甘肃高山细毛羊KRT-IF基因克隆、生物信息学分析及其遗传效应研究
|
摘要
本研究以甘肃高山细毛羊为研究对象,应用基因克隆、生物信息学、PCR-SSCP和HRM技术,克隆甘肃高山细毛羊KRT-IF 35基因,进行生物信息学分析,分析KRT-IF 35、KRT-IF 31基因的遗传效应及其与毛用性状的相关性,实验所获得结果如下：
1.首次克隆出绵羊KRT-IF 35基因,并对其进行生物信息学分析。绵羊KRT-IF 35基因全长4414bp (NCBI No:GU456629), CDS区全长1751bp；KRT-IF 35蛋白由455个氨基酸编码,存在两个跨膜区,二级结构以螺旋为主,且其存在1个结构域,为亲水性蛋白。
2.经PCR-SSCP分析和测序分析,KRT-IF 35 CDS区第145碱基处发生C→T的突变,产生一对等位基因A,B,存在AA、AB、BB三种基因型。AA基因型为细毛型、超细型品系的优势基因型；A等位基因为细毛型、超细型品系的优势等位基因。AA、AB基因型细度差异显著；AA、BB基因型细度离散差异显著；AA、BB基因型弯曲差异显著,这三个品系均处于Hardy-weinberg动态平衡状态(P>0.05)。推测此位点可能是与影响羊毛细度、弯曲度、细度离散的一个主效基因或调控羊毛性状的基因紧密连锁的分子遗传标记。
3.经HRM分析和测序分析,在KRT-IF 31基因CDS区166位发生A-G突变,产生一对等位基因C,D,存在CC、CD、DD三种基因型。C等位基因为细毛型和超细型品系的优势等位基因,CC基因型为细毛型和超细型品系优势基因型,CD基因型为毛肉兼用型品系优势基因型,细毛型品系处于Hardy-weinberg动态平衡状态。CC、CD基因型细度差异显著；CC、DD基因型细度差异显著,因此此位点可能与羊毛细度有关。
4.KRT-IF 31基因CDS区191位发生T-C突变,产生一对等位基因E,F,存在EE、EF、FF三种基因型。E等位基因为细毛型、超细型和毛肉兼用型品系的优势等位基因,而EE基因型为细毛型和毛肉兼用型品系优势基因型；EF基因型为超细型品系优势基因型。细毛型品系处于Hardy-weinberg动态平衡状态(P>0.05)。EF、FF基因型细度差异显著；EE、EF基因型细度离散差异显著,因此此位点可能与羊毛细度,细度离散有关。
This study taked Gansu Alpine Fine Wool Sheep as a research object,with the technology of clone, bioinformatic analysis,PCR-SSCP and HRM,cloned and determined the KRT-IF 35 of Gansu Alpine Fine Wool Sheep,the correlation was analyzed between mutation locus of KRT-IF 35、KRT-IF 31 and wool traits.Experimental results are as follows:
1.A 4414bp KRT-IF 35 gene was amplified by PCR technique from the genome of Gansu Alpine Fine Wool Sheep(NCBI No:GU456629),the coding region is 1751bp, contained 455 amino acids.It's a hydrophilic protein,has two transmembrane regions,the main secondary structure is helix and there is 1 structure domain.
2.The results of PCR-SSCP and DNA sequencing showed:there is a novel C to T mutation in KRT-IF 35 CDS area 145bp. A pair alleles A and B and three genotype AA, BB, AB were dectected; A allele is the advantage allele of Fine strains and Super fine strains and AA genotype is the advantage genotype of Fine strains and Super fine strains, the fineness of AA, AB genotype significantly different, the fineness discrete of AA, BB genotype significant different, the bending of AA, BB genotype significant different. Tests of Hardy-Weinberg equilibrium confirmed that three strains was in equilibrium (P>0.05). So this loucs maybe the molecular genetic markers linked with the major gene that affect wool fineness、bending or fineness discrete or control factor.
