| Background: Plague is one of the most deadly diseases caused by Y.pestis. Until now, its pathogenic factors have not been thoroughly understood. Large quantities of genetic material flow occured during Y. pestis genome evolution.By the horizontal gene transfer, genetic recombination, frame shift and point mutations, Y. pestis genome has acquired some large genetic fragments and plasmids, evolved many pseudogenes and undergone chromosomal rearrangements, resulting in the disparities of genomic structures and functions. The technique for genomic library construction can help to purify and store the genomic genes for further research. Bacterial artificial chromosome (BAC) library is a technique used widely in many fields of genome researches, including organism evolution, gene structure analysis, cloning of specific chromosomal genes, genome organization and gene expression, and construction of the genomic physical map and genome sequencing.Methods: The overnight cultures of Y. pestis 910706 was mixed with low-melting agarose gel for preparing plugs, which were then used for DNA purification and partial digestion with different restriction enzymes. Pulsed-field gel electrophoresis (PFGE) demonstrated that HindIII was the suitable enzyme for the BAC library construction. DNA fragments ranging from 50-300kb were collected and ligated to the vector pCC1BAC, and then transformed into E.coli EPI300. Terminal sequencing was employed to verify the source of the clones and evaluate the coverage. The plasimds extracted from different subcultures were digested by HindIII for stability's evaluation of the BAC clones. The average insert size and the ratio of the empty clones in the BAC library were assessed by PFGE.Results: The Y. pestis BAC library contains about 3248 clones with average insert size of of c.a. 105kb and empty clones of 2%. The inserts of BAC clones showed homologous with Y. pestis and good stability after subcultured for 100 generations. Based on the data mentioned above, the capacity of this BAC library is about 35Mb, 8 times of Yesinia pestis genome, with the genomic coverage of about 97%. Conclusions:The BAC library of Y. pestis 910706 was successfully constructed, laying the solid foundation for future research on functions of large chromosomal fragments of this pathogen. |
