Abstract
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.
Highlights
Recent advances in genetic engineering have rendered possible the transfer of PPT-tolerant genes to plants to produce PPT-tolerant crops, thereby ensuring that only weeds can be killed by PPT
Wild-type OsGS1S cannot be directly used for the development of transgenic PPT-tolerant plants because of its extreme PPT sensitivity
We performed DNA shuffling on OsGS1S, and one highly PPT-resistant mutant with four amino acids variations was isolated after three rounds of DNA shuffling and screening
Summary
Recent advances in genetic engineering have rendered possible the transfer of PPT-tolerant genes to plants to produce PPT-tolerant crops, thereby ensuring that only weeds can be killed by PPT. Biosafety concerns are being raised in relation to the transformation of the heterologous PPT-resistant gene from pathogenic bacteria into crop plants. In another way, the recently discovery of PPT-resistant transgenic plant with an insensitive mutant GS gene resourced from plants has provided an alternative to the bar/pat gene of microorganisms for crop engineering[8]. Obtaining novel and valid PPT-insensitive GS genes which is acceptable mentally by customer is critical for the development of PPT-resistant transgenic crops in future. We performed DNA shuffling on OsGS1S gene under selective pressure caused by high PPT concentrations with the aim of obtaining mutants with improved PPT tolerence. The mutant gene has been transformed into Saccharomyces cerevisiae and Arabidopsis to confirm its potential in developing PPT-resistant crops
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