大豆转录因子GmWRKY58亚细胞定位及在非生物胁迫下的表达分析

doi:10.7668/hbnxb.2018.02.008

摘要/Abstract

摘要： WRKY转录因子在植物响应非生物胁迫，调控抗逆基因的表达方面具有重要作用。为了解析大豆转录因子GmWRKY58基因在非生物胁迫中的功能，从大豆中克隆了GmWRKY58cDNA全长，推导其编码的氨基酸序列、生理生化特征与进化关系，并研究了其亚细胞定位和在不同组织及非生物胁迫下基因的表达变化。结果表明，GmWRKY58基因cDNA ORF全长为954 bp，编码317个氨基酸。亚细胞定位结果显示，GmWRKY58蛋白质定位于细胞核中。实时荧光定量PCR基因表达分析表明，GmWRKY58在大豆根、茎、叶、花和荚等组织均有表达，根、茎、叶中的表达量明显高于花和荚。在大豆根中，GmWRKY58基因受高盐、干旱、低N和缺Fe等非生物胁迫因子强烈诱导，在高盐胁迫下，基因表达量增加187.4倍；GmWRKY58基因受水杨酸（SA）、低温及低P和低K等诱导轻微，差异表达不显著。由此推测，GmWRKY58转录因子在大豆抗盐、抗旱、低N和缺Fe等非生物胁迫过程中起到重要的调控作用。

关键词: 大豆, 转录因子, GmWRKY58, 亚细胞定位, 非生物胁迫

Abstract: WRKY transcription factor plays an important role in plant response to abiotic stress and regulation of resistance gene expression. In order to reveal the role of a soybean transcription factor GmWRKY58 in response to abiotic stresses.The full length of GmWRKY58 cDNA was cloned from soybean,and the amino acid sequence,physiological and biochemical characteristics and evolutionary relationship were deduced. Subcellular localization and the changes of gene expression under different tissues and abiotic stress were studied. The results showed that GmWRKY58 had a 954 bp length open reading frame and encoded a total of 317 amino acids. Subcellular localization results showed that GFP signals combined GmWRKY58 was only detected in the nucleus. Real-time RT-PCR analysis showed that GmWRKY58 was expressed in soybean roots,stems,leave,flowers and pods,and the expression in each tissue of roots,stems and leave was significantly higher than flowers and pods. In soybean roots, GmWRKY58 gene can be significantly induced by abiotic stress factors such as high salinity,drought,low nitrogen and iron deficiency.Under high salt stress, GmWRKY58 gene expression was increased by more than 187.4 times. Moreover,this gene just slightly induced by other abiotic stresses shch as SA,low temperature,low phosphorus and low potassium. The results indicate that GmWRKY58 plays an important regulatory role in abiotic stress process of salt,drought,low nitrogen and iron deficiency in soybean.

Key words: Soybean, Transcription factor, GmWRKY58, Subcellular localization, Abiotic stresses

中图分类号:

Q78
S565.03

张红梅, 刘晓庆, 陈华涛, 袁星星, 崔晓艳, 张智民, 黄中文, 陈新. 大豆转录因子GmWRKY58亚细胞定位及在非生物胁迫下的表达分析[J]. 华北农学报, 2018, 33(2): 49-57. doi: 10.7668/hbnxb.2018.02.008.

ZHANG Hongmei, LIU Xiaoqing, CHEN Huatao, YUAN Xingxing, CUI Xiaoyan, ZHANG Zhimin, HUANG Zhongwen, CHEN Xin. Subcellular Localization and Expression Analysis of a Soybean Transcription Factor GmWRKY58 in Response to Abiotic Stresses[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2018, 33(2): 49-57. doi: 10.7668/hbnxb.2018.02.008.

