扁蓿豆SK2型脱水素基因MrDHN3的异源表达提高大肠杆菌对盐和高温胁迫的抗性

Heterologous expression of an SK2-type dehydrin gene(MrDHN3)from Medicago ruthenica enhances Escherichia coli tolerance under salt and high temperature stress

扁蓿豆为高原高寒地区优质豆科牧草,具有极强的抗旱、耐寒、抗盐碱的能力。脱水素(DHNs)是参与植物逆境应答的一类蛋白。根据前期RNA-seq的结果,从扁蓿豆幼苗中克隆到一个编码脱水素的基因MrDHN3。序列分析显示该基因含666bp的开放阅读框,编码221个氨基酸,为一个SK2型酸性脱水蛋白。氨基酸序列比对结果表明,MrDHN3与豆科植物白三叶和蒺藜苜蓿相似性最高,达83%。实时荧光定量PCR结果显示,MrDHN3基因受脱水、低温、高盐和脱落酸处理诱导表达,表明MrDHN3参与了扁蓿豆的非生物胁迫响应。通过构建原核表达载体,在大肠杆菌中过表达MrDHN3蛋白,检测重组菌在盐和高温胁迫处理下的生长存活情况。结果发现,在0.5mol/L NaCl和0.5mol/L KCl高盐胁迫条件下,重组大肠杆菌的存活率明显高于对照菌株;在55℃高温胁迫条件下,转化大肠杆菌的生长状态明显优于对照。表明MrDHN3对盐和高温引起的细胞损伤具有保护作用。为今后作物抗逆性遗传改良的研究提供了有用信息。

Medicago ruthenica ,is an excellent legume in highland and cold regions,and is highly resistant to drought,cold and high salinity.Dehydrins （DHNs）are stress proteins involved in plant protective reactions a-gainst environmental stress.According to our previous RNA-sequence data,a DHN gene,MrDHN3,was cloned from young seedlings of M.ruthenica.Sequence analyses showed that the MrDHN3 gene contained a 666 bp open reading frame,putatively translated to 221 amino acids,and was an SK2-type acidic DHN.Amino acid sequence alignment showed that MrDHN3 shared the highest similarity （83%）with TrDHN3 and Mt-DHN3.Quantitative RT-PCR analysis showed that the expression of MrDHN3 was induced by dehydration, cold,high salinity stress and abscisic acid （ABA）,which suggests that MrDHN3 is involved in abiotic stress responses.A prokaryotic expression vector was constructed and transferred to Escherichia coli so as to induce MrDHN3 over expression in E .coli.The survival and growth of the recombinant E .coli under salinity and high temperature stress conditions were determined.It was found that survival rates of recombinant E .coli af-ter exposure to high salinity （0.5 mol/L NaCl,0.5 mol/L KCl）and high temperature （55 ℃）stress were ob-viously higher than those of the control group.This suggests that MrDHN3 plays an important role in cell re-sponse to damage caused by salinity and high temperature stress.This research indicates a potential methodolo-gy for the genetic improvement of crops to improve stress tolerance.