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作物学报 ›› 2017, Vol. 43 ›› Issue (09): 1319-1327.doi: 10.3724/SP.J.1006.2017.01319

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

碱胁迫相关基因GsWRKY15的克隆及其转基因苜蓿的耐碱性分析

朱娉慧**,陈冉冉**,于洋,宋雪薇,李慧卿,杜建英,李强,丁晓东,朱延明*   

  1. 东北农业大学农业生物功能基因重点实验室, 黑龙江哈尔滨 150030
  • 收稿日期:2016-12-03 修回日期:2017-05-10 出版日期:2017-09-12 网络出版日期:2017-05-22
  • 通讯作者: 朱娉慧, E-mail: zhupinghui@outlook.com, Tel: 15604601520
  • 基金资助:

    本研究由国家自然科学基金项目(31171578), 黑龙江省高校科技创新团队建设计划(2011TD005)和东北农业大学学科团队建设项目(团队1)资助。

Cloning of Gene GsWRKY15 Related to Alkaline Stress and Alkaline Tolerance of Transgenic Plants

ZHU Ping-Hui**,CHEN Ran-Ran**,YU Yang,SONG Xue-Wei,LI Hui-Qing,DU Jian-Ying,LI Qiang,DING Xiao-Dong,ZHU Yan-Ming*   

  1. Key Laboratory of Agricultural Biological Functional Genes, Northeast Agricultural University, Harbin 150030, China
  • Received:2016-12-03 Revised:2017-05-10 Published:2017-09-12 Published online:2017-05-22
  • Contact: 朱娉慧, E-mail: zhupinghui@outlook.com, Tel: 15604601520
  • Supported by:

    This study was supported by National Natural Science Foundation of China (31171578),Heilongjiang Provincial Higher School Science and Technology Innovation Team Building Program (2011TD005), and the Northeast Agricultural University Discipline Team Construction Project (Group 1).

摘要:

WRKY蛋白属于锌指型转录调控因子, 能够参与植物多种逆境响应。本研究利用前期野生大豆盐碱胁迫RNA-seq测序数据, 从构建的碱胁迫基因调控网络中筛选并克隆到GsWRKY15基因。分析GsWRKY15在碱胁迫下野生大豆根中的表达模式, 发现该基因受碱胁迫诱导显著上调表达, 且在胁迫后1 h表达量最高。分析GsWRKY15基因在野生大豆各组织中的表达特异性, 发现该基因在各组织中均有表达, 花中表达量最高。采用根癌农杆菌侵染苜蓿子叶节方法, 将GsWRKY15转化肇东苜蓿, 获得39株抗性植株。通过PCR、Southern blot和RT-PCR方法分析抗性植株, 获得了超量表达GsWRKY15基因的转基因株系并对其进行了耐碱性分析。在150 mmol L–1 NaHCO3处理2周后转基因苜蓿生长状态良好, 而非转基因苜蓿出现萎蔫、变黄、甚至死亡; 非转基因苜蓿的相对质膜透性和丙二醛含量显著高于转基因苜蓿, 而叶绿素含量显著低于转基因苜蓿; 同时分析碱胁迫下转基因植株中胁迫相关基因的表达模式, 发现H+-Ppase、NADP-ME、KIN1、RD29A基因的表达量高于非转基因苜蓿。结果表明GsWRKY15基因的超量表达能够显著增强苜蓿的耐碱能力。

关键词: 野生大豆, GsWRKY15, 肇东苜蓿, 农杆菌, 耐碱性

Abstract:

