WP2深海细菌基因组文库的构建和克隆子测序比对分析

The Genomic Library Construction and Clone Sequence BLAST of Deep-sea Bacterium WP2

以pUC19质粒DNA作为载体，用BamH1酶切、碱性磷酸酶处理后，分别与Sau3A Ⅰ部分酶切的Shewanella sp．（WP2）基因组DNA 2-6kb的DNA片段连接，电击转化大肠杆菌DH5 α感受态，在含有Amp的LB平板上筛选重组子，得到转化子约5～6×10^3；随机挑取100个克隆子，经DNA测序，通过在网络数据库中进行BLASTX分析，得到一系列相对保守蛋白的比对结果．这些结果为遗传背景不清楚的细菌的研究，提供了一条从分子水平上探讨的技术路线．

A genomic library of a deep-sea bacterium was constructed by using pUC19 plasmid as vector. PUC19 was digested by BamH1 enzyme, treated with CIP, and then ligated with 2-6 Kb fragments of Shewanella sp. （WP2） digested by Sau3A 1. These ligation mixtures were electransformed to E. coli DH5 α. The recombinants were screened on the LB plate with Amp and 5～6×10^3 recombinants were obtained. Then 100 positive clones were randomly selected and then sequenced. The sequences were searched in NCBI using BLASTX tool and the homology of 49 sequences were obtained for further study. Our results suggest that our strategy may be an approach for study of a bacterium without genetic knowledge at molecular level.