棉花胚珠与纤维蛋白质的两种提取方法比较研究

Effect of Two Methods of Protein Extraction from Cotton Ovule and Fiber

分别用三氯乙酸丙酮沉淀和苯酚抽提两种方法,提取棉花胚珠和纤维总蛋白质,发现用三氯乙酸-丙酮沉淀法提取得到的蛋白质,获得率为2.3 mg·g-1,双向电泳后,用胶体考马斯亮蓝染色法染色后检测的蛋白质点数极少;银染色后,发现蛋白质点数没有明显的增加.而用苯酚抽提法得到的蛋白质获得率达到10.4 mg·g-1,是三氯乙酸-丙酮沉淀法的5倍,双向电泳后,用胶体考马斯亮蓝染色法染色后检测的蛋白质点数可以达到923个;银染色,可以检测到1061个蛋白质点.等电点pI从pH 3～10都有分布,但是集中在pH 4.3～8.6.结果表明:酚抽提法具有获得蛋白质种类更多、pI分布范围较宽,蛋白质容易溶解等优点,而且该方法操作简便,是适用于棉花胚珠和纤维的蛋白质组学研究的理想方法.

Total protein of cotton ovule and fiber were extracted using Trichloroacetic acid （TCA）-acetone precipitation method and phenol isolation method, respectively, we found that for the TCA-ace tone precipitation method, the protein harvest rate was 2.3 mg·g^-1. The protein sample was separated with 2-dimension gel electrophoresis （2-DE） and visualized with colloidal Coomassie Brilliant Blue G-250 （cCBB G-250） and silver staining methods respectively, in cCBB G-250 gels, few protein spots were detected, no considerable change observed in silver stained gel as well. For phenol isolation method, however, the harvest rate reached 10.4 mg·g^-1,which was as 5 folds high as that of TCA- acetone precipitation method, 923 spots were detected on cCBB stained 2-DE gel, and 1061 spots detected on silver stained 2-DE gel, the isoelectric poit （pI） values of the proteins ranged from 3 to 10, focusing between 4.3 and 8. 6. The results demonstrated that phenol isolation method, which may harvest more kinds of protein with wider pI distribution range and better dissolvability, is easy-handling and thus is optimal for extracting cotton ovule and fiber protein in proteomic research.