二穗短柄草BdDREB1H基因的克隆和表达分析

Cloning and Expression Analysis of BdDREB1H Gene in Brachypodium distachyon

植物DREB转录因子在应答干旱、低温和高盐等非生物胁迫过程中发挥着重要作用,为了探讨二穗短柄草BdDREB1H转录因子在应答逆境胁迫的作用,对其序列进行了克隆、生物信息学和转录激活分析。然后利用qRT-PCR法对其在根、茎、叶3种组织和冷、干旱、盐和过氧化氢4种非生物胁迫条件下的表达模式进行了研究。结果显示,BdDREB1H转录因子基因全长编码框为735 bp,编码244个氨基酸,相对分子量26.34 ku,理论等电点5.63,不稳定指数(Ⅱ)64.17,为不稳定蛋白质;总亲水平均值-0.261,为亲水蛋白。多序列比对与结构域分析结果表明,BdDREB1H转录因子含有一个典型的AP2结构域,属于AP2/EREBP(APETALA2/ethylene-responsive element binding protein)超家族的DREB亚家族成员。进化分析显示,它与普通小麦的CBF蛋白亲缘关系较近,序列相似性64.44%。酵母单杂分析显示,BdDREB1H转录因子具有转录活性。组织表达分析显示,BdDREB1H基因在根、茎、叶中均有表达,其中在茎中表达量最高。胁迫处理表达分析结果表明,冷处理条件下,BdDREB1H的表达先升高后下降;干旱和氧化胁迫处理条件下,BdDREB1H在2 h后表达水平均显著高于对照;而盐胁迫条件下,BdDREB1H在2 h后表达水平均显著低于对照。这些结果表明,BdDREB1H转录因子可能参与了植物逆境胁迫反应,为进一步探索其在二穗短柄草中的抗逆分子机制奠定了基础。

Plant DREB transcription factor plays an important role in response to abiotic stress such as drought,low temperature and high salinity.In order to investigate the role of DREB in response to stress,we cloned a BdDREB1H gene and analyzed its sequence by bioinformatics and transcriptional activation.Then qRT-PCR was used to study its expression patterns in three tissues of root,stem and leaf and under four abiotic stress conditions of cold,drought,salt and hydrogen peroxide.The results showed that the full-length reading frame of BdDREB1H transcription factor was 735 bp,encoding 244 amino acids,and its relative molecular weight was 26.34 ku.The theoretical isoelectric point was 5.63.The instability index(Ⅱ)was 64.17,indicating it was an unstable protein.The mean total affinity level was-0.261 and was hydrophilic protein.Multiple sequence alignment and domain analysis indicated that BdDREB1H transcription factor contained a typical AP2 domain,which belonged to the DREB subfamily of AP2/EREBP(APETALA2/ethylene responsive element binding protein)superfamily.Evolutionary analysis showed that it was closely related to the CBF of ryegrass,and their sequence similarity was 64.44%.Tissue expression analysis showed that BdDREB1H gene was expressed in all the three tested tissues of root,stem and leaf and had the highest expression level in stem.The expression analysis of stress treatment showed the transcripts of BdDREB1H transcription factor increased firstly and then decreased with time under cold treatment.Under drought and oxidative stresses,the expression level of BdDREB1H was significantly higher than that of the control after 2 hours,while under salt stress,the expression level of BdDREB1H transcription factor was significantly lower than that of control.These results indicated that BdDREB1H transcription factor may be involved in plant stress response,which would lay a foundation for further exploring the molecular mechanism of stress resistance in Brachypodium distachyon.