欢迎访问作物学报，今天是
作物学报

作物学报 ›› 2023, Vol. 49 ›› Issue (5): 1272-1281.doi: 10.3724/SP.J.1006.2023.24070

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

CBL互作蛋白激酶GmCIPK10增强大豆耐盐性

李慧1,2(), 路依萍1, 汪小凯1, 王璐瑶1, 邱婷婷1, 张雪婷1, 黄海燕1, 崔晓玉1,*()   

  1. 1临沂大学农林科学学院, 中国山东临沂 276000
    2菲律宾克里斯汀大学国际学院, 菲律宾 1004
  • 收稿日期:2022-03-29 接受日期:2022-07-21 出版日期:2023-05-12 网络出版日期:2022-08-18
  • 通讯作者: *崔晓玉, E-mail: cuixiaoyu@lyu.edu.cn
  • 作者简介:E-mail: lihuiqau@163.com
  • 基金资助:
    国家自然科学基金项目(32001459);山东省自然科学基金项目(ZR2020QC123)

GmCIPK10, a CBL-interacting protein kinase promotes salt tolerance in soybean

LI Hui1,2(), LU Yi-Ping1, WANG Xiao-Kai1, WANG Lu-Yao1, QIU Ting-Ting1, ZHANG Xue-Ting1, HUANG Hai-Yan1, CUI Xiao-Yu1,*()   

  1. 1College of Agriculture and Forestry Sciences, Linyi University, Linyi 276000, Shandong, China
    2Center for International Education, Philippine Christian University, 1004, the Philippines
  • Received:2022-03-29 Accepted:2022-07-21 Published:2023-05-12 Published online:2022-08-18
  • Contact: *E-mail: cuixiaoyu@lyu.edu.cn
  • Supported by:
    National Natural Science Foundation of China(32001459);Natural Science Foundation of Shandong Province(ZR2020QC123)

RichHTML

30

PDF

2784

摘要：

盐胁迫严重威胁大豆产量和品质。类钙调磷酸酶B亚基互作蛋白激酶(CIPKs)在植物应对环境胁迫过程中发挥重要作用。但是, 目前对大豆CIPKs的生物学功能知之甚少。本研究从大豆基因组克隆到GmCIPK10。生物信息学分析结果表明, GmCIPK10属于不含内含子型CIPKs, 包含一个丝氨酸(Ser)/苏氨酸(Thr)蛋白激酶结构域和NAF/FISL基序。表达模式分析结果表明, 在盐(NaCl)、甲基紫精(MV)和过氧化氢(H2O2)处理下, GmCIPK10的转录水平升高。在拟南芥和大豆毛状根中过表达GmCIPK10能够提高转基因植株的抗盐性。进一步的生理指标测定发现, 在盐胁迫下, 过表达GmCIPK10能够降低转基因植株中丙二醛(MDA)和H2O2积累, 增强抗氧化酶活性以及降低钠离子(Na+)/钾离子(K+)比值。此外, qRT-PCR分析发现GmCIPK10促进抗氧化和耐盐相关基因表达响应盐胁迫。酵母双杂交、Pull-down和双分子荧光互补试验结果证明GmCIPK10与钙离子(Ca2+)感应器GmCBL4相互作用。这些结果为解析CBL-CIPK信号通路在大豆盐胁迫应答的作用提供了参考。

关键词: GmCIPK10, 耐盐性, ROS清除, Na+/K+稳态, 大豆

Abstract:

Salt stress seriously restricts the yield and quality of soybean. Calcineurin B subunit-interacting protein kinases (CIPKs) play a vital role in response to environmental stresses in plant. However, few study is known about the biological function of soybean CIPKs. In this study, GmCIPK10 was cloned from soybean genome. Bioinformatics analysis exhibited that GmCIPK10 encoded an intron-poor type CIPKs with a serine (Ser)/threonine (Thr) protein kinase domain and a NAF/FISL motif. The relative expression pattern levels showed that the transcript level of GmCIPK10 was increased under NaCl, MV, and H2O2 treatments. Overexpression of GmCIPK10 in Arabidopsis and soybean hairy roots improved salt tolerance of transgenic plants. Further physiological indicator assays demonstrated that GmCIPK10 overexpression could decrease the accumulation of MDA and H2O2, enhance the activity of antioxidant enzymes, and reduce sodium (Na+)/potassium (K+) in transgenic plants under salt stress. In addition, qRT-PCR illustrated that GmCIPK10 promoted the relative expression level of antioxidant- and salt tolerance-related gene in salt stress. The yeast two-hybrid, pull-down, and bimolecular fluorescence complementation experiments confirmed that GmCIPK10 interacts with the Ca2+ sensor GmCBL4. These results provide a reference for investigating the role of the CBL-CIPK signaling pathway in response to salt stress in soybean.

