欢迎访问作物学报，今天是
作物学报

作物学报 ›› 2026, Vol. 52 ›› Issue (2): 480-493.doi: 10.3724/SP.J.1006.2026.55038

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

大豆GmARA6a的克隆及响应盐胁迫的功能分析

张晴1(), 杨昱2, 郭茜2, 岳霈尧2, 殷丛丛1, 牛景萍3, 赵晋忠1, 杜维俊2, 岳爱琴2,*()   

  1. 1山西农业大学基础部, 山西太谷030801
    2山西农业大学农学院, 山西太谷030801
    3山西农业大学生命科学学院, 山西太谷030801
  • 收稿日期:2025-06-24 接受日期:2025-10-30 出版日期:2026-02-12 网络出版日期:2025-11-10
  • 通讯作者: *岳爱琴 E-mail:yueaiqinnd@126.com
  • 作者简介:E-mail: sunny001110@163.com
  • 基金资助:
    山西省科技重大专项计划揭榜挂帅项目子课题(202201140601025-3-06);山西省农业关键核心技术攻关子课题(NYGG27-04-01);山西农业大学科技创新提升工程项目(CXGC2023004);山西省现代农业产业技术体系建设项目(2025CYJSTX05-07);山西农业大学育种工程项目(YZGC096);山西“1331工程”作物科学一级学科建设项目

Cloning and functional analysis of the soybean GmARA6a gene in response to salt stress

Zhang Qing1(), Yang Yu2, Guo Qian2, Yue Pei-Yao2, Yin Cong-Cong1, Niu Jing-Ping3, Zhao Jin-Zhong1, Du Wei-Jun2, Yue Ai-Qin2,*()   

  1. 1College of Basic Sciences, Shanxi Agricultural University, Taigu 030801, Shanxi, China
    2College of Agriculture, Shanxi Agricultural University, Taigu 030801, Shanxi, China
    3College of Life Sciences, Shanxi Agricultural University, Taigu 030801, Shanxi, China
  • Received:2025-06-24 Accepted:2025-10-30 Published:2026-02-12 Published online:2025-11-10
  • Contact: *Yue Ai-Qin E-mail:yueaiqinnd@126.com
  • Supported by:
    Sub-project of the Shanxi Province Science and Technology Major Special Plan Unveiling Project(202201140601025-3-06);Sub-project on Key Core Technology Breakthroughs in Agriculture of Shanxi Province(NYGG27-04-01);Shanxi Agricultural University Scientific and Technological Innovation Enhancement Program(CXGC2023004);the Construction Project of Modern Agricultural Industrial Technology System of Shanxi Province(2025CYJSTX05-07);Breeding Project of Shanxi Agricultural University(YZGC096);Shanxi “1331 Project” Crop Science First-Class Discipline Construction Project

RichHTML

14

PDF

199

摘要：

囊泡运输在植物响应盐胁迫过程中起关键作用, ARA6 (RabF1)通过促进内体与质膜间囊泡介导的物质转运, 在调节植物的耐盐能力上发挥积极影响。本研究对大豆GmARA6a基因进行克隆及生物信息学分析; 通过BFA、WM处理以及与AtARA6共定位探究GmARA6a的亚细胞定位; 探究该基因在不同组织及盐胁迫下的表达模式; 利用拟南芥ara6突变株、GmARA6a回补株系和过表达株系研究GmARA6a基因在调控植株耐盐性方面的作用。结果表明, 大豆中克隆的GmARA6a基因长603 bp, 编码200个氨基酸, 具有ARA6家族的4段共有序列、1段效应结合区以及N端独特的“MGCXSS”结构域。GmARA6a定位于细胞膜以及多囊泡体(MVB)上。在大豆根中GmARA6a被盐胁迫诱导上调表达。盐胁迫下, 拟南芥ara6突变株出现盐敏感表型; 而GmARA6a过表达株系生长良好, 抗氧化能力增强, 膜损伤、脂质过氧化程度降低, H2O2、O2?积累量下降; 通过分析相关基因表达水平, 表明GmARA6a可能通过SOS信号通路、囊泡运输途径调控植株耐盐性。本研究结果为进一步探究GmARA6a的功能提供参考。

