Discovery and expression of unigenes involved in triterpenesaponin biosynthesis in <i>Hedera helix L</i>.

<p>Discovery and expression of unigenes involved in triterpenesaponin biosynthesis in <i>Hedera helix L</i>.</p

RT-qPCR validation of selected unigenes involved in triterpene saponin biosynthesis.

<p>Columns indicate relative expression obtained by RT-qPCR (left y-axis); lines indicating the expression level were calculated by FPKM method (right y-axis). All data are presented as mean value of three repeats.</p

Differentially expressed gene analysis of six libraries in <i>H</i>. <i>helix</i>.

<p>(A) Expressed higher unigenes in leaf samples. (B) Expressed lower unigenes in leaf samples.</p

<i>De novo</i> leaf and root transcriptome analysis to identify putative genes involved in triterpenoid saponins biosynthesis in <i>Hedera helix</i> L.

<div><p><i>Hedera helix</i> L. is an important traditional medicinal plant in Europe. The main active components are triterpenoid saponins, but none of the potential enzymes involved in triterpenoid saponins biosynthesis have been discovered and annotated. Here is reported the first study of global transcriptome analyses using the Illumina HiSeq^™ 2500 platform for <i>H</i>. <i>helix</i>. In total, over 24 million clean reads were produced and 96,333 unigenes were assembled, with an average length of 1385 nt; more than 79,085 unigenes had at least one significant match to an existing gene model. Differentially Expressed Gene analysis identified 6,222 and 7,012 unigenes which were expressed either higher or lower in leaf samples when compared with roots. After functional annotation and classification, two pathways and 410 unigenes related to triterpenoid saponins biosynthesis were discovered. The accuracy of these <i>de novo</i> sequences was validated by RT-qPCR analysis and a RACE clone. These data will enrich our knowledge of triterpenoid saponin biosynthesis and provide a theoretical foundation for molecular research on <i>H</i>. <i>helix</i>.</p></div

COG function classification of <i>H</i>. <i>helix</i> transcriptome.

<p>A total of 33,205 unigenes showed significant homology (E-value ≤1.0E-5) to genes in one of the 25 categories (A-W, Y and Z) in the NCBI COGs database.</p

Length distribution frequency of unigenes in <i>H</i>. <i>helix</i>.

<p>Length distribution frequency of unigenes in <i>H</i>. <i>helix</i>.</p

Summary of data output quality of various libraries.

<p>Summary of data output quality of various libraries.</p

Summary of functional annotations of <i>H</i>. <i>helix</i> unigenes.

<p>Summary of functional annotations of <i>H</i>. <i>helix</i> unigenes.</p

Gene similarity of unigenes against the Nr database.

<p>(A) E-value distribution of top BLAST hits for each unigene (E-value of 1.0E-5). (B) Similarity distribution of best BLAST hits for each unigene. (C) Distribution of BLAST results by species shown as percentage of total homologous sequences (E-value ≤1.0E-5). All plant proteins in the NCBI Nr database were used for homology search and the best hit of each sequence was used for analysis.</p

<i>De Novo</i> Characterization of a <i>Cephalotaxus hainanensis</i> Transcriptome and Genes Related to Paclitaxel Biosynthesis

<div><p><i>Cephalotaxus hainanensis</i>, an endangered plant, is known to contain several metabolites with anti-cancer activity. Despite its clinical impact, the alkaloid metabolism of this species has remained largely uncharacterized. The potential of <i>Cephalotaxus</i> for metabolic engineering of medically interesting compounds has, so far, not been exploited, due to the almost complete lack of molecular information. We have therefore performed a high throughput RNA-seq analysis and assembled the transcriptome <i>de novo</i>. Raw reads comprising 4.3 Gbp were assembled <i>de novo</i> into 39,416 unique sequences (unigenes) with a mean length of 1,089.8 bp and a total assembly size of 45.8 Mbp, which equals to more than 50 times the number of <i>Cephalotaxaceae</i> sequences currently deposited in the GenBank (as of August 2013). As proof of principle for medically interesting pathways, gene fragments related to paclitaxel biosynthesis were searched and detected. To verify their functionality, the metabolic product paclitaxel, and its precursor baccatin III, were identified in the leaves of <i>C. hainanensis</i> by HPLC, and shown to be induced by MeJA. This finding demonstrates exemplarily the potential of the annotated transcriptome as information resource for the biotechnological exploitation of plant secondary metabolism.</p></div