绵羊角蛋白关联蛋白2基因的克隆表达与生物信息学分析
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摘要
本研究旨在克隆新吉细毛羊和小尾寒羊的角蛋白关联蛋白2(keratin-associated protein 2,KAP2)基因并对其进行生物信息学分析,并初步探讨KAP2基因与羊毛毛用性状分子的关系。分别提取新吉细毛羊和小尾寒羊皮肤组织中总RNA,用RT-PCR方法扩增KAP2基因,将PCR产物克隆于pMD19-T载体,转入大肠杆菌DH5α感受态细胞,筛选阳性克隆。提取扩增后的重组质粒pMD19-T-KAP2并进行PCR扩增、双酶切鉴定、序列测定及蛋白质结构预测。结果表明,试验成功克隆了大小为463bp的KAP2基因,开放阅读框架(ORF)长414bp,编码137个氨基酸。测序结果比对发现,与小尾寒羊KAP2基因相比,新吉细毛羊的KAP2基因中存在1个由G→A的碱基突变,编码氨基酸由精氨酸(Arg)变为谷氨酰胺(Gln),属于错义突变。新吉细毛羊和小尾寒羊的KAP2蛋白的分子质量分别为14.81和14.84ku,等电点分别是9.67与9.82,两种绵羊KAP蛋白的基本理化性质无明显差异,同属于碱性疏水性、跨膜蛋白;预测到蛋白二级结构均由α螺旋、延伸链、无规则卷曲等构成,新吉细毛羊和小尾寒羊KAP2蛋白三级结构中存在一个由α螺旋引起的空间结构的差异位点。综合以上结果推测KAP2基因可能是影响羊毛性状的差异基因,本试验结果可为探讨两种绵羊毛用性状表型差异的分子遗传奠定理论基础。
The keratin-associated protein 2(KAP2)gene was cloned from Xinji Fine-wool sheep and Small-tail Han sheep,the KAP2 proteins was analyzed by bioinformatics,and the relationship between KAP2 gene and molecules trait of wool hair was preliminary analyzed in this study.The total RNA was extracted from wool follicles of Xinji Fine-wool sheep and Small-tail Han sheep.The KAP2 gene was cloned by RT-PCR,and the PCR product was cloned into pMD19-T vector,then was transferred to Escherichia coli DH5αcompetent cells and the requisite clones were filtered,and the recombinant plasmid was extracted.Identification of PCR and double enzyme digestion and sequencing were operated,then the prediction of protein structure was analyzed by bioinformatics.The results showed that the length of KAP2 gene was 463 bp,containing an ORF that was 414 bp and encoded 137 amino acid residues.The comparison of sequencing resultsshowed that compared with the KAP2 gene in Small-tail Han sheep,there was a base mutation from G to A in KAP2 gene of Xinji Fine-wool sheep which was a missense mutation causing the encoded amino acid changed from arginine(Arg)to glutamine(Gln).The molecular weight of KAP2 protein in Xinji Fine-wool sheep and Small-tailed Han sheep were 14.81 and 14.84 ku,and the isoelectric points of KAP2 protein were 9.67 and 9.82,respectively.The KAP2 protein of the two breeds was no obvious differences in physical and chemical properties,which was transmembrane,alkaline and hydrophobic protein.It was predicted that the secondary structure of KAP2 protein was composed ofα-helix,extend chain and random coil.The tertiary structure of KAP2 protein in Xinji Fine-wool sheep and Small-tail Han sheep had a different spatial structure that came fromα-helix.Therefore,it could be speculated that KAP2 gene might be a differential gene affecting wool quality.The study of KAP2 gene provided a theoretical basis for exploring the molecular inheritance of phenotypic differences between two wool species.
The keratin-associated protein 2(KAP2)gene was cloned from Xinji Fine-wool sheep and Small-tail Han sheep,the KAP2 proteins was analyzed by bioinformatics,and the relationship between KAP2 gene and molecules trait of wool hair was preliminary analyzed in this study.The total RNA was extracted from wool follicles of Xinji Fine-wool sheep and Small-tail Han sheep.The KAP2 gene was cloned by RT-PCR,and the PCR product was cloned into pMD19-T vector,then was transferred to Escherichia coli DH5αcompetent cells and the requisite clones were filtered,and the recombinant plasmid was extracted.Identification of PCR and double enzyme digestion and sequencing were operated,then the prediction of protein structure was analyzed by bioinformatics.The results showed that the length of KAP2 gene was 463 bp,containing an ORF that was 414 bp and encoded 137 amino acid residues.The comparison of sequencing resultsshowed that compared with the KAP2 gene in Small-tail Han sheep,there was a base mutation from G to A in KAP2 gene of Xinji Fine-wool sheep which was a missense mutation causing the encoded amino acid changed from arginine(Arg)to glutamine(Gln).The molecular weight of KAP2 protein in Xinji Fine-wool sheep and Small-tailed Han sheep were 14.81 and 14.84 ku,and the isoelectric points of KAP2 protein were 9.67 and 9.82,respectively.The KAP2 protein of the two breeds was no obvious differences in physical and chemical properties,which was transmembrane,alkaline and hydrophobic protein.It was predicted that the secondary structure of KAP2 protein was composed ofα-helix,extend chain and random coil.The tertiary structure of KAP2 protein in Xinji Fine-wool sheep and Small-tail Han sheep had a different spatial structure that came fromα-helix.Therefore,it could be speculated that KAP2 gene might be a differential gene affecting wool quality.The study of KAP2 gene provided a theoretical basis for exploring the molecular inheritance of phenotypic differences between two wool species.