关键词: 大豆, GmARA6a, 亚细胞定位, 盐胁迫, 功能分析

Abstract:

Vesicle trafficking plays a crucial role in plant responses to salt stress. ARA6 (RabF1) positively regulates salt tolerance by mediating vesicle transport between endosomes and the plasma membrane. In this study, GmARA6a was cloned from soybean and analyzed using bioinformatics approaches. Subcellular localization was examined through BFA and WM treatments, as well as colocalization with AtARA6. The expression pattern of GmARA6a was assessed across various tissues and under salt stress conditions. To evaluate its functional role in salt tolerance, we used Arabidopsis ara6 mutants, complemented lines (Com-1, Com-2), and overexpression lines (OE-1, OE-2). The GmARA6a gene, with a full-length coding sequence of 603 bp encoding a 200-amino-acid protein, was successfully cloned. The protein contains four conserved ARA6-family domains, an effector-binding region, and a unique N-terminal “MGCXSS” motif. Subcellular localization analysis revealed that GmARA6a localizes to the plasma membrane and multivesicular bodies (MVBs). Its expression was induced by salt stress, particularly in soybean roots. Under salt stress conditions, Arabidopsis ara6 mutants exhibited a salt-sensitive phenotype compared to wild-type plants, while GmARA6a overexpression lines showed improved growth, enhanced antioxidant enzyme activity, reduced membrane damage and lipid peroxidation, and lower levels of H2O2 and O2?accumulation. Expression analysis of related genes suggested that GmARA6a may contribute to salt tolerance by modulating the SOS signaling pathway and vesicle trafficking. These findings provide new insights into the functional role of GmARA6a in plant salt stress responses.

Key words: soybean, GmARA6a, subcellular localization, salt stress, functional analysis

表1

本研究所用引物序列"

引物名称
Primer name		 引物序列
Primer sequence (5′-3′)		 用途
Purpose
GmARA6a-F TCTCCTCTCGCTATGGGTTG 基因克隆
Gene cloning
GmARA6a-R TGCATGGGCGTCTCACTT
KpnI-GmARA6a-F GCTTTCGCGAGCTCGGTACCATGGGTTGCGGCTCCT 亚细胞定位载体构建
Construction of subcellular localization vector
KpnI-GmARA6a-R ACCATGGATCCCCGGGTACCACTAACTGATGGGCGAGGC
SalI-AtARA6-F GAATTCTGCAGTCGACATGGGATGTGCTTCTTCTCTTCCAG
AtARA6-BamHI-R CTCACCATCACGGATCCTGACGAAGGAGCAGGACGAG
GmARA6a-qF CTTGAGAAGCGGGAAGTTGC 实时荧光定量PCR
Quantitative real-time PCR
GmARA6a-qR TCCCTTCCCTTACTGAACTGCT
XbaI-GmARA6a-F CCGGGGATCCTCTAGAATGGGTTGCGGCTCCT 过表达载体构建
Construction of overexpression vector
XbaI-GmARA6a-R GCAGGTCGACTCTAGAACTAACTGATGGGCGAGGCA
AtSOS1-qF GCAAACACTTTGATATTTATCCTCAG 耐盐相关基因
Salt tolerance related genes
AtSOS1-qR CATGAATTCCCTTGGTAGGC
AtSOS3-qF TCAATGGAATTTGGCTTTGGTT
AtSOS3-qR CACCCACCAAAATCGAACCG
AtVAMP727-qF TTCGTGTTTCTCGAACGGGT
AtVAMP727-qR GCAACGCTAAACCGATCTCC
AtVPS9A-qF GGTCTGGTTGTTCTGAGGAGG
AtVPS9A-qR ATCTTCTGGAGCTCTTTTTGAGC
AtActin-qF GGTAACATTGTGCTCAGTGGTGG 内参基因
Reference gene
AtActin-qR AACGACCTTAATCTTCATGCTGC
GmActin-qF GGTGGTTCTATCTTGGCATC
GmActin-qR CTTTCGCTTCAATAACCCTA

图1

GmARA6a基因的克隆及生物信息学分析 A: GmARA6a基因的克隆; B: GmARA6a蛋白的三级结构; C: GmARA6a蛋白的二级结构; D: GmARA6a氨基酸序列比对; E: GmARA6a蛋白的保守基序分析。"

