小麦14-3-3蛋白TaGRF3-D基因克隆和功能分析

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。本研究克隆了小麦(Triticum aestivum) 14-3-3蛋白TaGRF3-D基因, TaGRF3-D基因编码261氨基酸残基的蛋白, 在单子叶植物中高度保守, 与乌拉尔图小麦(Triticum 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。通过转化获得TaGRF3-D基因过表达拟南芥(Arabidopsis thaliana)转基因株系, 对转基因株系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试验(yeast two-hybrid, Y2H)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。以上研究结果表明, 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干旱胁迫的耐受性。本研究为小麦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: The 14-3-3 proteins are a highly conserved protein family that can specifically recognize phosphorylated target proteins playing a crucial role in plant abiotic stress responses. By interacting with AREBs/ABFs (ABA-responsive element binding proteins/ABA-responsive element binding factors) transcription factors, the 14-3-3 proteins can participate in ABA signal transduction and regulate abiotic stress tolerance. TaGRF3-D is a 14-3-3 protein in wheat (Triticum aestivum), our previous studies showed that the expression of this gene was up-regulated under ABA and abiotic stress.

RATIONALE: To explore the function of TaGRF3-D gene, the gene was cloned, and its subcellular localization and function on drought stress were investigated.

RESULTS: The results showed that the TaGRF3-D is highly conserved in monocotyledonous plants and is located in the nucleus and cell membrane. Compaired with wild type, the Arabidopsis thaliana transgenic lines overexpressed TaGRF3-D had significantly longer roots under PEG and ABA treatments, and demonstrated a significantly higher survival rate after drought stress. Further yeast two-hybrid analysis showed that the TaGRF3-D could interact with wheat TaABF3-B, TaABF4-A, TaABF15-D, TaABF16-B, TaABF17-D and TaABF18-B, but not with TaABF1-D, TaABF2-A and TaABF19-A.

CONCLUSION: These results indicate that TaABF3-D may respond to ABA signals by interacting with wheat TaABFs transcription factors, thereby enhancing drought stress tolerance of transgenic plants.

The phenotypes (A) and survival rates (B) of the TaGRF3-D transgenic lines and the wild type under drought stress

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基因克隆和功能分析. 植物学报, 2025, 60(6): 1-0.

Yue Sun, Shujuan Guo, Huixian Zhao, Meng Ma, Xiangli Liu. Cloning and Functional Analysis of 14-3-3 Protein gene TaGRF3-D in wheat (Triticum aestivum). Chinese Bulletin of Botany, 2025, 60(6): 1-0.

