利用DDRT-PCR技术筛选新疆超细型细毛羊毛用性状相关基因
摘要
羊毛的生产具有重要的经济价值。羊毛纤维直径是评定羊毛品质的重要性状,在同一品种,同一年龄,相同的环境和饲养管理条件下,个体的遗传差异对羊毛的生长起到了决定性的作用。
新疆是我国重要的生产羊毛的地区,新吉细毛羊是我国生产优质羊毛的主要品种。本实验测定了184只2岁新疆超细型细毛羊(新吉细毛羊)的羊毛纤维直径,选取其中纤维直径极粗和极细的细毛羊各6只,取其皮肤组织组成RNA池作为实验研究对象,应用mRNA差异显示技术筛选不同纤维直径的绵羊肩部皮肤的差异表达基因,以期为细毛羊早期选种提供理论和技术上的参考。
实验过程中,选用3种锚定引物,8种随机引物,经过24对引物组合进行差异显示反转录PCR,扩增产物经过聚丙烯凝胶电泳分析、回收差异表达的基因片段,共筛选出53条差异表达片段,进行二次扩增,利用pGM-T载体将扩增后的片段克隆,经过反Northern Blot分析后,筛除假阳性片段,将13条阳性差异片段测序。在13条测序的片段中,有3条片段未找到引物序列,2条片段与切胶时片段大小不一致。将其余8条片段的测序结果在GenBank里分析,其中6条为新序列,2条较高相似性功能基因,其中一条与LaminB基因(LMNB)同源,另一条与SHROOM2基因和GPR143基因同源。
本实验同时用半定量逆转录-聚合酶链式反应(RT-PCR)方法测定BⅢB4基因在细毛羊皮肤中的mRNA表达水平,以18SrRNA为内标,分析相对表达量及其与羊毛纤维直径的关系。用Independent Samples T Test分析BⅢB4 PCR产物与18SrRNA PCR产物电泳后的灰度比值,可知BⅢB4基因mRNA表达量对羊毛纤维直径的影响不显著。
Wool production has an important economic value.Wool fiber diameter is an important assessment of the wool quality.in the same species,the same age,the same feeding and the growth environment,genetic differences between individuals on the growth of wool play a decisive role.
Xinjiang is an important area of wool production,XinJi fine-wool sheep is the Chinese major variety of high quality wool production.184 two-year-old of XinJiang superfine wool sheep (XinJi fine-wool sheep) fibre diameter were detected,the study select the fiber diameter of the very crude and the very fine of the six fine-wool sheep,take their skin composing the RNA pool as an experimental sample,the application of mRNA differential display reverse transcription PCR technique to separate differential expressed genes in differential fiber fineness of the sheep shoulder skin,with a view to early selection for fine-wool sheep to provide theoretical and technical reference.
The experiment choose three anchor primers,eight random primers,the 24 primer pairs be composed to DDRT-PCR.PCR products were analyzed by polyacrylamide gel electrophoresis,and the differential displayed genes were reclaimed,separating 53 differential expressed genes as templates of second PCR.The purified genes were cloned by pGM-T vector and verified by reverse Northern blot analysis,screening out false-positive fragments,13 positive fragments will be sequenced.In 13 sequenced fragments,there are three fragments not found primer sequences,two fragments are inconsistent with their length when cutting from polyacrylamide gel.The sequences of the remaining eight fragments are analysed in GenBank,including six unknown function genes,two putative function genes.One similar to LaminB gene(LMNB),and another sequence blast reveal that it homology to SHROOM2 genes and GPR143 gene.
At the same time in this experiment,the gene expression of BⅢB4 in Xinji fine-wool sheep skin was measured by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) method,The relationship between relative gene expression of BⅢB4 and fineness wool were analyzed by using 18SrRNA as internal standard.The ratio of integrated optical density (IOD) of BⅢB4 PCR and 18SrRNA PCR were analyzed by Independent Samples T Test.The result indicated how gene expression of BⅢB4 influence fineness of wool is negative significant correlation.
新疆是我国重要的生产羊毛的地区,新吉细毛羊是我国生产优质羊毛的主要品种。本实验测定了184只2岁新疆超细型细毛羊(新吉细毛羊)的羊毛纤维直径,选取其中纤维直径极粗和极细的细毛羊各6只,取其皮肤组织组成RNA池作为实验研究对象,应用mRNA差异显示技术筛选不同纤维直径的绵羊肩部皮肤的差异表达基因,以期为细毛羊早期选种提供理论和技术上的参考。
实验过程中,选用3种锚定引物,8种随机引物,经过24对引物组合进行差异显示反转录PCR,扩增产物经过聚丙烯凝胶电泳分析、回收差异表达的基因片段,共筛选出53条差异表达片段,进行二次扩增,利用pGM-T载体将扩增后的片段克隆,经过反Northern Blot分析后,筛除假阳性片段,将13条阳性差异片段测序。在13条测序的片段中,有3条片段未找到引物序列,2条片段与切胶时片段大小不一致。将其余8条片段的测序结果在GenBank里分析,其中6条为新序列,2条较高相似性功能基因,其中一条与LaminB基因(LMNB)同源,另一条与SHROOM2基因和GPR143基因同源。
本实验同时用半定量逆转录-聚合酶链式反应(RT-PCR)方法测定BⅢB4基因在细毛羊皮肤中的mRNA表达水平,以18SrRNA为内标,分析相对表达量及其与羊毛纤维直径的关系。用Independent Samples T Test分析BⅢB4 PCR产物与18SrRNA PCR产物电泳后的灰度比值,可知BⅢB4基因mRNA表达量对羊毛纤维直径的影响不显著。
Wool production has an important economic value.Wool fiber diameter is an important assessment of the wool quality.in the same species,the same age,the same feeding and the growth environment,genetic differences between individuals on the growth of wool play a decisive role.
