| Isoflavones are phenylpropanoids that are found mainly in legumes, particularly soybean. In plants, they help in establishing a relationship with the nitrogen-fixing bacteria Rhizobium, act as chemoattractants, antifeedants, and anti-microbials that provide disease resistance in plants. When consumed in the human diet, they can reduce menopausal symptoms, cardiovascular disease symptoms, aid in the prevention of prostate and breast cancer, and also reduce osteoporosis. Cowpea is a highly nutritious crop, being rich in proteins and carbohydrates, but the isoflavone content is negligible. So, it would be beneficial for millions of people in developing countries such as Africa and Asia where cowpea is consumed as a staple crop, if the isoflavone content could be increased. The first committed step in the production of isoflavones is performed by a cytochrome P450-like enzyme, isoflavone synthase, which catalyzes a 2, 3 aryl migration of flavanones to isoflavones. We have cloned and characterized two isoflavone synthase genes (VuIFS1 & VuIFS2 ), with their respective promoters, from cowpea (Vigna unguiculata L. Walp.). We found that there are three poly A signals in the 3'UTR of VuIFS1 mRNA, potentially resulting in three transcripts of different lengths. RT-PCR analysis indicated that both were expressed in stem and leaf tissues only. Both gene products were able to produce daidzein and genistein from liquiritigenin and naringenin, respectively, in a yeast in vivo experiment. After introducing soybean IFS1 with its native promoter, daidzein and genistein levels increased in transgenic cowpea calli. Conjugated forms of isoflavones such as daizin, malonyl-glucosyl daidzin, genistin, malonyl-glucosyl genistin, glycitein (O-methylated form of liquiritigenin) were formed, which indicates that cowpea endogenous glucosyltrasferase enzymes were able to recognize newly formed isoflavones. These results suggest that, once transgenic plants are obtained, cowpea with enhanced isoflavone levels may be produced. |
