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作物学报 ›› 2013, Vol. 39 ›› Issue (03): 440-448.doi: 10.3724/SP.J.1006.2013.00440

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

分离和鉴定油菜D型MAP激酶基因BnMPK9

余舜武1,张利达2,刘国兰1,罗利军1,*   

  1. 1 上海市农业生物基因中心，上海 201106; 2 上海交通大学农业与生物学院，上海 200240
  • 收稿日期:2012-07-26 修回日期:2012-11-16 出版日期:2013-03-12 网络出版日期:2012-12-11
  • 通讯作者: 罗利军, E-mail: lijun@sagc.org.cn, Tel: 021-52230526
  • 基金资助:

    本研究由上海市自然科学基金项目(12ZR1427700), 国家科技重大专项课题项目(2011ZX08001-003)和上海市启明星计划(11QA1405900)资助。

Isolation and Characterization of a Rape MAP Kinase BnMPK9 from Group D

YU Shun-Wu1,ZHANG Li-Da2,LIU Guo-Lan1,LUO Li-Jun1,*   

  1. 1 Shanghai Agrobiological Gene Center, Shanghai Academy of Agricultural Sciences, Shanghai 201106, China; 2 Plant Biotechnology Research Center, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 200240, China
  • Received:2012-07-26 Revised:2012-11-16 Published:2013-03-12 Published online:2012-12-11
  • Contact: 罗利军, E-mail: lijun@sagc.org.cn, Tel: 021-52230526

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摘要：

为了应对不良的外界环境，植物形成了一整套复杂的信号网络体系以适应变化的外界环境。其中MAPK级联是其中一个重要而保守的信号传导系统。采用RT-PCR策略，从干旱处理的油菜cDNA中克隆出全长的细胞分裂原激活的蛋白激酶基因。该基因命名为BnMPK9(GenBank登录号为AY737714)，包含一个蛋白激酶区和一个保守的CD区。该基因与拟南芥AtMPK9高度同源，其激酶的磷酸化位点为TDY，同为D型MAPK亚家族基因。Southern杂交结果表明该基因在油菜基因组中拷贝数不少于2个。Northern杂交结果显示该基因能在不同的植物组织中表达。其表达受到甘露醇、紫外线和双氧水诱导上调表达，但低温和水杨酸处理后下调表达。实时RT-PCR分析表明甘露醇和双氧水能长时间诱导该基因高表达。在根中，甘露醇也能促进其上调表达。BnMPK9连接到pYES2.1酵母表达载体中转化酵母，发现增强了酵母对600 mmol L–1甘露醇和0.2 mmol L-1 tBuOOH的抗性。以上结果说明BnMPK9是MAPK基因家族中的一员，涉及真核生物细胞渗透和活性氧胁迫，并增强其抗性。

关键词: BnMPK9, 油菜, 非生物胁迫, 酵母表达

Abstract:

Under environmental stresses, plant has developed complex signaling networks to respond environmental signals and adapt to unfavorable conditions. Mitogen-activated protein kinase (MAPK) signaling pathways are one of the most important and conserved system to control cellular response and growth. A gene encoding Mitogen-activated protein kinase was isolated from oilseed rape (Brassica napus) treated with drought based on RT-PCR, designated as BnMPK9 (GenBank accession number AY737714). The cloned gene contains a Pkinase domain and a conserved CD domain. BnMPK9 is highly homologous to AtMPK9 gene from Arabidopsis thaliana and belongs to the D-Subgroup of mitogen-activated protein kinase with phosphorylation motif (TDY). Southern bolt analysis revealed that there were at least two copies in rape genome. Northern bolt analyses showed that BnMPK9 could express in different tissues. The expression level of BnMPK9 transcript was up-regulated by mannitol, UV, and H2O2,but down-regulated by low temperature and salicylic acid (SA). Through Real-time RT-PCR, mannitol and H2O2 treatments could induce its high expression under different time. In root, mannitol treatment could enhance its expression. BnMPK9 was ligated into pYES2.1 and transformed into yeasts, and BnMPK9’s expression enhanced yeasts’ resistance to mannitol and peroxide in 600 m mol L–1 mannitol and 0.2 mmol L–1 tBuOOH drug assays. The present studies show that BnMPK9 is a member of MAPK family involved in eukaryotic responses and resistance to osmotic and ROS stresses.