Xinjiang is an important area of wool production,XinJi fine-wool sheep is the Chinese major variety of high quality wool production.184 two-year-old of XinJiang superfine wool sheep (XinJi fine-wool sheep) fibre diameter were detected,the study select the fiber diameter of the very crude and the very fine of the six fine-wool sheep,take their skin composing the RNA pool as an experimental sample,the application of mRNA differential display reverse transcription PCR technique to separate differential expressed genes in differential fiber fineness of the sheep shoulder skin,with a view to early selection for fine-wool sheep to provide theoretical and technical reference.
The experiment choose three anchor primers,eight random primers,the 24 primer pairs be composed to DDRT-PCR.PCR products were analyzed by polyacrylamide gel electrophoresis,and the differential displayed genes were reclaimed,separating 53 differential expressed genes as templates of second PCR.The purified genes were cloned by pGM-T vector and verified by reverse Northern blot analysis,screening out false-positive fragments,13 positive fragments will be sequenced.In 13 sequenced fragments,there are three fragments not found primer sequences,two fragments are inconsistent with their length when cutting from polyacrylamide gel.The sequences of the remaining eight fragments are analysed in GenBank,including six unknown function genes,two putative function genes.One similar to LaminB gene(LMNB),and another sequence blast reveal that it homology to SHROOM2 genes and GPR143 gene.
At the same time in this experiment,the gene expression of BⅢB4 in Xinji fine-wool sheep skin was measured by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) method,The relationship between relative gene expression of BⅢB4 and fineness wool were analyzed by using 18SrRNA as internal standard.The ratio of integrated optical density (IOD) of BⅢB4 PCR and 18SrRNA PCR were analyzed by Independent Samples T Test.The result indicated how gene expression of BⅢB4 influence fineness of wool is negative significant correlation.
引文
[1] Allain D,Lantier I,Elsen JM,et al. A design aiming at detecting QTL controlling wool traits and other traits in the INRA401 sheep line[J].Genet.,1998:51-54.
[2] Henry HM,Dodds KG,Wuliji T,et al. A genome screen for QTL for wool traits in aMerino×Romney backcross flock[J].Wool Technology and Sheep Breeding,1998.46:213-217.
[3] Ponz R,Moreno C,Allain D,et al. Assessement of genetic variation explained by markers for wool traits in sheep via a segment mapping approach[J].Mamm.Genome,2001,12:569-572.
[4] F.Bidinost. Quantitative trait loci related to merino sheep wool quality[C].8th World Congress on Genetics Applied to Livestock Production,2006,8:13-18.
[5] Vitezica ZG. Quantitative trait loci linked to PRNP gene controlling health and production traits in INRA 401 sheep[J].Genet.,2007,39(4):421-30.
[6]张明亚.凉山半细毛羊1号染色体遗传连锁图和羊毛性状QTL定位研究[D].四川:四川农业大学硕士学位论文,2005.
[7]马玉萍.绵羊主要经济性状的微卫星标记研究[D].石河子:石河子大学硕士学位论文,2003.
[8]徐新明.超细型细毛羊遗传多样性及羊毛性状的微卫星分析[D].新疆:新疆农业大学硕士学位论文,2007.
[9]沙莎.绵羊微卫星标记与部分毛用性状的关系研究[J].新疆农业职业技术学院学报,2006(3):49-53.
[10]赵宗胜,王根林,马玉萍.绵羊微卫星标记与部分毛用性状的关系研究[J].畜牧兽医学报,2006(9):28-33.
[11] McLaren RJ,Rogers GR,Davies KP,et al. Linkage mapping of wool keratin and keratin-associated protein genes in sheep[J].Mammalian Genome,1997,8:938-940.
[12] Powell BC,Rogers GE. Analysis of hair follicle proteins in:Keratinocyte Methods.Leigh I.Cambridge University Press,UK,1994:149-155.
[13] Rogers GR,Hickford JGH,Bickerstaffe R,et al. Polymorphism in two genes for B2 sulfur proteins of wool[J].Anim. Genet.,1994,25:407-415.
[14] Parsons YM,Piper LR,Cooper DW,et al. Linkage relation-ships between keratin-associated protein (KRTAP) genes and growth hormone in sheep[J].Genomics,1994,20(3):500-502.
[15] Plowman JE,Bryson WG,Jordan TW,et al. Application of proteomics for determining protein markers for wool quality traits[J].Electrophoresis,2000,5,21(9):1899-1906.