http://www.hbnxb.net/CN/Y2018/V33/I2/49

参考文献

[1] Choudhury S,Panda P,Sahoo L,et al.Reactive oxygen species signaling in plants under abiotic stress[J].Plant Signaling&Behavior,2013,8(4):e23681.
[2] Nakashima K,Yamaguchi-Shinozaki K,Shinozaki K.The transcriptional regulatory network in the drought response and its crosstalk in abiotic stress responses including drought,cold,and heat[J].Frontiers in Plant Science,2014,5:170.
[3] Jiang J,Ma S,Ye N,et al.WRKY transcription factors in plant responses to stresses[J].Journal of Integrative Plant Biology,2017,59(2):86-101.
[4] Ishiguro S,Nakamura K.Characterization of a cDNA encoding a novel DNA-binding protein,SPF1,that recognizes SP8 sequences in the 5'upstream regions of genes coding for sporamin and β-amylase from sweet potato[J].Molecular and General Genetics,1994,244(6):563-571.
[5] Rushton P J,Macdonald H,Huttly A K,et al.Members of a new family of DNA-binding proteins bind to a conserved cis-element in the promoters of α-Amy2 genes[J].Plant Molecular Biology,1995,29(4):691-702.
[6] Rushton P J,Torres J T,Parniske M,et al.Interaction of elicitor-induced DNA-binding proteins with elicitor response elements in the promoters of parsley PR1 genes[J].The EMBO Journal,1996,15(20):5690-5700.
[7] Abuqamar S,Chen X,Dhawan R,et al.Expression profiling and mutant analysis reveals complex regulatory networks involved in Arabidopsis response to Botrytis infection[J].The Plant Journal,2006,48(1):28-44.
[8] Dai X,Wang Y,Zhang W H.OsWRKY74,a WRKY transcription factor,modulates tolerance to phosphate starvation in rice[J].Journal of Experimental Botany,2015,67(3):947-960.
[9] Guan Y,Meng X,Khanna R,et al.Phosphorylation of a WRKY transcription factor by MAPKs is required for pollen development and function in Arabidopsis[J].PLoS Genetics,2014,10(5):e1004384.
[10] Zhou L,Wang N N,Gong S Y,et al.Overexpression of a cotton (Gossypium hirsutum) WRKY gene,GhWRKY34, in Arabidopsis enhances salt-tolerance of the transgenic plants[J].Plant Physiology and Biochemistry,2015,96:311-320.
[11] Hui D,Iqbal J,Lehmann K,et al.Molecular interactions between the specialist herbivore Manduca sexta(lepidoptera,sphingidae) and its natural host Nicotiana attenuata:V.microarray analysis and further characterization of large-scale changes in herbivore-induced mRNAs[J].Plant Physiology,2003,131(4):1877-1893.
[12] Liu D,Leib K,Zhao P,et al.Phylogenetic analysis of barley WRKY proteins and characterization of HvWRKY1 and-2 as repressors of the pathogen-inducible gene HvGER4c[J].Molecular Genetics and Genomics,2014,289(6):1331-1345.
[13] Niu C F,Wei W,Zhou Q Y,et al.Wheat WRKY genes TaWRKY2 and TaWRKY19 regulate abiotic stress tolerance in transgenic Arabidopsis plants[J].Plant,Cell&Environment,2012,35(6):1156-1170.
[14] Chi Y,Yang Y,Li G,et al.Identification and characterization of a novel group of legume-specific,Golgi apparatus-localized WRKY and Exo70 proteins from soybean[J].Journal of Experimental Botany,2015,66(11):3055-3070.
[15] Zhou Q Y,Tian A G,Zou H F,et al.Soybean WRKY-type transcription factor genes,GmWRKY13,GmWRKY21,and GmWRKY54,confer differential tolerance to abiotic stresses in transgenic Arabidopsis plants[J].Plant Biotechnology Journal,2008,6(5):486-503.
[16] Suttipanta N,Pattanaik S,Kulshrestha M A,et al.The transcription factor CrWRKY1 positively regulates the terpenoid indole alkaloid biosynthesis in Catharanthus roseus[J].Plant Physiology,2011,157(4):2081-2093.
