苦荞FtWRKY28基因克隆及其在低磷与激素诱导下的表达分析

Cloning and expression of the FtWRKY28 gene from Fagopyrum tataricum under low phosphorus and hormone treatment

[目的]WRKY转录因子参与植物对低磷胁迫反应的调控。基于前期苦荞在低磷胁迫下的转录组数据,克隆FtWRKY28基因,预测基因及其编码蛋白的序列结构特征,分析基因在各器官中以及在低磷和激素处理下的表达模式、蛋白的亚细胞定位与转录激活活性,为阐明基因的生物学功能奠定基础。[方法]基于苦荞基因组数据库注释设计特异引物,用逆转录PCR从低磷处理的苦荞根RNA样中克隆FtWRKY28的完整编码序列,用生物信息学方法分析基因与蛋白的结构以及同源蛋白的进化关系,用实时荧光定量分析基因的表达模式,用拟南芥原生质体瞬时表达体系分析蛋白的亚细胞定位,用酵母单杂交分析蛋白的转录激活活性。[结果]FtWRKY28的完整编码序列长876bp,编码1个含291个氨基酸、有1个WRKY结构域、锌指结构域为C2H2型的蛋白,归属WRKY家族Ⅱ组。FtWRKY28绝大部分定位于细胞核,具有转录激活活性。FtWRKY28在根中表达水平最高,受低磷、吲哚乙酸、赤霉素3和6-苄氨基嘌呤显著诱导。[结论]FtWRKY28具有转录因子的基本结构与生物化学特征,可能通过生长素、赤霉素、细胞分裂素信号网络的交互作用,调控苦荞在低磷胁迫下的响应过程。

[Objective]WRKY transcription factors are involved in response to low phosphorus stress in plants.Based on transcriptome data of Tartary buckwheat(Fagopyrum tataricum)under low phosphorus stress,the aim of this study is to isolate FtWRKY28 gene,to predict the structure of the gene and its de-duced protein,to analyze the subcellular localization and transcription-activating activity,and to investigate gene expression in different organs under low phosphorus stress and hormone application,providing a basis for function identification of the gene.[Methods]Primer sequences were designed according to the Tartary buckwheat genome sequence.RT-PCR was used to amplify the CDS of FtWRKY28 from the RNAs generated from Tartary buckwheat roots at low phosphorus.Bioinformatical tools were employed to analyze the structure of the gene and protein and the phylogenetic relationship of the homologous proteins.qRT-PCR was used to investigate gene expression pattern.Transient expression system of the Arabidopsis protoplast was used to analyze the subcellular localization of the protein.Yeast-one-hybrid was employed to analyze the transcription-activating activity of the protein.[Results]The obtained CDS of Ft-WRKY28 was 876 bp in length,encoding a polypeptide of 291 amino acid residues consisting of one conserved WRKY domain with a zinc finger motif of C2H2,belonging to WRKY groupⅡ.FtWRKY28 was predominantly localized in nucleus,which had transcription-activating activity.The transcript abundance of FtWRKY28 was relatively higher in roots,and was induced by low phosphorus and hormones such as indole acetic acid,gibberellin 3,and 6-benzylamino purine.[Conclusion]FtWRKY28 has basic structural and biochemical characteristics of a putative transcription factor,and involves in response to low phosphorus,which may crosstalk with auxin,gibberellin,and cytokinin signaling networks.