Normal and lysine-containing zeins are unstable in transgenic tobacco seeds

Abstract

Chimeric genes composed of the beta-phaseolin promoter, an alpha-zein coding sequence and its modified versions containing lysine codons, and a beta-zein polyadenylation signal were inserted into the genome of tobacco by Agrobacterium-mediated transformation. alpha-Zein mRNA levels in the transgenic tobacco seeds 20 days after self-pollination varied between 1.0% and 2.5% of the total mRNA population. At 25 days after pollination the 19 kDa alpha-zein was immunologically detected with a polyclonal antiserum in protein extracts from the seeds of transgenic plants. The transgenic plant with the highest level of zein gene expression had an alpha-zein content that was approximately 0.003% of the total seed protein. The amount of alpha-zein in other transgenic plants varied between 1 x 10(-4)% and 1 x 10(-5)% of the total seed protein. The differences in the amounts of mRNA and protein did not correlate with the lysine substitutions introduced into the alpha-zein protein. Polysomes translating alpha-zein mRNA isolated from tobacco seeds contained fever ribosomes than those from maize endosperm, but this did not appear to be the cause of the inefficient protein synthesis. In vivo labelling and immunoprecipitation indicated that newly synthesized alpha-zein was degraded in tobacco seeds with a half-life of less than 1 hour.