鲤lipea基因表达的时空特征及与脂肪沉积相关性分析
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摘要
本文旨在克隆鲤(Cyprinus carpio)激素敏感性脂肪酶a (hormone-sensitive lipase a, lipea)基因,探究其时空表达特征及其与脂肪沉积的关系,以期为鲤脂肪沉积分子调控机制研究奠定理论基础。本实验采用RACE技术克隆获得鲤lipea基因,其cDNA全长为3379 bp,包括230 bp的5′非翻译区(5′-UTR)、1067 bp的3′非翻译区(3′-UTR)和编码693个氨基酸的开放阅读框。多序列比对显示,鲤lipea与斑马鱼(Danio rerio)、牙鲆(Paralichthys olivaceus)和虹鳟(Oncorhynchusmykiss)的氨基酸序列相似性分别为88.17%、64.94%和63.35%,不同物种间序列差异主要集中在C-端的多肽序列上。蛋白质结构分析显示该蛋白含有3个功能域分别具有脂肪酶活性、乙酰基水解酶活性和水解酶活性。应用实时荧光定量PCR技术分析了lipea基因在鲤各组织、不同发育时期以及脂肪含量极端差异个体的肌肉和脂肪组织中mRNA表达量的变化。结果显示, lipea在各组织中均有表达,腹腔脂肪中相对表达量最高,其次是腹部肌肉,血液中最少。随着胚胎的发育, lipea的表达水平呈现下降趋势, 8细胞期表达量最高,其次是囊胚晚期,开口期最少。lipea表达量与背部肌肉脂肪含量呈正相关,但差异不显著(P>0.05),与腹腔脂肪量呈负相关,且差异极显著性(P<0.01),推测lipea基因对鲤腹腔脂肪沉积可能具有抑制作用。本实验结果可为进一步解析鲤lipea基因的功能和基因调控脂肪沉积分子机制研究提供参考。
The objectives of this study were to investigate the expression pattern of lipea gene in different tissues and developmental stages of common carp(Cyprinus carpio), to lay the foundation for studying and understanding the molecular regulation mechanism of fat deposition in common carp. In this study, a full-length cDNA sequence of lipea gene in common carp was cloned by reverse transcription PCR and rapid-amplification of cDNA ends(RACE) technology, and the expression pattern of lipea gene in different tissues of common carp and during its different developmental stages. cDNA of the lipea gene of common carp was 3379 bp, with an open reading frame of 2079 bp, 5? untranslated region was 230 bp, 3? untranslated region was 1067 bp, encoding 693 amino acids.Multiple sequences alignment revealed that the amino acid sequences of lipea have 88.17%, 64.94%, and 63.35%identity with Danio rerio, Paralichthys olivaceus, and Oncorhynchus mykiss, respectively. It was determined that the sequence differences were mainly concentrated on the C-terminal polypeptide sequence between different species, and the structural analysis of proteins showed that the protein contains three functional domains with lipase activity, acetyl hydrolase activity, and hydrolase activity, respectively. Real-time quantitative PCR showed that the lipea gene was expressed in all tested tissues, and the relative expression level was the highest in visceral fat, followed by abdominal muscles, and lowest in the blood. Its expression declined during the development of embryo;it was the highest in the 8-cell stage, followed by the late blastula stage. It was the lowest in the feeding period.The lipea mRNA expression in different fat tissues negatively correlated with adipose tissue in the abdominal cavity; the highest and the lowest expression levels differed significantly(P<0.01). The expression also positively correlated with dorsal muscle but it was not significant(P>0.05). Knowledge of the lipea gene highlights its function and molecular regulation mechanism of fat development in common carp.