图2

GmARA6a系统发育进化树分析"

图3

BFA和WM对烟草叶片表皮细胞中GmARA6a-GFP瞬时表达的影响 A: 烟草叶表皮细胞中空载GFP与GmARA6a-GFP的瞬时表达; 物镜放大倍数: 20×。B: 未经处理的烟草叶表皮细胞经50 μmol L-1 BFA处理、200 μmol L-1 WM处理后的烟草叶表皮细胞中GmARA6a-GFP的瞬时表达(白色箭头表示囊泡状细胞器); BF: 明场; Merged: 合并场; 物镜放大倍数: 40×。图A和图B标尺为20 μm。C: 不同处理下囊泡细胞器的直径大小。"

图4

BFA和WM对烟草叶表皮细胞中GmARA6a-GFP、AtARA6-mCherry瞬时共表达的影响 A: 未经处理的烟草叶表皮细胞中GmARA6a-GFP、AtARA6-mCherry的瞬时共表达; 物镜放大倍数: 20×。B: 未处理、经50 μmol L-1 BFA处理、200 μmol L-1 WM处理后的烟草叶表皮细胞中GmARA6a-GFP、AtARA6-mCherry瞬时共表达(白色箭头表示共定位的囊泡状细胞器); BF: 明场; Merged: 合并场; 物镜放大倍数: 40×; PCC: 皮尔逊相关系数。标尺为20 μm。"

图5

GmARA6a的表达模式分析 A: GmARA6a在大豆不同组织中的表达; B: 盐胁迫下GmARA6a在大豆根和叶中的表达。不同小写字母表示差异水平显著(P < 0.05)。"

图6

萌发期盐胁迫下拟南芥各株系发芽率测定 不同小写字母表示差异水平显著(P < 0.05)。"

图7

盐胁迫下拟南芥各株系生长情况及生长指标测定 A: 盐胁迫下拟南芥幼苗期各株系生长情况; B~D: 盐胁迫下拟南芥幼苗期各株系根长、鲜重及干重测定; E: 盐胁迫下拟南芥苗期各株系生长情况: F: 盐胁迫下拟南芥苗期各株系莲座叶面积测定。不同小写字母表示差异水平显著(P < 0.05)。"

图8

转基因拟南芥株系在盐胁迫下叶片生理指标的检测 不同小写字母表示差异水平显著(P < 0.05)。"

图9

转基因拟南芥株系在盐胁迫下根中生理指标的检测 不同小写字母表示差异水平显著(P < 0.05)。"

图10

转基因拟南芥叶片经盐胁迫后的NBT、DAB染色及染色面积百分比的统计 A: 拟南芥不同株系叶片的NBT染色; B: NBT染色面积百分比; C: 拟南芥不同株系叶片的DAB染色; D: DAB染色面积百分比。不同小写字母表示差异水平显著(P < 0.05)。"

图11

转基因拟南芥耐盐相关基因的表达 不同小写字母表示差异水平显著(P < 0.05)。"

