藏鸡KLF15基因克隆、组织表达谱及其表达与肌内脂肪含量相关性的研究
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摘要
旨在获得藏鸡KLF15基因序列,阐明其组织和时序表达谱,并分析该基因表达与肌内脂肪(IMF)含量的关系。本研究选取1、81、119、154、210日龄健康公母藏鸡各5只为试验动物,利用RT-PCR技术克隆KLF15基因,利用生物信息学软件分析基因生物学特征,利用实时荧光定量PCR(fluorescence quantitative PCR,qPCR)检测KLF15基因在藏鸡不同组织、不同发育阶段的表达谱,并分析其表达水平与肌内脂肪(intramuscular fat,IMF)含量的相关性。结果显示,克隆得到藏鸡KLF15基因序列长度为1 288bp(GenBank登录号:KY747450),开放阅读框为1 212bp,编码403个氨基酸,是具有3个锌指结构的亲水不稳定碱性蛋白质。藏鸡KLF15基因与原鸡的核苷酸和氨基酸的同源性均为99%。KLF15mRNA在藏鸡各个组织中广泛表达,但在肺组织中表达水平最高,极显著高于其他组织(P<0.01)。KLF15mRNA在1日龄藏鸡胸肌组织中的表达水平高于其他各日龄段,并随着日龄的增长呈下降趋势;在119日龄腿肌组织中的表达水平最高,且整体表达水平的变化都呈先下降后上升再下降的趋势。藏鸡公鸡154日龄前KLF15基因的表达水平与其对应的IMF含量呈正相关,154日龄以后则呈负相关;藏鸡母鸡在各个日龄段则主要表现为微弱的负相关。上述结果为进一步揭示KLF15基因在藏鸡IMF沉积中的作用提供重要数据。
The objective of this study was to obtain the sequence of KLF15 gene of Tibetan chicken,clarify its tissue and temporal expression profiles and to analyze the relationship between its expression and the intramuscular fat(IMF)content.Ten healthy Tibetan chicken(5 cock and 5 hen)on 1,81,119,154 and 210 days old were selected,respectively as experimental animals.RT-PCR technology was used to clone KLF15 gene of Tibetan chicken,and bioinformatics softwares were used to analyze the biological characteristics of KLF15.The expression profiles of KLF15 gene in different tissues and different developmental stages was detected by fluorescence quantitative PCR,and its correlation with IMF content was analyzed.The result showed that the cloned Tibetan chicken KLF15 gene sequence was 1 288 bp in length(GenBank accession number:KY747450),an open reading frame of 1 212 bp,encoding 403 amino acids,and it was a hydrophilic unstable basic protein with 3 zinc finger structures.The homology between the nucleotide and amino acid sequences of KLF15 gene of Tibetan chicken and Gallus gallus were 99%.KLF15 mRNA was widely expressed in all detected tissues of Tibetan chicken,and the expression level was the highest in lung,which is significantly higher than that in other tissues(P<0.01).The expression level of KLF15 gene in breast muscle of Tibetan chicken of 1 day old was higher than that of the other age groups,and declined with the increasing age.The expression level of KLF15 gene in leg muscle of Tibetan chicken of 119 days old was higher than that of the other age groups,and the change of the overall expression level showed a trend of decreasing first,then rising,finally falling.The expression level of KLF15 gene before 154 days old was positively correlated with IMF content in Tibetan cocks,after 154 days old,the correlation was reverse,and the Tibetan hens mainly showed a weak negative correlation in all age groups.These results provide the basic data for further research on the role of KLF15 in IMF deposition of Tibetan chicken.
