叶绿体蛋白OsPPR-N1和OsPPR-N2在水稻围绕光系统I循环电子传递中的调控机理研究

Cyclic electron flow around photosystem I is suggested to be essential for efficient photosynthesis. However, the regulative mechanism is remained to be clarified. We isolated two mutants defective in cyclic electrron flow from a mutation library using detection of a transient increase in chlorophyll fluorescence after termination of actinic light. We found that the mutations caused a defect in mature oligocistronic RNAs of NDH subunits, while the amounts of their precursor transcripts were significantly increased. Map-based cloning and genetic analysis indicate that the two proteins are novel nucleus-encoded PPR proteins and localize to the chloroplast. There are no homologous between them. We name the proteins as OsPPR-N1 and OsPPR-N2. So far, there is no related report about these genes. In the project, we focus on the study of cyclic electron flow and then investigate the regulative roles of OsPPR-N1 and OsPPR-N2 in NDH and other chloroplastic genes by genetic and biochemical methods. Further study the regulative mechanism and look for the proteins involved in these regulations by co-immunoprecipitation, yeast two-hybrid, in vitro pull down, RNA-protein binding and bioinformation methods. The goal of the project is to reveal the regulative roles of these novel proteins in NDH and photosynthesis.

围绕光系统I的循环电子传递为高效光合作用所必需，然而其调控机制尚有待于揭示。我们从水稻突变体库中筛选到两个循环电子传递失活的突变体，这两个突变都导致介导循环电子传递的NADPH脱氢酶（NDH）复合体相关亚基的成熟RNA的缺失以及其转录本前体的积累，从而影响这些蛋白的积累。图位克隆和遗传学功能验证表明，这两个新基因都是编码功能未知、定位于叶绿体的PPR蛋白，它们之间没有同源性，我们命名为OsPPR-N1和OsPPR-N2。本项目拟利用分子遗传学和生化方法，研究OsPPR-N1和OsPPR-N2对NDH等叶绿体基因的调控作用。并进一步利用免疫共沉淀、酵母双杂交、体外蛋白结合和RNA-蛋白结合实验，生物信息学等技术手段，深入研究OsPPR-N1和OsPPR-N2调控叶绿体NDH相关亚基的机理，并寻找到参与这些调控途径的相互作用蛋白, 以揭示这两个新功能蛋白在NDH以及光合作用中的网络调控机制。

围绕光系统I的循环电子传递为高效光合作用所必需，然而其调控机制尚有待于揭示。我们从水稻突变体库中筛选到两个循环电子传递失活的突变体，这两个突变都导致介导循环电子传递的NADPH脱氢酶（NDH）复合体相关亚基的成熟RNA的缺失以及其转录本前体的积累，从而影响这些蛋白的积累。图位克隆和遗传学功能验证表明，这两个新基因都是编码功能未知、定位于叶绿体的PPR蛋白，它们之间没有同源性，我们命名为OsPPR-N1和OsPPR-N2。本项目研究发现，OsPPR-N1参与核糖体基因内含子的剪接，进而使成熟的mRNA核糖体能够正常组装；而OsPPR-N2参与rpl20 3’-258到3’-66的剪切加工，使叶绿体的翻译机构中的叶绿体蛋白能够正常地合成。因此，OsPPR-N1和OsPPR-N2都参与叶绿体翻译系统的功能，在叶绿体基因表达中发挥重要的作用。