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本研究以百合组培苗叶片为试验材料,研究不同蛋白提取方法的得率、电泳上样量及聚焦除盐时间对双向电泳图谱的影响。结果表明:采用三氯乙酸(TCA)/丙酮沉淀法提取百合叶片蛋白质比Tris-HCl法效果好,蛋白得率为38.5 mg/g,且颜色较白,获得的2-DE图谱较好;上样量为600μg时得到的图谱分辨率高、蛋白斑点分布均匀、清晰;等电聚焦(Iso-electrofocusing,IEF)中的2个除盐步骤的时间分别为2 h和2.5 h时,可以较好地去除盐分,顺利完成聚焦并得到较好的双向电泳图谱。该体系的建立将为下一步百合蛋白质组学研究及利用分子育种技术培育抗病品种提供技术资料。
In this study, the leaves of lily tissue culture seedlings were used as experimental materials to study the influence of different protein extraction methods, electrophoresis sample volume and focused desalting time on the two-dimensional electrophoresis pattern. The results showed that the extraction of lily leaves protein by trichloroacetic acid (TCA) / acetone precipitation was better than that of Tris-HCl solution, the yield of protein was 38.5 mg / g and the color was white. The 2-DE pattern obtained was better; At 600 μg, the pattern resolution and protein spot distribution were uniform and clear. The time of 2 desalting steps in iso-electrofocusing (IEF) was 2 h and 2.5 h, respectively, which could be better To remove salt, successfully completed the focus and get better two-dimensional electrophoresis. The establishment of this system will provide technical information for the next lily proteomics research and the use of molecular breeding techniques to cultivate resistant varieties.