小麦14-3-3蛋白TaGRF3-D基因克隆及功能分析

doi:10.11983/CBB24156

摘要/Abstract

摘要： 14-3-3蛋白广泛参与植物生长发育、代谢和非生物逆境信号转导过程。该研究克隆了小麦(Triticum aestivum) 14-3-3蛋白TaGRF3-D基因, TaGRF3-D基因编码261个氨基酸残基, 在单子叶植物中高度保守, 其与乌拉尔图小麦(T. urartu)的TuGF14d和大麦(Hordeum vulgare)的HvGF14a氨基酸序列完全相同; TaGRF3-D启动子区含有脱落酸等激素响应元件和多个非生物胁迫响应元件。亚细胞定位结果显示, TaGRF3-D蛋白主要定位于细胞膜与细胞核。对过表达TaGRF3-D基因的拟南芥(Arabidopsis thaliana)转基因株系ABA敏感性及干旱胁迫耐受性分析发现, TaGRF3-D过表达拟南芥在PEG和ABA处理下根长显著大于野生型, 干旱胁迫后存活率显著高于野生型。进一步利用酵母双杂交(yeast two-hybrid, Y2H)实验对TaGRF3-D蛋白与小麦AREBs/ABFs (ABA-responsive element binding proteins/ABA-responsive element binding factors)蛋白进行互作分析, 结果表明TaGRF3-D蛋白与TaABF3-B、TaABF4-A、TaABF15-D、TaABF16-B、TaABF17-D和 TaABF18-B存在相互作用; 而与TaABF1-D、TaABF2-A和TabABF19-A不互作。综上表明, TaABF3-D可能通过与TaABFs蛋白互作响应ABA信号, 从而提高转基因植株对干旱胁迫的耐受性。研究结果为小麦TaGRF3-D基因逆境胁迫响应功能研究奠定了基础。

关键词: 14-3-3蛋白, 干旱胁迫, ABA响应, 蛋白互作

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

孙月, 郭树娟, 赵惠贤, 马猛, 刘香利. 小麦14-3-3蛋白TaGRF3-D基因克隆及功能分析. 植物学报, 2025, 60(6): 863-874.

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). Chinese Bulletin of Botany, 2025, 60(6): 863-874.

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链接本文: https://www.chinbullbotany.com/CN/10.11983/CBB24156

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

图/表 10

表1 TaGRF3-D基因克隆和载体构建引物信息

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

图1 TaGRF3-A/B/D基因结构分析(A)及TaGRF3-A/B/D蛋白与其它物种同源蛋白的氨基酸序列比对(B) B图中黑色方框表示14-3-3蛋白家族保守结构域。

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.

图2 TaGRF3-A/B/D启动子主要顺式作用元件

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

图3 TaGRF3-D cDNA PCR扩增 M: DL 2000 bp DNA marker; 1: cDNA扩增产物

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

图4 TaGRF3-D在烟草叶片中的亚细胞定位分析 Bars=100 μm

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

图5 TaGRF3-D转基因株系和野生型PCR鉴定(A)以及TaGRF3-D基因表达分析(B) M: DL 2000 DNA marker; H2O: 空白对照; WT: 野生型; Plasmid: pCAMBIA1304-TaGRF3-D; OE1-OE3: 转基因株系

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

图6 脱落酸(ABA)和聚乙二醇(PEG)处理下TaGRF3-D转基因拟南芥根长分析 (A) ABA处理下的表型; (B) ABA处理下的根长统计; (C) PEG处理下的表型; (D) PEG处理下的根长统计。不同小写字母表示根据ANOVA分析结果(n=3)数据间存在显著性差异(P<0.05)。WT: 野生型; Bars=1 cm

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

图7 干旱胁迫下TaGRF3-D转基因拟南芥和野生型(WT)的表型(A)和存活率(B) 不同小写字母表示根据ANOVA分析结果(n=3)数据间存在显著性差异(P<0.05)。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

图8 诱饵蛋白的自激活验证和3-AT浓度的选择

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

图9 TaGRF3-D蛋白与ABF蛋白互作分析

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

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