欢迎访问作物学报,今天是

作物学报 ›› 2016, Vol. 42 ›› Issue (04): 469-481.doi: 10.3724/SP.J.1006.2016.00469

• 作物遗传育种·种质资源·分子遗传学 •    下一篇

大豆中一个WRKY28-like基因的克隆与功能分析

王婷婷,丛亚辉,柳聚阁,王宁帅,琴李艳*,盖钧镒*   

  1. 南京农业大学作物遗传与种质创新国家重点实验室 / 国家大豆改良中心 / 农业部大豆生物学与遗传育种重点实验室 / 江苏省现代作物生产协同创新中心, 江苏南京210095
  • 收稿日期:2015-08-21 修回日期:2016-01-11 出版日期:2016-04-12 网络出版日期:2016-01-27
  • 通讯作者: 李艳, E-mail: yanli1@njau.edu.cn; 盖钧镒,E-mail:sir@njau.edu.cn
  • 基金资助:

    本研究由国家高技术研究发展计划(863计划)(2013AA102602)项目,教育部长江学者和创新团队发展计划(PCSIRT13073)项目,中央高校基本科研业务费专项资金,教育部新世纪优秀人才支持计划(NCET-12-0891)和农业部大豆生物学与遗传育种创新团队和江苏省双创计划资助。

Cloning and Functional Analysis of a WRKY28-like Gene in Soybean

WANG Ting-Ting,CONG Ya-Hui,LIU Ju-Ge,WANG Ning,SHUAI Qin,LI Yan*,GAI Jun-Yi*   

  1. National Key Laboratory of Crop Genetics and Germplasm Enhancement / National Center for Soybean Improvement / Key Laboratory for Biology and Genetic Improvement of Soybean (General), Ministy of Agriculture /Jiangsu Collaborative Innovation Center for Modern Crop Production,Nanjing Agricultural University, Nanjing 210095, China
  • Received:2015-08-21 Revised:2016-01-11 Published:2016-04-12 Published online:2016-01-27
  • Contact: 李艳, E-mail: yanli1@njau.edu.cn; 盖钧镒,E-mail:sir@njau.edu.cn
  • Supported by:

    This research was supported bythe National High-tech R & D Program of China (2013AA102602), theProgram for Changjiang Scholars and Innovative Research Team in University of Ministry of Education of China(PCSIRT13073), the Fundamental Research Funds for the Central Universities, Program for New Century Excellent Talents in University of Ministry of Education of China(NCET-12-0891), Program for Soybean Biology and Genetic Breeding Innovative Research Team of Ministry of Agriculture of China, and Program for High-level Innovative and Entrepreneurial Talents in Jiangsu Province.

摘要:

WRKY转录因子参与调节植物生长发育、生物与非生物胁迫应答等多种过程,AtWRKY28是拟南芥中与抗病和耐逆相关的重要转录因子。为探讨大豆中一个AtWRKY28同源基因GmWRKY28-like(Glyma.14G028900)的生物学功能,本文对该基因进行了克隆、生物信息学分析、亚细胞定位、组织表达等试验,并对其在ABA、PEG、NaCl胁迫下的表达水平进行了分析。结果显示,GmWRKY28-like基因的编码区(CDS)为1008bp,编码335个氨基酸。GmWRKY28-like蛋白具有保守的WRKY结构域,含有22个丝氨酸(Serine)、1个苏氨酸(Threonine)、2个酪氨酸(Tyrosine),不含跨膜结构与信号肽;进化树分析表明大豆GmWRKY28-like与菜豆(Phaseolus vulgaris)WRKY28的相似性最高;亚细胞定位显示GmWRKY28-like定位在细胞核中。该基因在根、种子中表达量很低,在真叶、花、及茎尖分生组织表达量较高。GmWRKY28-like启动子中含有多种与生物和非生物逆境胁迫应答相关的元件,如MBS、W-box、ABRE、Box-W1等,且表达受到ABA、PEG、NaCl的诱导。此外,过表达GmWRKY28-like显著增强了拟南芥的耐盐性。

关键词: GmWRKY28, 进化树分析, 亚细胞定位, 非生物胁迫

Abstract:

