Analysis of expressed sequence tags from theHuperzia serrataleaf for gene discovery in the areas of secondary metabolite biosynthesis and development regulation

Abstract

Huperzia serrata produces various types of lycopodium alkaloids, especially the huperzine A (HupA) that is a promising drug candidate for Alzheimer's disease. Despite the medicinal importance of H. serrata, little genomic or transcriptomic data are available from the public databases. A cDNA library was thus generated from RNA isolated from the leaves of H. serrata. A total of 4012 clones were randomly selected from the library, and 3451 high-quality expressed sequence tags (ESTs) were assembled to yield 1510 unique sequences with an average length of 712 bp. The majority (79.4%) of the unique sequences were assigned to the putative functions based on the BLAST searches against the public databases. The functions of these unique sequences covered a broad set of molecular functions, biological processes and biochemical pathways according to GO and KEGG assignments. The transcripts involved in the secondary metabolite biosynthesis of alkaloids, terpenoids and flavone/flavonoids, such as cytochrome P450, lysine decarboxylase (LDC), flavanone 3-hydroxylase, squalene synthetase and 2-oxoglutarate 3-dioxygenase, were well represented by 34 unique sequences in this EST dataset. The corresponding peptide sequence of the LDC contained the Pfam 03641 domain and was annotated as a putative LDC. The unique sequences encoding transcription factors, phytohormone biosynthetic enzymes and signaling components were also found in this EST collection. In addition, a total of 501 potential SSR-motif microsatellite loci were identified from the 393 H. serrata leaf unique sequences. This set of non-redundant ESTs and the molecular markers obtained in this study will establish valuable resources for a wide range of applications including gene discovery and identification, genetic mapping and analysis of genetic diversity, cultivar identification and marker-assisted selections in this important medicinal plant.