Cloning and Functional Analysis of the 14-3-3 Protein-encoding Gene TaGRF3-D in Wheat (Triticum aestivum)

doi:10.11983/CBB24156

Abstract

Abstract: INTRODUCTION: 14-3-3 proteins are a highly conserved protein family that specifically recognize phosphorylated target proteins and play crucial roles in plant abiotic stress responses. By interacting with AREB/ABF (ABA-responsive element binding protein/ABA-responsive element binding factor) transcription factors, 14-3-3 proteins participate in ABA signal transduction and regulate abiotic stress tolerance. TaGRF3-D is a 14-3-3 protein gene in wheat (Triticum aestivum), and our previous studies revealed that the expression of this gene was upregulated under ABA and abiotic stress.
RATIONALE: To explore the biological function of the TaGRF3-D gene, we cloned the gene, and investigated its subcellular localization and function under drought stress.
RESULTS: The results revealed that TaGRF3-D is highly conserved in monocotyledonous plants and is localizes in the nucleus and plasma membrane. Compared with the wild type, the Arabidopsis thaliana transgenic lines overexpressing TaGRF3-D presented significantly longer roots under PEG and ABA treatments and showed a markedly greater survival rate after drought stress. Yeast two-hybrid analysis revealed that TaGRF3-D interacted with wheat TaABF3-B, TaABF4-A, TaABF15-D, TaABF16-B, TaABF17-D, and TaABF18-B, but not with TaABF1-D, TaABF2-A or TaABF19-A.
CONCLUSION: These results suggest that TaABF3-D responds to ABA signaling by interacting with wheat TaABF transcription factors, thereby increasing the drought stress tolerance of transgenic plants.

Phenotypes of the TaGRF3-D transgenic lines and the wild type (WT) under drought stress (A) and interaction between the TaGRF3-D protein and the ABF protein (B). Bars=1 cm

Key words: 14-3-3 protein, drought stress, ABA response, protein interaction

Yue Sun, Shujuan Guo, Huixian Zhao, Meng Ma, Xiangli Liu. Cloning and Functional Analysis of the 14-3-3 Protein-encoding Gene TaGRF3-D in Wheat (Triticum aestivum)[J]. Chinese Bulletin of Botany, 2025, 60(6): 863-874.

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URL: https://www.chinbullbotany.com/EN/10.11983/CBB24156

https://www.chinbullbotany.com/EN/Y2025/V60/I6/863

Figures/Tables 10

Table 1 Primer information for TaGRF3-D gene cloning and vector construction

Primers	Primer sequence (5′-3′)	Primer usage
TaGRF3-D-F	ATGTCTACCGCTGAGGCAAC	TaGRF3-D gene cloning
TaGRF3-D-R	TCAGTGCCCCTCTCCCTCAG	TaGRF3-D gene cloning
1304-R	GTATCTTGAAAAGCATTGAACACC	Genomic PCR detection
Actin-F	TCGCTGACCGTATGAGCAAAG	Actin gene semi quantitative analysis
Actin-R	TGTGAACGATTCCTGGACCTG	Actin gene semi quantitative analysis
TaGRF3-D-RT-F	TCCTGAACTCTCCAGACCGT	TaGRF3-D gene semi quantitative analysis
TaGRF3-D-RT-R	CCTCTGCGTTATCGGAGGTC	TaGRF3-D gene semi quantitative analysis
TaGRF3-D-BD-F	atggccatggaggccgaattcATGTCTACCGCTGAGGCAACC	BD-TaGRF3-D vector construction
TaGRF3-D-BD-R	ttatgcggccgctgcaggtcgacTCAGTGCCCCTCTCCCTCA	BD-TaGRF3-D vector construction

Figure 1 Structural analysis of the TaGRF3-A/B/D gene (A) and alignment of the amino acid sequences of TaGRF3-A/B/D with those of homologous proteins of other species (B) The black box in Figure B indicates the conserved domain of the 14-3-3 protein family.

Figure 2 The main cis-acting elements of the TaGRF3-A/B/D gene promoter

Figure 3 Amplification of TaGRF3-D cDNA via PCR M: DL 2000 bp DNA marker; 1: Amplification product of cDNA

Figure 4 Subcellular localization analysis of TaGRF3-D in tobacco leaves Bars=100 μm

Figure 5 PCR identification (A) and TaGRF3-D gene expression analysis (B) of the TaGRF3-D transgenic lines and the wild- type M: DL 2000 DNA marker; H2O: Blank control; WT: Wild type; Plasmid: pCAMBIA1304-TaGRF3-D; OE1-OE3: Transgenic lines

Figure 6 Analysis of the root length of the TaGRF3-D Arabidopsis thaliana transgenic lines under abscisic acid (ABA) and polyethylene glycol (PEG) treatment (A) Growth phenotype under ABA treatment; (B) Root length statistics under ABA treatment; (C) Growth phenotype under PEG treatment; (D) Root length statistics under PEG treatment. Different lowercase letters indicate significant differences (P<0.05) among the data according to the results of ANOVA analysis (n=3). WT: Wild type; Bars=1 cm

Figure 7 Phenotype (A) and survival rate (B) of the TaGRF3-D transgenic Arabidopsis thaliana and the wild type (WT) under drought stress Different lowercase letters indicate significant differences (P<0.05) among the data according to the results of ANOVA (n=3). Bars=1 cm

Figure 8 Self-activation verification of bait proteins and selection of 3-AT concentrations

Figure 9 Interaction analysis between the TaGRF3-D protein and the ABF protein

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