Barbi Florian, Bragalini Claudia, Vallon Laurent, Prudent Elsa, Dubost Audrey, Fraissinet-Tachet Laurence, Marmeisse Roland, Luis Patricia
Ecologie Microbienne, UMR CNRS 5557, USC INRA 1364, Université de Lyon, Université Lyon 1, Villeurbanne, France.
Ecologie Microbienne, UMR CNRS 5557, USC INRA 1364, Université de Lyon, Université Lyon 1, Villeurbanne, France; Department of Life Sciences and Systems Biology, University of Turin, Turin, Italy.
PLoS One. 2014 Dec 29;9(12):e116264. doi: 10.1371/journal.pone.0116264. eCollection 2014.
Plant biomass degradation in soil is one of the key steps of carbon cycling in terrestrial ecosystems. Fungal saprotrophic communities play an essential role in this process by producing hydrolytic enzymes active on the main components of plant organic matter. Open questions in this field regard the diversity of the species involved, the major biochemical pathways implicated and how these are affected by external factors such as litter quality or climate changes. This can be tackled by environmental genomic approaches involving the systematic sequencing of key enzyme-coding gene families using soil-extracted RNA as material. Such an approach necessitates the design and evaluation of gene family-specific PCR primers producing sequence fragments compatible with high-throughput sequencing approaches. In the present study, we developed and evaluated PCR primers for the specific amplification of fungal CAZy Glycoside Hydrolase gene families GH5 (subfamily 5) and GH11 encoding endo-β-1,4-glucanases and endo-β-1,4-xylanases respectively as well as Basidiomycota class II peroxidases, corresponding to the CAZy Auxiliary Activity family 2 (AA2), active on lignin. These primers were experimentally validated using DNA extracted from a wide range of Ascomycota and Basidiomycota species including 27 with sequenced genomes. Along with the published primers for Glycoside Hydrolase GH7 encoding enzymes active on cellulose, the newly design primers were shown to be compatible with the Illumina MiSeq sequencing technology. Sequences obtained from RNA extracted from beech or spruce forest soils showed a high diversity and were uniformly distributed in gene trees featuring the global diversity of these gene families. This high-throughput sequencing approach using several degenerate primers constitutes a robust method, which allows the simultaneous characterization of the diversity of different fungal transcripts involved in plant organic matter degradation and may lead to the discovery of complex patterns in gene expression of soil fungal communities.
土壤中植物生物量的降解是陆地生态系统碳循环的关键步骤之一。真菌腐生群落通过产生对植物有机物质主要成分具有活性的水解酶，在这一过程中发挥着重要作用。该领域的悬而未决的问题涉及所涉及物种的多样性、主要的生化途径以及这些途径如何受到凋落物质量或气候变化等外部因素的影响。这可以通过环境基因组学方法来解决，该方法涉及使用土壤提取的RNA作为材料，对关键酶编码基因家族进行系统测序。这种方法需要设计和评估与高通量测序方法兼容的基因家族特异性PCR引物。在本研究中，我们开发并评估了用于特异性扩增真菌CAZy糖苷水解酶基因家族GH5（亚家族5）和GH11的PCR引物，GH5和GH11分别编码内切-β-1,4-葡聚糖酶和内切-β-1,4-木聚糖酶，以及担子菌纲II类过氧化物酶，对应于CAZy辅助活性家族2（AA2），对木质素具有活性。这些引物通过从包括27个已测序基因组的多种子囊菌和担子菌物种中提取的DNA进行了实验验证。与已发表的用于编码对纤维素具有活性的酶的糖苷水解酶GH7的引物一起，新设计的引物被证明与Illumina MiSeq测序技术兼容。从山毛榉或云杉林土壤中提取的RNA获得的序列显示出高度的多样性，并均匀分布在具有这些基因家族全球多样性的基因树中。这种使用多种简并引物的高通量测序方法构成了一种强大的方法，它允许同时表征参与植物有机物质降解的不同真菌转录本的多样性，并可能导致发现土壤真菌群落基因表达中的复杂模式。