导出引用管理器 EndNote|Ris|BibTeX

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

https://www.chinbullbotany.com/CN/Y2025/V60/I6/1

参考文献

[1]Aitken A(2006).proteins: a historic overview.Semin Cancer Biol, 16:162-172.
[2]Campo S, Peris-Peris C, Montesinos L, Pe?as G, Messeguer J, San Segundo B(2012).Expression of the maize ZmGF14-6 gene in rice confers tolerance to drought stress while enhancing susceptibility to pathogen infection.J Exp Bot, 63:983-99.
[3]Guo SJ, Sun Y, Zheng HY, Zhao HX, Ma M, Liu XL(2023).Genome-wide identification and expression analysis of ABFAREB ABI5 gene family in wheat (Triticum aestivum).J Agric Biotechnol, 31:667-681.郭树娟, 孙月, 郑昊元, 赵惠贤, 马猛, 刘香利(2023).小麦基因家族全基因组鉴定与表达分析.农业生物技术学报, 31:667-681.
[5]He Y, Zhang Y, Chen LH, Wu CL, Luo QC, Zhang F, Wei QH, Li KX, Chang JL, Yang GX, He GY (2017)(2017).A member of the 14-3-3 gene family in Brachypodium distachyon, BdGF14d, confers salt tolerance in transgenic tobacco plants. Front Plant Sci 8, 340..Front Plant Sci , 8:340-.
[6]Ho SL, Huang LF, Lu CA, He SL, Wang CC, Yu SP, Chen J, Yu SM(2013).Sugar starvation- and GA-inducible calcium-dependent protein kinase 1 feedback regulates GA biosynthesis and activates a 14-3-3 protein to confer drought tolerance in rice seedlings.Plant Mol Biol, 81:347-361.
[7]Huang Y, Wang WS, Yu H, Peng JH, Hu ZR, Chen L(2022).The role of 14-3-3 proteins in plant growth and response to abiotic stress.Plant Cell Rep, 41:833-852.
[8]Jiang W, Tong T, Li W, Huang ZH, Chen G, Zeng FR, Riaz A, Amoanimaa-Dede H, Pan R, Zhang WY, Deng FL, Chen ZH(2023).Molecular evolution of plant 14-3-3 proteins and function of Hv14-3-3A in stomatal regulation and drought tolerance.Plant Cell Physiol, 63:1857-1872.
[9]Johnson RR, Shin M, Shen JQ(2008).The wheat PKABA1-interacting factor TaABF1 mediates both abscisic acid-suppressed and abscisic acid-induced gene expression in bombarded aleurone cells.Plant Mol Biol, 68:93-103.
[10]Kaundal A, Ramu VS, Oh S, Lee S, Pant B, Lee HK, Rojas CM, Senthil-Kumar M, Mysore KS(2017).GENERAL CONTROL NONREPRESSIBLE4 degrades 14-3-3 and the RIN4 complex to regulate stomatal aperture with implications on nonhost disease resistance and drought tolerance.Plant Cell, 29:2233-2248.
[11]Kobayashi F, Maeta E, Terashima A, Takumi S(2008).Positive role of a wheat HvABI5 ortholog in abiotic stress response of seedlings.Physiol Plant, 134:74-86.
[12]Latz A, Becker D, Hekman M, Müller T, Beyhl D, Marten I, Eing C, Fischer A, Dunkel M, Bertl A, Rapp UR, Hedrich R(2008).TPK1,a Ca2+-regulated Arabidopsis vacuole two-pore K+ channel is activated by 14-3-3 proteins.Plant J, 54:963-963.
[13]Latz A, Mehlmer N, Zapf S, Mueller TD, Wurzinger B, Pfister B, Csaszar E, Hedrich R, Teige M, Becker D(2013).Salt stress triggers phosphorylation of the Arabidopsis vacuolar K+ channel TPK1 by calcium-dependent protein kinases (CDPKs).Mol plant, 6:1274-1289.
[14]Liu JP, Sun XJ, Liao WC, Zhang JH, Liang JS, Xu WF(2019).Involvement of OsGF14b Adaptation in the Drought Resistance of Rice Plants. Rice 12, 82..Rice, 12:82-.
[15]Ma YM, Wu ZY, Dong JF, Zhang SH, Zhao JL, Yang TF, Yang W, Zhou L, Wang J, Chen JS, Liu Q, Liu B(2023).The 14-3-3 protein OsGF14f interacts with OsbZIP23 and enhances its activity to confer osmotic stress tolerance in rice.Plant Cell, 35:4173-4189.
[16]Ren YR, Yang YY, Zhang R, You CX, Zhao Q, Hao YJ(2019).MdGRF11, an apple 14-3-3 protein, acts as a positive regulator of drought and salt tolerance. Plant Sci 288: 110219..Plant Sci , 288:110219-.
[17]Schoonheim PJ, Costa Pereira DD, De Boer AH(2009).Dual role for 14-3-3 proteins and ABF transcription factors in gibberellic acid and abscisic acid signalling in barley (Hordeum vulgare) aleurone cells.Plant Cell Environ, 32:439-447.
[18]Shao WN, Chen W, Zhu XG, Zhou XY, Jin YY, Zhan C, Liu GS, Liu X, Ma DF, Qiao YL(2021).Genome-wide identification and characterization of wheat 14-3-3 genes unravels the role of TaGRF6-A in salt stress tolerance by binding MYB transcription factor. Int J Mol Sci 22, 1904..Int J Mol Sci , 22:1904-.
[19]Shen YX (2018).Identification and Experssion Analysis of the 14-3-3 Gene Family in Triticum aestivum (L.). Master’s thesis. Yangling: Northwest A& F University. pp. 30–36. (in Chinese)., :-.
[20]申玉霞 (2018).小麦14-3-3蛋白基因家族鉴定和表达分析. 硕士论文. 杨凌: 西北农林科技大学. pp. 30–36.., :-.
[21]Sun XL, Sun MZ, Jia BW, Chen C, Qin ZW, Yang KJ, Shen Y, Meiping Z, Mingyang C, Zhu YM(2015).A 14-3-3 family protein from wild soybean (Glycine Soja) regulates ABA sensitivity in Arabidopsis. PLoS One 10, e0146163..PLoS One, 10:e0146163-.
[22]Yang L, You J, Wang YP, Li JZ, Quan WL, Yin MZ, Wang QF, Chan ZL(2017).Systematic analysis of the G-box Factor 14-3-3 gene family and functional characterization of GF14a in Brachypodium distachyon.Plant Physiol Biochem, 117:1-11.
[23]Zhang X, Henriques R, Lin SS, Niu QW, Chua NH(2006).Agrobacterium-mediated transformation of Arabidopsis thaliana using the floral dip method.Nat Protoc, 1:641-646.
[24]Zhang Y, He Y, Zhao HY, Zhang Y, Yang J, Ou XQ, Zhang JL, Zhu QD( 2023).A 14-3-3 protein-encoding gene, BdGF14g, confers better drought tolerance by regulating ABA biosynthesis and signaling. Plants 12, 3975..Plants , 12:3975-.
[25]Zhang Y, Zhao HY, Zhou SY, He Y, Luo QC, Zhang F, Qiu D, Feng JL, Wei QH, Chen LH, Chen MJ, Chang JL, Yang GX, He GY(2018).Expression of TaGF14b,a 14-3-3 adaptor protein gene from wheat,enhances drought and salt tolerance in transgenic tobacco.Planta, 248:117-137.
[26]Zhao X, Li F, Li K(2021).The 14-3-3 proteins: regulators of plant metabolism and stress responses.Plant Biol, 23:531-539.

