水稻甲基结合蛋白基因MBD701的克隆及原核表达

甲基结合域蛋白MBD作为与甲基化位点特异结合的重要反式作用因子,在植物生长发育中发挥着重要的调控作用.为了探讨MBD基因的结构与功能,利用RT-PCR方法克隆了水稻中编码甲基结合蛋白基因MBD701,构建了MBD701的原核表达载体pGEX-MBD701,并在大肠杆菌BL21(DE3)工程菌株中实现了融合蛋白GST-MBD701的表达.结果表明,MBD701除了包含典型的甲基结合域(第138～212)外,还包含CW的锌指结构(第73～132);在37℃,1 mmol/L IPTG浓度条件下成功诱导表达了大小为65.87 kD的GST-MBD701融合蛋白,这为进一步开展MBD701的蛋白纯化和功能分析奠定了基础.

Cloning of Methyl-binding Domain Protein Gene MBD701 and Its Prokaryotic Expression

MBD is a kind of trans-acting factor which binding with DNA methylation,which plays important regulation roles during the growth and development in plant.In order to study the structure and function,the MBD701 in rice was cloned by using the RT-PCR,the prokaryotic expression vector pGEX-MBD701 was constructed and transformed to E.coli.BL21(DE3).The amino acid sequence analysis showed that MBD701 contained a conserved methyl-CpG binding domain,and CW-zinc finger as well.The fusion protein GST-MBD701 with molecular weight of 65.87 kD was effectively expressed under 1 mmol/L IPTG(Isopropyl-beta-D-thiogalactopyranoside) concentrations at 37 ℃,which provides a foundation for further purification and functional study of the MBD701 protein.