西藏绒山羊KAP9.2基因CDS克隆、外显子区多态性及表达分析
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摘要
KAP9.2基因是角蛋白关联蛋白(keratin associated protein, KAP)中的一员,在毛发的形成过程中有重要的调控作用。本研究对西藏绒山KAP9.2基因CDS进行了克隆;采用直接测序法对绒山羊200个个体KAP9.2基因外显子区的遗传变异情况进行分析;并利用Real-time PCR分析了KAP9.2基因在不同海拔山羊中的表达。结果显示,西藏绒山羊KAP9.2基因CDS序列为576 bp,编码191个氨基酸;KAP9.2基因外显子存在25处SNP位点及一处30 bp的缺失突变,其中12处SNP为错义突变,其他13处为同义突变。遗传多态性分析表明KAP9.2基因多态性丰富,遗传变异大;连锁分析发现12与388位点、54与93位点、153与159位点、273与279位点完全连锁,H1为优势单倍型;Real-time PCR显示西藏绒山羊KAP9.2基因在高海拔地区m RNA表达水平显著高于特高海拔地区,推测该基因可能促进绒毛的生长。本研究结果揭示了西藏绒山羊KAP9.2基因的遗传多态性及其在不同海拔的表达,为进一步研究KAP9.2基因潜在的功能位点提供一定的理论依据。
KAP9.2 gene is one of members of keratin associated protein(KAP), which plays a key role in regulation of hair formation. In this study, we have cloned the KAP9.2 CDS from Tibetan cashmere goat, analyzed genetic variation of exocrine regions of KAP9.2 gene in 200 cashmere goats by direct sequencing method, and analyzed the expression levels of KAP9.2 gene at different altitudes by Real-time PCR. The results showed that gene. And there were 25 SNP loci and one 30 bp deletion mutation in the exons of KAP9.2 gene, including 12 nonsynonymous mutations and 13 syn onymous mutations. Genetic polymorphism analysis showed that the polymorphism of KAP9.2 gene was rich and the genetic variation was large. Linkage disequilibrium analysis showed that 12 site and388 site, 54 site and 93 site, 153 site and 159 site, 273 site and 279 site were complete linkage. And H1 was the dominant haplotype. Real-time PCR show ed that KAP9.2 gene of Tibetan cashmere goat was significantly expressed in high altitude than it in extra high altitude. We presumed that the KAP9.2 gene may promote the growth of the wool. The results of this study revealed the genetic polymorphism and the different expression in imparity altitudes of KAP9.2 gene of Tibetan cashmere goat, which provides a theoretical basis for the study of the potential functional loci of the KAP9.2 gene in the further.
KAP9.2 gene is one of members of keratin associated protein(KAP), which plays a key role in regulation of hair formation. In this study, we have cloned the KAP9.2 CDS from Tibetan cashmere goat, analyzed genetic variation of exocrine regions of KAP9.2 gene in 200 cashmere goats by direct sequencing method, and analyzed the expression levels of KAP9.2 gene at different altitudes by Real-time PCR. The results showed that gene. And there were 25 SNP loci and one 30 bp deletion mutation in the exons of KAP9.2 gene, including 12 nonsynonymous mutations and 13 syn onymous mutations. Genetic polymorphism analysis showed that the polymorphism of KAP9.2 gene was rich and the genetic variation was large. Linkage disequilibrium analysis showed that 12 site and388 site, 54 site and 93 site, 153 site and 159 site, 273 site and 279 site were complete linkage. And H1 was the dominant haplotype. Real-time PCR show ed that KAP9.2 gene of Tibetan cashmere goat was significantly expressed in high altitude than it in extra high altitude. We presumed that the KAP9.2 gene may promote the growth of the wool. The results of this study revealed the genetic polymorphism and the different expression in imparity altitudes of KAP9.2 gene of Tibetan cashmere goat, which provides a theoretical basis for the study of the potential functional loci of the KAP9.2 gene in the further.