WRKY transcription factors play important roles in regulating plant growth and development, andparticipate in plant response to abiotic and biotic stresses.Arabidopsis WRKY28 is an essential transcription factor for plant response to necrotrophic pathogen and abiotic stress. To explore the function of the homologues gene of AtWRKY28 in soybean, we cloned GmWRKY28-like (Glyma.14G028900) and performed bioinformatics analysis. Its sub-cellular localization and tissue expression patternswere further investigated. We also analyzed the expression levels of GmWRKY28-like under ABA, PEG and NaCl stress treatments. Our results showed that the coding DNA sequence (CDS) of GmWRKY28-like gene is 1008 bp in length, encoding 335 amino acids. GmWRKY28-like contains a conserved WRKY domainwith22 serine, one threonine and two tyrosine, without any transmembrane domain or signal peptide. Phylogenetic analysis showed that the WRKY28 from Phaseolus vulgariswas most similar to GmWRKY28-like. GmWRKY28-like was verified to be located in the nucleus. This gene expressed at low levels in root and seed while high levels in true leaf, flower and shoot apical meristem. The 1500 bp upstream region of GmWRKY28-like contains a variety of cis-elements, such as MBS, W box, ABRE, and box-W1, which are involved in the response to biotic and abiotic stresses, andits expression was induced by ABA, PEG and NaCl treatments.In addition, overexpressing GmWRKY28-like in Arabidopsis thaliana enhanced plant tolerance to NaCl.This study would provide reference basis to further study the functions of GmWRKY28-like in soybean tolerance to abiotic stresses.

Key words: GmWRKY28, Phylogenetic analysis, Subcellular location, Abiotic stress

[1]von Koskull-Döring P, Scharf KD, Nover L. The diversity of plant heat stress transcription factors. Trends Plant Sci, 2007, 12: 452–457

[2]Nakashima K, Yamaguchi-Shinozaki K. Molecular studies on stress-responsive gene expression in Arabidopsis and improvement of stress tolerance in crop plants by regulon biotechnology. Jpn Agric Res Quart, 2005, 39: 221–229

[3]Zhou Q Y, Tian A G, Zou H F, Xie Z M, Lei G, Huang J, Wang C M, Wang H W, Zhang J S, Chen S Y. Soybean WRKY-type transcription factor genes, GmWRKY13, GmWRKY21, and GmWRKY54, confer differential tolerance to abiotic stresses in transgenic Arabidopsis plants. Plant Biotechnol J, 2008, 6: 486–503

[4]Jiang Y, Deyholos M K. Functional characterization of Arabidopsis NaCl-inducible WRKY25 and WRKY33 transcription factors in abiotic stresses. Plant Mol Biol, 2009, 69: 91–105

[5]Eulgem T, Somssich I E. Networks of WRKY transcription factors in defense signaling. Curr Opin Plant Biol, 2007, 10: 366–371

[6]Christian A. Ross, Yue Liu, Qingxi J. The WRKYgene family in rice (Oryza sativa). J Integr Plant Biol, 2007, 49: 827–842 DOI: 10.1111/j.1744-7909.2007.00504.x

[7]Eulgem T , Rushton P J, Robatzek S, Somssich I E. The WRKY superfamily of plant transcription factors. Trends Plant Sci, 2000, 5: 199–206

[8]Dong J, Chen C, Chen Z. Expression profiles of the Arabidopsis WRKY gene superfamily during plant defense response. Plant Mol Biol, 2003, 51: 21–37

[9]Brand L H, Kirchler T, Hummel S, Chaban C, Wanke D. DPI-ELISA: a fast and versatile method to specify the binding of plant transcription factors to DNA in vitro. Plant Methods, 2010, 6: 25

[10]Lagacé M, Matton D P. Characterization of a WRKY transcription factor expressed in late torpedo-stage embryos of Solanum chacoense. Planta, 2004, 219: 185-189

[11]Johnson C S, Kolevski B, and Smyth D R. TRANSPARENT TESTA GLABRA2, a trichome and seed coat development gene of Arabidopsis, encodes a WRKY transcription factor. Plant Cell, 2002, 14: 1359–1375

[12]Huang T, Duman J G. Cloning and characterization of a thermal hysteresis (antifreeze) protein with DNA-binding activity from winter bittersweet nightshade, Solanum dulcamara. Plant Mol Biol, 2002, 48: 339–350

