植物学报

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

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

小麦14-3-3蛋白TaGRF3-D基因克隆及功能分析

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孙月 , 郭树娟 , 赵惠贤 , 马猛 , 刘香利 ^*

植物学报 | 研究论文 2025,60(6): 863-874

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植物学报 |研究论文 2025 , 60 (6) : 863 -874

小麦14-3-3蛋白TaGRF3-D基因克隆及功能分析

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孙月, 郭树娟, 赵惠贤, 马猛, 刘香利^*

作者信息

西北农林科技大学生命科学学院, 杨凌 712100

通讯作者:

^* 刘香利, 博士, 副教授, 硕士生导师。一直从事小麦产量和抗旱相关基因功能及遗传改良基础研究。主持完成国家自然科学基金(No.31101205)、杨凌示范区产学研用协同创新重大项目“高产、优质、耐旱小麦分子设计育种实用技术研究及应用”(No.2017CXY-01)、基本科研业务费专项资金项目2项以及校长基金项目1项。主持陕西省自然科学基础研究计划(No.2024JC-YBMS-171)。先后参加国家自然科学基金5项, 国家转基因生物新品种培育科技重大专项课题等4项。以第一作者/通讯作者发表论文20余篇。E-mail: liuxianglii@163.com

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

Yue Sun, Shujuan Guo, Huixian Zhao, Meng Ma, Xiangli Liu^*

Affiliations

College of Life Sciences, Northwest A&F University, Yangling 712100, China

Corresponding Author:

^* E-mail: liuxianglii@163.com

出版时间: 2025-11-10 doi: 10.11983/CBB24156

文章导航

摘要

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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

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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

引用本文

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 . DOI: 10.11983/CBB24156

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 . DOI: 10.11983/CBB24156

正文

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14-3-3蛋白是广泛存在于所有真核生物中高度保守的蛋白家族, 其通过特异性识别磷酸化的靶蛋白, 与之相互作用并改变靶蛋白的定位、稳定性、磷酸化状态和活性, 参与多种细胞过程的调控, 包括代谢调节、信号转导、细胞周期调控以及胁迫响应等(Fu et al., 2000; Aitken, 2006; Zhao et al., 2021)。植物14-3-3蛋白被命名为G-box因子14-3-3 (G-box factor 14-3-3 homologs, GF14)或通用调节因子(general regulatory factor, GRF) (Lu et al., 1992), 在植物非生物胁迫(如干旱和盐胁迫)和激素信号转导中发挥关键作用(周颖等, 2012; Huang et al., 2022)。拟南芥(Arabidopsis thaliana) 14-3-3蛋白GRF6 (GF14-λ)与磷酸化的双孔钾离子通道蛋白TPK1 (two-pore K⁺channel 1)相互作用并增强其活性, 促进K⁺流向细胞质基质, 增强转基因株系的耐盐性(Latz et al., 2007, 2013)。拟南芥GRF11 (GF14-o)可通过调节质膜质子泵(PM H⁺-ATPase)活性影响耐旱性(Kaundal et al., 2017)。在水稻(Oryza sativa)中过表达玉米(Zea mays) 14-3-3蛋白基因ZmGF14-6能够提高转基因水稻对干旱胁迫的耐受性(Campo et al., 2012)。过表达水稻14-3-3蛋白编码基因OsGF14c和OsGF14f可提高转基因水稻的干旱胁迫耐受性(Ho et al., 2013; Ma et al., 2023)。

