Increasing Tryptophan Synthesis in a Forage Legume Astragalus sinicus by Expressing the Tobacco Feedback-Insensitive Anthranilate Synthase (ASA2) Gene

Abstract

Abstract A cDNA clone that encodes a feedback-insensitive anthranilate synthase (AS), ASA2, isolated from a 5-methyl-tryptophan (Trp) (5MT)-resistant tobacco cell line under the control of the constitutive cauliflower mosaic virus 35S promoter, was introduced into the forage legume Astragalus sinicus byAgrobacterium rhizogenes with kanamycin selection. The 35S-ASA2 gene was expressed constitutively as demonstrated by northern-blot hybridization analyses and the presence of feedback-insensitive AS. Hairy root lines transformed with 35S-ASA2 grew in concentrations of up to 100 μm 5MT, whereas the controls were completely inhibited by 15 μm 5MT. Expression of the feedback-insensitive ASA2 resulted in a 1.3- to 5.5-fold increase in free Trp. Kinetic studies of the AS activity demonstrate the Trp feedback alterations and indicate that the ASA2 α-subunit can interact with the native A. sinicus β-subunit to form an active enzyme. TheASA2 transcript and high free Trp were also detected in the leaves, stems, and roots of plants regenerated from the transformed hairy roots. Thus, we show for the first time that ASA2can be used to transform plants of a different species to increase the levels of the essential amino acid Trp and impart 5MT resistance.