黄瓜叶片胞间隙蛋白质双向电泳体系的建立

Construction of Protein Two-Dimensional Polyacrylamide Gel Electrophoresis System for Intercellular Washing Fluid of Cucumber Leaves

以黄瓜种质‘PI088’幼苗为材料,提取胞间隙液,制备蛋白样品,通过对不同IPG胶条、等电聚焦条件、分离胶浓度、上样量等条件的探索,建立适合黄瓜叶片胞间隙蛋白质组的双向电泳体系。结果显示:(1)用pH 3～10的非线性IPG胶条,等电聚焦时间为70 000Vh,分离胶浓度为10%,上样量为800μg时,能够得到较好的2-DE图谱。(2)利用所建立的双向电泳体系找到了对照及接种霜霉菌后2d的黄瓜叶片胞间隙差异蛋白,其中的12个上调表达点和10个下调表达点的表达量变化在1.5倍以上。并选取一个差异点成功进行了质谱分析。(3)质谱分析结果显示,所找的差异点为一种酸性的几丁质酶,等电点为4.27。可见,采用所建立的双向电泳体系可获得分辨率高、重复性好的2-DE图谱并能很好地用于质谱分析。

The purpose of this research was to establish a suitable two-dimensional electrophoresis system by optimizing the parameters including the different IPG strips,isoelectric focusing conditions,different gel concentrations and sample loading for intercellular washing fluid of cucumber leaves to analyze proteomics.Cucumber seedling leaves of the variety PI088 was used in the experiment,extracting the intercellular washing fluid and preparing for protein samples.（1）As shown by the results,with pH 3~10 nonlinear IPG strips,under the conditions of isoelectric focusing 70 000 Vh,10% separating gel concentration,sample volume 800 μg,we got a clear two-dimensional electrophoresis map.（2）The proteomics profiling of intercellular washing fluid of cucumber leaves in different inoculation time was analyzed by using the previous established protocol.We found 12 up-regulated and 10 down-regulated proteins with more than 1.5 times changes in 2 days after inoculating with Pseudoperonospora cubensis compared with control and one of the different protein spots were successfully identified.（3）Mass analysis showed that the different point is an acidic chitinase and the isoelectric point is 4.27.Reproducible profiles with high resolution were obtained by this 2-DE system.