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 . Site-directed mutagenesis of RePAT was performed, and the wild-type ( RePAT ) and mutant ( RePAT V120I ) 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 ( RePAT V120I ) was lower than that of plants containing the wild-type gene, indicating that the missense mutation in RePAT V120I had a significant effect on its glufosinate resistance.
Conclusions
Our study provided improvement result for knowing the transferring herbicide resistance gene RePAT .
RePAT; Site-directed mutagenesis; Glufosinate resistance; Arabidopsis thalia
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The Val120 is the mutant site
a. The plant expression vector. RePAT and RePAT V120I transcriptions were initiated by the CaMV 35S Pro promoter and terminated by NOS polyA. Kanamycin was used for screening plasmid-positive T1 plants. NcoI and PmlI were the two restriction enzyme digestion sites used to construct the vector. LB, left border; RB, right border; b. Glufosinate resistance assays of transgenic Arabidopsis plants. A. Germination of the Arabidopsis group containing RePAT V120I was slightly inhibited on half-strength MS medium containing 1 mg/L glufosinate, whereas that of the Arabidopsis group containing RePAT was normal. B. Germination of the Arabidopsis group containing RePAT V120I was moderately inhibited on half-strength MS medium containing 5 mg/L glufosinate, whereas that of the Arabidopsis group containing RePAT was normal. C. Germination of the Arabidopsis group containing RePAT V120I was completely inhibited on half-strength MS medium containing 10 mg/L glufosinate, whereas that of the Arabidopsis group containing RePAT was normal. D. Germination of the Arabidopsis group containing RePAT V120I was completely inhibited on half-strength MS medium containing 20 mg/L glufosinate, whereas that of the Arabidopsis group containing RePAT was certainly inhibited. NOS, nopaline synthase; MS, Murashige and Skoog
a. The transmembrane structure of the RePAT protein. b. Prediction of the functional domain. The red arrow indicates the amino acid site of the coenzyme A-binding pocket. c. Prediction of the conserved domain