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Pyrosequencing of the <it>Camptotheca acuminata </it>transcriptome reveals putative genes involved in camptothecin biosynthesis and transport

By Sun Yongzhen, Luo Hongmei, Li Ying, Sun Chao, Song Jingyuan, Niu Yunyun, Zhu Yingjie, Dong Liang, Lv Aiping, Tramontano Enzo and Chen Shilin

Abstract

<p>Abstract</p> <p>Background</p> <p><it>Camptotheca acuminata </it>is a Nyssaceae plant, often called the "happy tree", which is indigenous in Southern China. <it>C. acuminata </it>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 <it>C. acuminata </it>and the mechanism of CPT biosynthesis, as only few nucleotide sequences are included in the GenBank database.</p> <p>Results</p> <p>From a constructed cDNA library of young <it>C. acuminata </it>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 <it>De Novo </it>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 <it>Arabidopsis thaliana </it>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 (<it>CaPG10H</it>), secologanin synthase (<it>CaPSCS</it>), and strictosidine synthase (<it>CaPSTR</it>) were cloned and analyzed. The expression level of the three genes was also detected using qRT-PCR in <it>C. acuminata</it>. 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.</p> <p>Conclusion</p> <p>This study produced a large amount of transcriptome data from <it>C. acuminata </it>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 <it>C. acuminata</it>. This study will facilitate further identification of key enzymes and transporter genes in <it>C. acuminata</it>.</p

Topics: Genetics, QH426-470, Biology (General), QH301-705.5, Science, Q, DOAJ:Genetics, DOAJ:Biology, DOAJ:Biology and Life Sciences, Biotechnology, TP248.13-248.65
Publisher: BioMed Central
Year: 2011
DOI identifier: 10.1186/1471-2164-12-533
OAI identifier: oai:doaj.org/article:1bde3fae43234aab9115228a597c3c37
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