半滑舌鳎雌鱼基因组Fosmid文库构建及分析

Construction of a Fosmid Library of Female Half Smooth Tongue Sole and Its Preliminary Characterization

半滑舌鳎（Cynoglossus semilaevis）是我国的大型经济鱼类,其养殖发展迅速。有关半滑舌鳎的基因组文库构建和分析等方面的研究,目前尚未见报道。用流式细胞仪检测雌性半滑舌鳎基因组大小。从半滑舌鳎雌鱼肌肉提取基因组DNA,选取大小合适的DNA片段,经末端修复并回收后,再以Fosmid作为载体,构建半滑舌鳎雌鱼基因组文库。经检测,雌性半滑舌鳎基因组大小约为606.36 Mb。所构建的Fosmid文库含有49 920个克隆,重组率为96.88%;插入片段长度分布在33-45 kb之间,平均为39.2 kb;覆盖雌性半滑舌鳎基因组3.12倍;从文库中筛选得到单拷贝DNA序列的概率为95.6%。对培养第1天和第6天的克隆进行比较,结果表明文库稳定性高,培养过程中没有发生变化。

Half smooth tongue sole（Cynoglossus semilaevis） is an important marine flatfish especially in north China where the cultivation has been developed quickly in recent years.The DNA content of female half smooth tongue sole blood cell was（0.620±0.044） pg as was determined with flow cytometry method,and its genome size was deduced as about 606.36 Mb.The genomic DNA prepared from muscle with phenol-chloroform extract method was end-repaired with the fragment longer than 36 kb recovered using GelaseTM.The recovered fragments were inserted into fosmid vector,packaged and transferred into host bacterium,creating a library containing 49 920 clones with an insert size of 39.2 kb in average.The library covers the haploid genome of female half tonge sole 3.12 folds.About 96.88% of the clones contained the DNA fragment.Any single copy fragment can be selected out from the library at a possibility of 95.6%.