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： 《中国水产科学》 2006年第2期187-192,共6页

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Sau 3AⅠ限制性内切酶消化后，选取400～900bp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2000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，完美础占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。 The CA DNA sequnces in Bluntnose black bream （ Megalobrama arnblycephala ） 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 brearn, and digested with Sau 3A Ⅰ ,and 400- 900 bp 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 2 000 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.

关 键 词： 团头鲂 微卫星 磁珠富集

领　　域： [生物学]