扁蓿豆MrERF1的转录激活活性、亚细胞定位及表达分析

Transcriptional activation,subcellular localization,and expression analysis of MrERF1 from Medicago ruthenica

乙烯应答因子(ethylene responsive factor,ERF)广泛存在于植物中,在植物生长、发育和逆境胁迫响应的调节中具有重要作用。本研究根据扁蓿豆(Medicago ruthenica)低温胁迫转录组测序信息,设计特异性引物,利用反转录聚合酶链式反应(reverse transcription-polymerase chain reaction,RT-PCR)技术克隆了一个受低温胁迫诱导表达的ERF转录因子基因MrERF1。序列分析表明,MrERF1基因包含了一个948 bp的开放阅读框,编码由316个氨基酸组成的序列中含有一个高度保守的AP2结构域,具有典型的ERF转录因子特征。遗传进化分析表明其与蒺藜苜蓿(Medicago truncatula)MtABR1蛋白的同源性最高。构建表达MrERF1融合增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)的载体,本氏烟草(Nicotiana benthamiana)叶片下表皮细胞瞬时表达结果证明Mr ERF1蛋白定位于细胞核。结果表明,构建pGBKT7-MrERF1载体转化AH109酵母感受态细胞具有转录激活活性。对MrERF1在不同组织中的表达分析发现,该基因在扁蓿豆的茎、叶、芽和花序中均有表达,但在根中不表达。进一步分析发现,MrERF1基因的转录与光周期和生物节律无关。此外,MrERF1的表达受高盐、干旱、水淹、低温和脱落酸(abscisic acid,ABA)诱导。本研究表明,MrERF1能够响应多种非生物胁迫,为进一步分析MrERF1的功能奠定了基础。

Ethylene responsive factors(ERFs)are widespread in plants and play a critical role in plant growth,development,and stress response.Here,based on RNA-sequence data of Medicago ruthenica under low temperature stress,we designed specific primers and cloned the MrERF1 gene by reverse transcription-polymerase chain reaction(RT-PCR).Sequence analysis showed that the MrERF1 gene contained a 948 bp open reading frame,encoding 316 amino acids.One highly conserved AP2 domain was found in MrERF1,indicating that it is a typical ERF transcription factor.Phylogenetic analysis showed that MrERF1 had the highest homology with MtABR1 in M.truncatula.A 35 S::MrERF1:EGFP vector was constructed.Transient expression analysis in Nicotiana benthamiana demonstrated that MrERF1 is located in the nucleus.A pGBKT7-MrERF1 vector was constructed,and its transcriptional activation activity was verified in AH109 yeast cells.Spatial expression analysis revealed that MrERF1 was expressed in the stems,leaves,buds,and inflorescences,but not in the roots.Further analysis showed that the transcription of MrERF1 was not related to photoperiod or biological rhythm.The expression of MrERF1 was induced under cold,salinity,dehydration,submergence,and abscisic acid(ABA)treatment.The present study demonstrates that MrERF1 can respond to a variety of abiotic stresses,which paves the way for further study of the functions of MrERF1.