Overexpression of D-amino acid oxidase from Bradyrhizobium japonicum, enhances resistance to glyphosate in Arabidopsis thaliana

Transgenic glyphosate-resistant crops have a high percentage in the total area devoted to transgenic crops worldwide. d-amino acid oxidase (DAAO) can metabolize glyphosate by oxidative cleavage of the carbon-nitrogen bond on the carboxyl side and yield aminomethyl phosphonic acid and glyoxylate, which are less toxic to plants than glyphosate. To date, reports on the use of DAAO to enhance glyphosate resistance in plants are lacking. In this paper, we report synthesis, and codon usage optimization for plant expression, of the DAAO gene by successive polymerase chain reaction from Bradyrhizobium japonicum. To confirm the glyphosate resistance of the DAAO gene, the recombinant plasmid pYPX251 (GenBank Accession No: AY178046) harboring the wild-type DAAO gene was transformed into DH5 alpha. The positive transformants grew well both on solid and in liquid M9 medium containing 200 mM glyphosate. The optimized DAAO gene was transformed into Arabidopsis and 9 days after application of 10 mM glyphosate, the 4-week-old wild-type plants all died; by contrast, transgenic plants could grow normally. The proline content and peroxidase activity showed that glyphosate could induce proline accumulation and produce reactive oxygen species.