Biosynthesis and Regulation of Bioactive Compounds In a Medicinal Plant—Epimedium

Epimedium, also known as Horny Goat Weed or Ying Yang Huo, has been extensively used as tradationanl medicinal plant in China for more than 2000 years. In the last decades, although the focuses of Epimedium studies were restricted to extract the bioactive compounds such as epimedin A, epimedin B, epimedin C, and icariin, little is known about the potential molecular mechanism for the biosynthesis and regulation of these bioactive compounds in Epimedium. An EST dataset corresponding for 50.9 Mb was generated by 454 GX-FLS sequencing red-magenta fully expanded leaves of E. sagittatum Maxim. Consequently, 76,459 consensus sequences were obtained and submitted to bioinformatic analysis. Based on the KEGG analysis, 511, 102, and 383 consensus sequences were annotated and involved in flavonoid, carotenoid, and terpenoid pathways, respectively. Furthermore, these gene fragments and other flavonoid gene fragments isolated by SSH and homolog cloning were used as core fragments for cloning full-length flavonoid genes by RACE technology. Subsequently, all of the structural genes involved in Epimedium flavonoid pathway were obtained and several full-length transcriptional factors, such as MYB, bHLH, and WD, were also isolated. Moreover, several key genes have been functionally characterized. In addition, an EsaCyc database was established and the pathway of icariin biosynthesis was predicted, which was partly confirmed by experimentally cloning of three copies of isopentenyltransferase. All these results of bioinformatics analysis, 454 EST dataset, and traditional functional characterization facilitate the study toward uncovering flavonoid molecular mechanism in Epimedium. Therefore, combination of bioinformatic methods, new generation of sequencing technology (454 technology), and common molecular biology technologies was a great strategy to study non-model plants with huge genome, for example Epimedium.