Identification of a phosphinothricin-resistant mutant of rice glutamine synthetase using DNA shuffling.
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-resistant 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.
Biotechnology Research Institute of Shanghai Academy of Agricultural Sciences, Shanghai, 201106, China.|Shanghai Ruifeng Agricultural Science and Technology Co., Ltd, Shanghai, 201106, China.|College of horticulture, Nanjing Agricultural University, Nanjing 210095, China.
Yong-Sheng
YS
Tian
Biotechnology Research Institute of Shanghai Academy of Agricultural Sciences, Shanghai, 201106, China.
Jing
J
Xu
Biotechnology Research Institute of Shanghai Academy of Agricultural Sciences, Shanghai, 201106, China.
Wei
W
Zhao
College of horticulture, Shanxi Agricultural University, Taigu 030801, China.
Xiao-Juan
XJ
Xing
Biotechnology Research Institute of Shanghai Academy of Agricultural Sciences, Shanghai, 201106, China.
Xiao-Yan
XY
Fu
Biotechnology Research Institute of Shanghai Academy of Agricultural Sciences, Shanghai, 201106, China.
Ri-He
RH
Peng
Biotechnology Research Institute of Shanghai Academy of Agricultural Sciences, Shanghai, 201106, China.
Quan-Hong
QH
Yao
D000613:Aminobutyrates / Q000494:pharmacology*; D001483:Base Sequence; D005974:Glutamate-Ammonia Ligase / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D012275:Oryza / Q000201:enzymology*; D030821:Plants, Genetically Modified; D012689:Sequence Homology, Nucleic Acid
D000613:Aminobutyrates; C003121:phosphinothricin; D005974:Glutamate-Ammonia Ligase