引文
Fang Y.,Liu W.J.,Zhang F.Q.,Shao Y.J.,and Yu S.G.,2010,The polymorphism of a novel mutation of KAP13.1 gene and its associations with cashmere traits on Xinjiang local goat breed in China,Asian Journal of Animal and Veterinary Advances,5(1):34-42
Guryev V.,Smits B.M.,Van de Belt J.,Verheul M.,Hubner N.,and Cuppen E.,2006,Haplotype block structure is conserved across mammals,PLoS Genetics,2(7):e121
Gong H.,Zhou H.,Dyer J.M.,Plowman J.E.,and Hickford J.G.H.,2011,Identification of the keratin-associated protein 13-3(KAP13-3)gene in sheep,Open Journal of Genetics,1(3):60-64
Gong H.,Zhou H.T.,Forrest R.H.J.,Li S.B.,Wang J.Q.,Dyer J.M.,Luo Y.Z.,and Hickford J.G.H.,2016,Wool keratin-associated protein genes in Sheep-A review,Genes,7(6):24
Jin M.,Cao Q.,Wang R.,Piao J.,Zhao F.,and Piao J.A.,2017,Molecular characterization and expression pattern of a novel Keratin-associated protein 11.1 gene in the Liaoning cashmere goat(Capra hircus),Asian-Australasian Journal of Animal Sciences,30(3):328-337
Jin M.,Wang L.,Li S.,Xing M.X.,and Zhang X.,2011,Characterization and expression analysis of KAP7.1,KAP8.2 gene in Liaoning new-breeding cashmere goat hair follicle,Molecular Biology Reports,38(5):3023-3028
Liu H.,Li N.,Jia C.,Zhu X.,and Jia Z.,2007,Effect of the polymorphisms of keratin associated protein 8.2 gene on fibre traits in Inner Mongolia cashmere goats,Asian Australasian Journal of Animal Sciences,20(6):821-826
Liu H.,Yue C.W.,Zhang W.,Zhu X.P.,Yang G.Q.,and Jia Z.H.,2011,Association of the KAP 8.1 gene polymorphisms with Fibre Traits in Inner Mongolian Cashmere Goats,Asian Australasian Journal of Animal Sciences,24(10):1341-1347
Li S.,Zhou H.,Gong H.,Zhao F.,Wang J.,Liu X.,Luo Y.,and Hickford J.G.,2017,Identification of the ovine keratin-associated protein 22-1(KAP22-1)gene and its effect on wool traits,Genes,8(1):27
Plowman J.E.,2003,Proteomic database of wool components,Journal of Chromatography B,787(1):63-76
Powell B.C.,Sleigh M.J.,Ward K.A.,and Rogers G.E.,1983,Mammalian keratin gene families:organization for genes coding for the B2 high-sulfur proteins in sheep wool,Nucleic Acids Research,11(16):5327-5346
Qu L.,Li X.C.,Jing X.Q.,Yan H.L.,and Gao Y.,2009,Affecting factors of cashmere diameter and control measures of shanbei White Cashmere Goat,Jiachu Shengtai Xuebao(Acta E-cologiae Animalis Domastici),30(3):109-112(屈雷,李新春,敬晓棋,闫海龙,高晔,2009,陕北白绒山羊羊绒细度的主要影响因素及控制措施,家畜生态学报,30(3):109-112)
Sauna Z.E.,and Kimchi-Sarfaty C.,2011,Understanding the contribution of synonymous mutations to human disease,Nature Reviews Genetics,12(10):683-691
Suo L.D.,Yi X.D.J.,Wu Y.J.,Ba G.,Ci R.D.J.,and Song T.Z.,2015,Effects of light control and melatonin embedment on the cashmere growth of northwest Tibet cashmere goats during the Cashmere non-growing period,Zhongguo Shicao Dongwu Kexue(Chinese Herbivore Science),35(2):15-17(索朗达,益西多吉,吴玉江,巴贵,次仁德吉,宋天增,2015,非产绒期控制光照和埋植褪黑激素对藏西北绒山羊增绒效果的影响,中国草食动物科学,35(2):15-17)
Thompson L.G.,Yao T.,Mosley-Thompson E.,Davis M.E.,Henderson K.A.,and Lin P.N.,2000,A high-resolution millennial record of the south asian monsoon from himalayan ice cores,Science,289(5486):1916-1920
Wang J.Q.,Che L.J.,Hickford J.G.H.,Zhou H.T.,Hao Z.Y.,Luo Y.Z.,Hu J.,Liu X.,and Li S.B.,2017,Identification of the caprine keratin-associated protein 20-2(KAP20-2)gene and its effect on cashmere traits,Genes,8(11):328
Wang Y.,Wang J.,Zi X.D.,Huatai C.R.,Ouyang X.,and Liu L.D.,2011,Genetic diversity of Tibetan goats of Plateau type using microsatellite markers,Archiv.Fur.Tierzucht.,54(2):188-197
