云嶺牛和文山牛背最長肌肉質(zhì)特性及DNA甲基化分析
發(fā)布時(shí)間：2019-06-03 03:25
【摘要】：本研究以南方黃牛云嶺牛和文山牛的背最長肌組織為實(shí)驗(yàn)材料,以西門塔爾牛的背最長肌組織為對照,比較不同牛品種的肉品質(zhì)的差異,利用甲基化修飾依賴性內(nèi)切酶測序法(MethylRAD-seq)和亞硫酸氫鹽測序PCR(BSP)技術(shù)分析比較不同品種牛背最長肌的甲基化圖譜,分析甲基化對不同品種牛肌肉肉質(zhì)的調(diào)控影響。結(jié)合MethylRAD-seq和實(shí)驗(yàn)室已有的RNA-seq的數(shù)據(jù),分析甲基化對不同牛種肉質(zhì)基因的表達(dá)調(diào)控,進(jìn)一步研究甲基化對牛背最長肌的調(diào)控機(jī)制。背最長肌肉品質(zhì)測定結(jié)果表明,牛肉中pH值和失水率在云嶺牛、文山牛和西門塔爾牛之間差異不顯著(P0.05),云嶺牛和文山牛的脂肪百分含量顯著高于西門塔爾牛(P0.05),西門塔爾牛的蛋白質(zhì)含量顯著高于文山牛(P0.05)。本次實(shí)驗(yàn)測定的4種常見脂肪酸(肉豆蔻酸、棕櫚酸、棕櫚油酸和硬脂酸)在云嶺牛,文山牛和西門塔爾牛之間的含量差異不顯著(P0.05)。MethylRAD-seq分析結(jié)果表明,云嶺牛、文山牛和西門塔爾牛分別有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%)個reads可以比對上牛參考基因組的唯一位置,這三種牛基因組中主要的甲基化模式是CpG型。唯一比對上的reads主要分布在gene和mRNA上。不同牛品種之間比較,以西門塔爾牛為對照組,在云嶺牛肌肉組織中共檢測到39個甲基化差異基因,在文山牛肌肉組織中共檢測到123個甲基化差異基因,以文山牛為對照組,云嶺牛肌肉組織中共檢測到104個甲基化差異基因,只有一個差異基因(ALDH3A1)在西門塔爾牛、文山牛和云嶺牛之間甲基化水平均顯著差異(P0.05)。KEGG pathway分析表明,這些甲基化差異基因顯著富集的信號通路主要是氨基酸代謝,Ras信號通路、α-亞麻酸代謝、血管平滑肌收縮、脂肪消化吸收、醚類脂質(zhì)代謝等(P0.05)。MethylRAD-seq和實(shí)驗(yàn)室已有的RNA-seq結(jié)果的聯(lián)合分析表明,西門塔爾牛和文山牛之間有21個基因甲基化程度和表達(dá)量均差異,西門塔爾牛和云嶺牛之間有24個差異甲基化和表達(dá)基因,文山牛和云嶺牛之間有80個差異甲基化和表達(dá)基因。本研究利用亞硫酸氫鹽測序技術(shù)(BSP)檢測3個候選基因(ACAD11,FADS6,FASN)啟動子區(qū)甲基化程度,并分析基因表達(dá)量與啟動子區(qū)甲基化程度之間的關(guān)系。結(jié)果表明,FADS6和FASN整體的DNA甲基化程度與表達(dá)量呈現(xiàn)負(fù)相關(guān),西門塔爾牛FASN的DNA甲基化程度顯著高于云嶺牛和文山牛(P0.05),FADS6的DNA甲基化程度在三個品種的牛之間差異不顯著,FADS6的CpG4位點(diǎn)的甲基化程度西門塔爾牛品種顯著高于云嶺牛品種,CpG7位點(diǎn)的甲基化程度西門塔爾牛和云嶺牛品種顯著高于文山牛品種,除了 CpG7、CpG9、CpG19、CpG14、CpG15位點(diǎn)以外,其余位點(diǎn)的甲基化水平和表達(dá)量之間的關(guān)系為負(fù)相關(guān)。FASN的CpG1、CpG2、CpG3、CpG5、CpG7、CpG10位點(diǎn)甲基化水平和表達(dá)量之間的關(guān)系為負(fù)相關(guān)。
[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, 偽-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.
【學(xué)位授予單位】：揚(yáng)州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：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, 偽-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.
【學(xué)位授予單位】：揚(yáng)州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S823
【參考文獻(xiàn)】
相關(guān)期刊論文 前10條
1 張亞偉;趙麗萍;耿春銀;孟慶翔;任麗萍;;中國牛肉食用品質(zhì)評價(jià)方法研究及應(yīng)用[J];中國農(nóng)業(yè)大學(xué)學(xué)報(bào);2017年02期
2 金顯棟;王安奎;N惪，
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