[16] Flanagan LM,Plowman JE,Bryson WG,et al. The high sulphur proteins of wool:Towards an understanding of sheep breed diversity[J].Proteomics,2002 Sep,2(9):1240-1246.
[17] Rufaut NW,Pearson AJ,Nixon AJ,et al. Identification of differentially expressed genes during a wool follicle growth cycle induced by prolactin[J].Invest.Dermatol,1999,113:865-872.
[18]刘桂芬.优质细毛羊羊毛细度的候选基因分析[J].遗传,2007,29(1):70-74.
[19]张亚妮,张恩平,吴迪. KAP基因的多态性与辽宁绒山羊经济性状的关系研究[J].中国农业科学,2007,40(9):2062-2067.
[20]张亚妮,张恩平,吴迪.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(05):33-37
[21] Parsons YM,Fleet MR,Cooper Des W,et al. Isolation of the ovine agouti coding sequence[J].Pigment Cell Res,1999,12:394-397.
[22] Smit MA,Shay TL,Beever JE,et al. Identification of an agouti-like locus in sheep[J].Anim.Genet.,2002,33:383-385.
[23] Sander GR,Powell BC. Structure and expression of the ovine Hoxc-13 gene[J].Gene,2004,2(18),327(1):107-116.
[24] Liang P,Pardee AB. Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction[J].Science,1992,257:967-971.
[25]迪芬巴赫,德维克斯勒著,黄培堂等译. PCR技术实验指南[M].北京:北京科学出版社,1999.
[26] Liang P,Averboukh L,Pardee AB,et al. Distribution and cloning of eukaryotic mRNAs by means of differential display:refinements and optimization[J].Nucleic Acids Res,1993,21:3269-3275.
[27] Liang P,Zhu WM,Zhang XY,et al. Differential display using one-base anchored oligo-dT primers[J].Nucleic Acids Res,1994,22:5763-5764.
[28] Ito T,Kito K,Adati N,et al. Fluorescent differential display:arbitrarily primed RT-PCR fingerprinting on an automated DNA sequencer.FEBS Lett,1994,351(2):231-236.
[29] GenHunter Corporation,RNA imageTM,kit for mRNA differential display,1996,April.
[30] Jones SW,Cai D,Weislow OS,et al. Generation of multiple mRNA fingerprints using fluorescence-based differential display and an automated DNA sequencer[J].Biotechniques,1997,22:536-543.
[31] Luehrsen KR,Marr LL,van der Knaap E,et al. Analysis of differential display RT-PCR products using fluorescent primers and GENESCANTM software[J].Biotechniques,1997,22:168-174.
[32] Smith NR,Aldersley M,Li A,et al. Automated differential display using a fluorescently labeled universal primer[J].Biotechniques,1997,23:274-279.
[33]陈东,高冬玲.非放射性同位素mRNA差异显示技术[M].国外医学:生理、病理科学与临床分册,2000,20:518-520.
[34] Weaver KR,Caetano-Anolles G,Gresshoff PM,et al. Isolation and cloning of DNA amplification products from silver-stained ployacrylamide gels[J].Biotechniques,1994,16:226-227.
[35] Doss RP. Differential display without radioactivity a modified procedure[J].Biotechniques,1996,21:408-412.
[36] Ponsuksili S,Wimmers K,Schmoll F,et al. Porcine ESTs detected by differential display representing possible candidates for the trait“eye muscle area”[J].Anim.Breed.Genet.,2000,117:25-35.
[37] Bhattacharjee A,Abrahamsen MS,Lappi VR,et al. Refinements in re-amplification and cloning of DDRT-PCR products[J].Biotechniques,1994,16:1096-1103.
[38] Liang P,Pardee AB. Differential display[J].Molecular Biotechnology,1998,10:261-267.
[39] Liang P,Bauer D,Averboukh L,et al. Analysis of altered gene expression by differential display[J].Methods in enaymology,1995,254:304-320.
[40] Sompayrac L,Jane S,Burn TC,et al. Overcoming limitations of the mRNA differential display technique[J].Nucleic Acids Res,1995,23:4738-4739.
[41] Guimaraes MJ,Lee F,Zlotnik A,et al. Differential display by PCR:novel findings and applications[J].Nucleic Acids Res,1995,23:1832-1833.
[42] Li ZK,Yao KT,Cao Y. Molecular cloning of a novel tissue-specific gene from human nasopharyngeal epithelium[J].Gene,1999,237:235-240.
[43] Clouscard-Martinato C,Mulsant P,Robic A,et al. Characterization of FSH-regulated genes isolated by mRNA differential display from pig ovarian granulose cells[J].Anim Genet.,1998,29:98-106.
[44] Robert C,Gagne D. Differential display and suppressive subtractive hybridization used to identify granulose cell messenger ma associated with bovine oocyte developmental competence[J].Biol Reprod,2001,64(6):1812-1820.
[45] Boardman PE. A comprehensive collection of chicken cDNAs.Curr Biol,2002,12(22):1965-1969.
[46] Schwerin M. Application of disease-associated differentially expressed genes-mining for functional candidate genes for mastitis resistance in cattle[J].Genet Sel Evol,2003,35 Suppl:19-34.
