动物学研究2008，Vol.29Issue(3)：245-252,8.
黑斑狗鱼部分基因组文库构建和微卫星位点的筛选
Construction Fractional Genomic Libraries and Screening Microsatellites DNA of Esox reieherti Dybowski
摘要
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 3A Ⅰ, 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 DHSα 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 fur optimizing E. reieherti breeding strain in order to detect the genetic diversity and construct a genetic map.关键词
黑斑狗鱼/微卫星/磁珠富集Key words
Esox reieheri Dybowsk/ Microsatellite DNA/ Magnetic beads enrichment protocol分类
生物科学引用本文复制引用
王洪哲,殷倩茜,冯志纲,李大宇,孙效文,李婵..黑斑狗鱼部分基因组文库构建和微卫星位点的筛选[J].动物学研究,2008,29(3):245-252,8.基金项目
国家科技基础条件平台建设(2005DKA30470-005)Supported by Platform Construction of National Science and Technology Basic Research. (2005DKA30470-005) （2005DKA30470-005）