3.The results of HRM and DNA sequencing showed:there is a novel A to G mutation in KRT-IF 31 CDS area 166bp,a pair alleles C and D and three genotypes CC、CD and DD were dectected. C allele is the advantage allele of Fine strains and Super fine strains;CC genotype is the advantage genotype of Fine strains and Super fine strains, CD genotype is the advantage genotype of Mutton-wool strains, tests of Hardy-Weinberg equilibrium confirmed that Fine strains was in equilibrium(P>0.05). CC, CD genotype significantly different, the fineness of CC, DD genotype significantly different. Therefore this Locus may be concerned with the wool fineness.
4. The results of HRM and DNA sequencing showed:there is a novel T to C mutation in KRT-IF 31 CDS area 191bp, a pair alleles E and F and three genotypes EE、EF and FF were dectected. E allele is the advantage allele of Fine strains、Super fine strains and Mutton-wool strains, EE genotype is the advantage genotype of Fine strains and Mutton-wool strains and EF genotype is the advantage genotype of Super fine strains. Tests of Hardy-Weinberg equilibrium confirmed that Fine strains was in equilibrium (P>0.05). EE, EF and EF, FF genotype significantly different, the fineness discrete of EE, EF genotype significant different. Therefore this Locus may be concerned with the wool fineness and fineness discrete.
1.首次克隆出绵羊KRT-IF 35基因,并对其进行生物信息学分析。绵羊KRT-IF 35基因全长4414bp (NCBI No:GU456629), CDS区全长1751bp；KRT-IF 35蛋白由455个氨基酸编码,存在两个跨膜区,二级结构以螺旋为主,且其存在1个结构域,为亲水性蛋白。
2.经PCR-SSCP分析和测序分析,KRT-IF 35 CDS区第145碱基处发生C→T的突变,产生一对等位基因A,B,存在AA、AB、BB三种基因型。AA基因型为细毛型、超细型品系的优势基因型；A等位基因为细毛型、超细型品系的优势等位基因。AA、AB基因型细度差异显著；AA、BB基因型细度离散差异显著；AA、BB基因型弯曲差异显著,这三个品系均处于Hardy-weinberg动态平衡状态(P>0.05)。推测此位点可能是与影响羊毛细度、弯曲度、细度离散的一个主效基因或调控羊毛性状的基因紧密连锁的分子遗传标记。
3.经HRM分析和测序分析,在KRT-IF 31基因CDS区166位发生A-G突变,产生一对等位基因C,D,存在CC、CD、DD三种基因型。C等位基因为细毛型和超细型品系的优势等位基因,CC基因型为细毛型和超细型品系优势基因型,CD基因型为毛肉兼用型品系优势基因型,细毛型品系处于Hardy-weinberg动态平衡状态。CC、CD基因型细度差异显著；CC、DD基因型细度差异显著,因此此位点可能与羊毛细度有关。
4.KRT-IF 31基因CDS区191位发生T-C突变,产生一对等位基因E,F,存在EE、EF、FF三种基因型。E等位基因为细毛型、超细型和毛肉兼用型品系的优势等位基因,而EE基因型为细毛型和毛肉兼用型品系优势基因型；EF基因型为超细型品系优势基因型。细毛型品系处于Hardy-weinberg动态平衡状态(P>0.05)。EF、FF基因型细度差异显著；EE、EF基因型细度离散差异显著,因此此位点可能与羊毛细度,细度离散有关。
This study taked Gansu Alpine Fine Wool Sheep as a research object,with the technology of clone, bioinformatic analysis,PCR-SSCP and HRM,cloned and determined the KRT-IF 35 of Gansu Alpine Fine Wool Sheep,the correlation was analyzed between mutation locus of KRT-IF 35、KRT-IF 31 and wool traits.Experimental results are as follows:
1.A 4414bp KRT-IF 35 gene was amplified by PCR technique from the genome of Gansu Alpine Fine Wool Sheep(NCBI No:GU456629),the coding region is 1751bp, contained 455 amino acids.It's a hydrophilic protein,has two transmembrane regions,the main secondary structure is helix and there is 1 structure domain.
