转座因子Tn917随机诱变变形链球菌UA159

Tn917 Insertional Mutagenesis of Multiple Genes on the Chromosome of Streptococcus Mutans UA159

目的 :诱导转座因子Tn917随机插入变形链球菌UA15 9染色体 ,产生在不同位点突变的突变株库。方法 :用含转座因子Tn917的质粒pTV1 OK转化变形链球菌UA15 9,诱导转座因子发生转座后 ,产生大量的突变株。随机挑选 6株突变株 ,提取基因组DNA ,EcoRⅠ酶切 ,以质粒pTV1 OK的 4 .3kbKpnⅠ片段 (含Tn917部分序列 )为探针 ,做Southern杂交 ,以野生株为对照。结果 :野生株无杂交带 ,突变株均有且只有一条杂交带 ,且杂交带的位置不尽相同。结论 :转座因子Tn917可以单拷贝随机诱变变形链球菌野生株 ,产生在不同位点突变的突变株 ,是从基因水平研究变形链球菌的有效手段。

Objective: To generate mutant pools of Streptococcus mutans(S.mutans) UA159 by Tn917-mediated insertional mutagenesis. Methods: S.mutans UA159 was transformed with temperature-sensitive plasmid pTV1-OK that harbors Tn917. The saturated culture of one transformant was diluted with fresh medium and incubated at the higher restrictive temperature in order to induce the transposition of Tn917. Souhtern blot analysis was performed with EcoRⅠ(no cut within Tn917)digests in wild S.mutans UA159 and 6 randomly chosen transposants genomic DNA,with DIG-labeled probe of 4.3 kb KpnⅠ fragment of pTV1-OK containing Tn917. Results: The probe hybridized to one EcoRⅠfragment of each transposant,although each hybridization fragment had different molecular size. The probe did not hybridize to the wild S.mutans UA159 DNA. Conclusion:Transposon Tn917 mutagenesis results in single chromosome insertions with apparent randomness in S.mutans UA159.