Genome-Wide Identification, Characterization, and Expression Analysis Related to Low-Temperature Stress of the CmGLP Gene Family in Cucumis melo L

Abstract

Germin-like protein (GLP) participates in plant growth and development and plays an important role in plant stress. In the present study, 22 CmGLPs belonging to five classes were identified in the melon genome. Each member of the CmGLPs family contains a typical Cupin_1 domain. We conducted a genome-wide analysis of the melon GLP gene family characterization. CmGLPs were randomly distributed in the melon chromosomes, with the largest number on chromosome 8, having eight family members. Gene duplication events drive the evolution and expansion of the melon GLP gene family. Based on the phylogenetic tree analysis of GLP proteins in melon, rice, Arabidopsis, and cucumber, it was found that the GLP gene families of different species have diverged in evolution. Based on qRT-PCR results, all members of the CmGLP gene family could be expressed in different tissues of melon. Most CmGLP genes were up-regulated after low-temperature stress. The relative expression of CmGLP2-5 increased by 157.13 times at 48 h after low-temperature treatment. This finding suggests that the CmGLP2-5 might play an important role in low-temperature stress in melon. Furthermore, quantitative dual LUC assays indicated that CmMYB23 and CmWRKY33 can bind the promoter fragment of the CmGLP2-5. These results were helpful in understanding the functional succession and evolution of the melon GLP gene family and further revealed the response of CmGLPs to low-temperature stress in melon.

Keywords: Cucumis melo L.; GLP gene family; gene structure; low-temperature stress.

Figure 1

Chromosomal localization and collinear distribution of melon GLP genes. The gray line indicates the collinear block between the melon genome; the green line indicates the tandem duplication gene pairs of the CmGLP, gene names marked in red and connected with red lines indicate segmented duplication genes.

Figure 2

Phylogenetic tree of GLPs from melon (Cm), Arabidopsis thaliana (At), rice (Os), and cucumber (Cs). To distinguish different subfamilies by the color of evolutionary branches.

Figure 3

Gene structure (A) of CmGLPs and the conserved motifs (B,C) in their encoded proteins. (A) Gene structures of CmGLP genes in melon. UTR, CDS, Cupin_1 and introns were represented by green boxes, orange boxes, pink boxes and black lines, respectively. The size of CDS and introns can be estimated using the ratio of the bottom. The numbers of 0, 1, and 2 represent the splicing phase of intron. (B) Schematic representation of the conserved motifs predicted in the CmGLP proteins. The different colours represent different conserved motifs. The length of the amino acid was inferred by ruler at bottom. (C) Length and amino acid species of the seven conserved motifs of the CmGLPs protein sequence. The size of letters represents the frequency of amino acid occurrence.

Figure 4

Predicted cis-elements in CmGLP promoters of melon. Different cis-elements were identified and plotted against a bar diagram. The abundance of different regulatory elements on each of the promoters is shown in different colors.

Figure 5

Heat map of relative expression differences of GLP genes in different tissues of melon based on qRT-PCR analysis. Each column represents the relative expression levels of CmGLP genes in roots, stems, leaves, flowers, seeds. The expression level of each gene in the root was used as a control, with log2 processing on the qRT-PCR value. Blue indicates lower expression levels and red indicates higher expression levels.

Figure 6

Relative expression of melon GLP gene family members in melon leaves under low-temperature (LT) stress. (A–V) Relative expression levels of melon GLP gene family members in leaves of melon seedlings after low-temperature stress. Data are expressed as the mean ± standard error of three independent biological replicates. Different letters above the bars indicate a significant difference at p < 0.05.

Figure 7

Dual luciferase assay CmMYB23 and CmWRKY33 binding to CmGLP2-5 promoter. (A) Schematic diagram of CmGLP2-5 promoter, black rectangle indicates the W-box and MYB-binding elements in the partial promoter fragment (P). (B) Schematic diagrams of effector and reporter constructs used for dual-LUC transient assay. (C) The various combinations of vectors were performed on Dual-LUC transient expression assays. Asterisks indicate that the value is significantly different from that of the control (*** p < 0.001).