应用转LUX报告基因重组发光菌监测评价DNA损伤毒性污染物

Evaluation of DNA Damage Toxic Pollutants by Recombinant Light Emitting Bacteria Expressing LUX Reporter Gene

为研究对DNA损伤污染物MNNG、MMC特异性响应作用,构建2株分别含有PUCD-uvr A、PUCD-alk A载体的重组发光菌THSH1711及THSH1712。uvr A、alk A PCR产物经双酶切后与PUCD615载体连接,测序结果表明,基因已正确插入到PUCD615的多克隆位点,方向和读码框正确,重组载体构建成功。THSH1711及THSH1712均对MNNG及MMC有特异响应作用。2菌株对MNNG的响应起始浓度均为5 mg/L,最佳反应浓度为50 mg/L,在3.5~5.5 h达到反应高峰,超过该浓度则反应效应下降。THSH1711对MMC的响应起始浓度为0.1 mg/L,最佳反应浓度为5 mg/L,在作用3 h后达到反应高峰。相比之下,THSH1712对MMC的反应较弱,需MMC浓度达到10 mg/L以上才有反应。构建重组发光菌用于水质等毒性污染物检测,具有快速、经济、稳定可靠等优点,能实现连续在线及自动化检测,达到早起预警目的,发展前景广阔。

In order to research the damage of DNA pollutants MNNG,MMC specific response,2 strains of recombinant bioluminescent bacteria THSH1711 and THSH1712 were constructed containing PUCD-uvr A and PUCD-alk A. The PCR products were digested by restriction enzymes and ligated with PUCD615 vector. The sequencing results revealed that the gene had been correctly inserted into the multiple cloning sites of PUCD615,the direction and the reading frame was correct,the recombinant vector was successfully constructed. Both THSH1711 and THSH1712 had specific response to MNNG and MMC. The initial response concentration of MNNG for the two strains was 5 mg/L,the optimum concentration of 50 mg/L reached the peak at 3.5 h to 5.5 h. The reaction effect decreased when the concentration was higher. The initial response concentration of MMC for THSH1711 was 0.1 mg/L,and the optimum reaction concentration was 5 mg/L,which reached the peak after 3 h. On the other hand,The reaction of THSH1712 to MMC was weaker,and the reaction could be achieved only when the concentration of MMC reached above 10 mg/L. The construction of recombinant luminous bacteria for the detection of toxic pollutants,has the advantages of fast,economic,stable and reliable,can achieve continuous online and automatic detection,early warning purposes,and has broad prospects for development.