De novo assembly of Euphorbia fischeriana root transcriptome identifies prostratin pathway related genes

Abstract

Background: Euphorbia fischeriana is an important medicinal plant found in Northeast China. The plant roots contain many medicinal compounds including 12-deoxyphorbol-13-acetate, commonly known as prostratin that is a phorbol ester from the tigliane diterpene series. Prostratin is a protein kinase C activator and is effective in the treatment of Human Immunodeficiency Virus (HIV) by acting as a latent HIV activator. Latent HIV is currently the biggest limitation for viral eradication. The aim of this study was to sequence, assemble and annotate the E. fischeriana transcriptome to better understand the potential biochemical pathways leading to the synthesis of prostratin and other related diterpene compounds.

Results: In this study we conducted a high throughput RNA-seq approach to sequence the root transcriptome of E. fischeriana. We assembled 18,180 transcripts, of these the majority encoded protein-coding genes and only 17 transcripts corresponded to known RNA genes. Interestingly, we identified 5,956 protein-coding transcripts with high similarity (> = 75%) to Ricinus communis, a close relative to E. fischeriana. We also evaluated the conservation of E. fischeriana genes against EST datasets from the Euphorbeacea family, which included R. communis, Hevea brasiliensis and Euphorbia esula. We identified a core set of 1,145 gene clusters conserved in all four species and 1,487 E. fischeriana paralogous genes. Furthermore, we screened E. fischeriana transcripts against an in-house reference database for genes implicated in the biosynthesis of upstream precursors to prostratin. This identified 24 and 9 candidate transcripts involved in the terpenoid and diterpenoid biosyntehsis pathways, respectively. The majority of the candidate genes in these pathways presented relatively low expression levels except for 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase (HDS) and isopentenyl diphosphate/dimethylallyl diphosphate synthase (IDS), which are required for multiple downstream pathways including synthesis of casbene, a proposed precursor to prostratin.

Conclusion: The resources generated in this study provide new insights into the upstream pathways to the synthesis of prostratin and will likely facilitate functional studies aiming to produce larger quantities of this compound for HIV research and/or treatment of patients.

Figure 1

The effect of query sequence length on the distribution of significant matches against NCBI non-redundant (nr) peptide database. The number of transcripts with matches (cut-off E-value of 1e^-05) in NCBI peptide database (nr) is greatest with the longer assembled sequences.

Figure 2

Statistics of homology search of transcripts against nr peptide database. A) E-value distribution of the top BLASTx hits with a cut-off E-value of 1e^-05. B) Similarity distribution of the top BLASTx hits with a cut-off E-value of 1e^-05. C) Species distribution of the top BLASTx hits is shown as a percentage of the total homologous sequences with an E-value greater than or equal to 1e^-05.

Figure 3

Frequencies and mean expression levels of transcripts matching GO terms. The percentage of transcripts matching GO terms is show for each category as grey bars and the normalized mean expression levels of transcripts matching each of these GO terms are shown as black diamonds.

Figure 4

Frequencies and mean expression levels of transcripts matching KEGG pathways. The percentage of transcripts matching to KEGG pathways within each high level category are shown as grey bars, while the normalized mean expression levels of transcripts matching each KEGG pathways are indicated as black diamonds. B) Number of transcripts for each secondary metabolite pathways is shown.

Figure 5

Euphorbiaceae comparative transcriptome analysis. The BLAST program tBLASTx was used in conjunction with OrthoMCL using a threshold E-value of 1e^-20 to identify orthologous genes between E. fischeriana and related species. Sequence datasets from related species were made non-redundant using CD-HIT-EST [33]. The number of orthologous or putative species-unique gene clusters is shown for all comparisons.

Figure 6

Mean expression levels within the Terpenoid Backbone, Diterpenoid and Zeatin Biosynthesis pathways. A) Normalized mean expression levels for enzymes within the Terpenoid Backbone Biosynthesis (TBB) pathways, namely, plastidic 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway, Diterpenoid Biosynthesis (DB) pathway and Zeatin Biosynthesis (ZB) pathway are provided. B) Normalized mean expression levels for enzymes within the TBB pathways, namely, cytosolic mevalonic acid (MVA) pathway, DB and ZB pathways. Number of E. fischeriana transcripts, from distinct gene clusters, matching each enzyme are shown between brackets in panels A and B. Abbreviations: AACT, acetoacetyl-coenzyme A (CoA) thiloase; CMS, 2-C-methyl-erythritol 4-phosphate cytidyl transferase; DXR, 1-deoxy-D-xylulose 5-phosphate reductoisomerase; DXS, 1-deoxy-D-xylulose-5-phosphate synthase; FPPS, farnesyl diphosphate synthase; GGPPS, geranylgeranyl diphosphate synthase; GPPS, geranyl diphosphate synthase; NA, Not Available; HMGR, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase; IPI, isopentenyl diphosphate isomerase; MK, mevalonate kinase; MPK, mevalonate-5-phosphate kinase; CMK, 4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol kinase; MDD, mevalonate diphosphate decarboxylase; IDS, isopentenyl diphosphate/dimethylallyl diphosphate synthase; MCS, 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase; HDS, 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase; HMGS, HMG-CoA synthase; HMG-CoA, 3S-hydroxy-3-methylglutaryl coenzyme A; DXP, 1-deoxy-D-xylulose 5-phosphate; MVA, 3R-Mevalonic acid; M5P, Mevalonate-5-phosphate; MPP, Mevalonate diphosphate; MEP, 2-C-methyl-D-erythritol 4-phosphate; CDP-ME, 4-(cytidine 5'-diphospho)-2C-methyl-D-erythritol; CDP-MEP, 4-(cytidine 5'-diphospho)-2C-methyl-D-erythritol 2-phosphate; cMEPP, 2C-methyl-D-erythritol 2,4-cyclodiphosphate; DMAPP, Dimethylallyl diphosphate; HMBPP, 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate; IPP, isopentenyl diphosphate; G3P, Glyceraldehyde 3-phosphate; GPP, geranyl diphosphate; GGPP, geranylgeranyl diphosphate; FPP, farnesyl diphosphate; GGPPS, geranylgeranyl diphosphate; ent-KSA, ent-Kaurene synthase A; ent-KSB, ent-Kaurene synthase B; ent-Kox, ent-Kaurene oxidase; CS, Casbene synthase; tRNA-DMAT, tRNA Dimethylallyltransferase; cis-ZOG, cis-Zeatin O-beta-D-glucosyltransferase; ent-CPP, ent-Copalyl diphosphate; ent-K, ent-Kaurene; UDP, Uridine 5'-diphosphate.