中国农业科学 ›› 2013, Vol. 46 ›› Issue (21): 4603-4611.doi: 10.3864/j.issn.0578-1752.2013.21.024

• 研究简报 • 上一篇    下一篇

白菜型冬油菜铁超氧化物歧化酶(Fe-SOD) 基因的克隆及表达分析

 曾秀存12, 孙万仓1, 孙佳3, 许耀照2, 方彦1, 史鹏辉1, 杨刚1, 孔德晶1, 武军艳1, 刘自刚1   

  1. 1.甘肃农业大学农学院,中国兰州 730070
    2.河西学院农业与生物技术学院,中国甘肃张掖734000
    3.Department of Plant Sciences,University of Saskatchewan,Saskatoon,Saskatchewan,Canada S7N5A8
  • 收稿日期:2013-06-18 出版日期:2013-11-01 发布日期:2013-08-14
  • 通讯作者: 孙万仓,Tel:18293121851;E-mail:18293121851@163.com
  • 作者简介:曾秀存,Tel:0936-8362180;E-mail:xiucunzeng@126.com
  • 基金资助:

    国家“863”高技术研究发展计划(2011AA10A104)、国家公益性行业(农业)科研专项(200903002-04)、现代农业产业技术体系建设专项(CARS-13)、河西学院校长科研基金项目计划(XZ2011-02)

Cloning and Expression Analysis of Fe Superoxide Dismutase (Fe-SOD) Gene from Winter Turnip Rape (Brassica rapa L.)

 ZENG  Xiu-Cun-1, 2 , SUN  Wan-Cang-1, SUN  Jia-3, XU  Yao-Zhao-2, FANG  Yan-1, SHI  Peng-Hui-1, YANG  Gang-1, KONG  De-Jing-1, WU  Jun-Yan-1, LIU  Zi-Gang-1   

  1. 1.Agricultural College, Gansu Agricultural University, Lanzhou 730070, China
    2.College of Agronomy and Biotechnology, Hexi University, Zhangye 734000, Gansu, China
    3.Department of Plant Sciences, University of Saskatchewan, Saskatoon, Saskatchewan,  Canada S7N5A8
  • Received:2013-06-18 Online:2013-11-01 Published:2013-08-14

摘要: 【目的】了解SOD酶蛋白家族Fe-SOD在超强抗寒冬油菜中的表达情况及其在低温胁迫下的作用。【方法】采用RT-PCR技术克隆超强抗寒白菜型冬油菜陇油7号Fe-SOD的cDNA序列。对该序列进行生物信息学分析,采用半定量以及相对定量RT-PCR研究Fe-SOD在低温胁迫下的表达模式,采用氮蓝四唑(NBT)光还原法测定冬油菜叶片和根中的总SOD酶活性。【结果】获得Fe-SOD,GenBank登录号为KF178713,该基因cDNA片段全长645 bp,包含一个639 bp完整的开放阅读框,编码212个氨基酸的蛋白质,与白菜(Chiifu)的蛋白质氨基酸序列同源性高达99%。该基因编码的酶蛋白是1个主要由α-螺旋组成的亲水性稳定蛋白,无信号肽,无跨膜结构域。Fe-SOD表达模式分析显示初期低温胁迫下(4℃),该基因上调表达,继续低温胁迫处理(-4℃和-8℃),Fe-SOD表达量受到抑制。总SOD酶活性测定显示冬油菜根中酶活性高于叶片,以利于冬油菜安全越冬。【结论】从白菜型冬油菜克隆的Fe-SOD具有已知物种Fe-SOD的特征。Fe-SOD在冬油菜品种陇油7号抗寒过程中起作用。

关键词: 白菜型油菜 , 低温 , Fe-SOD , 表达分析 , SOD活性

Abstract: 【Objective】The objectives of the present study were to clone the Fe-SOD gene of SOD family from an extremely low temperature (-32℃) resistant winter turnip rape (B. rapa L.) cultivar Longyou 7 and analyze its expression under low temperature conditions .【Method】The cDNA sequence of Fe-SOD was isolated by RT-PCR, and the obtained cDNA sequence and the deduced amino acid sequence was analyzed. Semi-quantitative and real time RT-PCR were used to assess the expression of Fe-SOD in response to low temperature stress. The superoxide dismutase enzyme activity was measured by NBT deoxidization method in leaves and roots. 【Result】The Fe-SOD gene was isolated from winter turnip rape (GenBank accession number. KF178713). The cDNA sequence of this gene was 645 bp in length, containing a 639 bp opening reading frame (ORF) , encoded a polypeptide of 212 amino acid,and with 99 % of amino sequence similarity to a vegetable cultivar of B. rapa (Chiifu). The protein encoded by this gene was a hydrophilic protein without signal-peptide and transmembrane region. The prediction of the second structures indicated that the Fe-SOD was a steady protein with more α-helices. Semi-quantitative and real time RT-PCR result showed that Fe-SOD was expressed upregulatedly in response to early low temperature stress (4℃). However, the expression of this gene was inhibited at the lower temperature stress (-4℃ and -8℃). The result of measured enzyme activity showed that superoxide dismutase enzyme activity in roots was higher than that of leaves,which could make winter turnip rape successfully overwinter. 【Conclusion】The Fe-SOD gene cloned from winter turnip rape had genetic characteristics similar with other known species and it might play a role in cold tolerance of the B. rapa cultivar Longyou 7.

Key words: winter rape (Brassica rapa L.) , lower temperature , Fe-SOD , expression analysis , activity of SOD