[47] Bertani GR. Evaluation of gene expression in pigs selected for enhanced reproduction using differential display PCR:Ⅱ.Anterior pituitary[J].Anim Sci,2004,82(1):32-40.
[48] Wesolowski SR. Developmental changes in the fetal pig transcriptome[J].Physiol Genomics,2004 Jan 15;16(2):268-74.
[49] M Clinton. Identification of disease markers by differential display:prion disease[J].Methods Mol Biol,January 1,2006,317:157-78.
[50] S.G.Roh. Identification of differentially expressed transcripts in bovine rumen and abomasum using a differential display method[J].Anim Sci,Feb 2007;85:395-403.
[51]俞英.鸡心肌和肝脏组织基因差异表达与杂种优势关系的研究[D].北京:中国农业大学博士学位论文,2003.
[52]李亮.乌骨鸡肝脏差异显示(DD)表达序列标签(ESTs)的筛选及分析鉴定[D].四川:四川农业大学博士学位论文,2004.
[53]任竹青.杂种和纯种猪之间脂肪组织差异表达基因的分离、克隆和序列分析[J].农业生物技术学报,2005,13(5):601-607.
[54]康文霞.高产与低产蛋鸡产蛋高峰期卵巢组织基因差异表达的研究[D].山东:山东农业大学硕士学位论文,2006.
[55]沙莎.绵羊毛囊差异表达基因的定为研究[D].石河子:石河子大学硕士学位论文,2006.
[56] Dalal SS,Welsh J,Tkachenko A,et al. Rapid isolation of tissue-specific and developmentally regulated brain cDNAs using RNA arbitrarily primed PCR(RAP-PCR)[J].Mol Neurosci,1994,5(2):93-104.
[57] Lamar EE,Palmer E.Y-encoded,species-specific DNA in mice:evidence that the Y chromosome exist in two polymorphic forms in inbred strains[J].Cell,1984,37(1):171-177.
[58] Diatchenko L,Lau YF,Campbell A P,et al. Suppression subtractive hybridization:a method for generating differentially regulated or tissue-specific cDNA probes and libraries[J].Proc Natl Acad Sci USA,1996,93(12):6025-6030.
[59]田连福,郭新红,姜孝成,等.抑制消减杂交技术(SSH)及其在植物基因克隆上的应用[J].生物学杂志,2002,19(4):29-31.
[60] Von Stein OD,Thies WG,Hofmann M. A high throughput screening for rarely transcribed differentially expressed genes[J].Nucleic Acids Res,1997,25(13):2598-2602.
[61] Lisitsyn NA,Rosenberg MV,Launer GA,et al. A method for isolation of sequences missing in one of two related genomes[J].Mol Gen Mikrobiol Virusol,1993,(3):26-29.
[62] Hubank M,Schatz DG. Identifying differences in mRNA expression by representational diference analysis of cDNA[J].Nucleic Acids Res,1994,22(25):5640-5648.
[63] Yuan BZ,Miller MJ,Keck CL,et al. Cloning,characterization and chromosomal localization of a gene frequently deleted in human liver cancer(DIC1)homologous to rat RhoGAP[J].Cancer Res,1998,58(10):2196-2199.
[64] Bachem CW,Vander Hoeven RS,de Bruijn SM,et al. Visualization of differential gene expression using a novel method of RNA finger-printing based on AFLP:analysis of gene expression duing potato tuber development[J].Plant,1996,9(5):745-753.
[65] Vos P,Hogers M,Bleeker M,et al. AFLP:a new technique for DNA fingerprinting[J].Nucleic Acids Res,1995,23:4407-4414.
[66] Bachem CW,Oomen RJFJ,Visser RGF. Transcript imaging with cDNA-AFLP:a step-by-step protocol[J].Plant Mol Biol,1998,16:157-173.
[67] Velculescu VE,Zhang L,Vogelstein B,et al. Serial analysis of gene expression[J].Science,1995,270(5235):484-287.
[68]张锦超,俞炜源.基因表达系列性分析技术及其应用[J].生物技术通讯,2002,13(5):395-399.
[69] Schena M,Shalon D,Davis RW,et al. Quantitative monitoring of gene expression patterns with a complementary DNA microarray[J].Science,1995,270(5235):467-470.
[70] Wang SM,Rowley JD. A strategy for genome-wide gene analysis:integrated procedure for gene identification[C].Proc Natl Acad Sci USA,1998,95(20):11909-11914.
[71]史春梦,粟永萍.全基因组基因表达频谱研究的新方法——SAGE和IPGI[J].生物工程进展,2001,21(2):65-67.
[72]贺三刚.利用cDNA-AFLP技术筛选与羊毛细度相关的分子标记[D].新疆:新疆农业大学硕士学位论文,2008.
[73] Zhang H,Zhang R,Liang P. Differential screening of gene expression difference enriched bydifferential display[J].Nucleic Acids Research,1996,24(12):2454-2455.
[74]刘红,任笑蒙,陈兰英.一种快速筛选阳性克隆的方法——反向Northern印迹杂交技术[J].基础医学与临床,2002,22(3)278-280.
[75]蔡树涛等. Nuclear Lamina(核纤层)——一种重要的细胞核结构[J].细胞生物学杂志,1990,12(l):15.
