Identification and Expression Characteristics Analysis of CmRGLG Gene Family in Melon

doi:10.13560/j.cnki.biotech.bull.1985.2025-0307

Abstract

Abstract:

Objective RGLG proteins belong to the RING (Really Interesting New Gene, RING) type E3 ubiquitin ligases, which participate in plant growth, development, and abiotic stress responses by ubiquitinating and degrading other proteins. By identifying the members of the CmRGLGs gene family (CmRGLGs) in melon and analyzing their expression patterns, this study lays a theoretical foundation for further investigation into their potential functions. Method Bioinformatics methods were employed to analyze the chromosomal localization, gene structure, physicochemical properties of encoded proteins, phylogenetic evolution, syntenic relationships, and protein-protein interactions of CmRGLGs. The expression levels of these genes in different organs, under varying phytohormone concentrations, and after abiotic stress treatments were analyzed using RT-qPCR in young melon leaves. Result A total of six CmRGLGs members (CmRGLG1-CmRGLG6) were identified in the melon genome, named based on their chromosomal positions. The encoded proteins were hydrophilic, with amino acid lengths ranging from 364 to 596 aa. Phylogenetic analysis classified CmRGLGs into three distinct branches. Intraspecific and interspecific collinearity analysis revealed no gene duplication events among CmRGLGs. CmRGLG1, CmRGLG2, CmRGLG4, and CmRGLG5 showed collinear genes in Arabidopsis thaliana, Cucumis sativus, and Solanum lycopersicum. The promoter regions contained cis-acting elements associated with phytohormones and abiotic stress responses. Protein-protein interaction network prediction revealed that CmRGLGs-interacting proteins were primarily enriched in ubiquitin-protein transferase activity, protein metabolism, biosynthesis, and organic cyclic compound binding pathways. Expression analysis showed that CmRGLG5 and CmRGLG6 were expressed in the stems at the lowest level, while CmRGLG1, CmRGLG2, CmRGLG3, and CmRGLG4 were more abundant in the flowers. Additionally, each gene presented varying expression under different phytohormone treatments and abiotic stress conditions. Conclusion Under different phytohormone treatments, most CmRGLG members in melon showed downregulated expression trends. Under abiotic stress treatments, the majority of members demonstrated an initial upregulation followed by downregulation in their expressions.

Key words: Cucumis melo L., ubiquitin ligase, RGLG gene family, bioinformatics, protein-protein interaction, phytohormones, abiotic stress, expression analysis

WANG Ya-ping, JIN Lan, HAO Jin-feng, CHANG Ming, WANG Yan-dan, GAO Feng. Identification and Expression Characteristics Analysis of CmRGLG Gene Family in Melon[J]. Biotechnology Bulletin, 2025, 41(12): 156-167.

Figures/Tables 11

Table 1 RT-qPCR primer sequences

引物名称 Primer name	正向引物序列 Forward primer sequence （5′‒3′）	反向引物序列 Reverse primer sequence （5′‒3′）	产物大小 Product size （bp）
CmGAPDH	ATCATTCCTAGCAGCACTGG	TTGGCATCAAATATGCTTGACCTG	278
CmRGLG1	AGTGGGGTCATTATGGTTATCCG	CCTCTTTCTTGTCTCAGGGGCT	116
CmRGLG2	GGTCAATCCCCCCCTTAT	GCTCCTGTTACCTCCTCCAA	221
CmRGLG3	CTTGTTTCGGTTTTGGTGAT	CCAGGTGGTGTTTTAGGATTTC	262
CmRGLG4	ATATCTCCCCAGAGAGTTGCA	AAGACCAGGTAGGCTTTGAAT	97
CmRGLG5	CAAACTCCACAACAACCACGG	GAGATTAGATGACTCAAGTCCAGCG	123
CmRGLG6	CGATTCTCCTAATCCCTACCA	TTGCCCACCACTCTTCTCTA	280

Fig. 1 Gene structure and conservative motif analysis of CmRGLGs in melonA: Gene structure. B: Conserved motifs. C: Motif sequence logo (The height of each letter indicates the frequency of amino acid occurrence at that position)

Fig. 2 Phylogenetic tree of RGLG protein family in melon and other speciesCm: Cucumis melo; At: Arabidopsis thaliana; Os: Oryza sativa; Pa: Populus alba; Ta: Triticum aestivum; Gs: Glycine soja; Gh: Gossypium hirsutum; Bn: Brassica napus; Hs: Hibiscus syriacus; Zo: Zingiber officinale

Fig. 3 Chromosomal localization of CmRGLGs in melon

Fig. 4 Intraspecific (A) and interspecific synteny (B) analysis of CmRGLGs in melonGray lines indicate collinear blocks between the melon genome and the genomes of the analyzed species; red lines highlight RGLG gene pairs

Fig. 5 Cis-acting elements analysis in the promoter region of CmRGLGs in melon

Fig. 6 Statistical results of KEGG and GO annotation in protein-protein interaction analysis by Cytoscape 3.10.2A: KEGG pathway enrichment analysis. B: GO functional enrichment analysis (X-axis: percentage of enriched genes per Term; numbers at the end of bars indicate the specific gene count in each Term; Y-axis: significantly enriched GO Terms). *P<0.05, **P<0.01

Fig. 7 Protein-protein interaction network of CmRGLGs in melonHexagons indicate biological processes； triangles indicate molecular functions； circles indicate proteins

Fig. 8 Analysis of expression characteristics of CmRGLGs in different organs of melonDifferent lowercase letters indicate significant differences (P<0.05). The same below

Fig. 9 Analysis of expression characteristics of CmRGLGs under phytohormone (A) and abiotic stress treatments (B) in melon

Fig. 10 Analysis of expression characteristics of CmRGLGs in the transcriptome data of melon fruits35, 40, and 45 DAP indicate 35, 40, and 45 d after pollination (DAP), respectively

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