苏淮猪VRTN基因克隆、组织表达特征与多态性分析
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摘要
【目的】获得苏淮猪VRTN基因编码区序列,了解其序列特征和组织表达特征,分析苏淮猪VRTN基因ins291位点的多态性。【方法】以苏淮猪卵巢组织c DNA为模板,采用克隆测序技术分离获得苏淮猪VRTN基因编码区序列。利用Bio Edit 7.0软件分析其序列特征和蛋白理化性质。利用Clustal W软件进行核苷酸和蛋白质序列比对分析。利用UCSC基因组浏览器与NCBI基因组数据库进行猪VRTN基因的染色体定位和基因组结构分析。利用SMART软件预测蛋白质功能域,CPHmodels软件预测蛋白质三级结构。随机选取3头成年苏淮猪母猪,屠宰后立即采集心、肝、脾、肺、肾、下丘脑、肌肉和卵巢等组织,提取组织总RNA,采用RT-PCR技术分析苏淮猪VRTN基因的组织表达谱。采集106头成年苏淮猪繁殖母猪耳组织样,提取基因组DNA,采用PCR-凝胶电泳分析苏淮猪VRTN基因ins291位点的多态性。【结果】苏淮猪VRTN基因编码区序列全长为2 097bp,与其它哺乳动物如人、小鼠、牛和羊的一致性分别为84.98%、74.53%、85.84%和86.45%,而与鸡的一致性只有54.42%。基因组结构分析发现猪VRTN基因由2个外显子和1个内含子构成,定位在猪7号染色体上。苏淮猪VRTN基因编码蛋白含有698个氨基酸残基,与人、小鼠、牛和羊等的一致性分别为85.55%、70.07%、86.20%和86.06%,但与鸡的一致性只有51.17%,可见哺乳动物VRTN基因在进化过程中比较保守。氨基酸组分分析显示苏淮猪VRTN蛋白氨基酸序列存在全部20种氨基酸,其中Leu(亮氨酸)含量最高,达到10.44%,而Asp天冬氨酸含量最低,只有1.43%。蛋白结构分析发现苏淮猪VRTN蛋白含有HTH结构域等典型结构域,由6个α螺旋、5个β折叠以及若干无规则卷曲组成。RT-PCR分析表明VRTN基因在苏淮猪心、肝、脾、肺、肾、下丘脑、卵巢和肌肉组织等组织中均有表达,说明VRTN基因是一个广泛表达的基因。在苏淮猪群体中检测到VRTN基因ins291位点的3种基因型,其中仅有93bp条带的为野生纯合型(wt/wt),仅有384bp条带的为突变纯合型(Q/Q),含有384和93bp条带的为杂合型(wt/Q)。在苏淮猪群体中wt/wt型为优势基因型,基因型频率为0.717;wt为优势等位基因,基因频率是0.816;高脊椎数等位基因Q的频率为0.184。遗传多态性分析发现苏淮猪VRTN基因ins291位点的多态信息含量为0.331,为中度多态位点,杂合度为0.40,变异程度相对较低。【结论】获得了苏淮猪VRTN基因序列,其表达无组织特异性;苏淮猪群体中存在高脊椎数等位基因Q。
【Objective】The aim of this study is to obtain the coding region sequence of Suhuai VRTN gene, identify its characteristics and expression patterns, analyze the polymorphism of VRTN ins291 in Suhuai pig population. 【Method】The coding sequence of Suhuai pig VRTN gene was isolated by cloning and sequencing. The Bio Edit 7.0 software was used to analyze the molecular characteristics and physicochemical properties of Suhuai pig VRTN. Nucleotide and amino acid sequences alignment was performed using Clustal W. Genomic organization and chromosomal locations were investigated by comparing the c DNA and corresponding genomic sequence(UCSC server and NCBI database). Motif analysis was performed using the online programs SMART, and protein tertiary structure was predicted by CPHmodels server. Tissue samples of heart, liver, spleen, lung, kidney, hypothalamus, ovary and skeleton muscle were obtained from adult female Suhuai pigs(n=3). Total RNA was extracted using a Trizol kit, and the expression patterns of VRTN gene of Suhuai pig were analyzed by RT-PCR. The ear samples were obtained from adult female Suhuai pigs(n=106), and genomic DNA was extracted by a conventional phenol-chloroform extraction method. PCR-Gel electrophoresis was performed to analyze the polymorphism of VRTN ins291. 【Result】The full length of the coding sequences of Suhuai pig VRTN gene is 2097 bp, which shared similarities of 84.98%, 74.53%, 85.84% and 86.45% to human, mouse, cattle and sheep, respectively, but only 54.42% to chicken. Genomic structure analysis showed that pig VRTN gene includes 2 exons and 1 intron, and is located on chromosome 7. The Suhuai pig VRTN gene encodes 698 amino acid residues, which shared similarities of 85.55%, 70.07%, 86.20% and 86.06% to human, mouse, cattle and sheep, respectively, but only 51.17% to chicken, indicated that VRTN is conserved in the evolution process of mammalian. Protein structure analysis found that VRTN protein of Suhuai pig contains several typical domains including HTH domain, and comprised 6 α-helices, 5 β-sheets and many random coils. Real-time PCR analysis revealed that the VRTN gene is expressed in heart, liver, spleen, lung, kidney, hypothalamus, ovary and skeleton muscle of Suhuai pig, suggesting that VRTN is widely expressed in various tissues of pig. Genotyping ins291 of VRTN gene in 106 Suhuai pigswas done, and three genotypes were detected. In Suhuai pigs, the frequencies of wt/wt, wt/Q and Q/Q genotypes were 0.717, 0.198 and 0.085. The frequencies of high vertebrate number of Q allele was 0.184. The polymorphic information content of VRTN ins291 in Suhuai pigs was 0.331. The heterozygosity of VRTN ins291 in Suhuai pigs was 0.40. 