Key words: GmCIPK10, salt tolerance, ROS scavenging, Na+/K+ homeostasis, soybean

表1

本研究所使用的引物"

引物
Primer ID		 正向序列
Forward sequence (5'-3')		 反向序列
Reverse sequence (5'-3')
AtActin GAAATCACAGCACTTGCACC TGGAATGTGCTGAGGGAAGC
GmTubulin GAGGCAAGATGAGCACCAAG ACGGAACATTTCCTGAATGGAG
GmCIPK10 CAAGCCTGTTTTTGCATCTGAAT GATAGTGTGTTTGGATCCCAGC
GmCAT1 AGCTAGCGCAAAGGGTTTCT AAGGTTTCAGGGCTACCACG
GmPOD21 CCGTTTCGTGGGTCAGAAATCT- CCGACGCCTGCTCCGACACTA
GmZAT10 AAGCCTTCTCCTCTTACCAAGCA TCGACGCCGA ACTCGTTGT
GmMYB118 ATCATACTGTTCGGAGTCAC CAGACACTGTAGAGACCTTGTT
GmLEA5 CCGATGTATCGGTAAGAGT AGGCTTTTGA ACCATCTC
GmNHX1 GTCGGGGCACACTTCACTAA GGATGCTGCTTGGACGATGA
GmSALT3 AAGCAGGTGCTTAACGACGA CAAATCTGTTAGCCGCGACG
GmSOS1 ATCGGCTGGGAAAGATTGGG CACCAGGGCCAGCTAGTAAG

图1

GmCIPK10的蛋白结构和进化关系分析"

图2

GmCIPK10响应盐胁迫和氧化胁迫的表达模式 以GmTubulin为内参基因, 每组3棵, 3次生物学重复。MV: 甲基紫精。*: P < 0.05。"

图3

过表达GmCIPK10提高转基因拟南芥的耐盐性 A和B: GmCIPK10-OE和WT拟南芥植株经盐胁迫(75 mmol L-1 NaCl)处理10 d后的表型和主根长; C: GmCIPK10基因表达量; D: MDA含量; E: H2O2含量; 每组4棵, 3次生物学重复; WT: 野生型; OE: 过表达。*: P < 0.05。"

图4

过表达GmCIPK10提高转基因大豆发状根的耐盐性 A和B: GmCIPK10-OE和VC大豆植株的表型经盐胁迫(200 mmol L-1 NaCl)处理10 d后的表型和成活率; C: GmCIPK10基因表达量; GmCIPK10-OE和VC大豆植株经盐胁迫处理7 d后的; D: MDA含量; E: H2O2 含量; F: CAT活性; G: POD活性; H: K+离子含量; I: Na+离子含量; J: Na+/K+比值; 每组10 棵, 3次生物学重复; VC: 载体对照; OE: 过表达; FW: 鲜重; DW: 干重。*: P < 0.05。"

图5

GmCIPK10调节盐胁迫和氧化胁迫相关基因表达响应盐胁迫 每组3棵, 3次生物学重复。VC: 载体对照; OE: 过表达。*: P < 0.05。"

图6

GmCBL4与GmCIPK10互作 A: 酵母双杂交检测GmCBL4和GmCIPK10互作; B: Pull-down检测GmCBL4和GmCIPK10互作; C: BiFC检测GmCBL4和GmCIPK10互作。BiFC: 双分子荧光互补; YFP: 黄色荧光蛋白。标尺为12 μm。"

