The Application of Gene Recombinant Luminescent Bacteria to Environmental Sample Toxicity Test
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摘要: 选取基因重组发光菌E.coli HB101 pUCD607和费氏弧菌为试验生物，采用急性毒性微孔板法，发光抑制率为测试指标，分别用锌离子、汞离子作为阳性对照，研究8种重金属分别对基因重组发光菌和费氏弧菌产生的生物毒性，结果显示：锌离子毒性小、稳定性高、变异系数小，更适合作为阳性对照.利用基因重组发光菌比较了离子液体和有机溶剂的生物毒性效应，同等条件下的毒性测试结果表明：基因重组发光菌半数效应质量浓度(EC50)比费氏弧菌高，灵敏度更高；在铜离子、锰离子、铁离子和镉离子的毒性作用下，作为受试生物，基因重组发光菌的灵敏度显著高于费氏弧菌(EC50之间存在显著性差异)；离子液体对基因重组发光菌的毒性显著高于有机溶剂.综上所述，基因重组发光菌应用于环境样品的毒性检测可行性较高.Abstract: Gene recombinant luminescent bacteria(E.coli HB101 pUCD607)and Vibrio fischeri were used as the experimental organisms in toxicity test, and luminescence inhibition rate was used as the endpoint. The toxic micropore-plate method, in which Zn2+ replaced Hg2+ as positive control, was used to study the effects of eight heavy metals on toxicity tests of recombinant luminescent bacteria and Vibrio fischeri. The results showed that Zn2+ was of high stability and had the advantages of being soluble, stable and less harmful to people and the environment. Therefore, Zn2+ could replace the traditional Hg2+ as the positive reference. The toxicity test results (EC50) of the recombinant bioluminescent bacteria were more sensitive than Vibrio fischeri. Under the toxic effects of copper ions, manganese ions, iron ions and cadmium ions, the sensitivity of the recombinant bioluminescent bacteria was significantly higher than that of Vibrio fischeri. The toxicity of ionic liquids to luminescent bacteria was significantly higher than that of organic solvents. So, it is feasible to apply recombinant luminescent bacteria to the toxicity test of chemicals.
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图 1 阳性对照物质锌离子对基因重组发光菌的毒性效应
Figure 1. The toxic effects of positive control substance zinc ions on gen recombinant luminescent bacteria
图 2 8种重金属对基因重组发光菌和费氏弧菌的毒性比较
Figure 2. The comparison of the toxicity of eight heavy metals to gene recombinant luminescent bacteria and Vibrio fischeri
图 3 6种有机溶剂对基因重组发光菌的剂量-效应曲线
Figure 3. The dose-effect curve of six organic solvents to gene recombinant luminescent bacteria
图 4 6种不同烷基链长度离子液体对基因重组发光菌的剂量-效应曲线
Figure 4. Theose-effect curve of six different alkyl chain length ionic liquids to gene recombinant luminescent bacteria
图 5 有机溶剂和离子液体对基因重组发光菌的毒性比较
Figure 5. The comparison of toxicity of organic solvents and ionic liquids to gene recombinant luminescent bacteria
表 1 8种重金属对费氏弧菌和基因重组发光菌的剂量-效应曲线拟合信息
Table 1. The dose-effect curve fitting information of eight heavy metals on Vibrio fischeri and gene recombinant luminescent bacteria
重金属化合物 菌种类型 模型 a b c EC50/(mg·L-1) R2 ZnSO4 费氏弧菌 Boltzmann Sigmoidal -0.148 0.801 0.803 1.978 0.962 重组发光菌 0.055 0.929 -2.745 1.203 0.993 CuSO4 费氏弧菌 Boltzmann Sigmoidal 0.020 0.891 5.798 17.860 0.995 重组发光菌 -0.078 0.909 0.376 1.646 0.996 FeCl3 费氏弧菌 Boltzmann Sigmoidal -0.095 0.988 1.780 5.214 0.999 重组发光菌 -0.115 0.875 0.135 1.226 0.995 HgCl2 费氏弧菌 Boltzmann Sigmoidal -0.136 0.996 0.055 0.150 0.999 重组发光菌 0.023 0.983 0.006 0.659 0.999 MnCl2 费氏弧菌 Boltzmann Sigmoidal -0.004 0.985 10.680 47.370 1.000 重组发光菌 -0.001 0.922 4.945 9.935 0.995 CdCl2 费氏弧菌 Boltzmann Sigmoidal -0.464 1.011 22.070 18.290 0.987 重组发光菌 -0.084 1.058 -0.815 8.718 0.978 CoCl2 费氏弧菌 Boltzmann Sigmoidal 0.050 0.819 4.744 2.898 0.989 重组发光菌 -0.204 1.272 -0.676 1.045 0.987 K2Cr2O7 费氏弧菌 Boltzmann Sigmoidal -0.605 1.009 8.431 11.980 0.983 重组发光菌 0.036 0.970 1.715 10.550 0.999 注：a、b和c分别表示Boltzmann Sigmoidal的拟合参数，EC50表示半数效应质量浓度，R2表示相关系数的平方. 
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表 2 6种有机溶剂对基因重组发光菌的剂量-效应曲线拟合信息
Table 2. The dose-effect curve fitting information of six organic solvents on gene recombinant luminescent bacteria
有机溶剂 模型 ZEC/(g·L-1) EC/(g·L-1) EC50/(g·L-1) R2 甲醇 LSSVM 10.110 2.319 36.120 0.985 乙醇 LSSVM 54.050 9.246 52.140 1.000 丙酮 LSSVM 26.050 4.687 10.270 0.992 二甲基亚砜 LSSVM 122.500 12.890 101.800 0.999 甲醛 Boltzmann Sigmoidal 4.168 1.194 5.122 0.995 乙腈 LSSVM 38.700 11.510 47.880 0.985 注：ZEC表示零效应点质量浓度，EC表示最大刺激效应点质量浓度，EC50表示半数效应质量浓度，R2表示相关系数的平方，表 3同.
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表 3 6种离子液体对基因重组发光菌的剂量-效应曲线拟合信息
Table 3. The dose-effect curve fitting information of six ionic liquids on gene recombinant luminescent bacteria
离子液体 模型 ZEC/(g·L-1) EC/(g·L-1) EC50/(g·L-1) R2 IM-2 LSSVM 22.010 4.687 20.780 0.999 IM-4 Boltzmann Sigmoidal - - 9.131 0.996 IM-6 Boltzmann Sigmoidal - - 0.219 0.992 IM-8 Boltzmann Sigmoidal - - 0.012 0.988 IM-10 Boltzmann Sigmoidal - - 0.024 0.982 IM-12 Boltzmann Sigmoidal - - 0.016 0.997
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