Increasing tryptophan synthesis in a forage legume Astragalus sinicus by expressing the tobacco feedback-insensitive anthranilate synthase (ASA2) gene

A cDNA clone that encodes a feedback-insensitive anthranilate synthase (AS) , ASA2, isolated from a 5-methyl-tryptophan (Trp) (5MT)-resistant tobacco c ell line under the control of the constitutive cauliflower mosaic virus 35S promoter, was introduced into the forage legume Astragalus sinicus by Agro bacterium rhizogenes with kanamycin selection. The 35S-ASA2 gene was expres sed constitutively as demonstrated by northern-blot hybridization analyses and the presence of feedback-insensitive AS. Hairy root lines transformed w ith 35S-ASA2 grew in concentrations of up to 100 mu M 5MT, whereas the cont rols were completely inhibited by 15 mu M 5MT. Expression of the feedback-i nsensitive ASA2 resulted in a 1.3- to 5.5-fold increase in free Trp. Kineti c studies of the AS activity demonstrate the Trp feedback alterations and i ndicate that the ASA2 alpha-subunit can interact with the native A. si beta -subunit to form an active enzyme. The ASA2 transcript and high free Trp we re also detected in the leaves, stems, and roots of plants regenerated from the transformed hairy roots. Thus, we show for the first time that ASA2 ca n be used to transform plants of a different species to increase the levels of the essential amino acid Trp and impart 5MT resistance.