旱柳根系蛋白质双向电泳体系的建立

A two-dimensional electrophoresis system of Salix matsudana root proteomics

林木抗逆机制的研究是林木抗逆育种的重要基础。为了解旱柳Salix matsudana耐盐机制,实验采用蛋白质双向电泳技术研究盐碱胁迫前后旱柳蛋白表达变化,分离鉴定旱柳耐盐相关基因,建立一套适合于旱柳根系蛋白质的双向电泳技术体系是蛋白质组学的基础。用改进的酚抽法抽提旱柳根总蛋白,此方法不仅能很好地分离蛋白,而且能有效去除样品中的盐分。通过比较分析了双向电泳不同条件对电泳结果的影响,确立了900μg.胶条-1的最佳上样量,10 min平衡时间以及10万V.h的聚焦时间的双向电泳条件。还比较了氯化钠胁迫后双向电泳图谱差异,发现了39个差异表达蛋白,其中15个下调,24个上调(11个新诱导表达),为下一步通过质谱鉴定分析旱柳耐盐分子机制及抗性基因分离奠定了基础。

To establish a suitable two-dimensional electrophoresis technology,for bettering the use of proteomic technology in discovering new proteins of Salix matsudana roots induced by salt-stress and in revealing differences of protein expression of the roots with fresh water and salt-stress,this study adopted an improved phenol extraction method to extract total proteins from S.matsudana roots.Results were compared for different treatment conditions,and then two-dimensional electrophoresis maps of roots with treatments of 1/4 stregth Hoagland hydroponic medium and 1/4 stregth Hoagland hydroponic medium containing 100 mmol·L-1 NaCl,were compared after 24 h of salt-stress.Results showed that the improved phenol extraction method could not only extract root proteins enough for 2-D,but it could also effectively remove salt from samples.Results showed that 900 μg per immobilized pH gradient（IPG） gel,10 min equilibrium time,and 100 000 V·h isoelectric focusing（IEF） time were the best conditions combination for the protein isolation.With the two-dimensional electrophoresis maps,39 proteins were differentially expressed,of which 15 were down-regulated,and 24 were up-regulated with 11 newly induced.