Genome-wide characterization of the Zn(II)2Cys6 zinc cluster-encoding gene family in Pleurotus ostreatus and expression analyses of this family during developmental stages and under heat stress

Abstract

Pleurotus ostreatus is one of the most widely cultivated mushrooms in China. The regulatory mechanisms of fruiting body formation and the response to heat stress in P. ostreatus are main research focuses. The Zn(II)₂Cys₆ family is one of the largest families of transcriptional factors and plays important roles in multiple biological processes in fungi. In this study, we identified 66 zinc cluster proteins in P. ostreatus (PoZCPs) through a genome-wide search. The PoZCPs were classified into 15 types according to their zinc cluster domain. Physical and chemical property analyses showed a huge diversity among the PoZCPs. Phylogenetic analysis of PoZCPs classified these proteins into six groups and conserved motif combinations and similar gene structures were observed in each group. The expression profiles of these PoZCP genes during different developmental stages and under heat stress were further investigated by RNA-sequencing (RNA-seq), revealing diverse expression patterns. A total of 13 PoZCPs that may participate in development or the heat stress response were selected for validation of their expression levels through real-time quantitative PCR (RT-qPCR) analysis, and some developmental stage-specific and heat stress-responsive candidates were identified. The findings contribute to our understanding of the roles and regulatory mechanisms of ZCPs in P. ostreatus.

Keywords: Fruiting body development; Heat stress; Pleurotus ostreatus; Zinc cluster protein.

Figure 1. The distribution of conserved domains in 66 members of the PoZCP family.

The domain organization of the zinc cluster proteins was determined from the Pfam and NCBI conserved domain databases. The X-axis represents the amino acid position from the N-terminus to the C-terminus. The gray rectangles represent amino acid regions with no identified domain, and the rectangles in different colors represent different domains, as shown in the key.

Figure 2. The motif logos of major types of zinc cluster domains in the PoZCP family.

The capital letters (A–D, F, I, J) on the left side of the logos represent the PoZCP family types shown in Table 1. The X-axis represents the positions beginning with the first cysteine of the zinc cluster domain, and the Y-axis represents the number of bits for each amino acid. The amino acids are shown with one-letter abbreviations.

Figure 3. Phylogenetic tree, motif composition and gene structures of the PoZCP family.

(A) Phylogenetic tree obtained using the maximum likelihood (ML) method by IQ-TREE with best fitting model branch support computed with 1000 UltraFast Bootstrap. Different colors of nodes represent different ranges of bootstrap support values. The classification of different types of PoZCPs is shown with polygons of different shapes and colors. (B) Motif composition of the PoZCP proteins, obtained by the MEME program. The gray line represents the length of the proteins, and the rectangles in different colors represent different motifs from motif 1 to motif 10. (C) Gene structures of PoZCP genes. The exons are represented by red rectangles, and the black lines connecting two exons represent introns.

Figure 4. Expression levels of PoZCPs under different conditions using RNA-seq analysis.

(A) Expression levels of PoZCPs during different developmental stages. M, mycelia (control); P, primordia; F, fruiting bodies. The heatmap was constructed based on RNA-seq analysis of PoZCP genes. The color scale at the top right corner represents high to low expression levels of each gene from red to blue. Different colors of clades represent different expression patterns. (I), Downregulated during developmental stages; (II), upregulated at the fruiting body stage; (III), upregulated at the primordia stage; (IV), upregulated during developmental stages. (B) Expression levels of PoZCPs under heat stress at different time points. The heatmap was constructed based on RNA-seq analysis of PoZCP genes. Mycelia without heat treatment (0 h) were analyzed as a control. The color scale at the top right corner represents high to low expression levels of each gene from red to green. Different colors of clades represent different expression patterns. (I), Downregulated during heat stress; (II), upregulated during 0.5–1 h of heat treatment. (III), upregulated during 2–3 h of heat treatment; (IV), downregulated during 0.5–1 h of heat treatment.

Figure 5. Expression analysis of 13 PoZCP genes using RT-qPCR.

(A–I) The expression levels of six PoZCP genes during different developmental stages. M, mycelia (control); P, primordia; F, fruiting bodies. (J–P) The expression levels of 10 PoZCP genes under heat stress. Mycelia without heat treatment (0 h) were analyzed as a control. The data are representative of three independent experiments. The error bars represent the standard deviations of three replicates. Statistical analysis was performed using the t test. An asterisk (*) represents p < 0.05 and two asterisks (**) represent p < 0.01.