Isolation Of An Organophosphate Degrading Bacterial Strain And Molecular Cloning Of Degrading Gene

Organophosphate pesticides are mostly broad-spectrum and highly effective insecticides. High residua resulting from the extensively and long use in China has been the critical threaten to the environment and the food safety.Microorganism in soil degrading pesticides can clear up the residua and eliminate the pesticide effects on the ecosystem .This research is focused on the degrading characters of microorganism and molecular cloning the pesticide-degrading gene applying in the crops which will resolve the organophosphate pesticide residual problem in the agriculture and be a new way for non-pollution agricultural manufacture.This research includes two parts. The first part is focused on isolation and the degrading characters of organophosphate pesticides degrading bacterial strains. Three strains screened X1, X2, X3 can utilize organophosphate pesticides such as dimethoate and dichlorvos as sole carbon resource.X2 is gram positive, other strains are gram negative. Most microorganism degrading pesticides are gram negative, and gram positive strain could hardly been found. So we chose X2 as the material. According to the analysis of 16s rDNA sequence and biological and chemical identification, strain X2 was characterized taxonomically as Arthrobacter flavescens. High performance liquid chromatography (HPLC) results showed that the efficiency of X2 to degrade dimethoate and dichlorvos in 72 hours was 88.11% and 96.32% respectively. The optimal growth temperature and PH are 28 °C and 7.0. The highest tolerance concentration to dimethoate and dichlorvos was 1600 mg/L and 800 mg/L. Strain X2 has obviously degrading effect on organophosphate pesticides, but no degrading effect on glyphosate. Strain X2 possesses no plasmids.The second part is to obtain the degrading gene by shot-gun cloning. Chromosomal DNA from strain X2 was completely digested with BamHI, and 2-5 kb DNA fragments were extracted from agarose gels after electrophoresis. The fragments were ligated to dephosphorylated pBluescript KS at the BamHI site and transformed into competent E.coli DH5a cells. Rapid preparation of plasmid DNA, restriction enzyme digestion, agarose gel electrophoresis and transformation were performed by standard procedures. Three clones (termed EpKS-B1, EpKS-B2 and EpKS-B3) was screened by the plate containing 800 mg/L dimethoate and 100μg/ ml Amp.pKS-B1, pKS-B2 and pKS-B3 are...