亲免蛋白在光合大分子蛋白复合体组装过程中的功能研究

Improving photosynthetic efficiency by biotechnological means is a new frontier of plant biology. Currently, the precise processes of photosynthesis are well known; the composition, function, and structure of participating multi-protein complexes are also pretty clear; however, our knowledge about the assembly and maintenance of the photosynthetic multi-protein complexes is very limited. It hinders our further understanding of photosynthesis, also dampens the possibility of increasing photosynthetic capacity by genetic engineering approaches. Previously we reported that two chloroplast lumenal immunophilins (FKBP20-2 and CYP38) play significant roles in assembly and maintenance of photosystem II (PSII). It opens a door to understand the physiological function of chloroplast lumenal proteins. In the chloroplast lumen space, there are other 14 immunophilins with unknown functions. We hypothesize that those lumenal immunophilins may also play vital roles in the assembly processes of photosynthetic multi-protein complexes. In this proposal, our major goal is to elucidate the function of lumenal immunophilins during assembly process of photosynthetic complexes. To reach the purpose, firstly, we aim to create mutants of each immunophilin gene throughT-DNA mutagenesis and/or RNAi method; and then, we will dissect photosynthetic deficiency in each mutant line. After pinpointing which photosynthetic complexes were affected by lack of certain immunophilins, we plan to identify which subunit in the affected complex interacts with corresponding immunophilins via yeast two hybrid, co-immuno-precipitation, and in vitro pull-down methods. By using all those means, we can gain a deep insight on how immunophilins assist the assembly of photosynthetic complexes. For further understanding the full picture of photosynthetic complex assembly, we propose to build a protein-to-protein interaction network in the lumen with immunophilins serving as hubs, and to identify more assembly factors besides luminal proteins via forward genetics approaches. We believe that the results from this research program will enlarge current understanding on the biogenesis of photosynthetic apparatus. It will also enhance the chance to improve photosynthetic efficiency via bioengineering technologies, especially via synthetic biology strategies.

通过遗传工程提高光合效率是植物学的一个研究热点。光合作用的各个生化反应，以及参与其中的大分子蛋白复合体的结构与功能已有深入研究。但光合大复合体如何由其蛋白亚基组成的分子机制，我 们知之甚少。这严重影响了对光合机器的认识，也降低了对光合作用进行遗传操作的可能性。我们发现两个亲免蛋白参与光合系统 II的组装。叶绿体lumen中还有14个亲免蛋白功能未知。它们也可能在光合复合体的组装过程中有重要作用。本项目旨在从鉴定亲免蛋 白的突变体入手，揭示它们的生理功能和在光合复合体组装过程中的作用；再利用分子生物学的手段探讨亲免蛋白与靶蛋白的相互作用，分析其如何协助光合复合体组装；再以亲免蛋白为中心，构建Lumenal蛋白的互作关系网；并进一步用顺向遗传学方法鉴定更多的复合体组装因子，进而探索复合体组装的分子途径。 本项目不仅能增强对光合作用的了解，也能为提高光合效率、应对粮食和能源需求提供技术储备。

在植物叶绿体中有一组名为亲免蛋白的蛋白质, 它们可能在光合作用中有重要的生理功能, 但我们不解大部分的亲免蛋白的功能, 也对它们的作用机制一无所知，了解这些蛋白的功能与作用方式十分必要。. 本项目利用反向遗传学的手法, 从鉴定叶绿体亲免蛋白的突变体入手，发现了亲免蛋白CYP38, CYP28, FKBP20-2等对植物的叶绿体的功能有重要影响。细致的分析发现CYP38影响了PSII的组装与稳定，它可能是以一种分子间自抑制的方式来和PSII发生作用，它在PSII上的靶点可能是PSII的CP43和CP47亚基。CYP28 的缺失使植物早开花，非常有意思的是，CYP28的突变体比野生型的植株有更多地PSII超级复合物，CYP28可能是影响内囊体膜朵叠和PSII与PSI状态转换的重要因子。FKBP20-2的突变体生长矮小，植物对光强敏感， 是一个典型的PSII功能受损的突变体。有一些亲免蛋白的突变体和野生型植株生长相似，没有明显的不正常表型，表明这些亲免蛋白可能对植物的生命不是必须的。但它们在叶绿体中有很高的表达，说明它们对光合作用有一定的影响。. 有一些亲免蛋白没有现成的突变体，如CYP37，CYP26-2和FKBP17-2等。我们应用新一代的基因编辑技术CRISPR-CAS9成功地得到了这三个蛋白缺失的纯合突变体。CYP37的突变体表现出生长矮小和微黄的表型，光合膜上的大分子复合物的含量比野生型少。CYP26的突变体也比野生型的植株生长矮小，光合作用的效率更低，分析发现CYP26-2是一个内囊体膜的附着蛋白，它极有可能是内囊体膜上某一重要复合物的内囊体腔中的亚基。FKBP17-2的敲除突变体没有明显的不正常表现，但又意思的是:FKBP27-2有两种肽段的存在，它们的功能正在鉴定中。. 本项目还利用顺向遗传学的方法：T-DNA随机插入突变的方法来创建一个大规模的突变体库，为进一步研究大分子复合体的组装因子奠定基础。本项目不仅能增强对光合作用的了解，也能为提高植物光合效率、应对粮食和能源需求提供技术储备。