Isolation,identification Of Acetamiprid Degradation Strains And Cloning,expression Of Nitrile Hydratase Gene

Acetamiprid is a new broad spectrum nicotinyl insecticide and a new agricultural chemicals, which has been put used in more and more countries, and the annual production of acetamiprid in our country has already exceeded 5000 tons. Acetamiprid is a kind of insecticide which has acaricidal activity, and the mode of its action is systematical insecticide of soil, branches and leaves. It has been widely used in paddies, especially in vegetables, fruit trees, aphids in tea leaves, plant hoppers, thrips and prevention of part of lepidopterous insect pests. But it’s hypotonic as a kind of insecticide and it can be extensively detected in ecological system, which has raised wide concern on this society that pay more and more attention to ecology. Bio-remediation is deemed to an effective, reliable and no re-pollution way of dealing with acetamiprid pollution. Therefore, screening the microbial resources which can effectively biodegrade acetamiprid, exploring the mechanism of action and making full use of its biodegrade ability has important theoretical significance and practical value.The aim of this paper is to screen and isolate bacterial strains which can effectively degrade acetamiprid, and study the characteristics of acetamiprid degradation by bacterial strains under different circumstances. It will provide theoretical basis for bio-remediation of acetamiprid pollution. Meanwhile, the gene of key enzyme of acetamiprid degradation was cloned and characteristics of was studed.By the means of enrichment culture, this paper separated 5 acetamiprid degrading bacterial strains from acetamiprid polluted soil, which was named 22-5, DF-1, Y-3, DY-17, YF-20. The most effective bacterial strain DCM DY-17 was chosen to further. It’s been determined as a gram-negative bacillus through Gram staining and pictures token by electron microscope. High-quality DNA of acetamiprid degradation bacterial DY-17 was extracted by SDS-CTAB method, and as a template, it’s been identified as a new member of Pseudomonas fluorescence sp. through analytics of 16 S rRNA gene phylogeny, and phylogenetic tree was built according to results comparison.The biological dcharacteristics of acetamiprid degradation bacillus DCM DY-17 was studied, and the optimum condition of DCM DY-17 to degrade acetamiprid was found at 37℃, pH 7, concentration of acetamiprid is 200 mg/L; the research results provide strain resources of bioremediation of acetamiprid polluted environment.Genomic DNA library was constructed by the shotgun method. One acetamiprid degradation positive clone was screened out of approximately 12000 transformants. It’s been identified as a nitrile hydratase structural gene through software analysis. Nitrile hydratase structural gene of acetamiprid and the activator protein of downstream gene of nitrile hydratase structural gene were ligated into the expression vector pET29 a, and transformed to Ecoli.BL21(DE3) respectively to express; SDS-PAGE analysis, indicated that the weight of α subunit of nitrile hydratase structural gene is 26 kDa, the weight of β subunit is 25 kDa, and the weight of activator protein is 48 kDa through electrophoretogram. Through the experiment, we can make the conclusion that the acetamiprid nitrile hydratase structural gene has low activitywhen expressed individually, soluble expression level wass low, the activity of enzyme was cow; when coexpressed with activator protein, soluble expression level and activity of enzyme increased, which illustrates that activator protein may work as a molecular chaperones that help nitrile hydratase fold correctly to construct functional spatial configuration. This will offer basic research of the microbial remediation of neonicotiniod insecticides pollution.