盐生草HgWRKY19和HgWRKY23基因的克隆及表达分析

Cloning and expression analysis of HgWRKY19 and HgWRKY 23 genes in Halogeton glomeratus

【目的】WRKY转录因子在响应植物逆境胁迫过程中发挥重要的生物学功能。克隆盐生草(Halogeton glomeratus)HgWRKY19和HgWRKY23转录因子,分析其亚细胞定位及表达模式,为进一步探究其在盐胁迫响应中的分子机制奠定基础。【方法】基于盐生草全长转录组数据,采用RT-PCR法克隆得到HgWRKY19和HgWRKY23全长序列,通过烟草瞬时转化试验明确其亚细胞定位,并使用qRT-PCR方法分析其在不同盐胁迫时间下(0、6、24和48 h)盐生草叶片和根系中的相对表达量。【结果】成功克隆得到HgWRKY19和HgWRKY23基因,分别编码389和378个氨基酸;其编码蛋白均包含1个典型的WRKYGQK保守结构域和C2H2型(CX5C23HXH)的锌指结构,具碱性、不稳定性、亲水性、无信号肽和跨膜结构等特征;有65个磷酸化位点;二级结构预测均以无规则卷曲占比最大。系统进化分析发现HgWRKY19和HgWRKY23与藜麦、菠菜、甜菜头等藜科草本植物的WRKY蛋白同源性最高。亚细胞定位显示二者主要在细胞核和细胞膜中表达。qRT-PCR分析表明HgWRKY19和HgWRKY23主要在根系表达,具组织特异性,且表达量均在盐胁迫24 h达到峰值。【结论】HgWRKY19和HgWRKY23编码蛋白具典型的WRKY家族结构特征,属于Ⅱd亚类WRKY蛋白,进化高度保守,定位于细胞核和细胞膜上。HgWRKY19和HgWRKY23在根系中受盐胁迫诱导表达,且在不同胁迫时间下表达量差异显著(P<0.05),推测其在盐胁迫响应中发挥重要作用。

【Objective】 TheWRKY transcription factor has an important biological function in response to plant stress.The transcription factors HgWRKY19 and HgWRKY23 of Halogeton glomeratus were cloned,followed by analysis of their subcellular localization and expression patterns to provide a basis for further elucidation of their molecular mechanisms in response to salt stress.【Method】 From the full-length transcriptome data of H.glomeratus,the full-length sequences of HgWRKY19 and HgWRKY23 were cloned by the method of RT-PCR,and their subcellular localization was carried out by the transient transformation test of tobacco leaves.Their relative expression levels in leaves and roots of H.glomeratus were then determined by qRT-PCR under different salt stress times(0,6,24 and 48 h).【Result】 The HgWRKY19 and HgWRKY23 genes,encoding 389 and 378 amino acids,were successfully cloned.Their encoded proteins all contained a typical WRKYGQK conserved domain and a zinc finger structure of the C2H2 type(CX5C23HXH) and were characterized by alkaline,unstable,hydrophilic,no signal peptide and transmembrane structure.There were 65 phosphorylation sites.,and the secondary structure was dominated by random curls.Based on phylogenetic analysis,HgWRKY19 and HgWRKY23 had the highest homology with the WRKY protein of Chenopodium quinoa,Spinacia oleracea,Beta vulgaris subsp.Vulgaris and other herbs of Chenopodiaceae.Subcellularly,HgWRKY19 and HgWRKY23 were mainly expressed in the nucleus and cell membrane.The qRT-PCR results indicated that HgWRKY19 and HgWRKY23 were mainly expressed in the root system with tissue specificity,and the expression levels reached the peak at 24h after salt stress.【Conclusion】 HgWRKY19 and HgWRKY23 have typical structural features of the WRKY family and belong to the Ⅱd subclass of WRKY proteins.They are highly conserved during evolution and are localized in the nucleus and cell membrane.The expressions of HgWRKY19 and HgWRKY23genes were induced by salt stress in roots,and the expression levels of them were significantly different at different stress times,suggesting that they play an important role in salt stress response.