利用cdnaaflp技术筛选与羊毛细度相关的分子标记-screening molecular markers related to wool fineness by cdnaaflp technology.docxVIP

摘要羊毛细度的改良一直是细毛羊育种的重要目标之一，新吉细毛羊是我国具有很好羊 毛品质的细毛羊，其主体为细型，部分个体达到了超细型，为了初步了解造成这种表型 差异的遗传本质，进而能筛选出超细型细毛羊的分子标记，本研究利用AFLP技术对超 细型细毛羊的基因组进行检测，利用cDNA．AFLP技术对羊毛细度极端个体(超细型和 细型)皮肤组织的mRNA进行了差异基因的比较分析，结果如下：1．经过大量摸索，建立了一套银染AFLP技术体系。采用Taq I和EcoR I双酶切， 利用6对引物对24只细毛羊基因组DNA进行分析，共获得226个AFLP标记，平均一 对引物获得的标记数在29～50之间，研究结果表明：AFLP技术适合检测绵羊的遗传多 态性，新吉超细型细毛羊相似系数AFLP研究结果为O．914--O．982。2．依据所建立的AFLP技术体系，共使用64对选择性引物组合对羊毛细度极端个 体皮肤组织的总RNA进行了差异显示研究，检测了近1200条cDNA带，发现了21条 差异片段，其中16条在超细型细毛羊的表达量比细毛羊上高，其它在细毛羊上的表达 量的比在超细型细毛羊上的高。成功克隆测序8条差异片段，得到的序列与Gerd3ank 中的序列进行同源性比对后发现其中4个ESTs在GenBank中无同源性，其他的ESTs 分别与羊的膜联蛋白的mRNA(annexin 2)，牛的不均一核蛋白(heterogeneous nuclearribonucleoprotein H2)的mRNA，羊的BIIIB4高硫角蛋白的mRNA，牛的钙蛋白酶抑制蛋 白(calpastatin)的mRNA高度同源。本研究有助于对超细型，细型细毛羊差异形成原因的分子基础的认识，为羊毛基因组的研究增加了新的资料。 关键词：超细型细毛羊，AFLP，cDNA．AFLP，基因表达ⅡIAbstractTo Improve wool fineness quality is always one of the important goals for fine sheep breeding．XinJi fine．wool sheep is which cail product good quality wool in China,the main types of wool are fine wool， other individuals are the super．fine w001．In order to research the genetic factors causing the difference between fine wool sheep and super fine wool sheep，AFLP techniques were used to analysis genomic DNA，eDNA．AFLP techniques were used to display the mRNAs expression status in skin between tworepresentative sheep group which were the fine wool sheep and super-fine wool sheep．The main results of this study were presented as following：1．The condition of related techniques was optimized by a series of experiment and the silver-staining AFLP experimental system WaS established．Six pairs AFLP primers were used，genomic DNA from 24 sheeps were restrictived by Taq I and EcoR I，226 AFLP markers were obtained，the average obtain markernumbers of individual primer were between 29～50．The results indicated as the following：AFLP marker issuitable for analysis genetic polymorphism in sheep；The similarity index of population in Xin Ji SuperfineWool Sheep WaS 0．9 14-4)．982．2．In this study，64 pair primers wer