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To date, only  bar/pat  gene derived from  Streptomyces  has been used to generate the commercial PPT-resistant crops currently available in the market. The limited source of  bar/pat  gene is probably what has caused the decrease in PPT-tolerance, which has become the main concern of those involved in field management programs. Although glutamine synthetase (GS) is the target enzyme of PPT, little study has been reported about engineering PPT-resisitant plants with  GS  gene. Then, the plant-optimized  GS  gene from  Oryza sativa  ( OsGS1S ) was chemically synthesized in the present study by PTDS to identify a  GS  gene for developing PPT-tolerant plants. However,  OsGS1S  cannot be directly used for developing PPT-tolerant plants because of its poor PPT-resistance. Thus, we performed DNA shuffling on  OsGS1S , and one highly PPT-resistant mutant with mutations in four amino acids (A63E, V193A, T293A and R295K) was isolated after three rounds of DNA shuffling and screening. Among the four amino acids substitutions, only R295K was identified as essential in altering PPT resistance. The R295K mutation has also never been previously reported as an important residue for PPT resistance. Furthermore, the mutant gene has been transformed into  Saccharomyces cerevisiae  and  Arabidopsis  to confirm its potential in developing PPT-resistant crops.

Identification of a phosphinothricin-resistant mutant of rice glutamine synthetase using DNA shuffling