WRKY proteins are members of a transcription factor family with Zinc-finger structure in higher plant, which participate in various responses to multiple stresses.In this study,we constructed a gene expression profile under alkaline treatment using RNA-seq data, from which we cloned a gene GsWRKY15.We analyzedtheexpression pattern of GsWRKY15 in roost of Glycine soja under alkaline stress, and found that this gene was up-regulated by alkaline stress,wirh the highest expression at one hour after alkaline treatment. We analyzed theexpression pattern of GsWRKY15 in different tissues of Glycine soja, and found that this gene could express in all tissues, with the highest expression level in flowers.GsWRKY15 was transformed into Zhaodong alfalfa by Agrobacteriumtumefaciens-mediated infection of alfalfa cotyledonary nodes, and 39 resistant plants were obtained. The results of PCR, Southern blot and RT-PCR showed that GsWRKY15 was integrated into the genome of Zhaodong alfalfa and expressed in transgenic plants. Alkaline tolerance analysis showed that the growth of transgenic alfalfa after two weeks of treatment with 150 mmol L–1 NaHCO3 was better than those of non-transgenic alfalfa. MDA content and relative membrane permeability were significantly higher while chlorophyll content was significantly lower in non-transgenic alfalfa than in transgenic alfalfa. And by analyzing t some stress response marker genes , we found that he expression levels of H+-Ppase, NADP-ME, KIN1, RD29A were higher in transgenic alfalfa than in non-transgenic alfalfa.Taken together we suggest that the expression of GsWRKY15 gene can enhance the alkaline-resistant ability of alfalfa.

Key words: Glycine soja, GsWRKY15, Zhaodong alfalfa, Agrobacterium tumefaciens, Alkaline tolerance