2.The results of PCR-SSCP and DNA sequencing showed:there is a novel C to T mutation in KRT-IF 35 CDS area 145bp. A pair alleles A and B and three genotype AA, BB, AB were dectected; A allele is the advantage allele of Fine strains and Super fine strains and AA genotype is the advantage genotype of Fine strains and Super fine strains, the fineness of AA, AB genotype significantly different, the fineness discrete of AA, BB genotype significant different, the bending of AA, BB genotype significant different. Tests of Hardy-Weinberg equilibrium confirmed that three strains was in equilibrium (P>0.05). So this loucs maybe the molecular genetic markers linked with the major gene that affect wool fineness、bending or fineness discrete or control factor.
3.The results of HRM and DNA sequencing showed:there is a novel A to G mutation in KRT-IF 31 CDS area 166bp,a pair alleles C and D and three genotypes CC、CD and DD were dectected. C allele is the advantage allele of Fine strains and Super fine strains;CC genotype is the advantage genotype of Fine strains and Super fine strains, CD genotype is the advantage genotype of Mutton-wool strains, tests of Hardy-Weinberg equilibrium confirmed that Fine strains was in equilibrium(P>0.05). CC, CD genotype significantly different, the fineness of CC, DD genotype significantly different. Therefore this Locus may be concerned with the wool fineness.
4. The results of HRM and DNA sequencing showed:there is a novel T to C mutation in KRT-IF 31 CDS area 191bp, a pair alleles E and F and three genotypes EE、EF and FF were dectected. E allele is the advantage allele of Fine strains、Super fine strains and Mutton-wool strains, EE genotype is the advantage genotype of Fine strains and Mutton-wool strains and EF genotype is the advantage genotype of Super fine strains. Tests of Hardy-Weinberg equilibrium confirmed that Fine strains was in equilibrium (P>0.05). EE, EF and EF, FF genotype significantly different, the fineness discrete of EE, EF genotype significant different. Therefore this Locus may be concerned with the wool fineness and fineness discrete.
引文
[1]张明星,付大治,杜伟等.细毛羊与羊毛产业问题研究[J].吉林畜牧兽,2004(3)：6-9.
[2]夏新山.我国细毛羊产业现状及发展对策[J].动物生产,2007(43)：57,62-63.
[3]华太才让.世界养羊业的发展现状与发展趋势[J].四川草原,2004(12)：43-45.
[4]张明新,杜伟,王春昕等.我国细毛羊育种与羊毛产业状况诌议[J].中国畜牧兽医学会养羊学分会,全国养羊生产与学术研讨会议论文集.兰州：中国草食动物杂志社,2004：25-27.
[5]楚晓,叶得明,陈秉谱等.中国羊毛生产现状与市场分析[J].甘肃农业大学学报,2005(10)：708-712.
[6]潘君乾.我国细毛羊业的发展趋势[J].动物科学与动物医学,2003(20)：17-20.
[7]赵有璋,关于当前我国养羊业中若干热点问题的商榷[J].中国畜牧兽医学会养羊学分会,全国养羊生产与学术研讨会议论文集兰州：中国草食动物杂志社,2004：4-51.
[8]李文辉,保国俊,黄殿选等.甘肃高山细毛羊优质毛品系选育研究[J].遗传育种,2008(44)：9-13.
[9]梁永彦.对加快甘肃细毛羊发展的思考与对策[J].甘肃农业,2004(10)：39
[10]李文辉.高寒地区超细毛羊品系选育技术试验研究[D].甘肃农业大学硕士学位论文,2006.
[11]保国俊,李文辉.甘肃高山细毛羊育种现状及发展建议[J].甘肃畜牧兽医,2008,38(6)：43-48.
[12]李鸿浩,岳文斌.DNA多态性与绵羊遗传育种的研究进展[J].草食家畜,2005(2)：25-29.
[13]金海国,姜成国.分子遗传标记及其分析技术的研究进展[J].延边大学农学学报,2002,24(1)：55-59.
[14]张宇红0PCR-SSCP分析技术的研究进展及应用前景[J].中国优生与遗传杂志,2002,10(5)：126-127.
[15]欧阳建华,黄建华.PCR-SSCP技术的研究进展[J].上海畜牧兽医通讯,2002(4)：10-11.
[16]薛燕,常洪,常国斌.PCR-SSCP技术在动物育种中的研究进展[J].畜牧兽医杂志,2005,24(3)：21-24.
