Pyrosequencing of the Camptotheca acuminata transcriptome reveals putative genes involved in camptothecin biosynthesis and transport

Abstract

BackgroundCamptotheca acuminata is a Nyssaceae plant, often called the "happy tree", which is indigenous in Southern China. C. acuminata produces the terpenoid indole alkaloid, camptothecin (CPT), which exhibits clinical effects in various cancer treatments. Despite its importance, little is known about the transcriptome of C. acuminata and the mechanism of CPT biosynthesis, as only few nucleotide sequences are included in the GenBank database.ResultsFrom a constructed cDNA library of young C. acuminata leaves, a total of 30,358 unigenes, with an average length of 403 bp, were obtained after assembly of 74,858 high quality reads using GS De Novo assembler software. Through functional annotation, a total of 21,213 unigenes were annotated at least once against the NCBI nucleotide (Nt), non-redundant protein (Nr), Uniprot/SwissProt, Kyoto Encyclopedia of Genes and Genomes (KEGG), and Arabidopsis thaliana proteome (TAIR) databases. Further analysis identified 521 ESTs representing 20 enzyme genes that are involved in the backbone of the CPT biosynthetic pathway in the library. Three putative genes in the upstream pathway, including genes for geraniol-10-hydroxylase (CaPG10H), secologanin synthase (CaPSCS), and strictosidine synthase (CaPSTR) were cloned and analyzed. The expression level of the three genes was also detected using qRT-PCR in C. acuminata. With respect to the branch pathway of CPT synthesis, six cytochrome P450s transcripts were selected as candidate transcripts by detection of transcript expression in different tissues using qRT-PCR. In addition, one glucosidase gene was identified that might participate in CPT biosynthesis. For CPT transport, three of 21 transcripts for multidrug resistance protein (MDR) transporters were also screened from the dataset by their annotation result and gene expression analysis.ConclusionThis study produced a large amount of transcriptome data from C. acuminata by 454 pyrosequencing. According to EST annotation, catalytic features prediction, and expression analysis, novel putative transcripts involved in CPT biosynthesis and transport were discovered in C. acuminata. This study will facilitate further identification of key enzymes and transporter genes in C. acuminata.

De novo characterization of the root transcriptome of a traditional Chinese medicinal plant Polygonum cuspidatum

“…Such studies are imperative to build a foundation for the development of natural medicines and for the selection of cultivars with good agricultural traits, as well as to raise the study of traditional Chinese medicine to the frontiers of the modern life sciences. Using Roche 454 pyrosequencing, transcriptome datasets of nine medicinal plants, T. cuspidata [25], Ginkgo biloba [26], Huperzia serrata, Phlegmariurus carinatus [27], Panax quinquefolius [29], Panax ginseng [30], Salvia miltiorrhiza [31], Camptotheca acuminata [32], and Glycyrrhiza uralensis [35] have been obtained. Illumina second generation sequencing was used previously to obtain the transcriptome dataset of T. mairei [11].…”

“…The OrthoMCL algorithm was applied to generate orthologous groups for the transcriptome datasets of 19 non-model plants, P. cuspidatum (this study), Fagopyrum esculentum, F. tataricum [15], T. mairei [11], Korea T. cuspidata [24], China T. cuspidata [25], Ginkgo biloba [26], Huperzia serrata, Phlegmariurus carinatus [27], Pteridium aquilinum [28], Panax quinquefolius [29], Panax ginseng [30], Salvia miltiorrhiza [31], Camptotheca acuminata [32], Artemisia annua [33], Cucurbita pepo [34], Glycyrrhiza uralensis [35], Eucalyptus hybrid [36], and Oryza longistaminata [37]. These plants cover a broad range of ferns, gymnosperms, monocots, and eudicots.…”

Identification of the ligand of Pru p 3, a peach LTP

“…Camptothecin is synthesized through a modified terpenoid indole alkaloid pathway in which there is a key enzyme: strictosidine synthase (STR), which is involved in the final step in the formation of the strictosidine backbone (Lorence and Nessler 2004). Gene expression involved in STR synthesis was found in leaves (more active in the young one), flowers (specially in petioles) and fruit and in most plant tissues (Isah and Mujib 2015;Sun et al 2011;WenZhe 2004;Zhao et al 2010). It is produced in pre-vacuol compartiments before being secreted through glandular trichomes.…”

Application of virus-induced gene silencing approach in Camptotheca acuminata

“…From IPP to secologanin, there were about ten steps of specific reactions that were responsible for secologanin biosynthesis. Among genes involved in this iridoid pathway, 10-hydroxygeraniol oxidoreductase (CaHGO), whose encoding enzyme converted 10-hydroxygeraniol to 10-oxogeranial, and geraniol-10-hydroxylase (CaG10H), whose encoding enzyme converted geraniol to 10-hydroxygeraniol, were deeply investigated for both cellular localizaion and expression partern (Valletta et al 2010;Sun et al 2011). After strictosidine was formed by coupling tryptamine and secologanin, it's transformed into strictosamide by Strictosidine beta-glucosidase (CaSGD) and then CPT was finally synthesized through several unknown specific catalytic steps.…”