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采用蓝绿温和胶凝胶电泳(blue-nativepolyacrylamidegel-electrophoresis,BN-PAGE),以及改进的第二向SDS-PAGE分离了水稻低叶绿素b突变体ZH249-Y和野生型ZH249-W类囊体膜蛋白复合物,系统比较了突变体和野生型各复合物亚基的表达差异.结果显示,第一向BN-PAGE分离了PSⅠ-LHCⅠ、LHCⅠ缺失的PSⅠ、ATP合成酶、细胞色素b6f、CP43缺失的PSⅡ及LHCⅡ六种复合物.上述各复合物经第二相SDS-Urea-PAGE分离后,利用胶内酶解,高效液相层析分离肽段,电喷雾串联质谱鉴定了复合物的亚基.结合免疫印迹研究,证明和野生型相比,突变体光系统Ⅱ捕光天线复合体的表达量适度下降,但光系统Ⅰ捕光天线破坏严重,同时光系统Ⅱ核心蛋白和ATP合成酶的表达量上升.研究结果对揭示低叶绿素b水稻突变体较高光化学效率和光稳定性的分子基础提供了线索,同时也表明,改进的BN/SDS-PAGE双向电泳不仅可以有效地分离膜蛋白复合物及亚基,也可以进行不同生理条件下,或野生型和突变体之间膜蛋白质组的比较研究.
The low chlorophyll b mutant ZH249-Y and the wild type ZH249-W thylakoid membrane were isolated by blue-native polyacrylamide gel-electrophoresis (BN-PAGE) and modified secondary SDS-PAGE. Protein complex, the differences in the expression of subunits of mutant and wild-type complexes were compared systematically.The results showed that PSⅠ-LHCⅠ, PSⅠ, ATP synthase, cytochrome b6f, CP43 Lack of PS Ⅱ and LHC Ⅱ complexes.After the second phase SDS-Urea-PAGE separation of the second phase, using in-gel digestion, high performance liquid chromatography separation of peptides, electrospray ionization tandem mass spectrometry identified the complex Subunit.According to the Western blot analysis, it was proved that the expression level of mutant light system Ⅱ light harvesting antenna complex decreased moderately compared with the wild type, but light system Ⅰ light harvesting antenna was severely damaged, while the photosystem Ⅱ core protein and ATP synthesis The results of this study provide clues for revealing the molecular basis of higher photochemical efficiency and photostability of low chlorophyll b mutants, and also indicate that the improved BN / SDS-PAGE two-dimensional electrophoresis can not only effectively separate the membrane Protein complexes and subunits can also be compared under different physiological conditions, or between the wild-type and mutant membrane proteome comparison.