基于黄河鲤新品系IGF2b基因SNPs获取体系的建立及在该品系选育中的应用
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摘要
为了建立鲤IGF2b (insulin-like growth factor 2)基因全基因组信息SNPs (single nucleotide polymorphism)的获取体系,并验证该获取体系在黄河鲤新品系选育中运用的适用性,本研究首先对10个不同鲤品种的33个鲤个体重测序数据进行分析,整体上了解IGF2b基因的单核苷酸位点及其在基因组上的分布情况。然后在基因组DNA水平上分段获取IGF2b基因的SNPs信息,并以不同的鲤品种验证引物的适用性,最后在黄河鲤新品系中检测其应用。研究结果:获得8对聚合酶链式反应(polymerase chain reaction, PCR)引物进行分段克隆的结果,并通过直接测序和限制性片段长度多态性来获取相应的SNPs。经过直接测序,本文对黄河鲤、建鲤、黄河鲤和建鲤的正反交后代进行PCR检测发现条带单一,目地片段正确;结合黄河鲤新品系的体重数据,本文又以IGF2b~(3#)引物检测到一个与体重明显相关的SNPs,同时检测到一个与该基因表达量相关的另一个SNPs。本研究的方法能够在该基因全基因组范围内开展鲤分子育种中SNPs的检测,并验证发现黄河鲤新品系IGF2b~(3#)引物227号位点处,出现的突变位点使体重降低,且有一处SNPs与其表达量有关。这将为其他物种、其他功能基因的多态性分子标记研究提供思路。
In order to establish an SNP(single nucleotide polymorphism) acquisition system for the IGF2b (insulin-like growth factor 2) gene, a total of 33 DNA samples extracted from 10 different carp species were sequenced and the data were analyzed to verify the function and efficacy of the breeding process for the new Huanghe carp strain. By determining the position and distribution of SNPs on the genome and obtaining the IGF2b gene by segmentation at the genomic DNA level in this way, the applicability of primers from different carp species for use in Huanghe carp new strains was verified. As a result of the segmentation at the genomic DNA level, eight pairs of polymerase chain reaction(PCR) primers were obtained, and the corresponding SNPs were determined by direct sequencing and restriction fragment length polymorphisms. Furthermore, direct sequencing showed a single band from the PCR of Huanghe carp and Jian carp as well as the progenies resulting from their reciprocal crosses, which allowed us to confirm the validity of the target fragment. Finally, analysis of the Huanghe carp body weight data combined with the latest results allowed the detection of an SNP significantly associated with the body weight using IGF2b~(3#) primers. Likewise, another SNP was detected in association with the expression level of this gene.The methods used in this study were able to detect the SNPs of genes in the molecular breeding of carp within the range of the whole genome, to verify that the mutation at the No. 227 site on the IGF2b~(3#) primer of Huanghe carp led to reduced body weight, and to identify another SNP related to the expression level of IGF2b. This will provide the background for the study of polymorphic molecular markers and other functional genes in other species.
In order to establish an SNP(single nucleotide polymorphism) acquisition system for the IGF2b (insulin-like growth factor 2) gene, a total of 33 DNA samples extracted from 10 different carp species were sequenced and the data were analyzed to verify the function and efficacy of the breeding process for the new Huanghe carp strain. By determining the position and distribution of SNPs on the genome and obtaining the IGF2b gene by segmentation at the genomic DNA level in this way, the applicability of primers from different carp species for use in Huanghe carp new strains was verified. As a result of the segmentation at the genomic DNA level, eight pairs of polymerase chain reaction(PCR) primers were obtained, and the corresponding SNPs were determined by direct sequencing and restriction fragment length polymorphisms. Furthermore, direct sequencing showed a single band from the PCR of Huanghe carp and Jian carp as well as the progenies resulting from their reciprocal crosses, which allowed us to confirm the validity of the target fragment. Finally, analysis of the Huanghe carp body weight data combined with the latest results allowed the detection of an SNP significantly associated with the body weight using IGF2b~(3#) primers. Likewise, another SNP was detected in association with the expression level of this gene.The methods used in this study were able to detect the SNPs of genes in the molecular breeding of carp within the range of the whole genome, to verify that the mutation at the No. 227 site on the IGF2b~(3#) primer of Huanghe carp led to reduced body weight, and to identify another SNP related to the expression level of IGF2b. This will provide the background for the study of polymorphic molecular markers and other functional genes in other species.
