Dissertation > Environmental science, safety science > The basic theory for the Environment and Science > Environmental Biology > Environmental Microbiology

Characterization of an Atrazine-Degrading Strain, Cloning of Key Degrading Related Genes and Construction of a Gene Cluster

Author ZuoCaiFang
Tutor LiShunPeng;HongQing
School Nanjing Agricultural College
Course Microbiology
Keywords atrazine biodegradation isolation & Characterization gene cluster GEM
CLC X172
Type Master's thesis
Year 2011
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Atrazine is a kind of triazine herbicide, which is widely used at home and abroad, it plays an important role in the weed control. Although it has low toxity, its residue could damage subsequent crops, such as wheat, soybean and rice. So the bioremediation of the atrazine-contaminated soil has become the hot research topic in the scope of pesticide pollution control. Investigations of microbial degradation are useful in the development of strategies for bioremediation.A bacteria strain X-4 capable of degrading atrazine was isolated from atrazine-contaminated soil. It was preliminarily identified as Arthrobacte sp. according to its physical and biochemical characteristics and the analysis of its 16S rRNA gene sequence.Biological characteristics of strain X-4 was investigated and the results showed that, Strain X-4 could grow well from pH 6.0-10.0, the optimal pH for its growth was 6.0, the optional temperature for its growth was 30℃; maltose and beef extract were the best carbon source and nitrogen source its growth, respectively. The degradation rate against 100 mg·L-atrazine of strain X-4 was 95.7% within 42 h,30℃was the optimal degrading temperature. On the concentration of 1 mmol·L-1, strain X-4 was resistant to heavy metal ions, except Cu2+, Mn2+and Hg2+, which showed its potential in treating the co-contamination of atrazine and heavy metals.The primers were designed according to the published sequences of atrazine degrading related genes. The complete trzN gene of strain X-4 was cloned by PCR, it showed 100% sequence identity to that of stain C190. The conserved fragments of atzB and atzC gene were also amplified by PCR and sequenced; they showed 100% and 99% sequence identity to the reported corresponding ones respectively, which revealed that the degrading related genes of strain X-4 were the combination of trzN, atzB and atzC.On the base of plasmid pETatzA and strain X-4, complete gene of atzA, atzB and atzC were amplified by PCR and cloned to plasmid pUC18, and the expressing related elements were equipped, then the recombinant plasmid pUCatzABC with the gene cluster atzABC was constructed by combination. E.coli/pUCatzABC could transform atrazine to cyanuric acid and undertake the key catalyzing steps of the atrazine- degrading pathway, which established the foundation for the construction of atrazine-degrading genetically engineered microorganism (GEM).

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