Construction Of New Genetically Engineered Bacteria Containing Flr-2 Fluoride-resistant Gene And Its Enrichment Behavior And Mechanism For U(Ⅵ)

As a clean renewable energy source,nuclear energy has been extensively developed in recent years.However,the mining of uranium,the main raw material for nuclear energy,is gradually increasing,and uranium resources are gradually scarce.In addition,uranium has a long half-life and has certain chemical and radiotoxicity.Discharge into the environment poses a potential threat to environmental safety and human health.Therefore,it is urgent to increase the removal and recovery of uranium in the environment.As the acid leaching uranium mining technology is mostly used nowadays,the acidic conditions make F^-be leached in large quantities while leaching uranium.In order to achieve the purpose of sustainable treatment of uranium-containing wastewater by living microorganisms,it is of great practical significance to construct an integrated genetic engineering bacteria with multiple resistances,so that it can enrich uranium and have multiple ion resistances.Chapter Two:The flr-2 fluoride-resistant gene was synthesized from the whole gene,amplified by PCR,and cloned seamlessly with the linear vector p RADK after double enzyme digestion to construct the p RADK-flr-2 recombinant vector.The p RADK-flr-2 recombinant vector was transferred to E.coli Top10.Through double enzyme digestion,colony PCR identification,gene sequencing and Blast comparison,the results showed that the E.coli Top10-flr-2 clone was successfully constructed.Chapter Three:In order to verify the expression of flr-2 fusion protein and explore the enrichment effect of bacteria on uranium,the E.coli expression strain BL21-flr-2 was constructed.Western blot detection,amino acid comparison and the growth curve of BL21-flr-2 in F^-containing medium all indicated that the flr-2 gene was expressed normally and BL21-flr-2 expressing bacteria had great fluoride resistance.The single-factor uranium enrichment experiment of E.coli expressing bacteria BL21-flr-2was carried out,and the enrichment reaction was 180min at p H 4,the initial uranium concentration of 20 mg L^-1,and the addition ratio of the bacteria solution was 33.3%.The enrichment rate of BL21-flr-2 for uranium can reach about 88%,and the unit uranium enrichment capacity at equilibrium was 31.88 mg g^-1.The structure of BL21-flr-2 dry cells before and after enrichment was characterized by SEM,EDS and FTIR.The results showed that uranium was successfully enriched on the surface of BL21-flr-2.Active functional groups such as phosphoryl,carboxyl and hydroxyl participated in the enrichment process of uranium by BL21-flr-2,and there might be biological macromolecules involved in the enrichment process of uranium.Chapter Four:In order to solve the lack of resistance of active microorganisms to the complex ion environment in the treatment of uranium wastewater,a Deino-flr-2 radiation-tolerant genetic engineering bacteria containing flr-2 fluorine-resistant gene was constructed.It has multiple resistance to fluoride ion,radiation and heavy metal environment.Utilizing the reductase activity of Deinococcus radiodurans itself and the enrichment of uranium by the surface active functional groups of the bacteria,the treatment of simulated low-concentration uranium-containing wastewater will be realized.PCR identification,gene sequencing and BLAST comparison of p RADK-flr-2 recombinant plasmid in Deino-flr-2were conducted.The results showed that the Deino-flr-2 radiation-tolerant genetic engineering strain was successfully constructed.The single-factor uranium enrichment experiment of Deino-flr-2 radiation-resistant genetically engineered bacteria was carried out,and the enrichment reaction was 75 min at p H 5,the initial uranium concentration of 25 mg L^-1,and the bacterial solution dosage ratio of 33.3%.The enrichment rate of Deino-flr-2 for uranium can reach about 90%,and the unit uranium enrichment capacity at equilibrium is 47.87 mg g^-1.The structure of Deino-flr-2 dry cells before and after enrichment was characterized by SEM,EDS and FTIR.The results showed that uranium was successfully enriched on the surface of Deino-flr-2.The bioconcentration of uranium by Deino-flr-2 is a complex process that combines passive and active mechanisms,including rapid cell surface biosorption and subsequent enzyme-mediated bioreduction.