虹鳟HMGCR基因克隆及组织差异表达分析
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摘要
[目的]阐述虹鳟β-羟-β-甲基戊二酰辅酶A还原酶(HMGCR)基因的mRNA序列,并比较其在虹鳟不同组织器官中的表达差异。[方法]通过PCR技术扩增虹鳟HMGCR基因的mRNA序列,采用荧光定量PCR技术,比较HMGCR基因在虹鳟肝脏、肠道、胃、心脏、鳃、脾脏、肾脏、肌肉8种组织器官中的表达差异。[结果]虹鳟HMGCR基因的mRNA序列与大西洋鲑的同源性达97%,其蛋白序列与脊椎动物的同源性都在70%以上,表明该基因在物种进化过程中比较保守;该基因在虹鳟肝脏、肠道、胃、心脏、鳃、脾脏、肾脏、肌肉8种组织器官中均有表达;其中,该基因在肝脏和心脏中的表达量较高,而在胃中的表达量最低。[结论]该研究克隆出虹鳟胆固醇合成关键限速酶HMGCR的部分mRNA序列,分析了该基因在不同组织中的表达情况,为该酶的深入研究奠定基础。
[Objective]The purpose of this study was to reveal the mRNA sequence of 3-hydroxy-3-methylglutaryl-coenzyme A reductase(HMGCR) and compare the gene expression levels in different tissues of rainbow trout(Oncorhynchus mykiss).[Method]The partial sequence of HMGCR gene was amplified by PCR technology and the gene expression differences among tissues such as liver,intestine,stomach,heart,gill,spleen,kidney and muscle were revealed by fluorescence quantitative PCR technology.[Result]The amino acid sequence shared 97% homology with Salmo salar,and the deduced protein sequence shared more than 70% homology with vertebrate,indicating that the gene was more conservative in the process of species evolution.The gene expression could be detected in the liver,intestines,stomach,heart,gill spleen,kidney and muscle of rainbow trout.The expression level of HMGCR gene in liver and heart was the highest,whereas which was the lowest in the stomach.[Conclusion]We cloned the partial RNA sequence of HMGCR,a key rate-limiting enzyme for cholesterol synthesis in rainbow trout,and analyzed the expression of HMGCR in different tissues,which laid a foundation for further study of the enzyme.
[Objective]The purpose of this study was to reveal the mRNA sequence of 3-hydroxy-3-methylglutaryl-coenzyme A reductase(HMGCR) and compare the gene expression levels in different tissues of rainbow trout(Oncorhynchus mykiss).[Method]The partial sequence of HMGCR gene was amplified by PCR technology and the gene expression differences among tissues such as liver,intestine,stomach,heart,gill,spleen,kidney and muscle were revealed by fluorescence quantitative PCR technology.[Result]The amino acid sequence shared 97% homology with Salmo salar,and the deduced protein sequence shared more than 70% homology with vertebrate,indicating that the gene was more conservative in the process of species evolution.The gene expression could be detected in the liver,intestines,stomach,heart,gill spleen,kidney and muscle of rainbow trout.The expression level of HMGCR gene in liver and heart was the highest,whereas which was the lowest in the stomach.[Conclusion]We cloned the partial RNA sequence of HMGCR,a key rate-limiting enzyme for cholesterol synthesis in rainbow trout,and analyzed the expression of HMGCR in different tissues,which laid a foundation for further study of the enzyme.
引文
[1] 黄雨薇,叶元土,蔡春芳,等.氧化鱼油对草鱼(Ctenopharyngodon idelluspond)肝胰脏、肠道胆固醇、胆汁酸合成代谢的影响[J].基因组学与应用生物学,2015,34(8):1636-1646.
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[3] KLIEWER S A,MOORE J T,WADE L,et al.An orphan nuclear receptor activated by pregnanes defines a novel steroid signaling pathway[J].Cell,1998,92(1):73-82.
[4] 汪留全,李海洋,胡王,等.饲料中胆固醇水平对幼蟹生长和饲料利用率影响的研究[J].淡水渔业,2004,34(1):13-15.
[5] 温小波,陈立侨.磷脂和胆固醇在虾蟹类营养中的研究进展[J].淡水渔业,2000,30(5):25-27.
[6] 安光全.鸡HMGCR基因多态性与血清VLDL浓度和繁殖性状的关联研究[D].雅安:四川农业大学,2010:1-5.
[7] 魏杨.鸡HMGCR基因多态性及其与经济性状相关性研究[D].郑州:河南农业大学,2012:4-6.
[8] 丛日华.品种和母猪日粮蛋白水平对仔猪肝脏胆固醇代谢的影响及其机制[D].南京:南京农业大学,2011:2-4.
