Isolation And Characterization Of Glyphosate-Degrading Ochrobacterium Sp. Strain G-1and Cloning Of Gox Gene

Glyphosate have genetoxic to human. The level of DNA damage in exposed cells at2.5-7.5mM showed a significant dose-dependent effect, and AMPA has statistically significant clastogenic effect at1.8mM in human lymphocytes. An USA health examination survey showed that Glyphosate and its main metabolite can induce multiple myeloma, as for the connection with other cancers need more envidence.There are vast microbial resources degrading glyphosate and glyphosate-degrading genes have broad use in anti-herbicide transgenic crops.Glyphosate-degrading strain G-lwas isolated from contaminated sample, the bacterium was preliminarily identified as Ochrobacterum sp.(GeneBank Accession No.GU214710) based on the physiological and biochemical analysis and16S rRNA gene sequence analysis. It was designated as strain G-1. The colonial morphology of G-1on LB medium is sphere-cal, appearance convex, catalase positive, urease positive and esterase negative. Strain G-1can not hydrolyze gelatin. Biological properties of strain G-1were analyzed. The optimal pH value for the growth of G-1was7.0, the optimal temperature was30℃, and grow well under aerobic condition. The strain G-1was resistant to ampicil, kanamycin, cidomycin; actinospectacin, sensitive to ambramycin, chloramphenicol, etal. G-1can degradate glypho-sate which the concentration is300mg/L in24h, and the efficiency was much higher than bacteriums isolated before. The optimal pH value for the glyphosate-degrading of G-l was7.0, the optimal temperature was30℃.No significant effect of K+、Fe3+、Mn2+on glyphosate-degrading,whereas Cu2+. Ni2+showed obviously inhibitory effect.The primers were designed based on the published sequences of the glyphosate oxidoreductase gene. The glyphosate oxidoreductase gene was cloned by PCR from G-1. The analysis of the glyphosate oxidoreductase gene revealed that this gene has1296bases, which coding for the organophosphate pesticides hydrolase contained431amino acids and a stop codon. The similarity between glyphosate oxidoreductase gene and Chelativorans sp. BNC1(CP000389) was99%. The gox gene was linked to the expression plasmid vector pET-29a, the expression strains were gained by transfering the expression plasmid into strain BL21(DE3).