引文
[1]张立春,孙福亮,金海国,等.小尾寒羊与新吉细毛羊羊毛及毛囊性状比较研究[J].中国畜牧杂志,2017,53(9):52-56.ZHANG L C,SUN F L,JIN H G,et al.Comparison of wool and hair follicle traits in Small-tail Han sheep and Xinji fine wool sheep[J].Chinese Journal of Animal Science,2017,53(9):52-56.(in Chinese)
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[4]ROGERS M A,WINTER H,LANGBEIN L,et al.Characterization of human KAP24.1,a cuticular hair keratin-associated protein with unusual amino-acid composition and repeat structure[J].Journal of Investigative Dermatology,2007,127(5):1197-1204.
[5]ROGERS M A,LANGBEIN L,PRAETZELWUNDER S,et al.Human hair keratin-associated proteins(KAPs)[J].International Review of Cytology,2006,251(3):209-263.
[6]ROGERS M A,LANGBEIN L,WINTER H,et al.Characterization of a cluster of human high/ultrahigh sulfur keratin-associated protein genes embedded in the typeⅠkeratin gene domain on chromosome17q12-21[J].Journal of Biological Chemistry,2001,276(22):19440-19451.
[7]李明娜.绒山羊KAP13.1和KAP13.3基因的遗传变异及经济性状相关性分析[D].兰州:甘肃农业大学,2013.LI M N.Correlation analysis of genetic variation and economic traits for KAP13.1and KAP13.3genes in cashmere goats[D].Lanzhou:Gansu Agricultural University,2013.(in Chinese)
[8]朱建勋.5个中国绵羊群体KAPs基因的遗传变异研究[D].兰州:甘肃农业大学,2016.ZHU J X.Genetic variation of KAPs gene in five sheep populations of China[D].Lanzhou:Gansu Agricultural University,2016.(in Chinese)
[9]朱汇,张红琳,代蓉,等.绵羊高硫角蛋白KAP1.3基因启动子活性分析[J].生物技术通报,2012,9:74-79.ZHU H,ZHANG H L,DAI R,et al.Promoter activity analysis of KAP1.3gene of sheep[J].Biotechnology Bulletin,2012,9:74-79.(in Chinese)
[10]刘桂芬,田可川,张恩平,等.优质细毛羊羊毛细度的候选基因分析[J].遗传,2007,29(1):70-74.LIU G F,TIAN K C,ZHANG E P,et al.Candidate gene analysis of wool fineness for fine wool sheep[J].Genetic,2007,29(1):70-74.(in Chinese)
[11]赵金山,李和刚,柳楠,等.羊毛毛囊发育机理及细度育种研究进展[J].畜牧与兽医,2012,44(5):101-106.ZHAO J S,LI H G,LIU N,et al.Research progress of development mechanism and fineness breeding in wool hair follicle[J].Animal Husbandry&Veterinary Medicine,2012,44(5):101-106.(in Chinese)
[12]王小佳,贺建宁,程明,等.BMPR1B基因在细毛羊皮肤毛囊中的表达及与羊毛性状关系的研究[J].中国畜牧杂志,2017,53(1):33-38.WANG X J,HE J N,CHENG M,et al.Expression of BMPR1Bgene in skin and its association with wool traits in fine wool sheep[J].Chinese Journal of Animal Science,2017,53(1):33-38.(in Chinese)
[13]桓蕾.阿尔巴斯白绒山羊胸腺素β4毛囊发育相关信号通路的研究[D].呼和浩特:内蒙古大学,2015.HUAN L.The research of hair follicle developmental related signal pathways of thymosin beta 4inArbas cashmere goat[D].Hohhot:Inner Mongolia University,2015.(in Chinese)
[14]杨洁,付雪峰,黄锡霞,等.细毛羊皮肤组织中毛囊蛋白质2-DE图谱的建立与初步分析[J].中国畜牧兽医,2015,42(1):124-130.YANG J,FU X F,HUANG X X,et al.Establishment and preliminary studies on the skin tissue hair follicles proteins of 2-DE map building in fine-wool sheep[J].China Husbandry&Veterinary Medicine,2015,42(1):124-130.(in Chinese)
[15]张文建,马广伟,张潇飞,等.羊毛品质性状相关基因CCNY的验证和分析[J].畜牧兽医学报,2017,48(8):1389-1400.ZHANG W J,MA G W,ZHANG X F,et al.Verification and analysis of wool quality traits-associated CCNYgene[J].Acta Veterinaria et Zootechnica Sinica,2017,48(8):1389-1400.(in Chinese)
[16]朱建勋,王继卿,胡江,等.五个绵羊群体KAP11-1基因核苷酸序列变异分析[J].华北农学报,2016,5(31):101-107.ZHU J X,WANG J Q,HU J,et al.Nucleotide sequence variations of KAP11-1 gene in 5 Chinese sheep populations[J].Acta Agriculturae Borealisinca,2016,5(31):101-107.(in Chinese)
[17]ZHANG L C,SUN F L,JIN H G,et al.A comparison of transcriptomic patterns measured in the skin of Chinese fine and coarse wool sheep breeds[J].Scientific Reports,2017,7:14301.
