沙葱提取物对杜寒杂交羊肌肉脂代谢相关基因表达及甲基化的影响
|
摘要
本试验旨在研究沙葱提取物对杜寒杂交羊肌肉脂代谢相关基因表达及甲基化的影响。采用单因素完全随机设计,选取60只体重(35～40 kg)相近、4.5月龄的杜寒杂交母羊,随机分为4组,每组15只。对照组(T1组)饲喂基础饲粮,试验组在基础饲粮中每只分别添加沙葱粉(10.0 g/d,T2组)、沙葱水溶性提取物(3.2 g/d,T3组)、沙葱脂溶性提取物(2.8 g/d,T4组)。预试期为15 d,正试期为60 d。正试期结束后,每组随机选取3只羊屠宰,采集背最长肌样品。采用目的区域甲基化水平测序(二代测序)检测乙酰辅酶A羧化酶α(ACACA)、硬脂酰辅酶A去饱和酶(SCD)、固醇调节元件结合转录因子1(SREBF1)基因转录起始位点上游2K至第一外显子下游1K区域内胞嘧啶-磷酸-鸟嘌呤(CpG)岛甲基化水平,实时荧光定量PCR法测定其基因相对表达量。结果表明:1)试验第1～30天,T2组干物质采食量显著低于其他3组(P <0.05)。2)与T1组相比,T2组、T3组、T4组ACACA、SCD基因相对表达量均有不同程度的提高,其中T3组>T2组>T4组>T1组。T3组ACACA、SCD、SREBF1基因相对表达量均显著高于T1组(P <0. 05),T2组ACACA基因相对表达量显著高于T1组(P <0. 05),T3组ACACA、SREBF1基因相对表达量显著高于T4组(P<0.05)。3) ACACA、SCD、SREBF1基因各CpG岛及位点均呈现高度去甲基化状态,且各组间CpG岛的平均甲基化水平差异不显著(P> 0.05)。ACACA、SCD、SREBF1的基因表达与CpG岛甲基化水平不相关(P>0.05)。综上所述,沙葱及其提取物可以上调杜寒杂交羊背最长肌ACACA、SCD、SREBF1的基因表达,饲粮添加沙葱水溶性提取物的上调幅度最大,沙葱粉次之,沙葱脂溶性提取物最小,且这种上调与特定区域的DNA甲基化修饰无关。
This experiment was conducted to investigate the effects of Allium mongolicum Regel extracts on lipid metabolism related gene expression and methylation in muscle of Dorper×thin-tailed Han crossbred sheep. A single factor complete random design was used in the current study,sixty 4. 5-month-old Dorper × thin-tailed Han crossbred ewes with similar body weight( 35 to 40 kg) were assigned to 4 groups with 15 ewes in each group. Sheep in the control group( T1 group) were fed a basal diet,and the others in experimental groups were fed the basal diet supplemented with Allium mongolicum Regel powder( 10. 0 g/d each sheep,T2 group),Allium mongolicum Regel water-soluble extract( 3.2 g/d each sheep,T3 group),Allium mongolicum Regel fat-soluble extract( 2.8 g/d each sheep,T4 group),respectively. The pre-trial lasted for 15 days,and the trial lasted for 60 days. After the trial,three sheep were randomly selected from each group and slaughtered to collect the longissimus dorsi muscle samples. The cytosine-phosphoric acid-guanine( CpG) island methylation level of acetyl-CoA carboxylase α( ACACA),stearoyl-CoA desaturase( SCD) and sterol regulatory element binding transcription factor 1( SREBF1) genes from 2 K upstream of transcriptional start site to 1 K downstream of the first exon were measured according to methylation level sequencing of target region( second generation sequencing),and the gene relative expression levels were determined by real-time quantitative polymerase chain reaction. The results showed as follows: 1) during the 1 to 30 days of the experiment,the dry matter intake in T2 group was significantly lower than that in other 3 groups( P<0.05). 2) Compared with T1 group,the relative gene expression levels of ACACA and SCD in T2 group,T3 group and T4 group were increased at different degrees,among them,T3 group>T2 group>T4 group>T1 group. The relative expression levels of ACACA,SCD and SREBF1 genes in T3 group were significantly higher than those in T1 group( P<0.05),the relative expression level of ACACA gene in T2 group was significantly higher than that in T1 group( P<0.05),and the relative expression levels of ACACA and SREBF1 genes in T3 group were significantly higher than those in T4 group( P<0.05). 3) A state of high demethylation was observed for each CpG island and site of ACACA,SCD and SREBF1 genes,and no significant differences were found in mean methylation level of CpG islands among four groups( P>0.05). The gene expressions of CACA,SCD and SREBF1 were not correlated with methylation level of CpG islands( P>0.05). In conclusion,Allium mongolicum Regel and its extracts can up regulate ACACA,SCD and SREBF1 gene expressions in longissimus dorsi muscle of Dorper×thin-tailed Han crossbred sheep,diet supplemented with Allium mongolicum Regel water-soluble extract has the largest range of up-regulation,followed by Allium mongolicum Regel powder,Allium mongolicum Regel fat-soluble extract is the lowest,and this up-regulation is not associated with DNA methylation modification of specific regions.[Chinese Journal of Animal Nutrition,2019,31( 5) : 2340-2349]
