鸡OBR基因g.7851G>A单核苷酸多态性功能性鉴定及分析
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摘要
瘦蛋白受体(OBR)对脂肪沉积和体重调节有重要影响。基于前期发现鸡(Gallus gallus)OBR基因g.7851G>A单核苷酸多态性(Single nucleotide polymorphism,SNP)与鸡腹脂重极显著相关(P=0.0076)。生物信息学分析显示,该SNP位于多个转录因子结合区域,可能与某个或某些转录因子结合,影响OBR基因转录效率,为功能性SNP。为确定SNP功能性,研究该基因在高、低脂两系肉鸡各组织中差异表达;构建含该SNP位点A和G等位基因荧光素酶报告基因载体,利用双荧光素酶报告系统分析转录因子C/EBPα和PPARα对该SNP位点不同等位基因荧光素酶活性的影响。Real-time RT-PCR结果表明,肝脏和腹部脂肪中,高脂系公鸡OBR基因表达量显著高于低脂系公鸡;在大脑中,高脂系公鸡OBR基因表达量显著低于低脂系公鸡(P<0.05)。报告基因结果表明,A等位基因荧光素酶活性显著高于G等位基因(P<0.05)。此外,转录因子PPAR录对G等位基因荧光素酶活性抑制作用显著强于A等位基因(P<0.05),转录因子C/EBα因对G等位基因荧光素酶活性抑制作用显著弱于A等位基因(P<0.05)。研究表明,OBR基因g.7851G>A可能是功能性SNP位点,确定为优质鸡育种功能性分子标记。研究为深入探讨OBR基因对脂肪代谢分子机制的影响提供试验依据。
The leptin receptor gene(OBR) plays an important role in fat deposition and body weight regulation. Previously, it was found that genotypes of chicken OBR g.7851 G>A(SNP) was significantly associated with abdominal fat weight in chickens. Bioinformatics analysis revealed that the SNP was located in multiple transcription factor binding regions, which might affect the transcription efficiency of the OBR gene by binding to a certain(some) transcription factor(factors). The SNP might be a functional SNP. To determine the functionality of the SNP, differential m RNA expressions of the gene in various tissues of fat and lean line broilers were carried out, respectively; the luciferase reporter gene containing the A and G alleles of the SNP was constructed. The dual luciferase reporter system was used to analyze the effects of candidate transcription factors C/EBPα and PPARα on the luciferase activity of different alleles of this SNP. qRT-PCR results showed that both in the liver and abdominal fat,the expression level of OBR gene in fat cocks was significantly higher than that in lean cocks. However,the opposite results were observed in the cerebrum. Reporter gene results showed that the luciferase activity of allele A was significantly higher than that of allele G(P<0.05). In addition, the luciferase activity of allele G inhibited by the transcription factor PPARa was stronger than that of allele A, the luciferase activity of allele G inhibited by the transcription factor C/EBPα was weaker than that of allele A. The results of this study suggested that the OBR gene g.7851 G>A might be a functional SNP, which could be initially identified as a functional molecular marker for high quality chicken breeding. The results provided an experimental basis for further investigating the molecular regulation mechanism of OBR gene on fat metabolism.
