Screening Of Cyt F Interacting Proteins By Yeast Two Hybrid In Arabidopsis Thaliana

Arabidopsis(Arabidopsis thaliana.) is a model organism and ideal material for plant researches, which’s characteristics are fast growth, short multiplication cycle, blameless organs, diverse phenotypes and so on. As one of the important components for chloroplast electron transport chain, cytochrome f (Cyt f) locus itself on the chloroplast thylakoid membrane by its C-terminal tansmembrane region, and N terminal part is free in thylakoid matrix. Related paper reported that, chloroplast could induce Arabidopsis suspension cells’ progress cell death (PCD); the Cyt b6f complex has peroxidase activity, which could induce the HR of cells. Plants PCD is a research hotspot in recent years, and develops very fast. Plants PCD is throughout the whole life among seed germination, tissues or organs’ differentiation and development, immune defense against disease, stress reaction. Compared with animal cell apoptsis, plants PCD has some different characteristics, such as little apoptsis bodies, vacuoles taking part in, vacuole membrane fusion, pH falling in internal vacuole and so on.Arabidopsis Cyt f contains 320 amino acids, whose C terminal 18-20 residues form a transmembrane domain. Wild type (WT) Arabidopsis would be the material for experiments. The gene AtPetA which codes Cyt f was amplify by PCD with WT Arabidopsis cDNA as template. The fragment which codes transmembrane domain would be removed by primers design, and the last obtained fragment’s length was 861bp. We successfully constructed vector pGBKT7-AtPetA by making the fragment insert into bait vector pGBKT7. The results of toxic test and autoactivition identification for pGBKT7-AtPetA indicate that BD-AtCyt f fusion protein producted from Y2H Gold yeasts has no toxic effects on yeast propagation, and also couldn’t active transcription of the report genes HIS3, ADE2, MEL1, AURI-C. We use the recombinant bait vector for screening Arabidopsis cDNA library. Primarily,39 positive clones were obtained from SD/-Leu/-Trp/-His/-Ade plates. The 39 positive clones would be cultured on SD/-Leu/-Trp/-His/-Ade/X-a-gal/AbA plates repeatedly by streaking.6 positive clones:c-4, c-9, c-24, c-25, c-27, c-32 which could transform to blue clones and grow well would be remained. Recombinant prey vectors which were extracted from the 6 positive clones would be sequenced, and 5 candidate interacting proteins were discovered, which are P-loop containing nucleoside triphosphate hydrolases AtP-NTPase,26S proteasome subunit AtRPN9b, E3-ubiquitin ligase AtATL72, nuclear factor Y β subunit AtNF-YB13, calcium-dependent protein kinase AtCDPK32. Some papers reports shows that the candidate interacting proteins are associated with plant PCD or take part in the regulation of PCD.To identify the interactings between candidate proteins and AtCyt f, the recombinant prey vectors were co-transform into Y2H Gold strain with pGBKT7’-AtPetA. We observed the co-transformed yeast culture on SD/-Leu/-Trp/-His/-Ade/X-a-gal/AbA plates and discovered that AtATL72 showed the strongest ability to interact with AtCyt f; interactings between AtCyt f and AtP-NTPase, AtNF-YB13 were relatively weaker; AtCyt f had no interacting with AtRPN9b, AtCDPK32. Combining Arabidopsis cDNA library screening with the co-transforms results, we could get conclusion:AtCyt f interactings with AtATL72 was strongest, with AtP-NTPase, AtNF-YB13 were relatively weaker. AtATL72 could be AtCyt f interacting protein in PCD pathway.These results suggestted that Arabidopsis E3-ubiquitin ligase AtATL72 interacts with AtCyt f. Due to AtCyt f which was used for yeast two hybrid system was lack of C terminal transmembrane domain, we speculated that the interacting between AtCyt f and AtATL72 in plant cell cytoplasm, therefore the interacting may occur after AtCyt f released from chloroplast.