This experiment was conducted to investigate the effects of Allium mongolicum Regel extracts on lipid metabolism related gene expression and methylation in muscle of Dorper×thin-tailed Han crossbred sheep. A single factor complete random design was used in the current study,sixty 4. 5-month-old Dorper × thin-tailed Han crossbred ewes with similar body weight( 35 to 40 kg) were assigned to 4 groups with 15 ewes in each group. Sheep in the control group( T1 group) were fed a basal diet,and the others in experimental groups were fed the basal diet supplemented with Allium mongolicum Regel powder( 10. 0 g/d each sheep,T2 group),Allium mongolicum Regel water-soluble extract( 3.2 g/d each sheep,T3 group),Allium mongolicum Regel fat-soluble extract( 2.8 g/d each sheep,T4 group),respectively. The pre-trial lasted for 15 days,and the trial lasted for 60 days. After the trial,three sheep were randomly selected from each group and slaughtered to collect the longissimus dorsi muscle samples. The cytosine-phosphoric acid-guanine( CpG) island methylation level of acetyl-CoA carboxylase α( ACACA),stearoyl-CoA desaturase( SCD) and sterol regulatory element binding transcription factor 1( SREBF1) genes from 2 K upstream of transcriptional start site to 1 K downstream of the first exon were measured according to methylation level sequencing of target region( second generation sequencing),and the gene relative expression levels were determined by real-time quantitative polymerase chain reaction. The results showed as follows: 1) during the 1 to 30 days of the experiment,the dry matter intake in T2 group was significantly lower than that in other 3 groups( P<0.05). 2) Compared with T1 group,the relative gene expression levels of ACACA and SCD in T2 group,T3 group and T4 group were increased at different degrees,among them,T3 group>T2 group>T4 group>T1 group. The relative expression levels of ACACA,SCD and SREBF1 genes in T3 group were significantly higher than those in T1 group( P<0.05),the relative expression level of ACACA gene in T2 group was significantly higher than that in T1 group( P<0.05),and the relative expression levels of ACACA and SREBF1 genes in T3 group were significantly higher than those in T4 group( P<0.05). 3) A state of high demethylation was observed for each CpG island and site of ACACA,SCD and SREBF1 genes,and no significant differences were found in mean methylation level of CpG islands among four groups( P>0.05). The gene expressions of CACA,SCD and SREBF1 were not correlated with methylation level of CpG islands( P>0.05). In conclusion,Allium mongolicum Regel and its extracts can up regulate ACACA,SCD and SREBF1 gene expressions in longissimus dorsi muscle of Dorper×thin-tailed Han crossbred sheep,diet supplemented with Allium mongolicum Regel water-soluble extract has the largest range of up-regulation,followed by Allium mongolicum Regel powder,Allium mongolicum Regel fat-soluble extract is the lowest,and this up-regulation is not associated with DNA methylation modification of specific regions.[Chinese Journal of Animal Nutrition,2019,31( 5) : 2340-2349]
引文
[1] BAIK M,KANG H J,PARK S J,et al. Triennial grow th and development symposium:molecular mechanisms related to bovine intramuscular fat deposition in the longissimus muscle[J]. Journal of Animal Science,2017,95(5):2284-2203.
