拟南芥PPR蛋白FBA2调控叶绿体ATP合酶基因表达的分子机理研究

Chloroplast is a semiautomous organelle, and it retained about 100 protein-coding genes. The gene expression of chloroplast genome requires fine regulation by a number of nuclear-encoded factors involving in multiple aspects, including gene transcription, RNA editing and translation. It plays crucial roles in regulating the biogenesis and content of chloroplast thylakoid protein complexes. We have isolated and characterized an Arabidopsis mutant fba2 with low accumulation of ATP synthase by forward genetics, and the mutant gene encoding a chloroplast-encoded PPR protein with unknown function. The accumulation of atpF transcript was detective in fba2 mutants. In this project, we aiming to use the following research strategies to clarify the molecular function of FBA2 in regulating target gene content: 1) Dissect the FBA2 target sites and specific sequence by Northern hybridization, RNA co-immunoprecipitation, gene microarray or high throughout sequencing and electrophoretic mobility shift assay technology. 2) Determine the protein interacting with FBA2 by using of yeast two hybridization system and immune affinity chromatography in combination with mass spectrometry. The results obtained from this study will provide new insights into molecular mechanism of chloroplast gene expression, the biogenesis and content regulation of ATP synthase.

叶绿体是一个半自主型细胞器，其携带约100个基因，这些基因的表达需要大量核基因编码的调控因子在基因转录、RNA编辑以及翻译等各个阶段进行精密调节，这对于叶绿体类囊体膜蛋白复合物的生物发生和含量调节是至关重要的。我们采用正向遗传学策略筛选获得了一个ATP合酶含量特异性降低的拟南芥突变体fba2，其突变基因编码一个含有PPR结构域的新型叶绿体蛋白，突变体中atpF的累积缺失。本项目拟利用核酸杂交法、RNA免疫共沉淀（RIP）、基因芯片或高通量测序技术以及凝胶阻滞手段确定FBA2作用的靶基因位点与特异性序列；通过酵母双杂或免疫亲和层析结合质谱分析鉴定相互作用蛋白，阐明FBA2调节靶基因含量的分子机制。相关结果对于我们深入认识叶绿体基因表达调控以及ATP合酶生物发生与含量调节机制具有重要的科学意义。

作为复杂的分子马达，叶绿体ATP合酶由九个亚单位组成，分别由核基因组和叶绿体基因组共同编码。由于其亚基化学计量的不均匀性，ATP合酶的生物发生和质体编码ATP合酶的基因表达需要核基因编码的调控因子在转录、转录后和翻译水平分别进行精密调控。本项目报道了一个P亚族PPR蛋白FBA2，其对于拟南芥叶绿体中atpH/F双顺反子的聚集是必须的。我们采用正向遗传学策略筛选获得了一个ATP合酶含量特异性降低的拟南芥突变体fba2 ，FBA2降低至野生型的3/4 。FBA2蛋白由22个PPR结构域组成，定位于叶绿体。生物信息学和EMSA实验结果表明FBA2结合atpF-atpA区保守序列。综上所述，FBA2蛋白阻止atpH/F转录本的降解，而且我们发现atpH/F双顺反子和加工的atpB/E双顺反子在ATP合酶含量不同的突变体中明显受到影响。结果表明ATP合酶复合体含量和叶绿体中atp基因的mRNA水平之间存在一定关系。相关结果对于我们深入认识叶绿体基因表达调控以及ATP合酶生物发生与含量调节机制具有重要的科学意义。