The objective of this study was to obtain the sequence of KLF15 gene of Tibetan chicken,clarify its tissue and temporal expression profiles and to analyze the relationship between its expression and the intramuscular fat(IMF)content.Ten healthy Tibetan chicken(5 cock and 5 hen)on 1,81,119,154 and 210 days old were selected,respectively as experimental animals.RT-PCR technology was used to clone KLF15 gene of Tibetan chicken,and bioinformatics softwares were used to analyze the biological characteristics of KLF15.The expression profiles of KLF15 gene in different tissues and different developmental stages was detected by fluorescence quantitative PCR,and its correlation with IMF content was analyzed.The result showed that the cloned Tibetan chicken KLF15 gene sequence was 1 288 bp in length(GenBank accession number:KY747450),an open reading frame of 1 212 bp,encoding 403 amino acids,and it was a hydrophilic unstable basic protein with 3 zinc finger structures.The homology between the nucleotide and amino acid sequences of KLF15 gene of Tibetan chicken and Gallus gallus were 99%.KLF15 mRNA was widely expressed in all detected tissues of Tibetan chicken,and the expression level was the highest in lung,which is significantly higher than that in other tissues(P<0.01).The expression level of KLF15 gene in breast muscle of Tibetan chicken of 1 day old was higher than that of the other age groups,and declined with the increasing age.The expression level of KLF15 gene in leg muscle of Tibetan chicken of 119 days old was higher than that of the other age groups,and the change of the overall expression level showed a trend of decreasing first,then rising,finally falling.The expression level of KLF15 gene before 154 days old was positively correlated with IMF content in Tibetan cocks,after 154 days old,the correlation was reverse,and the Tibetan hens mainly showed a weak negative correlation in all age groups.These results provide the basic data for further research on the role of KLF15 in IMF deposition of Tibetan chicken.
引文
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[14]GRAY S,FEINBERG M W,HULL S,et al.The Krüppel-like factor KLF15regulates the insulin-sensitive glucose transporter GLUT4[J].J Biol Chem,2002,277(37):34322-34328.
[15]FISCH S,GRAY S,HEYMANS S,et al.38Kruppellike factor 15is a novel regulator of cardiomyocyte hypertrophy[J].J Invest Med,2007,55(2):S354.
[16]HALDAR S M,LU Y,JEYARAJ D,et al.Klf15deficiency is a molecular link between heart failure and aortic aneurysm formation[J].Sci Transl Med,2010,2(26):26ra26.
[17]WANG B Q,HALDAR S M,LU Y,et al.The Kruppel-like factor KLF15 inhibits connective tissue growth factor(CTGF)expression in cardiac fibroblasts[J].J Mol Cell Cardiol,2008,45(2):193-197.
[18]GRAY S,WANG B Q,ORIHUELA Y,et al.Regulation of gluconeogenesis by Krüppel-like factor 15[J].Cell Metab,2007,5(4):305-312.
[19]JEYARAJ D,SCHEER F A J L,RIPPERGER J A,et al.Klf15orchestrates circadian nitrogen homeostasis[J].Cell Metab,2012,15(3):311-323.
[20]H ALDAR S M,JEYARAJ D,ANAND P,et al.Kruppel-like factor 15regulates skeletal muscle lipid flux and exercise adaptation[J].Proc Natl Acad Sci U S A,2012,109(17):6739-6744.
[21]李青莹.KLF15对猪前体脂肪细胞分化及通过其含量预测脂肪沉积能力的研究[D].长春:吉林大学,2015.LI Q Y.Research on effects of KLF15in porcine preadipocytes differentiation and prediction of fat deposition capability[D].Changchun:Jilin University,2015.(in Chinese)
[22]林亚秋,徐亚欧,张润锋,等.藏鸡Chemerin和ChemR23基因时序表达及其与肌内脂肪含量的相关性研究[J].畜牧兽医学报,2015,46(8):1290-1299.LIN Y Q,XU Y O,ZHANG R F,et al.Association analysis of sequential expression of Chemerin and ChemR23with intramuscular fat contents in Tibetan chicken[J].Acta Veterinaria et Zootechnica Sinica,2015,46(8):1290-1299.(in Chinese)
[23]MAN C L,LI X,ZHAO D D.Cloning,sequence identification,and tissue expression analysis of novel chicken NYGGF4gene[J].Mol Cell Biochem,2011,346(1-2):117-124.
[24]LIVAK K J,SCHMITTGEN T D.Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method[J].Methods,2001,25(4):402-408.
