棉花脱氢抗坏血酸还原酶基因的克隆、原核表达与纯化

Cloning,Prokaryotic Expression and Purification of a Cotton Dehydroascorbate Reductase Gene

为进一步探讨脱氢抗坏血酸还原酶的生物学功能,克隆棉花的脱氢抗坏血酸还原酶基因,对该基因进行了原核表达,并对重组蛋白进行纯化和分析。通过RT-PCR方法扩增脱氢抗坏血酸还原酶基因全长,利用BamH I和Xhol I酶切位点将其克隆至组氨酸(histidine,His)融合蛋白表达载体pET28a中,转化大肠杆菌BL21(DE3)后,经异丙基硫代--βD-半乳糖苷(isopropy--βD-5-thiogalactoside,IPTG)诱导表达,用SDS-PAGE鉴定表达产物,并用亲和层析柱纯化重组表达的pET28a-DHAR蛋白。结果表明:重组体PET28a-DHAR经测序和酶切鉴定证实构建成功。导入大肠杆菌BL21进行表达,SDS-PAGE分析目的蛋白高效表达,相对分子量为26 kD左右,并获得了纯化的His-DHAR融合蛋白。

A cotton dehydroascorbate reductase gene was cloned,and expression was performed using prokaryotic expression vector.The full-length cDNA of GhDHAR was cloned by RT-PCR,and then was constructed into pET28a vector containing histidine.Recombinant pET28a-DHAR plasmid was obtained after digestion using BamH I and Xhol I restriction endonuclease sites.Recombinant fusion protein was produced after transformation into E.coli BL21（DE3） with subsequent IPTG induction,and was purified utilizing a histrap chromatogram column.SDS-PAGE identification was also used to analysis the recombinant protein expression,and a particular 26 kD protein band visualized successfully.The results establish a basis for further functional research of DHAR gene and molecular mechanism elucidation of ascorbate participating cotton fiber fast elongation.