棉花单脱氢抗坏血酸还原酶基因的克隆及原核表达

Cloning and Prokaryotic Expression of a Cotton Monodehydroascorbate Reductase Gene

以棉花纤维组织为材料,根据NCBI棉花EST数据库中MDAR基因序列的已知信息,利用5′-RACE获得GhMDAR基因全长。结果显示,该基因开放读码框为1 305bp,编码包含435个氨基酸的蛋白质,分子质量约为52ku。构建原核表达载体pET28a-GhMDAR并转化大肠杆菌,经过测序和酶切鉴定后,成功构建重组体pET28a-GhMDAR;将重组体导入大肠杆菌BL21诱导表达,经过SDS-PAGE分析,显示成功获得分子质量为52ku左右的诱导表达蛋白GhMDAR。

A cotton GhMDAR full-length cDNA was cloned from fiber tissue by searching the sequence information in GenBank database and utilizing 5＇-RACE method. The results showed that GhMDAR gene contains a 1305 bp open reading frame and codes a 52 ku protein of 435 amino acids. Prokaryotic expression vector pET28a-GhMDAR was constructed and confirmed by sequencing and restriction endonuclease digestion. After detection by SDS-PAGE analysis, a 52 ku recombinant protein was expressed through induction in Escherichia coli BL21 （DE3）. These results establish the basis in elucid b ating mechanisms of ascorbic acid involved in cotton fiber cell elongation and in improving cotton fier quality using genetic engineering technique