南阳牛ATP5B基因启动子区域多态性与生长性状的关联性研究
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摘要
ATP合成酶亚基β(ATP synthase subunit beta,ATP5B)作为ATP合成的重要关键蛋白之一,参与多种生物代谢活动。南阳牛(Bos taurus)是中国本土五大黄牛良种之一,以良好的抗逆性闻名于世,是重要的黄牛种质资源。本研究以南阳牛为研究对象,探究ATP5B基因启动子区域的多态性与南阳牛生长性状相关关系。利用生物信息学技术分析了不同物种ATP5B蛋白功能保守性,检测了南阳牛不同组织ATP5B基因m RNA表达量,并利用DNA测序方法检测南阳牛ATP5B基因启动子区域的多态性,分析了单个多态位点不同基因型与南阳牛生长性状的关联性,随后对具有多态性位点的不同基因型启动子活性做了双荧光素酶活性检测。结果发现牛的ATP5B和猪(Sus scrofa)等物种存在14个相似的motif结构和5个保守性功能域;南阳牛ATP5B基因在内脏组织和肌肉、脂肪组织中广泛表达,其中,在肌肉组织中表达量最高且显著高于其他组织(P<0.05),脂肪中次之;在南阳牛ATP5B基因启动子区域检测到了3个SNP位点,分别为g.-428T>A,g.-390T>C和g.-322C>G。将南阳牛生长性状与SNPs进行关联性分析,结果表明g.-428T>A位点上TT基因型个体的体重、体高和胸围显著高于AA基因型个体(P<0.01或P<0.05);g.-390T>C位点上TT基因型个体的体高和胸围与CC基因型个体存在极显著性差异(P<0.01);g.-322C>G位点CC基因型个体的体重和体长显著高于GG基因型个体(P<0.01或P<0.05)。双荧光素酶活性报告显示,各SNP位点的不同基因型启动子活性略有差别,但未达到显著水平(P>0.05)。因此,南阳牛ATP5B基因启动子区域的这3个SNP可考虑作为南阳牛生长性状相关分子标记的候选基因位点,用于南阳牛的分子育种工作,为相关分子育种工作提供参考。
The ATP synthase subunit beta(ATP5 B) is one of key factors in ATP synthesis,participating in various metabolic activities.Nanyang cattle(Bos taurus) is the one of the five major Chinese native cattle varieties,famous for its adaptability.This study investigated the genetic variation in ATP5 B promoter regions and the association of Nanyang cattle growth traits; estimated the conservation of ATP5 B among different species,and examined the m RNA expression.It was sequenced the ATP5 B promoter region to determine thegenetic variants and their association with Nanyang cattle growth traits.To find the reason of this association,It was detected the promoter activity of each genotype promoter at these SNPs site using dual luciferase assay.In the results,there were 14 similar motifs and 5 conservative functional domains in the ATP5 B of 7 species,such as,Bos taurus,Sus scrofa,Ruttus and so on.The ATP5 B was widely expressed in many tissues in Nanyang cattle,and the muscle tissue had the significant high m RNA level than other tissues(P<0.05),the ATP5 B m RNA in fat tissue was following,the next was in heart,kidney,lung,and liver; the ATP5 B m RNA in spleen was the lowest compared with other tissues(P<0.05).3 SNPs in ATP5 B promoter regions were identified,named as g.-428 T>A,g.-390 T>C and g.-322 C>G,respectively.After association analysis,there were 3 mutations fell into Hardy-Weinberg equilibrium significantly(Chi-Squared test,P>0.05).The polymorphism information content(PIC) classification indicated that the 3 SNPs were moderately polymorphic(0.25A locus,individuals with TT genotype exhibited significantly higher body weight,body height and chest circumference than those with AA genotype(P<0.01).Such as,TA individuals had greater body weight than AA individuals(P<0.01),but not than TT individuals(P>0.05).In body height,the individuals with TT or TA had greater level than AA individuals(P<0.05).In chest circumference,the TT individuals had greater level than the individuals with TA or AA(P<0.01).So,the TT was the preferable genotype.At g.-390 T>C locus,the body height and chest circumference of animals with TT genotype were significantly higher(P<0.01) than these traits of animals with CC genotype.TC individuals had greater body height than CC individuals(P<0.05),but less than TT individuals(P<0.01).TC individuals had less chest circumference than TT individuals(P<0.01).So,the TT was the preferable genotype.Moreover,the g.