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

作　　者：李学宁[1] 杜军伟[2] 李鸿彬[1] 

机构地区：[1]石河子大学生命科学学院/石河子大学农业生物技术重点实验室,石河子832003 [2]石河子大学动物科技学院,石河子832003

出　　处：《石河子大学学报（自然科学版）》2010年第5期542-545,共4页Journal of Shihezi University(Natural Science)

基　　金：国家自然科学基金项目(30860031)

摘　　要：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.

关 键 词：棉花 脱氢抗坏血酸还原酶基因 克隆 原核表达 

分 类 号：S513[农业科学—作物学]