缺铁逆境胁迫下水稻叶片蛋白的双向电泳及其蛋白质组图谱分析

水稻幼苗经缺铁胁迫诱导分别处理1、3、5天后,用酚法和TCA/丙酮法提取叶片中的可溶性蛋白进行双向电泳分析,从而研究在缺铁条件下叶片中蛋白表达的动态变化规律.结果显示1.不同pHIPG胶条分离蛋白的效果不同.用pH3-10的IPG胶条进行双向电泳,经考马斯亮蓝染色后,可在胶面上检测到大约450个蛋白点,其中约有89%的蛋白是酸性蛋白.如果用pH4-7的IPG胶条进行双向电泳,则可检测到大约600个蛋白点,其中有29个蛋白是上调表达,1个蛋白是下调表达,5个蛋白是诱导特异表达.2.不同方法提取的可溶性蛋白质量不同.TCA法简单易操作,似乎对于碱性蛋白的抽提效果更好,在2-DE图像上,减性端显示的蛋白点多;但此方法所得蛋白的再溶性差.酚法提取的蛋白再溶性好,所抽提的蛋白量较大,纯度较高.

2-DE Method and proteomic map analysis in rice leaves under iron deficient stress

Rice seedlings were treated in Fe-deficient hydroponic solution for 1d,3d and 5 d respectively. Then soluble proteins in rice leaves were extracted with phenolic or TCA / acetone method, and protein dynamic changes were studied on the Fe-deficient stress by 2-DE analysis. The results depicted as follows:. First, using different pH IPG gel strip leaded to different separation effectiveness. Stained with coomassie brilliant blue, nearly 450 protein spots in a 2-DE gel were detected with the pH 3～10 gradient IPG strip, among which about 89% proteins were acidic ones. However, about 600 spots were tested in a 2-DE gel with the pH4～7 gradient IPG strip. In the 2-DE gel with pH4～7 IPG strip, 29 proteins were up-regulated, only 1 protein was down-regulated, 5 proteins were specially displayed. Second, the quality and quantity of the soluble proteins were different with different extraction methods. TCA technique was easy to operate and effective to distribute basic proteins on the basic apex of 2-DE gels. But the proteins extracted by TCA were difficult to resolve thoroughly. The proteins extracted by phenolic method were more quantitative and purer than that by TCA technique.