Wang X.,Zhao Z.D.,Xu H.R.,Qu L.,Zhao H.B.,Li T.,and Zhang Z.Y.,2012,Variation and expression of KAP9.2 gene affecting cashmere trait in goats,Molecular Biology Reports,39(12):10525-10529
Wang X.,Xu H.R.,Li T.,Qu L.,Zhao Z.D.,and Zhang Z.Y.,2014,Expression analysis of KAP9.2 and Hoxc13 genes during different cashmere growth stages by qRT-PCRmethod,Molecular Biology Reports,41(9):5665-5668
Xuan J.L.,Ma X.M.,Wang H.H.,Cao J.X.,Wei C.H.,Zhao F.P.,Ma Y.J.,Zhang L.,and Du L.X.,2016,Study on exon polymorphism of seasonal breeding related gene TSHR in sheep,Xumu Shouyi Xuebao(Acta Veterinaria et Zootechnica Sinica),47(7):1342-1353(轩俊丽,马晓萌,王慧华,曹家雪,魏彩虹,赵福平,马友记,张莉,杜立新,2016,绵羊季节性繁殖相关基因TSHR外显子多态性研究,畜牧兽医学报,47(7):1342-1353)
Yu H.,2009,The genetic variation of KAPs and HOX genes and their genetic effects analysis on cashmere traits in cashmere goat,Thesis for M.S.,Northwest A&F University,Supervisor:Chen H.,pp.1-58(于辉,2009,绒山羊KAP基因家族与Hoxc13基因遗传变异及其对产绒性状的影响,硕士学位论文,西北农林科技大学,导师:陈宏,pp.1-58)
Zhao Z.D.,2012,Popolation genntic variation of KAP9.2 gene and expression analysis in anagen and telogen in shanbei white cashmere goat,Thesis for M.S.,Northwest A&F University,Supervisor:Wang X.,pp.1-32(赵志东,2012,KAP9.2基因的群体遗传变异及羊绒生长期和休止期基因的表达研究,硕士学位论文,西北农林科技大学,导师:王昕,pp.1-32)
Zhou H.,Gong H.,Li S.,Luo Y.,and Hickford J.G.,2015,A57-bp deletion in the ovine KAP6-1 gene affects wool fibre diameter,J.Anim.Breed.Genet.,132(4):301-307
Zhou H.,Gong H.,Yan W.,Luo Y.,Hickford J.G.,2012,Identi fication and sequence analysis of the keratin-associated protein 24-1(KAP24-1)gene homologue in sheep,Gene,511(1):62-65
Zhu Y.X.,Li Y.,Zheng X.F.,and Guo H.W.,eds.,2013,Modern Molecular Biology,4th Edition,Higher Education Press,Beijing,China,pp.108-163(朱玉贤,李毅,郑晓峰,郭红卫,编著,2013,现代分子生物学,第4版,高等教育出版社,中国,北京,pp.108-163)
Guryev V.,Smits B.M.,Van de Belt J.,Verheul M.,Hubner N.,and Cuppen E.,2006,Haplotype block structure is conserved across mammals,PLoS Genetics,2(7):e121
Gong H.,Zhou H.,Dyer J.M.,Plowman J.E.,and Hickford J.G.H.,2011,Identification of the keratin-associated protein 13-3(KAP13-3)gene in sheep,Open Journal of Genetics,1(3):60-64
Gong H.,Zhou H.T.,Forrest R.H.J.,Li S.B.,Wang J.Q.,Dyer J.M.,Luo Y.Z.,and Hickford J.G.H.,2016,Wool keratin-associated protein genes in Sheep-A review,Genes,7(6):24
Jin M.,Cao Q.,Wang R.,Piao J.,Zhao F.,and Piao J.A.,2017,Molecular characterization and expression pattern of a novel Keratin-associated protein 11.1 gene in the Liaoning cashmere goat(Capra hircus),Asian-Australasian Journal of Animal Sciences,30(3):328-337
Jin M.,Wang L.,Li S.,Xing M.X.,and Zhang X.,2011,Characterization and expression analysis of KAP7.1,KAP8.2 gene in Liaoning new-breeding cashmere goat hair follicle,Molecular Biology Reports,38(5):3023-3028
Liu H.,Li N.,Jia C.,Zhu X.,and Jia Z.,2007,Effect of the polymorphisms of keratin associated protein 8.2 gene on fibre traits in Inner Mongolia cashmere goats,Asian Australasian Journal of Animal Sciences,20(6):821-826
Liu H.,Yue C.W.,Zhang W.,Zhu X.P.,Yang G.Q.,and Jia Z.H.,2011,Association of the KAP 8.1 gene polymorphisms with Fibre Traits in Inner Mongolian Cashmere Goats,Asian Australasian Journal of Animal Sciences,24(10):1341-1347
Li S.,Zhou H.,Gong H.,Zhao F.,Wang J.,Liu X.,Luo Y.,and Hickford J.G.,2017,Identification of the ovine keratin-associated protein 22-1(KAP22-1)gene and its effect on wool traits,Genes,8(1):27