AREBs/ABFs转录因子属于碱性亮氨酸拉链(basic leucine zipper, bZIP)转录因子A亚家族, 能够特异性识别并结合下游基因启动子区的脱落酸(abscisic acid, ABA)响应元件(ABA-responsive element, ABRE), 激活相关基因表达(Choi et al., 2000)。14-3-3蛋白可通过与AREBs/ABFs转录因子相互作用, 调节ABA信号通路相关基因的转录水平和气孔闭合, 进而对逆境条件做出响应(Sun et al., 2015; Zhang et al., 2023)。大麦(Hordeum vulgare) 5种14-3-3蛋白质与HvABFs相互作用, 在ABA和赤霉素(gibberellin, GA)信号转导中发挥调节功能(Schoonheim et al., 2009)。大豆(Glycine soja) 14-3-3蛋白GsGF14o与AREBs/ABFs转录因子相互作用, 拟南芥中GsGF14o的异位表达可增强ABA对转基因株系种子萌发和幼苗生长的抑制作用, 促进ABA诱导的气孔关闭(Sun et al., 2015)。嘎拉苹果(Malus × domestic ‘Royal Gala’) 14-3-3蛋白MdGRF11可与4种ABFs转录因子互作, 参与ABA信号转导, 正调控盐和干旱胁迫耐受性(Ren et al., 2019)。二穗短柄草(Brachypodium distachyon) BdGF14d蛋白通过与AREBs/ABFs转录因子BdbZIP62和BdbZIP71相互作用参与ABA信号通路, 调节气孔关闭, 提高干旱胁迫耐受性(He et al., 2017); BdGF14g可与烟草(Nicotiana tabacum) NtABF2相互作用, 增强ABA相关基因的转录, 提高BdGF14g过表达烟草的干旱胁迫耐受性(Zhang et al., 2023)。

小麦(Triticum aestivum)是重要的粮食作物, 非生物胁迫严重影响其生长发育, 降低产量。因此, 研究小麦逆境胁迫响应相关基因功能对于小麦分子育种具有重要意义。相比其它物种, 关于小麦14-3-3蛋白的研究较少。小麦14-3-3蛋白编码基因TaGF14b过表达烟草转基因株系, 通过增强ABA诱导的气孔关闭和活性氧清除能力, 显著提高对干旱和盐胁迫的耐受性(Zhang et al., 2018)。小麦TaGRF6-A通过与MYB (myeloblastosis)转录因子TaMYB64相互作用, 正向调控小麦耐盐性(Shao et al., 2021)。本课题组前期研究发现, 位于小麦第4部分同源群染色体上的14-3-3蛋白基因TaGRF3-A/B/D在ABA、干旱、高盐和冷处理后上调表达, 说明其可能与胁迫响应有关(申玉霞, 2018)。本研究对过表达TaGRF3-D基因的拟南芥转基因株系进行干旱胁迫响应分析, 并对TaGRF3-D与小麦TaABFs转录因子进行酵母双杂交互作分析, 旨在为小麦TaGRF3-D基因逆境胁迫响应功能研究奠定基础。

1 材料与方法

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1.1 材料

供试植物材料为小麦(Triticum aestivum L.)中国春(Chinese Spring, CS)与拟南芥Columbia (Col-0)。改良的植物表达载体pCAMBIA1304 (载体上原有的hpt基因替换为除草剂抗性bar基因, 35S启动子序列后引入多个酶切位点)由本实验室保存。

1.2 序列比对和启动子顺式作用元件分析

从NCBI数据库下载小麦TaGRF3-A/B/D同源蛋白序列, 使用MEGA软件构建系统进化树, 对TaGRF3-D蛋白与聚于同一分支的同源蛋白用DNAMAN进行多重比对。使用PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/)预测TaGRF3-A/ B/D基因启动子区域(编码区上游延伸2 kb)的顺式作用元件。

1.3 TaGRF3-D基因克隆

提取小麦幼苗叶片总RNA, 利用反转录试剂盒Evo M-MLV Reverse Transcriptase (艾科瑞)反转录成cDNA, 以cDNA为模板, 利用特异性引物TaGRF3- D-F和TaGRF3-D-R (表1) PCR扩增TaGRF3-D基因编码区, 反应体系: 25 μL 2× Phanta Max Master Mix, 10 µmol∙L^-1上、下游引物各2 μL, 2 μL cDNA, 用ddH₂O补足体积至50 μL。扩增程序: 94°C3分钟; 94°C15秒, 60°C15秒, 72°C30秒, 32个循环; 72°C5分钟。将扩增产物构建至T载体, 测序验证正确后将其命名为pMD19-T-TaGRF3-D。

