沙鞭PvDREB基因克隆与表达特异性分析

Cloning and Expression Characteristics of PvDREB Gene in Psammochloa villosa (Poaceae)

基于沙鞭的三代转录组数据,该研究利用PCR技术克隆DREB基因,并对其进行生物信息学分析;采用实时荧光定量PCR分析该基因的表达模式以及在20%PEG-6000模拟干旱胁迫处理下的表达特征,以探讨干旱胁迫下沙鞭DREB转录因子的功能和作用,为揭示PvDREB基因响应沙鞭的耐旱分子机制奠定基础。结果表明:(1)成功克隆获得一个沙鞭DREB基因,命名为PvDREB;PvDREB基因编码区长度831 bp、编码276个氨基酸,含有典型的AP2转录因子保守结构域;PvDREB蛋白是亲水性蛋白,不具有信号肽结构,存在跨膜结构和可能的糖基化及磷酸化位点。(2)系统进化分析显示,PvDREB基因与毛竹的DREB亲缘关系较近。(3)亚细胞定位预测表明,PvDREB蛋白定位于线粒体和细胞核中。(4)qRT-PCR显示,沙鞭根、茎和叶中PvDREB均可诱导表达但差异较大,且在茎中表达量最高,叶中次之,根中最低,具有明显的组织特异性;20%PEG-6000模拟干旱下,PvDREB基因在叶中的表达量随干旱胁迫时间增加而增加,12 h时达到最高,之后逐渐下降。研究推测,沙鞭PvDREB基因受干旱胁迫诱导表达,且该基因可能在沙鞭响应干旱胁迫的过程中起重要作用。

In the present study, we cloned a DREB gene by PCR based on the third-generation transcriptomic data of Psammochloa villosa and analyzed by bioinformatics. We analyzed the expression pattern of PvDREB gene and its expression characteristics under the drought stress of 20% PEG-6000 through real-time quantitative PCR to explore the function and role of DREB transcription factor under drought stress. It lays a foundation for revealing the molecular mechanism of PvDREB gene of P. villosa response to drought stress. The results showed that:(1) we have successfully cloned a DREB gene named PvDREB. The coding sequence length of PvDREB gene was 831 bp, which encoding 276 amino acids and containing a typical conserved domain of AP2 transcription factor. It was a stable hydrophobic protein without signal peptide structure, and had possible transmembrane domain, glycosylation, and phosphorylation sites.(2) The result of phylogenetic analysis indicated that PvDREB gene of P. villosa had the closer relationship with DREB gene of Phyllostachys edulis.(3) The prediction of subcellular localization showed that PvDREB protein was localized in mitochondria and nucleus.(4) qRT-PCR analysis suggested that PvDREB could be induced and expressed in the roots, stems and leaves of P. villosa, but the difference was large, and the expression level was the highest in stems, followed by leaves, and the lowest in roots, with obvious tissue specificity. Under 20% PEG-6000 simulated drought, the expression of PvDREB gene in leaves increased with the increase of drought stress time, reached the highest at 12 h, and then decreased gradually. It was speculated that the expression of PvDREB gene was induced by drought stress, and this gene may play an important role in the response of P. villosa to drought stress.