引文
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[7]Yuan Y,Hong Y.Medaka insulin-like growth factor-2 supports self-renewal of the embryonic stem cell line and blastomeres in vitro[J].Scientific Reports,2017,7:Article No.78.
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[10]Knoll A,PutnováL,Dvo?ák J,et al.A Ncii PCR-RFLPwithin intron 2 of the porcine insulin-like growth factor 2b(IGF2)gene[J].Animal Genetics,2000,31(2):150.
[11]Wang G Y,Yan B X,Deng X M,et al.Effects of IGF2 gene on growth and carcass traits of chickens and their imprinting status[J].Science in China Ser.C:Life Sciences,2004,34(5):429-435.[王根宇,颜炳学,邓学梅,等.IGF2基因对鸡生长及屠体性状的影响及印记状况的研究[J].中国科学:C辑,生命科学,2004,34(5):429-435.]
[12]Chen J Y,Chen J C,Chang C Y,et al.Expression of recombinant tilapia insulin-like growth factor-I and stimulation of juvenile tilapia growth by injection of recombinant IGFs polypeptides[J].Aquaculture,2000,181(3-4):347-360.
[13]Bachner-Melman R,Zohar A H,Nemanov L,et al.Association between the insulin-like growth factor 2 gene(IGF2)and scores on the Eating Attitudes Test in nonclinical subjects:A family-based study[J].American Journal of Psychiatry,2005,162:2256-2262.
[14]Petry C J,Seear R V,Wingate D L,et al.Associations between paternally transmitted fetal IGF2 variants and maternal circulating glucose concentrations in pregnancy[J].Diabetes,2011,60(11):3090-3096.
[15]Van Laere A S,Nguyen M,Braunschweig M,et al.A regulatory mutation in IGF2 causes a major QTL effect on muscle growth in the pig[J].Nature,2003,425(6960):832-836.
[16]Sun Y,Fei S Q,Tang H,et al.Polymorphism of G3072A in IGF2 gene and its association with birth weight and early growth traits in Yorkshire pigs[J].Acta Veterinaria et Zootechnica Sinica,2007,38(12):1306-1310.[孙亿,费思清,唐辉,等.猪IGF2基因G3072A位点多态性及其与大白猪初生重和早期生长的关系[J].畜牧兽医学报,2007,38(12):1306-1310.]
[17]Fassa V B,Carden T R,Marini S J,et al.Analysis of the effects of five genes(IGF2,CTSD,TBC1D1,MC4R and FABP3)on feed conversion,growth rate and the content of subcutaneous fat in pigs of the Landrace breed[J].RIA,Revista de Investigaciones Agropecuarias,2015,41(3):282-288.
[18]Zou S M,Kamei H,Modi Z,et al.Zebrafish IGF genes:gene duplication,conservation and divergence,and novel roles in midline and notochord development[J].PLo S ONE,2009,4(9):e7026.
[19]Su S Y,Dong Z J,Qu J Q,et al.Molecular cloning and single nucleotide polymorphism analysis of IGF2a genes in the common carp(Cyprinus carpio)[J].Genetics and Molecular Research,2012,11(2):1327-1340.
[20]Xu P,Zhang X F,Wang X M,et al.Genome sequence and genetic diversity of the common carp,Cyprinus carpio[J].Nature Genetics,2014,46:1212-1219.
[21]Su S Y,Dong Z J,Yuan X H,et al.Clone of IGF2b introns,analysis of its genomic sequence and.construction of lentiviral vector(Lenti-IGF2-IRES-EGF)in common carp(Cyprinus carpio L.)[J].Acta Scientiarum Naturalium Universitatis Sunyatseni,2012,51(2):77-85.[苏胜彦,董在杰,袁新华,等.鲤IGF2b基因内含子的克隆,基因组序列分析及慢病毒载体构建[J].中山大学学报(自然科学版),2012,51(2):77-85.]
