Expressed sequence tags from Madagascar periwinkle (Catharanthus roseus)

Abstract

The Madagascar periwinkle (Catharanthus roseus) is well known to produce the chemotherapeutic anticancer agents, vinblastine and vincristine. In spite of its importance, no expressed sequence tag (EST) analysis of this plant has been reported. Two cDNA libraries were generated from RNA isolated from the base part of young leaves and from root tips to select 9824 random clones for unidirectional sequencing, to yield 3327 related sequences and 1696 singletons by cluster analysis. Putative functions of 3663 clones were assigned, from 5023 non-redundant ESTs to establish a resource for transcriptome analysis and gene discovery in this medicinal plant.

The Leaf Epidermome ofCatharanthus roseusReveals Its Biochemical Specialization

“…To demonstrate the value of the CROLF1NG data set, a full length candidate gene, obtained by overlapping 12 separate ESTs (C57Contig1; see Supplemental Table 5 online), was cloned and functionally characterized. This unique sequence was not found in the EST data sets obtained from cDNA libraries produced from young leaves or from root tips (CrUniGene database; Murata et al, 2006) nor from other Catharanthus tissues described in the public database. The clone was 1396 bp long and exhibited moderate similarities (44% amino acid sequence identity) to a putative carboxyl methyltransferase from Medicago truncatula (ABE92230) (see Supplemental Table 5 online).…”

“…By contrast, this EST was not represented in the CrUniGene set established from sequencing of cDNA libraries prepared from the base part of the leaf and from the root tip (Murata et al, 2006). The public database did contain one EST sequence (EG555799) from a C. roseus flower bud cDNA library identical to CL314Contig1, Among ;30 enzymatic steps involved in vindoline biosynthesis, 17 genes encoding known MIA biosynthesis enzymes and transcription factors and the recently cloned and characterized 16OMT (Levac et al, 2008) and LAMT described in this article are represented in the CROLF1NG data set.…”

“…that have partly been submitted to the GenBank database (Murata et al, 2006;Rischer et al, 2006;Shukla et al, 2006). While these efforts identified only four known MIA biosynthesis pathway genes (Table 2), this study, using a cDNA library prepared from leaf epidermis-enriched mRNA, identified ESTs for virtually all known MIA biosynthesis and regulatory genes up to and including T16H (Figure 1) and 16OMT (Levac et al, 2008).…”

“…Random sequencing of Catharanthus cDNA libraries prepared from whole organs (Murata et al, 2006;Shukla et al, 2006) or from induced cell cultures (Rischer et al, 2006) have produced potentially valuable candidate genes for MIA biosynthesis; virtually no MIA pathway genes have been identified and functionally characterized using these approaches. These results suggest that the MIA pathway was poorly represented in the RNA populations derived from leaf, root, and induced cell cultures and that new approaches like LCM and CA technique (Murata and De Luca, 2005;Murata et al, 2006) were required to harvest cell types specialized in MIA biosynthesis.…”

A Picrinine N-Methyltransferase Belongs to a New Family of γ-Tocopherol-Like Methyltransferases Found in Medicinal Plants That Make Biologically Active Monoterpenoid Indole Alkaloids

“…Other uncharacterized g-TLMT-like genes (Cr2551 [HM584930], Cr7756; Liscombe et al, 2010) were not represented in this database. However, Cr7756, which was identified from a C. roseus root EST database (Liscombe et al, 2010), also was present in a previously described C. roseus root EST database (KF934425; Murata et al, 2006). The other Catharanthus g-TLMT from our pyrosequencing database, Cr706 (KC708453), is not represented in the medicinal plant genomic resource database (medicinalplantgenomics.msu.edu).…”