木薯EIL基因克隆和蛋白诱导表达及纯化

ETHYLENE INSENSITIVE3-LIKE(EIL)基因家族是乙烯信号通道重要的正调控因子,在乙烯信号传递过程中启动抗病相关基因表达参与植物免疫应答提高植物的抗性。为了研究EIL基因编码蛋白与下游靶基因特异性结合传递乙烯信号,是否参与木薯抗逆境胁迫的调控,本研究从木薯中克隆一个EIL同源基因,命名为MeEIL。分析MeEIL启动子所包含的各种功能元件,结果显示该启动子中含有G-box、W box和MYC等元件。对MeEIL基因的ORF分析结果表明该基因编码含621个氨基酸残基的蛋白质。使用同源重组的方法将MeEIL的CDS序列连接到蛋白表达载体pET32a上。SDS-PAGE检测表明在28℃和16℃都可成功诱导蛋白表达,且28℃诱导效果更佳,通过大量诱导蛋白表达并用His标签纯化试剂盒纯化,Western blot分析验证MeEIL蛋白被成功的诱导出来。

Cloning,Protein Induction Expression and Purification of Cassava EIL Gene

ETHYLENE INSENSITIVE3-LIKE(EIL)gene family is an important positive regulator of ethylene signaling pathway.During ethylene signaling,the expression of disease-resistant genes is activated to participate in plant immune response and improve plant resistance.In order to study whether the specific binding of EIL gene coding protein and downstream target genes transmits ethylene signal,which may be involved in the regulation of cassava against stress,this study cloned an EIL homologous gene,named MeEIL.Throught analysised the various functional elements containing in the MeEIL promoter,we found the promoter contained G-box,W-box and MYC elements.The ORF analysis of MeEIL gene showed that it encodes a protein containing 621 amino acid residues.The CDS sequence of MeEIL was linked to the expression vector pET32 a by homologous recombination.SDS-PAGE test showed that protein expression could be successfully induced at 28℃and 16℃,and the induction effect was better at 28℃.Through a large amount of induced protein expression and purification kit of His label,Western blot analysis verified that MeEIL protein was successfully induced.