Molecular Characterization， Expression Profiling， and Protein Interaction Analysis of the EuGIF1 Gene in Eucommia ulmoides

doi:10.13560/j.cnki.biotech.bull.1985.2025-0438

Abstract

Abstract:

Objective To systematically identify the GIF gene family in Eucommia ulmoides and analyze their expression patterns and protein interaction characteristics, aiming to decipher the regulatory network of organ development and optimize genetic transformation strategies. Method HMMER and BLASTPwere used to identify GIF genes in Eucommia ulmoides. Bioinformatics tools were employed to analyze protein physicochemical properties, 3D structures, and promoter cis-elements. RT-qPCR was adapted to validate the tissue-specific and GA₃-induced expression of EuGIF1. Yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays were conducted to verify the interaction between EuGIF1 and EuGRF5. Additionally, the overexpression vector carrying the EuGIF1 gene was to genetically complement the gif1 mutant of Arabidopsis thaliana. Result Three EuGIF genes EuGIF1/2/3 with conserved SSXT/SNH domains were identified in E. ulmoides. EuGIF1 and EuGIF2 were located on chromosome 7 of the E. ulmoides genome, and EuGIF3 was located on chromosome 17. The proteins encoded by EuGIF1,EuGIF2 and EuGIF3 all contained conserved SSXT/SNH domain.Promoter sequence analysis revealed that the promoter region of the EuGIF1 gene contained two gibberellin-response elements. EuGIF1 had high expression in apical meristems and rapid induction by GA₃. 3D structural prediction of the protein revealed potential interaction interfaces between the SSXT/SNH domains. Co-expression analysis suggested functional synergy between EuGIF1 and EuGRF1/3/5, but Y2H and BiFC assays showed no direct interaction between EuGIF1 and EuGRF5. The overexpression of EuGIF1 in A. thalianagif1 mutant failed to complement its narrow-leaf phenotype. Conclusion EuGIF1 may play a significant role in gibberellin-mediated organ development in E. ulmoides, whereas its interaction with GRFs shows species specificity. Furthermore, the functional conservation of this gene appears limited across divergent species.

Key words: Eucommia ulmoides, GIF gene family, gibberellin, protein 3D-structure, GRF protein, protein-protein interaction

WANG Ruo-Ruo, QU Peng-Kun, ZHANG Xin, WANG Luo, ZHU Ying, TIAN Shuang-Yi, ZHAO De-Gang. Molecular Characterization， Expression Profiling， and Protein Interaction Analysis of the EuGIF1 Gene in Eucommia ulmoides[J]. Biotechnology Bulletin, 2025, 41(12): 267-279.

Figures/Tables 11

Table 1 Table of primer sequences

引物名称 Primer name	序列 Sequence （5′‒3′）
cdsEuGIF1-F	ctagaggatctcgaggcATGCAGCAGCACCTGATG
cdsEuGIF1-R	cgtctgtacacctaggTCAGTTTCCATCATCAGAA
EuGIF1gfp-F	ttggagaggacacgcATGCAGCAGCACCTGATG
EuGIF1gfp-R	cttctcccttacccatGTTTCCATCATCAGAAGCCT
YN-GIF1-F	gcatttaaatctcgaggATGCAGCAGCACCTGATG
YN-GIF1-R	gtggcgatggatcttctGTTTCCATCATCAGAAGC
YC-GRF5-F	gaggaggacctgctttATGAACGTGAACGCGATGA
YC-GRF5-R	acgccggacgggtaccgCCAGTAGGGTCTGGAGT
AD-GIF1-F	ggccatggaggccagtATGCAGCAGCACCTGATG
AD-GIF1-R	gcagctcgagctcgatGTTTCCATCATCAGAAGCC
BD-GRF5-F	tatggccatggaggccATGAACGTGAACGCGATGA
BD-GRF5-R	gccgctgcaggtcgacCCAGTAGGGTCTGGAGTT
YS22-GFP-R	aggaagctttccagtagtgca
termi-R	aagaccggcaacaggattc
cdsEuGIF1-F2	CAGCTGGGAATGAGCTCTGG
EuGIF1-qRT-F	CAGCAGTATTCGGCACTACAG
EuGIF1-qRT--R	CCTCCACTCCCACCCATT
EuGIF2-qRT--F	CCATCACCAGCAGCATCAAT
EuGIF2-qRT--R	CACCGCCACCTCCGATAT
EuGIF3-qRT--F	ACAATGGCTCAGCAACAACA
EuGIF3-qRT--R	GTGGTGGAAGTGGAGTAGGT
EuACTIN-qRT--F	GTGTTATGGTTGGGATGGG
EuACTIN-qRT--R	TGCTGACTATGCCGTGTTC

