Production of Hypoallergenic Rice by Gene Engineering

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Several studies on allergic reaction to cereal grains have suggested that sera from some patients with atopic food allergy contain immunoglobulin E (IgE) reacting with rice proteins, and that soluble proteins, albumins and globulins have a high degree of allergenic activity. We isolated a rice seed protein of about 16kDa with reactivity for IgE in rice-allergic patients, and the 16kDa-protein was later identified as one of the major allergenic in rice seed.A cDNA clone (RA17) encoding the rice major allergenic protei (RAP) was isolated from cDNA library of maturing rice seeds. An antisence RNA strategy was applied to repress RAP gene expression in maturing rice seeds. Some chimeric genes encoding antisence RNAs of RAP under the control of CaMV 35S promoter, rice prolamin promoter and RAP promoter were introduced into rice by electroporation. Protophasts isolated from rice suspension cells were co-transformed with each antisence gene and a hygromycin phosphotransferase (HPT) gene linked to the CaMV 35S promoter, and rice plants were independently regenerated from hygromysin-resistant calli. Total DNA isolated from leaves of the regenerated plants was digested with some restriction enzymes, and Southern blot analyzes were done using an RAP cDNA as a probe. In several clones, an additional band with an expected size of the antisence gene was observed. Selfed seeds from these transgenic plants with the antisence gene were tested for RAP content by SDS-PAGE/immunoblotting and ELISA using a monoclonal antibody specific for RAP.The RAP bands stained with the antibody of seeds from some transgenic plants were much weaker than those of the wild-type control. ELISA analysis for proteins extracted from the trangenic and wild-type rice seeds suggested that RAP content of rice seeds was reduced to about 30% of the original one. Studies on stable transmission and expression of the introduced antisence gene among the transformant progeny are in progress.