牛伟萍-梅花鹿10.9.28.docVIP

梅花鹿微卫星DNA富集文库的构建与鉴定 牛伟萍 张丽颖 杨润军 张嘉保 赵志辉* 张明军* （吉林大学畜 牧兽医学院，吉林 长春 130062） 中图分类号：S603.7 文献标识码：A 摘要：梅花鹿基因组DNA经限制性内切酶MboI酶切，用1.5%的琼脂糖凝胶电泳回收400~1 000 bp的DNA片段，与MboI连接接头相连接，连接产物与生物素标记的（AC）15微卫星探针变性及退火，再通过链亲和素偶联磁珠亲和捕捉，经吸附，洗涤及洗脱，然后以洗脱产物为模版，以其中一个接头为引物进行 PCR扩增，将扩增产物回收纯化并与pMD18-T载体相连接，转化大肠杆菌DH5a，构建梅花鹿 (AC)n 微卫星DNA富集文库，经测序分析表明该文库含重组克隆约为1 380个，其中计算得到阳性克隆率为82.9%。梅花鹿微卫星文库的建立将为一步进行梅花鹿基因组结构的分析、梅花鹿遗传连锁图谱的构建、分子进化和系统发育研究、标记辅助选择以及经济性状的QTL定位提供大量的微卫星标记。 关键词：梅花鹿；微卫星；遗传标记 Construction and identification of DNA libraries enriched for microsatellite repeat sequences in sika deer （NIU Wei-ping, ZHANG Li-ying, YANG Runjun, ZHANG Jia-bao, ZHAO Zhihui*，ZHANG Ming-jun*） （College of Animal Science and Veterinary Medicine, Jilin University, Changchun 130062,China） Abstract: Genomic DNAs from sika deer were cleaved with restriction enzymesMboI. The fragments in size from 400 bp to 1 000 bp were recovered by 1.5% agarose gel electrophoresis and ligated with adaptor, and then denatured and hybridized biotinylated (AC)n oligonucleotide. The biotinylated hybrids were retained on the magnetic particles according to the strong affinity between biotin and strep tavidin. The eluate was amplified by PCR and cloned into pMD18-T plasmid vector, and then transformed into Escherichia Ecoli DH5αto construct DNA libraries enriched for microsatellite repeat sequences of sika deer. Sequence analysis shows that the library contains 1 380 clones and 82.9% of the clones with positive. Microsatellite library of deer will step to the analysis of genome structure deer, sika deer genetic linkage mapping, molecular evolution and phylogenetic studies, marker-assisted selection and QTL localization economic traits to provide a large number of microsatellite markers. Key Words: sika deer; microsatellite; genetic markers 梅花鹿为东亚特有的珍贵动物，历史上我国梅花鹿资源非常丰富，分布范围也极为广泛，但由于自然因素及人为因素的影响，中国梅花鹿6个亚种中仅存3个亚种：四川亚种， 华南亚种和东北亚种，其分布范围也大为缩减，种群数量急剧减少，已处于濒危的状态[1-2]。微卫星（Microsatellite）又称简单序列重复（SSR），一般以1~6个碱基为核心序列，首尾相 连组成的串联重复序列。这种序列存在于几乎所有真核生物的基因组中，且成