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作物学报 ›› 2013, Vol. 39 ›› Issue (08): 1400-1408.doi: 10.3724/SP.J.1006.2013.01400

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

白菜型冬油菜抗坏血酸过氧化物酶(APX)基因的克隆、表达及其活性分析

曾秀存1,2,孙万仓1,*,方彦1,刘自刚1,董云3,孙佳4,武军艳1,张鹏飞1,史鹏辉1,孔德晶1,张腾国5,何丽1,赵彩霞1   

  1. 1甘肃农业大学农学院, 甘肃兰州 730070; 2河西学院农业与生物技术学院, 甘肃张掖734000; 3甘肃省农业科学院作物研究所, 甘肃兰州 730070;
    4 Department of Plant Sciences, University of Saskatchewan, Saskatoon, Saskatchewan, Canada. S7N5A8; 5西北师范大学生命科学学院, 甘肃兰州 730070
  • 收稿日期:2012-12-23 修回日期:2013-04-22 出版日期:2013-08-12 网络出版日期:2013-05-22
  • 通讯作者: 孙万仓, E-mail: wangcangsun@yahoo.com.cn
  • 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, Expression, and Activity Analysis of Ascorbate Peroxidase (APX) Gene from Winter Turnip Rape (Brassica campestris L.)

ZENG Xiu-Cun1,2,SUN Wan-Cang1,*,FANG Yan1,LIU Zi-Gang1,DONG Yun3,SUN Jia4,WU Jun-Yan1,ZHANG Peng-Fei1,SHI Peng-Hui1,KONG De-Jing1,ZHANG Teng-Guo5,HE Li1,ZHAO Cai-Xia1   

  1. 1 Agricultural College, Gansu Agricultural University, Lanzhou 730070, China; 2 College of Agronomy and Biotechnology, Hexi University, Zhangye 734000, China; 3 Crop Research Institute, Gansu Academy of Agricultural Sciences, Lanzhou 730070 China; 4 Department of Plant Sciences, University of Saskatchewan, Saskatoon, Saskatchewan, Canada. S7N5A8; 5 College of Life Science, Northwest Normal University, Lanzhou 730070, China
  • Received:2012-12-23 Revised:2013-04-22 Published:2013-08-12 Published online:2013-05-22
  • Contact: 孙万仓, E-mail: wangcangsun@yahoo.com.cn

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摘要：

抗坏血酸过氧化物酶(APX)是一种在逆境条件下清除细胞内氧自由基、增强植物抗逆性的关键酶。本研究根据已发表植物抗坏血酸过氧化物酶基因APX的同源保守序列设计引物, 采用RT-PCR扩增超强抗寒白菜型冬油菜陇油7号的DNA, 获得APX基因开放阅读框, 长度为753 bp, 编码250个氨基酸残基, 推导的氨基酸序列具有APX蛋白的典型特征。生物信息学分析显示, 与已报道的大白菜的APX氨基酸序列同源性达到99%, 该酶蛋白具有高度的进化保守性, 其保守序列属于植物的POD超家族, APX相对分子质量和理论等电点依次为27.7 kD和5.58；APX基因无信号肽, 是一个亲水性蛋白, 可推测其定位于细胞质中；二级结构预测表明陇油7号的APX是由不规则卷曲和α-螺旋组成的稳定蛋白。实时荧光定量PCR和酶活性分析显示, 该基因表达和酶活性受低温胁迫诱导, 表明该基因在白菜型冬油菜陇油7号适应低温胁迫过程中发挥重要作用。

关键词: 白菜型油菜, 低温, APX基因克隆, 表达分析, APX活性

Abstract:

Ascorbate Peroxidase (APX) is a key enzyme which eliminates oxygen free radicals and increases plant resistance in adverse circumstances. The complete open reading frame of APX gene was cloned from an extremely low temperature (–32℃) resistant winter turnip rape (Brassica campestris L.) cultivar Longyou 7 using the method of reverse transcription PCR (RT-PCR). The primers were designed according to the published APX cDNA sequences. The sequence of APX from B. campestris L. was 753 bp, encoding a protein of 250 amino acid residues with a predicted molecular weight of 27.7 kD and a theoretical pI of 5.58. Bioinformatics analysis showed that the predicted APX protein contained a conserved amino acid sequence corresponding to the plant peroxidase (POD) superfamily. The APX had no signal peptide and was a hydrophilic protein locating in cytoplasm. The prediction of the second structures indicated theAPX was a steady protein with more random coils and α-helices. The expression analysis of APX gene using real time RT-PCR and the activity of APX in response to lower temperature showed that the APX could be induced and the activity of APX could be changed by lower temperature. In conclusion, APX gene might play a role in cold tolerance of the B. campestris L. cultivar Longyou 7.

Key words: Winter rape (Brassica compestris L.), Lower temperature, APX gene cloning, Expression analysis, Activity of APX

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