The leptin receptor gene(OBR) plays an important role in fat deposition and body weight regulation. Previously, it was found that genotypes of chicken OBR g.7851 G>A(SNP) was significantly associated with abdominal fat weight in chickens. Bioinformatics analysis revealed that the SNP was located in multiple transcription factor binding regions, which might affect the transcription efficiency of the OBR gene by binding to a certain(some) transcription factor(factors). The SNP might be a functional SNP. To determine the functionality of the SNP, differential m RNA expressions of the gene in various tissues of fat and lean line broilers were carried out, respectively; the luciferase reporter gene containing the A and G alleles of the SNP was constructed. The dual luciferase reporter system was used to analyze the effects of candidate transcription factors C/EBPα and PPARα on the luciferase activity of different alleles of this SNP. qRT-PCR results showed that both in the liver and abdominal fat,the expression level of OBR gene in fat cocks was significantly higher than that in lean cocks. However,the opposite results were observed in the cerebrum. Reporter gene results showed that the luciferase activity of allele A was significantly higher than that of allele G(P<0.05). In addition, the luciferase activity of allele G inhibited by the transcription factor PPARa was stronger than that of allele A, the luciferase activity of allele G inhibited by the transcription factor C/EBPα was weaker than that of allele A. The results of this study suggested that the OBR gene g.7851 G>A might be a functional SNP, which could be initially identified as a functional molecular marker for high quality chicken breeding. The results provided an experimental basis for further investigating the molecular regulation mechanism of OBR gene on fat metabolism.
引文
[1] Demeure O, Duclos M J, Bacciu N, et al. Genome-wide interval mapping using SNPs identifies new QTL for growth, body composition and several physiological variables in an F2 intercross between fat and lean chicken lines[J]. Genetics Selection Evolution, 2013, 45:36.
[2] Ramiah S K, Meng G Y, Sheau W T, et al. Dietary conjugated linoleic acid supplementation leads to downregulation of PPAR Transcription in broiler chickens and reduction of adipocyte cellularity[J]. PPAR Research, 2014, 2014:1-10.
[3]陈耀峰,谢可嘉,张长超,等.鸡ApoB基因g.-112A>G单核苷酸多态性的功能性鉴定与分析[J].农业生物技术学报, 2018,26(11):103-111.
[4]王桂华,李辉,王启贵,等.鸡OBR基因在肉鸡和蛋鸡肝脏组织中差异表达的研究[J].东北农业大学学报, 2006, 37(5):650-655.
[5] Rancourt R C, Schellong K, Ott R, et al. Acquired alterations of hypothalamic gene expression of insulin and leptin receptors and glucose transporters in prenatally high-glucose exposed threeweek old chickens do not coincide with aberrant promoter DNA methylation[J]. PLoS One, 2015, 10(3):e0119213.
[6]徐淑静,徐明彤,傅祖植.肥胖基因和瘦素[J].中华内分泌代谢杂志, 1999, 15(2):111-113.
[7] Gohda T, Tanimoto M, Kaneko S, et al. Minor gene effect of leptin receptor variant on the body weight in KK/Ta mice[J]. Diabetes,Obesity and Metabolism, 2006, 8(5):581-584.
[8] Balatsky V, Bankovska I, Pena R N, et al. Polymorphisms of the porcine cathepsins, growth hormone-releasing hormone and leptin receptor genes and their association with meat quality traits in Ukrainian Large White breed[J]. Molecular Biology Reports,2016, 43(6):517-526.
[9]王颖,李辉,顾志良,等.鸡瘦蛋白受体(OBR)基因内含子8单核苷酸多态性与体脂性状的相关研究[J].遗传学报, 2004, 31(3):265-269.
[10] Lei M M, Wu S Q, Shao X B, et al. Creating leptin-like biofunctions by active immunization against chicken Leptin receptor in growing chickens[J]. Domestic Animal Endocrinology, 2015, 50:55-64.
[11] Fong T M, Huang R R, Tota M R, et al. Localization of leptin binding domain in the leptin receptor[J]. Molecular Pharmacology, 1998, 53(2):234-240.
[12] Yang C, Cui H X, Fan P. Expression of OBR and NPY in mouse hypothalamus[J]. Basic and Clinical Medicine, 2009, 29(2):152-155.
[13] Pan W, Hsuchou H, He Y, et al. Astrocyte leptin receptor(OBR)and leptin transport in adult-onset obese mice[J]. Endocrinology,2013, 149(6):2798-2806.
[14]王桂华.鸡OBR基因组织表达规律的研究[D].哈尔滨:东北农业大学, 2004.
