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作物学报 ›› 2014, Vol. 40 ›› Issue (04): 636-643.doi: 10.3724/SP.J.1006.2014.00636

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

白菜型冬油菜铜锌超氧化物歧化酶(Cu/Zn-SOD)基因的克隆及其在低温条件下的表达分析

曾秀存1,2,刘自刚2,史鹏辉2,许耀照1,孙佳3,方彦4,杨刚2,武军艳2,孔德晶2,孙万仓2,*   

  1. 1河西学院农业与生物技术学院, 甘肃张掖734000; 2甘肃农业大学农学院/甘肃省油菜工程技术研究中心, 甘肃兰州 730070; 3 Department of Plant Sciences, University of Saskatchewan, Saskatoon, Saskatchewan S7N5A8, Canada; 4甘肃农业大学研究测试中心, 甘肃兰州 730070
  • 收稿日期:2013-07-07 修回日期:2013-11-24 出版日期:2014-04-12 网络出版日期:2014-01-16
  • 通讯作者: 孙万仓, E-mail: 18293121851@163.com
  • 基金资助:

    本研究由国家高技术研究发展计划(863计划)项目(2011AA10A104), 国家公益性行业(农业)科研专项经费项目(200903002-04)和国家现代农业产业技术体系建设专项(CARS-13)资助。

Cloning and Expression Analysis of Copper and Zinc Superoxide Dismutase (Cu/Zn-SOD) Gene from Brassica campestris L.

ZENG Xiu-Cun1,2,LIU Zi-Gang2,SHI Peng-Hui2,XU Yao-Zhao1,SUN Jia3,FANG Yan4,YANG Gang2,WU Jun-Yan2,KONG De-Jing2,SUN Wan-Cang2,*   

  1. 1 College of Agronomy and Biotechnology, Hexi University, Zhangye 734000, China; 2 The College of Agronomy Gansu Agricultural University / Gansu Engineering Research Center of Rapeseed, Lanzhou 730070, China; 3 Department of Plant Sciences, University of Saskatchewan, Saskatoon, Saskatchewan S7N5A8, Canada; 4 Research and Testing Center of Gansu Agricultural University, Lanzhou 730070, China
  • Received:2013-07-07 Revised:2013-11-24 Published:2014-04-12 Published online:2014-01-16
  • Contact: 孙万仓, E-mail: 18293121851@163.com

摘要:

超氧化物歧化酶(SOD)是一种逆境条件下清除细胞内活性氧的关键酶,而铜锌超氧化物歧化酶(Cu/Zn-SOD)是该酶系中最主要的活性氧清除剂,与植物的抗逆性关系密切。本研究根据已发表的十字花科植物Cu/Zn-SOD基因的编码区设计引物,采用RT-PCR克隆超强抗寒冬油菜陇油7Cu/Zn-SODcDNA序列,该基因全长459 bp。生物信息学分析表明,该基因与甘蓝型油菜(Brassica napus) Cu/Zn-SOD基因同源性高达99%编码1个亲水性稳定蛋白,无跨膜结构域和信号肽,具有胞质Cu/Zn-SOD超基因家族特有的序列特征和保守结构区域。半定量RT-PCR以及SOD酶活性分析表明,低温诱导条件下Cu/Zn-SOD基因差异表达,在白菜型冬油菜适应低温胁迫过程中发挥重要作用。超强抗寒冬油菜陇油6号品种低温诱导蛋白的SDS-PAGE分析以及蛋白串联质谱技术鉴定表明Cu/Zn-SOD是受低温诱导表达的抗逆基因。

关键词: 白菜型油菜, Cu/Zn-SOD基因克隆, 低温, 表达分析, SOD活性, 低温诱导蛋白

Abstract:

Superoxide Dismutase (SOD) is a key enzyme eliminating reactive oxygen species (ROS), and the copper and zinc superoxide dismutase (Cu/Zn-SOD) is the most important active oxygen scavenger in SOD family, which can improve plant tolerance to environmental stresses. The cDNA of Brassica campestris L. cultivar Longyou 7 was cloned by RT-PCR, using the primers designed according to the published crucifer Cu/Zn-SOD cDNA sequences. The sequence of Cu/Zn-SOD from B. campestris L. was 459 bp, encoding a predicted protein of 152 amino acid residues. Bioinformatics analysis showed that amino sequence similarity with Brassica napus was 99%, and the predicted Cu/Zn-SOD protein was a hydrophilic protein without signal-peptide as well as transmembrane region. It contained specific sequence characteristics and conserved domain of copper and zinc superoxide dismutase (Cu/Zn-SOD) superfamily. The expression analysis of Cu/Zn-SOD gene using semi-quantitative RT-PCR and results of SOD activity in response to lower temperature showed that the Cu/Zn-SOD was a differential expressed gene induced by lower temperature. Results of separating low-temperature-induced proteins from Brassica campestris L. cultivar Longyou 6 by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry further showed that Cu/Zn-SOD was a stress-responsed gene, whose expression was induced by lower temperature.

Key words: Winter rape (Brassica compestris L.), Cu/Zn-SOD gene cloning, Lower temperature, Expression analysis, Activity of SOD, Low-temperature-induced protein

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