Tryptophan synthesis block-associated sugar response, a physiological marker for rapid selection of Arabidopsis thaliana transformants, marked with feedback-inhibition-insensitive anthranilate synthase gene |
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| Authors: | T Nonomura S Komaki L Xu N Moriura H Ioroi S Takashima K Kakutani Y Takikawa Y Matsuda and H Toyoda |
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| Institution: | Laboratory of Plant Protection Science and Biotechnology, Faculty of Agriculture, Kinki University, 3327-204 Nakamachi, Nara 631-8505, Japan;;School of Life Science, East China Normal University, Shanghai 200062, China;;Pharmaceutical Research and Technology Institute, Kinki University, 3-4-1 Kowakae, Higashi-Osaka, Osaka 577-8502, Japan;;Plant Center, Institute of Advanced Technology, Kinki University, Wakayama 644-0025, Japan;;Corresponding author, E-mail: |
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| Abstract: | The feedback-insensitive anthranilate synthase (AS) gene was used as a selection marker for transformants of Arabidopsis thaliana . The mutant gene ( mAS1-2 ) was constructed by substituting nucleotide at the effector-binding site of the intrinsic AS gene via PCR-mediated site-directed mutagenesis and flanked with the myrosinase promoter pyk10 to drive its expression during initial root elongation. This inducible gene cassette was first introduced into Agrobacterium tumefaciens and then delivered into A. thaliana by floral-dip inoculation. 5-methyltryptophan (5-MT) inhibited AS and suppressed seedling growth of wild type plants as a result of tryptophan starvation. With the addition of sucrose (10 mg/ml), 5-MT inhibited cotyledon opening and caused anthocyanin to accumulate in juvenile seedlings. The present mutant reversed the tryptophan starvation caused by 5-MT and blocked subsequent sugar responses. The sugar responses were detected in non-transformed plants grown on a selection medium containing 10 mg/ml of sucrose and 10 μg/ml of 5-MT after 3 days of incubation. Thus, true transformants could be selected after a short incubation, compared to the conventional kanamycin-selection method that did not eliminate all non-transformed plants. |
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| Keywords: | anthranilate synthase gene 5-methyltryptophan selection marker sugar response feedback-inhibition-insensitive mutant |
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