Structural characterization of phosphinothricin N-acetyltransferase (RePAT) and the glufosinate-resistant analysis for site-directed mutagenesis of RePAT in Arabidopsis thaliana

Abstract

Background: Transferring herbicide resistance genes to plant cultivars is one of the most effective methods for managing weed growth in agricultural land. The RePAT gene, previously isolated from the marine bacterium Rhodococcus sp. strain YM12, was found to provide glufosinate resistance to plants. Objective: In this study, we further investigated the protein structure and function of RePAT isolated from the marine. Methods: The physicochemical properties, two-dimensional structure, three-dimensional structure, and functional domains of the RePAT protein were analyzed and predicted using bioinformatics tools, and RePAT was optimized according to codon bias present in Arabidopsis thaliana. Sitedirected mutagenesis of RePAT was performed, and the wild-type (RePAT) and mutant (RePATV120I) genes were successfully transferred into A. thaliana. Results: Our results showed that RePAT was an unstable hydrophilic protein, and six phosphorylation sites and two N-glycosylation sites were predicted. In addition, conserved domains containing the NAT_SF super family and coenzyme A-binding pocket were predicted in RePAT. Transgenic experiments and glufosinate resistance assays showed that the glufosinate resistance of A. thaliana containing the mutant gene (RePATV120I) was lower than that of plants containing the wild-type gene, indicating that the missense mutation in RePATV120I had a significant effect on its glufosinate resistance. Conclusions: Our study provided improvement result for knowing the transferring herbicide resistance gene RePAT.