miRNA样RNA(milRNAs)已被认为是真核生物中基因表达转录后调节的序列特异性调节因子。然而,数百种真菌milRNAs在代谢成分的生物合成中的功能是模糊的。三黄孢菌产生多种生物活性化合物,在亚洲国家被广泛使用。这里,编码两个Dicers的基因,四个Argonautes,并在三黄孢菌中鉴定和表征了四个RdRP。由于缺乏高效的基因操纵系统,喷雾诱导的基因沉默(SIGS)的功效在S.vanini,这显示了有效的双链RNA(dsRNA)摄取和基因沉默效率。SIGS介导的基因敲减表明SVRDRP-3、SVRDRP-4、SVDICER-1和SVDICER-2对菌丝生物量至关重要,类黄酮,三萜类,和多糖生产。进行Illumina深度测序以表征来自S.vanini菌丝体和子实体的milRNA。总共鉴定了31个milRNAs,其中,SvmilR10、SvmilR17和SvmilR33是Svrdrp-4-和Svdicer-1-依赖性milRNA。重要的是,SIGS介导的SvmilR10和SvmilR33的过表达导致类黄酮产量的显著变化,三萜类,和多糖。进一步的分析表明,这些编码逆转录转座子衍生的蛋白质PEG1和组蛋白-赖氨酸N-甲基转移酶的milRNA靶基因在milRNA过表达菌株中可能被下调。我们的结果揭示了S.vanini具有高的外部dsRNA和小RNA摄取效率,并且milRNAs可能在生物活性化合物的生物合成中起着至关重要的调节作用。重要性真菌可以吸收环境RNA,可以通过RNA干扰沉默真菌基因,这促进了SIGS的发展。在S.vanini中有效的dsRNA和milRNA摄取,成功的dsRNA靶向基因阻断,细胞内miRNA丰度的增加表明SIGS技术是真菌基因和millRNAs功能解剖的有效和强大的工具。我们发现RdRP,Dicer,和Argonaute基因对于菌丝体生物量和生物活性化合物的生产至关重要。我们的研究还表明,过度表达的SVRDRP-4-和SVDICER-1依赖性milRNAs(SvmilR10和SvmilR33)导致三种活性化合物的产量发生显着变化。这项研究不仅提供了基于SIGS的基因和milRNA功能探索的第一个报告,而且也为探索参与真菌代谢化合物生物合成的milRNAs的功能提供了理论平台。
miRNA-like RNAs (milRNAs) have been recognized as sequence-specific regulators of posttranscriptional regulation of gene expression in eukaryotes. However, the functions of hundreds of fungal milRNAs in the biosynthesis of metabolic components are obscure. Sanghuangporus produces diverse bioactive compounds and is widely used in Asian countries. Here, genes encoding two Dicers, four Argonautes, and four RdRPs were identified and characterized in Sanghuangporus vanini. Due to the lack of an efficient gene manipulation system, the efficacy of spray-induced gene silencing (SIGS) was determined in S. vanini, which showed efficient double-stranded RNA (dsRNA) uptake and gene silencing efficiency. SIGS-mediated gene knockdown showed that SVRDRP-3, SVRDRP-4, SVDICER-1, and SVDICER-2 were critical for mycelial biomass, flavonoid, triterpenoid, and polysaccharide production. Illumina deep sequencing was performed to characterize the milRNAs from S. vanini mycelium and fruiting body. A total of 31 milRNAs were identified, out of which, SvmilR10, SvmilR17, and SvmilR33 were Svrdrp-4- and Svdicer-1-dependent milRNAs. Importantly, SIGS-mediated overexpression of SvmilR10 and SvmilR33 resulted in significant changes in the yields of flavonoids, triterpenoids, and polysaccharides. Further analysis showed that these milRNA target genes encoding the retrotransposon-derived protein PEG1 and histone-lysine N-methyltransferase were potentially downregulated in the milRNA overexpressing strain. Our results revealed that S. vanini has high external dsRNA and small RNA uptake efficiency and that milRNAs may play crucial regulatory roles in the biosynthesis of bioactive compounds. IMPORTANCE Fungi can take up environmental RNA that can silence fungal genes with RNA interference, which prompts the development of SIGS. Efficient dsRNA and milRNA uptake in S. vanini, successful dsRNA-targeted gene block, and the increase in intracellular miRNA abundance showed that SIGS technology is an effective and powerful tool for the functional dissection of fungal genes and millRNAs. We found that the RdRP, Dicer, and Argonaute genes are critical for mycelial biomass and bioactive compound production. Our study also demonstrated that overexpressed SVRDRP-4- and SVDICER-1-dependent milRNAs (SvmilR10 and SvmilR33) led to significant changes in the yields of the three active compounds. This study not only provides the first report on SIGS-based gene and milRNA function exploration, but also provides a theoretical platform for exploration of the functions of milRNAs involved in biosynthesis of metabolic compounds in fungi.