[76] Stick R. The Cytoskeleton.Greenwieh JAJ Press,1995,257-296.
[77] Moir RD,et al. The dynamic properties and possible functions of nuclear Lamins.Int Rev Cytol,1995,162B:141.
[78] Stuurman N,et al. Nuclear lamins:Their structure,assembly and interactions[J].Struct Biol,1998,122:42.
[79] Broers JL,Madicels BM,et al. A and B-type lamins are differentially expressed in normal human tissues[J].Histochem Cell Biol.1997,107:505-517.
[80] Duband-Goulet I,Courvalin JC. LBR,a chromatin and lamin binding protein from the inner nuclear membrane is proteolyzed at late stages of apoptosis[J].Cell Sei.1998,111:1441-1451.
[81]狄江,李勇,拉扎提,等.羊毛纤维直径与其他经济性状的相关性研究[J].中国草食动物,2004,5:44-46.
[82] Parry DAD,Steinert PM. IFs:moleeular,architecture,assembly,dynamics and polymer-phism[J].Q Rev Biophys,1999,32:99-187.
[83] Monteiro MJ,Hicks C,Gu L,et al. Determinants for intracellular sorting of cytoplasmic and nuclear intermediate filaments[J].Cell Biol,1994,127:1327-1343.
[84] Lee MK,Xu ZS,Wong PC,et al. Neurofilaments are obligate heteropolymers in vivo[J].Cell Biol,1993,122:1337-1350.
[85] Pamela DF,Chanjae Lee,Avegiyel Ellis,et al. Shroom2 (APXL) regulates melanosome biogenesis and localization in the retinal pigment epithelium[J].Development,2006,133,4109-4118.
[86] Ilaria Palmisano,Paola Bagnato,Angela Palmigiano,et al. The ocular albinism type 1 protein,an intracellular G protein-coupled receptor,regulates melanosome transport in pigment cells[J].Hum Mol Genet.2008 November 15,17(22):3487–3501.
[87] Mathias C,Martine B,Anne V C. A semi-quantitative RT-PCR method to readily compare expression levels within Botrytis cinerea multigenic families in vitro and in planta[J].Curr Genet,2003,43(1):303-309.
[88] Rolland V G. Semi-quantitative analysis of gene expression incultured chondrocytes by RT-PCR[J].Methods Mol Med,2004,100(1):69-78.
[89] Kuddus R H,Lee T H,Valdivea L A. A semi-quantitative PCR technique for detecting chimerism in hamster-to-rat bone marrow xenotransplantation[J]Immunol Methods,2004,285(1):245-251.
[90] Fellenr M D,Durand K,Correa M. A semi quantitative PCR method(SQ-PCR)to measure Epstein-Barr virus(EBV)load:its ap-plication in transplant patients[J].Biotechniques,1997,22(1):630-634,636.
[91]张翔,王寒正,龚岳亭,等. RT-PCR测定mRNA的荧光定量分析[J].生物化学与生物物理学报,1998,30(1):70-73.
[92] Chen D,Klebe RJ. Controls forvalidation ofrelative reverse tran-scription-polymerase chain reaction assays[J].PCR Methods App,1993,3(2):127-129.
[93]徐春兰,汪以真.半定量RT-PCR法分析猪肌肉组织细胞谷胱甘肽过氧化物酶mRNA表达水平[J].中国兽药杂志,2005,39(8):3-6.
[94]王荣,李红菊.半定量RT-PCR法在检测基因表达水平中的应用[J].阜阳师范学院学报.2007,3(24):49-52.
[95] Frenkel MJ,Powell BC,Ward KA. The keratin BIIIB gene family:isolation of cDNA clones and structure of a gene and a related pseudogene[J].Genomics.1989 Feb,4(2):182-91.
[96] Flanagan LM,et al. The high sulphur proteins of wool:Towards an understanding of sheep breed diversity[J].Proteomics,2002,2(9):1240-1246.
[2] Henry HM,Dodds KG,Wuliji T,et al. A genome screen for QTL for wool traits in aMerino×Romney backcross flock[J].Wool Technology and Sheep Breeding,1998.46:213-217.
[3] Ponz R,Moreno C,Allain D,et al. Assessement of genetic variation explained by markers for wool traits in sheep via a segment mapping approach[J].Mamm.Genome,2001,12:569-572.
[4] F.Bidinost. Quantitative trait loci related to merino sheep wool quality[C].8th World Congress on Genetics Applied to Livestock Production,2006,8:13-18.
[5] Vitezica ZG. Quantitative trait loci linked to PRNP gene controlling health and production traits in INRA 401 sheep[J].Genet.,2007,39(4):421-30.
[6]张明亚.凉山半细毛羊1号染色体遗传连锁图和羊毛性状QTL定位研究[D].四川:四川农业大学硕士学位论文,2005.
[7]马玉萍.绵羊主要经济性状的微卫星标记研究[D].石河子:石河子大学硕士学位论文,2003.
[8]徐新明.超细型细毛羊遗传多样性及羊毛性状的微卫星分析[D].新疆:新疆农业大学硕士学位论文,2007.