-322 C>G locus were significantly affected body weight and body length(P<0.01 or P<0.05),including that,the CC individuals had greater body weight than CG individuals(P<0.05) and GG individuals(P<0.01),meanwhile,CG individuals had greater body weight than GG individuals(P<0.05).In body length,the individuals with CC or CG had greater level than GG individuals(P<0.05).So that,CC was the preferable genotype.Last,according to the report of dual luciferase assay,although there were many difference between each promoter style at each SNP site,but all of them not reached at significant level(P>0.05).The molecular mechanism of regulation between these SNPs and growth traits need to further search.To sum up,these results suggested that the SNPs of ATP5 B promoter region,the TT at-428 T>A locus,TT at-390 T>C locus,and CC at-322 C>G locus,affected the growth traits of Nanyang cattle,and they could be used as candidate genotype for breeding programs.
The ATP synthase subunit beta(ATP5 B) is one of key factors in ATP synthesis,participating in various metabolic activities.Nanyang cattle(Bos taurus) is the one of the five major Chinese native cattle varieties,famous for its adaptability.This study investigated the genetic variation in ATP5 B promoter regions and the association of Nanyang cattle growth traits; estimated the conservation of ATP5 B among different species,and examined the m RNA expression.It was sequenced the ATP5 B promoter region to determine thegenetic variants and their association with Nanyang cattle growth traits.To find the reason of this association,It was detected the promoter activity of each genotype promoter at these SNPs site using dual luciferase assay.In the results,there were 14 similar motifs and 5 conservative functional domains in the ATP5 B of 7 species,such as,Bos taurus,Sus scrofa,Ruttus and so on.The ATP5 B was widely expressed in many tissues in Nanyang cattle,and the muscle tissue had the significant high m RNA level than other tissues(P<0.05),the ATP5 B m RNA in fat tissue was following,the next was in heart,kidney,lung,and liver; the ATP5 B m RNA in spleen was the lowest compared with other tissues(P<0.05).3 SNPs in ATP5 B promoter regions were identified,named as g.-428 T>A,g.-390 T>C and g.-322 C>G,respectively.After association analysis,there were 3 mutations fell into Hardy-Weinberg equilibrium significantly(Chi-Squared test,P>0.05).The polymorphism information content(PIC) classification indicated that the 3 SNPs were moderately polymorphic(0.25
引文
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田万强,梅楚刚,付常振,等.2016.牛PRKAA2基因SNP检测及与其生长和肉质性状的关联分析[J].西北农林科技大学学报:自然科学版,44(7):1-9.(Tian W Q,Mei C G,Fu C Z,et al.2016.SNPs of bovine PRKAA2gene and its association with growth and meat quality traits[J].Journal of Northwest A&F University(Natural Science Edition),44(7):1-9.)
肖凯,王庆松.2002.PTSD样情感行为异常大鼠脑组织ATP酶活性改变[J].西南国防医药,12(3):204-207.(Xiao K,Wang Q S.2002.Alteration of ATPase activities in hippocampi of rats with ptsd-like behavior[J].Medicial Journal of National Defending Forces in Southwest China,12(3):204-207.)
王梦琦,倪玮,张慧敏,等.2017.中国荷斯坦牛TLR1基因启动子区SNP多态与乳房炎抗性和泌乳性状的关联分析[J].农业生物技术学报,25(3):397-404.(Wang M Q,Ni W,Zhang H M,et al.2017.Correlation Between the Mutation of SNPs in the Promoter Region of TLR1 and Mastitis Resistance and Milking Traits in Chinese Holstein(Bos taurus)[J].Journal of Agricultural Biotechnology,25(3):397-404.)
