Micrococcus Luteus-derived Rpf Protein Enhances The Rhizosphere Remediation Of PCBs Contaminated Soil By Alfalfa

Polychlorinated biphenyls（PCBs）are one of the most representative persistent organic pollutants.How to remove PCBs from polluted environment in a green and efficient way has always been a research hotspot.Rhizoremediation technology has great potential for PCBs degradation in soil.However,PCBs can inhibit the growth of degrading microorganisms and make them to enter the dormancy state,limiting PCBs biodegradation efficiency.To improve rhizoremediation remediation efficiency,this study selected the perennial alfalfa as the phytoremediation material,and the exogenous recovery promoting factor Rpf to promote the degrader growth and metabolism of PCBs in plant rhizosphere soil.Combining recovery factor Rpf and alfalfa stimulated the interaction of plant and rhizosphere microorganisms to improve the degradation efficiency of PCBs.The main contents and conclusions are as follows:（1）Purification and identification of Rpf protein and validation of its resuscitation function.The rpf gene subcloned from Micrococcus luteus was ligated with the vector p ET-28 a and transferred into E.coli BL21（DE3）p Lys S cells to screen the positive strains.The Rpf protein was induced using IPTG,purified using affinity chromatography with nickel column and identified using western blotting.In shaking experiment,adding 5% Rpf protein（v/v）significantly increased the growth rate of Pseudomonas Monteilii PN1,which was mainly reflected in shortening the delay period of PN1 and making it rapidly to enter the exponential growth stage and stable stage.The Rpf showed a promoting effect on the growth of PN1.（2）In pot experiments,phytoremediation effeciency of the alfalfa is determined for PCBs contaminated soil in the present of protein Rpf.After 40 days,the removal rates of PCB 3,PCB 5,PCB 28,PCB 52 and PCB 101 were 35%、40%、31%、37% and29% respectively in the present of Rpf protein at 5%（v/w,m L/g）.Compared with negative treatment with only planting the alfalfa,removal rates increased by 2.4times、0.5 times、1.7 times、0.9 times and 1.6 times,respectively.When the concentration of Rpf protein added was 0.5%（v/w,m L/g）,removal rates increased by2.4 times、0.7 times、2.2 times、0.8 times and 1.4 times,respectively,compared with the control group that only planted alfalfa.The results indicated that the combined remediation of Rpf protein and alfalfa improved the removal efficiency of PCBs in soil.（3）Effects of Rpf protein on expression of degradation genes and enzyme activities in rhizosphere soil.The expression level of bph A and bph C genes increased by 0.6times and 0.8 times respectively.The results showed that the addition of Rpf protein increased the gene expression of rhizosphere PCBs degradation.The addition of Rpf protein significantly increased the activities of DHA and GST in rhizosphere soil.These results suggested that Rpf protein enhanced the activities of rhizosphere degraded microorganisms,and then strengthened the remediation efficiency of PCBs by rhizosphere microorganisms.（4）Effects of Rpf protein on plant growth and enzyme activities.The fresh weight and chlorophyll content of alfalfa treated with Rpf were significantly higher than those of negative control group.The addition of Rpf protein significantly increased the activities of SOD、POD and GST in plants.These results suggested that Rpf protein enhanced the tolerance and the metabolism of PCBs in alfalfa,and reduced the toxic inhibition of PCBs on alfalfa growth.（5）Effects of Rpf protein on rhizosphere microbial community.The results of high-throughput sequencing in soil microbial community showed that the addition of Rpf protein affected the dominant populations of bacteria and fungi at phylum and genus level.Rpf protein stimulated the growth of PCBs degradation strains such as Pseudomonas and Rhizobium and plant growth-promoting bacteria such as Streptomyces and Bacillu.Rpf protein promoted the growth of PCBs degrader and alfalfa.The above results provide a new idea for studying the effect of Rpf protein on rhizoremediation.Rpf protein may stimulate the PCBs metabolic activities of rhizosphere degraded microorganisms and improve the combined remediation efficiency of plants and microorganisms.