北柴胡转录因子BcbZIP179的基因克隆及原核表达

Gene Cloning and Prokaryotic Expression of Transcription Factor BcbZIP179 from Bupleurum chinense DC

目的克隆北柴胡转录因子基因BcbZIP179的全长编码序列,原核表达融合蛋白。为进一步研究该基因在北柴胡转录调控中的功能提供理论依据。方法以北柴胡转录组数据为基础,设计特异性引物,PCR扩增BcbZIP179基因的全长编码序列,利用生物信息学分析软件分析序列特征。利用qPCR方法分析BcbZIP179基因在不同组织器官中的表达及MeJA处理条件下的表达情况。构建原核表达载体,在不同温度和IPTG浓度下,采用2种菌株诱导表达目的蛋白。结果BcbZIP179编码序列全长432 bp,编码144个氨基酸,BcbZIP179受MeJA诱导,在MeJA处理的北柴胡不定根中,处理前24 h内,表达量逐渐升高,随后表达量降低;BcbZIP179在北柴胡的根、茎、叶、花及果实中均有表达,在果实中的表达量最高;利用大肠杆菌原核表达系统,在16℃和37℃不同温度条件下均获得了带有6×His标签的BcbZIP179融合蛋白。结论克隆到了北柴胡中的1个bZIP家族的转录因子基因BcbZIP179,在16℃、IPTG 0.05 mmol·L^(-1)条件下,可获得体外表达的蛋白,为制备抗体、分离互作蛋白和调控靶基因进而研究其功能奠定基础。

Objective In this work,the encoding sequence cloning of a transcription factor in Bupleurum chinense DC.,BcbZIP179,and the prokaryotic expression of its recombinant protein were presented.It provides a theoretical basis for further study on the transcriptional regulatory function of this gene in B.chinense.Methods Based on the transcriptome data of B.chinense,the specific primers were designed and the encoding sequence of BcbZIP179 was cloned by PCR.The sequence characteristics were assayed with bioinformatics software.The expression profile of BcbZIP179 was assayed by qPCR in different organs and MeJA-treated adventitious roots.The prokaryotic expression vectors for BcbZIP179 were constructed.Two E.coli strains were used to express recombinant protein with different IPTG contents and in different temperatures.Results The sequence is 432 bp in length,encoding 144 amino acids.The expression of BcbZIP179 was induced by MeJA in adventitious roots of B.chinense.The expression of BcbZIP179 increased gradually within 24 hours after treatment and then decreased.The gene was expressed in roots,stems,leaves,flowers and fruits of B.chinense with the highest level in fruits.BcbZIP179 fusion proteins with 6*His label were obtained with induction of IPTG in E.coli at 16℃or 37℃.Conclusion The encoding sequence of a member of bZIP transcription factors,BcbZIP179,was cloned in B.chinense.BcbZIP179 protein can be obtained by the induction of IPTG 0.05 mmol·L^(-1) at16℃after prokaryotic expression vector transformation.This will lay a foundation for the preparation of antibodies,isolation of interacting proteins,regulation of target genes,and further study on their functions.