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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

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