The Overexpression of a Feedback -Insensitive Anthranilate Synthase in Arabidopsis and Tobacco
Tsai, Fei-Yi
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https://hdl.handle.net/2142/85006
Description
Title
The Overexpression of a Feedback -Insensitive Anthranilate Synthase in Arabidopsis and Tobacco
Author(s)
Tsai, Fei-Yi
Issue Date
2002
Doctoral Committee Chair(s)
Widholm, Jack M.
Department of Study
Crop Sciences
Discipline
Crop Sciences
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Biology, Plant Physiology
Language
eng
Abstract
Anthranilate synthase (AS) is the controlling enzyme of the Trp biosynthetic pathway. The activity of AS is feedback inhibited by Trp and its analogs such as 5-methyl Trp (5-MT). Trp analogs are toxic to cells because they cause false feedback inhibition of AS and hinder the normal production of Trp. A feedback insensitive AS gene (ASA2) was previously cloned from a 5-MT-resistant tobacco cell culture. In this study, ASA2 was expressed under the control of the CaMV 35S promoter in Arabidopsis and tobacco to determine the effect of expression of ASA2 on Trp production and Trp-associated secondary metabolism. Transgenic Arabidopsis showed AS activity that was insensitive to inhibition caused by Trp, and had up to 4-times higher free Trp levels than the wild type plants. The levels of the Trp-derived compounds indole glucosinolates were not altered in transgenic plants, suggesting that the increased Trp pool did not affect the synthesis of indole glucosinolates. Transgenic Arabidopsis also exhibited greater resistance than the wild type to all of the Trp and anthranilate analogs tested (4-MT, 5-MT, 6-MT, alpha-MT, 3-MA, 5-MA, 6-MA, 5-FA). alpha-MT effectively inhibited the germination of wild type seeds at 15 muM while the growth of the transgenic seedlings was not affected. Direct selection of T1 ASA2 transgenic seeds on alpha-MT allowed identification of the putative transformants, indicating that ASA2 and alpha-MT could be an effective selectable marker system for Arabidopsis transformation. Similarly, transgenic tobacco plants that overexpress ASA2 exhibited higher AS activity, elevated free Trp levels, and greater resistance to the Trp analogs tested (4-MT, 5-MT, alpha-MT). The transgenic leaf explants were able to regenerate shoots and roots on medium containing alpha-MT while the wild type were not, indicating that alpha-MT can be used for the selection and regeneration of tobacco transformants. Another objective of this study was to determine if a deplicate set of Trp pathway enzymes exists in the cytosol. ASA2 was targeted to the cytosol of Arabidopsis and tobacco. Preliminary results showed that Trp levels were not altered in these transgenic plants. More experiments are needed before one can draw conclusions about this study.
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