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Tian Y.-S.,Biotechnology Research Institute of Shanghai Academy of Agricultural science | Tian Y.-S.,Shanghai Ruifeng Agricultural Science and Technology Co. | Tian Y.-S.,Nanjing Agricultural University | Xu J.,Biotechnology Research Institute of Shanghai Academy of Agricultural science | And 5 more authors.
Molecular Breeding | Year: 2015

To date, only AroA variant derived from Agrobacterium tumefaciens CP4 has been used to generate the commercial glyphosate-resistant crops currently available in the market. This single source of the EPSPS gene may have caused the decrease in herbicide tolerance, which has become a major concern of those involved in field management programs. Therefore, it is of interest to increase aroA/EPSPS gene diversity and seek new glyphosate-tolerant genes for developing glyphosate-tolerant crops. In the current study, EPSPS gene from Vitis vinifera (VvEPSPS) was cloned using reverse transcription polymerase chain reaction. However, wild type VvEPSPS cannot be directly used for developing transgenic crops because of its extreme glyphosate sensitivity. Recent studies have demonstrated that DNA shuffling is an effective strategy in producing multi-mutated EPSPS resourced from plants (EPSPSplant) with improved glyphosate resistance in bacteria and plants. After performing DNA shuffling on VvEPSPS gene, one highly glyphosate-resistant mutant with seven amino acid variations was isolated after five rounds of shuffling and screening. The mutant showed seven amino acid changes in the EPSPS gene, namely, Q93R, T113A, P117L, G126A, C160Y, N239H, and V343A. The assay of glyphosate resistance further confirmed the potential of the VvEPSPS mutant in developing glyphosate-resistant crops. © 2015, Springer Science+Business Media Dordrecht.


Gao J.-J.,Nanjing Agricultural University | Gao J.-J.,Shanghai Academy of Agricultural science | Shen X.-F.,Nanjing Agricultural University | Shen X.-F.,Shanghai Academy of Agricultural science | And 9 more authors.
Plant Cell, Tissue and Organ Culture | Year: 2011

The R2R3-Myb proteins comprise a large family of plant transcription factors that regulate the expression of multiple drought-responsive and cold-responsive genes by binding special cis-elements at the promoter. There have been previous studies in transgenic plant over-expression of the myb protein. In this article, we analyzed the function of MdMYB6, an R2R3-type Myb transcription factor from apples (Malus × domestica), in transgenic Arabidopsis plants. In contrast to wild type plants, these transgenic lines accumulated less anthocyanin. Equimolar concentrations of sorbitol did not alter the anthocyanin accumulation. Real-time polymerase chain reaction (PCR) indicated that MdMYB6 over-expression suppressed enzymes involved in anthocyanin biosynthesis and some Arabidopsis basic helix-loop-helix (bHLH) genes. The MdMYB6 transcription faction may be an important repressor of anthocyanin biosynthesis in plants. © 2011 Springer Science+Business Media B.V.


PubMed | Biotechnology Research Institute of Shanghai Academy of Agricultural science and Shanxi Agricultural University
Type: | Journal: Scientific reports | Year: 2015

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.


Tian Y.-S.,Biotechnology Research Institute of Shanghai Academy of Agricultural science | Peng R.-H.,Biotechnology Research Institute of Shanghai Academy of Agricultural science | Xu J.,Biotechnology Research Institute of Shanghai Academy of Agricultural science | Zhao W.,Biotechnology Research Institute of Shanghai Academy of Agricultural science | And 4 more authors.
World Journal of Microbiology and Biotechnology | Year: 2010

We applied in vitro mutagenesis and colony screening, using the wild type phyI1s gene from Aspergillus niger 113 as the template, and obtained two mutant phyI1s (gene products) after one round of screening. The two mutants had mutations at two nucleic acid sites, resulting in changes in two amino acids: K41E, E121F. None of the amino acid substitutions in the two mutants was in a position reported to be important for catalysis or substrate binding. Kinetic analysis of the phytase activity of the two mutants indicated that the substitutions gave rise to 2.5- and 3.1-fold increased specific activity, and a 1.78- and 3.24-fold reduced affinity for sodium phytate. In addition, the overall catalytic efficiency (kcat/Km) of the two mutants was changed by 0.52-fold and 0.68-fold compared to that of the wild type. Such mutants will be instrumental for the structure-function study of the enzyme and for industrial application. © Springer Science+Business Media B.V. 2009.


PubMed | Biotechnology Research Institute of Shanghai Academy of Agricultural science
Type: Journal Article | Journal: Applied and environmental microbiology | Year: 2010

Applying the genomic library construction process and colony screening, a novel aroA gene encoding 5-enopyruvylshikimate-3-phosphate synthase from Ochrobactrum anthropi was identified, cloned, and overexpressed, and the enzyme was purified to homogeneity. Furthermore, site-directed mutagenesis was employed to assess the role of single amino acid residues in glyphosate resistance.


PubMed | Biotechnology Research Institute of Shanghai Academy of Agricultural science
Type: Evaluation Studies | Journal: Molecular biology reports | Year: 2010

Thermal hysteresis proteins (Thps) known as antifreeze proteins for their antifreeze activity, depress the freezing point of water below the melting point in many polar marine fishes, terrestrial arthropods and plants. For the purpose of breeding cold-resistant plants, we designed to introduce the Thp gene into the plants. The physiological and biochemical effect of high-lever expression of the modified Choristoneura fumiferana Thp (ThpI) in Arabidopsis thaliana plants was analyzed. Under low temperature stress, the ThpI transgenic plants exhibited stronger growth than wild-type plants. The elevated cold tolerance of the ThpI over-expressing plants was confirmed by the changes of electrolyte leakage activity, malonyldialdehyde and proline contents. These results preliminarily showed that the Thp possibly be used to enhance the low temperature-tolerant ability of plants.

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