【作者】 劳荃蘅；

【导师】 田可川； 黄锡霞；

【作者基本信息】 新疆农业大学 ， 动物遗传育种与繁殖， 2009， 硕士

【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。更多还原

【Abstract】 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.更多还原