[17]丁建松,曹毅,童建.PCR-SSCP研究进展[J].辐射防护通讯,2004,24(5)：27-31.
[18]陈斌,雷秀霞,周小棉.高分辨率溶解曲线及其在分子诊断中的应用进展[J],分子诊断与治疗杂志,2009(7)：120-124.
[19]Cameron N.Gundry, Joshua G.Vandersteen, Gudrun H.Reed, et al.Amplicon Melting Analysis with Labeled Primers A Closed-Tube Method for Differentiating Homozygotes and Heterozygotes [J]. Clinical Chemistry,2003:396-406.
[20]谭昌渊,罗九甫,任兆瑞.高分辨率溶解曲线分析——分子诊断的新技术[J].生命科学研究,2009(3)：268-271.
[21]沈晓红,高分辨率溶解曲线在地中海贫血检测中的应用展望[J].检验医学,2010(25)：61=62.
[22]贾妤,黄国庆,高分辨率溶解——SNP及突变研究的最新工具[J].生命的化学,2007(27)：573-576.
[23]Dellmann H D,Brown E M.Textbook of Veterinary Histology[M]秦鹏春,聂其灼主译,北京：中国农业出版社出版,1989年5月第一版.
[24]王春昕,汪孟丽,张明新.羊毛性状在分子水平的研究[J].全国养羊生产与学术研讨会议论文集,2007-2008：81-83
[25]贾斌,新疆绵羊微卫星遗传分析及羊毛生长的神经内分泌调控[D].南京农业大学,2002.
[26]曾献存,陈韩英,彭林泽等.影响羊毛性状和羊毛品质的主要基因和QTL的研究进展[J].甘肃畜牧兽医,2006(5)：39-41.
[27]尹俊,扈庭茂,李金泉等.与绵羊KAP6-1相似的6个绒山羊全长cDNA的克隆与序列分析[J].遗传学报,2004,31(5)：502-507.
[28]McLaren RJ,Rogers GR,Davies KP,et al.Linkage mapping of wool keratin and keratin-associated protein genes in sheep[J].Mamm Genome,1997,8(12):938-940.
[29]刘桂芬,田可川,黄锡霞.羊毛细度候选基因的研究进展[J].生命科学仪器,2007(5)：19-22.
[30]于辉,王昕,陈宏等,绒山羊KAP9.2基因多态性分析[J].2007-2008年全国养羊生产与学术研讨会议论文集：171-173.
[31]韩黔峰,周总光,陈尧.角蛋白家族特性及其与恶性肿瘤的关系[J].中国普外基础与临床杂志,2005,9(12)：527-529.
[32]王志有.藏系绵羊KAP基因与部分经济性状的遗传相关性研究[D],西北农林科技大学硕士论文,2008.
[33]Powell BC, Rogers GE, Hair keratin.Composition, structure and biogenesis [A].Biology of the Integument[C].Berlin:Springer-Verlag,1986,696-721.
[34]Pruett,N.D.,Tkatchenko,T.V.,Jave-Suarez,L,et al.Krtapl6,characterization of a new hairkeratin-associated protein(KAP)gene complex on mouse chromosome 16 and evidence for regulation by Hoxcl3[J].Biol.Chem.2004,279:51524-51533.
[35]Crewther, WG, Primary structure and chemical properties of wool [J].Proceedings of 5th International Wool Textile Research Conference,1976 (1):1-101.
[36]Dolling, C.H.S, Brooker, M.G.A viable hypotrichosis in Polled Dorset Sheep [J].Hered,1996, 57:87-90.
[37]Coulombe, PA, Omary, MB.'Hard'and'soft'principles defining the structure, function and regulation of keratin intermediate filaments [J].Curr Opin Cell Biol,2002(14):110-122.
[38]Deramaudt,TB,Sachdeva,MM,Wescott,MP,et al.The PDX1 homeodomain transcription factor negatively regulates the pancreatic ductal cell-specific keratin 19 promoter[J].Biol Chem,2006, 281:38385-38395.
[39]Ponz R,Moreno C,Allain D.Assessment of genetic variation explained by markers for wool traits in sheep via a segment mapping approach[J].Manunalian Genome,2001(12):569-572.