[2]解小莉,俞英,袁志发,等.奶牛产奶性状与乳房炎相关基因CpG含量及分布特征的比较分析[J].遗传,2012,34(4):437-444.
[3] LILLYCROP K A,PHILLIPS E S,JACKSON A A,et al. Dietary protein restriction of pregnant rats induces and folic acid supplementation prevents epigenetic modification of hepatic gene expression in the offspring[J]. The Journal of Nutrition,2005,135(6):1382-1386.
[4] AMARAL C L,CRISMA A R,MASI L N,et al.DNA methylation changes induced by a high-fat diet and fish oil supplementation in the skeletal muscle of mice[J]. Journal of Nutrigenetics and Nutrigenomics,2014,7(4/5/6):314-326.
[5] TIAN P,LUO Y W,LI X,et al. Negative effects of long-term feeding of high-grain diets to lactating goats on milk fat production and composition by regulating gene expression and DNA methylation in the mammary gland[J]. Journal of Animal Science and Biotechnology,2017,8:74.
[6] FUKUOKA H,KUBOTA T. One-carbon metabolism and lipid metabolism in DOHaD[M]//KUBOTA T,FUKUOKA H. Developmental origins of health and disease(DOHaD). Singapore:Springer,2018,1012:3-9.
[7] GERAGHTY A A,LINDSAY K L,ALBERDI G,et al.Nutrition during pregnancy impacts offspring’s epigenetic status-evidence from human and animal studies[J].Nutrition and Metabolic Insights,2016,8(Suppl.1):41-47.
[8]木其尔.沙葱黄酮对肉羊机体抗氧化和免疫功能的影响及其机理研究[D].博士学位论文.呼和浩特:内蒙古农业大学,2016:32-52.
[9]张巧娥.沙葱提取物的分离鉴定及其对绵羊消化道共轭亚油酸含量和胴体脂肪沉积影响的研究[D].博士学位论文.呼和浩特:内蒙古农业大学,2007:94-116.
[10]张巧娥,敖长金.沙葱提取物中营养成分的分析[J].畜牧与兽医,2008,40(5):53-54.
[11]赵国芬.沙葱和油料籽实对绵羊瘤胃发酵、营养物质消化流通及胴体品质影响的研究[D].博士学位论文.呼和浩特:内蒙古农业大学,2005:130-144.
[12]卢媛.沙葱、地椒风味活性成分及其对绵羊瘤胃发酵和羊肉风味的影响[D].硕士学位论文.呼和浩特:内蒙古农业大学,2002:36-64.
[13]刘洁.肉用绵羊饲料代谢能与代谢蛋白质预测模型的研究[D].博士学位论文.北京:中国农业科学院,2012:41-42.
[14] VAN SOEST P J,ROBERTSON J B,LEWIS B A.M ethods for dietary fiber,neutral detergent fiber,and nonstarch polysaccharides in relation to animal nutrition[J]. Journal of Dairy Science,1991,74(10):3583-3597.
[15] DERVISHI E,SERRANO C,JOY M,et al.The effect of feeding system in the expression of genes related w ith fat metabolism in semitendinous muscle in sheep[J].Meat Science,2011,89(1):91-97.
