Isolation And Identification Of Herbicide Diclofop-methyl Degradation Bacteria And Analysis Of Metabolic Pathway

This test is based on diclofop-methyl(DCM),which is one of the aroxyphenylpropanoic acid herbicides.Two highly effective degradation bacteria were obtained by enrichment,screening,purification and identification from pesticide contaminated areas.The growth characteristics and metabolic pathway were studied,and an unreported metabolic pathway of DCM was proposed.This study provides theoretical basis of environmental pollution control and genetically modified crops resistanting to DCM and microbial resource foundation.By enrichment,we screened and purified two biodegradable bacteria,named JT-3 and JT-9.Based on the culture characteristics of the two strains and the homology of the 16 S rRNA sequence,JT-3 bacteria were identified as Rhodococcus and JT-9 bacteria as Brevundimononas.The growth characteristics of JT-3 and JT-9 were studied from four aspects:growth temperature,pH,tolerance to NaCl,and optimal carbon source.It was found that the optimum growth temperature of two graminoglyphs was 30 °C.The optimum growth pH is around 7.0;Low concentrations of NaCl are conducive to their growth,and high concentrations inhibit growth;The best carbon source is glucose,followed by fructose,lactose,ethanol,and starch.Three metabolites were detected by HPLC and LC-ITMS.It was identified as diclofop acid(DC),2,4-dichlorophenol(DCP)and 2-(4-hydroxyphenoxy)propionic acid(HPP)respectively.Then we determined that the relationship between the two bacteria was synergistic metabolism.Because JT-9 bacteria could degrade DC to produce DCP and HPP,JT-3 bacteria could not use DC.However,the strain JT-3 can further degrade DCP and JT-9 could not.In this paper,the pathway of the strain JT-3 metabolic DCP was studied with DCP as the substrate.Based on the results of the GC-MS,we further discovered the new products 4-chlorophenol(4-CP)and 4-chlorocatechol(4-CC).This study sequenced the draft genome of JT-3 degradation bacteria.Based on the genome sequence sketches,annotations,the key enzyme genes that may participate in the reaction were found.The transcription levels of the genes dcmA,dcmB1B2,dcmC,dcmD and dcmE were detected by RT-qPCR test when the substrate DCM or DCP was added to the reaction mixture.The results showed that the transcription levels of genes dcmA,dcmB1B2,dcmC,dcmD and dcmE increased by 345,271,57,49 and 82 times,respectively.It shows that these genes involved in the metabolism of DCM.Then,prokaryotic expression vectors with a histidine label and transfer to E.coli BL21(DE3)were constructed,expressed by IPTG induction.In this paper,crude enzyme solution is obtained by high pressure crushing of cells.The target protein was purified by Ni-NTA-Sefinose Colum and the pure enzyme solution was analyzed by polypropylene gel electrophoresis.The electrophoresis results are consistent with our calculations based on the enzyme gene sequence.Then to verify that these enzymes did participate in the DCP metabolic reaction,This study purchased the products and degraded them with DcmA,DcmB1B2 and DcmC,respectively.However,based on thehomology analysis of the purified proteins DcmC,DcmD and DcmE,we have completed the metabolism pathway of DCM in this paper.