Cloning And Expression Of Atrazine-degrading Related Genes From Bacteria, Shinella Sp. And Staphylococcus Sp.

As a s-triazine herbicide, Atrazine was widely used in controlling corn, sorghum, sugar cane, one-year-old orchard grass weeds and other broadleaf weeds. Although it played an important role in agricultural production, Atrazine existed lots of adverse effects in ecological environment because it had long residues in the soil and sensitive influence to the stubble crop. In this study, the Shinella sp. BZB-1 strain and Staphylococcus sp. X-700 materials were used to study the residue and degradation problems of atrazine. Also, the experiments in degradation gene cloning, location and expression were studied.The results were as follow:.1.Identification of degradation strainsThe morphological, biochemical and molecular identification of two degradation strains, including Staphylococcus and X-700 strain, were used in this experiment. The results showed that the 16S rDNA sequence from X-700 strain was 99% similarity with that from Staphylococcus based on the morphological appearances and 16S rDNA sequence analysisanalysis, which were indicated that X-700 strain was identified as Staphylococcus.sp.2.Degrading gene cloning of degradation bacteriaThe conservative sequences of atzA,atzB,atzC and tnp genes were used to clone the gene of BZB-1, X-700 strain. The results showed that the conservative sequences of atzA,atzB,atzC and tnp genes from Shinella sp. BZB-1 were similar with Pseudomonas sp.ADP And the gene similarity was more than 99%. But only the conservative sequence of atzA was cloned from Staphylococcus sp. X-700, and the gene similarity was also beyond 99% compared with Pseudomonas sp. ADP, which indicated that the gene from Staphylococcus sp. X-700 and Pseudomonas sp.ADP were homologous.3.The orientation of degradation geneThe orientation was analyzed by Staphylococcus sp. X-700 and Shinella sp. BZB-1. The results showed that there were two big plasmids in the BZB-1 with one was about 23kb and the other was bigger than 23kb. The genes of atzA,atzB,atzC and tnp were consisted in the 23kb plasmid, however, there was no degradation gene in the bigger plasmid. There was no plasmid in X-700 strain. The results indicated that the degrading-gene was in the genome of X-700 strain.4.The analysis of reading frame sequence of Atrazine chlorohydrolase geneThe open reading frame (ORF) of the atrazine chlorohydrolase(atzA) gene in BZB-1 strain was cloned , according to the gene of Pseudomonas sp. ADP. Their similarity was 99.65% . The difference was observed in 5 nucleotides through 1425 nucleotides. Through translating the ORF of atzA from BZB-1 strain to the amino acid sequence, there was only 1 different amino acid in 474 amino acids. The 93th amino acid was phenylalanine instead of tyrosine.5.The expression of Atrazine chlorohydrolase geneThe Atrazine chlorohydrolase in BZB-1 was connected with the expression vector of PET-42a, and then transferred into E.coli BL21(DE3). The SDS-PAGE electrophoresis method was measured by 1mmol/L IPTG to induce the atzA. The results showed that the expression protein was about 53kDa.6.Bioassay the degradability of expressing products of degradation geneThe degradability of BZB-1 and Atrazine chlorohydrolase activities were measured in this experiment by wheat sensitivity in Aatrazine. The results showed that there were strong degradation ability among BZB-1 strain, engineering strain and crude enzyme solution of engineering strain. Which could make the atrazine to lose its activity completely , for strain responses within 48 hours and enzyme reaction for 12 hours in 300mg/kg Aatrazine substrate.Upon all the results above, it was sure that the strain X-700 was Staphylococcus sp.. The conservative sequences of atzA,atzB and atzC genes were cloned from the smaller plasmid of Shinella sp. strain BZB-1, and only the conservative sequences of atzA was cloned from the genome of strain X-700. There were only 5 different nucleotides out of 1425, only 1 different amino acid out of 474 amino acids between strain BZB-1 and Pseudomonas sp.ADP. At the end the ORF of the atzA was expressed successfully in E.coli, and the expressing protein of the atzA showed its activity.