The objectives of this study were to investigate the expression pattern of lipea gene in different tissues and developmental stages of common carp(Cyprinus carpio), to lay the foundation for studying and understanding the molecular regulation mechanism of fat deposition in common carp. In this study, a full-length cDNA sequence of lipea gene in common carp was cloned by reverse transcription PCR and rapid-amplification of cDNA ends(RACE) technology, and the expression pattern of lipea gene in different tissues of common carp and during its different developmental stages. cDNA of the lipea gene of common carp was 3379 bp, with an open reading frame of 2079 bp, 5? untranslated region was 230 bp, 3? untranslated region was 1067 bp, encoding 693 amino acids.Multiple sequences alignment revealed that the amino acid sequences of lipea have 88.17%, 64.94%, and 63.35%identity with Danio rerio, Paralichthys olivaceus, and Oncorhynchus mykiss, respectively. It was determined that the sequence differences were mainly concentrated on the C-terminal polypeptide sequence between different species, and the structural analysis of proteins showed that the protein contains three functional domains with lipase activity, acetyl hydrolase activity, and hydrolase activity, respectively. Real-time quantitative PCR showed that the lipea gene was expressed in all tested tissues, and the relative expression level was the highest in visceral fat, followed by abdominal muscles, and lowest in the blood. Its expression declined during the development of embryo;it was the highest in the 8-cell stage, followed by the late blastula stage. It was the lowest in the feeding period.The lipea mRNA expression in different fat tissues negatively correlated with adipose tissue in the abdominal cavity; the highest and the lowest expression levels differed significantly(P<0.01). The expression also positively correlated with dorsal muscle but it was not significant(P>0.05). Knowledge of the lipea gene highlights its function and molecular regulation mechanism of fat development in common carp.
引文
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[11]Kittilson J D,Reindl K M,Sheridan M A.Rainbow trout(Oncorhynchus mykiss)possess two hormone-sensitive lipase-encoding mRNAs that are differentially expressed and independently regulated by nutritional state[J].Comparative Biochemistry and Physiology Part A:Molecular&Integrative Physiology,2011,158(1):52-60.
[12]Chen Q L,Luo Z,Song Y F,et al.Hormone-sensitive lipase in yellow catfish Pelteobagrus fulvidraco:Molecular characterization,m RNA tissue expression and transcriptional regulation by leptin in vivo and in vitro[J].General and Comparative Endocrinology,2014,206:130-138.
[13]Khieokhajonkhet A,Kaneko G,Hirano Y,et al.Different effects of growth hormone and fasting on the induction patterns of two hormone-sensitive lipase genes in red seabream Pagrus major[J].General and Comparative Endocrinology,2016,236:121-130.
[14]Han C Y,Wen X B,Zheng Q M,et al.Effects of dietary lipid levels on lipid deposition and activities of lipid metabolic enzymes in hybrid tilapia(Oreochromis niloticus×O.aureus)[J].Journal of Animal Physiology and Animal Nutrition,2011,95(5):609-615.
[15]Ai Q H,Yuan Y H,Mai K S,et al.Lipolysis related genes clone and regulation by dietary lipid level in half-smooth tongue sole(Cynoglossus Semilaevis)larvae[J].Periodical of Ocean University of China,2014,44(10):65-71.[艾庆辉,袁禹惠,麦康森,等.半滑舌鳎稚鱼脂肪分解关键酶基因的克隆及饲料中脂肪水平对其表达的调控[J].中国海洋大学学报:自然科学版,2014,44(10):65-71.]
[16]Zhang Y G,Wang Q R,Wang Z Y.Research advance on flesh quality improvement and evaluation of farmed fish[J].Journal of Fujian Fisheries,2015,37(2):172-178.[张亚光,王秋荣,王志勇.养殖鱼类肉质改良与评价的研究进展[J].福建水产,2015,37(2):172-178.]
[17]Robb D,Kestin S C,Warriss P D,et al.Muscle lipid content determines the eating quality of smoked and cooked Atlantic salmon(Salmo salar)[J].Aquaculture,2002,205(3-4):345-358.
[18]Du Z Y.Causes of fatty liver in farmed fish:a review and new perspectives[J].Journal of Fisheries of China,2014,38(9):1628-1638.[杜震宇.养殖鱼类脂肪肝成因及相关思考[J].水产学报,2014,38(9):1628-1638.]
[19]Zheng X H,Kuang Y Y,Lv W H,et al.Genome-wide association study for muscle fat content and abdominal fat traits in common carp(Cyprinus carpio)[J].PLoS ONE,2016,11(12):e0169127.
