鮸鱼微卫星标记的开发及筛选【毕业论文】.doc

本科毕业论文 （20 届） 鮸鱼微卫星标记的开发及筛选摘要 I Abstract II 引言 1 材料与方法 3 1.1 材料 3 1.1.1 仪器与设备 3 1.1.2 仪药品与试剂 4 1.2 实验方法 5 1.2.1 鮸鱼基因组DNA的提取 5 1.2.2 酶切 5 1.2.3 接头的制备与连接 5 1.2.4 鮸鱼基因组文库的建立 6 1.2.5 探针杂交和磁珠富集 6 1.2.6 富含微卫星序列的PCR产物及测序 6 1.2.7 微卫星PCR引物和电泳 7 1.2.8 聚丙烯酰胺凝胶银染 8 结果与分析 9 2.1 DNA提取结果 9 2.2 酶切及适宜基因组片段的获得 10 2.3 菌落PCR检测重组率 11 2.4 测序结果及其序列分析 13 讨论 15 3.1 测序结果及序列分析 15 3.2 目标产物显色法的选取 16 3.3 PCR引物的优点 17 小结 18 参考文献 19 致谢 21 摘要 [摘要] 为研究鮸鱼Miichthys miiuy）的群体遗传结构，本实验采用富集文库法构建鮸鱼的部分基因组文库，将鮸鱼基因组DNA用Mse酶切，回收纯化300bp-1000bp左右的片段，用生物素标记的简单重复序列作探针与其杂交，杂交复合物结合到有链霉亲和素包被的磁珠上，经一系列的洗涤，去除磁珠表面含微卫星的片段。将吸附在磁珠上的片段洗脱，PCR仪扩增放大，再进行克隆和测序，根据微卫星两端的保守序列设计引物，得到微卫星分子标记。根据自行分离得到的序列设计44对引物，对舟山地区的鮸鱼进行群体遗传多样性，其中12对引物扩增出清晰条带，多态性高的目的片段。鮸鱼微卫星研究工作提供更加详细和深入的信息，可以促进鮸鱼资源保护人工养殖的可持续发展。 [关键词] 微卫星；鮸鱼；种群多样性；多态性；分子标记Abstract [Abstract] Partial genomic library was constructed in order to study the genetic structure of Miichthys miiuy. In this experiment, we isolated microsatellite DNA from Miichthys miiuy genome with this method. Miichthys miiuy genomic DNA was extracted and digested with restriction enzyme MseI. Fragments over 300bp were isolated and purified and then ligated with short linkers. This genomic DNA was hybridized with a biotin-labeled microsatellite probe (CA) 13. The hybrid mixture was incubated with magnetic beads coated with streptavidin. After washing to remove the non-SSR fragments, the eluted single-stranded DNA contains the selected microsatellite DNA. From the primers designed for the 44 microsatellite loci, 12 could amplify expected PCR products. The developing and screening of microsatellites in Miichthys miiuy will provide more information for the research of heredity source analysis and heredity evolution, also benefits the future development of Miichthys miiuy protection and Miichthys miiuy artificial breeding to some extent. [Key words] Microsatellite; Miichthys miiuy; Population diversity; Polymorph; Molecular markers. 引言 鮸鱼Miichthys miiuy）属鲈形目（Perciformes）、石首鱼科（Sciaenidae）、鱼属（Miichth