编码棉花胞质铜锌超氧物歧化酶基因的克隆与表达分析

 【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。

Cloning and Expressing of a Gene Encoding Cytosolic Copper/Zinc Superoxide Dismutase in the Upland Cotton

A gene encoding a superoxide dismutase was isolated from senescent leave of cotton(Gossypium hirsutum) cv CRI36 by utilizing rapid amplification of cDNA ends polymerase chain reaction(RACE-PCR),and set of consensus oligonucleotide primers was designed to anneal the conserved sequences of plant Cu/Zn SOD genes. the cDNA ,which designated Cu/Zn-SOD gene,contained 682 bp terminating in a poly(A)+signal. and the opening reading frame (ORF)456bp encodes polypeptide 152 amino acid with the predicted molecular mass of 15.03 Kd and theoretical Pi of6.09.A comparison of the deduced amino sequence with the sequence in the Genbank database has shown considerable sequence similarity to a family of plant Cu/Zn SOD. This putative cotton Cu/Zn SOD shows from 82% to 87% identity to the other plants. All of them share catalytic tetrad of residues, which are highly conserved in four redions. A characteristic feature of Cu/Zn SOD is the inexistence of the signal peptide at the N-terminal region. that suggests the Cu/Zn SOD localizes to the cytoplasm. the Cu/Zn SOD is encoded by nucleolus gene, southern blotting shown that gene had different copies in different cotton cultivars ; only one copy in upland cotton .the expression of the Cu/Zn SOD gene was determined using northern blotting analysis. the Cu/Zn SOD mRNA levels show regular changing in the total development stages：lower in prophase, higher in upper stage and highest at the opening flower stage. the curve of the Cu/Zn SOD mRNA levels is consistent with that of Cu/Zn SOD enzyme activity. the Cu/Zn SOD mRNA levels of different organs shows that the gene are higher in the root, next lesf, lower in the flower.