Promoter Analysis of a Rice Seed Storage Protein Glutelin Gene and Expression of Bean 7S Seed Storage Protein Beta-Phaseolin in Transgenic Rice Plants.

It was demonstrated, using a homologous transgenic rice system, $5\sp\prime$ distal and proximal cis-acting transcriptional regulatory elements were required for developmental control of a rice seed storage protein glutelin (Gt1) gene. The functional importance of proximal elements in the context of the 1.8 kb promoter was demonstrated by single substitution mutations in the TATA box ($-$28/$-$23), AACA motif ($-$73/$-$61), and protein-binding Boxes I ($-$103/$-$86), II ($-$124/$-$110), III ($-$175/$-$158), and IV ($-$200/$-$217). A simultaneous mutation of five protein-binding sites ($-$410/$-$86) essentially eliminated the activity of the 1.8 kb promoter. To understand the function of the $5\sp\prime$ distal cis-acting upstream sequence, eighteen deletions were made in the region between $-$5.1k and $-$507, relative to the transcriptional initiation site. The results showed that the long distal cis-acting upstream sequence ($-$5.1k/$-$508) greatly enhanced quantitative expression of GUS reporter gene in developing endosperm and increased the number of plants showing high accumulation of pea 11S legumin in mature endosperm. As an initial approach to increasing the lysine content of rice seed proteins for nutritional quality improvement, the bean 7S storage protein $\beta$-phaseolin gene was introduced into rice. The highest quantity of phaseolin detected by ELISA was 4% of the total endosperm protein in the transgenic rice seeds under the direction of the 5.1 kb Gt1 promoter. The lysine content of the soluble protein fraction was significantly improved by 30% in these seeds. Phaseolin was stably deposited in mature rice seeds. Both phaseolin genomic and cDNA coding sequences produced phaseolin polypeptides with apparent molecular weights of 50.5, 47.7, 46.9 and 44.9 kd. Differential glycosylation contributed to the apparent isoforms. The results indicated a promising avenue to improving the nutritional quality of rice