Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, PR China.
PLoS One. 2013 Aug 13;8(8):e72705. doi: 10.1371/journal.pone.0072705. eCollection 2013.
Dendrobiumofficinale (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.
铁皮石斛（兰科）是世界上最濒危的植物之一，具有很高的药用价值。在自然界中，铁皮石斛种子必须与真菌建立共生关系才能发芽。然而，在这个过程中真菌与植物之间相互作用所涉及的分子事件还知之甚少。为了分离与共生发芽相关的基因，构建了共生发芽的铁皮石斛种子抑制性消减杂交（SSH） cDNA 文库。从该文库中，聚类出 1437 条表达序列标签（EST），得到 1074 个 Unigenes（包括 902 个单基因和 172 个基因簇），这些基因与 NCBI 非冗余（NR）蛋白质数据库（E 值截止值，e(-5)）进行比对。根据与已知蛋白质的序列相似性，在铁皮石斛中鉴定出 579 个差异表达基因，并通过基因本体论（GO）、直系同源簇的蛋白质（COG）和京都基因与基因组百科全书（KEGG）途径将其分为不同的功能类别。通过实时定量 PCR 验证了 15 个具有代表性的共生发芽基因的表达水平。这些基因分为防御和应激反应、代谢、转录调控、运输过程和信号转导途径等不同类别。所有转录本在共生发芽种子（SGS）中均上调。预测了这些基因在共生发芽中的功能。此外，还从铁皮石斛中克隆了两个真菌诱导的钙依赖性蛋白激酶（CDPK），它们在 SGS 中的表达水平分别比未发芽种子（UGS）上调了 6.76 倍和 26.69 倍。这两个基因首次被克隆并进行了特征描述。本研究首次提供了与铁皮石斛共生种子发芽相关的假定基因的全面概述，并为进一步研究兰花共生关系提供了基础。
