陆地棉叶绿体铜锌超氧化物歧化酶基因的克隆与表达(英文)  

Cloning and Expression of the Chloroplast Copper/Zinc-Superoxide Dismutase Gene in Upland Cotton (Gossypium hirsutum L.)

作　　者：胡根海[1] 喻树迅[1] 范术丽[1] 宋美珍[1] 

机构地区：[1]中国农业科学院棉花研究所

出　　处：《植物生理与分子生物学学报》2007年第3期197-204,共8页Journal Of Plant Physiology and Molecular Biology

基　　金：国家"863"计划项目(No.2002AA241021);国家"973"项目(No.2004CB117300)资助~~

摘　　要：以陆地棉‘CRI36’的叶片为材料,使用RACE技术克隆到了棉花叶绿体Cu/Zn-SOD酶基因。基因序列全长共1043bp,含有完整的开放阅读框。推导的氨基酸序列分析显示含有叶绿体信号肽,和已知植物的叶绿体Cu/Zn-SOD酶蛋白的氨基酸残基的同源性在66%～74%之间。基因的表达谱分析显示:棉花叶绿体Cu/Zn-SOD酶基因主要在叶片、茎中表达,根、花和下胚轴中没有检测到信号,即基因的表达主要在棉花的绿色组织。不同生育期的表达谱结果证实:该基因主要在苗期表达,以后表达逐渐减少。用pET-2la(+)构建了原核表达载体,在大肠杆菌BL21(DE3)的表达结果显示:表达后得到一个29.0kD的新蛋白,其分子量与预期目标一致。对SOD酶活性的分析证实,重组菌的酶活性显著增加,证明克隆的基因具有活性。A full-length 1 043-base-pair cDNA clone encoding a chloroplast copper/zinc superoxide dismutase （Cu/Zn-SOD） of upland cotton was first isolated by rapid amplification of cDNA ends （RACE） from the leaves of the cotton （Gossypium hirsutum L.） variety ‘CRI36＇. Nucleotide sequence analysis of the clone revealed that it contained the complete coding sequence of the mature SOD isozyme subunit, along with a 60-amino acid transit peptide at N-terminal. The amino acid sequence predicted from the full-length clone showed 66%-74% homology with the amino acid sequences of Cu/Zn-SOD from several other plants. This gene was found to be expressed in the leaves and stems, but not in roots, flowers, and hypocotyls, indicating that the gene was expressed only in green tissues. Also, its expression was found to be most active at seedling stage and declined gradually in later development stages. Expression of this cotton Cu/Zn-SOD gene by using the pET-21a （＋） expression vector in E. coli BL21 （DE3） led to the production of a novel 29 kD polypeptide with SOD enzyme activity, confn-rning that the cloned cotton Cu/Zn-SOD cDNA was indeed encoding a functioning SOD enzyme.

关 键 词：棉花 铜锌超氧化物歧化酶 CDNA末端快速扩增 基因表达 

分 类 号：S562[农业科学—作物学] Q943.2[生物学—植物学]