[25]聂晓庆,林亚秋,徐亚欧,等.藏鸡磷酸酪氨酸互作结构域1(PID1)的克隆及组织表达谱研究[J].畜牧兽医学报,2016,47(6):1102-1111.NIE X Q,LIN Y Q,XU Y O,et al.Research on cloning and tissue expression profile of phosphotyrosine interaction domain containing 1(PID1)in Tibetan chicken[J].Acta Veterinaria et Zootechnica Sinica,2016,47(6):1102-1111.(in Chinese)
[26]S AKANE N,YOSHIDA T,YOSHIOKA K,et al.β3-adrenoreceptor gene polymorphism:a newly identified risk factor for proliferative retinopathy in NIDDMpatients[J].Diabetes,1997,46(10):1633-1636.
[27]PROSDOCIMO D A,JOHN J E,ZHANG L L,et al.KLF15and PPARαcooperate to regulate cardiomyocyte lipid gene expression and oxidation[J].PPARRes,2015,2015:201625.
[28]王杰,陈婷,沈清武.KLF15基因表达特征及其腺病毒超表达载体的构建[J].西北农林科技大学学报:自然科学版,2015,43(7):35-40.WANG J,CHEN T,SHEN Q W.Expression characteristics of KLF15gene and construction of its adenovirus vector[J].Journal of Northwest A&F University:Natural Science Edition,2015,43(7):35-40.(in Chinese)
[29]朱江江,林亚秋,左璐璐,等.牦牛KLF5、KLF6、KLF7基因的克隆表达及其与肌内脂肪含量的相关性分析[J].畜牧兽医学报,2017,48(3):416-424.ZHU J J,LIN Y Q,ZUO L L,et al.Molecular cloning,tissue expression of KLF5,KLF6,KLF7genes,and their correlations with intramuscular fat content in Yak[J].Acta Veterinaria et Zootechnica Sinica,2017,48(3):416-424.(in Chinese)
[30]吕世杰.鸡KLF15基因的组织表达特性及多态性分析研究[D].郑州:河南农业大学,2014.LS J.Study of polymorphisms and gene expression pattern of the chicken KLF15gene[D].Zhengzhou:Henan Agricultural University,2014.(in Chinese)
[31]柴孟龙,李青莹,姜昊,等.锌指蛋白15在猪前体脂肪细胞分化中的作用[J].吉林农业大学学报,2016,38(3):325-329.CHAI M L,LI Q Y,JIANG H,et al.Role of zinc finger protein15in differentiation of porcine preadipocyte[J].Journal of Jilin Agricultural University,2016,38(3):325-329.(in Chinese)
[32]MORI T,SAKAUE H,IGUCHI H,et al.Role of Krüppel-like factor 15(KLF15)in transcriptional regulation of adipogenesis[J].J Biol Chem,2005,280(13):12867-12875.
[33]GUO H F,KHAN R,RAZA S H A,et al.KLF15promotes transcription of KLF3gene in bovine adipocytes[J].Gene,2018,659:77-83.
[34]ZHAO Z D,ZAN L S,LI A N,et al.Characterization of the promoter region of the bovine long-chain acylCoA synthetase 1gene:Roles of E2F1,Sp1,KLF15,and E2F4[J].Sci Rep,2016,6:19661.
[2]张学余.我国优质鸡种资源---藏鸡[J].中国家禽,2003,25(10):44-45.ZHANG X Y.Zangji chicken:a Chinese high quality chicken resource[J].Chinese Poultry,2003,25(10):44-45.(in Chinese)
[3]WANG Y H,BYRNE K A,REVERTER A,et al.Transcriptional profiling of skeletal muscle tissue from two breeds of cattle[J].Mamm Genome,2005,16(3):201-210.
[4]GEAY Y,BAUCHART D,HOCQUETTE J F,et al.Effect of nutritional factors on biochemical,structural and metabolic characteristics of muscles in ruminants,consequences on dietetic value and sensorial qualities of meat[J].Reprod Nutr Dev,2001,41(1):1-26.
