and expression of other loci. CpG1,CpG2, of FASN There was a negative correlation between the level of methylation at CpG3,CpG5,CpG7,CpG10 site and the expression level.
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S823
[Abstract]:In this study, the longissimus dorsi muscle tissue of Yunling cattle and Wenshan cattle in Southern Yellow cattle and Wenshan cattle were used as experimental materials, and the longest muscle tissues of Simmental cattle were used as control to compare the differences of meat quality among different breeds. The methylated patterns of longissimus dorsi muscle of different breeds were analyzed and compared by MethylRAD-seq and bisulfite sequencing PCR (BSP), and the effects of methylation on muscle quality of different breeds were analyzed. Based on the data of MethylRAD-seq and RNA-seq in laboratory, the regulation of methylated meat quality gene expression in different beef breeds was analyzed, and the mechanism of methylated regulation on bovine longissimus dorsi muscle was further studied. The results of the longest muscle quality of the back showed that there was no significant difference in pH value and water loss rate among Yunling cattle, Wenshan cattle and Simmental cattle (P 0.05). The percentage of fat in Yunling cattle and Wenshan cattle was significantly higher than that in Simmental cattle (P 0.05), and the protein content in Simmental cattle was significantly higher than that in Wenshan cattle (P 0.05). There was no significant difference in the contents of four common fatty acids (myristic acid, palmitic acid and stearic acid) among Yunling cattle, Wenshan cattle and Simmental cattle (P 0.05). The results of MethylRAD-seq analysis showed that Yunling cattle, Wenshan cattle and Simmental cattle were 7027017 (57.74%), 6405515 (56.37%), 6734119 (58.12%), 6092960 (54.82%), 5510367 (56.94%), 7204540 (62.06%), 6829702 (61.38%), 6759345 (54.58%), respectively. 6901684 (57.14%) of the reads could be compared to the unique position of the bovine reference genome. the main methylation pattern of the three bovine genomes was CPG type. The reads on the only comparison is mainly distributed on gene and mRNA. Compared with different cattle breeds, 39 different genes were detected in muscle tissue of Yunling cattle and 123 genes in muscle tissue of Wenshan cattle, with Simmental cattle as control group and Wenshan cattle as control group. A total of 104 differentially methylated genes were detected in muscle tissue of Yunling cattle, and only one differential gene (ALDH3A1) was significantly different among Simmental cattle, Wenshan cattle and Yunling cattle (P 0.05). KEGG pathway analysis showed that there was a significant difference in the level of methylation between Simmental cattle, Wenshan cattle and Yunling cattle. The main signal pathways of these methylated differentially enriched genes are amino acid metabolism, Ras signal pathway, 伪-linolenic acid metabolism, vascular smooth muscle contraction, fat digestion and absorption. The combined analysis of MethylRAD-seq and RNA-seq results showed that there were differences in the degree of methylation and expression of 21 genes between Simmental cattle and Wenshan cattle. There were 24 differentially methylated and expressed genes between Simmental cattle and Yunling cattle, and 80 differentially methylated and expressed genes between Wenshan cattle and Yunling cattle. In this study, bisulfite sequencing technique (BSP) was used to detect the degree of methylation in the promoter region of three candidate genes (ACAD11,FADS6,FASN), and the relationship between the expression of the gene and the degree of promoter region was analyzed. The results showed that the degree of DNA methylation of FADS6 and FASN was negatively correlated with the expression of DNA. The degree of DNA methylation of Simmental cattle FASN was significantly higher than that of Yunling cattle and Wenshan cattle (P 0.05). There was no significant difference in the degree of DNA methylation of FADS6 among the three breeds, but the degree of CpG4 methylation of FADS6 in Simmental cattle breeds was significantly higher than that in Yunling cattle breeds. The degree of methylation of CpG7 sites in Simmental cattle and Yunling cattle breeds was significantly higher than that in Wenshan cattle breeds. Except for CpG7,CpG9,CpG19,CpG14,CpG15 loci, there was a negative correlation between the methylated level and expression of other loci. CpG1,CpG2, of FASN There was a negative correlation between the level of methylation at CpG3,CpG5,CpG7,CpG10 site and the expression level.
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S823
【参考文献】
相关期刊论文 前10条
1 张亚伟;赵丽萍;耿春银;孟庆翔;任丽萍;;中国牛肉食用品质评价方法研究及应用[J];中国农业大学学报;2017年02期
2 金显栋;王安奎;N惪，
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