[9]沙莎.绵羊微卫星标记与部分毛用性状的关系研究[J].新疆农业职业技术学院学报,2006(3):49-53.
[10]赵宗胜,王根林,马玉萍.绵羊微卫星标记与部分毛用性状的关系研究[J].畜牧兽医学报,2006(9):28-33.
[11] McLaren RJ,Rogers GR,Davies KP,et al. Linkage mapping of wool keratin and keratin-associated protein genes in sheep[J].Mammalian Genome,1997,8:938-940.
[12] Powell BC,Rogers GE. Analysis of hair follicle proteins in:Keratinocyte Methods.Leigh I.Cambridge University Press,UK,1994:149-155.
[13] Rogers GR,Hickford JGH,Bickerstaffe R,et al. Polymorphism in two genes for B2 sulfur proteins of wool[J].Anim. Genet.,1994,25:407-415.
[14] Parsons YM,Piper LR,Cooper DW,et al. Linkage relation-ships between keratin-associated protein (KRTAP) genes and growth hormone in sheep[J].Genomics,1994,20(3):500-502.
[15] Plowman JE,Bryson WG,Jordan TW,et al. Application of proteomics for determining protein markers for wool quality traits[J].Electrophoresis,2000,5,21(9):1899-1906.
[16] Flanagan LM,Plowman JE,Bryson WG,et al. The high sulphur proteins of wool:Towards an understanding of sheep breed diversity[J].Proteomics,2002 Sep,2(9):1240-1246.
[17] Rufaut NW,Pearson AJ,Nixon AJ,et al. Identification of differentially expressed genes during a wool follicle growth cycle induced by prolactin[J].Invest.Dermatol,1999,113:865-872.
[18]刘桂芬.优质细毛羊羊毛细度的候选基因分析[J].遗传,2007,29(1):70-74.
[19]张亚妮,张恩平,吴迪. KAP基因的多态性与辽宁绒山羊经济性状的关系研究[J].中国农业科学,2007,40(9):2062-2067.
[20]张亚妮,张恩平,吴迪.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(05):33-37
[21] Parsons YM,Fleet MR,Cooper Des W,et al. Isolation of the ovine agouti coding sequence[J].Pigment Cell Res,1999,12:394-397.
[22] Smit MA,Shay TL,Beever JE,et al. Identification of an agouti-like locus in sheep[J].Anim.Genet.,2002,33:383-385.
[23] Sander GR,Powell BC. Structure and expression of the ovine Hoxc-13 gene[J].Gene,2004,2(18),327(1):107-116.
[24] Liang P,Pardee AB. Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction[J].Science,1992,257:967-971.
[25]迪芬巴赫,德维克斯勒著,黄培堂等译. PCR技术实验指南[M].北京:北京科学出版社,1999.
[26] Liang P,Averboukh L,Pardee AB,et al. Distribution and cloning of eukaryotic mRNAs by means of differential display:refinements and optimization[J].Nucleic Acids Res,1993,21:3269-3275.
[27] Liang P,Zhu WM,Zhang XY,et al. Differential display using one-base anchored oligo-dT primers[J].Nucleic Acids Res,1994,22:5763-5764.
[28] Ito T,Kito K,Adati N,et al. Fluorescent differential display:arbitrarily primed RT-PCR fingerprinting on an automated DNA sequencer.FEBS Lett,1994,351(2):231-236.
[29] GenHunter Corporation,RNA imageTM,kit for mRNA differential display,1996,April.
[30] Jones SW,Cai D,Weislow OS,et al. Generation of multiple mRNA fingerprints using fluorescence-based differential display and an automated DNA sequencer[J].Biotechniques,1997,22:536-543.
[31] Luehrsen KR,Marr LL,van der Knaap E,et al. Analysis of differential display RT-PCR products using fluorescent primers and GENESCANTM software[J].Biotechniques,1997,22:168-174.
[32] Smith NR,Aldersley M,Li A,et al. Automated differential display using a fluorescently labeled universal primer[J].Biotechniques,1997,23:274-279.
[33]陈东,高冬玲.非放射性同位素mRNA差异显示技术[M].国外医学:生理、病理科学与临床分册,2000,20:518-520.
[34] Weaver KR,Caetano-Anolles G,Gresshoff PM,et al. Isolation and cloning of DNA amplification products from silver-stained ployacrylamide gels[J].Biotechniques,1994,16:226-227.
[35] Doss RP. Differential display without radioactivity a modified procedure[J].Biotechniques,1996,21:408-412.
[36] Ponsuksili S,Wimmers K,Schmoll F,et al. Porcine ESTs detected by differential display representing possible candidates for the trait“eye muscle area”[J].Anim.Breed.Genet.,2000,117:25-35.
[37] Bhattacharjee A,Abrahamsen MS,Lappi VR,et al. Refinements in re-amplification and cloning of DDRT-PCR products[J].Biotechniques,1994,16:1096-1103.
[38] Liang P,Pardee AB. Differential display[J].Molecular Biotechnology,1998,10:261-267.
[39] Liang P,Bauer D,Averboukh L,et al. Analysis of altered gene expression by differential display[J].Methods in enaymology,1995,254:304-320.
