蒺藜苜蓿MtLEA5B的克隆和功能分析

Cloning and Functional Analysis of the MtLEA5B Gene from Medicago truncatula

胚胎发育晚期丰富蛋白(Late embryogenesis abundant protein,LEA protein)是一类参与植物抗逆性应答的亲水蛋白。旨在揭示异源表达LEA蛋白在非生物胁迫条件下对宿主的保护作用。从蒺藜苜蓿(Medicago truncatula)幼苗中克隆到一个编码LEA_5蛋白的基因,命名为MtLEA5B,通过半定量RT-PCR分析MtLEA5B的表达模式。利用SDS-PAGE蛋白电泳检测重组蛋白的耐热性和可溶性。构建原核表达载体,转化大肠杆菌过表达,检测大肠杆菌的生长存活情况。MtLEA5B的表达模式受到低温(4℃)、脱水、150 mmol/L NaCl胁迫和ABA处理的调控。SDS-PAGE蛋白电泳出现一条相比理论预测值较大,且沸水条件下可溶性未降低的条带。在大肠杆菌中过表达MtLEA5B蛋白能明显提升宿主菌在高温(55℃)和冷冻(-20℃)条件下的生存率。MtLEA5B是诱导性表达基因;MtLEA5B蛋白具有较高的亲水性和耐热性。在大肠杆菌中过表达可以明显提升其对温度胁迫的耐受性。

Late embryogenesis abundant protein（LEA protein）is hydrophilic one that is involved in the responses to stress in plants.This work is aimed to unravel the protective effect of heterologous expressed LEA protein on host under abiotic stress. A novel gene encodingLEA_5 protein was cloned from Medicago truncatula seedlings,and designated as MtLES5B,and its expression patterns were analyzed bysemi-quantitative RT-PCR. Then the heat resistance and solubility of recombinant protein were detected by SDS-PAGE protein electrophoresis.Further,the prokaryotic expression vector was constructed and transferred to Escherichia coli so as to induce MtLEA5B over expression in E. coli. The expression patterns of Mt LEA5 B gene was regulated by dehydration,low temperature,high salt stress,and ABA induction. Therewas a band by SDS-PAGE protein electrophoresis,which was larger than the theoretical prediction value and did not decrease in solubilityunder boiling water condition. The overexpression of Mt LEA5 B protein in E. coli obviously increased the survival rate of hosts at heat（55℃）and freeze（-20℃）. Moreover,Mt LEA5 B was an inducible expressed gene,Mt LEA5 B protein had high hydrophily and heat resistance,overexpression in E. coli may significantly enhance its tolerance to temperature stress.