SIX1基因表达及其多态性与绵羊季节性繁殖和产羔数的关联分析
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摘要
为探究繁殖组织中SIX1基因的表达水平及其多态性与绵羊季节性繁殖和产羔数之间的关系,本实验通过实时荧光定量PCR(qPCR)研究SIX1基因在不同产羔数(单羔组和多羔组)小尾寒羊卵巢、子宫体、输卵管组织中的表达情况,同时利用Sequenom MassARRAY?SNP技术检测多羔品种(小尾寒羊、湖羊、策勒黑羊共560只)和单羔品种(滩羊、苏尼特羊、草地型藏羊共204只)中SIX1基因g.69738971 T>G的多态性,并与其中有产羔数记录的380只小尾寒羊产羔数进行关联分析。结果表明:SIX1基因在小尾寒羊卵巢、子宫体、输卵管组织中均表达,且单羔组的子宫体中SIX1基因表达量显著高于多羔组(P<0.05);分型结果表明,SIX1基因g.69738971 T>G位点共存在TT、GT和GG 3种基因型,且基因型频率和等位基因频率在单、多羔品种(即高、低繁殖性能的母羊)间差异均极显著(P<0.01);卡方适合性检验表明,该位点在6个绵羊品种中均处于Hardy-Weinberg平衡状态(P>0.05);该位点多态性与小尾寒羊第1、2及第3胎产羔数均显著相关(P<0.05),即野生型(TT)各胎产羔数均显著高于突变型(GT和GG)。综上,SIX1基因表达与绵羊繁殖有关,该基因表达水平与绵羊产羔数可能存在一定程度的负相关,其g.69738971 T>G位点可作为控制绵羊季节性繁殖和产羔数性状的一个潜在分子标记位点。
To explore expression level of SIX1 gene in reproductive tissues and the association between polymorphism and seasonal reproduction, litter size in sheep, real-time fluorescent quantitative PCR(qPCR) was used to detect the expression of SIX1 gene in ovary, uterine body and oviduct of Small Tail Han sheep(STH) at different litter sizes(uniparous group and multiparous group). Sequenom MassARRAY?SNP was applied for genotyping of g.69738971 T>G locus in SIX1 gene in multiparous(total 560 for STH, Hu and Cele black sheep) and uniparous(total 204 for Tan, Sunite and Prairie Tibetan sheep) sheep breeds, then the association was analyzed between SIX1 and litter size in STH. The results of qPCR showed that SIX1 gene was expressed in ovary, uterine body and oviduct in STH, and expression of SIX1 gene in uterine body of uniparous group was higher than that in multiparous group(P<0.05). The result of genotyping indicated that three genotypes(TT, GT and GG) were found at g.69738971 T>G locus of SIX1 gene, genotype frequency and allele frequency were extremely different between uniparous and multiparous sheep breeds(ewes of high and low reproductive capacity)(P<0.01). The χ2 test indicated that g.69738971 T>G locus of SIX1 gene in six breeds was under Hardy-Weinberg equilibrium(P>0.05). Polymorphism of g.69738971 T>G had correlation with litter size of the first, second and third parity in STH(P<0.05), while litter size of TT genotype was higher than GT or GG genotype for each parity. Above results implied that SIX1 gene expression may be related to reproduction in sheep, there be a certain negative correlation between the expression level of SIX1 and litter size in sheep, the g.69738971 T>G locus might be considered as a potential molecular marker which controls seasonal reproduction and litter size in sheep.
