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  

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

作　　者：曾秀存[1,2] 孙万仓[1] 孙佳 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] 

机构地区：[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

出　　处：《中国农业科学》2013年第21期4603-4611,共9页Scientia Agricultura Sinica

基　　金：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)

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。[ 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 a-helices. Semi-quantitative and real time RT-PCR resultshowed 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.

关 键 词：白菜型油菜 低温 FE-SOD 表达分析 SOD活性 

分 类 号：S565.4[农业科学—作物学]