[13]Yoda H, Ogawa M, Yamaguchi Y, Koizumi N, Kusano T, Sano H. Identification of early-responsive genes associated with the hypersensitive response to tobacco mosaic virus and characterization of a WRKY-type transcription factor in tobacco plants. Mol Genet Genom, 2002, 267: 154–161

[14]Qiu D, Xiao J, Ding X, Xiong M, Cai M, Cao Y, Li X, Xu C, Wang S. OsWRKY13 mediates rice disease resistance by regulating defense-related genes in salicylate-and jasmonate-dependent signaling. Mol Plant, Mol Plant Microbe Interact, 2007, 20: 492-499

[15]Qiu D, Xiao J, Xie W, Liu H, Li X, Xiong L, Wang S. Rice gene network inferred from expression profiling of plants overexpressing OsWRKY13, a positive regulator of disease resistance. Mol Plant, 2008, 1: 538–551

[16]Yu F,Huaxia Y,Lu W,Wu C,Cao X,Guo X. GhWRKY15, a member of the WRKY transcription factor family identified from cotton (Gossypium hirsutum L.), is involved in disease resistance and plant development.BMC Plant Biol, 2012, 12: 144

[17]Wang X,Yan Y,Li Y,Chu X,Wu C,Guo X. GhWRKY40, a multiple stress-responsive cotton WRKY gene, plays an important role in the wounding response and enhances susceptibility to ralstonia solanacearum infection in transgenic Nicotiana benthamiana.PLoS One, 2014, 18: e93577

[18]Chen L, Zhang L, Li D, Wang F, Yu D. WRKY8 transcription factor functions in the TMV-cg defense response by mediating both abscisic acid and ethylene signaling in Arabidopsis. Proc Natl Acad Sci USA, 2013, 110: 1963–1971

[19]Lan A,Huang J,Zhao W,Peng Y,Chen Z,Kang D. A salicylic acid-induced rice (Oryza sativa L.) transcription factor OsWRKY77 is involved in disease resistance of Arabidopsis thaliana. Plant Biol (Stuttg), 2013, 15: 452–461

[20]Peng Y, Bartley L E, Chen X, Dardick C, Chern M, Ruan R, Canlas P E, Ronald P C. OsWRKY62 is a negative regulator of basal and Xa21-mediated defense against Xanthomonas oryzae pv. oryzae in rice. Mol Plant, 2008, 1: 446–458

[21]Pandey S P, Somssich I E. The role of WRKY transcription factors in plant immunity. Plant Physiol, 2009, 150: 1648–1655

[22]Zheng Z, Qamar S A, Chen Z, Mengiste T. Arabidopsis WRKY33 transcription factor is required for resistance to necrotrophic fungal pathogens. Plant J, 2006, 48: 592–605

[23]Wang D, Amornsiripanitch N, Dong X. A genomic approach to identify regulatory nodes in the transcriptional network of systemic acquired resistance in plants. PLoS Pathog, 2006, 2: 1042–1050

[24]Lai Z, Vinod K, Zheng Z, Fan B, Chen Z. Roles of Arabidopsis WRKY3 and WRKY4 transcription factors in plant responses to pathogens. BMC Plant Biol, 2008, 8:68

[25]Hu Y, Dong Q, Yu D. Arabidopsis WRKY46 coordinates with WRKY70 and WRKY53 in basal resistance against pathogen Pseudomonas syringae. Plant Sci, 2012, 185: 288–297

[26]钟贵买, 伍林涛, 王健美, 杨毅, 李旭锋. 转录因子AtWRKY28亚细胞定位及在非生物胁迫下的表达分析. 中国农业科技导报, 2012, 14(5):57–63

Zhong G M, Wu L T, Wang J M, Yang Y, Li X F. Subcellular localization and expression analysis of transcription factor AtWRKY28 under biotic stresses. J Agric Sci Technol, 2012, 14(5): 57–63 (in Chinese with English abstract)

[27]Wu L T, Zhong G M, Wang J M, Li X F, Song X, Yang Y. Arabidopsis WRKY28 transcription factor is required for resistance to necrotrophic pathogen, Botrytis cinerea. Afr J Microbiol Res, 2011, 5: 5481–5488