[40] Sompayrac L,Jane S,Burn TC,et al. Overcoming limitations of the mRNA differential display technique[J].Nucleic Acids Res,1995,23:4738-4739.
[41] Guimaraes MJ,Lee F,Zlotnik A,et al. Differential display by PCR:novel findings and applications[J].Nucleic Acids Res,1995,23:1832-1833.
[42] Li ZK,Yao KT,Cao Y. Molecular cloning of a novel tissue-specific gene from human nasopharyngeal epithelium[J].Gene,1999,237:235-240.
[43] Clouscard-Martinato C,Mulsant P,Robic A,et al. Characterization of FSH-regulated genes isolated by mRNA differential display from pig ovarian granulose cells[J].Anim Genet.,1998,29:98-106.
[44] Robert C,Gagne D. Differential display and suppressive subtractive hybridization used to identify granulose cell messenger ma associated with bovine oocyte developmental competence[J].Biol Reprod,2001,64(6):1812-1820.
[45] Boardman PE. A comprehensive collection of chicken cDNAs.Curr Biol,2002,12(22):1965-1969.
[46] Schwerin M. Application of disease-associated differentially expressed genes-mining for functional candidate genes for mastitis resistance in cattle[J].Genet Sel Evol,2003,35 Suppl:19-34.
[47] Bertani GR. Evaluation of gene expression in pigs selected for enhanced reproduction using differential display PCR:Ⅱ.Anterior pituitary[J].Anim Sci,2004,82(1):32-40.
[48] Wesolowski SR. Developmental changes in the fetal pig transcriptome[J].Physiol Genomics,2004 Jan 15;16(2):268-74.
[49] M Clinton. Identification of disease markers by differential display:prion disease[J].Methods Mol Biol,January 1,2006,317:157-78.
[50] S.G.Roh. Identification of differentially expressed transcripts in bovine rumen and abomasum using a differential display method[J].Anim Sci,Feb 2007;85:395-403.
[51]俞英.鸡心肌和肝脏组织基因差异表达与杂种优势关系的研究[D].北京:中国农业大学博士学位论文,2003.
[52]李亮.乌骨鸡肝脏差异显示(DD)表达序列标签(ESTs)的筛选及分析鉴定[D].四川:四川农业大学博士学位论文,2004.
[53]任竹青.杂种和纯种猪之间脂肪组织差异表达基因的分离、克隆和序列分析[J].农业生物技术学报,2005,13(5):601-607.
[54]康文霞.高产与低产蛋鸡产蛋高峰期卵巢组织基因差异表达的研究[D].山东:山东农业大学硕士学位论文,2006.
[55]沙莎.绵羊毛囊差异表达基因的定为研究[D].石河子:石河子大学硕士学位论文,2006.
[56] Dalal SS,Welsh J,Tkachenko A,et al. Rapid isolation of tissue-specific and developmentally regulated brain cDNAs using RNA arbitrarily primed PCR(RAP-PCR)[J].Mol Neurosci,1994,5(2):93-104.
[57] Lamar EE,Palmer E.Y-encoded,species-specific DNA in mice:evidence that the Y chromosome exist in two polymorphic forms in inbred strains[J].Cell,1984,37(1):171-177.
[58] Diatchenko L,Lau YF,Campbell A P,et al. Suppression subtractive hybridization:a method for generating differentially regulated or tissue-specific cDNA probes and libraries[J].Proc Natl Acad Sci USA,1996,93(12):6025-6030.
[59]田连福,郭新红,姜孝成,等.抑制消减杂交技术(SSH)及其在植物基因克隆上的应用[J].生物学杂志,2002,19(4):29-31.
[60] Von Stein OD,Thies WG,Hofmann M. A high throughput screening for rarely transcribed differentially expressed genes[J].Nucleic Acids Res,1997,25(13):2598-2602.
[61] Lisitsyn NA,Rosenberg MV,Launer GA,et al. A method for isolation of sequences missing in one of two related genomes[J].Mol Gen Mikrobiol Virusol,1993,(3):26-29.
[62] Hubank M,Schatz DG. Identifying differences in mRNA expression by representational diference analysis of cDNA[J].Nucleic Acids Res,1994,22(25):5640-5648.
[63] Yuan BZ,Miller MJ,Keck CL,et al. Cloning,characterization and chromosomal localization of a gene frequently deleted in human liver cancer(DIC1)homologous to rat RhoGAP[J].Cancer Res,1998,58(10):2196-2199.
[64] Bachem CW,Vander Hoeven RS,de Bruijn SM,et al. Visualization of differential gene expression using a novel method of RNA finger-printing based on AFLP:analysis of gene expression duing potato tuber development[J].Plant,1996,9(5):745-753.
[65] Vos P,Hogers M,Bleeker M,et al. AFLP:a new technique for DNA fingerprinting[J].Nucleic Acids Res,1995,23:4407-4414.
[66] Bachem CW,Oomen RJFJ,Visser RGF. Transcript imaging with cDNA-AFLP:a step-by-step protocol[J].Plant Mol Biol,1998,16:157-173.
[67] Velculescu VE,Zhang L,Vogelstein B,et al. Serial analysis of gene expression[J].Science,1995,270(5235):484-287.
