High-quality RNA extraction and the regulation of genes encoding cellulosomes are correlated with growth stage in anaerobic fungi.
High-quality RNA extraction and the regulation of genes encoding cellulosomes are correlated with growth stage in anaerobic fungi.
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DOI：
10.3389/ffunb.2023.1171100
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发表时间：
2023
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厌氧真菌产生生物质降解酶和天然产物，这些对于利用几种生物技术应用是重要的。尽管在这些真菌的基因组和转录测序中提取核酸的方法已经取得了进展，但大多数研究都是有限的，因为它们不能在批培养中采样多个真菌生长阶段。在本研究中，我们建立了一种从真菌单一培养物和真菌-产甲烷菌共培养物中提取RNA的方法，并确定了从厌氧真菌中提取高质量RNA的最佳时间框架。根据RNA的质量和数量指标，收获厌氧真菌单一培养物和真菌-甲烷菌共培养物提取RNA的最佳时间框架为接种后生长2-5天。当在纤维素上生长时，与产甲烷菌Bryantii共培养的真菌菌株粗壮厌气霉菌在这段时间内相对于真菌单一培养物上调了编码真菌碳水化合物活性酶和其他纤维素体成分的基因，但表达模式在整个真菌生长阶段以24小时为间隔发生变化。这些结果证明了建立在分批培养过程中多个时间点从厌氧真菌中提取高质量RNA的方法的重要性。
Anaerobic fungi produce biomass-degrading enzymes and natural products that are important to harness for several biotechnology applications. Although progress has been made in the development of methods for extracting nucleic acids for genomic and transcriptomic sequencing of these fungi, most studies are limited in that they do not sample multiple fungal growth phases in batch culture. In this study, we establish a method to harvest RNA from fungal monocultures and fungal–methanogen co-cultures, and also determine an optimal time frame for high-quality RNA extraction from anaerobic fungi. Based on RNA quality and quantity targets, the optimal time frame in which to harvest anaerobic fungal monocultures and fungal-methanogen co-cultures for RNA extraction was 2-5 days of growth post-inoculation. When grown on cellulose, the fungal strain Anaeromyces robustus cocultivated with the methanogen Methanobacterium bryantii upregulated genes encoding fungal carbohydrate-active enzymes and other cellulosome components relative to fungal monocultures during this time frame, but expression patterns changed at 24-hour intervals throughout the fungal growth phase. These results demonstrate the importance of establishing methods to extract high-quality RNA from anaerobic fungi at multiple time points during batch cultivation.