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.
Highlights
Camptotheca acuminata is a Nyssaceae plant, often called the “happy tree”, which is indigenous in Southern China
Among them are tryptophan synthase (TSB) [4] and tryptophan decarboxylase (TDC) [5], which are involved in the synthesis of the indole precursor tryptamine, 3-hydroxy-3-methylglutaryl-CoA synthase (HMGR) [6], 1-deoxy-D-xylulose-5-phosphate reductoisomeras (DXR) [7], and 10-hydroxy geraniol oxidoreductase (10HGO) [8] are involved in secologanin synthesis
By searching the annotation information from the non-redundant protein (Nr), Swissprot, and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases, we found that transcripts of 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR), 10HGO, and secologanin synthase (SCS) were presented many Expressed sequence tags (ESTs), indicating that they are highly expressed in the young leaves of C. acuminata
Summary
Camptotheca 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. Camptothecin (CPT) was first extracted from the stems of Camptotheca acuminata in 1966 and subsequently from Nothapodytes foetida, Ophiorrhiza pumila, and Ophiorrhiza japonica [1]. CPT exhibits clinical antitumor activity by inhibiting DNA topoisomerase I, an candidate transcripts involved in CPT biosynthesis to further understand the CPT biosynthetic pathway. CPT is synthesized through a modified terpenoid indole alkaloid (TIA) pathway. The upstream biosynthesis pathways for all the TIA products are similar among alkaloid-producing plants, and involve a strictosidine backbone (Figure 1A). Several enzymes in the process of strictosidine biosynthesis in C. acuminata have been isolated and functionally identified. Among them are tryptophan synthase (TSB) [4] and tryptophan decarboxylase (TDC) [5], which are involved in the synthesis of the indole precursor tryptamine, 3-hydroxy-3-methylglutaryl-CoA synthase (HMGR) [6], 1-deoxy-D-xylulose-5-phosphate reductoisomeras (DXR) [7], and 10-hydroxy geraniol oxidoreductase (10HGO) [8] are involved in secologanin synthesis
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