1.4 TaGRF3-D蛋白亚细胞定位

将TaGRF3-D基因构建至空载体pCAMBIA1302- GFP, 获得pCAMBIA1302-TaGRF3-D-GFP载体并转化农杆菌GV3101。用携带pCAMBIA1302-GFP、pCAMBIA1302-TaGRF3-D-GFP以及核Marker mCherry载体的农杆菌侵染烟草表皮细胞, 48小时后利用激光共聚焦显微镜观察蛋白亚细胞定位情况。

1.5 TaGRF3-D拟南芥遗传转化和筛选鉴定

将TaGRF3-D基因构建至改造后的植物表达载体pCAMBIA1304, 获得pCAMBIA1304-TaGRF3-D载体并转化农杆菌GV3101, 采用花序侵染法转化拟南芥(Zhang et al., 2006)。向叶片喷施20%草胺磷溶液对转基因拟南芥逐代筛选, 获得T₃代转基因纯合株系, 进一步利用引物TaGRF3-D-F和1304-R (表1)对转基因株系进行PCR验证。将野生型和转基因拟南芥叶片总RNA反转录为cDNA后, 以Actin基因为内参进行半定量分析(半定量引物见表1)。反应体系: 10 μL 2× Accureate Taq Master Mix (dye plus), 0.4 μL 10 µmol∙L^-1 Primer F/R, 1 μL cDNA, 用ddH₂O补足体积至20 μL。扩增程序: 94°C30秒; 94°C30秒, 60°C30秒, 72°C30秒, 26个循环; 72°C2分钟。扩增产物用0.1%琼脂糖凝胶电泳进行检测。

1.6 转基因拟南芥表型观察与统计

将拟南芥野生型和3个转基因纯合株系种子消毒后, 点播在1/2MS培养基上, 4°C春化3天后, 在光周期为16小时光照/8小时黑暗、温度为22°C条件下培养5天。

根长统计: 选择长势一致的拟南芥幼苗, 分别移至1/2MS培养基、含20% PEG的1/2MS培养基和含10 µmol∙L^-1 ABA的1/2MS培养基中, 竖直培养14天后观察并统计根长。

存活率统计: 将拟南芥幼苗移栽到土壤中, 正常供水条件下培养4周后进行断水胁迫处理14天, 复水6天后统计存活率。

1.7 数据分析

利用SPSS软件进行数据单因素方差分析及差异显著性检验(One-way ANOVA, P<0.05表示差异显著; P<0.01表示差异极显著)。

1.8 诱饵载体构建及自激活检测

以质粒pMD19-T-TaGRF3-D为模板, 用引物TaGRF3- D-BD-F/R (表1)扩增TaGRF3-D基因后重组连接至pGBKT7 (BD)空载体中构建诱饵载体BD-TaGRF3-D。采用PEG/LiAc法(Gietz and Woods, 2002)将BD- TaGRF3-D+pGADT7 (AD)空载和阳性对照BD-p53+ AD-T分别共转化入Y2HGold酵母细胞, 涂布到含不同浓度HIS2竞争性抑制剂3-AT (3-amino-1,2,4-triazole)的SD/-Trp/-Leu/-His培养基上, 观察菌落的生长情况以筛选合适的3-AT浓度。

1.9 TaGRF3-D与TaABFs蛋白酵母双杂交分析

选择本课题组鉴定到的位于小麦7个不同同源群染色体的9个TaABFs蛋白(TaABF1-D、TaABF2-A、TaABF3-B、TaABF4-A、TaABF15-D、TaABF16-B、TaABF17-D、TaABF18-B和TabABF19-A) (郭树娟等, 2023)连接至pGADT7 (AD)空载体中。将构建好的9个AD-TaABFs质粒与BD-TaGRF3-D质粒共转化Y2HGold菌株, 长出的单菌落与阳性对照(BD-p53+ AD-T)和阴性对照(BD-Lam+AD-T)一起在DDO (SD/- Trp/-Leu)和添加金担子素(Aureobasidin A, AbA)的QDO (SD/-Trp/-Leu/-Ade/-His)/AbA/3-AT平板上滴度稀释培养, 观察菌株生长状况。