[5]GERBENS F,VERBURG F J,VAN MOERKERK HT B,et al.Associations of heart and adipocyte fatty acid-binding protein gene expression with intramuscular fat content in pigs[J].J Anim Sci,2001,79(2):347-354.
[6]KAKUMA T,LEE Y,HIGA M,et al.Leptin,troglitazone,and the expression of sterol regulatory element binding proteins in liver and pancreatic islets[J].Proc Natl Acad Sci U S A,2000,97(15):8536-8541.
[7]SHU J T,ZHANG M,SHAN Y J,et al.Analysis of the genetic effects of CAPN1gene polymorphisms on chicken meat tenderness[J].Genet Mol Res,2015,14(1):1393-1403.
[8]YE Y Q,LIN S M,MU H P,et al.Analysis of differentially expressed genes and signaling pathways related to intramuscular fat deposition in skeletal muscle of sexlinked dwarf chickens[J].Biomed Res Int,2014,2014:724274.
[9]HUANG Z G,XIONG L,LIU Z S,et al.The developmental changes and effect on IMF content of H-FABPand PPARγmRNA expression in sheep muscle[J].Acta Genet Sinica,2006,33(6):507-514.
[10]CHEN J,YANG X J,XIA D,et al.Sterol regulatory element binding transcription factor 1expression and genetic polymorphism significantly affect intramuscular fat deposition in the longissimus muscle of Erhualian and Sutai pigs[J].J Anim Sci,2008,86(1):57-63.
[11]PEARSON R,FLEETWOOD J,EATON S,et al.Krüppel-like transcription factors:A functional family[J].Int J Biochem Cell Biol,2008,40(10):1996-2001.
[12]WEI S J,ZHANG L F,ZHOU X,et al.Emerging roles of zinc finger proteins in regulating adipogenesis[J].Cell Mol Life Sci,2013,70(23):4569-4584.
[13]PEI J M,GRISHIN N V.A new family of predicted Krüppel-like factor genes and pseudogenes in placental mammals[J].PLoS One,2013,8(11):e81109.
[14]GRAY S,FEINBERG M W,HULL S,et al.The Krüppel-like factor KLF15regulates the insulin-sensitive glucose transporter GLUT4[J].J Biol Chem,2002,277(37):34322-34328.
[15]FISCH S,GRAY S,HEYMANS S,et al.38Kruppellike factor 15is a novel regulator of cardiomyocyte hypertrophy[J].J Invest Med,2007,55(2):S354.
[16]HALDAR S M,LU Y,JEYARAJ D,et al.Klf15deficiency is a molecular link between heart failure and aortic aneurysm formation[J].Sci Transl Med,2010,2(26):26ra26.
[17]WANG B Q,HALDAR S M,LU Y,et al.The Kruppel-like factor KLF15 inhibits connective tissue growth factor(CTGF)expression in cardiac fibroblasts[J].J Mol Cell Cardiol,2008,45(2):193-197.
[18]GRAY S,WANG B Q,ORIHUELA Y,et al.Regulation of gluconeogenesis by Krüppel-like factor 15[J].Cell Metab,2007,5(4):305-312.
[19]JEYARAJ D,SCHEER F A J L,RIPPERGER J A,et al.Klf15orchestrates circadian nitrogen homeostasis[J].Cell Metab,2012,15(3):311-323.
[20]H ALDAR S M,JEYARAJ D,ANAND P,et al.Kruppel-like factor 15regulates skeletal muscle lipid flux and exercise adaptation[J].Proc Natl Acad Sci U S A,2012,109(17):6739-6744.
