Key Laboratory of Bioactive Substances and Resource Utilization of Chinese Herbal Medicine from Ministry of Education, Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing 100193, PR China.
School of Pharmaceutical Sciences, Xiangnan University, Chenzhou, Hunan Province, PR China.
Fungal Genet Biol. 2020 Mar;136:103313. doi: 10.1016/j.fgb.2019.103313. Epub 2019 Nov 18.
MicroRNAs (miRNAs in animals and plants or milRNAs in fungi) are endogenous noncoding RNAs that can regulate gene expression. However, little information is known about milRNAs and their target genes in Ganoderma lucidum. Here, we systematically predicted and characterised the milRNAs and their target genes across the three developmental stages of G. lucidum. A total of 168 unique milRNAs were predicted using a small RNA sequencing method. For them, 1612 target sequences corresponding to 1311 unique genes were predicted by degradome sequencing. We selected 42 predicted milRNAs and performed RT-PCR amplification and Sanger sequencing of the products. Five products were found to have sequences similar to those predicted, confirming the presence of milRNAs in G. lucidum, and demonstrating the difficulty in their validation. Among the 168 milRNAs, 111 were found to be significantly differentially expressed across the three developmental stages (q ≤ 0.05). The expression levels of 12 milRNAs were measured by stem-loop quantitative real-time polymerase chain reaction. Eight of them were in line with the sequencing results (r ≥ 0.9, p ≤ 0.05). These 12 milRNAs and their target genes form 16 milRNA-target gene pairs. The expression profiles of 8 of these 16 miRNA-target pairs were negatively correlated, according to real-time quantitative analysis, whereas the other eight pairs were positively correlated. Furthermore, the results of functional enrichment analysis showed that the target genes of milRNAs mapped to the Gene Ontology terms 'GTP binding' and 'FAD binding' were enriched in specific developmental stages. These target genes were related to the biosynthesis of triterpenes and polysaccharides and lignin degradation pathway in G. lucidum. In summary, this study indicates that milRNAs may play crucial regulatory roles in various biological processes of G. lucidum and open up new avenues for research on milRNAs' biosyntheses and functions in basidiomycetes.
微生物 RNA（动植物中的 miRNAs 或真菌中的 milRNAs）是内源性非编码 RNA，可以调节基因表达。然而，关于灵芝中的 milRNAs 及其靶基因知之甚少。在这里，我们系统地预测和描述了灵芝三个发育阶段的 milRNAs 和它们的靶基因。使用小 RNA 测序方法预测了 168 个独特的 milRNAs。通过降解组测序，预测了 1612 个对应于 1311 个独特基因的靶序列。我们选择了 42 个预测的 milRNAs，并对产物进行 RT-PCR 扩增和 Sanger 测序。发现 5 个产物具有与预测序列相似的序列，证实了灵芝中存在 milRNAs，同时也证明了它们的验证存在困难。在这 168 个 milRNAs 中，有 111 个在三个发育阶段呈显著差异表达（q≤0.05）。通过茎环定量实时聚合酶链反应测量了 12 个 milRNAs 的表达水平。其中 8 个与测序结果一致（r≥0.9，p≤0.05）。这 12 个 milRNAs 和它们的靶基因形成了 16 个 milRNA-靶基因对。根据实时定量分析，这 16 个 miRNA-靶对中的 8 个对的表达谱呈负相关，而其他 8 个对呈正相关。此外，功能富集分析的结果表明，milRNAs 靶基因映射到 Gene Ontology 术语“GTP 结合”和“FAD 结合”在特定发育阶段富集。这些靶基因与灵芝三萜和多糖生物合成以及木质素降解途径有关。总之，这项研究表明 milRNAs 可能在灵芝的各种生物学过程中发挥重要的调节作用，为研究担子菌中 milRNAs 的生物合成和功能开辟了新的途径。