赵卫华,孟凡义,赖永榕,等.2017.急性白血病死亡相关蛋白激酶基因启动子的甲基化[J].南方医科大学学报,(3):407-410.(Zhao W H,Meng F Y,Lai Y R,et al.2017.Promoter methylation and expression of death-associated protein kinase gene in acute leukemia[J].Journal of Southern Medical University,(3):407-410.)
Alsiddig M A,Yu S G,Pan Z X,et al.2017.Association of single nucleotide polymorphism in melatonin receptor1A gene with egg production traits in Yangzhou geese[J].Animal Genetics,48(2):245-249.
Ahluwalia A,Busse B A,Thiruvengadam S S,et al.2011.Importins are critical for colorectal cancer(CRC)growth and are novel biomarkers of crc.underlying mechanisms include:Increased nuclear transport of P-CREBand P-STAT3,VEGF gene promoter activation and aberrant VEGF expression[J].Gastroenterology,140(5):S184-S184.
Bailey T L,Boden M,Buske F A,et al.2009.MEME Suite:Tools for motif discovery and searching[J].Nucleic Acids Research,37(Web Server issue):W202-W208.
Chafey P,Finzi L,Boisgard R,et al.2009.Proteomic analysis of beta-catenin activation in mouse liver by DIGE analysis identifies glucose metabolism as a new target of the Wnt pathway[J].Proteomics,9(15):3889-3900.
Chaze T,Meunier B,Chambon C,et al.2008.In vivo proteome dynamics during early bovine myogenesis[J].Proteomics,8(20):4236-4248.
Cosenza G,Iannaccone M,Pico B A,et al.2017.Molecular characterisation,genetic variability and detection of a functional polymorphism influencing the promoter activity of OXT gene in goat and sheep[J].Journal of Dairy Research,84(2):165.
Felsenstein J.1985.Confidence Limits on Phylogenies:An approach using the bootstrap[J].Evolution,39(4):783-791.
Garcia J.2011.The calcium channel alpha2/delta1 subunit interacts with ATP5bin the plasma membrane of developing muscle cells[J].American Journal of Physiologycell Physiology,30(1):44-52.
Garieri M,Delaneau O,Santoni F,et al.2017.The effect of genetic variation on promoter usage and enhancer activity[J].Nature Communications,8(1):9.
Gui L S,Hong J Y,Raza S H A,et al.2017.Genetic variants in SIRT3 transcriptional regulatory region affect promoter activity and fat deposition in three cattle breeds[J].Molecular and Cellular Probes,32(2017):40-45.
Huang Y Z,Li M X,Wang J,et al.2013.A 5′-regulatory region and two coding region polymorphisms modulate promoter activity and gene expression of the growth suppressor gene ZBED6 in cattle[J].PLo S One,8(11):e79744.
Komatsu Y,Sukegawa S,Yamashita M,et al.2016.Identification of genes showing differential expression profile associated with growth rate in skeletal muscle tissue of Landrace weanling pig[J].Journal of Genetics,95(2):341-347.
Kumar S,Stecher G,Tamura K.2016.MEGA7:Molecular evolutionary genetics analysis version 7.0 for bigger datasets[J].Molecular Biology&Evolution,33(7):1870.
Li A N,Mo D L,Zhao X,et al.2013.Comparison of the longissimus muscle proteome between obese and lean pigs at 180 days[J].Mammalian Genome,24(1-2):72-79.
Li Y,Bao C,Gu S,et al.2017.Associations between novel genetic variants in the promoter region of MALAT1 and risk of colorectal cancer[J].Oncotarget,8(54):92604-92614.
