De Novo Sequencing and Transcriptome Analysis of Pleurotus eryngii subsp. tuoliensis (Bailinggu) Mycelia in Response to Cold Stimulation

Abstract

Cold stimulation of Bailinggu's mycelia is the main factor that triggers primordia initiation for successful production of fruiting bodies under commercial cultivation. Yet, the molecular-level mechanisms involved in mycelia response to cold stimulation are still unclear. Here, we performed comparative transcriptomic analysis using RNA-Seq technology to better understand the gene expression regulation during different temporal stages of cold stimulation in Bailinggu. A total of 21,558 Bailinggu mycelia unigenes were de novo assembled and annotated from four libraries (control at 25 °C, plus cold stimulation treatments at -3 °C for a duration of 1-2 days, 5-6 days, and 9-10 days). GO and KEGG pathway analysis indicated that functional groups of differentially expressed unigenes associated with cell wall and membrane stabilization, calcium signaling and mitogen-activated protein kinases (MAPK) pathways, and soluble sugars and protein biosynthesis and metabolism pathways play a vital role in Bailinggu's response to cold stimulation. Six hundred and seven potential EST-based SSRs loci were identified in these unigenes, and 100 EST-SSR primers were randomly selected for validation. The overall polymorphism rate was 92% by using 10 wild strains of Bailinggu. Therefore, these results can serve as a valuable resource for a better understanding of the molecular mechanisms associated with Bailinggu's response to cold stimulation.

Keywords: Bailinggu; EST-SSR; cold stress; comparative transcriptomic analysis; qPCR-PCR.

Figure 1

Developmental stages of Bailinggu in year-round mushroom factory cultivation cycles. The vegetative (spawning, physiological ripening, cold stimulation) and fructification (primordia, fruiting body) phases are shown.

Figure 2

Unigene annotation. (A) Number of unigenes blasted to Non-Redundant (NR), Clusters of Orthologous Groups (COG), Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases (e < 0.00001); (B) COG classification. A total of 7576 unigenes were assigned to 25 classifications. The capital letters on the x-axis indicate the COG categories as listed on the right of the histogram.

Figure 3

Heat map representation of cluster analysis for the expression patterns of over 7000 differential gene expressions (DEGs) (p value: < 0.05, fold change: ≥ 2 fold (log2 |FC > 1|)) between control (CK = 25 °C) and different stages of cold stimulation (−3 °C for a duration of 1–2 d, 5–6 d, and 9–10 d) using the FPKM. The samples of the three stages of cold stimulation cluster together; notably, the 5–6 d and 9–10 d samples cluster particularly close.

Figure 4

qRT-PCR analysis of selected genes in mycelia during the physiological after-ripening stage (control (CK) = 25 °C) and different cold stimulation stages (1–2 d, 5–6 d, and 9–10 d at −3 °C). Transcript levels of five genes (PoH2-type hydrophobin, MAPK (mitogen-activated protein kinase hog1), catalase, Hsp70 (heat shock protein 70) and Hsp90 (heat shock protein 90)) that appeared to be up-regulated in all cold stimulation stages compared to the physiological after-ripening stage. The X-axis represents relative quantification of transcript levels of the three selected genes, and the Y-axis represents the control and the three sampling times after cold stimulation.

Figure 5

Amplification products obtained from PCR using EST-SSR primers and DNA from 10 Bailinggu wild strains using non-denaturing PAGE. EST-SSR primers are blgSSR22, blgSSR51, blgSSR65, and blgSSR74. Lines 1–10 are wild Bailinggu strains collected from Xinjiang Autonomous Region in China.