四环素与3,4-苯并芘复合污染对抗性基因tetA(C)产生高抗性突变的影响

Effect of tetracycline and benzo [a] pyrene combined pollution on the generation of resistance gene tetA(C) mutation

摘要:

[目的] 探究环境中同时存在低浓度四环素（tetracycline，TC）和3，4-苯并芘（benzo [a] pyrene，Bap）对抗性基因tetA（C）产生高抗性突变的影响。[方法] 以大肠杆菌（Escherichia coli，E.coli）为宿主菌株，pACYC184质粒作为载体，四环素抗性基因tetA（C）作为研究对象，采用易错PCR构建基因文库的方法，建立基因突变位点对应高抗性的关系密码表。同时设置添加低浓度TC且添加0-30 mg/L Bap以及仅添加0-30 mg/L Bap的处理组，培养携带pACYC184质粒的大肠杆菌14 d，每组中随机挑选10株获得高抗性的菌株，对其中的tetA（C）基因片段进行测序，再结合突变位点密码表，计算高抗性菌株中由基因突变产生高抗性菌株的比例。[结果] 测序结果显示在低浓度TC选择压力下，Bap浓度越高时，高抗性基因突变株占的比例也越高（P ≤ 0.01），而不添加TC时，Bap浓度与高抗性基因突变株占比之间无变化规律（P>0.05）。[结论] 当环境中同时存在Bap和低浓度TC时，高抗性突变基因易于通过选择压力保存下来。

关键词:四环素;3,4-苯并芘;抗性基因;基因突变;易错PCR

Abstract:

[Objective] Here, we explore the effect of the low concentration tetracycline (TC) and Benzo [a] pyrene (Bap) on the generation of high resistance gene tetA(C) mutations in the environment.[Methods] Escherichia coli, pACYC184 plasmid and tetracycline resistance gene tetA(C) was used as the host strain, the vector and the research object, respectively. Error-prone PCR was used to construct the gene library, the code table of high resistance mutation sites was established corresponding to high resistance mutants in the gene library. At the same time, the E. coli carrying pACYC184 were cultivated with 0-10 mg/L TC and 0-30 mg/L Bap 14 d. We randomly selected ten high resistance mutation from each group. The tetA(C) genes in these mutations were then sequenced. Sequencing results were compared with the code table of high resistance mutation sites, calculated the proportion of high resistance mutants caused by tetA(C) gene mutation in all high resistant strains.[Results] Sequencing results showed that under the selection pressure of low concentration TC, while the Bap concentration increases, the frequency of high resistance gene mutant increases (P ≤ 0.01). But without TC, there is no corelation between Bap concentration and the frequency of high resistance gene mutants (P>0.05).[Conclusion] When Bap and low concentration TC both in the environment, the high resistance mutants were easier survived by selection pressure.

Key words:tetracycline;benzo[a] pyrene;resistance gene;gene mutation;error-prone PCR