To explore expression level of SIX1 gene in reproductive tissues and the association between polymorphism and seasonal reproduction, litter size in sheep, real-time fluorescent quantitative PCR(qPCR) was used to detect the expression of SIX1 gene in ovary, uterine body and oviduct of Small Tail Han sheep(STH) at different litter sizes(uniparous group and multiparous group). Sequenom MassARRAY?SNP was applied for genotyping of g.69738971 T>G locus in SIX1 gene in multiparous(total 560 for STH, Hu and Cele black sheep) and uniparous(total 204 for Tan, Sunite and Prairie Tibetan sheep) sheep breeds, then the association was analyzed between SIX1 and litter size in STH. The results of qPCR showed that SIX1 gene was expressed in ovary, uterine body and oviduct in STH, and expression of SIX1 gene in uterine body of uniparous group was higher than that in multiparous group(P<0.05). The result of genotyping indicated that three genotypes(TT, GT and GG) were found at g.69738971 T>G locus of SIX1 gene, genotype frequency and allele frequency were extremely different between uniparous and multiparous sheep breeds(ewes of high and low reproductive capacity)(P<0.01). The χ2 test indicated that g.69738971 T>G locus of SIX1 gene in six breeds was under Hardy-Weinberg equilibrium(P>0.05). Polymorphism of g.69738971 T>G had correlation with litter size of the first, second and third parity in STH(P<0.05), while litter size of TT genotype was higher than GT or GG genotype for each parity. Above results implied that SIX1 gene expression may be related to reproduction in sheep, there be a certain negative correlation between the expression level of SIX1 and litter size in sheep, the g.69738971 T>G locus might be considered as a potential molecular marker which controls seasonal reproduction and litter size in sheep.
引文
[1]刘秋月,胡文萍,贺小云,等.绵羊多羔主效基因Fec B高通量检测方法的建立及应用[J].畜牧兽医学报,2017,48(1):39-51.
[2]程美玲,赵素梅,黄英.云岭黑山羊FSH基因表达量与其产羔数相关性研究[J].云南农业大学学报,2010,25(6):807-810.
[3]Xu S S,Gao L,Xie X L,et al.Genome-wide association analyses highlight the potential for different genetic mechanisms for litter size among sheep breeds[J].Front Genet,2018,9:118-131.
[4]潘章源,贺小云,刘秋月,等.全基因组测序(WGS)在畜禽群体进化和功能基因挖掘中的应用[J].农业生物技术学报,2016,24(12):1945-1954.
[5]周梅,曹晓涵,贺小云,等.绵羊FGF7基因组织表达及其多态性与产羔数之间的关系[J].畜牧兽医学报,2018,49(3):525-533.
[6]Liu Y,Han N,Zhou S,et al.The DACH/Eya/SIX gene network and its role in tumor initiation and progression[J].Int J Cancer,2015,138(5):1067-1075.
[7]Masumoto K,Ukaitadenuma M,Kasukawa T,et al.Acute induction of Eya3 by late-night light stimulation triggers TSHβexpression in photoperiodism[J].Curr Biol,2010,20(24):2199-2206.
[8]Sato S,Ikeda K,Shioi G,et al.Regulation of SIX1 expression by evolutionarily conserved enhancers in tetrapods[J].Dev Biol,2012,368(1):95-108.
[9]Wu W J,Liu K Q,Li B J,et al.Identification of an(AC)n microsatellite in the SIX1 gene promoter and its effect on production traits in Pietrain×Duroc×Landrace×Yorkshire pigs[J].J Anim Sci,2018,96(1):17-26.
[10]Wu W,Huang R,Wu Q,et al.The role of SIX1 in the genesis of muscle cell and skeletal muscle development[J].Int J Biol Sci,2014,10(9):983-989.
[11]李晓雨,贺小云,刘秋月,等.TAC1和PRLR基因在绵羊不同繁殖状态下的表达模式分析[J].畜牧兽医学报,2018,49(2):253-262.
[12]Johansen P,Aandersen J D,Borsting C,et al.Evaluation of the iPLEX?Sample ID Plus Panel designed for the Sequenom MassARRAY?system.A SNP typing assay developed for human identification and sample tracking based on the SNPforIDpanel[J].Forensic Sci Int Gen,2013,7(5):482-487.
[13]周梅,曹晓涵,贺小云,等.绵羊FSTL1基因组织表达及其多态性与产羔数之间的关系[J].农业生物技术学报,2018,26(5):801-810.
[14]Imarazene B,Andouche A,Bassaglia Y,et al.Eye development in sepia officinalis embryo:what the uncommon gene expression profiles tell us about eye evolution[J].Front Physiol,2017,8:613-627.
[15]Nie X,Xu J,Elhashash A,et al.SIX1 regulates grem1 expression in the metanephric mesenchyme to initiate branching morphogenesis[J].Dev Biol,2011,352(1):141-151.
[16]Konishi Y,Ikeda K,Iwakura Y,et al.SIX1 and SIX4 promote survival of sensory neurons during early trigeminal gangliogenesis[J].Brain Res,2006,1116(1):93-102.