[22]Li Z H,Wang Q G,Zhao J G,et al.Study on correlation analysis of single nucleotide polymorphism of IGF2 gene and body fatness traits in chicken[J].Scientia Agricultura Sinica,2004,37(4):600-604.[李志辉,王启贵,赵建国,等.类胰岛素生长因子II(IGF2)基因多态性与鸡体脂性状的相关研究[J].中国农业科学,2004,37(4):600-604.]
[23]Yu D B,He Z L,Zhang W F,et al.The genetic effects of IGF2 gene intron3 variance in pigs[J].Hereditas,2008,30(1):87-93.[虞德兵,何宗亮,张伟峰,等.猪IGF2基因内含子3变异的遗传效应分析[J].遗传,2008,30(1):87-93.]
[24]Zhang N,Su S Y,Yuan X H,et al.The effect of overexpression of IGF2b gene mediated by lentiviral vector in dorsal muscle of common carp on growth of common carp:Cyprinus carpio L.)[J].Journal of Yangzhou University(Agricultural and Life Science Edition),2015,36(3):31-35.[张宁,苏胜彦,袁新华,等.慢病毒载体介导的IGF2b基因在鲤鱼背部肌肉组织中过表达对鲤鱼生长的影响[J].扬州大学学报:农业与生命科学版,2015,36(3):31-35.]
[25]Younis S,Sch?nke M,Massart J,et al.The ZBED6-IGF2axis has a major effect on growth of skeletal muscle and internal organs in placental mammals[J].Proceedings of the National Academy of Sciences of the United States of America,2018,115(9):E2048-E2057.
[26]Msalya G,Shimogiri T,Ohno S,et al.Evaluation of PRNPexpression based on genotypes and alleles of two indel loci in the medulla oblongata of Japanese Black and Japanese Brown cattle[J].PLoS ONE,2011,6(5):e18787.
[27]Helmig S,Aliahmadi N,Stephan P,et al.TNF-α-308 genotypes are associated with TNF-αand TGF-β1 mRNA expression in blood leucocytes of humans[J].Cytokine,2011,53(3):306-310.
[28]Gossner A G,Bennet N,Hunter N,et al.Differential expression of Prnp and Sprn in scrapie infected sheep also reveals Prnp genotype specific differences[J].Biochemical and Biophysical Research Communications,2009,378(4):862-866.
[29]Lee E,Cho S,Kim K,et al.An integrated approach to infer causal associations among gene expression,genotype variation,and disease[J].Genomics,2009,94(4):269-277.
[30]Olsson M,Olsson B,Jacobson P,et al.Expression of the selenoprotein S(SELS)gene in subcutaneous adipose tissue and SELS genotype are associated with metabolic risk factors[J].Metabolism,2011,60(1):114-120.
[31]Yao J,Liao K,Li Q L,et al.m RNA expression of glutathione S-transferase Pi(GSTP1)under heat stress and association of genotypes with heat tolerance ability in Holstein[J].Journal of Animal and Veterinary Advances,2011,10(6):757-763.
[2]Su S Y,Zhang C F,Dong Z J,et al.The breeding gain of Huanghe carp(Cyprinus carpio hacmalopterus Temminck et Schlegel)new strain G3-The effect of higher carbohydrate diet on growth and fatty acid profile of Huanghe carp new strain[J].Journal of Yangzhou University(Agricultural and Life Science Edition),2018,39(2):63-66.[苏胜彦,张成锋,董在杰,等.黄河鲤新品系G3代选育进展--饲喂高碳水化合物饲料对其生长和脂肪酸组分的影响[J].扬州大学学报(农业与生命科学版),2018,39(2):63-66.]
[3]Rinderknecht E,Humbel R E.The amino acid sequence of human insulin-like growth factor I and its structural homology with proinsulin[J].Journal of Biological Chemistry,1978,253(8):2769-2776.
