Construction Of A Multi-Functional Genetically Engineering Stain And Its Use In Bioremediation Of Soil Contaminated By Pesticide And Cadmium

In this experiment we obtain an methyl parathion degrading bacterium Hn with a high degradation rate, which is identified as a Pseudomonas putida based on the results of phenotypic features, physiological and biochemical characteristics and analysis of 16SrRNA. The property of MP degradation of this wild strains and the molecular mechanism of its degradation. This degrading bacterium can completely mineralize as much as 100mg/L MP as the sole carbon and energy source in 6h, and its maximum tolerance concentration of the MP was 1000mg/L. The degradation gene mpd and p-nitrophenol complete degradation gene cluster were amplified by PCR and SEFA-PCR. mpd gene coding Methyl parathion hydrolase could decompose methyl parathion and generate an intermediate product p-nitrophenol PNP. Mpd was normally expressed in E.coli with a size of 35kDa by SDS-PAGE. As the other bacterium, MPD hydrolase has a high degradation activity in E. coli, and completely degrade 100mg/L MP within 6 hours. By SEFA-PCR we obtain the upstream and downstream sequence of the mpd gene, which located in a reverse insertion sequence IS6100 by BLAST analysis in NCBI. This result may imply that the mpd gene are widely existing in various microorganisms. The partial sequence analysis of PNP degrading gene cluster showed that decomposition approach of p-nitrophenol is the hydroxyquinol degradation pathwayA multi-functional engineering bacterium was constructed using enhanced green fluorescent protein (EGFP) as a label and Pseudomonas putida X4 as a host. This strain could degrade 100mg/L MP in 20h and had high resistance to heavy metal cadmium, copper, cobalt, zinc. There are series of functional test about this engineering strain X3 in pure culture condition were carried out. First, the enhanced green fluorescent protein in bacteria X3 has high level of fluorescence, and the intensity of fluorescence enhance accordingly with the increase in cell number. It seems feasible that X3 could remediate directly contaminated soil and be detected dynamic activity in situ. Secondly, X3 effectively combines the two functions of the wild strains, so it not only degrade 100mg/L MP within 20 hours, but also has high resistance to 7mM heavy metal cadmium.Finally, a simulated heavy metals and pesticide contaminated soil was used to study the bacterial ability dealing with environmental pollution in the actual application. X3 could degrade MP and change forms of cadmium in contaminated soil. It could reduce cadmium’s bioavailability and toxicity to the plants in the agricultural land. In this study, the egfp tag engineering bacterium was constructed for multi-purpose to monitor methyl parathion and cadmium composite pollution in the soil. The results of this preliminary study about X3 provide a theoretical basis for the use of engineering bacteria in cleanup of contaminated pesticides and heavy metals in the environment in the future.