高原鼠兔微卫星富集文库构建与引物筛选.doc

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
本研究采用分子生物学技术构建了高原鼠兔微卫星富集文库,并筛选出一套多态性较高的微卫星标记。该标记可对高原鼠兔个体间进行准确的身份鉴定,进而,为探讨高原鼠兔婚配制度、迁移扩散以及种群遗传多样性保持机制奠定了基础。
根据生物素与链霉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DN***段,获得单链目的片段,经PCR扩增形成双链,然后连接到pMD18-T载体上,转化至高效感受态细胞DH10B中,成功构建出高原鼠兔基因组微卫星富集文库。测序结果发现,%,说明构建的高原鼠兔基因组微卫星富集文库为一高质量的文库。从构建的微卫星文库中挑取克隆提取质粒进行测序,根据其侧翼的保守序列设计出120对引物,经过PCR扩增和STR扫描,最终筛选出13对多态性好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,,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和基因流动模式研究提供大量的微卫星标记。
关键词:高原鼠兔;微卫星;富集文库;多态性;等位基因
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 ic 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 ic beads,PCR amplification was performed to obtain double-stranded targeted fragments and ligated into pMD18-T vector and electransformed into petent cell. The sequencing results showed that % of clones contained microsatellite indicating a high degr