热处理重组质粒DNA的复性及其在水环境中的降解特性

Renaturation and degradation of thermo-treated waste plasmid DNA in an aquatic-environment

为了考察热处理后的重组DNA进入水环境后可能存在的环境风险,以pET-28b质粒为材料,以质粒相对转化效率为指标,考察了热变性重组质粒DNA的复性可能性,并在此基础上构建了人工模拟水环境系统,研究了热变性质粒DNA在水环境中的降解速率和影响因素.结果表明,热变性pET-28b质粒经过30min后,其转移活性可以得到恢复,在4～37℃之间,温度越高越有利于热变性质粒的复性.热处理过程中未被降解的质粒DNA进入水环境后,在pH值为7、8时降解速率相对较慢,在pH值为5、6、9的条件下,降解较快,但在任何pH下,1.0h后仍存在未降解的质粒DNA.水环境中的NaCl对热处理质粒DNA有一定的保护作用,而且这种作用是随着NaCl质量分数的提高而增强.进入水环境中的热变性DNA有足够的时间复性,从而可能发生基因转移.因此,这些热处理的质粒DNA进入环境后理论上存在一定的生态风险.

Plasmid pET-28b was used as a test plasmid to investigate the potential environmental risk caused by thermo-treated recombinant DNA. Firstly, plasmid DNA transformation was performed to evaluate the re-naturation efficiency of the thermo-treated plasmid DNA, and then simulated aquatic environments were constructed to determine the persistence of un-decayed plasmid DNAs in the aquatic environment after thermo-treatment. The results showed that the transforming activity of the thermo-treated plasmid DNA could be recovered to a certain extent after re-naturing for 30 min. From 4 - 37℃, the higher the temperature, the more transforming activity of denatured plasmid DNA will be recovered. When thermo-treated plasmid DNAs were discharged into simulated aquatic environments with pH from 5 - 9, the persistence of un-decayed plasmid DNAs at pH 7 and 8 exceeded those at 5, 6 or 9, but at any pH some could persist for 1.0 h or more. Higher ionic strengths further inhibited decay of the recombinant plasmid in the simulated aquatic environment. These results indicate that recombinant DNAs which are not destroyed during the thermo-treatment process cannot be degraded completely in a short time in the aquatic environment, which means when they are discharged into aquatic environment, they may have enough time to re-nature and transform, thus resulting in gene diffusion.