[40]Cockett NE, Shay TL, Smit M.Analysis of the sheep genome [J].Physiol Genomics,2001(7):69-81.
[41]赵俊星,任有蛇,岳文斌.3个山羊品种KAP6-2和KAP7基因的PCR-SSCP分析[J].中国畜牧兽医,2007,34(12)：42-45.
[42]张亚妮,张恩平,吴迪等.内蒙古绒山羊KAP基因与经济性状关系的研究[J].畜牧兽医学报,2007,38(5)：447-451.
[43]Bawden CS, Mclaughlan C, Nesci A, et al. A unique type I keratin intermediate filament gene family is aboundantly expressed in the inner root sheaths of sheep and human hair follicles[J].The Journal of Investigative Dermatology,2001,116(1):157-166.
[44]ZhidongYu, Steven W.G, Allan.J.N, et al.Expression patterns of keratin intermediate filament and keratin associated protein genes in wool follicles [J].Differentiation,2009,77:307-316.
[45]田春英,王凤,荣威恒.内蒙古细毛羊业可持续发展对策探讨[J].畜牧与饲料科学,2005,26(4)：48-50.
[46]Gasteiger E, Hoogland C, Gattiker A,et al. Protein Identification and Analysis Tools on the ExPASy Server [M],2005:571-607.
[47]薛庆中等编.DNA和蛋白质序列数据分析工具[M].科学出版社：75.
[48]Bendtsen JD, Nielsen H, von Heijne G, et al. Improved prediction of signal peptides:SignalP 3.0 [J]. J Mol Biol,2004,340:783-795.
[49]John B,Louise C S,Ross J,et al.TM base-A database of membrane spanning proteins segments[J] Biol. Chem,1993(374):166.
[50]管峰,石国庆,刘守仁等.角蛋白家族及其对羊毛生长发育的调控[J].生命的化学,2007,27(1)：92-94
[51]Arora R,Bhatia S,Sehrawat A,et al.Genetic Polymorphism of type Ⅰ intermediate filament wool keratin gene in native indian sheep breeds[J].Biochem Genet,2008,46:549-556.
[52]Rogers G R,Hickford J,Bickerstaffe R.A potential QTL for wool strength locsted on ovine chromosome[C].Proceedings of 5th World Congress Applied to livestock Production Guelp,1994: 291-294
[53]赵苗,绒山羊6个候选基因遗传变异及其与经济性状关系研究[D],西北农林科技大学,2008.
[54]熊元著.猪生化及分子遗传实验导论[M].北京：中国农业出版社,1999.165-70.
[55]赵兴波,储明星,李宁等.绵羊线粒体DNA控制区5端序列PCR-SSCP与序列分析[J].遗传学报,2001,28(3)：225-228.
[56]李其松,王金玉,沈华等.DNA聚丙烯酰胺凝胶电泳及银染方法的讨论[J].黑龙江畜牧兽医,2006(10)：79-80.
[57]徐颖,HRM技术在炎症性肠病患者TPMT基因多态性的检测中的应用性研究[J].北京协和医学院临床医学专业学生毕业论文,2008年6月.
[58]刘双春,张瑾,关明等.运用高分辨率溶解曲线技术检测痛风患者SLC22A12基因intron5 (+4668C/T)基因多态性[J].China Academic Journal Electronic Publishing House:148-152.
[59]王荣谈,刘冬儿,厉建盟.等实时荧光定量PCR技术及其在植物转基因产品检测中的应用[J].植物生理学通讯,45(6)：591-597
[60]Jobs.M., Howell.W.M, Stromqvist.L.et al.DASH-2:flexible, low cost and high-throughput SNP genotyping by dynamio allele-specific hybridization on memnrane arrays [J].Genome Res, 2003(13):916-124.
[61]Reed G H, Wittwer C T.Sensitivity and specificity of single-nucleotide polymorphism scanning by high-resolution melting analysis [J].Clin Chem,2004,19:1164-1169.
[62]Smith, Brad.High-resolution melting analysisi (HRMA):a highly sensitive inexpensive genotyping alternative for population studies [J].Molecular Ecology Resources:1-9.
