成功地构建了甘薯谷胱甘肽-S-转移酶基因IBGSTU1 的原核表达质粒pET-IbGST, 并在大肠杆菌 BL21(DE3)中进行IPTG 诱导表达。重组蛋白部分以包涵体形式存在, 部分以可溶性蛋白形式存在。酶活性测定表明可溶的重组蛋白具有GST的活性。纯化的重组蛋白质用于多克隆抗体的制备。半定量RT-PCR 和 Western blotting 分析结果显示, 在正常的生长条件下, 甘薯组织不启动IBGSTU1 基因的转录和翻译, 但是在冷胁迫或重金属离子等的作用下, 可以检测到该基因的mRNA和编码的蛋白质, 表明该基因在甘薯的胁迫耐受中行使重要功能, 并发现该基因的表达具有组织特异性。
A prokaryotic expression plasmid pET-IbGST, which contains the full encoding region of a glutathione-S- transferase (GST) gene of sweet potato, was constructed. The recombinant IBGSTU1 protein was expressed in Escherichia coli and found in the soluble fraction, as well as in insoluble inclusion bodies of lysed cells. Its enzymatic activity was de-tected using UV spectrophotometer. The protein was purified and used to prepare antibody. Semi-quantitative RT-PCR and Western bloting analyses demonstrated that IBGSTU1 gene was not expressed under normal conditions. When subjected to some environmental stresses such as cold-stress or heavy-medal stress, the organism switches on the expression of IBGSTU1 at both mRNA and protein levels, and its expression has tissue specificity.
