Abstract
Dendrobium officinale (Orchidaceae) is one of the world’s most endangered plants with great medicinal value. In nature, D . officinale seeds must establish symbiotic relationships with fungi to germinate. However, the molecular events involved in the interaction between fungus and plant during this process are poorly understood. To isolate the genes involved in symbiotic germination, a suppression subtractive hybridization (SSH) cDNA library of symbiotically germinated D . officinale seeds was constructed. From this library, 1437 expressed sequence tags (ESTs) were clustered to 1074 Unigenes (including 902 singletons and 172 contigs), which were searched against the NCBI non-redundant (NR) protein database (E-value cutoff, e-5). Based on sequence similarity with known proteins, 579 differentially expressed genes in D . officinale were identified and classified into different functional categories by Gene Ontology (GO), Clusters of orthologous Groups of proteins (COGs) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. The expression levels of 15 selected genes emblematic of symbiotic germination were confirmed via real-time quantitative PCR. These genes were classified into various categories, including defense and stress response, metabolism, transcriptional regulation, transport process and signal transduction pathways. All transcripts were upregulated in the symbiotically germinated seeds (SGS). The functions of these genes in symbiotic germination were predicted. Furthermore, two fungus-induced calcium-dependent protein kinases (CDPKs), which were upregulated 6.76- and 26.69-fold in SGS compared with un-germinated seeds (UGS), were cloned from D . officinale and characterized for the first time. This study provides the first global overview of genes putatively involved in D . officinale symbiotic seed germination and provides a foundation for further functional research regarding symbiotic relationships in orchids.
Highlights
Orchidaceae, which comprises an estimated 20000 to 35000 species, constitutes one of the most diverse families [1], and most species of this family have medicinal and ornamental properties [2]
One group of seeds was cultured with Sebacina sp., which was isolated from D. officinale symbiotic seeds [8] (Genbank number EU910926 and public culture collection accession number CGMCC No 3398) on oatmeal agar (OMA) medium [21] was germinated to the third stage, which is characterized by the appearance of protomeristem [22]
Compared with Sebacina, the sequence in Dendrobium is well known and the plant genotype can affect the extent of the fungi colonization and the response
Summary
Orchidaceae, which comprises an estimated 20000 to 35000 species, constitutes one of the most diverse families [1], and most species of this family have medicinal and ornamental properties [2]. Fungal hyphae invade and colonize the orchid root cortex and subsequently form elaborate intracellular coils called pelotons, which are structures distinctive in both appearance and distribution patterns with orchid tissues [5]. Several species of fungi that engage in symbiosis and provide nutrients, such as nitrogen (N) and carbon (C), during orchid seed germination have been isolated and characterized. Most of these species have been classified as orchid-associated Rhizoctonia-type fungi [6,7], in which Tulasnellaceae, Sebacinaceae and Ceratobasidiaceae have been reported to support seed germination and protocorm development [6,8,9]
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