Isolation and characterization of soybean chalcone reductase cDNA, which encodes the key enzyme for the biosynthesis of 4,2 0 ,4 0 -trihydroxychalcone in legumes

In plants, phytoalexins induced by patho- gen attack play an important role in disease resistance. In soybean (Glycine max L. (Merr.)), attack by pathogenic bacteria induces the synthesis of isoflavo- noids, especially daidzein. Chalcone reductase (CHR) is the key enzyme in the biosynthesis of daidzein. Along with chalcone synthase, it catalyzes the forma- tion of isoliquiritigenin, which is a necessary substrate for daidzein biosynthesis. In this study, a CHR gene, Gmchr2 (GenBank code: KF758395), was isolated from the soybean cultivar Jinong 17. The cDNA consisted of a 1,417-bp fragment that included an open reading frame of 969 bp. The gene is located on chromosome 9 of the soybean genome. Phytophthora sojae was inoculated onto soybean roots, and changes in the transcript levels of the chr genes and the catalytic activity of CHR were investigated in differ- ent soybean tissues by real-time fluorescence quanti- tative PCR and high-performance liquid chromatography (HPLC), respectively. The results showed that the isoliquiritigenin content in roots significantly increased after pathogen inoculation. The Gmchr2 gene was transformed into tobacco, and the presence of isoliquiritigenin in the transformants was confirmed by HPLC. Expression of the Gmchr2 gene under the control of the 35S CaMV promoter was also confirmed. This characterization of a chr gene encod- ing a soybean CHR helps to shed light on the biological synthesis and regulation of soybean iso- flavones and will be useful for manipulating the phenylpropanoid pathway leading to isoflavonoid phytoalexins.