【作者】 李炯；

【导师】 杜林方；

【作者基本信息】 四川大学 ， 生物化学与分子生物学， 2001， 硕士

【摘要】 磷酸化是调节蛋白功能最有效的方式之一。叶绿体中进行光能吸收、传递和转化的类囊体膜蛋白可以磷酸化。天线组分中主要捕光色素复合物Ⅱ（LHCⅡ）的LHCB1、LHCB2和次要捕光色素复合物CP29，以及四种PSⅡ反应中心蛋白D1、D2、CP43、psbH gene产物可以磷酸化。类囊体膜蛋白磷酸化对植物光合作用的调节机理已成为国际上研究热点。 本文建立了一种比通用的同位素[³²P]和磷酸化Thr抗体这两种磷酸化蛋白检测方法都更为先进的，用特异磷酸探针和PSⅡ蛋白抗体快速检测类囊体膜蛋白体内磷酸化的方法。 利用这种体内磷酸化检测方法和室温可见光吸收光谱、室温荧光光谱等技术研究未经光照和光强分别为50、100、200、400、800μmol·m^-2·s^-1光照处理的豌豆叶片PSⅡ蛋白的体内磷酸化特点。结果表明：随着光强增加，PSⅡ反应中心蛋白D1、D2的磷酸化增加；CP43的磷酸化也随着光强的增强而增加，但磷酸化形式没有未经光照的样品多：磷酸化LHCⅡ只在中等光强下表现出明显的积累，在高光强下，LHCⅡ的磷酸化反而减少。因此提出了D1、D2的磷酸化可能与光损伤的修复过程有关，CP43的磷酸化可能与光能的传递有关，验证了LHCⅡ可逆磷酸化调节光能在两个光系统之间的分配这一学说。 用同种方法研究在较高光强400μmol·m^-2·s^-1下，四叶期油菜野生型和黄化突变体的体内磷酸化差异。研究表明：光照使野生型的D1、D2磷酸化增加，突变体的D1、D2磷酸化减少；两种油菜光照后的磷酸化和非磷酸化CP43都比未经光照的少，光照使突变体CP43磷酸化减少多；光照后两种油菜的磷酸化LHCⅡ都有所增加。光照对突变体类囊体蛋白体内磷酸化影响较大，从而也说明了叶绿素缺乏对类囊体蛋白的结构功能产生的影响。更多还原

【Abstract】 Protein Phosphorylation is recognized to be a ubiquitous regulatory mechanism. The major function of the thylakoid membranes is to carry out photosynthetic electron transport and ATP synthesis. Several proteins of the thylakoid membranes can be phosphorylated. These phospho proteins include the light-harvesting chlorophyll a/b-binding protein (LHCII and CP29), four PSII core proteins (Dl and D2 reaction center proteins, chlorophyll a-binding protein CP43, and the psbH gene product). The study on physiological roles of the thylakoid phospho protein has received a great deal of attentionWe introduced an immunological approach using INDIA?phosphoprotein probe and Dl, D2, CP43 and LHCII antibody for the analysis of protein phosphorylation in vivo. This new analytical method is more suitable comparing with experiment with [32P] and phosphothreonine antibody.Take advantage of this new analytical tool, the PSII core protein and LHCII in the pea thylakoid were founded to have a different phosphorylation pattern in vivo. Accumulation of the photosystem II core phosphoproteins (Dl, D2) occurred with increasing irradiance, but maximal phosphorylation of LHCII only occurred at low light intensities and drastically decreased at high irradiance. Maximal phosphorylation of CP43 is found in thylakoids isolated from dark-adapted leaves. We used absorption spectroscopy and fluorescence spectroscopy to analyze our result. We proposed that the function established for reversible phosphorylation of PSII core proteins, including Dl and D2, is theprotection of PSII against photoinbibition. The physiological significance for phosphorylation of LHCII is implicated in the regulation of excitation energy distribution between PSII and PSI.We carried out a comparative study on thylakoid membrane phosphoprotein between a new oilseed rape (brassica napus) mutant Cr3529 and its wild type 3529 illuminated at 400 u mol-m-2-s-l. The result of the study indicated that phosphorylation of D1, D2 in the thylakoids of wild type isolated from illuminated leaves increased. Phosphorylation of Dl, D2 decreased in the thylakoids of mutant leaves isolated from illuminated leaves. Maximal phosphorylated and unphosphorylated CP43 of these two types is founded in the thylakoids of leaves isolated from dark-adapted leaves. Phosphorylation of LHCII increase under illuminated condition. So irradiance has a more great influence on the mutant.更多还原