基于高灵敏度全细胞传感器的汞转化基因元件评价体系

Evaluation system of mercury transformation genes based on high sensitivity whole-cell sensor

汞是最具毒性的重金属之一;不同形态的汞具有的毒性差异大,发掘高效的不同汞形态转化的基因元件,通过合成生物学手段构建汞的微生物转化减毒体系是未来汞污染治理的重要方向;因此,发展和建立一套基因元件功能和效率的评价体系,是筛选高效汞形态转化基因的关键.基于MerR这一汞离子特异性响应蛋白和绿色荧光蛋白(GFP)报告基因所构建的汞离子大肠杆菌细胞传感器,首先通过引入汞转运蛋白MerT提高汞离子的响应程度,构建含pUC57-MerT-MerR-GFP的细胞传感器.该传感器4h对氯化汞(HgCl_(2))的响应浓度为5-200μg/L,响应能力与汞离子浓度线性相关.在此基础上,将含有待测试汞转化基因的pACYC184质粒转入这一传感器,构建含相容性双质粒的大肠杆菌体系,通过GFP荧光的强度(胞内汞离子浓度)来评价待测基因是否具有将汞离子转化成其他形态的功能及其效率.抗生素处理下,相容性双质粒在大肠杆菌中拷贝数保持稳定,可用于基因元件评价.通过生物信息学分析,分别在原核生物蓝藻和单细胞真核生物四膜虫中鉴定和筛选了潜在的汞离子转化相关基因,包括半胱氨酸合成酶、胱硫醚-β-合成酶和半胱氨酸脱硫酶.利用所构建的大肠杆菌体系对蓝藻和四膜虫的基因元件进行评价,结果表明在100μg/L HgCl_(2)处理4 h下,含蓝藻半胱氨酸脱硫酶的大肠杆菌GFP荧光强度与对照组相比下降了79.5%,含四膜虫半胱氨酸脱硫酶的大肠杆菌GFP荧光强度与对照组相比下降了51.2%,说明两类生物中半胱氨酸脱硫酶能均能高效转化汞离子.本研究所构建的评价体系可用于上述原核生物基因元件和密码子优化后的真核生物基因元件,具备较强的响应速率、灵敏度和稳定性,为实现汞转化基因元件高通量筛选奠定了基础.

Mercury(Hg)is one of the most hazardous heavy metals.The toxicity of the different forms of mercury varies significantly.A promising method for mercury bioremediation is to convert highly toxic forms of mercury into less-toxic forms using microbes constructed using synthetic biology techniques.Therefore,developing a system to evaluate the function and efficiency of genes is crucial for screening high-efficiency genes involved in mercury transformation.In this study,a Hg^(2+)cell sensor in Escherichia coli was constructed using MerR,a Hg^(2+)-specific response protein,and a green fluorescent protein(GFP)reporter gene.The mercury transporter MerT was inserted to improve Hg^(2+)responsiveness,and a cell sensor incorporating pUC57-MerT-MerR-GFP was constructed.At 4 hours,the response concentration of this sensor to mercuric chloride(HgCl_(2))was 5–200μg/L,and the response was linearly correlated with the concentration of Hg^(2+).Based on this,the pACYC184 plasmid containing the Hg^(2+)transformation genes was introduced into the cell sensor as a second plasmid compatible with the pUC57-MerT-MerR-GFP plasmid.The fluorescence intensity of GFP(intracellular Hg^(2+)concentration)was used to evaluate whether a test gene had the function and efficiency to transform Hg^(2+)into other forms.The copy number of the compatible double plasmids remained stable in E.coli during antibiotic treatment,implying that it could be employed for gene evaluation.Potential Hg^(2+)transformation genes,including cysteine synthase,cystathionine-synthase,and cysteine desulfurase,were discovered in prokaryotic cyanobacteria and unicellular eukaryotic tetrahymena.The results showed that after 4 h of treatment with 100μg/L HgCl_(2),the fluorescence intensity of the GFP of E.coli containing cysteine desulfurase of Synechocystis decreased by 79.5%,and that of E.coli containing cysteine desulfurase of Tetrahymena thermophila decreased by 51.2%compared to the control group.These results indicated that cysteine desulfurases in both organisms were capable of transforming Hg^(2+)efficiently.These results suggested that the evaluation system constructed in this study can be applied to both prokaryotic and eukaryotic genes.In conclusion,the evaluation system was demonstrated to have good sensitivity and stability and may facilitate the high-throughput evaluation of genes involved in Hg^(2+)transformation in the future.