[63]Luming Zhou,Alesander N.Myers,Joshua Get al.,Closed-Tube Genotyping with Unlabeled Oligonucleotide Probes and a Saturating DNA Dye[J].Clinical Chemistry,2004:1328-1335.
[64]Virginie Dujols, Noriko Kusukawa, Jason T et al.High-resolution melting analysis for scanning and genotyping [J].Real time PCR:155-168.
[65]Maria Erali, Robert Palais, Carl Wittwer. SNP Genotyping by Unlabeled Probe Melting Analysis [J].Erali_Seitz,2007:199-206.
[66]Shale Dames,Rebecca L.Margraf,David C.Pattison.et al. Characterization of Aberrant Melting Peaks inUnlabeled Probe Assays [J].Journal of Molecular Diagnostics,2007(9):290-296
[67]姚毅,雷承志,尹君亮等.新疆细毛羊和陕北细毛羊羊毛细度候选基因的PCR-SSCP分析[J].西北农林科技大学学报,2006(12)：6-10.
[2]夏新山.我国细毛羊产业现状及发展对策[J].动物生产,2007(43)：57,62-63.
[3]华太才让.世界养羊业的发展现状与发展趋势[J].四川草原,2004(12)：43-45.
[4]张明新,杜伟,王春昕等.我国细毛羊育种与羊毛产业状况诌议[J].中国畜牧兽医学会养羊学分会,全国养羊生产与学术研讨会议论文集.兰州：中国草食动物杂志社,2004：25-27.
[5]楚晓,叶得明,陈秉谱等.中国羊毛生产现状与市场分析[J].甘肃农业大学学报,2005(10)：708-712.
[6]潘君乾.我国细毛羊业的发展趋势[J].动物科学与动物医学,2003(20)：17-20.
[7]赵有璋,关于当前我国养羊业中若干热点问题的商榷[J].中国畜牧兽医学会养羊学分会,全国养羊生产与学术研讨会议论文集兰州：中国草食动物杂志社,2004：4-51.
[8]李文辉,保国俊,黄殿选等.甘肃高山细毛羊优质毛品系选育研究[J].遗传育种,2008(44)：9-13.
[9]梁永彦.对加快甘肃细毛羊发展的思考与对策[J].甘肃农业,2004(10)：39
[10]李文辉.高寒地区超细毛羊品系选育技术试验研究[D].甘肃农业大学硕士学位论文,2006.
[11]保国俊,李文辉.甘肃高山细毛羊育种现状及发展建议[J].甘肃畜牧兽医,2008,38(6)：43-48.
[12]李鸿浩,岳文斌.DNA多态性与绵羊遗传育种的研究进展[J].草食家畜,2005(2)：25-29.
[13]金海国,姜成国.分子遗传标记及其分析技术的研究进展[J].延边大学农学学报,2002,24(1)：55-59.
[14]张宇红0PCR-SSCP分析技术的研究进展及应用前景[J].中国优生与遗传杂志,2002,10(5)：126-127.
[15]欧阳建华,黄建华.PCR-SSCP技术的研究进展[J].上海畜牧兽医通讯,2002(4)：10-11.
[16]薛燕,常洪,常国斌.PCR-SSCP技术在动物育种中的研究进展[J].畜牧兽医杂志,2005,24(3)：21-24.
[17]丁建松,曹毅,童建.PCR-SSCP研究进展[J].辐射防护通讯,2004,24(5)：27-31.
[18]陈斌,雷秀霞,周小棉.高分辨率溶解曲线及其在分子诊断中的应用进展[J],分子诊断与治疗杂志,2009(7)：120-124.
[19]Cameron N.Gundry, Joshua G.Vandersteen, Gudrun H.Reed, et al.Amplicon Melting Analysis with Labeled Primers A Closed-Tube Method for Differentiating Homozygotes and Heterozygotes [J]. Clinical Chemistry,2003:396-406.
[20]谭昌渊,罗九甫,任兆瑞.高分辨率溶解曲线分析——分子诊断的新技术[J].生命科学研究,2009(3)：268-271.
[21]沈晓红,高分辨率溶解曲线在地中海贫血检测中的应用展望[J].检验医学,2010(25)：61=62.
