橡胶树花序总蛋白双向电泳研究体系的建立与优化

Optimizing Two-Dimensional Gel Electrophoresis System for Proteins of Rubber Tree Inflorescence

以橡胶树热研7-33-97花序为试验材料,分别采取3种蛋白质提取方法(TCA/丙酮法,酚抽提法和Tris-丙酮-酚法)提取橡胶树花序总蛋白。对3种蛋白质提取方法的蛋白提取率、单向SDS-PAGE电泳和双向电泳结果进行分析比较,结果表明3种方法中TCA/丙酮法的提取率最高(6.09±0.22)mg/g,单向SDS-PAGE电泳条带清晰且较完整,双向电泳后获得的蛋白点最多(994±25),是建立橡胶树花序蛋白质组学研究体系的较好方法。进一步对基于TCA/丙酮法提取的橡胶树花序总蛋白质双向电泳体系进行优化,结果显示利用p H范围为4~7的IPG胶条,在聚焦时间为7.0×10~4 Vh、蛋白上样量为1.0×10~3μg,以及12.5%凝胶浓度条件下,考马斯亮蓝染色法获得的橡胶树花序蛋白质双向电泳图谱质量最好,可为橡胶树生殖发育相关蛋白质组学研究奠定良好基础。

In this research, using the inflorescence of Rubber tree(Reyan 7-33-97) as test material, the proteins were extracted from inflorescence in three ways(By using TCA-acetone method, phenol method and tris-acetone-phenol method). Comparing the results of protein extraction rate, SDS-PAGE electrophoresis and two-dimensional electrophoresis of three extraction methods, the results showed that TCA/acetone method had the highest extraction yield(6.09±0.22) mg/g, the bands of SDS-PAGE electrophoresis were clear and integrated, and the protein points of2-DE were of the most amount(994±25), which was the best method to establish the research system of rubber tree inflorescence proteomics. To further optimize the two-dimensional electrophoresis system based on the method of TCA-acetone, the results showed that using IPG strips with p H ranges from 4 to 7, 7.0×10~4Vh of IEF time, 1 000 μg loading amount, and 12.5% gel concentration, coomassie brilliant blue staining of rubber tree inflorescence 2-DE image could achieve the best quality, which can lay a good foundation for proteomics research of rubber tree reproductive development.