【作者】 彭连伟；

【导师】 张立新；

【作者基本信息】 兰州大学 ， 植物学， 2006， 博士

【摘要】 光系统Ⅱ(PSⅡ)是由多个亚基组成的色素蛋白复合体，它催化光驱动的水的裂解和醌的氧化。由于其结构的复杂性，PSⅡ的生物发生和组装需要核基因与叶绿体基因编码的蛋白以一定次序地合成并组装。这是一个多步骤的过程，需要大量核基因编码的辅助因子和调节蛋白的协助。到目前为止，对于PSⅡ生物发生的调节因子知之甚少。分离、鉴定拟南芥中这些核基因编码的调节因子以及研究它们的作用机制有助于我们认识高等植物PSⅡ复合物组装和功能调控的分子机理。基于这个原因，我们从T-DNA插入的拟南芥突变体库中筛选具有高叶绿素荧光表型的PSⅡ突变体并对其中的一个突变体lpal进行了详细的研究。主要结果如下： 1、通过对62，000个突变体株系进行高通量的筛选，获得了136个高叶绿素荧光的突变体。根据测定的叶绿素荧光慢诱导曲线，我们将突变体进行了简单的分类，其中一类属于PSⅡ突变体。对PSⅡ突变体进行SDS-urea-PAGE和蛋白免疫印记分析发现突变体中PSⅡ蛋白的累积量与野生型相比显著降低，而其它复合物含量的变化不大。这些低PSⅡ累积的突变体我们称之为lpa(low photosystemⅡ accumulation)突变体。 2、我们重点刻画了拟南芥突变体lpal。在突变体lpal中，其生长量、色素含量与野生型相比均显著降低。叶绿素荧光诱导和P700的氧化还原动力学分析表明在突变体中主要是PSⅡ的活性受到明显影响。蛋白免疫印记和BN-PAGE电泳分析表明，突变体中可以形成有功能的PSⅡ复合物，但是PSⅡ的累积量仅有野生型的20％左右。进一步用核酸杂交和多聚核糖体实验证明了PSⅡ累积量的降低不是由于某个PSⅡ亚基在转录和翻译起始水平受到影响而造成的，而可能是由于翻译后的组装或稳定性受到了影响。蛋白质标记实验表明突变体中D1、D2的合成量显著降低，而其他叶绿体编码蛋白的翻译速率与野生型相似。新合成的PSⅡ蛋白可以组装成有功能的复合物，但是组装效率没有野生型的高。除此之外，突变体中新合成的PSⅡ亚基D1、D2、CP43和CP47的周转速度比野生型的快。 3、用IPCR方法克隆到的LPA1基因编码一个含有TPR结构域的叶绿体膜蛋白，但它不是PSⅡ的一个组分。LPA1先于PSⅡ复合物而存在于黄花苗中并更多还原

【Abstract】 Photosystem II (PSII) is a multisubunit pigment-protein complex that catalyzes the light-driven water oxidation and reduction of plastoquinone. Because of the structural complexity of PSII, biogenesis and assembly of PSII requires the coordinated synthesis and assembly of both chloroplast- and nuclear-encoded proteins. These are likely to be a multi-step processes, which are assisted by many nuclear-encoded auxiliary and regulatory proteins. Until recently, the factors involved in regulating the biogenesis of PSII have remained largely unknown. Identification and characterization of these nuclear -encoded chloroplast proteins will increase our knowledge on molecular mechanisms of the PSII complexes biogenesis. To this purpose, we have screened PSII mutants with high chlorophyll fluorescence phenotypes from a collection of pSKI015 T-DNA-mutagenized Arabidopsis thaliana lines, and we further characterized the lpa1 mutant. The main results are summarized as follows:1. We have screened 62,000 T-DNA-mutagenized Arabidopsis thaliana lines and obtained 136 mutants with high chlorophyll fluorescence phenotype. Mutants with impaired energy transfer within PSII were identified based on the chlorophyll fluorescence induction kinetics. SDS-PAGE and immunoblot analyses indicated that the PSII subunits were reduced significantly and other complexes content were only affected slightly. These mutants with low photosystem II accumulation were nameded lpa mutants2. To gain insight into the processes involved in PSII biogenesis and maintenance, we characterized the lpa1 mutant of Arabidopsis thaliana, which generally accumulates lower than wild-type levels of the PSII complex. The pigment contents, relative growth were significantly reduced in the mutant. Analyses of the chlorophyll fluorescence induction and P700 redox kinetics showed that the mutants have defects in energy transfer within PSII. Immunoblot and BN-PAGE studies showed that the PSII can be assembled in the mutant, but the amounts of the PSII proteins were reduced to about 20% of wild-type levels. No alterations in the更多还原