Key words: BnMPK9, Brassica napus, Abiotic stress, Yeast expression

[1]Ichimura K, Tena G, Henry Y, Zhang Z, Hirt H, Wilson C, Morris P, Mundy J, Innes R, Ecker J, Scheel D, Klessig D F, Machida Y, Mundy J, Ohashi J, Walker J C. Mitogen-activated protein kinase cascades in plants: a new nomenclature. Trends Plant Sci, 2002, 7: 301–308



[2]Jonak C, Ökrész L, Bögre L, Hirt H. Complexity, cross talk and integration of plant MAP kinase signaling. Curr Opinion Plant Biol, 2002, 5: 415–424



[3]Yu S W, Tang K X. MAP Kinase cascades responding to environmental stress in plants. Acta Bot Sin, 2004, 46: 127–136



[4]Zhang S, Klessig D F. MAPK cascades in plant defense signaling. Trends Plant Sci, 2001, 6: 520–527



[5]Asai T, Tena G, Plotnikova J, Willmann M R, Chiu W L, Gomez-Gomez L, Boller T, Ausubel F M, Sheen J. MAP kinase signaling cascade in Arabidopsis innate immunity. Nature, 2002, 415: 977–983



[6]Moustafa K, Vos D, Leprince A, Savouré A, Laurière C. Analysis of the Arabidopsis mitogen-activated protein kinase families: organ specificity and transcriptional regulation upon water stresses. Scholarly Res Exchange, 2008, doi:10.3814/2008/143656



[7]Lian W, Tang Y, Gao S, Zhang Z, Zhao X, Zhao P. Phylogenetic analysis and expression patterns of the MAPK gene family in wheat (Triticum aestivum L.). J Integr Agric, 2012, 11: 1227–1235



[8]Jammes F, Song C, Shin D, Munemasa S, Takeda K, Gu D, Cho D, Lee S, Giordo R, Sritubtim S, Leonhardt N, Ellis B E, Murata Y, Kwak J M. MAP kinases MPK9 and MPK12 are preferentially expressed in guard cells and positively regulate ROS-mediated ABA signaling. Proc Natl Acad Sci USA, 2009, 106: 20520–20525



[9]Cheong Y H, Moon B C, Kim J K, Kim C Y, Kim M C, Kim I H, Park C Y, Kim J C, Park B O, Koo S C, Yoon H W, Chung W S, Lim C O, Lee S Y, Cho M J. BWMK1, a rice mitogen-activated protein kinase, locates in the nucleus and mediates pathogenesis-related gene expression by activation of a transcription factor. Plant Physiol, 2003, 132: 1961–1972



[10]Ge T-M(葛台明), Yu Y-J(余毓君). Research of nutrient demand about wheat regeneration during tissue culture. J Huazhong Agric Univ (华中农业大学学报), 1994, 13(4): 344–348 (in Chinese with English abstract)



[11]Jiang C-J(江昌俊), Chen Y(陈彦). A method of isolating genomic DNA from Brassica. Chin J Oil Crop Sci (中国油料作物学报), 1995, 17(4): 34–36 (in Chinese with English abstract)



[12]Dóczi R, Okrész L, Romero A E, Paccanaro A, Bögre L. Exploring the evolutionary path of plant MAPK networks. Trends Plant Sci, 2012, http://dx.doi.org/10.1016/j.tplants.2012.05.009



[13]Jammes F, Yang X, Xiao S, Kwak J M. Two Arabidopsis guard cell-preferential MAPK genes, MPK9 and MPK12, function in biotic stress response. Plant Signal Behav, 2011, 6: 1875–1877



[14]Singh R, Lee MO, Lee J E, Choi J H, Park J H, Yoo R H, Cho J I, Jeon J S, Rakwal R, Agrawal G K, Moon J S, Jwa N S. Rice MAPK interactome analysis using yeast two-hybrid system. Plant Physiol, 2012, DOI: 10.1104/pp.112.200071



[15]O’Rourke S M, Herskowitz I, O’Shea E K. Yeast go the whole HOG for the hyperosmotic response. Trends Genet, 2002, 18: 405–412



[16]Babiychuk E, Kushnir S, Belles-Boix E, Van Montagu M, Inzé D. Arabidopsis thaliana NADPH oxidoreductase homologs confer tolerance of yeasts toward the thiol-oxidizing drug diamide. J Biol Chem, 1995, 270: 26224–26231



[17]Belles-Boix E, Babiychuk E, Van Montagu M, Inzé D, Kushnir S. CEO1, a new protein from Arabidopsis thaliana, protects yeast against oxidative damage. FEBS Lett, 2000, 482: 19–24



[18]Quintero F J, Garciadeblás B, Rodríguez-Navarro A. The SAL1 gene of Arabidopsis, encoding an enzyme with 3'(2'), 5'-bisphosphate nucleotidase and inositol polyphosphate 1-phosphatase activities, increases salt tolerance in yeast. Plant Cell, 1996, 8: 529–537



[19]Piao H L, Pih K T, Lim J H, Kang S G, Jin J B, Kim S H, Hwang I. An Arabidopsis GSK3/shaggy-like gene that complements yeast salt stress-sensitive mutants is induced by NaCl and abscisic acid. Plant Physiol, 1999, 119: 1527–1534



[20]Yu S, Zhang L, Zuo K, Tang D, Tang K. Isolation and characterization of an oilseed rape MAP kinase BnMPK3 involved in diverse environmental stresses. Plant Sci, 2005, 169: 413–421
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