Transcriptional Dynamics of Genes Purportedly Involved in the Control of Meiosis, Carbohydrate, and Secondary Metabolism during Sporulation in Ganoderma lucidum

Abstract

Ganoderma lucidum spores (GLS), the mature germ cells ejected from the abaxial side of the pileus, have diverse pharmacological effects. However, the genetic regulation of sporulation in this fungus remains unknown. Here, samples corresponding to the abaxial side of the pileus were collected from strain YW-1 at three sequential developmental stages and were then subjected to a transcriptome assay. We identified 1598 differentially expressed genes (DEGs) and found that the genes related to carbohydrate metabolism were strongly expressed during spore morphogenesis. In particular, genes involved in trehalose and malate synthesis were upregulated, implying the accumulation of specific carbohydrates in mature G. lucidum spores. Furthermore, the expression of genes involved in triterpenoid and ergosterol biosynthesis was high in the young fruiting body but gradually decreased with sporulation. Finally, spore development-related regulatory pathways were explored by analyzing the DNA binding motifs of 24 transcription factors that are considered to participate in the control of sporulation. Our results provide a dataset of dynamic gene expression during sporulation in G. lucidum. They also shed light on genes potentially involved in transcriptional regulation of the meiotic process, metabolism pathways in energy provision, and ganoderic acids and ergosterol biosynthesis.

Keywords: Ganoderma lucidum; energy source; meiotic process; sporulation; transcription regulation.

Figure 1

Summary of transcriptome sequence datasets. (a–f) Developmental comparisons of pileus (a–c) and cross section of gill (d–f) at three spore developmental stages of YW-1 strain. Young fruiting body before sporulation (a,d), initial stage of sporulation (b,e), and nearly mature fruiting body with lots of spores (c,f) are referred to as YW1, YW2, and YW3, respectively. (g) Correlation among transcriptome datasets. Pearson’s correlation coefficient r was used to assess the reliability of every two RNA sequencing (RNA-Seq) libraries. A score of near 1 between biological replicates indicates faithful replication, while a much lower score between different developmental stages indicates globally different. (h) Distribution diagram shows the number of genes with different fragments per kilobase of transcript per million fragments mapped (FPKM) values across the three developmental stages. (i) Venn diagram shows the number of stage-specific expressed genes at each developmental stage and the shared expression genes between stages.

Figure 2

Gene Ontology (GO) functional enrichment of differentially expressed genes. (a) Venn diagrams show the number of differentially expressed genes (DEGs) between different developmental stages and the number of shared DEGs. The left Venn diagram indicates the upregulated genes, and the right one represents the downregulated genes. (b) GO enrichment for shared DEGs between different comparisons. Venn diagrams represent DEGs shared among different comparisons (from left to right): DEGs in YW2/YW1 and YW3/YW1; DEGs in YW3/YW1, but not in YW2/YW1. BP: biological process; MF: molecular function; CC: cellular component. The GO terms with p values < 0.01 are shown in color, otherwise, they are shown in grey. (c) Expression profiles of DEGs in carbohydrate metabolic process. The square represents the log₂(FPKM + 1) ratio of the expression level in different developmental stages.

Figure 3

Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment of differentially expressed genes. (a) KEGG enrichment for shared DEGs between different comparisons. Venn diagrams represent DEGs shared among different comparisons. Significantly enriched pathways with p < 0.05 are shown. (b) Expression of genes in the carbon metabolic pathway. (c) Expression of genes in the starch and sucrose metabolism pathway. The square represents the log₂(FPKM + 1) ratio of the expression level in different developmental stages.

Figure 4

DEGs predictably involved in triterpenoid and ergosterol biosynthesis pathways. (a) Heatmap shows the expression level of the genes coding for key enzymes, which are predictably involved in triterpenoid and ergosterol biosynthesis pathways. The square represents the log₂ ratio of the expression level in different developmental stages. Red, upregulated; blue, downregulated. (b) A schematic of triterpenoid and ergosterol biosynthesis pathways in Ganoderma lucidum.

Figure 5

Expression of genes coding for transcriptional regulators. (a) Transcriptional regulator family distribution and the proportions of developmentally regulated (orange) versus non-regulated (yellow) genes. (b) Heatmap of developmentally regulated transcription factor (TF)-coding genes. The square represents log₂(FPKM + 1) ratio of the transcript abundance change in different stages.

Figure 6

Expression level of selected genes in different developmental stages analyzed by qRT-PCR. qRT-PCR verification of the expression of seven differentially expressed genes (DEGs) and three non-differentially expressed genes (GL25739, GL20535, and GL24465) in three stages. Q: qRT-PCR; R: RNA-seq.