【Conclusion】 The coding region of VRTN gene was isolated, and its expression showed no tissue-specificity. The presence of high number of vertebrate Q allele in Suhuai pig population was confirmed.
【Objective】The aim of this study is to obtain the coding region sequence of Suhuai VRTN gene, identify its characteristics and expression patterns, analyze the polymorphism of VRTN ins291 in Suhuai pig population. 【Method】The coding sequence of Suhuai pig VRTN gene was isolated by cloning and sequencing. The Bio Edit 7.0 software was used to analyze the molecular characteristics and physicochemical properties of Suhuai pig VRTN. Nucleotide and amino acid sequences alignment was performed using Clustal W. Genomic organization and chromosomal locations were investigated by comparing the c DNA and corresponding genomic sequence(UCSC server and NCBI database). Motif analysis was performed using the online programs SMART, and protein tertiary structure was predicted by CPHmodels server. Tissue samples of heart, liver, spleen, lung, kidney, hypothalamus, ovary and skeleton muscle were obtained from adult female Suhuai pigs(n=3). Total RNA was extracted using a Trizol kit, and the expression patterns of VRTN gene of Suhuai pig were analyzed by RT-PCR. The ear samples were obtained from adult female Suhuai pigs(n=106), and genomic DNA was extracted by a conventional phenol-chloroform extraction method. PCR-Gel electrophoresis was performed to analyze the polymorphism of VRTN ins291. 【Result】The full length of the coding sequences of Suhuai pig VRTN gene is 2097 bp, which shared similarities of 84.98%, 74.53%, 85.84% and 86.45% to human, mouse, cattle and sheep, respectively, but only 54.42% to chicken. Genomic structure analysis showed that pig VRTN gene includes 2 exons and 1 intron, and is located on chromosome 7. The Suhuai pig VRTN gene encodes 698 amino acid residues, which shared similarities of 85.55%, 70.07%, 86.20% and 86.06% to human, mouse, cattle and sheep, respectively, but only 51.17% to chicken, indicated that VRTN is conserved in the evolution process of mammalian. Protein structure analysis found that VRTN protein of Suhuai pig contains several typical domains including HTH domain, and comprised 6 α-helices, 5 β-sheets and many random coils. Real-time PCR analysis revealed that the VRTN gene is expressed in heart, liver, spleen, lung, kidney, hypothalamus, ovary and skeleton muscle of Suhuai pig, suggesting that VRTN is widely expressed in various tissues of pig. Genotyping ins291 of VRTN gene in 106 Suhuai pigswas done, and three genotypes were detected. In Suhuai pigs, the frequencies of wt/wt, wt/Q and Q/Q genotypes were 0.717, 0.198 and 0.085. The frequencies of high vertebrate number of Q allele was 0.184. The polymorphic information content of VRTN ins291 in Suhuai pigs was 0.331. The heterozygosity of VRTN ins291 in Suhuai pigs was 0.40. 【Conclusion】 The coding region of VRTN gene was isolated, and its expression showed no tissue-specificity. The presence of high number of vertebrate Q allele in Suhuai pig population was confirmed.