[1]	樊蓓, 任敏, 王延峰, 党峰峰, 陈国梁, 程国亭, 杨金雨, 孙会茹. 番茄SlWRKY45转录因子在响应低温和干旱胁迫中的功能(长英文摘要)[J]. 植物学报, 2025, 60(2): 186-203.
[2]	赵来鹏, 王柏柯, 杨涛, 李宁, 杨海涛, 王娟, 闫会转. SlHVA22l基因调节番茄耐旱性[J]. 植物学报, 2024, 59(4): 558-573.
[3]	张盈川, 吴晓明玉, 陶保龙, 陈丽, 鲁海琴, 赵伦, 文静, 易斌, 涂金星, 傅廷栋, 沈金雄. Bna-miR43介导甘蓝型油菜响应干旱胁迫[J]. 植物学报, 2023, 58(5): 701-711.
[4]	邝嘉怡, 李洪清, 沈文锦, 高彩吉. 基于TurboID的植物蛋白邻近标记实验方法[J]. 植物学报, 2021, 56(5): 584-593.
[5]	车永梅, 孙艳君, 卢松冲, 侯丽霞, 范欣欣, 刘新. AtMYB77促进NO合成参与调控干旱胁迫下拟南芥侧根发育[J]. 植物学报, 2021, 56(4): 404-413.
[6]	李佳馨, 李霞, 谢寅峰. 外源海藻糖增强高表达转玉米C₄型PEPC水稻耐旱性的机制[J]. 植物学报, 2021, 56(3): 296-314.
[7]	徐重益. 植物中验证蛋白相互作用的Pull-down和Co-IP技术[J]. 植物学报, 2020, 55(1): 62-68.
[8]	张彤,郭亚璐,陈悦,马金姣,兰金苹,燕高伟,刘玉晴,徐珊,李莉云,刘国振,窦世娟. 水稻OsPR10A的表达特征及其在干旱胁迫应答过程中的功能[J]. 植物学报, 2019, 54(6): 711-722.
[9]	郜怀峰,张亚飞,王国栋,孙希武,贺月,彭福田,肖元松. 钼在桃树干旱胁迫响应中的作用解析[J]. 植物学报, 2019, 54(2): 227-236.
[10]	宋爱华, 张文斌, 孙姝兰, 李凌飞, 王小菁. 非洲菊原生质体制备及瞬时转化系统的建立[J]. 植物学报, 2017, 52(4): 511-519.
[11]	景艳军, 林荣呈. 我国植物光信号转导研究进展概述[J]. 植物学报, 2017, 52(3): 257-270.
[12]	刘小龙, 李霞, 钱宝云. 外源Ca²⁺对PEG处理下转C₄型PEPC基因水稻光合生理的调节[J]. 植物学报, 2015, 50(2): 206-216.
[13]	苗红霞, 王园, 徐碧玉, 刘菊华, 贾彩红, 张建斌, 王卓, 孙佩光, 金志强. 香蕉MaASR1基因的抗干旱作用[J]. 植物学报, 2014, 49(5): 548-559.
[14]	许凤莲, 李姣姣, 杜春晖, 王娜, 张素巧. 一个具有S位点的水稻类受体激酶OsSRL参与干旱胁迫反应[J]. 植物学报, 2012, 47(5): 474-482.
[15]	刘焱, 邢立静, 李俊华, 戴绍军. 水稻含有B-box锌指结构域的OsBBX25蛋白参与植物对非生物胁迫的响应[J]. 植物学报, 2012, 47(4): 366-378.