2 结果与分析

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2.1 TaGRF3-D基因结构和氨基酸序列比对

TaGRF3-A (TraesCS4A03G0697400.1)、TaGRF3- B (TraesCS4B03G0098100.1)和TaGRF3-D (TraesCS4D03G0082800.1)是位于小麦第4部分同源群上的3个14-3-3蛋白编码基因。基因结构分析显示, TaGRF3-A/B/D基因长度分别为2 702/2 714/2 647 bp, cDNA长度分别为1 498/1 440/1 380 bp, 均含有5个外显子和4个内含子(图1A), 虽然基因非编码区存在差异, 但CDS区高度相似, 长度均为786 bp, 编码261个氨基酸残基。

对与TaGRF3-A/B/D系统进化分析聚在同一分支的其它物种同源蛋白序列进行比对, 结果显示TaGRF3-A/B/D氨基酸序列完全相同, 与乌拉尔图小麦(T. urartu) TuGF14d和大麦HvGF14a氨基酸序列也完全相同, 与水稻OsGF14f和二穗短柄草BdGF14f序列相似度分别为95.4%和91.35%, 与山羊草(Aegilops tauschii) AetGF14c序列相似度为82.71%, 而与拟南芥GRF1相似度较低, 为62.89%, 表明TaGRF3-A/B/D在单子叶植物中高度保守, 单、双子叶植物蛋白保守区序列也高度相似, 仅在N端和C端可变区存在较大差异(图1B)。

TaGRF3-A/B/D启动子顺式作用元件分析结果显示, TaGRF3-A/B/D启动子区含有激素类响应元件, 包括ABA响应元件ABRE、茉莉酸甲酯(methyljasmonate)响应元件TGACG-motif和CGTCA-motif; 非生物胁迫相关元件, 包括低温响应元件LTR、干旱响应元件MYB以及干旱诱导MYB结合位点MBS (图2), 说明TaGRF3-A/B/D能响应激素和非生物逆境胁迫。

2.2 TaGRF3-D基因克隆

以小麦中国春幼苗叶片cDNA为模板, 对TaGRF3-D基因CDS进行扩增, 得到预期的786 bp单一条带(图3)。克隆后进行测序分析, 测序结果与小麦基因组数据库中碱基序列完全一致, 说明已成功克隆到小麦TaGRF3-D基因。

2.3 TaGRF3-D蛋白亚细胞定位分析

为确定TaGRF3-D蛋白的亚细胞定位, 用携带pCAMBIA1302-GFP、pCAMBIA1302-TaGRF3-D-GFP以及核Marker mCherry载体的农杆菌侵染烟草表皮细胞。瞬时表达分析结果显示, 空载体pCAMBIA1302- GFP在细胞核、细胞膜以及细胞质中均有绿色荧光蛋白信号; pCAMBIA1302-TaGRF3-D-GFP融合表达载体绿色荧光蛋白信号主要在细胞核和细胞膜中(图4)。

2.4 TaGRF3-D转基因拟南芥株系鉴定

为进一步阐明TaGRF3-D基因的功能, 通过转化筛选获得过表达TaGRF3-D基因的纯合T₃代拟南芥转基因株系, 选择3个纯合株系(OE1、OE2和OE3)幼苗叶片提取基因组DNA进行PCR检测, 提取RNA反转录成cDNA进行半定量分析。结果显示3个转基因株系幼苗均能扩增出1 134 bp的预期目的条带(图5A), 且转基因株系中TaGRF3-D基因均能够正常表达(图5B)。

