Analysis of the Dendrobium officinale transcriptome reveals putative alkaloid biosynthetic genes and genetic markers

Abstract

Dendrobium officinale Kimura et Migo (Orchidaceae) is a traditional Chinese medicinal plant. The stem contains an alkaloid that is the primary bioactive component. However, the details of alkaloid biosynthesis have not been effectively explored because of the limited number of expressed sequence tags (ESTs) available in GenBank. In this study, we analyzed RNA isolated from the stem of D. officinale using a single half-run on the Roche 454 GS FLX Titanium platform to generate 553,084 ESTs with an average length of 417 bases. The ESTs were assembled into 36,407 unique putative transcripts. A total of 69.97% of the unique sequences were annotated, and a detailed view of alkaloid biosynthesis was obtained. Functional assignment based on Kyoto Encyclopedia of Genes and Genomes (KEGG) terms revealed 69 unique sequences representing 25 genes involved in alkaloid backbone biosynthesis. A series of qRT-PCR experiments confirmed that the expression levels of 5 key enzyme-encoding genes involved in alkaloid biosynthesis are greater in the leaves of D. officinale than in the stems. Cytochrome P450s, aminotransferases, methyltransferases, multidrug resistance protein (MDR) transporters and transcription factors were screened for possible involvement in alkaloid biosynthesis. Furthermore, a total of 1061 simple sequence repeat motifs (SSR) were detected from 36,407 unigenes. Dinucleotide repeats were the most abundant repeat type. Of these, 179 genes were associated with a metabolic pathway in KEGG. This study is the first to produce a large volume of transcriptome data from D. officinale. It extends the foundation to facilitate gene discovery in D. officinale and provides an important resource for the molecular genetic and functional genomic studies in this species.

Keywords: 454-ESTs; Alkaloid biosynthesis; BLAST; Basic Local Alignment Search Tool; CYP450; Dendrobium officinale Kimura et Migo (Orchidaceae); EC; ESTs; G10H; GO; Gene Ontology; HQ; KEGG; Kyoto Encyclopedia of Genes and Genomes; MDR; MEP; MVA; NCBI; NCBI non-redundant nucleotide; NCBI non-redundant protein; NGS; National Center for Biotechnology Information; Nr; Nt; SCS; SSR; STR; Simple sequence repeats; TAIR; TDC; TFs; TIA; TSB; The Arabidopsis Information Resource; Transcriptome; cytochrome P450-dependent monooxygenase; enzyme commission; expressed sequence tags; geraniol 10-hydroxylase; high-quality; methylerythritol 4-phosphate; mevalonic acid; multidrug resistance protein; next-generation sequencing; qRT-PCR; quantitative real-time polymerase chain reaction; secologanin synthase; simple sequence repeat; strictosidine synthase; terpenoid indole alkaloid; transcription factors; tryptophan decarboxylase; tryptophan synthase.