[15] Beohar N, Kawamoto S. Transcriptional regulation of the human nonmuscle myosin II heavy chain-A gene:Identification of three clustered cis-elements in intron-1 which modulate transcription in a cell type-and differentiation state-dependent manner[J].Journal of Biological Chemistry, 1998, 273(15):9168-9178.
[16] Meyer K B, Maia A T, O'Reilly M, et al. Allele-specific up-regulation of FGFR2 increases susceptibility to breast cancer[J]. PLoS Biology, 2008, 6(5):108.
[17] Do T N, Ucisik-Akkaya E, Davis C F, et al. An intronic polymorphism of IRF4 gene influences gene transcription in vitro and shows a risk association with childhood acute lymphoblastic leukemia in males[J]. Biochimica Et Biophysica Acta, 2010, 1802(2):292-300.
[18] Pattison J M, Posternak V, Cole M D. Transcription factor KLF5binds a cyclin e1 polymorphic intronic enhancer to confer increased bladder cancer risk[J]. Molecular Cancer Research, 2016, 14(11):1078-1086.
[19] Gorshkova E V, Kaledin V I, Kobzev V F, et al. Lung cancerassociated SNP at the beginning of mouse k-ras gene intron 2 is essential for transcription factor binding[J]. Bulletin of Experimental Biology and Medicine, 2006, 141(6):731-733.
[20] Matsusue K, Gavrilova O, Lambert G, et al. Hepatic CCAAT/enhancer binding protein alpha mediates induction of lipogenesis and regulation of glucose homeostasis in leptin-deficient mice[J].Molecular Endocrinology, 2004, 18(11):2751-2764.
[21] Qiao L, Maclean P S, Schaack J, et al. C/EBPalpha regulates human adiponectin gene transcription through an intronic enhancer[J]. Diabetes, 2005, 54(6):1744-1754.
[22]贺綦.鸡L-FABP和L-BABP基因表达调控研究[D].哈尔滨:东北农业大学, 2014.
[23] Luo H, Zhang Y, Guo H, et al. Transcriptional regulation of the human,porcineandbovine OCTN2genebyPPARαviaaconserved PPRE located in intron 1[J]. BMC Genetics, 2014, 15(1):90.
[24] Armelin-Correa L M, Lin C J, Barbosa A, et al. Characterization of human Collagen XVIII promoter 2:Interaction of Sp1, Sp3 and YY1 with the regulatory region and a SNP that increases transcription in hepatocytes[J]. Matrix Biology, 2005, 24(8):550-559.
[2] Ramiah S K, Meng G Y, Sheau W T, et al. Dietary conjugated linoleic acid supplementation leads to downregulation of PPAR Transcription in broiler chickens and reduction of adipocyte cellularity[J]. PPAR Research, 2014, 2014:1-10.
[3]陈耀峰,谢可嘉,张长超,等.鸡ApoB基因g.-112A>G单核苷酸多态性的功能性鉴定与分析[J].农业生物技术学报, 2018,26(11):103-111.
[4]王桂华,李辉,王启贵,等.鸡OBR基因在肉鸡和蛋鸡肝脏组织中差异表达的研究[J].东北农业大学学报, 2006, 37(5):650-655.
[5] Rancourt R C, Schellong K, Ott R, et al. Acquired alterations of hypothalamic gene expression of insulin and leptin receptors and glucose transporters in prenatally high-glucose exposed threeweek old chickens do not coincide with aberrant promoter DNA methylation[J]. PLoS One, 2015, 10(3):e0119213.
[6]徐淑静,徐明彤,傅祖植.肥胖基因和瘦素[J].中华内分泌代谢杂志, 1999, 15(2):111-113.
[7] Gohda T, Tanimoto M, Kaneko S, et al. Minor gene effect of leptin receptor variant on the body weight in KK/Ta mice[J]. Diabetes,Obesity and Metabolism, 2006, 8(5):581-584.
