苦荞麦FtWRKY28基因克隆及其在低磷与激素诱导下的表达分析
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湖南科技大学

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科技部国家重点研发计划( 2017YFE0117600) ; 湖南省教育厅重点科研项目( 19A176)


The cloning and expression analysis of FtWRKY28 gene of Fagopyrum tataricum treated by low phosphorus and hormones
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National Key Research and Development Program of the Ministry of Science and Technology(2017YFE0117600);Key scientific research project of Hunan Provincial Department of Education(19A176)

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    摘要:

    【目的】WRKY转录因子参与植物对低磷胁迫反应的调控。文章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,分析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,采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,采用实时荧光定量PCR(qRT-PCR)分析基因的表达模式,采用拟南芥原生质体瞬时表达体系分析蛋白的亚细胞定位,采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876 bp,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。

    Abstract:

    【Objective】 WRKY transcription factors are involved in regulation of low phosphorus stress in plants. Based on previous 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 deduced protein, to analyze the subcellular localization and transcription activating activity of the protein, and to investigate the gene expression patterns in different organs and under low phosphorus stress and hormone application, thus providing a basis for the function identification of the gene. 【Methods】 Specific primer sequences were designed according to the annotated Tartary buckwheat genome database. Reverse transcription PCR was used to amplify the entire coding sequence (CDS) of FtWRKY28 from the root RNA pools generated from Tartary buckwheat stressed by low phosphorus. Bioinformatical tools were employed to analyze the structures of the gene and protein and the phylogenetic relationships of homologous proteins. Real-time fluorescence-based quantitative PCR (qRT-PCR) was used to investigate the gene expression patterns. Transient expression system of Arabidopsis proplasts 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 FtWRKY28 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, thus belonging to the WRKY group II. FtWRKY28 was localized in nucleus, and had transcription-activating activity. The transcript abundance of FtWRKY28 was relatively higher in roots, and was significantly induced by low phosphorus and hormones such as indole acetic acid, gibberellin 3, and 6-benzylamino purine in roots. 【Conclusion】 Taken together, FtWRKY28 possesses basic structural and biochemical characteristics as a putative transcription factor, and may be involved in low phosphorus response possibly by crosstalk of auxin, gibberellin and cytokinin signaling networks.

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文碧瑶,彭喜旭,田建红,等.苦荞麦FtWRKY28基因克隆及其在低磷与激素诱导下的表达分析[J].西北植物学报,2024,44(6):930-937

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  • 收稿日期:2023-05-15
  • 最后修改日期:2023-12-13
  • 录用日期:2024-01-05
  • 在线发布日期: 2024-04-24
  • 出版日期: 2024-06-06