兰州鲇(CAG)_n、(GATA)_n微卫星文库构建与鉴定  

Construction and identification of (CAG)_n and (GATA)_n microsatellites from the Silurus lanzhouensis genome

作　　者：魏大为[1,2,3] 连总强[1,2,3] 吴旭东[1,2,3] 张利平[1] 肖伟 赛清云 王发新 

机构地区：[1]甘肃农业大学动物科学技术学院,兰州730070 [2]宁夏回族自治区水产研究所,银川750001 [3]宁夏渔业工程技术研究中心,银川750001

出　　处：《淡水渔业》2014年第5期15-20,共6页Freshwater Fisheries

基　　金：国家自然科学基金项目(31360633);国家科技支撑计划项目(2012BAD25B09)

摘　　要：应用磁珠富集法构建兰州鲇（ Silurus lanzhouensis） CAG重复和GATA重复的微卫星文库，并分析其序列特征。兰州鲇基因组DNA经MseI酶切，选取200～800 bp的片段与生物素标记的探针（CAG）8和（GATA）6杂交，捕获到含有微卫星序列的目的DNA片段连接到pMD19-T载体，转化到大肠杆菌DH5α菌株中构建微卫星富集文库，经PCR检测筛选出阳性克隆进行测序。从126个阳性克隆中随机选取96个进行测序，获得59个微卫星序列（ GenBank登录号： KJ545973～KJ545998， KJ598088～KJ598120）。其中完美型31个（52.54％）、非完美型20个（33.9％）、混合型为8个（13.56％）。根据侧翼序列，成功设计48对引物，选取25对微卫星引物在10个个体进行扩增与多态性筛选，共获得10对多态性引物。结果表明，经优化的磁珠富集法能够高效地获得兰州鲇微卫星标记，这些标记将为兰州鲇种质资源保护、微卫星连锁图谱构建、经济性状的QTL定位及分子标记辅助选育奠定基础。A （ CAG） n and （ GATA） n microsatellite enriched library from Silurus lanzhouensis was constructed by the mag-netic beads enriched method .S.lanzhouensis genomic DNA was extracted with restriction enzyme MseI digestion.DNA fragments from 200 to 800 bp were collected and hybridized with biotinylated oligoprobes （ CAG） 8 and （ GATA） 6 .The se-lected DNA fragments were cloned into the pMD 19-T vector and transformed into competent Escherichia coli DH5αcells. 126 positive colonies were obtained through twice screens by colony PCR , 59 of which contained microsatellites （ GenBank Accession Number： KJ545973-KJ545998 , KJ598088-KJ598120 ） .According to Weber′s classification rules , these mic-rosatellites were divided into three categories： perfect type （52.54%）, imperfect repeat type （33.9%）, and compound repeat type （13.56%）.For each unique sequences of flanking motif , we designed 48 pairs of SSR-primers and synthe-sized 25 pairs among 10 individuals , 10 polymorphic microsatellite markers were screened out .The results indicated that magnetic bead enrichment was a good method for enhancing the efficiency of isolating microsatellite DNAs from S.lanzhouensis.These markers would offer solid foundation for S.lanzhouensis resources conservation and microsatellite ge-netic linkage mapping construction .Besides, they will also be helpful for marker-assisted selection and quantitative trait loci location analysis of S.lanzhouensis.

关 键 词：兰州鲇(Silurus lanzhouensis) 磁珠富集 微卫星 

分 类 号：S917.4[农业科学—水产科学]