[18]ZHOU H,GONG H,YAN W,et al.Identification and sequence analysis of the keratin-associated protein24-1(KAP24-1)gene homologue in sheep[J].Gene,2012,511(1):62-65.
[19]孙福亮,金海国,鲁承,等.新吉细毛羊KAP24.1基因多态性及羊毛性状的相关性分析[J].黑龙江畜牧兽医,2016,1:70-73.SUN F L,JIN H G,LU C,et al.Correlation analysis of the polymorphisms of KAP24.1gene and the wool traits in Xinji fine-wool sheep[J].Heilongjiang Animal Science and Veterinary Medicine,2016,1:70-73.(in Chinese)
[20]KU N O,LIAO J,CHOU C F,et al.Implications of intermediate filament protein phosphorylation[J].Cancer and Metastasis Reviews,1996,15(4):429-444.
[21]FENG L,ZHOU X J,LIAO J,et al.Pervanadate-mediated tyrosine phosphorylation of keratins 8and 19viaap38 mitogen-activated protein kinase-dependent pathway[J].Journal of Cell Science,1999,112(13):2081-2090.
[22]王丙萍.靶除FGF5基因体细胞克隆绒山羊的研究[D].呼和浩特:内蒙古农业大学,2014.WANG B P.The study on the cloned goat knockout FGF5 gene transferred somatic cell[D].Hohhot:Inner Mongolia Agricultural University,2014.(in Chinese)
[2]李丽娟.三个绒山羊品种KIFⅠ基因多态性与绒用性状相关性研究[D].兰州:甘肃农业大学,2009.LI L J.Relationship between KIFⅠgene polymorphisms and cashmere traits in three cashmere goat breeds[D].Lanzhou:Gansu Agricultural University,2009.(in Chinese)
[3]ROGERS M A,LANGBEIN L,WINTER H,et al.Characterization of a first domain of human high glycine-tyrosine and high sulfur keratin-associated protein(KAP)genes on chromosome 21q22.1[J].Journal of Biological Chemistry,2003,277(50):48993-49002.
[4]ROGERS M A,WINTER H,LANGBEIN L,et al.Characterization of human KAP24.1,a cuticular hair keratin-associated protein with unusual amino-acid composition and repeat structure[J].Journal of Investigative Dermatology,2007,127(5):1197-1204.
[5]ROGERS M A,LANGBEIN L,PRAETZELWUNDER S,et al.Human hair keratin-associated proteins(KAPs)[J].International Review of Cytology,2006,251(3):209-263.
[6]ROGERS M A,LANGBEIN L,WINTER H,et al.Characterization of a cluster of human high/ultrahigh sulfur keratin-associated protein genes embedded in the typeⅠkeratin gene domain on chromosome17q12-21[J].Journal of Biological Chemistry,2001,276(22):19440-19451.