[9] 黄雨薇.氧化鱼油、丙二醛对草鱼(Ctenopharyngodon idellus)肝胰脏、肠道胆固醇、胆汁酸合成代谢的影响[D].苏州:苏州大学,2015:2-4.
[10] NESS G C,WIGGINS L,ZHAO Z H.Insulin increases hepatic 3-hydroxy-methylglutaryl coenzyme A reductase mRNA and immunoreactive protein levels in diabetic rats[J].Archives of biochemistry & biophysics,1994,309(1):193-194.
[11] LATOUR M A,PEEBLES E D,DOYLE S M,et al.Broiler breeder age and dietary fat influence the yolk fatty acid profiles of fresh eggs and newly hatched chicks[J].Poult Science,1998,77(1):47-53.
[12] NESS G C,LOPEZ D,CHAMBERS C M,et al.Effects of L-triiodothyronine and the thyromimetic L-94901 on serum lipoprotein levels and hepatic low-density lipoprotein receptor,3-hydroxy-3-methylglutaryl coenzyme A reductase,and apo A-I gene expression[J].Biochemical pharmacology,1998,56(1):121-129.
[13] 李平华,黄阳,贺丽春,等.不同来源油脂及剂量对仔猪生长性能和血清脂质指标以及肝脏胆固醇代谢的影响[J].南京农业大学学报,2017,40(4):710-717.
[14] PLAT J,MENSINK R P.Relationship of genetic variation in genes encoding apolipoprotein A-IV,scavenger receptor BI,HMG-CoA reductase,CETP and apolipoprotein E with cholesterol metabolism and the response to plant stanol ester consumption[J].European journal of clinical investigation,2015,32(4):242-250.
[15] SONG B L,DEBOSEBOYD R A.Ubiquitination of 3-hydroxy-3-methylglutaryl-CoA reductase in permeabilized cells mediated by cytosolic E1 and a putative membrane-bound ubiquitin ligase[J].Journal of biological chemistry,2004,279(27):28798-28799.
[16] 魏健,江路易,宋保亮.胆固醇的内源合成与小肠吸收[J].生命科学,2015,27(7):847-858.
[17] NESS G C,HOLLAND R C.Degradation of HMG-CoA reductase in rat liver is cholesterol and ubiquitin independent[J].FEBS Letters,2005,579(14):3126-3130.
[18] DUCKWORTH P F,VLAHCEVIC Z R,STUDER E J,et al.Effect of hydrophobic bile acids on 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity and mRNA levels in the rat[J].Journal of biological chemistry,1991,266(15):9413-9414.
[19] BAKKER-ARKEMA R G,DAVIDSON M H,GOLDSTEIN R J,et al.Efficacy and safety of a new HMG-CoA reductase inhibitor,atorvastatin,in patients with hypertriglyceridemia[J].Jama the journal of the American medical association,1996,275(2):128-133.
[20] 甘玲.畜禽胆固醇合成关键酶HMGR的生物信息学分析[J].中国畜牧杂志,2010,46(7):5-8.
[21] 邹思湘.动物生物化学[M].北京:中国农业出版社,2005:201-205.
[22] 黄艳玲,罗绪刚.饲粮脂肪酸会对脂肪代谢酶有关基因表达的影响[J].动物营养学报,2005,17(4):1-5.
[23] 郭晓强,郭振清.SREBP介导的胆固醇生物合成反馈调节[J].生命的化学,2007,27(4):292-293.
[24] 王晓凤.猪HMGR基因的克隆、表达、染色体定位及多态性分析[D].北京:中国农业大学,2006:90-91.
[25] LANGE Y,STECK T L,YE J,et al.Regulation of fibroblast mitochondrial 27-hydroxycholesterol production by active plasma membrane cholesterol[J].Journal of lipid research,2009,50:1881-1888.
[26] 张燕萍,钟杰,周秋白,等.黄鳝PLA2基因的克隆及表达分析[J].淡水渔业,2017,47(3):16-24.
[27] LANGE Y,YE J,STECK T L.Activation of membrane cholesterol by displacement from phospholipids[J].Journal of biological chemistry,2005,280:36126-36131.
[2] SHEEN S S.Dietary cholesterol requirement of juvenile mud crab Scylla serrata[J].Aquaculture,2000,189(3):277-285.
[3] KLIEWER S A,MOORE J T,WADE L,et al.An orphan nuclear receptor activated by pregnanes defines a novel steroid signaling pathway[J].Cell,1998,92(1):73-82.
[4] 汪留全,李海洋,胡王,等.饲料中胆固醇水平对幼蟹生长和饲料利用率影响的研究[J].淡水渔业,2004,34(1):13-15.
