不同生态型芦苇叶片蛋白质双向电泳系统的筛选和优化

Selection and Optimization of 2-DE System for Leaf Proteome Profiling of Different Ecotypes of Reed Growing in Natural Habitats

通过优化组合植物蛋白质提取方法及与之匹配的蛋白质裂解液,采用改进的O′Farrel双向电泳系统,以自然生境野生芦苇叶片为材料,筛选出一种适合纤维含量高、革质化明显的4种不同生态型芦苇(水生芦苇、轻度盐化草甸芦苇、重度盐化草甸芦苇、沙丘芦苇)叶片蛋白质分析的双向电泳系统,即以饱和酚-醋酸铵/甲醇沉淀法提取叶片蛋白质样品,经裂解液[8mol/L尿素,2mol/L硫脲,4%CHAPS,65mmol/LDTT,2%Ampholine(pH3.5￣10∶pH5￣8=1∶4)]裂解后按80!g上样,银染后获得背景清晰、蛋白质分辨率较高的双向电泳图谱.该系统用于水稻等植物叶片蛋白质双向电泳分析,同样获得较好的电泳图谱和分辨率.

An optimized two-dimensional polyacrylamide gel electrophoresis （2-DE） system for analyzing plant proteins was developed by evaluating different reagents and concentrations used in the sample extraction solutions and lysis buffers. Two main sample preparation methods, referred to as trichloroacetic acid （TCA）-acetone method and phenol extraction-ammonium acetate/methanol （phenol-NH4Ac/methanol） precipitation method, were compared. Four ecotypes of reed plants （Phragmites communis Trin.） from the desert region of north-western China were used as experimental materials： （1） swamp reed （SR） which grows in water about 1 m deep; （2） dune reed （DR） which grows on 5-10 m high sand dunes; （3） heavy salt meadow reed （HSMR） which grows on low-lying salt flats; and （4） light salt meadow reed （LSMR） which grows in the transition area between DR and HSMR growing areas. The optimized phenol-NH4Ac/methanol precipitation method consisted of extracting leaf proteins of different ecotypes of reed with water-saturated phenol and then precipitating with a 5-fold volume of 0.1 mol/L NH4Ac in methanol, followed by dissolving in the lysis buffer. The optimized protein lysis buffer consisted of 7 mol/L urea, 2 mol/L thiourea, 4% CHAPS, 2% Ampholine（pH 3.5-10 ： pH 5-8 = 1 ： 4） and 65 mmol/L DTT. The prepared protein sample （80 μg） was then separated by 2-DE gel and detected by silver staining method. This improved 2-DE system resulted in a 2-D protein profile of higher resolution and higher protein yields as analyzed by PDQuest software. Good results were also obtained when this 2-DE system was used in 2-D analysis of proteins from other plant materials, such as rice leaves, indicating that it is a suitable 2-DE system for analyzing leaf proteins of different plant species.