摘要
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.
An improved two-dimensional blue native/SDS polyacrylamide gel-electrophoresis (BN/SDS-PAGE) followed by nano-HPLC-ESI-MS/MS was used to study thylakoid membrane protein complexes between a low chlorophyll b rice mutant (Oryza sativa L. Zhenhui 249Y) and its wild type (Oryza sativa L. Zhenhui 249W). The PSⅠ -LHC Ⅰ super-complexes, LHC Ⅰ -less PS Ⅰ, ATP synthase, Cytochrome b6t, CP43-1ess PS Ⅱ, trimeric and monomeric LHC Ⅱ were resolved. 24 proteins, which belong to the subunits of four major photosynthetic apparatus were identified. Comparative study indicated that the mutant had moderately decreased amount of LHC Ⅱ, greatly decreased amount of LHC Ⅰ, and increased amount of PS II reaction center and ATP synthase. Western blotting verified BN/SDS-PAGE analysis. These results provided clues about molecular foundation of higher photochemical efficiency and light stability of the mutant, and showed that the improved BN/SDS-PAGE is suitable for not only membrane protein complex separation, but also for comparative study of complex subunits in different physiological condition or between mutant and its wide type.
出处
《生物化学与生物物理进展》
SCIE
CAS
CSCD
北大核心
2006年第7期653-659,共7页
Progress In Biochemistry and Biophysics
基金
国家自然科学基金(30400030)
江苏省基础研究计划创新人才基金(BK2004416)资助项目~~