摘要： 为进一步研究转录因子TCP4在植物生长发育过程中的调控作用和下游靶基因。利用生物信息学分析转录因子TCP4家族及其蛋白序列，扩增AtTCP4基因CDS序列、构建原核表达载体并诱导纯化获得蛋白，利用凝胶电泳迁移试验（EMSA）验证AtTCP4蛋白与拟南芥功能基因LOX2启动子区域的顺式作用元件结合。生物信息学分析表明，拟南芥TCP蛋白含有非典型的螺旋-环-螺旋（bHLH）结构域；系统进化树表明，AtTCP4属于Ⅱ类TCP蛋白；蛋白三级结构预测表明：转录因子TCP4可以形成同源或异源二聚体。特异性引物扩增获得AtTCP4基因的CDS序列，其开放阅读框（ORF）长为1 263 bp。构建原核表达载体pET-28a-AtTCP4并转化到Rosetta （DE3）感受态中，经IPTG诱导、纯化和Western Blot检测证明获得AtTCP4蛋白。EMSA试验验证AtTCP4蛋白能够结合到功能基因LOX2的顺式作用元件。转录因子TCP4结合功能基因LOX2启动子区域，影响植物激素茉莉酸的生物合成，调控植物的生长发育过程。

关键词: 拟南芥, 转录因子, TCP, 基因克隆, 原核表达, 蛋白纯化

Abstract: To investigate the function of AtTCP4 downstream target genes and in plant growth and development. Bioinformatics analyzed transcription factor TCP4 family and its protein sequence.The prokaryotic expression vector was constructed and the protein was induced and purified. EMSA was done to analyze AtTCP4 protein can bind to the cis-acting elements in the promoter region of LOX2 gene.The bioinformatics analysis showed that the transcription factor TCPs of Arabidopsis thaliana contain a conserved bHLH domain, and the phylogenetic tree showed that AtTCP4 belonged to the class Ⅱ TCP protein. The prediction of protein tertiary structure indicated that TCP4 could form homodimer or heterodimer. Specific primers were used to obtain the CDS sequence of AtTCP4 gene from Arabidopsis thaliana, whose open reading frame (ORF) was 1 263 bp. The prokaryotic expression vector pET-28a-AtTCP4 was constructed and transformed into Rosetta (DE3) competence. After IPTG induction, protein purification and Western Blot detection proved that the AtTCP4 protein was obtained. EMSA experiment verified that the purified AtTCP4 protein could bind to the cis-acting elements of gene LOX2. It affected biosynthesis of plant hormone jasmonic acid and regulated the process of plant growth and development.

Key words: Arabidopsis thaliana, Transcription factor, TCP, Gene cloning, Prokaryotic expression, Protein purification

中图分类号: 

• Q78