[21]李青莹.KLF15对猪前体脂肪细胞分化及通过其含量预测脂肪沉积能力的研究[D].长春:吉林大学,2015.LI Q Y.Research on effects of KLF15in porcine preadipocytes differentiation and prediction of fat deposition capability[D].Changchun:Jilin University,2015.(in Chinese)
[22]林亚秋,徐亚欧,张润锋,等.藏鸡Chemerin和ChemR23基因时序表达及其与肌内脂肪含量的相关性研究[J].畜牧兽医学报,2015,46(8):1290-1299.LIN Y Q,XU Y O,ZHANG R F,et al.Association analysis of sequential expression of Chemerin and ChemR23with intramuscular fat contents in Tibetan chicken[J].Acta Veterinaria et Zootechnica Sinica,2015,46(8):1290-1299.(in Chinese)
[23]MAN C L,LI X,ZHAO D D.Cloning,sequence identification,and tissue expression analysis of novel chicken NYGGF4gene[J].Mol Cell Biochem,2011,346(1-2):117-124.
[24]LIVAK K J,SCHMITTGEN T D.Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method[J].Methods,2001,25(4):402-408.
[25]聂晓庆,林亚秋,徐亚欧,等.藏鸡磷酸酪氨酸互作结构域1(PID1)的克隆及组织表达谱研究[J].畜牧兽医学报,2016,47(6):1102-1111.NIE X Q,LIN Y Q,XU Y O,et al.Research on cloning and tissue expression profile of phosphotyrosine interaction domain containing 1(PID1)in Tibetan chicken[J].Acta Veterinaria et Zootechnica Sinica,2016,47(6):1102-1111.(in Chinese)
[26]S AKANE N,YOSHIDA T,YOSHIOKA K,et al.β3-adrenoreceptor gene polymorphism:a newly identified risk factor for proliferative retinopathy in NIDDMpatients[J].Diabetes,1997,46(10):1633-1636.
[27]PROSDOCIMO D A,JOHN J E,ZHANG L L,et al.KLF15and PPARαcooperate to regulate cardiomyocyte lipid gene expression and oxidation[J].PPARRes,2015,2015:201625.
[28]王杰,陈婷,沈清武.KLF15基因表达特征及其腺病毒超表达载体的构建[J].西北农林科技大学学报:自然科学版,2015,43(7):35-40.WANG J,CHEN T,SHEN Q W.Expression characteristics of KLF15gene and construction of its adenovirus vector[J].Journal of Northwest A&F University:Natural Science Edition,2015,43(7):35-40.(in Chinese)
[29]朱江江,林亚秋,左璐璐,等.牦牛KLF5、KLF6、KLF7基因的克隆表达及其与肌内脂肪含量的相关性分析[J].畜牧兽医学报,2017,48(3):416-424.ZHU J J,LIN Y Q,ZUO L L,et al.Molecular cloning,tissue expression of KLF5,KLF6,KLF7genes,and their correlations with intramuscular fat content in Yak[J].Acta Veterinaria et Zootechnica Sinica,2017,48(3):416-424.(in Chinese)
[30]吕世杰.鸡KLF15基因的组织表达特性及多态性分析研究[D].郑州:河南农业大学,2014.LS J.Study of polymorphisms and gene expression pattern of the chicken KLF15gene[D].Zhengzhou:Henan Agricultural University,2014.(in Chinese)
[31]柴孟龙,李青莹,姜昊,等.锌指蛋白15在猪前体脂肪细胞分化中的作用[J].吉林农业大学学报,2016,38(3):325-329.CHAI M L,LI Q Y,JIANG H,et al.Role of zinc finger protein15in differentiation of porcine preadipocyte[J].Journal of Jilin Agricultural University,2016,38(3):325-329.(in Chinese)
[32]MORI T,SAKAUE H,IGUCHI H,et al.Role of Krüppel-like factor 15(KLF15)in transcriptional regulation of adipogenesis[J].J Biol Chem,2005,280(13):12867-12875.
[33]GUO H F,KHAN R,RAZA S H A,et al.KLF15promotes transcription of KLF3gene in bovine adipocytes[J].Gene,2018,659:77-83.
[34]ZHAO Z D,ZAN L S,LI A N,et al.Characterization of the promoter region of the bovine long-chain acylCoA synthetase 1gene:Roles of E2F1,Sp1,KLF15,and E2F4[J].Sci Rep,2016,6:19661.