[17]Fonseca B F,Couly G,Dupin E.Respective contribution of the cephalic neural crest and mesoderm to SIX1-expressing head territories in the avian embryo[J].BMC Dev Biol,2017,17(1):13-27.
[18]Dardente H,Wyse C A,Birnie M J,et al.A molecular switch for photoperiod responsiveness in mammals[J].Curr Biol,2010,20(24):2193-2198.
[19]Camille M L,Maguire J.GABAergic regulation of the HPA and HPGA axes and the impact of stress on reproductive function[J].J Steroid Biochem Mol Biol,2016,160:196-203.
[2]程美玲,赵素梅,黄英.云岭黑山羊FSH基因表达量与其产羔数相关性研究[J].云南农业大学学报,2010,25(6):807-810.
[3]Xu S S,Gao L,Xie X L,et al.Genome-wide association analyses highlight the potential for different genetic mechanisms for litter size among sheep breeds[J].Front Genet,2018,9:118-131.
[4]潘章源,贺小云,刘秋月,等.全基因组测序(WGS)在畜禽群体进化和功能基因挖掘中的应用[J].农业生物技术学报,2016,24(12):1945-1954.
[5]周梅,曹晓涵,贺小云,等.绵羊FGF7基因组织表达及其多态性与产羔数之间的关系[J].畜牧兽医学报,2018,49(3):525-533.
[6]Liu Y,Han N,Zhou S,et al.The DACH/Eya/SIX gene network and its role in tumor initiation and progression[J].Int J Cancer,2015,138(5):1067-1075.
[7]Masumoto K,Ukaitadenuma M,Kasukawa T,et al.Acute induction of Eya3 by late-night light stimulation triggers TSHβexpression in photoperiodism[J].Curr Biol,2010,20(24):2199-2206.
[8]Sato S,Ikeda K,Shioi G,et al.Regulation of SIX1 expression by evolutionarily conserved enhancers in tetrapods[J].Dev Biol,2012,368(1):95-108.
[9]Wu W J,Liu K Q,Li B J,et al.Identification of an(AC)n microsatellite in the SIX1 gene promoter and its effect on production traits in Pietrain×Duroc×Landrace×Yorkshire pigs[J].J Anim Sci,2018,96(1):17-26.
[10]Wu W,Huang R,Wu Q,et al.The role of SIX1 in the genesis of muscle cell and skeletal muscle development[J].Int J Biol Sci,2014,10(9):983-989.
[11]李晓雨,贺小云,刘秋月,等.TAC1和PRLR基因在绵羊不同繁殖状态下的表达模式分析[J].畜牧兽医学报,2018,49(2):253-262.
[12]Johansen P,Aandersen J D,Borsting C,et al.Evaluation of the iPLEX?Sample ID Plus Panel designed for the Sequenom MassARRAY?system.A SNP typing assay developed for human identification and sample tracking based on the SNPforIDpanel[J].Forensic Sci Int Gen,2013,7(5):482-487.
[13]周梅,曹晓涵,贺小云,等.绵羊FSTL1基因组织表达及其多态性与产羔数之间的关系[J].农业生物技术学报,2018,26(5):801-810.
[14]Imarazene B,Andouche A,Bassaglia Y,et al.Eye development in sepia officinalis embryo:what the uncommon gene expression profiles tell us about eye evolution[J].Front Physiol,2017,8:613-627.
[15]Nie X,Xu J,Elhashash A,et al.SIX1 regulates grem1 expression in the metanephric mesenchyme to initiate branching morphogenesis[J].Dev Biol,2011,352(1):141-151.
[16]Konishi Y,Ikeda K,Iwakura Y,et al.SIX1 and SIX4 promote survival of sensory neurons during early trigeminal gangliogenesis[J].Brain Res,2006,1116(1):93-102.
[17]Fonseca B F,Couly G,Dupin E.Respective contribution of the cephalic neural crest and mesoderm to SIX1-expressing head territories in the avian embryo[J].BMC Dev Biol,2017,17(1):13-27.
[18]Dardente H,Wyse C A,Birnie M J,et al.A molecular switch for photoperiod responsiveness in mammals[J].Curr Biol,2010,20(24):2193-2198.
[19]Camille M L,Maguire J.GABAergic regulation of the HPA and HPGA axes and the impact of stress on reproductive function[J].J Steroid Biochem Mol Biol,2016,160:196-203.
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