用磁珠富集法快速制备银鲫微卫星标记

Rapid isolation of microsatellites from genome of silver crucian carp by magnetic beads

将磁珠富集法和用γ-32P标记的放射性探针法相结合,快速制备银鲫Carassius auratus gibelio微卫星。用限制性内切酶MboI对银鲫完整基因组DNA酶切,用不同蔗糖浓度梯度离心收集400-900 bp大小的片段,连接Brown接头,构建银鲫全基因组文库。用生物素标记的（CA）16寡核苷酸探针进行筛选,磁珠富集含有微卫星的DNA单链序列;对DNA模板进行PCR扩增,连接pMD18-T载体,转入用CaCl2制备的感受态大肠杆菌Escherichia coliDH5α中,得到微卫星序列文库;经用同位素γ-32P标记的探针（CA）16二次筛选,获得235个阳性克隆,对其测序,得224个含微卫星序列,完美型、非完美型、复合型序列分别占总数的52.8%、28.7%、18.5%。除探针中使用的（CA/GT）n重复单元外,还观察到（GA/CT）n、（ACTC）4的重复序列。设计163对引物,选择合成30对引物,经聚丙烯酰胺凝胶筛选,9对可以获得清晰条带,其中3对为单态,6对为多态。

Nine microsatellite markers of silver crucian carp Carassius auratus gibelio were constructed by combination of biotin capture method with radioactive labeling hybridization. Genomic DNA was extracted by normal methods and digested with restriction enzyme MboI. The fragments of 400- 900 bp were isolated by sucrose - density - gradient centrifugation and ligated to Brown adaptors and hybridized with biotin -labeled simple sequence repeats （SSR） probes（CA） 16. The target fragments were ligated into plasmid cloning pMD18 - T vector and transformed into competent Escherichia coli after collection by magnetic beads and PCR amplification, DH5α using the CaCl2 method. A microsatellite genomic library containing 1800 colonies was constructed in which 235 positive colonies were dected by radioactive γ- 32p - ATP labeling hybridization. Sequencing of 235 positive colonies confirmed that there were 224 colonies containing microsatellite loci in which 36 were compound repeat motifs（ 18.5% ）, 117 were perfect（52.8% ） and 71 were imperfect（28.7% ） respectively. 163 pairs of PCR primers were designed with the software primer 5.0 and 30 pairs were synthesized and screened. Nine loci can give bands, 6 had a degree of polymorphisms, and 3 were monomorphic.