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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。
Three rubber extracts (TCA / acetone, phenol extraction and Tris-acetone-phenol method) were used to extract the total protein from the inflorescences of Lycoris radiata 7-33-97 inflorescences. The protein extraction rate, one-way SDS-PAGE and two-dimensional electrophoresis results of three protein extraction methods were compared and analyzed. The results showed that TCA / acetone extraction rate of the three methods was the highest (6.09 ± 0.22) mg / The bands of SDS-PAGE electrophoresis were clear and complete, and the most spots were obtained after two-dimensional electrophoresis (994 ± 25), which was a good method to establish the research system of the rubber tree inflorescence proteomics. Furthermore, the two-dimensional electrophoresis system of total RNA extracted from TCA / acetone based on TCA / acetone method was optimized. The results showed that using IPG tape with pH range of 4 ~ 7, the focusing time was 7.0 × 10 ~ 4 Vh, Under the condition of 1.0 × 10 ~ 3μg and 12.5% gel concentration, the two-dimensional gel electrophoresis pattern of rubber tree inflorescence obtained by Coomassie Brilliant Blue dyeing method was the best, which laid a good foundation for the proteomics research of rubber tree reproductive development.