Fig. 1 Distribution of conserved domains in the EuGIFs protein sequences and chromosomal distribution of the genes in E. ulmoidesA: Distribution of conserved domains in the EuGIFs protein sequences. B: Amino acid sequence alignment of EuGIFs proteins. C: Chromosomal distribution of the EuGIFs genes

Table 2 Physicochemical properties of the EuGIFs proteins in E. ulmoides

基因

名称

Gene name

基因ID

Gene ID

氨基酸长度

Number of amino acids （aa）

蛋白分子量

Molecular weight （Da）

等电点

Theoretical pI

不稳定指数

Instability index

脂肪族氨基酸指数

Aliphatic index

疏水性平均值

Grand average of hydropathicity

亚细胞定位

Subcellular location

EuGIF1

EuGIF2

EuGIF3

Fig. 2 Three-dimensional spatial structure diagram of the EuGIFs proteinA: Cartoon representation of the 3D structure of EuGIFs protein. Regions corresponding to the SSXT/SNH domains are indicated by arrows. B: Surface representation of the 3D structure of EuGIFs protein. The arrows denote the amino acids with pLDDT (predicted local distance difference test) values>90, which correspond to the amino acids depicted as stick structures in Fig. A

Fig. 3 Number (A) and distribution (B) of response elements on the promoter of the EuGIFs gene in E. ulmoides

Fig. 4 Expression pattern of EuGIFs genes in E. ulmoidesA: Expression profiles of the EuGIFs gene in different tissues of E. ulmoides. B: Expression profiles of the EuGIFs gene in young leaves of seedlings following exogenous GA3 treatment. *** P<0.001, ns indicates no statistically significant difference (P>0.05)

Fig. 5 Phylogenetic analysis of GIF homologs and gene co-expression profiles of EuGIF and EuCRFsA: Neighbor-joining phylogenetic tree constructed using full-length protein sequences, with all proteins containing the conserved SNH/SSXT domain. The green-shaded region highlights AtGIF1 homologs, and EuGIF1 is marked in red. The species abbreviations in the protein names, Eu, Cc, At, Vv, Cl, Fve, Os, and Ta, correspond to Eucommia ulmoides, Citrus clementina, Arabidopsis thaliana, Vitis vinifera, Citrullus lanatus, Fragaria vesca, Oryza sativa, and Triticum aestivum, respectively. B: Expression patterns of EuGIFs and EuGRFs, with the red dashed box indicating genes having high and correlated expression in meristematic tissues. The color gradient (blue-yellow-red) indicate expression levels (low-medium-high) of EuGRFs

Fig. 6 Subcellular localization of GFP-tagged proteins in the epidermal cells of tobacco leafThe figure displays four imaging modalities: GFP fluorescence (green, excitation/emission: 488/510 nm); chloroplast autofluorescence (red, excitation/emission: 640/675 nm); bright field (grayscale) and merged image. Scale bar: 50 μm

Table 3 Structural analysis of EuGIF1-EuGRF5 protein-protein interaction predicted by HDOCK

蛋白模型编号 Number	结构偏差 RMSD （Å）	亲和力 Score
1	50.52	-809.33
2	31.22	-774.96
3	69.01	-764.03
4	60.97	-758.89
5	55.71	-755.58
6	79.41	-749.46
7	64.33	-747.77
8	62.85	-735.99
9	62.97	-732.66
10	68.86	-732.20

Fig. 7 Yeast two-hybrid assay for protein-protein interaction between EuGIF1 and EuGRF5SD/-LT: Synthetic dropout medium deficient in leucine (-L) and tryptophan (-T). SD/-LTAH/X-α-Gal: Stringent selection medium with quadruple dropout (-L/-T/-A/-H) supplemented with 5-bromo-4-chloro-3-indolyl-α-D-galactopyranoside (X-α-Gal). The indicated OD600 values (0.2, 0.02, 0.002, and 0.000 2) represent 10-fold serial dilutions of yeast cultures prior to spotting assay. Positive control: BD-P53+AD-T; negative control 1: BD-Lam+AD-T; test group: BD-EuGRF5+AD-EuGIF1; negative control 2: BD-EuGRF5+AD/BD+AD-EuGIF1

Fig. 8 Genetic complementation of the A. thalianagif1 mutant by EuGIF1A: Genotyping results of T3 generation transgenic plants for the complementation test. B: Phenotypic characterization of 12 d T3 transgenic seedlings grown in medium, with arrows indicating the wild type, mutant, and three representative T3 homozygous positive transgenic lines for juvenile leaves. Scale bar: 0.5 cm

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