Microbiology Department, College of Life Sciences, Nanjing Agricultural University, Key Laboratory of Microbiological Engineering of Agricultural Environment, Ministry of Agriculture, Nanjing, Jiangsu, P.R. China.
PLoS One. 2013 Jun 7;8(6):e65027. doi: 10.1371/journal.pone.0065027. Print 2013.
Ganoderma lucidum is a mushroom with traditional medicinal properties that has been widely used in China and other countries in Eastern Asia. Ganoderic acids (GA) produced by G. lucidum exhibit important pharmacological activities. Previous studies have demonstrated that methyl jasmonate (MeJA) is a potent inducer of GA biosynthesis and the expression of genes involved in the GA biosynthesis pathway in G. lucidum. To further explore the mechanism of GA biosynthesis, cDNA-Amplified Fragment Length Polymorphism (cDNA-AFLP) was used to identify genes that are differentially expressed in response to MeJA. Using 64 primer combinations, over 3910 transcriptionally derived fragments (TDFs) were obtained. Reliable sequence data were obtained for 390 of 458 selected TDFs. Ninety of these TDFs were annotated with known functions through BLASTX searching the GenBank database, and 12 annotated TDFs were assigned into secondary metabolic pathways by searching the KEGGPATHWAY database. Twenty-five TDFs were selected for qRT-PCR analysis to confirm the expression patterns observed with cDNA-AFLP. The qRT-PCR results were consistent with the altered patterns of gene expression revealed by the cDNA-AFLP technique. Additionally, the transcript levels of 10 genes were measured at the mycelium, primordia, and fruiting body developmental stages of G. lucidum. The greatest expression levels were reached during primordia for all of the genes except cytochrome b2 reached its highest expression level in the mycelium stage. This study not only identifies new candidate genes involved in the regulation of GA biosynthesis but also provides further insight into MeJA-induced gene expression and secondary metabolic response in G. lucidum.
灵芝是一种具有传统药用价值的蘑菇，在中国和东亚其他国家被广泛使用。灵芝产生的灵芝酸（GA）具有重要的药理活性。先前的研究表明，茉莉酸甲酯（MeJA）是灵芝中 GA 生物合成和参与 GA 生物合成途径的基因表达的有效诱导剂。为了进一步探讨 GA 生物合成的机制，使用 cDNA 扩增片段长度多态性（cDNA-AFLP）来鉴定对 MeJA 有差异表达的基因。使用 64 个引物组合，获得了超过 3910 个转录衍生片段（TDF）。从 458 个选定的 TDF 中，获得了 390 个可靠的序列数据。通过 BLASTX 搜索 GenBank 数据库，对其中的 90 个 TDF 进行了具有已知功能的注释，通过搜索 KEGGPATHWAY 数据库，对 12 个注释 TDF 进行了次级代谢途径的分配。选择了 25 个 TDF 进行 qRT-PCR 分析，以确认 cDNA-AFLP 观察到的表达模式。qRT-PCR 结果与 cDNA-AFLP 技术揭示的基因表达变化模式一致。此外，在灵芝菌丝体、原基和子实体发育阶段测量了 10 个基因的转录水平。除细胞色素 b2 在菌丝体阶段达到最高表达水平外，所有基因的最大表达水平都在原基期达到。本研究不仅鉴定了参与 GA 生物合成调控的新候选基因，还进一步了解了 MeJA 诱导灵芝基因表达和次级代谢反应。
