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为了获得植物在干旱胁迫反应中起关键作用的基因,本研究以大豆耐旱品种和敏感品种为试验材料,利用前期构建的数字基因表达谱,从中筛选出一个在两种材料间存在表达差异的基因,命名为GmbHLH25。采用实时定量PCR对表达谱的结果进行验证,并克隆得到该基因的全长序列。利用ExPASy的ProtParam工具分析发现GmbHLH25蛋白长度为368aa,含有由50aa组成的保守结构域bHLH。进化树分析表明GmbHLH25蛋白与百脉根、苜蓿中的同源蛋白关系最近,处于同一进化分枝。洋葱表皮亚细胞定位结果表明该蛋白在细胞核中表达。PCR和RT-PCR检测表明GmbHLH25基因已经整合到本生烟草的基因组中。在干旱、高盐胁迫下超表达GmbHLH25基因能提高烟草的耐旱、耐盐能力。本研究结果将为进一步分析该基因参与的耐旱信号传导途径,深入研究bHLH转录因子在植物耐逆反应中的分子机理奠定基础。
In order to obtain the genes that play key roles in drought stress response, this study selected soybean drought-tolerant cultivars and susceptible cultivars as experimental materials and used the digital gene expression profiles constructed in the earlier stage to screen out a gene that has differences in expression between the two materials Gene, named GmbHLH25. Real-time quantitative PCR was used to validate the expression profile, and the full-length sequence of the gene was cloned. Analysis of the ProtParam tool using ExPASy revealed that the length of the GmbHLH25 protein is 368 aa and contains the conserved domain bHLH consisting of 50 aa. Phylogenetic tree analysis showed that the protein of GmbHLH25 has the closest relationship with the homologous protein in Lotus japonicus and Medicago sativa and belongs to the same evolutionary branch. Onion epidermal subcellular localization results show that the protein is expressed in the nucleus. PCR and RT-PCR assays showed that the GmbHLH25 gene has been integrated into the genome of Nicotiana tabacum. Overexpression of the GmbHLH25 gene under drought and salt stress increased the drought tolerance and salt tolerance of tobacco. The results of this study will lay the foundation for further analysis of drought tolerance signal transduction pathways involved in this gene and in-depth study of the molecular mechanism of bHLH transcription factor in plant refractory response.