The Degradation Gene Function Analysis Of A High Efficient Chlorpyrifos Degraing Bacteria Of Cupriavidus Nantongensis X1

Chlorpyrifos is a moderately hazardous organophosphate pesticide.It is widely used as insecticides,fungicides,herbicides to against gall midge,plant hoppers and other crop pests,which are affect the growth of economically crops.It also have toxic effects for mammals and other non-target organisms.The biodegradation product of chlorpyrifos is 3,5,6-trichloro-2-pyridinol(TCP),which is classified as persistent and mobile by the U.S.EPA,with a half-life between 65 to 360 days in soil.It has greater water solubility than chlorpyrifos.Due to the long half-life,chlorpyrifos and TCP can remain in soil for a long time to make environment pollution.In this study,the Cupriavidus nantongensis X1‘s complete genome was sequenced.(X1 is an efficient chlorpyrifos degrading bacterium.And it has been reported as a novel species of cupriavidus genus.).The circle genome diagrams were drawn.Genes predicted involved in chlorpyrifos and TCP degradation pathway were analyzed.The whole gene cluster and complete pathway were discovered.And use the gene-knock of TcpA gene in TCP degradation.The res?lts were summarized as follows:1.This is the first time to report the complete genome sequence of Cupriavidus nantongensis species.The libraries were sequenced using an illumine Hi Seq2500 and MiSeq by PE125 strategy.Illumina PCR adapter reads and low quality reads were filtered.The filtered reads were assembled by SOAPdenovo to generate scaffolds.It res?lted in two circ?larized chromosomes and one circ?lar plasmid sequence.The whole genome has7136420 bp with a G+C content of 66.72%.A total of 6524 genes,57 tRNAs,15rRNAs were predicted.Genome information for the chromosome and plasmid of Cupriavidus nantongensis X1 were deposited in GenBank under the accession numbers CP014844,CP014845 and CP014846.2.Comparing with GO,KEGG,COG database,the encoding genes and corresponding products of strain X1 were analyzed.The circle genome diagrams was drawn with the information of assembling sequence,encoding genes,ncRNA,GC skew,COG,KEGG and GO functional analyze.3.Comparing with GeneBank database,one organophosphorus hydrolase gene(OPH gene)was identified in the px1 sequence,which was the key gene to degrade chlorpyrifos to TCP.One 2,4,6-trichlorophenol monooxygenase gene(TcpA gene)was located in thechromosome1 sequence,which was the key gene to degrade TCP to 3,6-dihydroxypyridine-2,5-Dione.It was the first time to identify the complete gene cluster of TCP degradation.4.The pathway of chlorpyrifos degradation in strain X1 was analyzed.First,the chlorpyrifos was degraded to TCP and TCMP by OPH gene.Then,the TCP was degraded to 3,6-dihydroxypyridine-2,5-Dione by oxidative dechlorination.The pyridine ring was opened by deacetylase.Then the ?-ketoadipate was generated by maleimide acetic acid reductase.And the last,the ?-ketoadipate was joined in the CAC cycle,eventually metabolized to carbon dioxide and water.5.Identified seven kinds of antibiotic(ampicillin,kanamycin,chloramphenicol,streptomycin,tetracycline,gentamicin,and erythromycin)resistance profile in strain X1.Using the gradient plate,the ampicillin MIC value was 20.67?g/ml,the tetracycline MIC value was 12.47?g/ml and the chloramphenicol MIC value was 2.35?g/ml,the streptomycin MIC value was 4.02?g/ml and the gentamicin MIC value was 2.82?g/ml.The strain X1 did not insensitive with 50?g/ml kanamycin and 100?g/ml erythromycin.6.Using the suicide plasmid PJQ200 SK to construct a recombinant vector PJQ-TY-TcpA.And then,using three pro way thro?gh to gain the strain X1-?TcpA,which TcpA gene was inactivated.Furthermore,verified the functional of TcpA gene.