[7]李明娜.绒山羊KAP13.1和KAP13.3基因的遗传变异及经济性状相关性分析[D].兰州:甘肃农业大学,2013.LI M N.Correlation analysis of genetic variation and economic traits for KAP13.1and KAP13.3genes in cashmere goats[D].Lanzhou:Gansu Agricultural University,2013.(in Chinese)
[8]朱建勋.5个中国绵羊群体KAPs基因的遗传变异研究[D].兰州:甘肃农业大学,2016.ZHU J X.Genetic variation of KAPs gene in five sheep populations of China[D].Lanzhou:Gansu Agricultural University,2016.(in Chinese)
[9]朱汇,张红琳,代蓉,等.绵羊高硫角蛋白KAP1.3基因启动子活性分析[J].生物技术通报,2012,9:74-79.ZHU H,ZHANG H L,DAI R,et al.Promoter activity analysis of KAP1.3gene of sheep[J].Biotechnology Bulletin,2012,9:74-79.(in Chinese)
[10]刘桂芬,田可川,张恩平,等.优质细毛羊羊毛细度的候选基因分析[J].遗传,2007,29(1):70-74.LIU G F,TIAN K C,ZHANG E P,et al.Candidate gene analysis of wool fineness for fine wool sheep[J].Genetic,2007,29(1):70-74.(in Chinese)
[11]赵金山,李和刚,柳楠,等.羊毛毛囊发育机理及细度育种研究进展[J].畜牧与兽医,2012,44(5):101-106.ZHAO J S,LI H G,LIU N,et al.Research progress of development mechanism and fineness breeding in wool hair follicle[J].Animal Husbandry&Veterinary Medicine,2012,44(5):101-106.(in Chinese)
[12]王小佳,贺建宁,程明,等.BMPR1B基因在细毛羊皮肤毛囊中的表达及与羊毛性状关系的研究[J].中国畜牧杂志,2017,53(1):33-38.WANG X J,HE J N,CHENG M,et al.Expression of BMPR1Bgene in skin and its association with wool traits in fine wool sheep[J].Chinese Journal of Animal Science,2017,53(1):33-38.(in Chinese)
[13]桓蕾.阿尔巴斯白绒山羊胸腺素β4毛囊发育相关信号通路的研究[D].呼和浩特:内蒙古大学,2015.HUAN L.The research of hair follicle developmental related signal pathways of thymosin beta 4inArbas cashmere goat[D].Hohhot:Inner Mongolia University,2015.(in Chinese)
[14]杨洁,付雪峰,黄锡霞,等.细毛羊皮肤组织中毛囊蛋白质2-DE图谱的建立与初步分析[J].中国畜牧兽医,2015,42(1):124-130.YANG J,FU X F,HUANG X X,et al.Establishment and preliminary studies on the skin tissue hair follicles proteins of 2-DE map building in fine-wool sheep[J].China Husbandry&Veterinary Medicine,2015,42(1):124-130.(in Chinese)
[15]张文建,马广伟,张潇飞,等.羊毛品质性状相关基因CCNY的验证和分析[J].畜牧兽医学报,2017,48(8):1389-1400.ZHANG W J,MA G W,ZHANG X F,et al.Verification and analysis of wool quality traits-associated CCNYgene[J].Acta Veterinaria et Zootechnica Sinica,2017,48(8):1389-1400.(in Chinese)
[16]朱建勋,王继卿,胡江,等.五个绵羊群体KAP11-1基因核苷酸序列变异分析[J].华北农学报,2016,5(31):101-107.ZHU J X,WANG J Q,HU J,et al.Nucleotide sequence variations of KAP11-1 gene in 5 Chinese sheep populations[J].Acta Agriculturae Borealisinca,2016,5(31):101-107.(in Chinese)
[17]ZHANG L C,SUN F L,JIN H G,et al.A comparison of transcriptomic patterns measured in the skin of Chinese fine and coarse wool sheep breeds[J].Scientific Reports,2017,7:14301.
[18]ZHOU H,GONG H,YAN W,et al.Identification and sequence analysis of the keratin-associated protein24-1(KAP24-1)gene homologue in sheep[J].Gene,2012,511(1):62-65.
[19]孙福亮,金海国,鲁承,等.新吉细毛羊KAP24.1基因多态性及羊毛性状的相关性分析[J].黑龙江畜牧兽医,2016,1:70-73.SUN F L,JIN H G,LU C,et al.Correlation analysis of the polymorphisms of KAP24.1gene and the wool traits in Xinji fine-wool sheep[J].Heilongjiang Animal Science and Veterinary Medicine,2016,1:70-73.(in Chinese)
[20]KU N O,LIAO J,CHOU C F,et al.Implications of intermediate filament protein phosphorylation[J].Cancer and Metastasis Reviews,1996,15(4):429-444.
[21]FENG L,ZHOU X J,LIAO J,et al.Pervanadate-mediated tyrosine phosphorylation of keratins 8and 19viaap38 mitogen-activated protein kinase-dependent pathway[J].Journal of Cell Science,1999,112(13):2081-2090.
[22]王丙萍.靶除FGF5基因体细胞克隆绒山羊的研究[D].呼和浩特:内蒙古农业大学,2014.WANG B P.The study on the cloned goat knockout FGF5 gene transferred somatic cell[D].Hohhot:Inner Mongolia Agricultural University,2014.(in Chinese)