Liu Z,Sun W,Zhao Y,et al.2014.The effect of variants in the promoter of BMPER on the intramuscular fat deposition in longissimus dorsi muscle of pigs[J].Gene,542(2):168-172.
Marchler-Bauer A,Bo Y,Han L,et al.2017.CDD/SPARCLE:Functional classification of proteins via subfamily domain architectures[J].Nucleic Acids Research,45(D1):D200.
Marchlerbauer A,Derbyshire M K,Gonzales N R,et al.2015.CDD:NCBI's conserved domain database[J].Nucleic Acids Research,43(Database issue):D222.
Marchler-Bauer A,Lu S,Anderson J B,et al.2011.CDD:AConserved Domain Database for the functional annotation of proteins[J].Nucleic Acids Research,39(Database issue):D225.
Neckelmann N,Warner C K,Chung A,et al.1989.The human ATP synthase beta subunit gene:sequence analysis,chromosome assignment,and differential expression[J].Genomics,5(4):829-843.
Pisignano G,Napoli S,Magistri M,et al.2017.A promoterproximal transcript targeted by genetic polymorphism controls E-cadherin silencing in human cancers[J].Nature Communications,8(15622):16.
Reggiani C,Coppens S,Sekhara T,et al.2017.Novel promoters and coding first exons in DLG2 linked to developmental disorders and intellectual disability[J].Genome Medicine,9(67):20.
Roth Z 2015.Physiology and endocrinology symposium:Cellular and molecular mechanisms of heat stress related to bovine ovarian function[J].Journal of Animal Science,93:2034-2044.
Saitou N,Nei M.1987.The neighbor-joining method:A new method for reconstructing phylogenetic trees[J].Molecular Biology&Evolution,4(4):406.
Seo H,Lee I,Chung H S,et al.2016.ATP5B regulates mitochondrial fission and fusion in mammalian cells[J].Animal Cells and Systems,20(3):157-164.
Wu C,Wei K,Chou J,et al.2015.945 Infection of H.pylori is associated with promoter methylation of a putative STAT3 targeted micro RNA,mi R-193a in gastric cancer[J].Gastroenterology,148(4):S182-S182.
Wu S,Wang Y,Ning Y,et al.2018.Genetic variants in STAT3promoter regions and their application in molecular breeding for body size traits in Qinchuan cattle[J].International Journal of Molecular Sciences,19(4):1035.
Xu H,Xu Y,Liang X,et al.2013.Porcine skeletal muscle differentially expressed gene ATP5B:Molecular characterization,expression patterns,and association analysis with meat quality traits[J].Mammalian Genome,24(3-4):142-150.
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段美艳,李安宁,赵志东,等.2015a.牛ATP5B基因启动子双荧光素酶报告基因载体构建与活性检测[J].西北农林科技大学学报自然科学版,43(8):39-45.(Duan M Y,Li A N,Zhao Z D,et al.2015a.Construction and identification of bovine ATP5B gene promoter dual luciferase report gene vector[J].Journal of Northwest A&F University(Natural Science Edition),43(8):39-45.)
段美艳,赵志东,李安宁,等.2015b.牛ATP5B基因启动子与组织差异性表达研究[J].畜牧兽医学报,46(3):382-387.(Duan M Y,Zhao Z D,Li A N,et al.2015b,Analysis of bovine ATP5B gene promoter and tissue expression[J].Chinese Journal of Animal and Veterinary Sciences,46(3):382-387.)
戴亚丽,蔡德鸿,张桦,等.2011.抑癌基因RAS启动子甲基化与乳头状甲状腺癌的关系[J].首都医科大学学报,32(1):135-138.(Dai Y L,Cai D H,Zhang H,et al.2011,Relationship between the promoter methylation of RASgene and human papillary thyroid[J].Journal of Capital Medical University.32(1):135-138.)