2.5 过表达TaGRF3-D提高了转基因拟南芥在PEG和ABA处理下的根长

TaGRF3-D基因启动子区含有ABA和干旱胁迫响应元件, 说明其可能响应ABA和干旱胁迫。TaGRF3-D基因过表达株系在ABA和PEG处理下的表型分析结果显示, 3个转基因纯合株系在1/2MS培养基上与野生型根长无显著差异, 在含10 µmol∙L^-1 ABA的1/2MS培养基中, 3个转基因株系的根长均显著大于野生型(图6A, B); 在含20% PEG的1/2MS培养基上, 转基因株系OE-1和OE-2根长显著大于野生型(图6C, D), 说明过表达TaGRF3-D基因可降低转基因株系对ABA的敏感性, 提高对干旱胁迫的耐受性。

2.6 TaGRF3-D转基因拟南芥干旱胁迫耐受性提高

进一步对转基因拟南芥株系和野生型进行断水干旱处理, 断水14天后野生型植株严重萎蔫并出现叶片干枯, 转基因株系萎蔫发黄(图7A)。复水6天后3个转基因株系(OE-1、OE-2和OE-3)的存活率分别为50.7%、52%和52%, 显著高于野生型(22.7%) (图7B), 说明TaGRF3-D基因在拟南芥中过表达可提高转基因拟南芥的干旱胁迫耐受性。

2.7 TaGRF3-D与TaABFs蛋白互作分析

研究表明, 14-3-3蛋白通过与AREBs/ABFs转录因子互作影响植物生长发育和胁迫响应(Sun et al., 2015; He et al., 2017)。为阐明TaGRF3-D与小麦TaABFs之间的互作, 我们构建了诱饵载体BD-TaGRF3-D。自激活分析结果显示, BD-TaGRF3-D+AD空载转化菌在TDO (SD/-Trp/-Leu/-His)平板上能正常生长, 说明TaGRF3-D有自激活活性; 在加入10 mmol∙L^-1和20 mmol∙L^-1 3-AT的TDO (SD/-Trp/-Leu-/His)培养基上阳性对照BD-p53+AD-T能正常生长, 含有BD-TaGRF3-D+AD质粒的酵母菌不能长出菌落(图8), 说明10 mmol∙L^-1 3-AT即可有效抑制自激活。

利用酵母双杂交实验对TaGRF3-D和TaABFs蛋白进行互作分析, 结果显示在DDO培养基上所有共转化质粒组合均能正常生长, 说明质粒转入成功且对细胞无明显毒性; 在QDO/AbA/3-AT培养基上, TaGRF3-D与TaABF3-B、TaABF4-A、TaABF15-D、TaABF16-B、TaABF17-D和TaABF18-B的共转化菌落正常生长, 说明其在酵母细胞中存在相互作用; TaGRF3-D与TaABF1-D、TaABF2-A和TabABF19-A菌落不生长, 说明它们之间不发生互作(图9)。有研究表明, TaABF4-A、TaABF17-D和TaABF18-A在植物ABA信号通路和胁迫响应中行使重要功能(Johnson et al., 2008; Kobayashi et al., 2008; Li et al., 2020)。因此, TaGRF3-D也可能通过与小麦TaABFs转录因子相互作用在干旱胁迫响应中发挥功能。

3 讨论

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14-3-3蛋白在植物干旱等非生物逆境响应中行使重要功能(He et al., 2017; Zhang et al., 2018; Huang et al., 2022)。二穗短柄草BdGF14f 在干旱、盐、低温和高温处理下上调表达(Yang et al., 2017); 水稻OsGF14f通过与OsbZIP23相互作用正向调节ABA反应, 增强其对下游靶基因的转录调控活性, 正向调节水稻对渗透胁迫的耐受性(Ma et al., 2023); 大麦Hv14-3-3A正向调控耐旱性, Hv14-3-3A沉默使大麦叶表皮气孔密度显著增大, 水分利用效率降低, 对干旱敏感(Jiang et al., 2023)。小麦TaGRF3-D基因在ABA和干旱胁迫下上调表达, 其启动子区存在激素和干旱胁迫响应元件; 过表达TaGRF3-D转基因拟南芥干旱胁迫耐受性提高, 说明TaGRF3-D与其不同物种中的同源蛋白功能相似, 可能均参与非生物逆境胁迫响应。