[1] Qi D H, Lee C F. Influence of soybean biodiesel content on basic properties of biodiesel-diesel blends. J Taiwan Inst Chem E, 2014, 45: 504-507.
doi: 10.1016/j.jtice.2013.06.021
[2] Shi D, Hang J Y, Neufeld J, et al. Effects of genotype, environment and their interaction on protein and amino acid contents in soybeans. Plant Sci, 2023, 337: 111891.
doi: 10.1016/j.plantsci.2023.111891
[3] Li S Z, Xu L, Li Y T, et al. Advances in salinity tolerance of soybean: molecular mechanism and breeding strategy. Food Energy Secur, 2025, 14: e70073.
[4] 毛韩成.耐盐大豆根际微生物群落特征及其耐盐效应. 南京农业大学硕士学位论文,江苏南京, 2023.
Mao H C. Characteristics of Rhizosphere Microbial Community in Salt Tolerant Soybean and Its Salt Tolerance Effect. MS Thesis of Nanjing Agricultural University, Nanjing, Jiangsu, China, 2023 (in Chinese with English abstract).
[5] Yu Z P, Duan X B, Luo L, et al. How plant hormones mediate salt stress responses. Trends Plant Sci, 2020, 25: 1117-1130.
doi: 10.1016/j.tplants.2020.06.008 pmid: 32675014
[6] Quan R D, Lin H X, Mendoza I, et al. SCABP8/CBL10, a putative calcium sensor, interacts with the protein kinase SOS2 to protect Arabidopsis shoots from salt stress. Plant Cell, 2007, 19: 1415-1431.
doi: 10.1105/tpc.106.042291
[7] Park H J, Kim W Y, Yun D J. A role for GIGANTEA: keeping the balance between flowering and salinity stress tolerance. Plant Signal Behav, 2013, 8: e24820.
[8] Zhao S S, Zhang Q K, Liu M Y, et al. Regulation of plant responses to salt stress. Int J Mol Sci, 2021, 22: 4609.
doi: 10.3390/ijms22094609
[9] Park H J, Kim W Y, Yun D J. A new insight of salt stress signaling in plant. Mol Cells, 2016, 39: 447-459.
doi: 10.14348/molcells.2016.0083 pmid: 27239814
[10] van Zelm E, Zhang Y X, Testerink C. Salt tolerance mechanisms of plants. Annu Rev Plant Biol, 2020, 71: 403-433.
doi: 10.1146/annurev-arplant-050718-100005 pmid: 32167791
[11] Wang K K, Zhu J, Xu X W, et al. Quantitative monitoring of salt stress in rice with solar-induced chlorophyll fluorescence. Eur J Agron, 2023, 150: 126954.
doi: 10.1016/j.eja.2023.126954
[12] Ismail A, Takeda S, Nick P. Life and death under salt stress: same players, different timing? J Exp Bot, 2014, 65: 2963-2979.
doi: 10.1093/jxb/eru159 pmid: 24755280
[13] Yamada N, Takahashi H, Kitou K, et al. Suppressed expression of choline monooxygenase in sugar beet on the accumulation of Glycine betaine. Plant Physiol Biochem, 2015, 96: 217-221.
doi: 10.1016/j.plaphy.2015.06.014
[14] Ren X L, Qi G N, Feng H Q, et al. Calcineurin B-like protein CBL10 directly interacts with AKT1 and modulates K+ homeostasis in Arabidopsis. Plant J, 2013, 74: 258-266.
doi: 10.1111/tpj.2013.74.issue-2
[15] Nieves-Cordones M, Alemán F, Martínez V, et al. K+ uptake in plant roots. The systems involved, their regulation and parallels in other organisms. J Plant Physiol, 2014, 171: 688-695.