胡建新,何坚,刘军,等.2011.氧自由基对生殖细胞三磷酸腺苷酶和生长抑素的影响[J].中国组织工程研究与临床康复,(18):3361-3364.(Hu J X,He J,Liu J,et al.2011.Effect of oxygen free radicals on adenosine triphosphatase and somatostatin in reproductive cells[J].Journal of Clinical Rehabilitative Tissue Engineering Research,(18):3361-3364.)
李圣彦,郎志宏,黄大昉.2014.真核生物启动子研究概述[J].生物技术进展,(3):158-164.(Li S Y,Lang Z H,Huang D F.2014.Research progress on eukaryotic promoter[J].Current Biotechnology,(3):158-164.)
田万强,梅楚刚,付常振,等.2016.牛PRKAA2基因SNP检测及与其生长和肉质性状的关联分析[J].西北农林科技大学学报:自然科学版,44(7):1-9.(Tian W Q,Mei C G,Fu C Z,et al.2016.SNPs of bovine PRKAA2gene and its association with growth and meat quality traits[J].Journal of Northwest A&F University(Natural Science Edition),44(7):1-9.)
肖凯,王庆松.2002.PTSD样情感行为异常大鼠脑组织ATP酶活性改变[J].西南国防医药,12(3):204-207.(Xiao K,Wang Q S.2002.Alteration of ATPase activities in hippocampi of rats with ptsd-like behavior[J].Medicial Journal of National Defending Forces in Southwest China,12(3):204-207.)
王梦琦,倪玮,张慧敏,等.2017.中国荷斯坦牛TLR1基因启动子区SNP多态与乳房炎抗性和泌乳性状的关联分析[J].农业生物技术学报,25(3):397-404.(Wang M Q,Ni W,Zhang H M,et al.2017.Correlation Between the Mutation of SNPs in the Promoter Region of TLR1 and Mastitis Resistance and Milking Traits in Chinese Holstein(Bos taurus)[J].Journal of Agricultural Biotechnology,25(3):397-404.)
赵卫华,孟凡义,赖永榕,等.2017.急性白血病死亡相关蛋白激酶基因启动子的甲基化[J].南方医科大学学报,(3):407-410.(Zhao W H,Meng F Y,Lai Y R,et al.2017.Promoter methylation and expression of death-associated protein kinase gene in acute leukemia[J].Journal of Southern Medical University,(3):407-410.)
Alsiddig M A,Yu S G,Pan Z X,et al.2017.Association of single nucleotide polymorphism in melatonin receptor1A gene with egg production traits in Yangzhou geese[J].Animal Genetics,48(2):245-249.
Ahluwalia A,Busse B A,Thiruvengadam S S,et al.2011.Importins are critical for colorectal cancer(CRC)growth and are novel biomarkers of crc.underlying mechanisms include:Increased nuclear transport of P-CREBand P-STAT3,VEGF gene promoter activation and aberrant VEGF expression[J].Gastroenterology,140(5):S184-S184.
Bailey T L,Boden M,Buske F A,et al.2009.MEME Suite:Tools for motif discovery and searching[J].Nucleic Acids Research,37(Web Server issue):W202-W208.
Chafey P,Finzi L,Boisgard R,et al.2009.Proteomic analysis of beta-catenin activation in mouse liver by DIGE analysis identifies glucose metabolism as a new target of the Wnt pathway[J].Proteomics,9(15):3889-3900.
Chaze T,Meunier B,Chambon C,et al.2008.In vivo proteome dynamics during early bovine myogenesis[J].Proteomics,8(20):4236-4248.
Cosenza G,Iannaccone M,Pico B A,et al.2017.Molecular characterisation,genetic variability and detection of a functional polymorphism influencing the promoter activity of OXT gene in goat and sheep[J].Journal of Dairy Research,84(2):165.
Felsenstein J.1985.Confidence Limits on Phylogenies:An approach using the bootstrap[J].Evolution,39(4):783-791.
Garcia J.2011.The calcium channel alpha2/delta1 subunit interacts with ATP5bin the plasma membrane of developing muscle cells[J].American Journal of Physiologycell Physiology,30(1):44-52.
