Genistein production in rice seed via transformation with soybean IFS genes.

To produce genistein in rice, the isoflavone synthase (IFS) genes, SpdIFS1 and SpdIFS2 were cloned from the Korean soybean cultivar, Sinpaldalkong II as it has a higher genistein content than other soybean varieties. SpdIFS1 and SpdIFS2 show a 99.6% and 98.2% identity at the nucleotide level and 99.4% and 97.9% identity at the amino acid level, respectively, with IFS1 and IFS2 from soybean (GenBank accession Nos. AF195798 and AF195819). Plant expression vectors were constructed harboring SpdIFS1 or SpdIFS2 under the control of a rice globulin promoter that directs seed specific expression, and used to transform two rice varieties, Heugnam, a black rice, and Nakdong, a normal rice cultivar without anthocyanin pigment. Because naringenin, the substrate of SpdIFS1 and SpdIFS2, is on the anthocyanin biosynthesis pathway, the relative production rate of genistein was compared between SpdIFS-expressing transgenic Heugnam and Nakdong. Southern blot analysis of eight of the resulting transgenic rice plants revealed that the T0 plants had one to three copies of the SpdIFS1 or SpdIFS2 gene. The highest level of genistein content found in rice seeds was 103 µg/g. These levels were about 30-fold higher in our transgenic rice lines than the genistein aglycon content of a non-leguminous IFS-expressing transgenic tobacco petal, equaling about 12% of total genistein content of Sinpaldalkong II. There were no significant differences found between the genistein content in Heugnam and Nakdong transgenic rice plants.