中国农业科学 2007,40(8):1602-1609 Scientia Agricultura Sinica
编码棉花胞质铜锌超氧物歧化酶基因的克隆与表达分析
胡根海
1,2
,喻树迅 ,范术丽 ,宋美珍
1
1
1
(1 中国农业科学院棉花研究所,安阳 455004;2 河南科技学院,新乡 453002)
摘要: 【目的】克隆编码棉花胞质铜锌超氧化物岐化酶基因并分析其表达特性. 【方法】采用 RACE 技术克隆 基因,Northern blotting 检测基因的表达谱;采用氮蓝四唑(NBT)光下还原法测定不同生育期的酶活性. 【结 果】获得了棉花胞质铜锌超氧化物岐化酶基因 cDNA 全长序列(GenBank 注册号:DQ445093) ;该基因 cDNA 全长 共 682 bp,开放阅读框 456 bp,编码 152 个氨基酸.分子结构预测结果:酶蛋白理论分子量约为 15.03 kD,理 论等电点为 6.09,与其它植物的蛋白质氨基酸序列同源性在 82%～87%之间.Southern blotting 显示不同棉种该 基因的拷贝数基本一致,均属于低拷贝基因.Northern blotting 显示该基因在不同的组织,不同的生育期表达 量不同;酶活性测定显示盛花期最高. 【结论】棉花胞质铜锌超氧化物岐化酶基因在陆地棉中属于低拷贝数基因; 在整个生育期中 mRNA 的含量呈规律性动态变化,前期较低,后期较高,在盛花期达到顶峰;变化曲线与不同时期 的酶活性变化一致;不同器官的基因表达检测结果显示:基因在根中表达量最高,叶片次之,花中的表达最低. 关键词:棉花;铜锌超氧物歧化酶;基因;克隆
Cloning and Expressing of a Gene Encoding Cytosolic Copper/Zinc Superoxide Dismutase in the Upland Cotton
HU Gen-hai1,2, YU Shu-xun1, FAN Shu-li1, SONG Mei-zhen1
(1Cotton research institute, Chinese Academy of Agricultural Sciences, Anyang 455004;
2
Henan Institute of Science and Technology, Xinxiang 453002)
Abstract: 【Objective】A gene encoding superoxide dismutase (SOD) was cloned from senescent leave of cotton (Gossypium hirsutum), and its expressing profile was analyzed. 【Method】 gene was cloned by Rapid Amplification of cDNA Ends (RACE) The method. Northern blotting was used to show the profile of the gene expression, and the enzyme activity was mensurated by NBT deoxidization method in different growth periods. 【Result】 full length of the gene of cytosolic copper/zinc superoxide dismutase The Cu/Zu-SODwas isolated from cotton (GenBank accession number.DQ445093). The sequence of cDNA contained 682 bp, the opening reading frame 456 bp, and encoded polypeptide 152 amino acids. The molecular construction was predicted including molecular mass of 15.03 kD and theoretical PI of 6.09, and amino sequence similarity with the other plants of 82% to 87%. Southern blotting showed that the gene had different number of copies in different cotton species. Northern blotting suggested that the gene had different expression in different tissues and development stages. The enzyme activity was the highest in peak flowering stage. 【Conclusion】The cotton cytosolic copper/zinc superoxide dismutase had lower copies in the upland cotton. The copper/zinc superoxide dismutase mRNA expressing level showed regular changing in the whole development stages: lower in the former stages, higher in latter stages and highest at the peak flowering stage. The curve of the copper/zinc superoxide dismutase mRNA expressing level was consistent with that of the copper/zinc superoxide dismutase enzyme activity. The copper/zinc superoxide dismutase mRNA expressing levels of different organs showed that the gene expression was high in root, medium in leaf, low in flower. Key words: Upland cotton; Copper/zinc superoxide dismutase; Gene; Cloning

下一页