引文
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[18]MA J,YANG J,ZHOU L,REN J,LIU X,ZHANG H,YANG B,ZHANG Z,MA H,XIE X,XING Y,GUO Y,HUANG L.A splice mutation in the PHKG1 gene causes high glycogen content and low meat quality in pig skeletal muscle.PLo S Genetics,2014,10(10):e1004710.
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[2]MIKAWA S,MOROZUMI T,SHIMANUKI S,HAYASHI T,UENISHI H,DOMUKAI M,OKUMURA N,AWATA T.Fine mapping of a swine quantitative trait locus for number of vertebrae and analysis of an orphan nuclear receptor,germ cell nuclear factor(NR6A1).Genome Research,2007.17(5):586-593.
[3]KING J,ROBERTS R.Carcass length in the bacon pig:its association with vertebrae numbers and prediction from radiographs of the young pig.Animal Production Science,1960,2:59-65.
[4]BERGE S.Genetical researches on the number of vertebrae in the pig.Animal Science,1948,7:233-238.
[5]MIKAWA S,HAYASHI T,NII M,SHIMANUKI S,MOROZUMI T,AWATA T.Two quantitative trait loci on Sus scrofa chromosomes 1and 7 affecting the number of vertebrae.Journal of Animal Science,2005,83(10):2247-2254.
[6]NAKANO H,SATO S,UEMOTO Y,KIKUCHI T,SHIBATAT,KADOWAKI H,KOBAYASHI E,SUZUKI K.Effect of VRTNgene polymorphisms on Duroc pig production and carcass traits,and their genetic relationships.Animal Science Journal,2015,86(2):125-131.
[7]WADA Y,AKITA T,AWATA T,FURUKAWA T,SUGAI N,INAGE Y,ISHII K,ITO Y,KOBAYASHI E,KUSUMOTO H,MATSUMOTO T,MIKAWA S,MIYAKE M,MURASE A,SHIMANUKI S,SUGIYAMAT,UCHIDA Y,YANAI S,YASUE H.Quantitative trait loci(QTL)analysis in a Meishan x Gottingen cross population.Animal Genetics,2000,31(6):376-384.
[8]SATO S,OYAMADA Y,ATSUJI K,NADE T,KOBAYASHI E,MITSUHASHI T,NIRASAWA K,KOMATSUDA A,SAITO Y,TERAI S,HAYASHI T,SUGIMOTO Y.Quantitative trait loci analysis for growth and carcass traits in a Meishan x Duroc F2resource population.Journal of Animal Science,2003,81(12):2938-2949.
[9]MIKAWA S,SATO S,NII M,MOROZUMI T,YOSHIOKA G,IMAEDA N,YAMAGUCHI T,HAYASHI T,AWATA T.Identification of a second gene associated with variation in vertebral number in domestic pigs.BMC Genetics,2011,12:5.
[10]REN D R,REN J,RUAN G F,GUO Y M,WU L H,YANG G C,ZHOU L H,LI L,ZHANG Z Y,HUANG L S.Mapping and fine mapping of quantitative trait loci for the number of vertebrae in a White Duroc×Chinese Erhualian intercross resource population.Animal Genetics,2012,43(5):545-551.
[11]FAN Y,XING Y,ZHANG Z,AI H,OUYANG Z,OUYANG J,YANGM,LI P,CHEN Y,GAO J,LI L,HUANG L,REN J.A further look at porcine chromosome 7 reveals VRTN variants associated with vertebral number in Chinese and Western pigs.Plo S One,2013,8(4):e62534.
