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

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7号Cu/Zn-SOD的cDNA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。

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

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.