[20]Haemmerle G,Zimmermann R,Strauss J G,et al.Hormone-sensitive lipase deficiency in mice changes the plasma lipid profile by affecting the tissue-specific expression pattern of lipoprotein lipase in adipose tissue and muscle[J].Journal of Biological Chemistry,2002,277(15):12946-12952.
[21]Fraher D,Sanigorski A,Mellett N A,et al.Zebrafish embryonic lipidomic analysis reveals that the yolk cell is metabolically active in processing lipid[J].Cell Reports,2016,14(6):1317-1329.
[22]Hu X C,Zhao Y L.Variation in the lipid content and fatty acid composition during embryonic and larval development of Odontobutis potamophila[J].Freshwater Fisheries,2008,38(3):46-50.[胡先成,赵云龙.河川沙塘鳢胚胎、仔鱼发育过程中脂类含量及脂肪酸组成的变化[J].淡水渔业,2008,38(3):46-50.]
[23]Desvilettes C,Bourdier G,Breton J C.Changes in lipid class and fatty acid composition during development in pike(Esox lucius L)eggs and larvae[J].Fish Physiology and Biochemistry,1997,16(5):381-393.
[24]Kazala E C,Petrak J L,Lozeman F J,et al.Hormone-sensitive lipase activity in relation to fat content of muscle in wagyu hybrid cattle[J].Livestock Production Science,2003,79(1):87-96.
[25]Luan Z J,Liu K D,He J N,et al.The FAM134B,PPARγ,HSL and FAS gene expression patterns and their asociation with intramuscular fat content in shep[J].Acta Veterinaria et Zotechnica Sinica,2016,47(12):2379-2389.[栾兆进,刘开东,贺建宁,等.绵羊FAM134B、PPARγ、HSL和FAS基因表达量及与肌内脂肪含量的关系[J].畜牧兽医学报,2016,47(12):2379-2389.]
[26]Li G J.Effect of dietary and carbohydrates on enzymes activities and relative gene expression of FAS and HSL in juvenile black sea bream,Acanthopagrus schlegelii[D].Hangzhou:Zhejiang University,2015.[李广经.不同脂肪和碳水化合物水平对黑鲷脂肪代谢关键酶FAS和HSL活性和基因表达的影响研究[D].杭州:浙江大学,2015.]
[27]Chen J,Yang X J,Tong H,et al.Expressions of FAS and HSL mRNA in longissimus dorsi muscle and their relation to intramuscular fat contents in pig[J].Journal of Agricultural Biotechnology,2004,12(4):422-426.[陈杰,杨晓静,佟辉,等.FAS和HSL m RNA在猪背最长肌的表达及其与肌内脂肪含量的关系[J].农业生物技术学报,2004,12(4):422-426.]
[28]Sztalryd C,Komaromy M C,Kraemer F B.Overexpression of hormone-sensitive lipase prevents triglyceride accumulation in adipocytes[J].Journal of Clinical Investigation,1995,95(6):2652-2661.
[29]Miao Z G,Xie H B,Wang Y Q,et al.Difference in fat metabolic enzymes and serum adiponectin between Huainan and Landrace pig[J].Guizhou Agricultural Sciences,2018,46(1):61-63.[苗志国,谢红兵,王永强,等.淮南猪与长白猪脂肪代谢酶及脂联素的发育差异[J].贵州农业科学,2018,46(1):61-63.]
[30]Liu J Z,Xing J Y,Cao S D,et al.Analysis of genetic and nutritious factors affecting domestic animal fat deposition[J].Livestock and Poultry Industry,2008(12):26-29.[刘京贞,邢晋祎,曹善东,等.影响畜禽脂肪沉积的遗传和营养因素分析[J].畜禽业,2008(12):26-29.]
[2]Kraemer F B,Shen W J.Hormone-sensitive lipase knockouts[J].Nutrition&Metabolism,2006,3(1):12-19.
[3]Holm C,Kirchgessner T G,Svenson K L,et al.Hormone-sensitive lipase:sequence,expression and chromosomal localization to 19 cent-q13.3[J].Science,1988,241(4872):1503-1506.
[4]Langin D,Laurell H,Hols L S,et al.Gene organization and primary structure of human hormone-sensitive lipase:possible significance of a sequence homology with a lipase of Moraxella TA144,an antarctic bacterium[J].Proceedings of the National Academy of Sciences of the United States of America,1993,90(11):4897-4901.
