作　　者： ; ; ; ; ; ; ; ;

机构地区： 中国水产科学研究院黑龙江水产研究所

出　　处： 《上海海洋大学学报》 2009年第2期129-135,共7页

摘　　要： 利用磁珠富集和放射性杂交相结合构建了中华绒螯蟹基因组微卫星文库。并利用生物素标记的(GA)12探针进行微卫星片段的富集。将得到的片段与pGEM-T载体连接后转入DH5α大肠杆菌中,然后利用γ-32P同位素标记的(GA)12探针进行第二次筛选。结果共获得微卫星基因组文库1 500个菌,杂交前菌落PCR检测阳性克隆率为62.5(;杂交后得到的阳性克隆为447个,占29.8(。经测序,有248个克隆含有重复次数大于5次的微卫星序列。在得到的微卫星序列中,重复单元除GA/CT外,还观察到三碱基、四碱基重复单元。根据侧翼序列设计引物164对,选择合成50对,通过优化PCR反应条件,结果有21对引物可扩增出清晰可重复的目的条带,15对具有多态性。本研究对中华绒螯蟹基因组资源的开发利用提供了相关资料。 In this experiment, microsatellite DNAs were isolated from Chinese mitten crab （Eriocheir sinensis） genome by combining biotin capture method and radioactive labeling hybridization. The purified ligated-DNA was hybridized with biotin-labeled simple sequence repeats （SSR） probes （GA）12, and the mixture was incubated with streptavidin-coated magnetic beads. The selected single-strand microsatellite DNA was obtained by removing the SSR-uncombined fragments. After capture of target fragments by magnetic beads and PCR amplification, the PCR products were ligated to T vector and transformed to E. coli competent ceils. A microsatellite genomic library 1 500 colonies was constructed. 447 positive colonies （29.8%） were obtained through twice screens by radioactive γ^-32 P labeling hybridization. Sequencing of 447 positive colonies confirmed that 248 contained 266 microsatellite loci. Besides the motif of GA/CT contained in the colonies, there were also CCT, TCC and AAGG, Trcc repeat motifs. 164 pairs of primers were designed with the software primer premier 3.0. In addition, 50 pairs of primers were composed and screened. 21 pairs were used successfully to amplify special fragments, among which 15 pairs were polymorphism within species. This research offers relevant data to explore genomes of Chinese mitten crab Eriocheir sinensis.

关 键 词： 中华绒螯蟹 微卫星 磁珠富集

领　　域： [农业科学]