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.

中文翻译：

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通过高通量测序和降解组分析鉴定灵芝中的milRNA及其靶基因。

微小RNA（动植物中的miRNA或真菌中的milRNA）是可以调节基因表达的内源性非编码RNA。但是，关于灵芝中的milRNA及其靶基因的信息知之甚少。在这里，我们系统地了解了灵芝三个发育阶段的milRNA及其靶基因。使用小型RNA测序方法预测总共有168个独特的milRNA。对于他们，通过降解基因组测序预测了对应于1311个独特基因的1612个靶序列。我们选择了42种预测的milRNA，并对产品进行了RT-PCR扩增和Sanger测序。发现五种产物具有与预测的序列相似的序列，证实了灵芝中存在milRNA，并证明了其验证的难度。在168个milRNA中，发现在三个发育阶段中有111个差异显着（q≤0.05）。通过茎环定量实时聚合酶链反应测量12 milRNA的表达水平。其中八个与测序结果一致（r≥0.9，p≤0.05）。这12个milRNA及其靶基因形成16个milRNA靶基因对。根据实时定量分析，这16个miRNA-靶标对中的8个表达谱呈负相关，而其他8个对则呈正相关。此外，功能富集分析的结果表明，映射到基因本体术语“ GTP结合”和“ FAD结合”的milRNA的靶基因在特定的发育阶段得到了富集。这些靶基因与灵芝中三萜和多糖的生物合成以及木质素降解途径有关。总而言之，这项研究表明，milRNAs在灵芝的各种生物过程中可能起着至关重要的调节作用，并为研究milRNAs在担子菌中的生物合成和功能开辟了新的途径。