[1] 李彬, 王志春, 孙志高, 陈渊, 杨福. 中国盐碱地资源与可持续利用研究. 干旱地区农业研究, 2005, 23(2): 154–158
Li B, Wang Z C, Sun Z G, Chen Y, Yang F. Resources and sustainable resource exploitation of salinized land in China. Agric Res Arid Res, 2005, 23(2): 154–158 (in Chinese with English abstract)
[2] 王鑫马, 马永祥, 李娟. 紫花苜蓿营养成分及主要生物学特性. 草业科学, 2003, 20(10): 39–41
Wang X M, Ma Y X, Li J. Alfalfa nutrient composition and main biological characteristics. Acta Pratac Sin, 2003, 20(10): 39–41 (in Chinese with English abstract)
[3] 乔建江, 王堃, 杨青川. 苜蓿转基因的研究现状和前景. 中国草地学报, 2006, 28(5): 98–103
Qiao J J, Wang K, Yang Q C. Research situation and future of transgenic alfalfa. Chin J Grassland, 2006, 28(5): 98–103 (in Chinese with English abstract)
[4] ülker B,Somssich I E. WRKY transcription factors: from DNA binding towards biological function. Curr Opin Plant Biol, 2004, 7: 491–498
[5] Li J, Brader G, PalvaE T. The WRKY70 transcription factor: a node of convergence for jasmonate-mediated and salicylate-mediated signals in plant defense. Plant Cell, 2004, 16: 319–331
[6] 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
[7] Eulgem T, Rushton P J, Robatzek S, Somssich I E. The WRKY superfamily of plant transcription factors. Trends Plant Sci, 2000, 5(5): 199–206
[8] 李福山. 中国野生大豆资源的地理分布及生态分化研究. 中国农业科学, 1993, 26(2): 47–55
Li F S.Studies on the ecological and geographical distribution of the chinese resources of wild soybean(G. soja). Sci Agric Sin, 1993, 26(2): 47–55 (in Chinese with English abstract)
[9] Bai X, Liu J, Tang L, Cai H, Chen M, Ji W, Liu Y, Zhu Y. Overexpression of GsCBRLK from Glycine soja enhances tolerance to salt stress in transgenic alfalfa (Medicago sativa). Funct Plant Biol, 2013, 40: 1048–1056
[10] 王臻昱, 才华, 柏锡, 纪巍, 李勇, 魏正巍, 朱延明. 野生大豆GsGST19基因的克隆及其转基因苜蓿的耐盐碱性分析. 作物学报, 2013, 38: 971–979
Wang Z Y, Cai H, Bai X, Ji W, Li Y, Wei Z W, Zhu Y M. Isolation of GsGST19 from Glycine soja and analysis of saline-alkaline tolerance for transgenic Medicago sativa. Acta Agron Sin, 2013, 38: 971–979 (in Chinese with English abstract)
[11] 魏正巍, 朱延明, 化烨, 才华, 纪巍, 柏锡, 王臻昱, 文益东. 转GsPPCK1基因苜蓿植株的获得及其耐碱性分析. 作物学报, 2013, 39: 68–75
Wei Z W, Zhu Y M, Hua Y, Cai H, Ji W, Bai X, Wang Z Y, Wen Y D. Transgenic alfalfa with GsPPCK1 and its alkaline tolerance analysis. Acta Agron Sin, 2013, 39: 68–75 (in Chinese with English abstract)
[12] 赵阳, 朱延明, 柏锡, 纪巍, 吴婧, 唐立郦, 才华. 转GsCBRLK/SCMRP双价基因苜蓿耐碱性及氨基酸含量分析. 作物学报, 2014, 40: 431–438
Zhao Y, Zhu Y M, Bai X, Ji W, Wu J, Tang L L, Cai H. Over-expressing GsCBRLK/SCMRP enhances alkaline tolerance and methionine content in transgenic Medicago sativa. Acta Agron Sin, 2014, 40: 431–438 (in Chinese with English abstract)
[13] Sun M Z, Jia B W, Cui N, Wen Y D, Duanmu H Z, Yu Q Y, Xiao J L, Zhu Y M. Functional characterization of a Glycine sojaCa2+ATPase in salt–alkaline stress responses. Plant Mol Biol, 2016, 90: 419–434
[14] Sun M Z, Sun X L, Zhao Y, Zhao C Y, Duanmu H Z, Yu Y, Ji W, Zhu Y M. Ectopic expression of GsPPCK3 and SCMRP in Medicago sativa enhances plant alkaline stress tolerance and methionine content. PLoS One, 2013, 9: e89578
[15] Duanmu H Z, Wang Y, Bai X, Cheng S F, Deyholos M K, Wong G K, Li D, Zhu D, Li R, Yu Y, Cao L, Chen C, Zhu Y M. Wild soybean roots depend on specific transcription factors and oxidation reduction related genesin response to alkaline stress. Funct Integr Genomics, 2015, 15: 651–660
[16] Rio D C, Ares M J, Hannon G J, Nilsen T W. Purification of RNA using TRIzol (TRI Reagent). Cold Spring Harbor Protocols, 2010(6): pdb.prot5439
[17] Cline J, Braman J C, Hogrefe H H. PCR fidelity of pfu DNA polymerase and other thermostable DNA polymerases. Nucl Acids Res, 1996, 24: 3546–3551