[1] Tang R J, Wang C, Li K, Luan S. The CBL-CIPK calcium signaling network: unified paradigm from 20 years of discoveries. Trends Plant Sci, 2020, 25: 604-617.
doi: 10.1016/j.tplants.2020.01.009
[2] Yu Q, An L, Li W. The CBL-CIPK network mediates different signaling pathways in plants. Plant Cell Rep, 2014, 33: 203-214.
doi: 10.1007/s00299-013-1507-1 pmid: 24097244
[3] Ma X, Li Q H, Yu Y N, Qiao Y M, Haq S U, Gong Z H. The CBL-CIPK pathway in plant response to stress signals. Int J Mol Sci, 2020, 21: 5668.
doi: 10.3390/ijms21165668
[4] Luan S. The CBL-CIPK network in plant calcium signaling. Trends Plant Sci, 2009, 14: 37-42.
doi: 10.1016/j.tplants.2008.10.005 pmid: 19054707
[5] Albrecht V, Ritz O, Linder S, Harter K, Kudla J. The NAF domain defines a novel protein-protein interaction module conserved in Ca2+-regulated kinases. EMBO J, 2001, 20: 1051-1063.
doi: 10.1093/emboj/20.5.1051 pmid: 11230129
[6] Kudla J, Xu Q, Harter K, Gruissem W, Luan S. Genes for calcineurin B-like proteins in Arabidopsis are differentially regulated by stress signals. Proc Natl Acad Sci USA, 1999, 96: 4718-4723.
doi: 10.1073/pnas.96.8.4718 pmid: 10200328
[7] Kudla J, Batistic O, Hashimoto K. Calcium signals: the lead currency of plant information processing. Plant Cell, 2010, 22: 541-563.
doi: 10.1105/tpc.109.072686
[8] Quan R, Lin H, Mendoza I, Zhang Y, Cao W, Yang Y, Shang M, Chen S, Pardo J M, Guo Y. 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
[9] Pandey G K, Kanwar P, Singh A, Steinhorst L, Pandey A, Yadav A K, Tokas I, Sanyal S K, Kim B G, Lee S C, Cheong Y H, Kudla J, Luan S. Calcineurin B-like protein-interacting protein kinase CIPK21 regulates osmotic and salt stress responses in Arabidopsis. Plant Physiol, 2015, 169: 780-792.
doi: 10.1104/pp.15.00623
[10] Cheong Y H, Pandey G K, Grant J J, Batistic O, Li L, Kim B G, Lee S C, Kudla J, Luan S. Two calcineurin B-like calcium sensors, interacting with protein kinase CIPK23, regulate leaf transpiration and root potassium uptake in Arabidopsis. Plant J, 2007, 52: 223-239.
doi: 10.1111/j.1365-313X.2007.03236.x pmid: 17922773
[11] D’Angelo C, Weinl S, Batistic O, Pandey G K, Cheong Y H, Schültke S, Albrecht V, Ehlert B, Schulz B, Harter K, Luan S, Bock R, Kudla J. Alternative complex formation of the Ca2+- regulated protein kinase CIPK1 controls abscisic acid dependent and independent stress responses in Arabidopsis. Plant J, 2006, 48: 857-872.
doi: 10.1111/tpj.2006.48.issue-6
[12] Yin X, Xia Y, Xie Q, Cao Y, Wang Z, Hao G, Song J, Zhou Y, Jiang X. The protein kinase complex CBL10-CIPK8-SOS1 functions in Arabidopsis to regulate salt tolerance. J Exp Bot, 2020, 71: 1801-1814.
doi: 10.1093/jxb/erz549
[13] Yasuda S, Aoyama S, Hasegawa Y, Sato T, Yamaguchi J. Arabidopsis CBL-interacting protein kinases regulate carbon/nitrogen- nutrient response by phosphorylating ubiquitin ligase ATL31 . Mol Plant, 2017, 10: 605-618.
doi: 10.1016/j.molp.2017.01.005
[14] Xiang Y, Huang Y, Xiong L. Characterization of stress-responsive CIPK genes in rice for stress tolerance improvement. Plant Physiol, 2007, 144: 1416-1428.
doi: 10.1104/pp.107.101295 pmid: 17535819
[15] M , X , Gai W X, Li Y, Yu Y N, Ali M, Gong Z H. The CBL-interacting protein kinase CaCIPK13 positively regulates defense mechanisms against cold stress in pepper. J Exp Bot, 2022, 73: 1655-1667.