14-3-3蛋白可与AREBs/ABFs转录因子相互作用, 通过ABA信号通路在植物非生物逆境响应中发挥功能(Schoonheim et al., 2009; Sun et al., 2015; Ren et al., 2019)。大豆GsGF14o蛋白可与AREBs/ ABFs转录因子发生相互作用, 参与ABA信号途径(Sun et al., 2015)。二穗短柄草BdGF14d和BdGF14a均可与AREB/ABF转录因子NtABF2相互作用, 通过ABA信号途径提高过表达转基因烟草的干旱胁迫耐受性(He et al., 2017; Zhang et al., 2024)。本研究发现, TaGRF3-D过表达拟南芥株系在添加外源ABA和PEG处理下根长大于野生型(图6), 干旱胁迫耐受性也高于野生型(图7), 说明TaGRF3-D也可能与小麦TaABFs相互作用, 参与胁迫响应。进一步通过酵母双杂交分析, 发现TaGRF3-D与TaABF3-B、TaABF4- A、TaABF5-D、TaABF16-B、TaABF17-D和TaABF18- B存在相互作用(图9)。TaABF4-A (TaABF1)可与蛋白激酶PKABA1 (ABA-induced protein kinase)相互作用, 参与ABA信号转导(Johnson et al., 2008); 大麦ABF转录因子HvABF1、HvABF2和HvABF3可与蛋白激酶PKABA1相互作用, 蛋白激酶PKABA1通过磷酸化ABF转录因子, 促使其与14-3-3蛋白相互作用, 参与赤霉素和ABA信号途径(Schoonheim et al., 2009)。TaGRF3-D与TaABF4-A (TaABF1)互作说明TaGRF3-D也有可能通过与TaABFs转录因子相互作用响应ABA信号。TaABF17-D (WABI5)在低温、干旱和ABA处理条件下被激活, 其过表达转基因烟草幼苗对外源ABA敏感性增强, 非生物胁迫耐受性增强(Kobayashi et al., 2008)。本课题组前期研究也表明, 干旱和盐处理后TaABF17、TaABF18和TaABF19基因均显著上调表达, 而ABA处理后TaABF18显著上调表达, TaABF17和TaABF19显著下调表达(郭树娟等, 2023)。TaGRF3-D蛋白与TaABF17-D和TaABF18-B互作, 进一步说明TaGRF3-D可能通过与小麦TaABFs蛋白互作响应ABA信号, 从而提高转基因植株对逆境胁迫的耐受性。

作者贡献声明

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孙月: 设计并完成实验, 分析数据并撰写论文; 郭树娟: 参与完成部分实验; 赵惠贤和马猛: 技术指导; 刘香利: 指导实验设计并审查修改论文。

基金

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陕西省自然科学基础研究计划(2024JC-YBMS-171)
国家自然科学基金(32072003)
国家自然科学基金(32372103)

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2025年第60卷第6期

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doi: 10.11983/CBB24156

接收时间：2024-10-17
首发时间：2026-10-27
出版时间：2025-11-10

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收稿日期：2024-10-17
录用日期：2025-03-18

基金

陕西省自然科学基础研究计划(2024JC-YBMS-171)

国家自然科学基金(32072003)

国家自然科学基金(32372103)

作者信息

西北农林科技大学生命科学学院, 杨凌 712100

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2种不同金属材料的力学参数

科 Family	属数 Number of genus	种数 Number of species	占总种数比例 Percentage of total species (%)	属 Genus	种数 Number of species	占总种数比例 Percentage of total species (%)
鹅膏菌科Amanitaceae	2	11	5.26	鹅膏菌属 Amanita	10	4.78
小菇科 Mycenaceae	2	12	5.74	丝盖伞属 Inocybe	5	2.39
多孔菌科 Polyporaceae	8	14	6.70	蜡蘑属 Laccaria	5	2.39
红菇科 Russulaceae	3	23	11.00	小皮伞属 Marasmius	6	2.87
				小菇属 Mycena	11	5.26
				光柄菇属 Pluteus	5	2.39
				红菇属 Russula	17	8.13
				栓菌属 Trametes	5	2.39

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