[16] ZHOU S Y,CAI B Y,ZHANG Z Z,et al. CDKN2B methylation and aortic arch calcification in patients w ith ischemic stroke[J]. Journal of Atherosclerosis and Thrombosis,2017,24(6):609-620.
[17]哈斯额尔敦.沙葱提取物对绵羊脂肪代谢的影响[D].硕士学位论文.呼和浩特:内蒙古农业大学,2007:60-62.
[18]郑彦楷.沙葱水溶性提取物对肉羊脂肪代谢相关基因表达量及甲基化的影响[D].硕士学位论文.呼和浩特:内蒙古农业大学,2017:21-25.
[19] NAFIKOV R A,SCHOONMAKER J P,KORN K T,et al. Sterol regulatory element binding transcription factor 1(SREBF1)polymorphism and milk fatty acid composition[J]. Journal of Dairy Science,2013,96(4):2605-2616.
[20] BOUCHARD-MERCIER A,RUDKOWSKA I,LEM IEUX S,et al.SREBF1 gene variations modulate insulin sensitivity in response to a fish oil supplementation[J].Lipids in Health and Disease,2014,13:152.
[21] CHEN J,YANG X J,XIA D,et al. Sterol regulatory element binding transcription factor 1 expression and genetic polymorphism significantly affect intramuscular fat deposition in the longissimus muscle of Erhualian and Sutai pigs[J]. Journal of Animal Science,2008,86(1):57-63.
[22]王波,罗海玲.DNA甲基化与去甲基化调控肌肉发育研究进展[J].动物营养学报,2017,29(8):2622-2629.
[23] CARRIOE,SUELVES M. DNA methylation dynamics in muscle development and disease[J].Frontiers in Aging Neuroscience,2015,7:19.
[24]赵海谕.奶山羊PITX1、PITX2基因的遗传变异、mRNA表达及DNA甲基化修饰对泌乳性能的影响[D].硕士学位论文.杨凌:西北农林科技大学,2014:36-38.
[25] OSORIO J S,JACOMETO C B,ZHOU Z,et al. Hepatic global DNA and peroxisome proliferator-activated receptor alpha promoter methylation are altered in peripartal dairy cow s fed rumen-protected methionine[J].Journal of Dairy Science,2016,99(1):234-244.
[26] PINNEY S E.Mammalian non-CpG methylation:stem cells and beyond[J].Biology,2014,3(4):739-751.
[2]解小莉,俞英,袁志发,等.奶牛产奶性状与乳房炎相关基因CpG含量及分布特征的比较分析[J].遗传,2012,34(4):437-444.
[3] LILLYCROP K A,PHILLIPS E S,JACKSON A A,et al. Dietary protein restriction of pregnant rats induces and folic acid supplementation prevents epigenetic modification of hepatic gene expression in the offspring[J]. The Journal of Nutrition,2005,135(6):1382-1386.
[4] AMARAL C L,CRISMA A R,MASI L N,et al.DNA methylation changes induced by a high-fat diet and fish oil supplementation in the skeletal muscle of mice[J]. Journal of Nutrigenetics and Nutrigenomics,2014,7(4/5/6):314-326.
[5] TIAN P,LUO Y W,LI X,et al. Negative effects of long-term feeding of high-grain diets to lactating goats on milk fat production and composition by regulating gene expression and DNA methylation in the mammary gland[J]. Journal of Animal Science and Biotechnology,2017,8:74.
[6] FUKUOKA H,KUBOTA T. One-carbon metabolism and lipid metabolism in DOHaD[M]//KUBOTA T,FUKUOKA H. Developmental origins of health and disease(DOHaD). Singapore:Springer,2018,1012:3-9.
[7] GERAGHTY A A,LINDSAY K L,ALBERDI G,et al.Nutrition during pregnancy impacts offspring’s epigenetic status-evidence from human and animal studies[J].Nutrition and Metabolic Insights,2016,8(Suppl.1):41-47.
[8]木其尔.沙葱黄酮对肉羊机体抗氧化和免疫功能的影响及其机理研究[D].博士学位论文.呼和浩特:内蒙古农业大学,2016:32-52.
