Expression Sequence Tag System And Application Of Medicinal Plants Based On High-throughput Sequencing Technology

EST analysis is a cost-effective and rapid tool used for the isolation of novel genes and characterization of the gene expressed profile. We used a cDNA library construction method and massive parallel pyrosequencing on the Roche 454-GS FLX Titanium platform to generate EST datasets of medicinal plants for transcriptome analysis. The EST labraries of Glycyrrhiza uralensis, Salvia miltiorrhiza, H. serrata and P. carinatus were constructed in our studies.Glycyrrhiza uralensis is one of the most popular medicinal plants in the world and is also widely used in the flavoring of food and tobacco. Due to limited genomic and transcriptomic data, the biosynthetic pathway of glycyrrhizin, the major bioactive compound in G. uralensis, is currently unclear. We used the 454 GS FLX platform and Titanium regents to produce a substantial expressed sequence tag (EST) dataset from the vegetative organs of G. uralensis. A total of 59,219 ESTs with an average read length of 409 bp were generated.454 ESTs were combined with the 50,666 G. uralensis ESTs in GenBank. The combined ESTs were assembled into 27,229 unique sequences (11,694 contigs and 15,535 singletons). A total of 20,437 unique gene elements representing approximately 10,000 independent transcripts were annotated using BLAST searches (e-value≤le-5) against the SwissProt, KEGG, TAIR, Nr and Nt databases. The assembled sequences were annotated with gene names and Gene Ontology (GO) terms. With respect to the genes related to glycyrrhizin metabolism, genes encoding 16 enzymes of the 18 total steps of the glycyrrhizin skeleton synthesis pathway were found. To identify novel genes that encode cytochrome P450 enzymes and glycosyltransferases, which are related to glycyrrhizin metabolism, a total of 125 and 172 unigenes were found to be homologous to cytochrome P450s and glycosyltransferases, respectively. The cytochrome P450 candidate genes were classified into 32 CYP families, while the glycosyltransferase candidate genes were classified into 45 categories by GO analysis. Finally,3 cytochrome P450 enzymes and 6 glycosyltransferases were selected as the candidates most likely to be involved in glycyrrhizin biosynthesis through an organ-specific expression pattern analysis based on real-time PCR.To investigate the profile of gene expression in Salvia miltiorrhiza and discover its functional gene, we used the 454 GS FLX platform and Titanium regent to produce a substantial expressed sequence tags (ESTs) dataset from the root of S. miltiorrhiza. A total of 46 722 ESTs with an average read length of 414 bp were generated. The 454 ESTs were combined with the S. miltiorrhiza ESTs from GenBank. These ESTs were assembled into 18 235 unigenes. Of these unigenes,454 sequencing identified 13 980 novel unigenes.73% of these unigenes (13 308) were annotated using BLAST searches (e-value≤le-5) against the SwissProt, KEGG, TAIR, Nr and Nt databases. We found 27 unigenes (encoding 15 enzymes) involved in tanshinones biosynthesis, and 29 unigenes (encoding 11 enzymes) involved in phenolic acids biosynthesis. We also found 70 putative genes encoding cytochromes P450 and 577 putative transcription factor genes.Plants of the Huperziaceae family, which comprise the two genera Huperzia and Phlegmariurus, produce various types of lycopodium alkaloids. The lycodine type alkaloid of Huperzine A (Hup A) has been used as an anti-Alzheimer's disease drug candidate, possessing good market prospects. Despite their medical importance, little genomic or transcriptomic data are available for the members of this family, which seriously hampered the development and use of new drugs. For H. serrata and P. carinatus 454-EST datasets,36763 and 31812 unigenes were generated from 140930 and 79920 reads, respectively. Approximately 115 H. serrata and 98 P. carinatus unigenes associated with the biosynthesis of triterpenoids, alkaloids and flavone/flavonoids were located in the 454-EST datasets. A total of 63 unigenes encoding cytochrome P450s (CYP450s) were present in both of H. serrata and P. carinatus. In particular,20 H. serrata CYP450s candidate genes, which are more abundant in leaves than in roots and might be involved in lycopodium alkaloid biosynthesis, were identified based on the comparison of H. serrata and P. carinatus 454-ESTs and real-time PCR analysis. The expressed profiles of these candidate genes were consistent with the organ-specific accumulation pattern of Hup A, which is accumulated higher in H. serrata leaves and lower in roots. In addition,2729 and 1573 potential SSR-motif microsatellite loci were identified from the H. serrata and P. carinatus 454-ESTs, respectively.The 454-EST resource allowed for the first large-scale EST project and transcriptome analysis for G. uralensis,S. miltiorrhiza,H. serrata and P. carinnatuas. We identified many candidate genes involved in the biosynthesis of bioactive compounds and developmental regulation. These results establish an essential resource for understanding secondary metabolite biosynthesis. This study will lay the foundation for the production of glycyrrhizin, Tanshinone, Salvianolic acid and Hup A using biotechnologies.