Screening Of Triazophos Degrading Bacteria And Cloning Of Degrading-related Gene

Organophosphorus pesticides (OPs) is one of the important pesticides. After the forbidden of high-toxic pesticide methamidophos, moderately toxic pesticides, such as triazophos(TAP), are widely used. With the widespread use of triazophos, however, high residue of triazophos in agricultural products and environmental pollution were leaded. At the same time, triazophos caused big threaten to non-target organisms and human health. Microbial remediation is an effective way to degrade or mineralize the pesticide residue, and especially it is the most promising method to deal with pesticide pollution. The purpose of this study is screening and isolating triazophos-degrading microorganisms, analyzing the degrading characteristics, cloning the degrading-related gene. These works would provide a scientific basis for microbial remediation of triazophos residues.We isolated a triazophos-degrading strain from the surrounding pond in Hunan Academy of Agricultural Sciences, which could use TAP as sole carbon source, and it was named TDB-1. We identified TDB-1as Klebsiella oxytoca by a series of physiological and biochemical tests and16S rDNA sequence analysis.TDB-1has a good tolerance to TAP with a maximum tolerated concentration of2g·L-1. Under the selection pressure of TAP, the optimum growth condition was at35℃, pH7.0and2.0%(V/V) inoculation. Strain TDB-1has an excellent tolerance of pH, arrange from pH4.0to10.0.The degradation kinetics of strain TDB-1to TAP is accord with the first-order model. The degradation constant K is0.6757and the half-life of TAP (t1/2) is9.7558min-1. Cell surface hydrophobicity (CSH) assays of strain TDB-1reflected that the strain TDB-1was higher hydrophobicity while grown on higher concentration of TAP.According to the reported conservative sequence of OPs-degrading-related genes, we designed specific primers and cloned a fragment of1134bp length. On the basis of this fragment, we adopted Self-formed adaptor PCR (SEFA PCR) to clone the3’-flanking sequence, and we obtained the downstream sequence of the known sequence,355bp length. The analysis of the sequence indicated that the sequence contains a fragment which has high homology to nitrilase gene and as well as its downstream sequence of the genes with unknown function. These results provide basis for deeply study of TAP-degrading gene and regulatory mechanism.