拟南芥叶绿体发育必需基因PAC的功能研究

Chloroplasts are essential places for photosynthesis and the production of lipids and other metabolites. The process of chloroplast biogenesis is tightly controlled both by nuclear genome and plastid genome complicatedly. Studies on chloroplast biogenesis is important for enhancement of solar energy utilization and photosynthetic efficiency of vascular plants.Once an essential gene for chloroplast biogenesis has been interrupted, the development of chloroplast would be halted, which lead to albino lethal plants. Some available mutants, which have low level expression of essential genes, are ideal materials to study the regulation mechanism of chloroplast biogenesis. In previous work, we had identified one variegated leaf color mutant in Arabidopsis thaliana, designated as var6-1, which was caused by the low PAC gene expression. PAC is an essential gene for chloroplast biogenesis with unknown function, and the completely PAC knock-out mutant is albino lethal. This study will focuses on var6-1 mutants. By analysis of chloroplast ultra-structure, accumulation of photosynthetic complexes, tissue expression pattern of the PAC gene, and whether PAC paricipate in protein complexes, meanwhile with screening and map-based cloning for suppressors of the variegated phenotype of the var6-1 mutant, we are going to explore the biological functions of the PAC gene in chloroplast biogenesis.

叶绿体是植物进行光合作用、脂类合成等重要物质代谢的场所，其发育是受核基因和叶绿体基因共同控制的复杂过程。对叶绿体发育过程的研究,将有助于提高绿色植物光能利用率和光合作用效率。叶绿体发育过程中的必需基因突变将导致叶绿体发育停滞、植株白化致死。而可稳定遗传的必需基因低表达突变株是研究叶绿体发育调控机制研究的理想材料。前期工作中，我们自主筛选得到了一个新的拟南芥花斑叶色突变体var6-1，该突变体的花斑叶色表型是由于PAC基因的低表达造成。PAC是一个功能未知的叶绿体发育的必需基因，其完全敲除突变体白化致死。本研究将以var6-1突变体为材料，进行叶绿体超微结构观察以及光合作用复合体积累的研究；研究 PAC基因的组织特异性；分析PAC是否形成蛋白复合体；筛选var6-1花斑表型的抑制突变体，并对感兴趣的抑制突变体进行基因克隆，来探索PAC基因在叶绿体发育过程中的生物学功能。

本项目以自主筛选得到的一个新的拟南芥花斑叶色突变体var6-1突变体为材料，研究其突变位点基因PAC (Pale Cress)参与叶绿体发育的生物学功能。已报道的PAC的敲除突变体白化致死。var6-1突变体的PAC表达量下降，导致了植物初生真叶白化，并在植物生长后期转化为花斑表型。var6-1突变体初生真叶中叶绿体编码蛋白如RbcL，D1和Cytf等严重下降，但其对应的mRNA量变化不明显。因此推测PAC下降影响了叶绿体内转录本的翻译过程。蔗糖梯度离心表明PAC存在一部分高分量复合体。利用酵母双杂交技术筛选得到了PAC潜在的互作蛋白，并结合BiFC验证了PAC与筛选得到的叶绿体核糖体大亚基蛋白RPLa和RPLb相互作用。var6-1 svr10-1双突变体表型增强。这些结果表明PAC可能通过叶绿体核糖体的组装过程参与到叶绿体基因组的表达调控过程。. .通过EMS对var6-1诱变，以第一对真叶白化为表型，筛选得到了一系列var6-1的抑制突变体sva（suppressor of var6-1）和增强突变体eva（enhancer of var6-1）。并对其中的sva 1（S01-03）进行了图位克隆及验证。对sva 1突变位点基因编码蛋白PRL1 (Pleiotropic Regulatory Locus 1)进行了初步的功能研究。这些抑制突变体的筛选及定位，为深入研究叶绿体基因组和细胞核基因组协同表达提供了新的材料与调控位点。