[12]DUIJVESTEIJN N,VELTMAAT J M,KNOL E F,HARLIZIUS B.High-resolution association mapping of number of teats in pigs reveals regions controlling vertebral development.BMC Genomics,2014,15:542.
[13]欧阳子璇.猪7号染色体上影响脊椎变异的VRTN因果突变的筛选和验证[D].南昌:江西农业大学,2012.OUYANG Z X.Identification of VRTN causal variations for vertebral numbers on pig chromosome 7[D].Nanchang:Jiangxi Agricultural University,2012.(in Chinese)
[14]吴井生,王金玉.FSHR基因第10外显子多态性及其与小梅山猪产仔数的相关性.中国农业科学,2012,45(13):2728-2736.WU J S,WANG J Y.Polymorphism of exon10 of FSHR gene and its relationship with litter size in Xiaomeishan pigs.Scentia Agricultura Sinica,2012,45(13):2728-2736.(in Chinese)
[15]VAN LAERE A S,NGUYEN M,BRAUNSCHWEIG M,NEZER C,COLLETTE C,MOREAU L,ARCHIBALD A L,HALEY C S,BUYS N,TALLY M,ANDERSSON G,GEORGES M,ANDERSSONL.A regulatory mutation in IGF2 causes a major QTL effect on muscle growth in the pig.Nature,2003,425(6960):832-836.
[16]KIM K S,LARSEN N,SHORT T,PLASTOW G,ROTHSCHILD M F.A missense variant of the porcine melanocortin-4 receptor(MC4R)gene is associated with fatness,growth,and feed intake traits.Mammalian Genome,2000,11:131-135.
[17]REN J,DUAN Y,QIAO R,YAO F,ZHANG Z,YANG B,GUO Y,XIAO S,WEI R,OUYANG Z,DING N,AI H,HUANG L.Amissense mutation in PPARD causes a major QTL effect on ear size in pigs.PLo S Genetics,2011,7(5):e1002043.
[18]MA J,YANG J,ZHOU L,REN J,LIU X,ZHANG H,YANG B,ZHANG Z,MA H,XIE X,XING Y,GUO Y,HUANG L.A splice mutation in the PHKG1 gene causes high glycogen content and low meat quality in pig skeletal muscle.PLo S Genetics,2014,10(10):e1004710.
[19]HIROSE K,MIKAWA S,OKUMURA N,NOGUCHI G,FUKAWAK,KANAYA N,MIKAWA A,ARAKAWA A,ITO T,HAYASHI Y,TACHIBANA F,AWATA T.Association of swine vertnin(VRTN)gene with production traits in Duroc pigs improved using a closed nucleus breeding system.Animal Science Journal,2013,84(3):213-221.
[20]赵永祥,刘吉英,潘增祥,张久峰,姚勇,周吉隆,谢庄,徐银学,刘红林,李齐发.二花脸猪Smad4基因的克隆与卵巢组织m RNA的表达水平.中国农业科学,2012,45(23):4883-4890.ZHAO Y X,LIU J Y,PAN Z X,ZHANG J F,YAO Y,ZHOU J,XIE Z,XU Y X,LIU H L,LI Q F.Cloning and mR NA expression of Smad4gene in ovaries of Erhualian pig.Scentia Agricultura Sinica,2012,45(23):4883-4890.(in Chinese)
[21]WANG Y D,ZHANG J,LI C H,XU H P,CHEN W,ZENG Y Q,WANG H.Molecular cloning,sequence characteristics,and tissue expression analysis of ECE1 gene in Tibetan pig.Gene,2015,571(2):237-244.
[22]HU X,LUO P,PENG X,SONG T,ZHOU Y,WEI H,PENG J,JIANGS.Molecular cloning,expression pattern analysis of porcine Rb1 gene and its regulatory roles during primary dedifferentiated fat cells adipogenic differentiation.General and Comparative Endocrinology,2015,214:77-86.
[23]ZHANG Y,LIANG J,ZHANG L,WANG L,LIU X,YAN H,ZHAOK,SHI H,ZHANG T,LI N,PU L,WANG L.Porcine methionine sulfoxide reductase B3:molecular cloning,tissue-specific expression profiles,and polymorphisms associated with ear size in Sus scrofa.Journal of Animal Science and Biotechnology,2015,6:60.
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