Abstract
BackgroundThe Himalayan or Indian Mayapple (Podophyllum hexandrum Royle) produces podophyllotoxin, which is used in the production of semisynthetic anticancer drugs. High throughput transcriptome sequences or genomic sequence data from the Indian Mayapple are essential for further understanding of the podophyllotoxin biosynthetic pathway.Results454 pyrosequencing of a P. hexandrum cell culture normalized cDNA library generated 2,667,207 raw reads and 1,503,232 high quality reads, with an average read length of 138 bp. The denovo assembly was performed by Newbler using default and optimized parameters. The optimized parameter generated 40, 380 assembled sequences, comprising 12,940 contigs and 27,440 singlets which resulted in better assembly as compared to default parameters. BLASTX analysis resulted in the annotation of 40,380 contigs/singlet using a cut-off value of ≤1E-03. High similarity to Medicago truncatula using optimized parameters and to Populus trichocarpa using default parameters was noted. The Kyoto encyclopedia of genes and genomes (KEGG) analysis using KEGG Automatic Annotation Server (KAAS) combined with domain analysis of the assembled transcripts revealed putative members of secondary metabolism pathways that may be involved in podophyllotoxin biosynthesis. A proposed schematic pathway for phenylpropanoids and podophyllotoxin biosynthesis was generated. Expression profiling was carried out based on fragments per kilobase of exon per million fragments (FPKM). 1036 simple sequence repeats were predicted in the P. hexandrum sequences. Sixty-nine transcripts were mapped to 99 mature and precursor microRNAs from the plant microRNA database. Around 961 transcripts containing transcription factor domains were noted. High performance liquid chromatography analysis showed the peak accumulation of podophyllotoxin in 12-day cell suspension cultures. A comparative qRT-PCR analysis of phenylpropanoid pathway genes identified in the present data was performed to analyze their expression patterns in 12-day cell culture, callus and rhizome.ConclusionsThe present data will help the identification of the potential genes and transcription factors involved in podophyllotoxin biosynthesis in P. hexandrum. The assembled transcripts could serve as potential candidates for marker discovery and conservation, which should form the foundations for future endeavors.
Highlights
The Himalayan or Indian Mayapple (Podophyllum hexandrum Royle) produces podophyllotoxin, which is used in the production of semisynthetic anticancer drugs
Podophyllum hexandrum Royle, commonly referred to as the Himalayan/Indian Mayapple, is an endangered perennial herb belonging to the family Berberidaceae, which is distributed on the lower slopes of the Himalayas in scrub and forest, from Afghanistan to central China [1]
454 sequencing of the Mayapple cell culture transcriptome Clonally amplified cDNA library beads obtained from emulsion-based clonal amplification reactions were subjected to two experimental runs on a Pico Titre Plate (PTP) for sequencing using Roche 454 GS FLX pyrosequencing chemistry
Summary
The Himalayan or Indian Mayapple (Podophyllum hexandrum Royle) produces podophyllotoxin, which is used in the production of semisynthetic anticancer drugs. Podophyllum hexandrum Royle, commonly referred to as the Himalayan/Indian Mayapple, is an endangered perennial herb belonging to the family Berberidaceae, which is distributed on the lower slopes of the Himalayas in scrub and forest, from Afghanistan to central China [1]. A crude resin extract of Podophyllum spp., podophyllin, was included in the US Pharmacopoeia in 1820, and this resin has been prescribed to treat venereal warts. A recent review [6] stated that species containing podophyllotoxin were traditionally used as folk-remedies in China, Japan, India and the United States to treat several illnesses, including gout, tuberculosis, syphilis, warts and various tumors. Peltatins do not contribute to the anti-cancer properties of the plant [9]
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