Plowman J.E.,2003,Proteomic database of wool components,Journal of Chromatography B,787(1):63-76
Powell B.C.,Sleigh M.J.,Ward K.A.,and Rogers G.E.,1983,Mammalian keratin gene families:organization for genes coding for the B2 high-sulfur proteins in sheep wool,Nucleic Acids Research,11(16):5327-5346
Qu L.,Li X.C.,Jing X.Q.,Yan H.L.,and Gao Y.,2009,Affecting factors of cashmere diameter and control measures of shanbei White Cashmere Goat,Jiachu Shengtai Xuebao(Acta E-cologiae Animalis Domastici),30(3):109-112(屈雷,李新春,敬晓棋,闫海龙,高晔,2009,陕北白绒山羊羊绒细度的主要影响因素及控制措施,家畜生态学报,30(3):109-112)
Sauna Z.E.,and Kimchi-Sarfaty C.,2011,Understanding the contribution of synonymous mutations to human disease,Nature Reviews Genetics,12(10):683-691
Suo L.D.,Yi X.D.J.,Wu Y.J.,Ba G.,Ci R.D.J.,and Song T.Z.,2015,Effects of light control and melatonin embedment on the cashmere growth of northwest Tibet cashmere goats during the Cashmere non-growing period,Zhongguo Shicao Dongwu Kexue(Chinese Herbivore Science),35(2):15-17(索朗达,益西多吉,吴玉江,巴贵,次仁德吉,宋天增,2015,非产绒期控制光照和埋植褪黑激素对藏西北绒山羊增绒效果的影响,中国草食动物科学,35(2):15-17)
Thompson L.G.,Yao T.,Mosley-Thompson E.,Davis M.E.,Henderson K.A.,and Lin P.N.,2000,A high-resolution millennial record of the south asian monsoon from himalayan ice cores,Science,289(5486):1916-1920
Wang J.Q.,Che L.J.,Hickford J.G.H.,Zhou H.T.,Hao Z.Y.,Luo Y.Z.,Hu J.,Liu X.,and Li S.B.,2017,Identification of the caprine keratin-associated protein 20-2(KAP20-2)gene and its effect on cashmere traits,Genes,8(11):328
Wang Y.,Wang J.,Zi X.D.,Huatai C.R.,Ouyang X.,and Liu L.D.,2011,Genetic diversity of Tibetan goats of Plateau type using microsatellite markers,Archiv.Fur.Tierzucht.,54(2):188-197
Wang X.,Zhao Z.D.,Xu H.R.,Qu L.,Zhao H.B.,Li T.,and Zhang Z.Y.,2012,Variation and expression of KAP9.2 gene affecting cashmere trait in goats,Molecular Biology Reports,39(12):10525-10529
Wang X.,Xu H.R.,Li T.,Qu L.,Zhao Z.D.,and Zhang Z.Y.,2014,Expression analysis of KAP9.2 and Hoxc13 genes during different cashmere growth stages by qRT-PCRmethod,Molecular Biology Reports,41(9):5665-5668
Xuan J.L.,Ma X.M.,Wang H.H.,Cao J.X.,Wei C.H.,Zhao F.P.,Ma Y.J.,Zhang L.,and Du L.X.,2016,Study on exon polymorphism of seasonal breeding related gene TSHR in sheep,Xumu Shouyi Xuebao(Acta Veterinaria et Zootechnica Sinica),47(7):1342-1353(轩俊丽,马晓萌,王慧华,曹家雪,魏彩虹,赵福平,马友记,张莉,杜立新,2016,绵羊季节性繁殖相关基因TSHR外显子多态性研究,畜牧兽医学报,47(7):1342-1353)
Yu H.,2009,The genetic variation of KAPs and HOX genes and their genetic effects analysis on cashmere traits in cashmere goat,Thesis for M.S.,Northwest A&F University,Supervisor:Chen H.,pp.1-58(于辉,2009,绒山羊KAP基因家族与Hoxc13基因遗传变异及其对产绒性状的影响,硕士学位论文,西北农林科技大学,导师:陈宏,pp.1-58)
Zhao Z.D.,2012,Popolation genntic variation of KAP9.2 gene and expression analysis in anagen and telogen in shanbei white cashmere goat,Thesis for M.S.,Northwest A&F University,Supervisor:Wang X.,pp.1-32(赵志东,2012,KAP9.2基因的群体遗传变异及羊绒生长期和休止期基因的表达研究,硕士学位论文,西北农林科技大学,导师:王昕,pp.1-32)
Zhou H.,Gong H.,Li S.,Luo Y.,and Hickford J.G.,2015,A57-bp deletion in the ovine KAP6-1 gene affects wool fibre diameter,J.Anim.Breed.Genet.,132(4):301-307
Zhou H.,Gong H.,Yan W.,Luo Y.,Hickford J.G.,2012,Identi fication and sequence analysis of the keratin-associated protein 24-1(KAP24-1)gene homologue in sheep,Gene,511(1):62-65
Zhu Y.X.,Li Y.,Zheng X.F.,and Guo H.W.,eds.,2013,Modern Molecular Biology,4th Edition,Higher Education Press,Beijing,China,pp.108-163(朱玉贤,李毅,郑晓峰,郭红卫,编著,2013,现代分子生物学,第4版,高等教育出版社,中国,北京,pp.108-163)