摘要
通过磁珠富集的方法分离池蝶蚌(Hyriopsis Schlegelii)的微卫星分子标记。将池蝶蚌基因组DNA酶切,连接Brown接头,采用PCR技术富集,与探针杂交后杂交复合物结合到包被有链霉亲和素的磁珠上,再将这些片段洗脱,PCR扩增,克隆构建含有微卫星片段的"基因组文库",所得到的阳性克隆进行测序。从所获得的212个阳性克隆中选取110个进行测序,其中54.5%含有微卫星序列,80%的重复序列重复数在10以上,微卫星序列中21.4%为完美型。除探针中使用的AC、AG等重复单元外,还观察到CT、GA、ATG等重复序列。设计获得60对微卫星引物,合成其中的30对经PCR筛选,结果有10对引物扩增出多态性条带。该实验表明:磁珠富集法是获得池蝶蚌微卫星分子标记的一种有效的方法,同时,制备出的微卫星标记可为今后研究池蝶蚌分子遗传多样性提供有用的遗传标记。
Microsatellites molecular markers of Hyriopsis Schlegelii were obtained by using magnetic beads enrichment.The genomic DNA was firstly cut by Sau3AI,Then,the purified fragment was linked to Brown adapter.These fragments were hybridized with a biotin-labeled SRS(simple repeat sequenced)probe(AC)12.The hybrid mixture was incubated with magnetic beads coated with streptavidin.After washing to remove the non-SSR fragments,the eluted single-stranded DNA contained the selected microsatellites DNA.The target DNA were amplified by using primers according to the linkers.The PCR product was cloned into the pMD19-T vector and transformed into DH5α.In this study,110of 212positive clones were sequenced and 60clones were identified as microsatellites sequences.In microsatellites sequences,the repeated number of 80% sequences were above 10repeats and 21.4% sequences were perfect.In addition,30pairs of primers were designed according to microsatellites sequences and 10pairs were used to amplify special fragments.These results showed that magnetic beads enrichment was an efficient method to acquire microsatellites.In a word,the microsatellites which were obtained can offer useful genetic markers for studying the diversity of Hyriopsis Schlegelii in the future.
出处
《南昌大学学报（理科版）》
CAS
北大核心
2014年第3期252-257,共6页
Journal of Nanchang University(Natural Science)
基金
国家公益性行业科研专项资助项目(HY200903028)
江西省科技支撑基金资助项目(2009BNA07400
2010BNA07700)
江西省农业厅农业新技术示范与推广资助项目(赣农计字[2010]230号)
江西省科技落地计划资助项目(KJLD12001)
关键词
池蝶蚌
微卫星
磁珠富集法
Hyriopsis schlegelii
microsatellites
enrichment by magnetic beads