[4]Vasilatosyounken R,Scanes C G.Growth hormone and insulin-like growth factors in poultry growth:Required,optimal,or ineffective?[J].Poultry Science,1991,70(8):1764-1780.
[5]Kouros-Mehr H,Werb Z.Candidate regulators of mammary branching morphogenesis identified by genome-wide transcript analysis[J].Developmental Dynamics,2006,235(12):3404-3412.
[6]Su S Y,Dong Z J,Qu J Q,et al.Molecular cloning and single,nucleotide polymorphism analysis of IGF2a genes in the common carp(Cyprinus carpio)[J].Genetics and Molecular Research,2012,11(2):1327-1340.
[7]Yuan Y,Hong Y.Medaka insulin-like growth factor-2 supports self-renewal of the embryonic stem cell line and blastomeres in vitro[J].Scientific Reports,2017,7:Article No.78.
[8]Andersson L,Haley C S,Ellegren H,et al.Genetic mapping of quantitative trait loci for growth and fatness in pigs[J].Science,1994,263(5154):1771-1774.
[9]Liu G L,Jiang S W,Xiong Y,et al.Association of PCR-RFLP polymorphisms of IGF2 gene with fat deposit related traits in pig resource family[J].Acta Genetica Sinica,2003,30(12):1107-1112.[刘桂兰,蒋思文,熊远,等.IGF2基因PCR-RFLP多态性与脂肪沉积相关性状的关联分析[J].遗传学报,2003,30(12):1107-1112.]
[10]Knoll A,PutnováL,Dvo?ák J,et al.A Ncii PCR-RFLPwithin intron 2 of the porcine insulin-like growth factor 2b(IGF2)gene[J].Animal Genetics,2000,31(2):150.
[11]Wang G Y,Yan B X,Deng X M,et al.Effects of IGF2 gene on growth and carcass traits of chickens and their imprinting status[J].Science in China Ser.C:Life Sciences,2004,34(5):429-435.[王根宇,颜炳学,邓学梅,等.IGF2基因对鸡生长及屠体性状的影响及印记状况的研究[J].中国科学:C辑,生命科学,2004,34(5):429-435.]
[12]Chen J Y,Chen J C,Chang C Y,et al.Expression of recombinant tilapia insulin-like growth factor-I and stimulation of juvenile tilapia growth by injection of recombinant IGFs polypeptides[J].Aquaculture,2000,181(3-4):347-360.
[13]Bachner-Melman R,Zohar A H,Nemanov L,et al.Association between the insulin-like growth factor 2 gene(IGF2)and scores on the Eating Attitudes Test in nonclinical subjects:A family-based study[J].American Journal of Psychiatry,2005,162:2256-2262.
[14]Petry C J,Seear R V,Wingate D L,et al.Associations between paternally transmitted fetal IGF2 variants and maternal circulating glucose concentrations in pregnancy[J].Diabetes,2011,60(11):3090-3096.
[15]Van Laere A S,Nguyen M,Braunschweig M,et al.A regulatory mutation in IGF2 causes a major QTL effect on muscle growth in the pig[J].Nature,2003,425(6960):832-836.
[16]Sun Y,Fei S Q,Tang H,et al.Polymorphism of G3072A in IGF2 gene and its association with birth weight and early growth traits in Yorkshire pigs[J].Acta Veterinaria et Zootechnica Sinica,2007,38(12):1306-1310.[孙亿,费思清,唐辉,等.猪IGF2基因G3072A位点多态性及其与大白猪初生重和早期生长的关系[J].畜牧兽医学报,2007,38(12):1306-1310.]
[17]Fassa V B,Carden T R,Marini S J,et al.Analysis of the effects of five genes(IGF2,CTSD,TBC1D1,MC4R and FABP3)on feed conversion,growth rate and the content of subcutaneous fat in pigs of the Landrace breed[J].RIA,Revista de Investigaciones Agropecuarias,2015,41(3):282-288.
[18]Zou S M,Kamei H,Modi Z,et al.Zebrafish IGF genes:gene duplication,conservation and divergence,and novel roles in midline and notochord development[J].PLo S ONE,2009,4(9):e7026.
