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天女木兰种子蛋白双向电泳体系的建立
张晓林1, 陆秀君2, 马蓓蓓1
1.沈阳农业大学 林学院;2.沈阳农业大学 林学院,沈阳农业大学 设施园艺省部共建教育部重点实验室
摘要:
【目的】探索一套适用于天女木兰种子蛋白的双向电泳体系,为研究其种子萌发过程中的蛋白表达差异奠定基础。【方法】比较蛋白的不同提取方法(酚提取法、三氯乙酸-丙酮-酚提取法、三氯乙酸-丙酮法和Tris-HCl法)、IPG胶条pH梯度(pH3~11 NL、pH3~10和pH4~7)、裂解液中的尿素浓度(6,7,8,9 mol/L)和分离胶浓度(10%和12.5%)等条件,对天女木兰种子蛋白双向电泳结果的影响,筛选适宜的双向电泳条件。【结果】以Tris HCl法提取种子蛋白,在IPG胶条pH为4~7、裂解液中尿素浓度为9 mol/L、分离胶浓度为12.5%的条件下,可以获得分辨率高、背景清晰、重复性好的天女木兰种子蛋白双向电泳图谱。【结论】建立了适用于天女木兰种子蛋白的双向电泳体系。
关键词:  天女木兰  种子  双向电泳  蛋白质
DOI:
分类号:
基金项目:国家自然科学基金面上项目(31070561);辽宁省百千万人才资助项目(2009921071)
Establishment of two-dimensional electrophoresis system of Magnolia sieboldii K.Koch seeds
Abstract:
【Objective】A two-dimensional electrophoresis(2-DE)system was established for proteomic analysis of Magnolia sieboldii K.Koch seeds,which will lay a strong foundation for further study of proteomics of seed germination.【Method】Different effects of 2-DE gels that were involved in the method of protein extraction (Phenol method,TCA-Acetone Phenol method,TCA-Acetone method and Tris HCl method) were compared,the pH gradient of IPG strip (pH3-11 NL,pH3-10 and pH4-7),concentration of urea (6,7,8,9 mol/L) and different separation gel concentrations (10% and 12.5%) were tested.【Result】The optimized system included the following steps:extracting the protein from M.sieboldii K.Koch seed by Tris-HCl precipitation,separating the proteins with pH4-7 IPG strips,dissolving proteins with lysis buffer containing 9 mol/L Urea and running SDS-PAGE with 12.5% gel concentration. Reproducible profiles with high resolution were obtained by this optimized 2-DE system of M.sieboldii seeds.【Conclusion】The optimized 2-DE system of protein from M.sieboldii’seed was established.
Key words:  Magnolia sieboldii  seed  two-dimensional electrophoresis(2-DE)  protein