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王洪哲, 殷倩茜, 冯志纲, 李大宇, 孙效文, 李 婵. 2008: 黑斑狗鱼部分基因组文库构建和微卫星位点的筛选(英文). 动物学研究, 29(3): 245-252. DOI: 10.3724/SP.J.1141.2008.03245
引用本文: 王洪哲, 殷倩茜, 冯志纲, 李大宇, 孙效文, 李 婵. 2008: 黑斑狗鱼部分基因组文库构建和微卫星位点的筛选(英文). 动物学研究, 29(3): 245-252. DOI: 10.3724/SP.J.1141.2008.03245
2008: Construction Fractional Genomic Libraries and Screening Microsatellites DNA of Esox reieherti Dybowski. Zoological Research, 29(3): 245-252. DOI: 10.3724/SP.J.1141.2008.03245
Citation: 2008: Construction Fractional Genomic Libraries and Screening Microsatellites DNA of Esox reieherti Dybowski. Zoological Research, 29(3): 245-252. DOI: 10.3724/SP.J.1141.2008.03245

黑斑狗鱼部分基因组文库构建和微卫星位点的筛选(英文)

Construction Fractional Genomic Libraries and Screening Microsatellites DNA of Esox reieherti Dybowski

  • 摘要: 采用磁珠富集与放射性杂交相结合的方法开发黑斑狗鱼(Esox reieherti Dybowski)基因组微卫星资源。基因组DNA 经Sau 3AⅠ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)12、(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-32 P标记的放射性同位素探针进行第二次杂交。结果,共获得微卫星基因组文库1 600个菌,杂交前菌落PCR检测阳性克隆率为90.91%;杂交后得到的阳性克隆为1 300个,占87.25%。从中挑出196个进行测序,192(97.96%)个含有微卫星序列。在得到的微卫星序列中,重复单元除CA/GT、GA/CT 外,还观察到单碱基、四碱基、五碱基重复单元。根据侧翼序列应用引物设计软件Primer Premier 5.0设计引物70对,选择合成32对,通过优化PCR反应条件,结果有28对引物可扩增出清晰可重复的目的条带。本研究旨在对黑斑狗鱼基因组资源的开发利用起到一定的促进作用,并为黑斑狗鱼养殖品系的优化、遗传多样性的检测及遗传图谱的构建等奠定基础。

     

    Abstract: Esox reieherti Dybowsk genomic microsatellites were developed by using enrichment protocols combined with radioactive hybridization protocol. Four hundred to nine hundred base pair fragments were selected for the whole genome. DNA PCR amplification after digestion with restriction endonuclease Sau 3AⅠ, and (CA)12, (GA)12 probes marked with biotin were used for microsatellite DNA enrichment. The product fragments were connected with carrier pGEM-T and transferred into DH5α Escherichia coli competent cells, and radioactive isotope probes marked with γ-32 P were used for the second hybridization. As a result, a total of 1600 bacteria were obtained in the microsatellite genomic libraries, positive clones accounted for 90.91% before hybridization and 81.25% after hybridization, amounting to 1300. One hundred and ninety-six positive clones were selected for sequencing, and 192 clones included microsatellite sequences. The microsatellite sequences obtained, mono-nucleotide, quad-nucleotide and quint-nucleotide repeat motifs were observed beside double-base-pairs CA/GT, GA/CT. Seventy primers were designed according to the flanking sequences by using software Primer Premier 5.0, and 32 primers were selected to be synthesized. After optimizing PCR reaction conditions, 28 primers were amplified and produced clear purpose bands. The aim of our research was to promote the development and utilization of E. reieherti genomic resource, and lay the foundation for optimizing E. reieherti breeding strain in order to detect the genetic diversity and construct a genetic map.

     

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