Modification of rice [Oryza sativa] grain components by recombinant DNA technology

An antisense gene strategy was applied to suppress expression of the allergen genes and waxy gene in maturing rice seeds. A 16 kDa albumin in rice grain has been identified as a major allergenic protein which sometimes causes severe atopic disease. In order to apply a cDNA for the antisense regulation of this gene expression, we initially constructed antisense genes using portions of the cDNA and RA promoter. They wee introduced into rice protoplasts by electroporation. RA content in the grain from transgenic plants was analyzed using mouse monoclonal antibody raised against RA by Western blot. Among these transgenic plants, several lines showed significantly lower level of RA content. This result suggested that the antisense gene efficiently repressed production of the rice allergenic proteins. However, degree of RA reduction was fluctuated between seeds and seeds even from the single plant. In order to achieve stable and high level of reduction of allergenic proteins, we constructed other antisense constructs utilizing the entire cDNA and either promoter of the RA, the rice starch branching enzyme, the rice prolamine and the rice glutelin genes, which were specifically expressed in developing seeds. These antisense genes were co-transferred into rice, and a transformant, that was confirmed to contain several kinds of antisense constructs, showed very low level of allergenic protein contents (more than 80% of reduction). The high levels of reduction observed were stably inherited in at least three generations. The waxy gene encodes a granule-bound starch synthase. A 1.0-kb portion of the sequence of the waxy gene was inserted in an antisense orientation between CaMV 35 S promoter and GUS gene of pBI 221. The resultant plasmid was introduced into rice protoplasts. Some of the rice seeds from these transformants showed a significant reduction of the amylose content of the grain starch