[16] Xu J, Li H D, Chen L Q, et al. A protein kinase, interacting with two calcineurin B-like proteins, regulates K+ transporter AKT 1 in Arabidopsis. Cell, 2006, 125: 1347-1360.
doi: 10.1016/j.cell.2006.06.011
[17] Sánchez-Barrena M J, Chaves-Sanjuan A, Raddatz N, et al. Recognition and activation of the plant AKT1 potassium channel by the kinase CIPK23. Plant Physiol, 2020, 182: 2143-2153.
doi: 10.1104/pp.19.01084 pmid: 32015077
[18] Echeverría E. Vesicle-mediated solute transport between the vacuole and the plasma membrane. Plant Physiol, 2000, 123: 1217-1226.
doi: 10.1104/pp.123.4.1217 pmid: 10938341
[19] Van Damme D, Inzé D, Russinova E. Vesicle trafficking during somatic cytokinesis. Plant Physiol, 2008, 147: 1544-1552.
doi: 10.1104/pp.108.120303 pmid: 18678745
[20] Ebine K, Miyakawa N, Fujimoto M, et al. Endosomal trafficking pathway regulated by ARA6, a RAB5 GTPase unique to plants. Small GTPases, 2012, 3: 23-27.
doi: 10.4161/sgtp.18299 pmid: 22710734
[21] Ebine K, Fujimoto M, Okatani Y, et al. A membrane trafficking pathway regulated by the plant-specific RAB GTPase ARA6. Nat Cell Biol, 2011, 13: 853-859.
doi: 10.1038/ncb2270 pmid: 21666683
[22] Ito E, Ebine K, Choi S W, et al. Integration of two RAB5 groups during endosomal transport in plants. eLife, 2018, 7: e34064.
[23] Sunada M, Goh T, Ueda T, et al. Functional analyses of the plant-specific C-terminal region of VPS9a: the activating factor for RAB5 in Arabidopsis thaliana. J Plant Res, 2016, 129: 93-102.
doi: 10.1007/s10265-015-0760-5
[24] Ueda T, Yamaguchi M, Uchimiya H, et al. Ara6, a plant-unique novel type Rab GTPase, functions in the endocytic pathway of Arabidopsis thaliana. EMBO J, 2001, 20: 4730-4741.
doi: 10.1093/emboj/20.17.4730 pmid: 11532937
[25] Hoepflinger M C, Geretschlaeger A, Sommer A, et al. Molecular and biochemical analysis of the first ARA6 homologue, a RAB5 GTPase, from green algae. J Exp Bot, 2013, 64: 5553-5568.
doi: 10.1093/jxb/ert322 pmid: 24127512
[26] Bottanelli F, Gershlick D C, Denecke J. Evidence for sequential action of Rab5 and Rab7 GTPases in prevacuolar organelle partitioning. Traffic, 2012, 13: 338-354.
doi: 10.1111/j.1600-0854.2011.01303.x pmid: 22004564
[27] Ito E, Uemura T. RAB GTPases and SNAREs at the trans-Golgi network in plants. J Plant Res, 2022, 135: 389-403.
doi: 10.1007/s10265-022-01392-x
[28] 田再民.马铃薯小G蛋白基因StRab5b的克隆及其调控晚疫病抗性的功能研究. 内蒙古农业大学博士学位论文, 内蒙古呼和浩特, 2020.
Tian Z M.Cloning and Functional Analysis of Small G Protein Gene StRab5b Regulation Potato Resistance Against Late Blight.PhD Dissertation of Inner Mongolia Agricultural University, Hohhot, Inner Mongolia, China , 2020 (in Chinese with English abstract).
[29] Hong Z P, Li Y, Zhao Y, et al. Heterologous expression of Arabidopsis AtARA6 in soybean enhances salt tolerance. Front Genet, 2022, 13: 849357.
doi: 10.3389/fgene.2022.849357
