卷柏总蛋白质提取方法及双向电泳条件的建立

The Establishment of Extraction Method and Two-Dimensional Electrophoresis Conditions for Proteome Analysis of Selaginella tamariscina Beauv. Spring

目的:建立复苏植物——卷柏总蛋白质的提取方法,以及可以对其蛋白质组进行阵列分离的双向电泳条件。方法:通过多种条件的组合与优化,建立了以物理和化学2种方法充分裂解植物组织细胞,并采用低温操作、加入PVP等去除植物组织中大量的酚类物质,最后离心去除不溶性杂质的卷柏蛋白质组双向电泳(2-DE)样品制备方法。第1向电泳为固相pH梯度等电聚焦,第2向电泳为垂直平板SDS-PAGE。结果:通过对样品制备、蛋白定量、第1向和第2向电泳、染色方法等各实验环节进行控制和优化,凝胶经考马斯亮蓝染色后,可分辨蛋白质斑点数约600个。结论:建立了卷柏总蛋白质的提取方法及蛋白质组双向电泳技术,为后续研究卷柏在干旱胁迫下的差异表达奠定了基础。

Objective： To establish extraction method and appropriate conditions for profiling cell proteome of Selaginella tamariscina （Beauv.） Spring which is a kind of resurrection plants. Methods： By combination and optimization of various conditions, a sample preparation method was established. The plant cells were sufficiently lysed by using physical and chemical techniques. Protein degradation was minimized by operation at lower temperature, adding PVP to wipe off mass of hydroxybenzene. Then the insoluble impurities were removed by centrifugation. The first-dimensional electrophoresis was immobilized pH gradients isoelectric focusing and second dimensional electrophoresis was vertical electrophoresis of SDSPAGE. Results： By control and optimization of many experimental conditions, after Commassie blue staining the technique could detect about 600 spots. Conclusion： The extraction method of proteome and technique of 2-DE, which have been established are the basis of work for study on the discrepancy expression of S.tamariscina （Beauv.） Spring under drought condition later on.