ABSTRACT: Screening and pathway analysis of genes related to biosynthesis of triterpenoid saponins in hairy root of Silene viscidula : high-throughput cloning method of comparative transcriptome based on elicitors induced chemical component difference phenotype
Project description:Scutellaria viscidula Bunge (Labiatae), a perennial herb, is an important medicinal plant that possesses broad pharmacological actions. S. viscidula contains flavonoids with good bioactivities (e.g. baicalin, wogonoside, baicalein and wogonin) mainly in its dry root, which is used as alternative to Scutellaria baicalensis in the north of China. Furthermore, S. viscidula also has flavones with interesting diverged structures such as panicolin, viscidulin I, viscidulin II and viscidulin III. Tracing the dynamic process of gene expression will help reveal the mechanism of flavonoid synthesis in S. viscidula, as well as the 4'-deoxyflavone biosynthesis in S. baicalensis. One way is to generate and analyze the expressed sequence tags (ESTs). However, little is known on the transcriptome information of S. viscidula, particularly the key genes involved in flavonoid biosynthesis. In this study, we conducted de novo transcriptome analysis of S. viscidula and obtained 42,310,834 reads and 40,052 unigenes, respectively. We revealed 177 genes relating to flavonoid biosynthesis, where 23 key enzyme encoding genes including CHS, CHI, F3H, PAL and 4CL were annotated. Furthermore, we investigated the dynamic expression of CHS, CHI, F3H, MYB2, and bHLH of stem, root and leaf of S. viscidula in May, July and September. Our results showed that these key genes had important regulatory function and exhibited positive correlation with total flavonoid content in different growth stages of S. viscidula. Collectively, this study provides high-quality transcriptome data of S. viscidula, and further gives significant information for understanding the molecular mechanism of gene expression and active ingredients in Scutellaria plants.
Project description:We identified two novel transcription factors ‘Triterpene Saponin Activation Regulator’ (TSAR) 1 and 2 that each activate a specific branch of saponin synthesis in M. truncatula. Therefore we generated three independent TSAR1 and three independent TSAR2 overexpression (OE) hairy root lines. As a control we raised three independent hairy root lines expressing a non-functional GUS gene. By qRT-PCR analyses and metabolite profiling of these lines, we found that TSAR1 OE leads to accumulation of non-haemolytic saponins whereas TSAR2 instigates accumulation of haemolytic saponins. To identify additional targets of TSAR1 and TSAR2 we carried out a genome-wide transcript profiling study by RNA-Seq.
Project description:Platycodon grandiflorus is a perennial traditional Chinese herb, which has more than 100 compounds including triterpenoid saponins, polysaccharides and phenolic acid etc. In this study, a (L9(34)) orthogonal experiment determined the optimal explants is stem with nodes and the optimum full medium is MS + NAA 1.0 mg/L + 6-BA 0.5 mg/L as best protocol for callus induction of P. grandiflorus.The content of platycodin D, platycoside E and total polysaccharides between callus and plant organs is significantly difference, and the distribution of platycodin D, platycoside E and total polysaccharide in whole plant has been clarified. Meanwhile, the content of platycodin D and total polysaccharide were found higher in calli than in leaves or the whole plant. While, the content of platycoside E in calli is very lower than in leaves. In order to screen candidate genes involved in platycoside E and platycodin D conversion, a RNA-Seq analysis between calli and leaves was performed. Correlating the content of platycodin D and platycoside E between calli and leaves with the gene expression level involving in triterpenoid saponin biosynthesis pathway, four cluster genes with high amino acid similarity were screened as the putative sequences of β-Glucosidase gene for converting platycoside E to platycodin D. It was found that the expression level of genes encoding enzymes involved in saponins and polysaccharides biosynthesis was co-expression with the content of the corresponding metabolite. Besides, some transcription factors predicted to regulate the expression of key enzymes involving in triterpernoid saponins and polysaccharides biosynthesis pathway of P. grandiflorus.
