【作者】 苑洁；

【导师】 徐艳春；

【作者基本信息】 东北林业大学 ， 动物学， 2005， 硕士

【摘要】 微卫星DNA在研究遗传多样性分析、基因组作图、QTL定位、疾病诊断与病理研究、亲缘关系鉴定及个体识别等方面发挥着巨大的作用。梅花鹿是重要的经济动物,也是备受关注的濒危物种。微卫星对于梅花鹿的保护遗传学、QTL定位、种群遗传管理等都非常重要。而目前对梅花鹿的微卫星分离的研究很少。本文以梅花鹿基因组DNA样品为材料,经Mbo Ⅰ酶切并电泳后回收1000bp～400bpDNA片段,一部分回收产物用于构建部分基因组文库,另一部分经过与生物素标记的（CA）₁₅探针杂交,再用磁珠(Dynabeads^? M-280 Streptavidin)富集和PCR扩增后,构建了微卫星富集文库。用（CA）₁₅、（TTC）₁₅等4种探针分别与富集文库和未富集的短片段部分基因组文库进行杂交。并随机挑选58个阳性克隆进行测序。从序列结果中发现,含有微卫星28个,目的克隆比例为43.75%。微卫星种类以与探针（CA）₁₅互补的（CA/GT）_n为主,同时还存在（CT/GA）_n、（ACTGA）_n等为重复单元的微卫星。利用Primer premier5.0和DNAstar软件对19个微卫星位点设计了PCR引物,并建立了PCR体系和程序。10个位点成功扩增,对各个位点等位基因数目、等位基因片段大小范围、等位基因频率分布、观察杂合度等指标的初步评价表明,7个具有良好的多态性。这些微卫星经过系统的遗传特征测试后,可以成为梅花鹿多方面研究的工具。更多还原

【Abstract】 Microsatellite DNAs play an important role in population genetic diversity analysis, genomic mapping, QTL allocation, disease clinics and pathology, paternity testing, individualization etc. The sika deer is an important economic species and endangered species. Microsatellites are of great significance in the conservation genetics, QTL allocation, and population management etc. of the sika deer. However, there have been very few studies on the microsatellite isolation from sika deer genome. This paper is filling this blank. Genomic DNA was digested with Mbo I, and isolated through electrophoresis of 1.5% agarose gel. Fragments of 1000bp⁴00bp was recovered and purified. A part of the recovered DNA was used to construct a partial genomic library, and the other part was hybridized with （CA） ₁₅ probe labeled with Biotin. A microsatellite enriched library was then constructed after an enrichment process using magnetic bead (Dynabeads^? M-280 Streptavidin) and PCR amplifications. Several oligo probes namely （CA） ₁₅, （TTC） ₁₅ etc. were applied to detect the microsatellite bearing clones from the two libraries. 58 positive clones were randomly picked up and subjected to sequencing. Sequences revealed 28 microsatellites being isolated, occupying 43.75% of the positive clones. Major types of motif of microsatellites isolated were included. Software Primer premier 5.0 and DNAstar were used to design PCR primers for 19 microsatellites, and corresponding PCR system and cycling programs were also established. 10 loci were effectively amplified. The primary assessment of them using parameters such as number and size range of alleles, allelic frequency, observed heterozygosity indicated that 7 loci were polymorphic, and potentially applicable to sika deer genetic analysis as powerful tools after systemaitic genetic characterization.更多还原