[28]张飞萃. 拟南芥WRKY28和WRKY42调控磷吸收和转运的机制研究.中国农业大学博士学位论文, 北京, 2015

Zhang F C. Regulatory mechanism of phosphate translocation and acquisition by AtWRKY28 and AtWRKY42 inArabidopsisplants. PhD Dissertation of ChinaAgriculturalUniversity, Beijing, China, 2015 (in Chinese with English abstract)

[29]Chen X, Liu J, Lin G, Wang A, Wang Z, Lu G. Overexpression of AtWRKY28 and AtWRKY75 in Arabidopsis enhances resistance to oxalic acid and Sclerotinia sclerotiorum. Plant Cell Rep, 2013, 32: 1589–1599

[30]Yoo S D, Cho Y H, Sheen J. Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nat Protoc, 2007, 2: 1565–1572

[31]Livak K J, Schmittgen T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(T)(-Delta Delta C (T)) method. Methods, 2001, 25: 402–408

[32]Uno Y, Furihata T, Abe H, Yoshida R, Shinozaki K, Yamaguchi-Shinozaki K. Arabidopsis basic leucine zipper transcription factors involved in an abscisic acid-dependent signal transduction pathway under drought and high-salinity conditions. Proc Natl Acad Sci USA, 2000, 97: 11632–11637
[1] 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.
[2] 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.
[3] 杨飚, 杜帅康, 张继旺, 石瑛, 张丽莉. 马铃薯III类POD基因家族的全基因组鉴定及其表达谱分析[J]. 作物学报, 2026, 52(2): 405-420.
[4] 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.
[5] 景秀清, 蔡永朵, 邓宁, 赵晓东, 翟飞红, 曾群. 藜麦RopGEF家族基因的鉴定及表达模式分析[J]. 作物学报, 2026, 52(1): 28-43.
[6] 闫知兰, 赵芹, 常甜达, 王一鸣, 王碧辉, 王鹏, 黄春国, 张会, 王利祥, 郝晓鹏, 赵波. 豆科作物AOX基因鉴定及其在普通菜豆响应非生物胁迫中的表达模式研究[J]. 作物学报, 2025, 51(7): 1769-1783.
[7] 潘炬忠, 韦萍, 朱德平, 邵胜雪, 陈珊珊, 韦雅倩, 高维维. 水稻转录因子OsERF104的克隆和功能研究[J]. 作物学报, 2025, 51(4): 900-913.
[8] 郭冰, 秦家范, 李娜, 宋梦瑶, 王黎明, 李君霞, 马小倩. 谷子SHMT基因家族全基因组鉴定与表达分析[J]. 作物学报, 2025, 51(3): 586-5897.
[9] 许睿, 何妙华, 王昊, 李卫, 任杰, 夏志强. 基于空间转录组技术解析大豆种胚对X射线辐射的响应机制[J]. 作物学报, 2025, 51(12): 3121-3132.
[10] 王玉娇, 王永乐, 添长久, 郁春旺, 吕佳斌, 朱加保. 薏苡VQ4基因的克隆及耐盐性初步分析[J]. 作物学报, 2025, 51(12): 3198-3210.
[11] 李万, 常紫锐, 卢瑶, 沈日敏, 赵永平, 白小东. 25种不同植物RAV家族的鉴定与马铃薯RAV基因分析[J]. 作物学报, 2025, 51(11): 2944-2957.
[12] 祁稼民, 许春苗, 肖斌. 马铃薯TIFY基因家族的全基因组鉴定及表达分析[J]. 作物学报, 2024, 50(9): 2297-2309.
[13] 高维东, 胡城祯, 张龙, 张艳艳, 张沛沛, 杨德龙, 陈涛. 小麦泛素结合酶TaUBC16基因的克隆与功能分析[J]. 作物学报, 2024, 50(8): 1971-1988.
[14] 刘宸铭, 赵克勇, 悦曼芳, 赵延明, 吴忠义, 张春. 玉米转录因子ZmEREB180调控根系生长发育及耐逆的功能研究[J]. 作物学报, 2024, 50(8): 1920-1933.
[15] 刘震, 陈丽敏, 李志涛, 朱金勇, 王玮璐, 齐喆颖, 姚攀锋, 毕真真, 孙超, 白江平, 刘玉汇. 马铃薯ARM基因家族的全基因组鉴定及表达分析[J]. 作物学报, 2024, 50(6): 1451-1466.
Viewed
Full text


Abstract

Cited

  Shared   
  Discussed   
No Suggested Reading articles found!