[5]Li Z,Sumida M,Birchbauer A,et al.Isolation and characterization of the gene for mouse hormone-sensitive lipase[J].Genomics,1994,24(2):259-265.
[6]Harbitz I,Langset M,Ege A G,et al.The porcine hormone-sensitive lipase gene:sequence,structure,polymorphisms and linkage mapping[J].Animal Genetics,1999,30(1):10-15.
[7]Ren S M,Zhao X T,Zhou C B,et al.Correlation of HSLgene polymorphisms with carcass traits and meat quality in pig breed Sujiang and its crossbreeds[J].Hunan Agricultural Sciences,2014(13):55-57.[任善茂,赵旭庭,周春宝,等.猪HSL基因多态性与胴体性状及肌肉品质的相关性分析[J].湖南农业科学,2014(13):55-57.]
[8]Qiao Y,Huang Z G,Li Q F,et al.Developmental changes of the FAS and HSL mRNA expression and their effects on the content of intramuscular fat in Kazak and Xinjiang sheep[J].Journal of Genetics and Genomics,2007,34(10):909-917.
[9]Wang Y P,Xu C C,Luo H L.Research progress in relevant candidate genes of animal intramuscular fat deposition[J].Chinese Journal of Animal Nutrition,2017,29(5):1475-1480.[王怡平,徐晨晨,罗海玲.动物肌内脂肪沉积相关候选基因的研究进展[J].动物营养学报,2017,29(5):1475-1480.]
[10]Khieokhajonkhet A,Kaneko G,Ohara K,et al.Hormone-sensitive lipase in Japanese flounder Paralichthys olivaceus:the potential function of the inclinator muscle of fin as a lipid storage site[J].Fisheries Science,2014,80(2):341-351.
[11]Kittilson J D,Reindl K M,Sheridan M A.Rainbow trout(Oncorhynchus mykiss)possess two hormone-sensitive lipase-encoding mRNAs that are differentially expressed and independently regulated by nutritional state[J].Comparative Biochemistry and Physiology Part A:Molecular&Integrative Physiology,2011,158(1):52-60.
[12]Chen Q L,Luo Z,Song Y F,et al.Hormone-sensitive lipase in yellow catfish Pelteobagrus fulvidraco:Molecular characterization,m RNA tissue expression and transcriptional regulation by leptin in vivo and in vitro[J].General and Comparative Endocrinology,2014,206:130-138.
[13]Khieokhajonkhet A,Kaneko G,Hirano Y,et al.Different effects of growth hormone and fasting on the induction patterns of two hormone-sensitive lipase genes in red seabream Pagrus major[J].General and Comparative Endocrinology,2016,236:121-130.
[14]Han C Y,Wen X B,Zheng Q M,et al.Effects of dietary lipid levels on lipid deposition and activities of lipid metabolic enzymes in hybrid tilapia(Oreochromis niloticus×O.aureus)[J].Journal of Animal Physiology and Animal Nutrition,2011,95(5):609-615.
[15]Ai Q H,Yuan Y H,Mai K S,et al.Lipolysis related genes clone and regulation by dietary lipid level in half-smooth tongue sole(Cynoglossus Semilaevis)larvae[J].Periodical of Ocean University of China,2014,44(10):65-71.[艾庆辉,袁禹惠,麦康森,等.半滑舌鳎稚鱼脂肪分解关键酶基因的克隆及饲料中脂肪水平对其表达的调控[J].中国海洋大学学报:自然科学版,2014,44(10):65-71.]
[16]Zhang Y G,Wang Q R,Wang Z Y.Research advance on flesh quality improvement and evaluation of farmed fish[J].Journal of Fujian Fisheries,2015,37(2):172-178.[张亚光,王秋荣,王志勇.养殖鱼类肉质改良与评价的研究进展[J].福建水产,2015,37(2):172-178.]
[17]Robb D,Kestin S C,Warriss P D,et al.Muscle lipid content determines the eating quality of smoked and cooked Atlantic salmon(Salmo salar)[J].Aquaculture,2002,205(3-4):345-358.