[22]贾妤,黄国庆,高分辨率溶解——SNP及突变研究的最新工具[J].生命的化学,2007(27)：573-576.
[23]Dellmann H D,Brown E M.Textbook of Veterinary Histology[M]秦鹏春,聂其灼主译,北京：中国农业出版社出版,1989年5月第一版.
[24]王春昕,汪孟丽,张明新.羊毛性状在分子水平的研究[J].全国养羊生产与学术研讨会议论文集,2007-2008：81-83
[25]贾斌,新疆绵羊微卫星遗传分析及羊毛生长的神经内分泌调控[D].南京农业大学,2002.
[26]曾献存,陈韩英,彭林泽等.影响羊毛性状和羊毛品质的主要基因和QTL的研究进展[J].甘肃畜牧兽医,2006(5)：39-41.
[27]尹俊,扈庭茂,李金泉等.与绵羊KAP6-1相似的6个绒山羊全长cDNA的克隆与序列分析[J].遗传学报,2004,31(5)：502-507.
[28]McLaren RJ,Rogers GR,Davies KP,et al.Linkage mapping of wool keratin and keratin-associated protein genes in sheep[J].Mamm Genome,1997,8(12):938-940.
[29]刘桂芬,田可川,黄锡霞.羊毛细度候选基因的研究进展[J].生命科学仪器,2007(5)：19-22.
[30]于辉,王昕,陈宏等,绒山羊KAP9.2基因多态性分析[J].2007-2008年全国养羊生产与学术研讨会议论文集：171-173.
[31]韩黔峰,周总光,陈尧.角蛋白家族特性及其与恶性肿瘤的关系[J].中国普外基础与临床杂志,2005,9(12)：527-529.
[32]王志有.藏系绵羊KAP基因与部分经济性状的遗传相关性研究[D],西北农林科技大学硕士论文,2008.
[33]Powell BC, Rogers GE, Hair keratin.Composition, structure and biogenesis [A].Biology of the Integument[C].Berlin:Springer-Verlag,1986,696-721.
[34]Pruett,N.D.,Tkatchenko,T.V.,Jave-Suarez,L,et al.Krtapl6,characterization of a new hairkeratin-associated protein(KAP)gene complex on mouse chromosome 16 and evidence for regulation by Hoxcl3[J].Biol.Chem.2004,279:51524-51533.
[35]Crewther, WG, Primary structure and chemical properties of wool [J].Proceedings of 5th International Wool Textile Research Conference,1976 (1):1-101.
[36]Dolling, C.H.S, Brooker, M.G.A viable hypotrichosis in Polled Dorset Sheep [J].Hered,1996, 57:87-90.
[37]Coulombe, PA, Omary, MB.'Hard'and'soft'principles defining the structure, function and regulation of keratin intermediate filaments [J].Curr Opin Cell Biol,2002(14):110-122.
[38]Deramaudt,TB,Sachdeva,MM,Wescott,MP,et al.The PDX1 homeodomain transcription factor negatively regulates the pancreatic ductal cell-specific keratin 19 promoter[J].Biol Chem,2006, 281:38385-38395.
[39]Ponz R,Moreno C,Allain D.Assessment of genetic variation explained by markers for wool traits in sheep via a segment mapping approach[J].Manunalian Genome,2001(12):569-572.
[40]Cockett NE, Shay TL, Smit M.Analysis of the sheep genome [J].Physiol Genomics,2001(7):69-81.
[41]赵俊星,任有蛇,岳文斌.3个山羊品种KAP6-2和KAP7基因的PCR-SSCP分析[J].中国畜牧兽医,2007,34(12)：42-45.
[42]张亚妮,张恩平,吴迪等.内蒙古绒山羊KAP基因与经济性状关系的研究[J].畜牧兽医学报,2007,38(5)：447-451.
[43]Bawden CS, Mclaughlan C, Nesci A, et al. A unique type I keratin intermediate filament gene family is aboundantly expressed in the inner root sheaths of sheep and human hair follicles[J].The Journal of Investigative Dermatology,2001,116(1):157-166.
[44]ZhidongYu, Steven W.G, Allan.J.N, et al.Expression patterns of keratin intermediate filament and keratin associated protein genes in wool follicles [J].Differentiation,2009,77:307-316.
