【摘要】 采用磁珠富集法与放射性杂交相结合开发团头鲂(Megalobrama amblycephala)基因组微卫星资源。以团头鲂基因组DNA为材料,经Sau3AⅠ限制性内切酶消化后,选取400~900 bp的片段进行PCR全基因组扩增,并利用生物素标记的(CA)15探针进行微卫星片段的富集。将得到的片段与T载体连接后转入DH5α大肠杆菌中,然后利用γ-32P标记的放射性同位素探针进行第二轮杂交。结果表明,共获得微卫星基因组文库2 000个菌,杂交前菌落PCR检测阳性克隆率为50%;杂交后得到的阳性克隆为230个,占11.5%。从得到的230个阳性克隆中挑出120个进行测序,有94个克隆含有重复次数大于5次的微卫星序列,其中46个(48.94%)有随机侧翼区,可以设计引物;14个缺乏足够的侧翼序列。在得到的微卫星序列中,重复单元除CA/GT外,还观察到CT、AG、CG、CAA、CTCA等重复单元。在单一型标记中,完美型占53.15%,非完美型为37.84%;混合型标记占9.01%。另外,微卫星重复次数主要集中在5~30次,占75.68%。本研究旨在对团头鲂基因组资源的开发利用起到一定的促进作用,并为团头鲂养殖品系的优化、遗传多样性的检测及遗传图谱的构建等奠定基础。更多还原

【Abstract】 The CA DNA sequnces in Bluntnose black bream（Megalobrama amblycephala） were isolated through combining biotin capture method and radioactive labeling hybridization,and the characteristics of the microsatellites were analyzed.DNA was extracted from a Bluntnose black bream,and digested with Sau 3A Ⅰ,and 400-900bp fragments were selected and ligated to different adaptors（Brown and Whitney）.Then the purified ligated-DNA was hybridized with biotin-labeled simple sequence repeats（SSR）probes（CA）_15.After capture of target fragments by magnetic beads and PCR amplification,a microsatellite genomic library containing 2000 colonies was constructed.Two hundred and thirty positive colonies（11.5%） were obtained through twice screens by radioactive γ-³²P-ATP labeling hybridization.Sequencing of 120 positive colonies confirmed that 94 contained 111 microsatellite loci（number of repeats≥5）.Of the 94 sequences,only 10 contained compound repeat motifs（9.01%）,and of the remaining sequences,59 perfect（53.15%） and 42 imperfect（37.84%） repeat motifs were included.Fourty-six（48.94%） pairs of PCR primers were designed through software package,while the remaining sequences were not chosen because of their insufficient flanking sequences.Besides the motif of CA/GT contained in the colonies,there were also CT/AG,CG,CAA and CTCA repeat motifs.In addition,the microsatellites with repeat times at 5-30 were more abundant than others.This research makes a positive contribution to explore genomes of Bluntnose black bream,offer genetic tools to check the genetic variations and construct genetic linkage map.更多还原