[19]Su S Y,Dong Z J,Qu J Q,et al.Molecular cloning and single nucleotide polymorphism analysis of IGF2a genes in the common carp(Cyprinus carpio)[J].Genetics and Molecular Research,2012,11(2):1327-1340.
[20]Xu P,Zhang X F,Wang X M,et al.Genome sequence and genetic diversity of the common carp,Cyprinus carpio[J].Nature Genetics,2014,46:1212-1219.
[21]Su S Y,Dong Z J,Yuan X H,et al.Clone of IGF2b introns,analysis of its genomic sequence and.construction of lentiviral vector(Lenti-IGF2-IRES-EGF)in common carp(Cyprinus carpio L.)[J].Acta Scientiarum Naturalium Universitatis Sunyatseni,2012,51(2):77-85.[苏胜彦,董在杰,袁新华,等.鲤IGF2b基因内含子的克隆,基因组序列分析及慢病毒载体构建[J].中山大学学报(自然科学版),2012,51(2):77-85.]
[22]Li Z H,Wang Q G,Zhao J G,et al.Study on correlation analysis of single nucleotide polymorphism of IGF2 gene and body fatness traits in chicken[J].Scientia Agricultura Sinica,2004,37(4):600-604.[李志辉,王启贵,赵建国,等.类胰岛素生长因子II(IGF2)基因多态性与鸡体脂性状的相关研究[J].中国农业科学,2004,37(4):600-604.]
[23]Yu D B,He Z L,Zhang W F,et al.The genetic effects of IGF2 gene intron3 variance in pigs[J].Hereditas,2008,30(1):87-93.[虞德兵,何宗亮,张伟峰,等.猪IGF2基因内含子3变异的遗传效应分析[J].遗传,2008,30(1):87-93.]
[24]Zhang N,Su S Y,Yuan X H,et al.The effect of overexpression of IGF2b gene mediated by lentiviral vector in dorsal muscle of common carp on growth of common carp:Cyprinus carpio L.)[J].Journal of Yangzhou University(Agricultural and Life Science Edition),2015,36(3):31-35.[张宁,苏胜彦,袁新华,等.慢病毒载体介导的IGF2b基因在鲤鱼背部肌肉组织中过表达对鲤鱼生长的影响[J].扬州大学学报:农业与生命科学版,2015,36(3):31-35.]
[25]Younis S,Sch?nke M,Massart J,et al.The ZBED6-IGF2axis has a major effect on growth of skeletal muscle and internal organs in placental mammals[J].Proceedings of the National Academy of Sciences of the United States of America,2018,115(9):E2048-E2057.
[26]Msalya G,Shimogiri T,Ohno S,et al.Evaluation of PRNPexpression based on genotypes and alleles of two indel loci in the medulla oblongata of Japanese Black and Japanese Brown cattle[J].PLoS ONE,2011,6(5):e18787.
[27]Helmig S,Aliahmadi N,Stephan P,et al.TNF-α-308 genotypes are associated with TNF-αand TGF-β1 mRNA expression in blood leucocytes of humans[J].Cytokine,2011,53(3):306-310.
[28]Gossner A G,Bennet N,Hunter N,et al.Differential expression of Prnp and Sprn in scrapie infected sheep also reveals Prnp genotype specific differences[J].Biochemical and Biophysical Research Communications,2009,378(4):862-866.
[29]Lee E,Cho S,Kim K,et al.An integrated approach to infer causal associations among gene expression,genotype variation,and disease[J].Genomics,2009,94(4):269-277.
[30]Olsson M,Olsson B,Jacobson P,et al.Expression of the selenoprotein S(SELS)gene in subcutaneous adipose tissue and SELS genotype are associated with metabolic risk factors[J].Metabolism,2011,60(1):114-120.
[31]Yao J,Liao K,Li Q L,et al.m RNA expression of glutathione S-transferase Pi(GSTP1)under heat stress and association of genotypes with heat tolerance ability in Holstein[J].Journal of Animal and Veterinary Advances,2011,10(6):757-763.