[45]田春英,王凤,荣威恒.内蒙古细毛羊业可持续发展对策探讨[J].畜牧与饲料科学,2005,26(4)：48-50.
[46]Gasteiger E, Hoogland C, Gattiker A,et al. Protein Identification and Analysis Tools on the ExPASy Server [M],2005:571-607.
[47]薛庆中等编.DNA和蛋白质序列数据分析工具[M].科学出版社：75.
[48]Bendtsen JD, Nielsen H, von Heijne G, et al. Improved prediction of signal peptides:SignalP 3.0 [J]. J Mol Biol,2004,340:783-795.
[49]John B,Louise C S,Ross J,et al.TM base-A database of membrane spanning proteins segments[J] Biol. Chem,1993(374):166.
[50]管峰,石国庆,刘守仁等.角蛋白家族及其对羊毛生长发育的调控[J].生命的化学,2007,27(1)：92-94
[51]Arora R,Bhatia S,Sehrawat A,et al.Genetic Polymorphism of type Ⅰ intermediate filament wool keratin gene in native indian sheep breeds[J].Biochem Genet,2008,46:549-556.
[52]Rogers G R,Hickford J,Bickerstaffe R.A potential QTL for wool strength locsted on ovine chromosome[C].Proceedings of 5th World Congress Applied to livestock Production Guelp,1994: 291-294
[53]赵苗,绒山羊6个候选基因遗传变异及其与经济性状关系研究[D],西北农林科技大学,2008.
[54]熊元著.猪生化及分子遗传实验导论[M].北京：中国农业出版社,1999.165-70.
[55]赵兴波,储明星,李宁等.绵羊线粒体DNA控制区5端序列PCR-SSCP与序列分析[J].遗传学报,2001,28(3)：225-228.
[56]李其松,王金玉,沈华等.DNA聚丙烯酰胺凝胶电泳及银染方法的讨论[J].黑龙江畜牧兽医,2006(10)：79-80.
[57]徐颖,HRM技术在炎症性肠病患者TPMT基因多态性的检测中的应用性研究[J].北京协和医学院临床医学专业学生毕业论文,2008年6月.
[58]刘双春,张瑾,关明等.运用高分辨率溶解曲线技术检测痛风患者SLC22A12基因intron5 (+4668C/T)基因多态性[J].China Academic Journal Electronic Publishing House:148-152.
[59]王荣谈,刘冬儿,厉建盟.等实时荧光定量PCR技术及其在植物转基因产品检测中的应用[J].植物生理学通讯,45(6)：591-597
[60]Jobs.M., Howell.W.M, Stromqvist.L.et al.DASH-2:flexible, low cost and high-throughput SNP genotyping by dynamio allele-specific hybridization on memnrane arrays [J].Genome Res, 2003(13):916-124.
[61]Reed G H, Wittwer C T.Sensitivity and specificity of single-nucleotide polymorphism scanning by high-resolution melting analysis [J].Clin Chem,2004,19:1164-1169.
[62]Smith, Brad.High-resolution melting analysisi (HRMA):a highly sensitive inexpensive genotyping alternative for population studies [J].Molecular Ecology Resources:1-9.
[63]Luming Zhou,Alesander N.Myers,Joshua Get al.,Closed-Tube Genotyping with Unlabeled Oligonucleotide Probes and a Saturating DNA Dye[J].Clinical Chemistry,2004:1328-1335.
[64]Virginie Dujols, Noriko Kusukawa, Jason T et al.High-resolution melting analysis for scanning and genotyping [J].Real time PCR:155-168.
[65]Maria Erali, Robert Palais, Carl Wittwer. SNP Genotyping by Unlabeled Probe Melting Analysis [J].Erali_Seitz,2007:199-206.
[66]Shale Dames,Rebecca L.Margraf,David C.Pattison.et al. Characterization of Aberrant Melting Peaks inUnlabeled Probe Assays [J].Journal of Molecular Diagnostics,2007(9):290-296
[67]姚毅,雷承志,尹君亮等.新疆细毛羊和陕北细毛羊羊毛细度候选基因的PCR-SSCP分析[J].西北农林科技大学学报,2006(12)：6-10.