[17] Yu F,Huaxia Y,Lu W,et al.GhWRKY15,a member of the WRKY transcription factor family identified from cotton (Gossypium hirsutum L.),is involved in disease resistance and plant development[J].BMC Plant Biology,2012,12:144.
[18] Jiang J,Ma S,Ye N,et al.WRKY transcription factors in plant responses to stresses[J].Journal of Integrative Plant Biology,2017,59(2):86-101.
[19] Ding Z J,Yan J Y,Li C X,et al.Transcription factor WRKY46 modulates the development of Arabidopsis lateral roots in osmotic/salt stress conditions via regulation of ABA signaling and auxin homeostasis[J].Plant Journal,2015,84(1):56-69.
[20] Jiang Y,Liang G,Yu D.Activated expression of WRKY57 confers drought tolerance in Arabidopsis[J].Molecular Plant,2012,5(6):1375-1388.
[21] Asai T,Tena G,Plotnikova J,et al.MAP kinase signalling cascade in Arabidopsis innate immunity[J].Nature,2002,415(6875):977-983.
[22] 李立芹.烟草WRKY12基因的分离、表达与多克隆抗体的制备[J].核农学报,2011,25(3):461-468.
[23] Jiang Y,Deyholos M K.Functional characterization of Arabidopsis NaCl-inducible WRKY25 and WRKY33 transcription factors in abiotic stresses[J].Plant Molecular Biology,2009,69(1-2):91-105.
[24] Li S J,Fu Q T,Chen L G,et al.Arabidopsis thaliana WRKY25,WRKY26,and WRKY33 coordinate induction of plant thermotolerance[J].Planta,2011,233(6):1237-1252.
[25] Li S J,Fu Q T,Huang W D,et al.Functional analysis of an Arabidopsis transcription factor WRKY25 in heat stress[J].Plant Cell Reports,2009,28(4):683-693.
[26] Raineri J,Wang S H,Peleg Z,et al.The rice transcription factor OsWRKY47 is a positive regulator of the response to water deficit stress[J].Plant Molecular Biology,2015,88(4/5):401-413.
[27] 钟贵买,伍林涛,王健美,等.转录因子AtWRKY28亚细胞定位及在非生物胁迫下的表达分析[J].中国农业科技导报,2012,14(5):57-63.
[28] 张飞萃.拟南芥WRKY28和WRKY42调控磷吸收和转运的机制研究[D].北京:中国农业大学,2015
[29] Xu Z L,Ali Z,Yi J X,et al.Over-expression of GmWRKY111 enhances NaCl tolerance of Salt-Sensitive genotype of Glycine max[J].International Journal of Agriculture and Biology,2014,16(1):153-159.
[30] Yang Y,Chi Y J,Wang Z,et al.Functional analysis of structurally related soybean GmWRKY58 and GmWRKY76 in plant growth and development[J].Journal of Experimental Botany,2016,67(15):4727-4742.
[31] Zhang G,Chen M,Li L,et al.Overexpression of the soybean GmERF3 gene,an AP2/ERF type transcription factor for increased tolerances to salt,drought,and diseases in transgenic tobacco[J].Journal of Experimental Botany,2009,60(13):3781-3796.
[32] 翟莹,雷婷婷,闫帆,等.大豆逆境诱导基因GmPRP的克隆与表达[J].作物学报,2011,37(12):2152-2157.
[33] Livak K J,Schmittgen T D.Analysis of relative gene expression data using real-time quantitative PCR and the 2(T)(-Delta Delta C) method[J].Methods,2001,25(4):402-408.
[34] Sparkes I A,Runions J,Kearns A,et al.Rapid,transient expression of fluorescent fusion proteins in tobacco plants and generation of stably transformed plants[J].Nature Protocols,2006,1(4):2019-2025.
[35] Hirayama T,Shinozaki K.Research on plant abiotic stress responses in the post-genome era:past,present and future[J].The Plant Journal:for Cell and Molecular Biology,2010,61(6):1041-1052.
[36] 徐影.大豆耐低磷相关转录因子GmWRKY75和GmWRKY6的克隆及功能分析[D].南京:南京农业大学,2014.
[37] Brand L H,Kirchler T,Hummel S,et al.DPI-ELISA:a fast and versatile method to specify the binding of plant transcription factors to DNA in vitro[J].Plant Methods,2010,6(1):25.

选择文件类型/文献管理软件名称

选择包含的内容