[8] Balatsky V, Bankovska I, Pena R N, et al. Polymorphisms of the porcine cathepsins, growth hormone-releasing hormone and leptin receptor genes and their association with meat quality traits in Ukrainian Large White breed[J]. Molecular Biology Reports,2016, 43(6):517-526.
[9]王颖,李辉,顾志良,等.鸡瘦蛋白受体(OBR)基因内含子8单核苷酸多态性与体脂性状的相关研究[J].遗传学报, 2004, 31(3):265-269.
[10] Lei M M, Wu S Q, Shao X B, et al. Creating leptin-like biofunctions by active immunization against chicken Leptin receptor in growing chickens[J]. Domestic Animal Endocrinology, 2015, 50:55-64.
[11] Fong T M, Huang R R, Tota M R, et al. Localization of leptin binding domain in the leptin receptor[J]. Molecular Pharmacology, 1998, 53(2):234-240.
[12] Yang C, Cui H X, Fan P. Expression of OBR and NPY in mouse hypothalamus[J]. Basic and Clinical Medicine, 2009, 29(2):152-155.
[13] Pan W, Hsuchou H, He Y, et al. Astrocyte leptin receptor(OBR)and leptin transport in adult-onset obese mice[J]. Endocrinology,2013, 149(6):2798-2806.
[14]王桂华.鸡OBR基因组织表达规律的研究[D].哈尔滨:东北农业大学, 2004.
[15] Beohar N, Kawamoto S. Transcriptional regulation of the human nonmuscle myosin II heavy chain-A gene:Identification of three clustered cis-elements in intron-1 which modulate transcription in a cell type-and differentiation state-dependent manner[J].Journal of Biological Chemistry, 1998, 273(15):9168-9178.
[16] Meyer K B, Maia A T, O'Reilly M, et al. Allele-specific up-regulation of FGFR2 increases susceptibility to breast cancer[J]. PLoS Biology, 2008, 6(5):108.
[17] Do T N, Ucisik-Akkaya E, Davis C F, et al. An intronic polymorphism of IRF4 gene influences gene transcription in vitro and shows a risk association with childhood acute lymphoblastic leukemia in males[J]. Biochimica Et Biophysica Acta, 2010, 1802(2):292-300.
[18] Pattison J M, Posternak V, Cole M D. Transcription factor KLF5binds a cyclin e1 polymorphic intronic enhancer to confer increased bladder cancer risk[J]. Molecular Cancer Research, 2016, 14(11):1078-1086.
[19] Gorshkova E V, Kaledin V I, Kobzev V F, et al. Lung cancerassociated SNP at the beginning of mouse k-ras gene intron 2 is essential for transcription factor binding[J]. Bulletin of Experimental Biology and Medicine, 2006, 141(6):731-733.
[20] Matsusue K, Gavrilova O, Lambert G, et al. Hepatic CCAAT/enhancer binding protein alpha mediates induction of lipogenesis and regulation of glucose homeostasis in leptin-deficient mice[J].Molecular Endocrinology, 2004, 18(11):2751-2764.
[21] Qiao L, Maclean P S, Schaack J, et al. C/EBPalpha regulates human adiponectin gene transcription through an intronic enhancer[J]. Diabetes, 2005, 54(6):1744-1754.
[22]贺綦.鸡L-FABP和L-BABP基因表达调控研究[D].哈尔滨:东北农业大学, 2014.
[23] Luo H, Zhang Y, Guo H, et al. Transcriptional regulation of the human,porcineandbovine OCTN2genebyPPARαviaaconserved PPRE located in intron 1[J]. BMC Genetics, 2014, 15(1):90.
[24] Armelin-Correa L M, Lin C J, Barbosa A, et al. Characterization of human Collagen XVIII promoter 2:Interaction of Sp1, Sp3 and YY1 with the regulatory region and a SNP that increases transcription in hepatocytes[J]. Matrix Biology, 2005, 24(8):550-559.
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