[9]张巧娥.沙葱提取物的分离鉴定及其对绵羊消化道共轭亚油酸含量和胴体脂肪沉积影响的研究[D].博士学位论文.呼和浩特:内蒙古农业大学,2007:94-116.
[10]张巧娥,敖长金.沙葱提取物中营养成分的分析[J].畜牧与兽医,2008,40(5):53-54.
[11]赵国芬.沙葱和油料籽实对绵羊瘤胃发酵、营养物质消化流通及胴体品质影响的研究[D].博士学位论文.呼和浩特:内蒙古农业大学,2005:130-144.
[12]卢媛.沙葱、地椒风味活性成分及其对绵羊瘤胃发酵和羊肉风味的影响[D].硕士学位论文.呼和浩特:内蒙古农业大学,2002:36-64.
[13]刘洁.肉用绵羊饲料代谢能与代谢蛋白质预测模型的研究[D].博士学位论文.北京:中国农业科学院,2012:41-42.
[14] VAN SOEST P J,ROBERTSON J B,LEWIS B A.M ethods for dietary fiber,neutral detergent fiber,and nonstarch polysaccharides in relation to animal nutrition[J]. Journal of Dairy Science,1991,74(10):3583-3597.
[15] DERVISHI E,SERRANO C,JOY M,et al.The effect of feeding system in the expression of genes related w ith fat metabolism in semitendinous muscle in sheep[J].Meat Science,2011,89(1):91-97.
[16] ZHOU S Y,CAI B Y,ZHANG Z Z,et al. CDKN2B methylation and aortic arch calcification in patients w ith ischemic stroke[J]. Journal of Atherosclerosis and Thrombosis,2017,24(6):609-620.
[17]哈斯额尔敦.沙葱提取物对绵羊脂肪代谢的影响[D].硕士学位论文.呼和浩特:内蒙古农业大学,2007:60-62.
[18]郑彦楷.沙葱水溶性提取物对肉羊脂肪代谢相关基因表达量及甲基化的影响[D].硕士学位论文.呼和浩特:内蒙古农业大学,2017:21-25.
[19] NAFIKOV R A,SCHOONMAKER J P,KORN K T,et al. Sterol regulatory element binding transcription factor 1(SREBF1)polymorphism and milk fatty acid composition[J]. Journal of Dairy Science,2013,96(4):2605-2616.
[20] BOUCHARD-MERCIER A,RUDKOWSKA I,LEM IEUX S,et al.SREBF1 gene variations modulate insulin sensitivity in response to a fish oil supplementation[J].Lipids in Health and Disease,2014,13:152.
[21] CHEN J,YANG X J,XIA D,et al. Sterol regulatory element binding transcription factor 1 expression and genetic polymorphism significantly affect intramuscular fat deposition in the longissimus muscle of Erhualian and Sutai pigs[J]. Journal of Animal Science,2008,86(1):57-63.
[22]王波,罗海玲.DNA甲基化与去甲基化调控肌肉发育研究进展[J].动物营养学报,2017,29(8):2622-2629.
[23] CARRIOE,SUELVES M. DNA methylation dynamics in muscle development and disease[J].Frontiers in Aging Neuroscience,2015,7:19.
[24]赵海谕.奶山羊PITX1、PITX2基因的遗传变异、mRNA表达及DNA甲基化修饰对泌乳性能的影响[D].硕士学位论文.杨凌:西北农林科技大学,2014:36-38.
[25] OSORIO J S,JACOMETO C B,ZHOU Z,et al. Hepatic global DNA and peroxisome proliferator-activated receptor alpha promoter methylation are altered in peripartal dairy cow s fed rumen-protected methionine[J].Journal of Dairy Science,2016,99(1):234-244.
[26] PINNEY S E.Mammalian non-CpG methylation:stem cells and beyond[J].Biology,2014,3(4):739-751.