[18]Du Z Y.Causes of fatty liver in farmed fish:a review and new perspectives[J].Journal of Fisheries of China,2014,38(9):1628-1638.[杜震宇.养殖鱼类脂肪肝成因及相关思考[J].水产学报,2014,38(9):1628-1638.]
[19]Zheng X H,Kuang Y Y,Lv W H,et al.Genome-wide association study for muscle fat content and abdominal fat traits in common carp(Cyprinus carpio)[J].PLoS ONE,2016,11(12):e0169127.
[20]Haemmerle G,Zimmermann R,Strauss J G,et al.Hormone-sensitive lipase deficiency in mice changes the plasma lipid profile by affecting the tissue-specific expression pattern of lipoprotein lipase in adipose tissue and muscle[J].Journal of Biological Chemistry,2002,277(15):12946-12952.
[21]Fraher D,Sanigorski A,Mellett N A,et al.Zebrafish embryonic lipidomic analysis reveals that the yolk cell is metabolically active in processing lipid[J].Cell Reports,2016,14(6):1317-1329.
[22]Hu X C,Zhao Y L.Variation in the lipid content and fatty acid composition during embryonic and larval development of Odontobutis potamophila[J].Freshwater Fisheries,2008,38(3):46-50.[胡先成,赵云龙.河川沙塘鳢胚胎、仔鱼发育过程中脂类含量及脂肪酸组成的变化[J].淡水渔业,2008,38(3):46-50.]
[23]Desvilettes C,Bourdier G,Breton J C.Changes in lipid class and fatty acid composition during development in pike(Esox lucius L)eggs and larvae[J].Fish Physiology and Biochemistry,1997,16(5):381-393.
[24]Kazala E C,Petrak J L,Lozeman F J,et al.Hormone-sensitive lipase activity in relation to fat content of muscle in wagyu hybrid cattle[J].Livestock Production Science,2003,79(1):87-96.
[25]Luan Z J,Liu K D,He J N,et al.The FAM134B,PPARγ,HSL and FAS gene expression patterns and their asociation with intramuscular fat content in shep[J].Acta Veterinaria et Zotechnica Sinica,2016,47(12):2379-2389.[栾兆进,刘开东,贺建宁,等.绵羊FAM134B、PPARγ、HSL和FAS基因表达量及与肌内脂肪含量的关系[J].畜牧兽医学报,2016,47(12):2379-2389.]
[26]Li G J.Effect of dietary and carbohydrates on enzymes activities and relative gene expression of FAS and HSL in juvenile black sea bream,Acanthopagrus schlegelii[D].Hangzhou:Zhejiang University,2015.[李广经.不同脂肪和碳水化合物水平对黑鲷脂肪代谢关键酶FAS和HSL活性和基因表达的影响研究[D].杭州:浙江大学,2015.]
[27]Chen J,Yang X J,Tong H,et al.Expressions of FAS and HSL mRNA in longissimus dorsi muscle and their relation to intramuscular fat contents in pig[J].Journal of Agricultural Biotechnology,2004,12(4):422-426.[陈杰,杨晓静,佟辉,等.FAS和HSL m RNA在猪背最长肌的表达及其与肌内脂肪含量的关系[J].农业生物技术学报,2004,12(4):422-426.]
[28]Sztalryd C,Komaromy M C,Kraemer F B.Overexpression of hormone-sensitive lipase prevents triglyceride accumulation in adipocytes[J].Journal of Clinical Investigation,1995,95(6):2652-2661.
[29]Miao Z G,Xie H B,Wang Y Q,et al.Difference in fat metabolic enzymes and serum adiponectin between Huainan and Landrace pig[J].Guizhou Agricultural Sciences,2018,46(1):61-63.[苗志国,谢红兵,王永强,等.淮南猪与长白猪脂肪代谢酶及脂联素的发育差异[J].贵州农业科学,2018,46(1):61-63.]
[30]Liu J Z,Xing J Y,Cao S D,et al.Analysis of genetic and nutritious factors affecting domestic animal fat deposition[J].Livestock and Poultry Industry,2008(12):26-29.[刘京贞,邢晋祎,曹善东,等.影响畜禽脂肪沉积的遗传和营养因素分析[J].畜禽业,2008(12):26-29.]