大豆GmbZIP71基因的克隆及EMSA分析

Expression Analysis and EMSA of Soybean GmbZIP71 Gene

通过PCR扩增的方法从大豆品种Harosoy中克隆到1个新的b ZIP基因(Gmb ZIP71),基因表达分析表明Gmb ZIP71在叶片、生长点、花、花芽、荚和根等多个器官中表达。将Gmb ZIP71构建到原核表达载体p ET29b上,导入大肠杆菌BL21(DE3)中,对其进行IPTG诱导。结果表明:在IPTG浓度为0.25 mmol·L-1,诱导时间为4 h,诱导温度为28℃时,重组蛋白得到表达,分子量大约为48 k Da,SDS-PAGE电泳结果表明重组蛋白主要以包涵体形式存在,用His蛋白纯化系统回收得到Gmb ZIP71-His重组蛋白,EMSA(electrophoretic mobility shift assay)实验表明Gmb ZIP71重组蛋白可以在体外与ACGT顺式作用元件结合。

Using PCR amplification method,we cloned a new b ZIP homolog which named Gmb ZIP71 in soybean cultivar Harosoy,RT-PCR showed that Gmb ZIP71 transcripted in multiple organs such as leaves,shoot apices,flowers,flower buds,pods and roots. Gmb ZIP71 was constructed into prokaryotic expression vector p ET29 b and then transformed into E. coli BL21（ DE3） to get the expression protein for further study. The results indicated that an 48 k Da recombinant protein was expressed with the treatment of 0. 25 mmol·L-1IPTG for 4 h at 28℃,the recombinant protein was confirmed to be mainly existed in inclusion body form by SDS-PAGE. The high-quality recombinant protein was obtained through His-tag nickle column purification system and the EMSA experiment indicated that Gmb ZIP71 protein could bind with ACGT cis-element in vitro.