[68]张锦超,俞炜源.基因表达系列性分析技术及其应用[J].生物技术通讯,2002,13(5):395-399.
[69] Schena M,Shalon D,Davis RW,et al. Quantitative monitoring of gene expression patterns with a complementary DNA microarray[J].Science,1995,270(5235):467-470.
[70] Wang SM,Rowley JD. A strategy for genome-wide gene analysis:integrated procedure for gene identification[C].Proc Natl Acad Sci USA,1998,95(20):11909-11914.
[71]史春梦,粟永萍.全基因组基因表达频谱研究的新方法——SAGE和IPGI[J].生物工程进展,2001,21(2):65-67.
[72]贺三刚.利用cDNA-AFLP技术筛选与羊毛细度相关的分子标记[D].新疆:新疆农业大学硕士学位论文,2008.
[73] Zhang H,Zhang R,Liang P. Differential screening of gene expression difference enriched bydifferential display[J].Nucleic Acids Research,1996,24(12):2454-2455.
[74]刘红,任笑蒙,陈兰英.一种快速筛选阳性克隆的方法——反向Northern印迹杂交技术[J].基础医学与临床,2002,22(3)278-280.
[75]蔡树涛等. Nuclear Lamina(核纤层)——一种重要的细胞核结构[J].细胞生物学杂志,1990,12(l):15.
[76] Stick R. The Cytoskeleton.Greenwieh JAJ Press,1995,257-296.
[77] Moir RD,et al. The dynamic properties and possible functions of nuclear Lamins.Int Rev Cytol,1995,162B:141.
[78] Stuurman N,et al. Nuclear lamins:Their structure,assembly and interactions[J].Struct Biol,1998,122:42.
[79] Broers JL,Madicels BM,et al. A and B-type lamins are differentially expressed in normal human tissues[J].Histochem Cell Biol.1997,107:505-517.
[80] Duband-Goulet I,Courvalin JC. LBR,a chromatin and lamin binding protein from the inner nuclear membrane is proteolyzed at late stages of apoptosis[J].Cell Sei.1998,111:1441-1451.
[81]狄江,李勇,拉扎提,等.羊毛纤维直径与其他经济性状的相关性研究[J].中国草食动物,2004,5:44-46.
[82] Parry DAD,Steinert PM. IFs:moleeular,architecture,assembly,dynamics and polymer-phism[J].Q Rev Biophys,1999,32:99-187.
[83] Monteiro MJ,Hicks C,Gu L,et al. Determinants for intracellular sorting of cytoplasmic and nuclear intermediate filaments[J].Cell Biol,1994,127:1327-1343.
[84] Lee MK,Xu ZS,Wong PC,et al. Neurofilaments are obligate heteropolymers in vivo[J].Cell Biol,1993,122:1337-1350.
[85] Pamela DF,Chanjae Lee,Avegiyel Ellis,et al. Shroom2 (APXL) regulates melanosome biogenesis and localization in the retinal pigment epithelium[J].Development,2006,133,4109-4118.
[86] Ilaria Palmisano,Paola Bagnato,Angela Palmigiano,et al. The ocular albinism type 1 protein,an intracellular G protein-coupled receptor,regulates melanosome transport in pigment cells[J].Hum Mol Genet.2008 November 15,17(22):3487–3501.
[87] Mathias C,Martine B,Anne V C. A semi-quantitative RT-PCR method to readily compare expression levels within Botrytis cinerea multigenic families in vitro and in planta[J].Curr Genet,2003,43(1):303-309.
[88] Rolland V G. Semi-quantitative analysis of gene expression incultured chondrocytes by RT-PCR[J].Methods Mol Med,2004,100(1):69-78.
[89] Kuddus R H,Lee T H,Valdivea L A. A semi-quantitative PCR technique for detecting chimerism in hamster-to-rat bone marrow xenotransplantation[J]Immunol Methods,2004,285(1):245-251.
[90] Fellenr M D,Durand K,Correa M. A semi quantitative PCR method(SQ-PCR)to measure Epstein-Barr virus(EBV)load:its ap-plication in transplant patients[J].Biotechniques,1997,22(1):630-634,636.
[91]张翔,王寒正,龚岳亭,等. RT-PCR测定mRNA的荧光定量分析[J].生物化学与生物物理学报,1998,30(1):70-73.
[92] Chen D,Klebe RJ. Controls forvalidation ofrelative reverse tran-scription-polymerase chain reaction assays[J].PCR Methods App,1993,3(2):127-129.
[93]徐春兰,汪以真.半定量RT-PCR法分析猪肌肉组织细胞谷胱甘肽过氧化物酶mRNA表达水平[J].中国兽药杂志,2005,39(8):3-6.
[94]王荣,李红菊.半定量RT-PCR法在检测基因表达水平中的应用[J].阜阳师范学院学报.2007,3(24):49-52.
[95] Frenkel MJ,Powell BC,Ward KA. The keratin BIIIB gene family:isolation of cDNA clones and structure of a gene and a related pseudogene[J].Genomics.1989 Feb,4(2):182-91.
[96] Flanagan LM,et al. The high sulphur proteins of wool:Towards an understanding of sheep breed diversity[J].Proteomics,2002,2(9):1240-1246.