doi: 10.1093/jxb/erab505
[16] Lu L, Chen X, Zhu L, Li M, Zhang J, Yang X, Wang P, Lu Y, Cheng T, Shi J, Yi Y, Chen J. NtCIPK9: a calcineurin B-like protein-interacting protein kinase from the halophyte Nitraria tangutorum, enhances Arabidopsis salt tolerance. Front Plant Sci, 2020, 11: 1112.
doi: 10.3389/fpls.2020.01112 pmid: 32973820
[17] Chen L, Ren F, Zhou L, Wang Q Q, Zhong H, Li X B. The Brassica napus calcineurin B-like 1/CBL-interacting protein kinase 6 (CBL1/CIPK6) component is involved in the plant response to abiotic stress and ABA signaling. J Exp Bot, 2012, 63: 6211-6222.
doi: 10.1093/jxb/ers273
[18] Cui X Y, Du Y T, Fu J D, Yu T F, Wang C T, Chen M, Chen J, Ma Y Z, Xu Z S. Wheat CBL-interacting protein kinase 23 positively regulates drought stress and ABA responses. BMC Plant Biol, 2018, 18: 93.
doi: 10.1186/s12870-018-1306-5
[19] Ma X, Li Y, Gai W X, Li C, Gong Z H. The CaCIPK3 gene positively regulates drought tolerance in pepper. Hortic Res, 2021, 8: 216.
doi: 10.1038/s41438-021-00651-7
[20] Lu L, Chen X, Wang P, Lu Y, Zhang J, Yang X, Cheng T, Shi J, Chen J. CIPK11: a calcineurin B-like protein-interacting protein kinase from Nitraria tangutorum, confers tolerance to salt and drought in Arabidopsis. BMC Plant Biol, 2021, 21: 123.
doi: 10.1186/s12870-021-02878-x pmid: 33648456
[21] Li R, Zhang J, Wu G, Wang H, Chen Y, Wei J. HbCIPK2, a novel CBL-interacting protein kinase from halophyte Hordeum brevisubulatum, confers salt and osmotic stress tolerance. Plant Cell Environ, 2012, 35: 1582-1600.
doi: 10.1111/pce.2012.35.issue-9
[22] Wang Y, Li T, John S J, Chen M, Chang J, Yang G, He G. A CBL-interacting protein kinase TaCIPK27 confers drought tolerance and exogenous ABA sensitivity in transgenic Arabidopsis. Plant Physiol Biochem, 2018, 123: 103-113.
doi: 10.1016/j.plaphy.2017.11.019
[23] Xu M, Li H, Liu Z N, Wang X H, Xu P, Dai S J, Cao X, Cui X Y. The soybean CBL-interacting protein kinase, GmCIPK2, positively regulates drought tolerance and ABA signaling. Plant Physiol Biochem, 2021, 167: 980-989.
doi: 10.1016/j.plaphy.2021.09.026
[24] Wang F, Chen H W, Li Q T, Wei W, Li W, Zhang W K, Ma B, Bi Y D, Lai Y C, Liu X L, Man W Q, Zhang J S, Chen S Y. GmWRKY27 interacts with GmMYB174 to reduce expression of GmNAC29 for stress tolerance in soybean plants. Plant J, 2015, 83: 224-236.
doi: 10.1111/tpj.12879
[25] Yu T F, Liu Y, Fu J D, Ma J, Fang Z W, Chen J, Zheng L, Lu Z W, Zhou Y B, Chen M, Xu Z S, Ma Y Z. The NF-Y-PYR module integrates the abscisic acid signal pathway to regulate plant stress tolerance. Plant Biotechnol J, 2021, 19: 2589-2605.
doi: 10.1111/pbi.v19.12
[26] Deng X, Hu W, Wei S, Zhou S, Zhang F, Han J, Chen L, Li Y, Feng J, Fang B, Luo Q, Li S, Liu Y, Yang G, He G. TaCIPK29, a CBL-interacting protein kinase gene from wheat, confers salt stress tolerance in transgenic tobacco. PLoS One, 2013, 8: e69881.
doi: 10.1371/journal.pone.0069881
[27] Sun T, Wang Y, Wang M, Li T, Zhou Y, Wang X, Wei S, He G, Yang G. Identification and comprehensive analyses of the CBL and CIPK gene families in wheat (Triticum aestivum L). BMC Plant Biol, 2015, 15: 269.
doi: 10.1186/s12870-015-0657-4