[5] 温小波,陈立侨.磷脂和胆固醇在虾蟹类营养中的研究进展[J].淡水渔业,2000,30(5):25-27.
[6] 安光全.鸡HMGCR基因多态性与血清VLDL浓度和繁殖性状的关联研究[D].雅安:四川农业大学,2010:1-5.
[7] 魏杨.鸡HMGCR基因多态性及其与经济性状相关性研究[D].郑州:河南农业大学,2012:4-6.
[8] 丛日华.品种和母猪日粮蛋白水平对仔猪肝脏胆固醇代谢的影响及其机制[D].南京:南京农业大学,2011:2-4.
[9] 黄雨薇.氧化鱼油、丙二醛对草鱼(Ctenopharyngodon idellus)肝胰脏、肠道胆固醇、胆汁酸合成代谢的影响[D].苏州:苏州大学,2015:2-4.
[10] NESS G C,WIGGINS L,ZHAO Z H.Insulin increases hepatic 3-hydroxy-methylglutaryl coenzyme A reductase mRNA and immunoreactive protein levels in diabetic rats[J].Archives of biochemistry & biophysics,1994,309(1):193-194.
[11] LATOUR M A,PEEBLES E D,DOYLE S M,et al.Broiler breeder age and dietary fat influence the yolk fatty acid profiles of fresh eggs and newly hatched chicks[J].Poult Science,1998,77(1):47-53.
[12] NESS G C,LOPEZ D,CHAMBERS C M,et al.Effects of L-triiodothyronine and the thyromimetic L-94901 on serum lipoprotein levels and hepatic low-density lipoprotein receptor,3-hydroxy-3-methylglutaryl coenzyme A reductase,and apo A-I gene expression[J].Biochemical pharmacology,1998,56(1):121-129.
[13] 李平华,黄阳,贺丽春,等.不同来源油脂及剂量对仔猪生长性能和血清脂质指标以及肝脏胆固醇代谢的影响[J].南京农业大学学报,2017,40(4):710-717.
[14] PLAT J,MENSINK R P.Relationship of genetic variation in genes encoding apolipoprotein A-IV,scavenger receptor BI,HMG-CoA reductase,CETP and apolipoprotein E with cholesterol metabolism and the response to plant stanol ester consumption[J].European journal of clinical investigation,2015,32(4):242-250.
[15] SONG B L,DEBOSEBOYD R A.Ubiquitination of 3-hydroxy-3-methylglutaryl-CoA reductase in permeabilized cells mediated by cytosolic E1 and a putative membrane-bound ubiquitin ligase[J].Journal of biological chemistry,2004,279(27):28798-28799.
[16] 魏健,江路易,宋保亮.胆固醇的内源合成与小肠吸收[J].生命科学,2015,27(7):847-858.
[17] NESS G C,HOLLAND R C.Degradation of HMG-CoA reductase in rat liver is cholesterol and ubiquitin independent[J].FEBS Letters,2005,579(14):3126-3130.
[18] DUCKWORTH P F,VLAHCEVIC Z R,STUDER E J,et al.Effect of hydrophobic bile acids on 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity and mRNA levels in the rat[J].Journal of biological chemistry,1991,266(15):9413-9414.
[19] BAKKER-ARKEMA R G,DAVIDSON M H,GOLDSTEIN R J,et al.Efficacy and safety of a new HMG-CoA reductase inhibitor,atorvastatin,in patients with hypertriglyceridemia[J].Jama the journal of the American medical association,1996,275(2):128-133.
[20] 甘玲.畜禽胆固醇合成关键酶HMGR的生物信息学分析[J].中国畜牧杂志,2010,46(7):5-8.
[21] 邹思湘.动物生物化学[M].北京:中国农业出版社,2005:201-205.
[22] 黄艳玲,罗绪刚.饲粮脂肪酸会对脂肪代谢酶有关基因表达的影响[J].动物营养学报,2005,17(4):1-5.
[23] 郭晓强,郭振清.SREBP介导的胆固醇生物合成反馈调节[J].生命的化学,2007,27(4):292-293.
[24] 王晓凤.猪HMGR基因的克隆、表达、染色体定位及多态性分析[D].北京:中国农业大学,2006:90-91.
[25] LANGE Y,STECK T L,YE J,et al.Regulation of fibroblast mitochondrial 27-hydroxycholesterol production by active plasma membrane cholesterol[J].Journal of lipid research,2009,50:1881-1888.
[26] 张燕萍,钟杰,周秋白,等.黄鳝PLA2基因的克隆及表达分析[J].淡水渔业,2017,47(3):16-24.
[27] LANGE Y,YE J,STECK T L.Activation of membrane cholesterol by displacement from phospholipids[J].Journal of biological chemistry,2005,280:36126-36131.