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The sequencing and analysis of expressed sequence tags has been a primary tool for the discovery of novel genes in plants, especially in non model plants for which UNC2250 full genome sequences are not currently available, EST sequencing represents a rapid and cost effective method for analyzing the transcribed regions of genomes. EST analysis is also a powerful tool for the discovery of genes involved in plant secondary metabolism. The 454 GS FLX sequencing technology has made EST based resources more readily accessible for non model organism tran scriptomes, Our experimental focus for this study was Glycyrrhiza uralensis Fisch. ex DC, which is one 4μ8C of the most ancient medicinal herbs and has been used as a Chinese herbal medicine to treat infectious diseases for over 3,000 years, This herb has been extensively stud ied and is widely used as a flavoring agent, medicament and tobacco additive.
Many of the biological activities of the bioactive constituents of G. uralensis have been inves tigated, including the protection against hepatotoxicity, anti ulcer effects, anti inflammatory and anti tumor promoting activities, This herb also exhib its antiviral activity against various DNA and RNA viruses, including herpes GSK525762A simplex virus, HIV and severe acute respiratory syndrome associ ated coronavirus, These biological activities of G. uralensis have been pri marily attributed to two of its components, flavonoids and saponins. Our research interests primarily concern glycyrrhizin, an oleanane type triterpene saponin and a well known natural sweetener that is fifty times sweeter than sugar, Although the various chemical and phar macological properties of glycyrrhizin in G.
uralensis have been extensively studied, the biosynthetic pathway of this compound remains poorly understood. Two func tional genes encoding squalene synthase have been isolated from G. uralensis, Two cytochrome P450 genes have also been isolated from G. uralensis Neuroblastoma based on the traditional EST sequencing method, CYP88D6, a cytochrome P450 monooxygenase, was characterized by in vitro enzymatic activity assays and was shown to cata lyze the oxidation of B amyrin at C 11 to produce 11 oxo B amyrin, a possible biosynthetic intermediate in the gly cyrrhizin biosynthetic pathway, Another cyto chrome P450 from G. uralensis, CYP93E3, GSK525762A possesses B amyrin 24 hydroxylase activity in in vitro enzymatic activity assays, A functional B amyrin synthase gene has been isolated from G.
glabra, Thus far, only one glycosyltransferase in the Glycyrrhiza UNC2250 genus, the isoflavonoid glucosyltransferase in G. echinata, has been identified, However, no progress has been made in the identification of the genes involved in the glycosyla tion of glycyrrhetinic acid to produce glycyrrhizin. Tran scriptome sequencing would provide a GSK525762A foundation for detailed studies of gene expression and genetic connec tivity with respect to plant secondary metabolism. In our study, we constructed a cDNA library using the vegetative organs of five year old wild G. uralensis culti vated from the city of Yanchi in the Ningxia province of China, one of the most famous areas for the production of wild G. uralensis.
The library was sequenced using the 454 GS FLX platform and Titanium reagents. There are currently 50,666 G. uralensis ESTs in the GenBank dbEST database, which were determined using conventional sequencing techniques, In our study, we increased this collection with an additional 59,219 ESTs generated from 454 GS FLX Titanium sequencing. UNC2250 Bioinformatic analyses indicated that almost all of the genes involved in the biosynthesis of the glycyrrhizin skeleton were within the combined EST database, except for mevalonate kinase and DXP synthase, Additionally, a pool of candidate genes for cytochrome P450s and glycosyltransferases was estab lished, containing 125 and 172 unigenes, respectively. Finally, using an organ specific expression pattern analy sis, a few GSK525762A unigenes were selected as the candidates most likely to be responsi