水稻花粉总蛋白质双向凝胶电泳方法建立及应用

Establishment and Application of Two-Dimensional Gel Electrophoresis Analysis of Proteins from Rice Pollen

采用石英砂加液氮研磨方法破碎花粉，然后用两种不同的方法提取水稻花粉总蛋白质，之后采用固相pH梯度等电聚焦／SDS-PAGE双向凝胶电泳对红莲型细胞质雄性不育水稻单核期花粉总蛋白质进行了分离。通过银染显色，PDOuest2DE软件可识别约1000-1500个蛋白质点。其中TCA-丙酮提取法更适合提取花粉总蛋白质进行双向凝胶电泳分离，并可获得分辨率和重复性较好的双向电泳图谱。对红莲型细胞质雄性不育水稻的不育系单核期和二核期花粉总蛋白进行了双向电泳分离，通过比较两个时期花粉蛋白质电泳图谱后发现，二核期的花粉蛋白质较单核期花粉蛋白质数目减少，并且部分蛋白质表达量下降。

To better understand the molecular mechanism of cytoplasmic male sterility （CMS） at the protein level during the development of microspores, proteomic technique was used to investigate the protein expression profile in uninucleatestage pollen of the rice CMS lines. Total pollen proteins were extracted using two different methods and then separated by two-dimensional gel electrophoresis （2-DE）. An efficient and economic method for high quality protein preparation from rice pollen for 2-DE was established. The silver-stained protein spots were analyzed by using software PDQuest 2DE, there were 1 500 detectable spots on the gel. These results showed that, the proteins extracted by this method were in good integrity and purity, and could meet the needs of proteomic analysis. Comparing with the 2-DE maps of the total proteins from the binucleate-stage and uninucleate-stage pollen, the number of total proteins and the volume of some proteins were decreased in the binucleate-stage pollen.