Overexpression of the feedback-insensitive anthranilate synthase gene in tobacco causes tryptophan accumulation.

Abstract

A total of 35 independent transgenic tobacco plants were produced using the Agrobacterium tumefaciens-leaf segment co-cultivation method followed by selection with kanamycin for the nptII gene. The vector also carried the tobacco feedback-insensitive anthranilate synthase gene (ASA2). Many of the lines showed increased ASA2 mRNA levels but only three contained increased free tryptophan (Trp) and many lines contained lower Trp than the untransformed control. The line with the highest Trp level (threefold that of the untransformed control) contained increased anthranilate synthase activity (AS) both in leaves and a cell suspension culture derived from the plant while the feedback insensitivity was most evident in the suspension culture. Other kinetic data also indicated that the ASA2 encoded AS alpha-subunit was more abundant in the tissue culture than in leaves. Progeny seedlings from this line were resistant to certain toxic Trp analogs, especially alpha-methyltryptophan (alphaMT) and less so to the most commonly used analog, 5-methyltryptophan. Shoots formed more readily from leaves of two of the transgenic lines than from leaves of the untransformed control on alphaMT, indicating that it might be possible to use ASA2 as a selectable marker gene and alphaMT as the selection agent.