Expression Analysis and Interaction Protein Screening of GmRLK19 in Soybean

doi:10.13560/j.cnki.biotech.bull.1985.2025-0903

Abstract

Abstract:

Objective GmRLK19, an important candidate gene for soybean 100-seed weight, was identified in our previous study. This study aims to analyze the expression pattern and screen for interacting proteins of GmRLK19, providing a basis for further investigation into its biological function. Method The bioinformatics was employed to analyze the structural characteristics of the GmRLK19 gene. The tissue expression pattern of GmRLK19 was analyzed using quantitative real-time PCR (RT-qPCR) technology. Subcellular localization was determined by fusing GmRLK19 with a fluorescent tag and observing with confocal microscopy. Proteins interacting with GmRLK19 were screened using yeast two-hybrid technology, and their potential functions were predicted using database information. Result The secondary structure of GmRLK19 protein is predominantly composed of random coils and α-helices, with a total of 145 predicted phosphorylation sites. Gene GmRLK19 is expressed ubiquitously across all tested tissues, showing the highest transcript levels in seeds at 30 d after flowering. Subcellular localization experiments confirmed that the GmRLK19 protein is primarily localized to the plasma membrane. Through yeast two-hybrid screening, a total of 187 positive clones were identified, among which 148 candidate interacting proteins were screened by PCR amplification of the inserted fragments after transformation. Functional analysis revealed that the interacting proteins function primarily in regulating plant growth and development, signal transduction, and metabolic processes. The GO enrichment analysis revealed significant enrichment for interacting proteins involved in photosynthesis and protein translation. Conclusion GmRLK19 is expressed in various tissues, and its encoded protein localizes primarily to the cell membrane. The 148 candidate interacting proteins are obtained via screening, suggesting that GmRLK19 influences soybean seed weight likely through multiple pathways, such as plant hormone signaling and photosynthesis.

Key words: soybean, GmRLK19, expression analysis, yeast two-hybrid, interacting protein

XU Meng-ge, SONG Huo-yan, LUO Jia, SU Yi, ZHOU Hui-wen, WANG Can, KONG Ke-ke. Expression Analysis and Interaction Protein Screening of GmRLK19 in Soybean[J]. Biotechnology Bulletin, 2026, 42(4): 92-100.

Figures/Tables 7

Fig. 1 Bioinformatics analysis of GmRLK19A: Secondary structure prediction; B: transmembrane structure prediction; C: hydrophilic/hydrophobic prediction; D: phosphorylation site prediction; E: signal peptide prediction

Fig. 2 Phylogenetic tree analysis of GmRLK19 and its homologous proteinPhylogenetic tree analysis of GmRLK19 and homologous proteins. The numbers in the nodes indicate 1 000-time Bootstrap. It is depicted using the full-length amino acid sequences of the 14 proteins that are homologous with GmRLK19, including Phacu.CVR.001G169400 (Phaseolus vulgaris); Phvul.001G137300 (Phaseolus acutifolius); Vigun01g119600 (Vigna unguiculata); Lalb Chr20g0110951 (Lupinus albus); Glyma.19G143300 (Glycine max); Lj1g0014304 (Lotus japonicus); Medtr7g092430 (Medicago truncatula); GhUA48.A07G027300 (Gossypium hirsutum); AT5G06940 (Arabidopsis thaliana); Glyma.03G140700 (Glycine max); Ca_07275 (Cicer arietinum); Sobic.006G116200 (Sorghum bicolor); LOC Os04g39650 (Oryza sativa); ZmPHB47.10G170400 (Zea mays); TraesCS2D03G0763100 (Triticum aestivum)

Fig. 3 Relative expressions of GmRLK19 gene in different soybean tissuesThe tissue samples were selected from the root, stem, leaf and 2 cm pod of the PI595843 of 14-day old seedlings, and the seeds at different development stages at 10-40 d after flowering were also collected. The relative expressions were normalized in the seed at 10DAF from PI595843, as the control (relative expression = 1). The error bars indicate the standard error (n = 3). Seed 10 DAF-Seed 40 DAF: the seed of the 10, 20, 30, and 40 d after flowering, respectively. * and ** indicate significant difference between tissue samples and seed at 10 DAF at 0.01 and 0.05 level (Student’s t-test, two-tail), respectively

Fig. 4 Subcellular localization of GmRLK19 proteinTransient expression of GFP protein or GmRLK19-GFP fusion protein under the control of CaMV 35S promoter in tobacco cells. GFP, green fluorescence protein. Chlorophyll, chloroplast spontaneous fluorescence. Bright field, brighten field. Merged, merged images of the GFP, chlorophyll, and bright field images. Scale bar = 10 μm

Fig. 5 Results of self-activation detectionFrom top to bottom, the groups are: positive control, negative control, functional validation group, and self-activation group. From left to right, the plates are: DDO, TDO/X, and QDO/X plate

Fig. 6 The 187 positive clones obtained double hybridization library screening“+” indicates positive control, pTSU2-APP+NubG-Fe65; “-” indicates negative control, pTSU2-APP+pPR3-N

Fig. 7 GO enrichment analysis of candidate interacting proteins of GmRLK19

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