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作物学报 ›› 2013, Vol. 39 ›› Issue (05): 789-805.doi: 10.3724/SP.J.1006.2013.00789

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

甘蓝型油菜MAPK7基因家族及其启动子的克隆与表达分析

朱斌1,2,3,**,陆俊杏1,2,3,**,彭茜1,2,3,翁昌梅1,2,3,王淑文1,2,3,余浩1,2,3,李加纳1,2,3,卢坤1,2,3,*,梁颖1,2,3,*   

  1. 1西南大学农学与生物科技学院,重庆400715;2重庆市油菜工程技术研究中心,重庆400715;3南方山地农业教育部工程研究中心,重庆400715
  • 收稿日期:2012-11-01 修回日期:2013-01-15 出版日期:2013-05-12 网络出版日期:2013-02-19
  • 通讯作者: 梁颖, E-mail: yliang@swu.edu.cn; 卢坤, E-mail: drlukun@swu.edu.cn
  • 基金资助:

    本研究由国家自然基金项目(31271756, 31101175)和高等学校学科创新引智计划(111计划)项目(B12006)资助。

Cloning and Analysis of MAPK7 Gene Family and Their Promoters from Brassica napus

ZHU Bin1,2,3,**,LU Jun-Xing1,2,3,**,PENG Qian1,2,3,WENG Chang-Mei1,2,3,WANG Shu-Wen1,2,3,YU Hao1,2,3,LI Jia-Na1,2,3,LU Kun1,2,3,*,LIANG Ying1,2,3,*   

  1. 1 College of Agronomy and Biotechnology, Southwest University, Chongqing 400715, China; 2 Chongqing Rapeseed Engineering & Technology Research Center, Chongqing 400715, China; 3Engineering Research Center of South Upland Agriculture, Ministry of Education, Chongqing 400715, China
  • Received:2012-11-01 Revised:2013-01-15 Published:2013-05-12 Published online:2013-02-19
  • Contact: 梁颖, E-mail: yliang@swu.edu.cn; 卢坤, E-mail: drlukun@swu.edu.cn

摘要:

甘蓝型油菜中克隆了MAPK7基因家族3个成员BnMAPK7-1BnMAPK7-2BnMAPK7-3的全长cDNA序列,它们最长标准mRNA分别为159314341547 bp。系统进化分析表明,甘蓝型油菜MAPK7基因全部来自白菜,而且BnMAPK7-1/-2的是由白菜Bra03723祖先基因加倍产生的,而BnMAPK7-3则由白菜中Bra037234的祖先基因进化形成的。同时,克隆获得BnMAPK7-1/-2的启动子序列,该启动子里含有多个与光诱导相关的元件、激素响应元件和逆境胁迫响应元件。实时荧光定量PCR (qRT-PCR)显示甘蓝型油菜MAPK7基因家族在所有的组织器官中均表达,并发现该基因家族的表达受植物激素(MeJAABASA)、信号分子(H2O2)逆境(高温)以及伤害(损伤和核盘菌)的诱导,初步证明甘蓝型油菜MAPK7基因在植物逆境胁迫应答中起一定作用。实现了甘蓝型油菜MAPK7-1/-3基因的原核表达,并证明它们编码的蛋白主要以包涵体形式存在,为后续的研究提供了基础。

关键词: 甘蓝型油菜, MAPK7基因, 实时荧光定量PCR, 原核表达

Abstract:

Mitogen-activated protein kinase (MAPK) is a large family of serine/threonine protein kinase, containing 11 conserved subdomains. MAPK pathway play a central role in transferring information from diverse receptors/sensors to a wide range of cellular responses in plants. Signaling through MAP kinase cascade can lead to cellular responses including cell division, development, hormone, physiology, as well as the response to a broad variety of biotic and abiotic stresses. In this study, the full-length cDNA of MAPK7 gene family was isolated from Brassica napus. There were three members in this gene family, including BnMAPK7-1, BnMAPK7-2, and BnMAPK7-3. Standard mRNA lengths of these genes were 1 593, 1 434 and 1 547 bp. Phylogenetic tree showed that BnMAPK7 gene family was all originated from Brassica rapa, and BnMAPK7-1/-2 were generated from ancestral gene of Bra03723, while BnMAPK7-3 was evolved from ancestral gene of Bra03724. We also cloned promoters of BnMAPK7-1/-2, which contain some light responsive elements, hormone responsive elements and stress response elements. Quantitative real-time PCR (qRT-PCR) showed that BnMAPK7 gene family expressed in all organs tested, and could be induced by phytohormones (MeJA, ABA, and SA), signaling molecules (H2O2), stress (heat) and injury (wounding and Sclerotinia sclerotiorum). This study preliminarily proved that the BnMAPK7 gene family plays certain roles in plant stress responses. In addition, the recombinant BnMAPK7-1/-3 proteins were successfully expressed in Escherichia coli, but the main form of proteins was inclusion body, this result would contribute to further discuss in the future.

Key words: Brassica napus, MAPK7 gene, Quantitative real-time PCR, Prokaryotic expression

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