扁蓿豆MrLEA2基因的克隆和原核表达
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中国科学院“西部之光”联合学者项目和青海省应用基础研究计划(2014-ZJ-764)


Cloning and Prokaryotic Expression of MrLEA2 from Medicago ruthenica
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    摘要:

    从扁蓿豆(Medicago ruthenica L.)幼苗中克隆到一个编码晚期胚胎发生丰富蛋白的基因MrLEA2,Pfam数据库检索表明其编码产物属于LEA_2蛋白家族。半定量RT-PCR分析发现MrLEA2在幼苗中表达水平不受非生物胁迫(脱水、高盐和低温)和脱落酸诱导。利用MrLEA2基因构建原核表达载体,在大肠杆菌中实现了过量表达。通过斑点试验和菌落计数实验,对过量表达MrLEA2蛋白的大肠杆菌细胞在高盐(0.5 mol/L NaCl和0.5 mol/L KCl)、55 ℃高温和-20 ℃冷冻胁迫处理下的生长存活情况检测发现,MrLEA2蛋白过量表达能够明显提高大肠杆菌对上述胁迫的耐受性。研究表明,MrLEA2蛋白对高盐和温度胁迫引起的细胞损伤具有保护作用。

    Abstract:

    A gene encoding a late embryogenesis abundant(LEA) protein was isolated from seedling of Medicago ruthenica L..Sequence analysis revealed that it belongs to LEA_2 family according to the Pfam classification of LEA proteins,here named as MrLEA2(Medicago ruthenica late embryogenesis abundant protein 2).Semi-quantitative RT-PCR analysis showed that the expression level of MrLEA2 did not response to dehydration,high salinity,cold stress treatments and ABA application.A prokaryotic expression vector was constructed and MrLEA2 was successfully overexpressed in E.coli.In order to get the clues of the function that MrLEA2 plays in protecting cells against abiotic stresses,growth and survival of E.coli cells overexpressing MrLEA2 protein under abiotic stresses,including high salinity,heating and freezing,was appraised.Spot assays and colony counting investigation showed that overexpression of MrLEA2 increased the tolerance of E.coli to high salinity(0.5 mol/L NaCl or 0.5 mol/L KCl),heating(55 ℃) and freezing(-20 ℃) stresses.The results suggest that MrLEA2 plays a role in protection of cell against damage caused by salinity and temperature stress.

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马 超,沈迎芳,吴小培,等.扁蓿豆MrLEA2基因的克隆和原核表达[J].西北植物学报,2014,34(10):1944-1950

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  • 在线发布日期: 2014-10-31
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