[28] Zhu K, Chen F, Liu J, Chen X, He T, Cheng Z M. Evolution of an intron-poor cluster of the CIPK gene family and expression in response to drought stress in soybean. Sci Rep, 2016, 6: 28225.
doi: 10.1038/srep28225 pmid: 27311690
[29] Luo Q, Wei Q, Wang R, Zhang Y, Zhang F, He Y, Zhou S, Feng J, Yang G, He G. BdCIPK31, a calcineurin B-like protein- interacting protein kinase, regulates plant response to drought and salt stress. Front Plant Sci, 2017, 8: 1184.
doi: 10.3389/fpls.2017.01184
[30] Chen X, Huang Q, Zhang F, Wang B, Wang J, Zheng J. ZmCIPK21, a maize CBL-interacting kinase, enhances salt stress tolerance in Arabidopsis thaliana. Int J Mol Sci, 2014, 15: 14819-14834.
doi: 10.3390/ijms150814819
[31] Su Y, Guo A, Huang Y, Wang Y, Hua J. GhCIPK6a increases salt tolerance in transgenic upland cotton by involving in ROS scavenging and MAPK signaling pathways. BMC Plant Biol, 2020, 20: 421.
doi: 10.1186/s12870-020-02548-4 pmid: 32928106
[32] Li M, Chen R, Jiang Q, Sun X, Zhang H, Hu Z. GmNAC06, a NAC domain transcription factor enhances salt stress tolerance in soybean. Plant Mol Biol, 2021, 105: 333-345.
doi: 10.1007/s11103-020-01091-y pmid: 33155154
[33] Chen Y, Han Y, Kong X, Kang H, Ren Y, Wang W. Ectopic expression of wheat expansin gene TaEXPA2 improved the salt tolerance of transgenic tobacco by regulating Na+/K+ and antioxidant competence. Physiol Plant, 2017, 159: 161-177.
doi: 10.1111/ppl.2017.159.issue-2
[34] Yang L, Han Y, Wu D, Yong W, Liu M, Wang S, Liu W, Lu M, Wei Y, Sun J. Salt and cadmium stress tolerance caused by overexpression of the Glycine max Na+/H+ antiporter (GmNHX1) gene in duckweed (Lemna turionifera 5511). Aquat Toxicol, 2017, 192, 127-135.
[35] Qu Y, Guan R, Bose J, Henderson S W, Wege S, Qiu L, Gilliham M. Soybean CHX-type ion transport protein GmSALT3 confers leaf Na+ exclusion via a root derived mechanism, and Cl- exclusion via a shoot derived process. Plant Cell Environ, 2021, 44: 856-869.
doi: 10.1111/pce.v44.3
[36] Deng J, Yang X, Sun W, Miao Y, He L, Zhang X. The calcium sensor CBL2 and its interacting kinase CIPK6 are involved in plant sugar homeostasis via interacting with tonoplast sugar transporter TST2. Plant Physiol, 2020, 183: 236-249.
doi: 10.1104/pp.19.01368
[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] 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.
[4] 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.
[5] 张晴, 杨昱, 郭茜, 岳霈尧, 殷丛丛, 牛景萍, 赵晋忠, 杜维俊, 岳爱琴. 大豆GmARA6a的克隆及响应盐胁迫的功能分析[J]. 作物学报, 2026, 52(2): 480-493.
[6] 亓青松, 牛翔雨, 刘冰可, 康禄, 王琛, 封德顺. 小偃麦辐射诱变种质芽期和苗期耐盐鉴定、筛选及耐盐指标评价[J]. 作物学报, 2026, 52(2): 389-404.
[7] 李诗晴, 王茜, 王素华, 张耀文, 王丽侠. 绿豆种质资源苗期耐盐性鉴定及相关基因发掘[J]. 作物学报, 2026, 52(2): 376-388.
[8] 黄丽霞, 张卫卫, 甄一越, 王秋宝, 田洪岭, 李国栋, 刘龙龙, 张丽君. 盐胁迫下苦荞萌发期耐盐碱性评价及种质筛选[J]. 作物学报, 2026, 52(2): 459-479.
[9] 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.
[10] 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.
[11] 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.
[12] 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.
[13] 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.
[14] 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.
[15] 蒋优, 马雪融, 张博, 李陈建. 苏丹草种子萌发期耐盐性评价及耐盐种质筛选[J]. 作物学报, 2025, 51(3): 835-844.
Viewed
Full text


Abstract

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