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为实现重金属污染环境中Cr6+的生物可利用度评价,进而为Cr6+的污染治理提供可靠依据,本研究采用分子生物技术,以重金属广谱抗性菌株Cupriavidus metallidurans CH34的pMOL28质粒上铬抗性调控系统的启动子和调控蛋白基因为传感器调控元件,以北美萤火虫Photinus pyralis的萤火虫荧光素酶基因luc为报告基因构建了一种可对Cr6+生物可利用度进行检测的微生物全细胞传感器CB10,并探究了其在不同检测条下的响应特征.结果表明,微生物全细胞传感器CB10在30 min内即可响应一定浓度的Cr6+;其对Cr6+的检测最低限LOD为2μmol·L-1;当Cr6+的浓度为20～200μmol·L-1时,CB10的响应能力与Cr6+浓度线性相关,进而传感器CB10可对该范围内的Cr6+生物可利用度进行定量分析(R2=0.980 55);当重金属诱导浓度为10～50μmol·L-1时,CB10对Cr6+具有较强的响应特异性;本研究也发现,较高浓度的Pb2+、Mn2+和Sb2+也可以产生对CB10的诱导能力;诱导温度为30℃,pH为7时,CB10的响应效率最高;诱导温度为15～30℃,pH为4～7时,CB10响应能力表现稳定.CB10具备较强的响应速率、灵敏度、特异性和稳定性,具备对水体重金属进行快速检测和土壤重金属的生物可利用进行评价的潜力.
In order to provide a reliable basis for the control of Cr6 + pollution in order to achieve the bioavailability of Cr6 + in heavy metal contaminated environment, molecular biological techniques were used in this study to control the chromium resistance of pMOL28 plasmid in the heavy-metal broad-spectrum resistant strain Cupriavidus metallidurans CH34 The promoter and regulatory protein genes were used as sensor regulatory elements. The luciferase gene luc of North American firefly Photinus pyralis was used as a reporter gene to construct a microbial whole cell sensor CB10 that can detect the bioavailability of Cr6 + The results showed that the microbial whole cell sensor CB10 could respond to a certain concentration of Cr6 + within 30 min, the detection limit of Cr6 + was 2 μmol·L-1. When the concentration of Cr6 + was The response of CB10 was linearly correlated with the concentration of Cr6 + at 20 ~ 200μmol·L-1, and the CB10 bioavailability was quantitatively analyzed by the sensor CB10 (R2 = 0.98055). When the concentration of heavy metals was 10 ~ CB10 had strong response specificity to Cr6 + at 50μmol·L-1, and higher concentrations of Pb2 +, Mn2 + and Sb2 + were also found in this study CB10 had the highest response efficiency at the induction temperature of 30 ℃ and pH of 7. CB10 had a stable response at the induction temperature of 15-30 ℃ and the pH of 4-7 and the CB10 had a strong response rate , Sensitivity, specificity and stability, has the potential to evaluate rapid detection of heavy metals in water and bioavailability of heavy metals in soil.