高原鼠兔微卫星富集文库构建与引物筛选.docVIP

硕士学位论文 高原鼠兔微卫星富集文库构建与引物筛选 指 导 教 师 申 请 学 位 级 别 学科专业名称 动物学 论 文 提 交 日 期 论文答辩日期 培 养 单 位 学 位 授 予 单 位  Dissertation of Master Degree, Northwest Institute of Plateau Biology, CAS Graduate School of the Chinese Academy of Sciences December, 2007, Xining, Qinghai, China Construction of enriched microsatellite libriaries of plateau pika and isolation of loci Kexin Li (Zoology) Directed By Professor Yanming Zhang 中文摘要 本研究根据生物素与链亲和素的强亲和性原理，用链霉亲和素包被的磁珠亲和捕捉与生物素标记的14种微卫星寡核苷酸探针退火结合的含有接头和高原鼠兔微卫星序列的单链DNA片段，获得单链目的片段，经PCR扩增形成双链，然后到pMD18-T载体上，转化至DH10B中，成功构建高原鼠兔基因组微卫星富集文库。测序结果发现，阳性克隆率为73.4%，说明构建的高原鼠兔基因组微卫星富集文库一高质量的文库。从构建的微卫星文库中挑取克隆提取质粒进行测序，设计出120对引物，经过PCR扩增和STR扫描，最终筛选出13对多态性的微卫星引物适合进行微卫星分析。在48个样本的13个微卫星位点上,等位基因的数目从6到16不等,等位基因的大小分布在147bp到302bp之间。对这些位点进行遗传学分析，发现这些微卫星的PIC值从0.598至0.870，说明这些位点的多态信息含量较高，达到了进行种群遗传学分析的要求。微卫星富集文库的建立以及引物的筛选将为高原鼠兔亲缘关系鉴定提供大量的微卫星标记。 Abstract The enriched micresatellite libraries of plateau pika were constructed and a suit of high polymorphic loci were isolated. The relationship among individuals within a family will be determined exactly using this marker suit. Furthermore, this will contribute to determine mating pattern, migration and dispurse and maintain of high genetic diversity. The microsatellite enriched library of plateau pika was constructed according to the strong affinity between biotin and streptavidin. First, genome DNA was cut up by using ultrasonicator which was innovation of our methods. Then the liker-ligated DNA fragments were annealed to biotinylated microsatellite oligo-probe, so microsatellite repeats can be captured by streptavidin-coated magnetic beads，PCR amplification was performed to obtain double-stranded targeted fragments and ligated into pMD18-T vector and electransformed into DH10B competent cell. The sequencing results show