低温胁迫小麦苗冰结构蛋白纯化的研究

焦宇知; 徐桃; 梁家冰; 朱云; 何志勇; 张晓晓; 刘永博; 孙芝杨; 翟玮玮

doi:10.13386/j.issn1002-0306.2017.03.011

低温胁迫小麦苗冰结构蛋白纯化的研究

Studies on purification of ice structuring protein from cold-acclimated wheat seedlings

摘要

摘要: 以研究低温胁迫小麦苗冰结构蛋白（ISP）纯化为目的,以小麦苗为对象,经低温胁迫,并采用粗分离、饱和硫酸铵溶液沉淀、DEAE-Cellulose 52层析、sephadex-G75凝胶层析4个步骤对其中ISP进行纯化,分别采用差示扫描量热法（DSC）、热休克处理法和聚丙烯酰氨凝胶电泳（SDS-PAGE）测定各纯化步骤所得ISP的热滞活性（THA）、冰晶重结晶抑制能力（ICP）和分子质量（MW）,并计算小麦苗存活率和各纯化步骤的得率。实验结果表明,梯度低温胁迫的小麦苗存活率与恒温低温胁迫相比提高了240%,小麦苗提取物（AWSE）中蛋白质含量与未低温胁迫小麦苗提取物（NAWSE）相比提高了7.8%;以THA表征的纯化倍数（T₁）和以ICP表征的纯化倍数（T₂）分别为451.15和137.60,得率为2.85%,THA为0.180℃（5 mg/mL）,ICP为70.03%,分子质量范围为20.1³1.0 ku;与THA相比,ISP在较低浓度下即可达到ICP的峰值。小麦苗可作为制备冰结构蛋白的天然植物资源。

Abstract: To investigate purification of ice structuring protein( ISP) from cold- acclimated wheat seedlings,wheat seedlings underwent cold acclimation and ISP from cold- acclimated wheat seedlings was purified by 4 sequential steps including coarse separation,saturated ammonium sulphate precipitation,DEAE- Cellulose 52 chromatography and sephadex- G75 gel filtration chromatography.Differential scanning calorimetry( DSC),heat shock treatments and polyacrylamide gel electro- phoresis( SDSPAGE) were employed respectively for determination of thermal hysteresis activity( THA),ice recrystallization inhibition power( ICP) and molecular weight( MW) of ISPs during process of purification.Survival rate of cold-acclimated wheat seedlings and purification times of ISP during each purification step were calculated as well.Results showed an increase of 240% in survival rate of wheat seedlings using gradient cold- acclimation compared with constant cold- acclimation. Protein content of cold-acclimated wheat seedlings extract( AWSE) was increased by 7.8% in comparison to non- cold- acclimated wheat seedlings extract( NAWSE). Eventual purification times based on THA and ICP were 451.15 and 137.60 respectively. Yield,THA at5 mg / m L,ICP and MW range of ISP were 2.85%,0.180 ℃,70.03% and 20.1 ~31.0 ku respectively. Maximal ICP of ISP was achieved at lower concentration compared with THA.Wheat seedlings can be a potential plant resource in production of ISP.