| The herbicide atrazine is one of the most important herbicides,which lead to the pollution of a large area of soil,ground water and underground water.Atrazine which is very persistent in the environment is able to exist in soil,surface and underground waters. In addition,it also has potential threat to the normal product of organisms.In this thesis, we tried to isolate degrading bacteria by the wayof enrichment isolation to isolate dominant in situ atrazine-degrading bacteria from polluted soil,and studied their characteristics, degrading genes and applied them to remove atrazine in simulated pollution soil on the base of others’ research achievements.Isolation and identification of high efficient atrazine-degrading bacteriaBy enrichment method,a bacteria strain ADH-2 capable of utilizing atrazine as sole nitrogen and carbon source was isolated from soil samples collected from maize field suffered long-time application of Atrazine It was preliminarily identified as Arthrobacter sp.according to its physiological-biochemical characters and the similarity analysis of its 16S rRNA gene sequence(GeneBank Accession No.EF373977).Studies of growth and degradation characteristicsADH-2 could use starch and sucrose well except maltose,.All the organic nitrogen source can be used wel,Strain ADH-2 could grow well at 25℃-37℃,and degraded atrazine well at pH 5-10.It’s degrading efficiency was not affected greatly by aeration. Strain ADH-2 could degrade 99%of 100mg/L atrazine within 10h.Compared with the other two atrazine-degrading bacterium isolated by our lab,ADH-2 showed good potentials in application.ADH-2 could grow on atrazine as solecarbon,nitrogen and energy source,cyanuric acid cumulated in liquid culture as end products.Studies of atrazine-degrading genesAtrazine-degrading related genes of ADH-2 were the combination of trzN atzB and atzC,with 99%,100%and 99%of sequence identity respectively to the reported ones. Complete trzN gene sequence could be amplified from ADH-2 by PCR,with 99% identity to the reported one of C190.In an effort to study the protein,the trzN gene was cloned into Escherichia coli.The gene was cloned downstream of a T7 promoter and an N-terminal six-His-tag clamp in the vectors pET29a,The constructs were transformed into E.coli BRL21(DE3),and their sequences were verified E.coli BL21(DE3) In our studies,most of the recombinant TrzN was localized to insoluble inclusion bodies and had low activity.To overcome these problems,the trzN was constructed under the control of the well-characterized P43 promoter and B.subtilis nprB signal peptide encoding sequence. The expression vector pP43T was introduced into B.subtilis WB800(deficient in eight proteases) and TrzN was continuously expressed.The enzyme activity maintained between 20℃and 30℃,or at pH higher than 6.0,with the highest activity at ph 7.3and 30℃.The enzyme activity could be enhanced due to addition of most metal ions at the concentration of 0.2mmol/L.Studies on the biodegradation of atrazine by ADH-2 in the soilADH-2 was inoculated in soils with atrazine concentration 50μg/g dry soil and ADH-2 cells 1.7×10~7 CFU /g dry soil.After 10 days’ incubation,much lower than the uninoculated control,This result showed that ADH-2 could degrade atrazine efficiently in these soils,Degrading efficiency was affected by soil water content,20%or larger of water content was needed for ADH-2 to degrading AT efficiently,but pH did not show much efficienc... |
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