[18] Willems E, Leyns L, Vandesompele J. Standardization of real-time PCR gene expression data from independent biological replicates. Anal Biochem, 2008, 379: 127–129
[19] Geu-Flores F, Nour-Eldin H H, Nielsen M T, Halkier B A. USER fusion: a rapid and efficient method for simultaneous fusion and cloning of multiple PCR products. Nucl Acids Res, 2007, 35: e55
[20] Nour-Eldin H H, Hansen B G, N?rholm M H, Jensen J K, Halkier B A. Advancing uracil-excision based cloning towards an ideal technique for cloning PCR fragments. Nucl Acids Res, 2006, 34(18): e122
[21] 盛慧, 朱延明, 李杰, 柏锡, 才华. DREB2A基因对苜蓿遗传转化的研究. 草业科学, 2007, 24(3): 40–45
Sheng H, Zhu Y M, Li J, Bai X, Cai H.Genetic transformation of DREB2A gene into alfalfa. Acta Pratac Sin, 2007, 24(3): 40–45
[22] 丁晓东, 吕柳新. 从顽拗植物荔枝中提取基因组DNA技术的研究. 应用与环境生物学报, 2000, 6(2): 142–145
Ding X D, Lyu L X. Study on genomic DNA extraction from recalcitrant litchi. Chinese J Appl Environ Biol, 2000, 6(2): 142–145 (in Chinese with English abstract)
[23] Mishra S, Bansal S, Sangwan R S, Sangwan N S. Genotype independent and efficient Agrobacterium-mediated genetic transformation of the medicinal plant Withania somnifera. J Plant Biochem Biot, 2016, 25:191–198
[24] Hodges D M, Delong J M, Forney C F, Prange R K. Improving the thiobarbituric acid-reactive-substances assay for estimating lipid peroxidation in plant tissues containing anthocyanin and other interfering compounds. Planta, 1999, 207: 604–611
[25] Gibon Y, Larher F. Cycling assay for nicotinamide adenine dinucleotides: NaCl precipitation and ethanol solubilization of the reduced tetrazolium. Anal Biochem, 1997, 251: 153–157
[26] Wellburn A R, Lichtenthaler H. Formulae and program to determinetotal carotenoids and chlorophylls a and b of leaf extracts in different solvents. Adv Photosynth Res, 1984: 9–12
[27] Shimono M, Sugano S J, Nakayama A, Jiang C J, Ono K, Toki S, Takatsuji H. Rice WRKY45 plays a crucial role in benzothiadiazole inducible blast resistance. Plant Cell, 2007, 19: 2064–2076
[28] Marè C, Mazzucotelli E, Crosatti C, Francia E, Stanca A M, Cattivelli L. Hv-WRKY38: a new transcription factor involved in cold and drought response in barley. Plant Mol Biol, 2004, 55: 399–416
[29] 秦伟, 赵光耀, 曲志才, 张立超, 段佳磊, 李爱丽, 贾继增, 孔秀英. 小麦白粉病菌诱导的TaWRKY34基因的鉴定与分析. 作物学报, 2010, 36: 249–255
Qin W, Zhao G Y, Qu Z C, Zhang L C, Duan J L, Li A L, Jia J Z, Kong X Y. Identification and analysis of TaWRKY34 gene induced by wheat powdery mildew (Blumeria graminis f. sp. tritici). Acta Agron Sin, 2010, 36: 249–255 (in Chinese with English abstract)
[30] 王瑞, 吴华玲, 王会芳, 黄珂, 霍春艳, 倪中福, 孙其信. 小麦TaWRKY44基因的克隆、表达分析及功能鉴定. 作物学报, 2013, 39: 1944–1951
Wang R, Wu H L, Wang H F, Huang K, Huo C Y, Ni Z F, Sun Q X. Cloning, characterization, and functional analysis of TaWRKY44 gene from wheat. Acta Agron Sin, 2013, 39: 1944–1951 (in Chinese with English abstract)
[31] 田云, 卢向阳, 彭丽莎, 方俊. 植物WRKY转录因子结构特点及其生物学功能. 遗传, 2006, 28: 1607–1612
Tian Y, Lu X Y, Peng L S, Fang J. The structure and function of plant WRKY transcription factors. Hereditas(Beijing), 2006, 28: 1607–1612 (in Chinese)
[32] 江淑琼, 周守标, 刘坤, 程龙玲. 干旱胁迫对中国石蒜叶片形态和部分生理指标的影响. 北方园艺, 2010, (7):16–19
Jiang S Q, Zhou S B, Liu K, Chen L L. Effects of drought stress on morphology and partial physiological indexes of leaves in lycoris chinensis. Northern Hort, 2010, (7):16–19
[33] Tardieu F, Davies W J. Stomatal response to abscisic acid is a function of current plant water status. Plant Physiol, 1992, 98: 540–545
[34] Sze H, Li X, Palmgren M G. Energization of plant cell membranes by H+-pumping ATPases regulation and biosynthesis. Plant Cell, 1999, 11: 677–690
[35] Davies D D. The fine control of cytosolic pH. Physiol Plant, 1986, 67:702–706

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