[30] Huang Y P, Hou P Y, Chen I H, et al. Dissecting the role of a plant-specific Rab5 small GTPase NbRabF1 in Bamboo mosaic virus infection. J Exp Bot, 2020, 71: 6932-6944.
doi: 10.1093/jxb/eraa422
[31] Kesawat M S, Satheesh N, Kherawat B S, et al. Regulation of reactive oxygen species during salt stress in plants and their crosstalk with other signaling molecules-current perspectives and future directions. Plants, 2023, 12: 864.
doi: 10.3390/plants12040864
[32] Ishitani M, Liu J, Halfter U, et al. SOS3 function in plant salt tolerance requires N-myristoylation and calcium binding. Plant Cell, 2000, 12: 1667-1678.
doi: 10.1105/tpc.12.9.1667 pmid: 11006339
[33] Nadarajah K K. ROS homeostasis in abiotic stress tolerance in plants. Int J Mol Sci, 2020, 21: 5208.
doi: 10.3390/ijms21155208
[34] Shen Y, Shen L K, Shen Z X, et al. The potassium transporter OsHAK21 functions in the maintenance of ion homeostasis and tolerance to salt stress in rice. Plant Cell Environ, 2015, 38: 2766-2779.
doi: 10.1111/pce.v38.12
[35] Liu G Y, Zeng Y L, Li B Y, et al. SOS 2 phosphorylates FREE1 to regulate multi-vesicular body trafficking and vacuolar dynamics under salt stress. Plant Cell, 37, 3, koaf012.
[1] 金昱何, 王雪菲, 徐张一娃, 缪怡宁, 蒋云杰, 伊莹, 缪德麟, 朱静仪, 钟一帆, 陈铭亨, 方芳, 刘鹏. 外源激素对低温胁迫下大豆叶片叶绿素荧光参数及抗氧化酶系统的影响[J]. 作物学报, 2026, 52(6): 1817-1829.
[2] 唐宽强, 李公允, 宋美毅, 赵雪, 常春玲. 大豆株高性状全基因组关联分析及预测模型构建[J]. 作物学报, 2026, 52(6): 1743-1756.
[3] 邹仪妹, 徐敏, 汪海洋, 姚辉, 王加峰, 刘浩, 任代胜. 两系不育系水稻幼苗根系响应盐胁迫的转录因子调控网络鉴定[J]. 作物学报, 2026, 52(6): 1728-1742.
[4] 姚术, 郭凯悦, 翟慧慧, 姚佳慧, 邓文琪, 闫玲, 黄驰, 高阳, 俞嫣然, 赵振邦, 李英慧, 王晓波, 李佳佳. 大豆苗期耐低铁综合评价及优异种质筛选[J]. 作物学报, 2026, 52(5): 1373-1387.
[5] 宋裕祯, Bheel Chander Kumar, 王跃, 张颖星, 郭娟, Khound Rituraj, Santra Dipak Kumar, 曹晓宁, 王瑞云. 糜子AP2亚家族全基因组鉴定及PmAP2-1和PmAP2-9耐盐功能分析[J]. 作物学报, 2026, 52(4): 1127-1139.
[6] 覃奕琰, 付瑶, 苏畅, 李娜, 徐静茹, 程笑然, 张琪, 赵明辉. OsST41调控水稻苗期耐盐性的功能分析[J]. 作物学报, 2026, 52(3): 802-812.
[7] 汪玲, 胡好, 宋家凤, 程洁蓝, 陈颖, 郑婷婷, 吕钊彦, 朱晓彪, 侯华兰. 马铃薯UDP-糖基转移酶基因StUGT52的克隆及功能验证[J]. 作物学报, 2026, 52(3): 665-676.
[8] 刘吉昌, 李思烨, 李雪婷, 王洪章, 刘鹏, 张吉旺, 赵斌, 任佰朝, 任昊. 盐胁迫对不同耐盐型夏玉米品种根系生长及养分吸收效率的影响[J]. 作物学报, 2026, 52(2): 565-577.
[9] 余开航, 周洪斌, 罗亮扎, 王玫郦, 姜瑞梅, 董陈文华, 李仕金, 毛孝强, 陈升位. 大麦亮氨酸富集重复型类受体激酶基因HvLRR-RLK-510的克隆和表达分析[J]. 作物学报, 2026, 52(2): 421-432.
[10] 王克晶, 李向华. 我国珍稀的大豆属多年生烟豆和短绒野大豆物种遗传资源濒危性评估分析[J]. 作物学报, 2025, 51(8): 2009-2019.
[11] 孟然, 李赵嘉, 冯薇, 陈悦, 刘路平, 杨春燕, 鲁雪林, 王秀萍. 大豆不同生育时期耐盐性综合评价及耐盐种质筛选[J]. 作物学报, 2025, 51(8): 1991-2008.
[12] 贺红利, 张雨涵, 杨静, 程云清, 赵杨, 李星诺, 司洪亮, 张兴政, 杨向东. 大豆e1-as基因突变体的创制及生理分析[J]. 作物学报, 2025, 51(8): 2228-2239.
[13] 胡蒙, 沙丹, 张晟瑞, 谷勇哲, 张世碧, 李静, 孙君明, 邱丽娟, 李斌. 大豆分枝数QTL定位及候选基因筛选[J]. 作物学报, 2025, 51(7): 1747-1756.
[14] 王琼, 邹丹霞, 陈兴运, 张威, 张红梅, 刘晓庆, 贾倩茹, 魏利斌, 崔晓艳, 陈新, 王学军, 陈华涛. 大豆开花时间和成熟期性状全基因组关联分析与候选基因预测[J]. 作物学报, 2025, 51(6): 1558-1568.
[15] 殷丛丛, 李睿琦, 岳霈尧, 李晨, 牛景萍, 赵晋忠, 杜维俊, 岳爱琴. 基于闭合哑铃介导等温扩增可视化检测大豆花叶病毒SC15方法的建立及应用[J]. 作物学报, 2025, 51(5): 1248-1260.
Viewed
Full text


Abstract

Cited
  Shared   
  Discussed   
No Suggested Reading articles found!
京ICP备15008789号-2