Screening Of Interacting Protein Of The PAC Protein:Essential Protein Of Chloroplast Development In Arabidopsis Thaliana

Photosynthesis of green plants has an essential value for the life of human. Therefore, chloroplast development research has attracted substantial attentions in the field of plant biology. Since chloroplast is the place where photosynthesis takes place, normal and healthy development of chloroplast is a prerequisite for plant in the process of establishing photosynthesis system. The Arabidopsis PALE CRESS(PAC) gene is one of the essential genes for chloroplast development. Loss of PAC leads to a seedling lethal phenotype. To elucidate the mechanism of how PAC is involved in regulating chloroplast development, we carried out yeast two hybrid screens for PAC interacting proteins. The main results obtained are as follows:1. Using an Arabidopsis leaf protoplast transient expression system, we determined that PAC-GFP fusion protein probably reside in chloroplast stoma.2. Taking advantage of the yeast two-hybrid system, we screened Arabidopsis cDNA library to identify potential interacting partners of the PAC protein. Two rounds of screening yielded a total of 214 positive clones, 78 of which harbor cDNA sequences that are in frame for Arabidopsis proteins. Among these in-frame cDNAs, 19 are part of the genes that encode chloroplast proteins.3. One of the possible PAC interacting proteins, Y79 is a key regulator during chloroplast development, so we verified the interaction of PAC and Y79 in the yeast.4. By constructing vector pET28a-PAC, we achieved the prokaryotic expression of PAC. Heterologously expressed PAC protein was purified via Nickel affinity chromatography, followed by electrophoretic elution from SDS-PAGE gel slices.5. To facilitate future study of PAC interacting proteins, we generated a polyclonal antibody against recombinant PAC.Taken together, this work laid the foundation for the future research on the biochemical mechanisms of how PAC exerts its role in chloroplast development.