藏羚羊(AAC)_n微卫星富集文库构建与引物筛选  

Construction of (AAC)_n Microsatellite enhanced library and Isolation of Microsatellite Markers in Tibetan Antelope

作　　者：徐永涛[1] 马建滨[1] 周智红[1] 谭春敏 张湑泽 郭松长[2] 都玉蓉[1] 

机构地区：[1]青海师范大学生命与地理科学学院,青海西宁810008 [2]中国科学院高原生物适应与进化重点实验室,青海西宁810001

出　　处：《青海师范大学学报（自然科学版）》2014年第1期41-46,共6页Journal of Qinghai Normal University(Natural Science Edition)

基　　金：青海省应用基础研究计划(2013-Z-750;2013-Z-751)

摘　　要：基于磁珠富集法，利用生物素标记的寡核苷酸探针（AAC）8从藏羚羊（Tibetan antelope）基因组酶切的300—1000bp片段中筛选微卫星位点，纯化的富集片段连接到pCR@2．1载体和转化到Trans5α感受态细胞．随机挑取180个白斑克隆，PCR检测到条带数大于或等于2的阳性克隆为56个，阳性率为31．1％．对阳性克隆测序，获得43条含微卫星座位的序列，微卫星重复次数在5～20次之间．舍弃不适宜设计引物的微卫星座位，试验设计并合成了20对微卫星引物；以3个样点（青海、新疆、西藏）的24个藏羚羊基因组为模板进行PCR扩增，获得8对可得到稳定扩增产物的微卫星引物，其中4对引物的扩增产物具有多态性，座位分别为AAC050、AAC056、AAC165和AAC477．上述引物可用于藏羚羊及近缘物种的遗传多样性、种群遗传结构等研究．Microsatellite-enhanced libraries of AAC-repeats in Tibetan Antelope were constructed using repeat-enrichment method with biotin-labeled oligos and streptavidin magnetic beads. The fragment of 300-1000bp were extracted using ristriction Mse-Ⅰ , cleaned products were ligated into a pCR@2.1 vector and transformed into Trans5αcompent ceils. 56 positive recombant clones with more than two bands were sequenced and got 43 microsatellite sequences ,the ratios of positive clones of （AAC） n microsatellite- enhanced libraries was 31.1%. The repeat time ranged from 5-20. There are 20 primers can be designed and got 4 high polymorphism primers（AAC050,AAC156,AAC165 ,AAC477） tested in 24 DNA samples using polyacrylamide gel electrophoresis among three sample sites containing Qinghai, Xinjiang, and Xizang. The results showed that the 4 microsatellite loci of Tibetan antelope are potential for further investigation of genetic diversity and population structure for tibetan antelope and its close species.

关 键 词：藏羚羊 微卫星 富集文库 (AAC)8探针 引物筛选 

分 类 号：Q149[生物学—生态学]