Garieri M,Delaneau O,Santoni F,et al.2017.The effect of genetic variation on promoter usage and enhancer activity[J].Nature Communications,8(1):9.
Gui L S,Hong J Y,Raza S H A,et al.2017.Genetic variants in SIRT3 transcriptional regulatory region affect promoter activity and fat deposition in three cattle breeds[J].Molecular and Cellular Probes,32(2017):40-45.
Huang Y Z,Li M X,Wang J,et al.2013.A 5′-regulatory region and two coding region polymorphisms modulate promoter activity and gene expression of the growth suppressor gene ZBED6 in cattle[J].PLo S One,8(11):e79744.
Komatsu Y,Sukegawa S,Yamashita M,et al.2016.Identification of genes showing differential expression profile associated with growth rate in skeletal muscle tissue of Landrace weanling pig[J].Journal of Genetics,95(2):341-347.
Kumar S,Stecher G,Tamura K.2016.MEGA7:Molecular evolutionary genetics analysis version 7.0 for bigger datasets[J].Molecular Biology&Evolution,33(7):1870.
Li A N,Mo D L,Zhao X,et al.2013.Comparison of the longissimus muscle proteome between obese and lean pigs at 180 days[J].Mammalian Genome,24(1-2):72-79.
Li Y,Bao C,Gu S,et al.2017.Associations between novel genetic variants in the promoter region of MALAT1 and risk of colorectal cancer[J].Oncotarget,8(54):92604-92614.
Liu Z,Sun W,Zhao Y,et al.2014.The effect of variants in the promoter of BMPER on the intramuscular fat deposition in longissimus dorsi muscle of pigs[J].Gene,542(2):168-172.
Marchler-Bauer A,Bo Y,Han L,et al.2017.CDD/SPARCLE:Functional classification of proteins via subfamily domain architectures[J].Nucleic Acids Research,45(D1):D200.
Marchlerbauer A,Derbyshire M K,Gonzales N R,et al.2015.CDD:NCBI's conserved domain database[J].Nucleic Acids Research,43(Database issue):D222.
Marchler-Bauer A,Lu S,Anderson J B,et al.2011.CDD:AConserved Domain Database for the functional annotation of proteins[J].Nucleic Acids Research,39(Database issue):D225.
Neckelmann N,Warner C K,Chung A,et al.1989.The human ATP synthase beta subunit gene:sequence analysis,chromosome assignment,and differential expression[J].Genomics,5(4):829-843.
Pisignano G,Napoli S,Magistri M,et al.2017.A promoterproximal transcript targeted by genetic polymorphism controls E-cadherin silencing in human cancers[J].Nature Communications,8(15622):16.
Reggiani C,Coppens S,Sekhara T,et al.2017.Novel promoters and coding first exons in DLG2 linked to developmental disorders and intellectual disability[J].Genome Medicine,9(67):20.
Roth Z 2015.Physiology and endocrinology symposium:Cellular and molecular mechanisms of heat stress related to bovine ovarian function[J].Journal of Animal Science,93:2034-2044.
Saitou N,Nei M.1987.The neighbor-joining method:A new method for reconstructing phylogenetic trees[J].Molecular Biology&Evolution,4(4):406.
Seo H,Lee I,Chung H S,et al.2016.ATP5B regulates mitochondrial fission and fusion in mammalian cells[J].Animal Cells and Systems,20(3):157-164.
Wu C,Wei K,Chou J,et al.2015.945 Infection of H.pylori is associated with promoter methylation of a putative STAT3 targeted micro RNA,mi R-193a in gastric cancer[J].Gastroenterology,148(4):S182-S182.
Wu S,Wang Y,Ning Y,et al.2018.Genetic variants in STAT3promoter regions and their application in molecular breeding for body size traits in Qinchuan cattle[J].International Journal of Molecular Sciences,19(4):1035.
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